Regulation of the 24-Hydroxylase Gene Promoter by 1,25-Dihydroxyvitamin D3 Adn Chemothereapeutics Drugs
Regulation of the 24-Hydroxylase Gene Promoter by 1,25-
Dihydroxyvitamin D3 and Chemotherapeutics Drugs.
JOSEPH TAN CHENG TA
BHlthSc, Honours Sc
Thesis submitted for the degree of Doctor of Philosophy
Department of Paediatrics
The University of Adelaide
(Faculty of Health Sciences)
October 2005 Declaration
This work contains no material which has been accepted for the award of any other degree or diploma in any university or other tertiary institution and, to the best of my knowledge and belief, contains no material previously published or written by another person, except where due reference has been made in the text.
I give consent to this copy of my thesis when deposited in the university library, being available for loan and photocopying.
…………………………………….. ………………..
Joseph TanCheng Ta Dated Acknowledgement
Firstly, I would like to take this opportunity to express my sincere thanks to my principal supervisor, Associate Professor Charles Hii for his continuous supervision, support, encouragement and friendship which has made my PhD study an enjoyable experience.
I would also like to thank my co-supervisor Professor Antonio Ferrante for his guidance, patience, encouragement, friendship and support.
My special thanks to Dr Prem Dwivedi, with whom I worked closely, for his valuable supervision, great discussions and long lasting friendship. Also, special thanks to
Professor Brian May, Professor Howard Morris and Dr Peter O’Loughlin for their guidance and for allowing me to use their laboratories to undertake some aspects of my study. Many thanks to Barbara Nutchey, Dr Yong Lee, Dr Sophia Gao and Dr Paul
Anderson for their endless help, great discussion and friendship. A special thanks to all the PhD and honours students in the Department of Immunopathology for their great friendship and jokes that we shared. Also, many thanks to Judy Ferrante, Trish Harvey,
Lily Zedda, Bernadette Miller, Jess Franks, Reneé Mackenzie and other members of the
Department of Immunology, WCH, for their help and in making the laboratory a wonderful place to work in.
Last but not least, I would like to express my gratitude and thanks to God for his support, wisdom and strength, my wonderful family: my parents, my brothers and sisters for their continuous support and encouragement, to my dearest wife Ya-Hui and daughter Catrina, for their endless love, support, encouragement and for making my PhD years a meaningful and worthwhile experience.
ii Summary
Chemotherapy in childhood cancer patients is associated with reduced bone density that can result in osteoporotic fracture in survivors. A significant proportion of paediatric patients experience a reduction in plasma 25-hydroxyvitamin D3 [25(OH)D3] and 1,25- dihydroxyvitamin D3 [1,25(OH)2D3] levels during treatment, the basis of which is unknown. A balance between the bioactivation and degradation of 1,25(OH)2D3 is responsible for maintaining homoeostatic levels of 1,25(OH)2D3 at the correct set-point.
Whereas the cytochrome P450 enzyme, CYP27B1 (25-hydroxyvitamin D3 1α- hydroxylase), catalyses the hydroxylation of the precursor 25(OH)D3 to generate
1,25(OH)2D3, catabolic inactivation and cleavage of 1,25(OH)2D3 is achieved by the mitochondrial cytochrome P450 enzyme, 25-hydroxyvitamin D3 24-hydroxylase
(CYP24), which is highly expressed in bone and kidney cells. Since many of the signalling pathways which regulate the expression of CYP24 are also activated by chemotherapeutic drugs, we hypothesised that the drugs could cause the degradation of plasma 25(OH)D3 and 1,25(OH)2D3 by increasing CYP24 expression, the principal means of facilitating the bio-inactivation and degradation of plasma 25(OH)D3 and
1,25(OH)2D3. Using the kidney cell-lines, COS-1 and HEK293T cells, we now report that chemotherapeutic drugs, represented by daunorubicin hydrochloride (an anthracycline antibiotics), etoposide and vincristine sulphate (vinca alkaloids and related compounds) and cisplatin (an alkylating agent), were able to enhance CYP24 promoter activity in kidney cell lines transfected with a CYP24 promoter-luciferase construct, either by themselves or in the presence of 1,25(OH)2D3. Dose-response studies with
iii daunorubicin hydrochloride and etoposide, two of the strongest inducers of CYP24 promoter activation under our experimental conditions, demonstrate that these drugs acted in a concentration-dependent manner. In addition to stimulating promoter activity on their own, the drugs also amplified the induction of the CYP24 promoter by
1,25(OH)2D3. Synergistic increases were generally observed when the cells were treated simultaneously with 1,25(OH)2D3 and a drug. The two kidney cell lines generally responded in a similar manner when challenged with the drugs, either in the presence or absence of 1,25(OH)2D3. Interestingly, the hydroxylated derivative of daunorubicin hydrochloride, doxorubicin hydrochloride which is also a commonly used chemotherapeutic drug, had no effect of promoter activity. Further studies with daunorubicin hydrochloride demonstrated that the effects of the drug per se were not mediated by oxidative stress and the vitamin D receptor was not required for daunorubicin hydrochloride per se to stimulate CYP24 promoter activity. However, daunorubicin hydrochloride caused a modest increase in the expression of the vitamin D receptor and this could contribute to its synergistic activity with 1,25(OH)2D3. In the presence of etoposide, there was also a tendency for the kidney cells to express higher levels of the vitamin D receptor. A key role for the extracellular signal-regulated protein kinase (ERK)1, ERK2 and ERK5 mitogen-activated protein (MAP) kinases was demonstrated for the inductive action of daunorubicin hydrochloride and etoposide, with
CYP24 promoter-specific transcription factors located in the first –298bp being likely targets of the ERK activity. Studies with a dominant negative mutant of MKK4, one of the two immediate upstream activators of the c-jun N-terminal kinase isoforms, demonstrated that this MAP kinase also played a crucial role in inductive actions of the
iv drugs. Consistent with their use in anti-cancer therapy, all of the above drugs killed the human promyelocytic HL60 leukaemic cells at very low concentrations but had no effect on the viability of kidney or liver cells, either at concentrations used in our experiments or at higher levels. Our data provide novel biochemical evidence that some of the commonly used chemotherapeutic drugs could cause an increase in the transcriptional activation of the promoter, most likely via the MAP kinases activating the transcription factors which bind to the CYP24 promoter. Such an effect could contribute to the reduction in plasma 25(OH)D3 and 1,25(OH)2D3 in some of the patients undergoing chemotherapy.
v Abreviations
1,25(OH)2D3/ 1,25D 1,25-dihydroxyvitamin D3
24,25(OH)2D2 24,25-dihydroxyvitamin D3
25(OH)D3 25-hydroxy vitamin D3
AP1 activator protein 1
BDNF brain-derived neurotrophic factor
CaT1 calcium transport protein
COS-1 African monkey kidney fibroblast cells
Cx43 connexin 43; a gap junction protein
CYP24 24-hydroxylase enzyme
CYP27b1 1α-hydroxylase enzyme
DN dominant negative
EBS Ets-1 binding site
ECaC2 epithelial calcium channel protein
EGF epithelial growth factor
ERK extracellular signal-regulated kinase
ERK5 Big MAP kinase 1
EtOH ethanol
Ets E26
FGF-2 fibroblast growth factor-2
HEK293T transform human embryonic kidney cells
HL60 human promyelocytic leukaemic
HPV human papillomaviruses
vi IL interleukin
JNK c-Jun NH2-terminal kinases
LY294002 PI3K inhibitor
MAPK mitogen-activated protein kinase
MEF2 myocyte enhancer factor 2
NCX1 Na+/Ca2+ exchanger pump
NF-Y transcription factor nuclear factor Y
NGF nerve growth factor
OPG osteoprotegerin gene
PDK phosphoinositide-dependent kinase
PI3K phosphatidylinositol 3-kinase
PKA protein kinase A
PKC protein Kinase C
PMA phorbol 12-myristate 13-acetate
PMCA plasma membrane calcium ATPase
PTH parathyroid hormone
RANKL NFkappa-B ligand
Ras small GTP-binding and hydrolyzing proteins
RXR retinoic X receptor
Sap1 Ets-domain transcription factor
SGK serum- and glucocorticoid-inducible kinase
Sp1 specific protein 1
vii TGF-β transforming growth factor-beta
Th T helper
Tpl tumor progression locus
TRPV transient receptor potential vanilloid
VDR vitamin D receptor
VDRE vitamin D responsive element
VEGF vascular endothelial growth factor
VP-16 etoposide
VSE vitamin D stimulatory element
viii CONTENTS
DECLARATION ...... ii ACKNOWLEDGEMENTS ...... iii THESIS SUMMARY...... iv ABREVIATIONS ...... vi
Chapter 1...... 1
Introduction ...... 1
1.1 General Introduction...... 2 1.2 Overview of the actions of vitamin D and its metabolism ...... 4 1.3 Biological Functions of vitamin D ...... 7 1.3.1 Vitamin D and bone...... 7 1.3.2 Vitamin D and Calcium Absorption ...... 14 1.3.3 Vitamin D and Cancer ...... 16 1.3.4 Vitamin D and the Immune system ...... 18 1.4 Vitamin D receptor...... 22 1.4.1 Nuclear VDR...... 22 1.4.2 Plasma membrane VDR ...... 23 1.4.3 Regulation of VDR expression...... 24 1.4.4 Non-classical VDRs ...... 25 1.4.4.1 Annexin II...... 25 1.4.4.2 1,25D3-MARRS ...... 26 1.5 Non-genomic action of vitamin D...... 28 1.6 Vitamin D and intracellular signaling ...... 29 1.6.1 Mitogen-activated protein kinases...... 29 1.6.1.1 ERK1/ERK2 ...... 30 1.6.1.3 JNK...... 34 1.6.2 Phosphathdylinositol 3-kinase...... 36 1.6.3 Protein Kinase C...... 41 1.6.4 Ras ...... 43 1.7 Enzymes involved in vitamin D Metabolism ...... 45
1 1.7.1 Hepatic 25-hydroxylase enzyme...... 45 1.7.2 Kidney 1α-hydroxylase enzyme...... 46 1.7.2.1 Role of 1α-hydroxylase...... 46 1.7.2.2 Regulation of the 1α-hydroxylase ...... 50 1.7.3 24-hydroxylase enzymes (CYP24) ...... 51 1.7.3.1 Regulation of the CYP24 promoter by vitamin D...... 53 1.7.3.2 Signaling pathways that regulate the CYP24 promoter in response to vitamin D...... 56 1.8 Regulation of CYP24 by compounds other than vitaminD...... 59 1.9 Chemotherapy and plasma vitamin D ...... 61 1.10 Chemotherapeutic drugs...... 63 1.10.1 Categories of chemotherapeutic drugs...... 64 1.10.2 Principal targets of Chemotherapeutic drugs: the cell cycle and apoptosis...... 65 1.10.3 Actions of anticancer drugs ...... 66 1.10.3.1 Daunorubicin hydrochloride...... 67 1.10.3.2 Etoposide ...... 69 1.10.3.3 Vincristine Sulfate ...... 70 1.10.3.4 Cisplatin...... 71 1.11 Activation of cell signaling molecules by chemotherapeutic drugs...... 72 1.12 Hypotheses ...... 78 1.13 Aims ...... 79 Chapter 2...... 80
Materials and Methods...... 80
2.1 Materials...... 81 2.1.1 General chemicals, drugs and reagents...... 81 2.1.2 Reagents for Western blots and Kinase assays...... 81 2.1.3 Chemotherapeutic drugs ...... 82 2.1.4 Reagents for lysis, washing and assay buffer, ...... 82 2.1.5 Reagents for transfection ...... 83 2.1.6 Reagents for Luciferase Assay ...... 83 2.1.7 Antibodies...... 83 2.1.8 Serum, Culture media and buffers ...... 83 2.1.9 Flasks and plates for culture ...... 84 2.1.20 Cell lines...... 84 2.1.21 Plasmids/Vectors ...... 84 2.1.22 Buffers and Solutions ...... 88 2.1.23 Reagents for RNA extraction and real time PCR ...... 89
2 2.2 Methods...... 90 2.2.1 Cell Culture ...... 90 2.2.1.1 Culture of HEK293T and COS-1 cells...... 90 2.2.1.2 Cell Culture for kinase assay and for western blotting...... 90 2.2.1.3 Culture of HL 60 cells...... 91 2.2.2 Cell Viability Assay using trypan blue...... 91 2.2.3 Plasmid DNA Preparation and Transfection...... 92 2.2.3.1 Midi-preparation using midi-plasmid DNA preparation kit (Qiagen) ...... 92 2.2.3.2 Large scale preparation by cesium-chloride (CsCl) density gradient procedure...... 92 2.2.3.4 Dual Luciferase assay ...... 94 2.2.4 Preparation of cell lysates...... 94 2.2.5 Lowry’s Protein determination ...... 95 2.2.6 Kinase Assay and Western Blotting ...... 95 2.2.6.1 Purification of GST-jun (1-79) ...... 95 2.2.6.2 JNK kinase assay ...... 96 2.2.6.3 ERK1/ERK2/ERK5 kinase assays...... 97 2.2.6.4 Western Blotting...... 97 2.2.6.5 Western Blot recycling ...... 99 2.2.7 Quantification of mRNA expression using real-time PCR...... 99 2.2.7.1 RNA Extraction ...... 99 2.2.7.2 Generation of cDNA...... 99 2.2.7.3 Real-time PCR...... 100 2.2.8 Statistics...... 100 Chapter 3...... 101 Effects of chemotherapeutic drugs on the induction of the
CYP24 promoter in kidney cells...... 101
3.1 Introduction ...... 102 3.2 Methods for investigating CYP24 promoter induction...... 103 3.3 Results ...... 104 3.3.1 Effects of chemotherapeutic drugs on the activity of the -1.4kb CYP24 promoter in COS-1 and HEK293T kidney cells...... 104 3.3.2 Effects of chemotherapeutic drugs on the activity of the -298 bp CYP24 promoter in COS-1 kidney cells...... 108 3.3.3 Daunorubicin hydrochloride stimulated the –1.4kb CYP24 promoter activity in a dose- dependent manner in COS-1 kidney cells...... 112
3 3.3.4 Dose-dependent effect of daunorubicin hydrochloride on the -298bp CYP24 promoter in COS-1 cells and HEK293T cells...... 114 3.3.5 Etoposide stimulated the -1.4kb CYP24 promoter activity in a dose-dependent manner in COS-1 kidney cells...... 117 3.3.6 Doxorubicin hydrochloride did not stimulate the activity of the -298bp CYP24 promoter or enhance CYP24 induction by 1,25(OH)2D3 in COS-1 cells...... 119 3.4 Summary ...... 121 Chapter 4 Mechanism I...... 124 Mechanism of action of daunorubicin hydrochloride and etoposide on CYP24 promoter activity: Role of reactive oxygen species (ROS), VDR, VDRE and Sp1...... 124
4.1 ROS are not required for CYP24 promoter induction by daunorubicin hydrochloride and etoposide in COS-1 cells...... 125 4.2 Over expression of VDR did not enhance CYP24 promoter induction by daunorubicin hydrochloride in COS-1 and HEK293T cells...... 127 4.3 VDRE and GC box are not involved in CYP24 promoter induction by daunorubicin hydrochloride in COS-1 cells...... 131 4.4 Daunorubicin hydrochloride did not alter the activity of the VDR in COS-1 cells...... 133 4.5 Daunorubicin hydrochloride and etoposide increased the expression of VDR in COS-1 and HEK293T cells...... 135 4.6 Summary ...... 138 Chapter 5 Mechanism II ...... 139 Role of MAP Kinases in 1,25(OH)2D3 mediated transactivation regulation of the CYP24 promoter in COS-1 cells...... 139
5.1 Introduction ...... 140 5.1.1 Vectors used: ...... 142 5.2 Results:...... 143 5.2.1 1,25(OH)2D3 stimulated phosphorylation of ERK1/ERK2 in COS-1 cells...... 143 5.2.2 1,25(OH)2D3 stimulated the activity of ERK5 activity in COS-1 cells ...... 145 5.2.3 Dominant negative ERK1 and MEK5 inhibited 1,25(OH)2D3 mediated induction of the CYP24 promoter...... 148
4 5.2.4 Dominant negative Ras inhibited the stimulation of ERK1/ERK2 activity by 1,25(OH)2D3 in COS-1 cells...... 151 5.2.5 Dominant negative Ras inhibited the stimulation of ERK5 activity by 1,25(OH)2D3 in COS- 1 cells. 152 5.2.6 Dominant negative Ras inhibited CYP24 promoter induction by 1,25(OH)2D3 ...... 155 5.2.7 ERK2 interacted with RXRα independently of 1,25(OH)2D3...... 155 5.2.8 Phosphorylation of RXRα was dependent on the interaction between activated ERK1/ERK2 and RXRα...... 158 5.2.9 RXR phosphorylation played an important role in CYP24 induction by 1,25(OH)2D3. ... 160 5.2.10 Interaction between ERK5 and Ets-1 was 1,25(OH)2D3-dependent...... 162 5.2.11 1,25(OH)2D3 stimulated the phosphorylation of Ets-1 protein...... 164 5.2.12 Phosphorylation of Ets-1 requires the activation of HA-ERK5 by MEK5 and Ras...... 166 5.2.13 CYP24 promoter induction by 1,25(OH)2D3 required the Ets binding site (EBS) and the ERK5 module...... 169 5.3 Summary ...... 171 Chapter 6 Mechanism III...... 173 Role of MAP kinases, PI3K and PKC in mediating CYP24 promoter induction by daunorubicin hydrochloride and etoposide...... 173
6.1 Introduction ...... 174 6.2 Results ...... 174 6.2.1 Dominant negative ERK1 and MEK5 prevent the induction of CYP24 promoter activity by daunorubicin hydrochloride in COS-1 cells...... 174 6.2.2 Daunorubicin hydrochloride stimulated the activities of ERK1/ERK2 and ERK5 in COS-1 cells. 175 6.2.3 Dominant negative MKK4 inhibited daunorubicin hydrochloride induction of CYP24 promoter activity in COS-1 cells...... 177 6.2.4 Daunorubicin hydrochloride stimulated JNK activity in COS-1 cells...... 180 6.2.5 Dominant negative PKCζ inhibited daunorubicin hydrochloride induction of CYP24 promoter activity in COS-1 cells...... 183 6.2.6 Inhibitor of PI3K (LY294002) suppressed daunorubicin hydrochloride induction of CYP24 promoter activity in COS-1 cells...... 186 6.2.7 Inhibition of daunorubicin hydrochloride-mediated transcriptional activation of the CYP24 promoter activity by dominant negative ERK1 and MEK5 in HEK293T cells...... 188
5 6.2.8 Daunorubicin hydrochloride stimulated ERK1/ERK2 and ERK5 activities in HEK293T cells. 191 6.2.9 Effect of dominant negative MKK4 on daunorubicin hydrochloride-and 1,25(OH)2D3- mediated induction of CYP24 promoter activity in HEK293T cells...... 194 6.2.10 Daunorubicin hydrochloride stimulated JNK activity in HEK293T cells...... 196 6.2.11 PKCζ was not required for the induction of the CYP24 promoter by daunorubicin hydrochloride and 1,25(OH)2D3 in HEK293T cells...... 196 6.2.12 Role of MAP Kinases and PKCζ in the induction of CYP24 promoter by etoposide in COS- 1 cells. 198 6.2.13 Dominant negative ERK1 and not MEK5 inhibited etoposide mediated induction of the CYP24 promoter activity in COS-1 cells...... 200 6.2.14 Etoposide stimulated ERK1/ERK2 activity in COS-1 cells...... 202 6.2.15 Dominant negative MKK4 but not PKCζ inhibited etoposide-mediated induction of the CYP24 promoter activity in COS-1 cells...... 202 6.2.16 Etoposide stimulated JNK activity in COS-1 cells...... 205 6.3 Summary ...... 208 Chapter 7...... 209 Effects of chemotherapeutic drugs on endogenous CYP24 mRNA expression in kidney cells ...... 209
7.1 Introduction ...... 210 7.2 Methods...... 210 7.3 Results ...... 211 7.3.1 Daunorubicin hydrochloride and etoposide inhibited the expression of endogenous CYP24 by 1,25(OH)2D3...... 211 7.3.2 Pre-treatment of 1,25(OH)2D3 prevented etoposide from suppressing the induction of CYP24 mRNA expression by 1,25(OH)2D3...... 211 3.3.3 Etoposide stimulated the degradation of CYP24 mRNA...... 214 7.4 Summary ...... 216 Chapter 8...... 217
Discussion ...... 217
8.1 Discussion ...... 218 8.1.1 Anti-cancer drugs and CYP24 promoter activity...... 218 8.1.2 Cytotoxic effects of the drugs in HL60 cells but not in kidney cells...... 220 8.1.3 Lack of a role for the VDR and VDREs in the actions of the drugs...... 221
6 8.1.4 Daunorubicin and etoposide increased the expression of the VDR...... 222 8.1.5 Reactive oxygen species and the promoter inducing effect of daunorubicin and etoposide223 8.1.6 Non-genomic actions of 1,25(OH)2D3 involving intracellular signalling molecules ...... 224 8.1.7 Anti-cancer drugs and intracellular signalling molecules in CYP24 promoter activation.. 227 8.1.8 Suppression of CYP24 mRNA induction by daunorubicin and etoposide ...... 230 8.1.9 Clinical implications of this work and future directions...... 231 8.2 Summary ...... 234 References...... 237
7
Chapter 1
Introduction
1 1.1 General Introduction
The biologically active form of vitamin D, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], also known as calcitriol, is a secosteroid hormone that plays important roles in a range of physiological processes, including calcium and phosphate homeostasis and bone remodelling (Holick et al., 2003), cellular proliferation and differentiation (Brown et al.,
1999) and immune regulation (Griffin et al., 2003). Thus, it is crucial that the circulating concentration of 1,25(OH)2D3 is maintained at an appropriate level.
Children undergoing chemotherapy has reduced bone mineral mass and density, and are likely to get fractures and skeletal defects during and at the end of the treatment period and beyond (Kaste, 2004). Recent studies have found that these children have reduced plasma levels of 25-hydroxy vitamin D3 (25(OH)D3) and 1,25(OH)2D3 and that the levels of vitamin D metabolites remain low even after the completion of chemotherapy
(Halton et al., 1996; Atkinson et al., 1998; Arikoski et al., 1999a, 1999b).
The homeostatic levels of 25(OH)D3 and 1,25(OH)2D3 are regulated by a series of enzymes which include the 25-hydroxylase, 1α-hydroxylase enzyme (CYP27b1) and 24- hydroxylase enzyme (CYP24). The 25 hydroxylase and the 1α-hydroxylase are involved in the biosynthesis of 25(OH)D3 and the 1,25(OH)2D3, respectively (Jones et al., 1998).
On the other hand, the 24-hydroxylase plays a role in the biodegradation of these hormones (Jones et al., 1998). There are many factors which could potentially lead to the low serum levels of vitamin D seen in these cancer patients. However, based on the
2 evidence that CYP24 biodegrades both 25(OH)D3 and 1,25(OH)2D3, it is possible that
the reduction in plasma 25(OH)D3 and 1,25(OH)2D3 seen in these children is the result
of increased CYP24 action/expression. Currently, it is not known whether
chemotherapeutic drugs alter CYP24 expression or action and this forms the focus of this
thesis.
The CYP24 promoter is highly sensitive to 1,25(OH)2D3 stimulation. The action of
1,25(OH)2D3 is initiated through the binding of the hormone to a vitamin D receptor
(VDR) located either in the plasma membrane or cytoplasm (Norman et al., 2001; Erben et al., 2002). The binding of 1,25(OH)2D3 to VDR in turn activates both genomic and non-genomic responses to induce the transcriptional activation of the CYP24 promoter
(Norman et al., 2001). The genomic response involves the binding of the VDR/retinoic X receptor (RXR) complex to the CYP24 promoter (Dwivedi et al., 2000). The non- genomic response is due to the activation of signaling molecules such as the extracellular signal-regulated kinase (ERK)1 and ERK2, Big MAP kinase 1 (ERK5), c-Jun NH2- terminal kinases (JNK), Protein Kinase C (PKC) and phosphatidylinositol 3-kinase
(PI3K) which have been recently shown to regulate the CYP24 promoter through the phosphorylation of a variety of transcription factors (Dwivedi et al., 2000; Pearson et al.,
2001; Dwivedi et al., 2002; Nutchey et al., 2005). Interestingly, chemotherapeutic drugs
can also stimulate the activities of these signaling molecules (Nowak, 2002; Boldt et al.,
2002; Hershberger et al., 2002; Hayakawa et al., 2003; Mas et al., 2003). Thus, it is
possible that these drugs, through their ability to stimulate the activities of the above
signaling molecules, could increase CYP24 expression.
3 This chapter will review the literature on the physiological significance of 1,25(OH)2D3, how 1,25(OH)2D3 acts and how 1,25(OH)2D3 homeostasis is achieved through a series of enzymes that synthesize and degrade it. This chapter will also provide a discussion on the molecular mechanisms that have been implicated in regulating the transcriptional activation of the CYP24 promoter via both genomic and non-genomic routes, the later involving a series of intracellular signaling molecules. An overview of chemotherapeutic drugs and their modes of action will also be presented, leading to the formulation of my hypothesis and aims of my studies.
1.2 Overview of the actions of vitamin D and its metabolism
1,25(OH)2D3 is crucial for maintaining proper physiological function in human. It acts
via the vitamin D receptor (VDR). A deficiency in 1,25(OH)2D3 can result in
osteomalacia in adults and rickets among children (Reginato and Coquia, 2003).
1,25(OH)2D3 has been shown to play an important role in maintaining calcium and
phosphate homeostasis, cell differentiation and proliferation, bone homeostasis,
regulation of immune cells, and many other important functions (DeLuca, 2004;
Cantorna et al., 2004, Christakos et al., 2003), each of which will be discussed in detail below.
The physiological levels of 1,25(OH)2D3 are regulated by a series of enzymes which belong to the class of mitochondrial and microsomal cytochrome P450-dependent hydroxylases that participate in the synthetic pathways of the adrenal and gonadal steroid
4 hormones (Henry, 2001). Figure 1.1 shows a cartoon of 1,25(OH)2D3 metabolism. The biologically inactive precursor, vitamin D3 is mainly synthesized from 7- dehydrocholesterol in the basal epidermal layer of the skin through exposure to UV radiation (290nm-315 nm) (Holick, 2004). Vitamin D3 is then transported to the liver by vitamin D binding protein (DBP) where it is hydroxylated by vitamin D3-25-hydroxylase at carbon atom 25 to form 25(OH)D3 (Jones et al., 1998). The 25(OH)D3 is transported to the kidney where it is further hydroxylated by 25-hydroxyvitamin D3 1α-hydroxylase
(CYP27B1 or 1α(OH)ase) at carbon atom 1 to form the biologically active
1,25(OH)2D3. 1,25(OH)2D3 is inactivated or degraded in the kidney by 25- dihydroxyvitamin D3 24-hydroxylase (CYP24) (Jones et al., 1998). Unlike the 25- hydroxylation in the liver which appears to be loosely regulated, both the 1α- hydroxylation and C23/C24 hydroxylation are tightly regulated and appears to play an important role in regulating ambient levels of serum and cellular 1,25(OH)2D3 (Omdahl et al., 2002). Although the CYP27B1 and CYP24 are found in almost all the cell types in the body, the kidney is the major site for the biosynthesis and degradation of circulating
1,25(OH)2D3 (Holick, 1997; Jones et al., 1998). These topics will be discussed in greater detail in the following sections.
5 Liver
UV light
25-hydroxylase Skin 7, 8-dehydrocholesterol Pre-Vitamin D3 Vitamin D3
25(OH)D3
Gastrointestinal Diet CYP27B1
Track OH 1,25(OH)2D3
Kidney CYP24 HO OH
24,25(OH)2D3 1,24,25(OH)3D3 Circulatory 1,25(OH)2D3 Calcitronic acid
Excreted
Figure 1.1 Summary of the biosynthesis and biodegradation of 1,25(OH)2D3
Vitamin D3 is obtained from dietary sources or produced in the skin by the photolytic
cleavage of 7-dehydrocholesterol followed by thermal isomerization. Vitamin D3 is
transported to the liver via the serum vitamin D binding protein, where it is converted to
25-hydroxyvitamin D3, the major circulating metabolite of vitamin D3. The final
activation step, 1α-hydroxylation is carried out by 1α-hydroxylase (CYP27B1) and
occurs primarily, but not exclusively, in the kidney, forming 1,25(OH)2D3, the
hormonally active form of the vitamin. Catabolic inactivation is carried out by 24-
hydroxylase (CYP24), which catalyzes a series of oxidation steps, resulting in side chain
cleavage.
6 1.3 Biological Functions of vitamin D
Traditionally, 1,25(OH)2D3 was considered to play an important role in regulating
calcium homeostasis and bone mineralization. However, more recent studies show that
1,25(OH)2D3 also regulate cell proliferation and differentiation. Additionally,
1,25(OH)2D3 has been demonstrated to regulate the immune system, central nervous
system, reproduction and plays a protective role against cancer (Christakos et al., 2003;
Henry and Norman, 1984).
1.3.1 Vitamin D and bone
1,25(OH)2D3 plays a crucial role in bone development. Bone development is dependent
on the properly regulated differentiation, function and interaction of various types of
bone cells, which include chondrocytes, osteoblasts and osteoclasts cells (Stains and
Civitelli, 2005; Provot and Schipani, 2005). Lack of vitamin D causes rickets in children and osteomalacia in adults.
Vitamin D deficiency in infants can result from a number of factors. These range from an abnormality in vitamin D metabolism, dietary deficiency, metabolic errors to end-organ resistance due to mutations in vitamin D receptor (VDR) (Berry et al., 2002; Pettifor,
2004; Malloy et al., 2004). These defects can result in rickets and osteomalacia. The symptoms include bone pain, failure to thrive, and deformity of the thorax, resulting from severe deformities of the long bone and spine (Wharton and Bishop, 2003). Bone lesions
7 mainly result from inadequate mineralization and deposition of calcium to the growth
plate. This allows the cartilage to continue proliferating, resulting in irregular formation
of under mineralized bone trabeculae in the region directly underlying the growth plate
cartilage. Abnormality in mineralization also occurs in the diaphysis, affecting both the
cortical and trabecular bone. This mineralization defect causes the widening of osteoid
seams, that is, an increase in the amount of un-mineralized bone matrix located between
the layer of osteoblasts and the fully mineralized bone (St-Arnaud and Glorieux, 1997).
Vitamin D deficiency in adults occurs mainly in the sick, the elderly and Middle Eastern
people who lack exposure to sunlight (Lyman, 2005; Richardson, 2005; Flicker et al.,
2003; Venning, 2005; Brock et al., 2004). Low levels of circulating 25(OH)D3 are
normally detected during winter time, especially in people living in Europe (Lamberg-
Allardt et al., 2001; Carnevale et al., 2001). However in Australia, with the heightened awareness of ozone depletion and increasing incidence of skin cancer; there is a general fear among Australians towards exposure to sunlight. With the use of sun creams and clothing to block out UV radiation, there is thus an increase in the number of people, especially people with darker skin, with insufficient levels of 25(OH)D3 (Nowson et al.,
2004)
The abnormality in bone mineralization in humans such as seen in patients with osteomalacia and rickets can be cured by treating the patients with vitamin D metabolites
(Prince and Glendenning, 2004; Harrell et al., 1985). It was observed that the healing of bone lesions only occurs after normalization of blood concentrations of calcium and
8 phosphate. Furthermore, experiments using vitamin D-deficient animals and VDR-null mice showed that normal cartilage and bone mineralization can be achieved after circulating levels of calcium, phosphate and lactose were normalized by intravenous infusion or dietry supplement (Weinstein et al., 1984; Underwood and DeLuca, 1984;
Marie et al., 1982; Holtrop et al., 1986; Amling et al., 1999; Li et al., 1998). Based on the above observations, it was concluded that vitamin D per se is not essential for bone mineralisation and that the main mechanism by which vitamin D restores bone mineralization is due to the restoration of circulating concentrations of calcium and phosphate (i.e. by maintaining intestinal calcium adbsorption). Other observations that support this hypothesis include studies on hereditary vitamin D rickets patients, who carry mutations in the VDR. These patients do not respond to vitamin D treatment.
However, treatment with intravenous calcium and oral phosphate supplementation to normalize their blood concentrations of calcium and phosphate cured the patients of rickets and osteomalacia (Bliziotes et al., 1988; Weisman et al., 1987; Hochberg et al.,
1992; al-Aqeel et al., 1993; Balsan et al., 1986). Thus, it can be concluded that vitamin D plays an important role in bone development by maintaining calcium and phosphate homeostasis.
Nevertheless, various in vitro and in vivo studies have shown that 1,25(OH)2D3 has some roles in bone formation and maintenance. A study by Dardenne et al., (2004) demonstrated that replacement therapy such as feeding with a diet containing calcium, phosphate and lactose to restore normocalcemia in CYP27b1-null mice was not completely effective at normalizing bone growth in a similar manner to 1,25(OH)2D3
9 replacement therapy. This finding suggests that beside maintaining calcium and
phosphate levels, 1,25(OH)2D3 has a direct role in bone growth. It has also been shown
that 1,25(OH)2D3 regulates the proliferation and differentiation of immature osteoblast
(osteoprogenitors cells) into mature osteoblast cells (Siddhanti and Quarles, 1994).
The maintenance of bone homeostasis is a balance between continuous bone formation by osteoblasts and bone resorption by osteoclasts. 1,25(OH)2D3 can also act directly on osteoblasts and osteoclasts to regulate this process. The evidence for the interaction between 1,25(OH)2D3 and bone cells comes from the identification of VDR in osteoblast and osteoclast precursor cells (Mee et al., 1996; van Leeuwen et al., 2001). Furthermore, it was shown that VDR in osteoblast was essential for 1,25(OH)2D3 mediated effect.
Studies by Takeda et al., (1999) demonstrated that osteoblasts from VDR knockout mice were unable to stimulate the differentiation of osteoclasts.
Beside the presence of VDR, bone cells have been shown to possess enzymes that can synthesize and inactivate 1,25(OH)2D3 (Anderson et al., 2005). Thus, CYP24, CYP27b1
and CYP25 have all been found in osteoblast (Staal et al., 1997; Ichikawa et al., 1995;
Panda et al., 2001), whereas, only CYP24 is found in osteoclast (Menaa et al., 2000). The
presence of these enzymes suggests the importance of maintaining 1,25(OH)2D3 levels
within bone cells and that these cell-types may directly regulate plasma 1,25(OH)2D3
levels.
10 1,25(OH)2D3 plays a distinct role at regulating osteoblast gene expression in different maturation stages of osteoblasts (Owen et al., 1991). It was shown that 1,25(OH)2D3 could stimulate osteoprogenitors cells (immature osteoblast) to differentiate into mature osteoblasts that are capable of producing matrix protein for bone tissue mineralization
(Aubin and Heersche, 1997). Stimulation of osteoblast differentiation by 1,25(OH)2D3 was found to involve increased expression of bone phenotypic genes such as bone sialoprotein, collagen type I, osteoprotegerin and transcriptional enhancement of osteocalcin genes in humans and rats and osteopontin gene in mice. (Chen et al., 1996;
Harrison et al., 1989; Demay et al., 1990; Markose et al., 1990; Kerner et al., 1989; Noda et al., 1990).
In contrast, recent in vitro studies that suggest that 1,25(OH)2D3 attenuates the differentiation of osteoprogenotors cells into mature osteoblast cells. A study using mouse osteoblast and rat osteosarcoma cells showed that 1,25(OH)2D3 attenuates expression of Runx2, a protein which is essential for osteoblast differentiation in vitro and skeletal development during embryogenesis (Drissi et al., 2002). This finding was further supported by another study using osteoblasts from VDR knockout mice, which demonstrated the importance of VDR in suppressing osteoblast differentiation (Sooy et al., 2005). Drissi et al., (2002) suggested that the suppression of Runx2 expression plays an important role in moderating 1,25(OH)2D3 induced up-regulation of osteocalcin gene expression. This allows 1,25(OH)2D3 to control the maturation of osteoblast cells in concert with other hormones.
11 Beside having a role in bone formation, 1,25(OH)2D3 plays an important role in inducing
osteoclastic bone resorption by stimulating osteoclast formation and osteoclast activity.
Studies have demonstrated that 1,25(OH)2D3 regulates osteoclasts in an indirect manner
(Suda et al., 2003). 1,25(OH)2D3 acts on pre-osteoblast and stromal cells to increase
production and expression of membrane-bound receptor for activation of NFkappa-B
ligand (RANKL) which binds to RANK, a cell-bound receptor on the osteoclast (see
Figure 1.2) (Anderson et al., 1997; Lacey et al., 1998; Dusso et al., 2005). Through cell- to-cell interaction, binding of RANKL to RANK promotes osteoclast differentiation, activation, survival and adherence to bone surface (Kong Y et al., 1999; Suda et al.,
2003). The up-regulation of RANKL mRNA expression by 1,25(OH)2D3 in human osteoblast cells has been shown to involve the interaction of VDR/retinoic X receptor
(RXR) complex to the vitamin D responsive element (VDRE) on the human RANKL gene promoter (Kitazawa et al., 2003). In mouse osteoblasts cells, 1,25(OH)2D3 has also been shown to inhibit the expression of the osteoprotegerin gene (OPG), a decoy receptor for RANKL that inhibits the stimulatory effect of RANKL (Kondo et al., 2004).
In addition to the direct effect which 1,25(OH)2D3 has on the osteoblast and osteoclast, a range of complex interactions between different vitamin D metabolites, endocrine hormones and various local regulatory systems can influence or alter the effect of
1,25(OH)2D3 on bone cells (Yamanaka et al., 2003; Fratzl-Zelman et al., 2003; Gurlek et
al., 2002 ). Thus it can be seen that 1,25(OH)2D3 has major effects either directly or
indirectly on bone formation and resorption
12
Figure 1.2 1,25(OH)2D3 regulates osteoclastogenesis by reciprocal regulation of
receptor activator of NF-κB (RANK) ligand (RANKL) and osteoprotegerin (OPG).
1,25(OH)2D3-VDR increases the expression of RANKL on the surfaces of the osteoblast. RANKL interaction with its receptor, RANK, promotes maturation of osteoclast progenitor cells to mature osteoclasts, the bone-resorbing cells. 1,25(OH)2D3-
VDR also represses the expression of OPG, a decoy receptor that binds RANKL and prevents RANK-mediated osteoclastogenesis (Dusso et al., 2005).
13 1.3.2 Vitamin D and Calcium Absorption
Calcium is crucial for maintaining cellular metabolic processes (Bianchi et al., 2004;
Olson, 1985) and neuromuscular function, amongst other functions (Gommans et al.,
2002). One of the major biological functions of 1,25(OH)2D3 is to maintain calcium and
phosphate homeostasis. (DeLuca, 2004). The regulation of serum calcium levels by
1,25(OH)2D3 involves increasing the efficiency of calcium and phosphate absorption in the intestine and the reabsorption of calcium from the distal renal tubules (DeLuca,
2004). Earlier studies demonstrated that the hormone enhance the diffusion of calcium in the gastrointestinal mucosa by increasing the fluidity of the microvillus membrane
(Brasitus et al.,1986; Lau et al., 1986). However, recent studies have shown that VDR knockout mice have very high 1,25(OH)2D3 levels without increasing calcium absorption suggesting that the increase in calcium absorption is not related to increase membrane fluidity. Figure 1.3 shows a model for calcium transport across intestinal mucosa. 1,25(OH)2D3 can also enhance the translocation of calcium from both intestinal and kidney cells by stimulating the expression of the cytosolic calcium-binding proteins, calbindins-D9K and D28K (Walters et al 1999; Cao et al., 2002; Hoenderop et al., 2002).
Calbindin is specifically induced by 1,25(OH)2D3 and is thought to be one of the major proteins responsible for altering the flux of calcium across the gastrointestinal mucosa and kidney (Bronner 2003; Zheng et al., 2004). Recently, it was shown that calcium channels, such as the calcium transport protein CaT1 and CaT2, are vitamin D receptor
(VDR)-dependent and their expression is strongly stimulated by 1,25(OH)2D3 (Van
Cromphaut et al., 2001; Okano et al., 2004). CaT1 and CaT2 play an important role in
14 3Na+ NCX1 Ca 2+
Paracellular pathway 1,25(OH) D Transcellular pathway VDR 2 3
ECaC2 ATP
Calbindins-D9K ECaC1 PMCA1b ADP + - - +
Figure 1.3 Molecular model for intestinal calcium absorption.
Calcium absorption from the lumen of intestinal track occurs mainly through the
paracellular pathway or via the transcellular pathway. The rate of passive diffusion of
calcium via the paracellular pathway is dependent on luminal calcium concentration and
the integrity of intracellular tight junction. The transcellular pathway involves the
epithelial calcium entry through specialised epithelial calcium channels (ECaC) such as
ECaC2 and to a much lesser extent ECaC1. The rate of absorption of calcium via these
channels are highly dependent on intracellular free calcium concentration, number of
channel and the constant buffering of intracellular calcium by intracellular calcium
binding proteins such as Calbindin-D9K. 1,25(OH)2D3 can increase the number of both calcium channel and calcium binding proteins to increase the rate of calcium absorption.
The extrusion of calcium into the plasma requires ATP dependent calcium pumps such as plasma membrane calcium ATPase (PMCA1b) and sodium calcium exchanger (NCX)
(Bouillon R.,2003).
15 active transport of calcium into the intestinal and kidney cells, respectively (Bronner,
2003). In vivo studies show considerably reduced expression of CaT1 in the duodenum of
VDR knockout mice. These VDR knockout mice display all the typical characteristics of vitamin D resistance: high circulating levels of 1,25-(OH)2D3, coincident with hypocalcemia, hypophosphatemia, secondary hyperparathyroidism, failure to thrive, rickets and a progressive alopecia. (Okano et al., 2004; Bouillon et al., 2003).
1.3.3 Vitamin D and Cancer
A number of epidemiological studies have demonstrated a protective role for 25(OH)D3 and 1,25(OH)2D3 in preventing the initiation and progression of cancer (Martinez and
Willett, 1998; Grant, 2004; Vanchieri, 2004). These studies found an inverse association between vitamin D intake and subsequent development of cancer. Prostate, breast and colon cancer cells have been found to have reduced 1α-hydroxylase (CYP27B1) activity
(hence lower ability to synthesise 1,25(OH)2D3 ) (Chen et al., 2003; Ma et al., 2004) and have high levels of 24-hydroxylase (CYP24) (greater capability to degrade 1,25(OH)2D3
) (Zhao et al., 1999) and thus have low levels of 1,25(OH)2D3 within the tumor cells.
Serum levels of 25(OH)D3 and 1,25(OH)2D3 have been used as a biomarker for colon cancer development (Guyton et al 2003). Lower levels of serum 1,25(OH)2D3 have also been noted in patients with acute lymphocytic leukemia (ALL) (Fukasawa et al., 2001;
Arikoski et al., 1999). Prostate and breast cancer cells that express high levels of CYP24 are also resistant to 1,25(OH)2D3 treatment (Miller et al., 1995; Miettinen et al., 2004) presumably due to the rapid inactivation of 1,25(OH)2D3. Low levels of CYP27B1
16 mRNA and over-expression of CYP24 have been found in poorly-differentiated colon
cancers (Bareis et al., 2001). Studies have shown that the chromosome 20q13.2-q13.3
region, where CYP24 is located, was amplified in a variety of cancers, such as ovarian
cancer, prostate cancer and gastric adenocarcinomas (Tanner et a., 2000; Wolter et al.,
2002; Weiss et al., 2003) Because of these observation, CYP24 has been proposed to be an oncogene (Albertson et al., 2000). Overexpression of CYP24 can abrogate the
1,25(OH)2D3-mediated growth control (Ly et al.,1999; Miller et al., 1995). Increasing the exposure to solar radiation (UV-B) or frequent diet of fish and soy can increase circulatory levels of 25(OH)D3 that has a protective role against cancer (Grant, 2002).
How does 1,25(OH)2D3 suppress cancer growth? In vitro experiments have shown that
1,25(OH)2D3 can function as an antiproliferative, prodifferentiating, and proapoptotic
agent in cancer cells. This anticancer property of 1,25(OH)2D3 is due to the ability of
1,25(OH)2D3 to alter cell cycle control proteins, growth factor signaling,
metalloproteinase activity, angiogenesis, and expression of apoptosis inhibitors (Liu et
al.,1996; Kobayashi et al., 1993; James et al., 1996; Majewski et al., 1993; Haugen et al.,
1996; Kim et al., 1992). Studies have also found that 1,25(OH)2D3 is able to block the growth-promoting pathways initiated by insulin-like growth factor I (IGF-I) and oestrogen-receptor (ER) pathway (Lowe et al., 2003)
It has been found that 1,25(OH)2D3 is effective at inhibiting the growth of certain cancer
types e.g prostate, breast, lung, colon, pancreas, bone, myeloid leukemia and melanoma
(Krishnan et al., 2003). This is due to the fact that prostate, breast, colon cancer and
17 leukemic cells express VDR and thus can respond to 1,25(OH)2D3 treatment. Well-
differentiated breast and colon cancer express higher levels of VDR mRNA than poorly
differentiated cancer cells and this may be used as an indicator for treatment and in
predicting the clinical outcome of the cancer patients (Bareis et al., 2002). However, the
potential use of 1,25(OH)2D3 in the treatment and prevention of cancer is hampered by
the tendency of 1,25(OH)2D3 to cause hypercalcaemia. To overcome this, numerous
synthetic analogues of 1,25(OH)2D3 have been synthesized (e.g 1,25 dihydro-16-ene-23-
yne-vitamin D3, CB1093 and EB1089, a dimethyl side-chain analogue) (Akhter et al.,
2001; Zhou et al., 1990; Lokeshwar et al., 1999). Such analogues have been reported to
exert the same anti-proliferative and pro-apoptotic effects as 1,25(OH)2D3 without
causing hypercalcaemia (Hansen et al., 2001). Other studies have identified the optimal
treatment duration and dosage of 1,25(OH)2D3 in animals models and human trials, and
investigated whether 1,25(OH)2D3 can be used in combination with other
chemotherapeutic agents, cytokines, growth factors, specific inhibitors of CYP24 and
steroid hormones to produce a greater anti-tumour effect. (Ravid et al., 1999; Ly et al.,
1999; Koren et al., 2000; Beer et al., 2001; Evans TR et al., 2002; Ordonez-Moran et al.,
2005). Thus, it appears promising that 1,25(OH)2D3 and its analogues may one day be
used as an effective form of anti-cancer therapy.
1.3.4 Vitamin D and the Immune system
The ability of 1,25(OH)2D3 to regulate immune cells was originally suggested with the identification of VDR in promyelocytes (Kizaki et al., 1991). VDR has subsequently
18 been detected in almost all immune cell-types with significant levels found in T-
lymphocytes, precursor monocytes and peripheral macrophages. CD8+ T-lymphocytes
and immature cells of the thymus have been shown to have the highest amount of VDR
(Veldman et al., 2000). Immune cells such as neutrophils, monocytes and macrophages
were found to have the same 1α-hydroxylase and 24-hydroxylase enzyme as kidney cells,
enzymes that produce and metabolize 1,25(OH)2D3, respectively (Cohen and Gray,
1984). Both enzymes found in these immune cells can be regulated by 1,25(OH)2D3 or cytokines such as interferon (IFN)-γ and tumour necrosis factor (TNF)-α (Reichel et al.,
1987; Pryke et al., 1990). Regulation of the 1α-hydroxylase enzyme by various
inflammatory cytokines has an important role in regulating the levels of 1,25(OH)2D3,
therefore acting as a negative feedback loop in regulating inflammation (Overbergh et al.,
2000). A defect in the enzymes that synthesize 1,25(OH)2D3 in the immune cells could
result in autoimmune disease. Studies have found that, macrophages of non-obese
diabetic (NOD) mice have low levels of 1α-hydroxylase enzyme (Overbergh et al.,
2000). This finding suggests that 1,25(OH)2D3 plays a crucial role in suppressing the
immune system to prevent an autoimmune reaction. Indeed, 1,25(OH)2D3 has been
found to prevent autoimmune disease and extending graft survival by regulating the T
cell-mediated immunity (Adorini, 2003; Zhang et al., 2004; Gysemans et al., 2005). The
suppression of T cell-mediated inflammatory response is achieved by redirecting the
immune system towards the T helper (Th)2 pattern. 1,25(OH)2D3 can inhibit the
production of interleukin (IL)-2 and the actions of IL-2 and IFN-γ, resulting in the
suppression of Th1 responses (Jordan et al., 1989; Daniel et al., 2005). In addition,
1,25(OH)2D3 can stimulate IL-4, IL-10 and transforming growth factor-beta (TGF-β)
19 production which constitutes the Th2 response (Hayes et al., 2003; Daniel et al., 2005).
IL-10 is also known to interfere with the induction of Th1 response (Jung et al., 2004). In other studies, 1,25(OH)2D3 was found to suppress the expression of cytokines such as
IL-1β, IL-6, IL-12 and TNF-α in stimulated monocytes/macrophages and inhibit IL-2 and interferon- γ in activated lymphocytes (Cohen et al., 2001; Muller et al., 1992;
Muller et al., 2003 ). 1,25(OH)2D3 can also inhibit the maturation of dendritic cells which play an important role as antigen presenting cells (Griffin et al., 2000).
1,25(OH)2D3 can inhibit adherence of neutrophils to endothelial cells by decreasing the expression of ICAM-1 and ELAM-1 on endothelial cells (Chen, 1995). Studies using murine peritoneal mast cells have demonstrated that 1,25(OH)2D3 can inhibit both the release of histamine from activated mast cells and proliferation of the mast cells (Toyota et al., 1996). In general, studies have shown that 1,25(OH)2D3 acts as an immuno- suppressant. However, this suppression of the immune system by 1,25(OH)2D3 seems to be specific towards the suppression of autoimmune response as tests done on animals have demonstrated that 1,25(OH)2D3 prevents autoimmune disease and prolongs graft survival without interfering with the ability of the animal to act defensively against opportunistic infections (Cantorna et al., 1998). In fact, a number of studies have suggested that, 1,25(OH)2D3 plays an important role in combating infections. Human studies have found that children with rickets or 1,25(OH)2D3 deficiency are susceptible to recurrent infection. These studies have implicated an action of 1,25(OH)2D3 on phagocytes as opposed to lymphocytes. Neutrophils and monocytes of these patients have a deficiency in chemotaxis, reduced motility and impaired phagocytosis (Etzioni et al.,
1989; Lorente et al., 1976). In vitro studies have found that 1,25(OH)2D3 can increase
20 chemotaxis activity, superoxide production and killing activity in human monocytes,
macrophages and neutrophils (Venezio et al., 1988; Deutsch et al., 1995; Levy et al.,
1990; Tokuda et al., 2000). Thus, it appears that while 1,25(OH)2D3 can inhibit the
inflammatory response by inhibiting the interaction between neutrophils and endothelial
cells (Chen, 1995), 1,25(OH)2D3 actually promotes neutrophil anti-microbial activity as
discussed above.
Overall, the above studies show that 1,25(OH)2D3 is important for the proper functioning of the immune system and has clinical potential to suppress or prevent organ rejection in recipients and for treating autoimmune diseases such as encephalomyelitis, rheumatoid arthritis, type I diabetes and inflammatory bowel disease (Hullett et al., 1998; Cantorna et al., 1998; DeLuca and Cantorna, 2001; Hayes et al., 2003).
21 1.4 Vitamin D receptor
The actions of the 1,25(OH)2D3 are mediated by the vitamin D receptor (VDR). VDR knockout mice exhibit similar characteristics to 1,25(OH)2D3-deficient mice such as, hypocalcemia, hyperparathyroidism, rickets, osteomalacia, impaired muscular and motor functions. The phenotype of the VDR knockout mice can be prevented with a high calcium, phosphate and lactose diet (Amling et al., 1999; Burne et al., 2005). In bone cells, the levels of 1,25(OH)2D3-mediated induction of the CYP24 enzyme activity has been shown to correspond to the levels of VDR expression (Staal et al., 1997).
1.4.1 Nuclear VDR
The VDR nuclear protein belongs to a large superfamily of nuclear receptors, which includes steroid, retinoid and thyroid hormone receptors. As with other members of the superfamily, the VDR has an N-terminal DNA binding domain that contains two zinc finger DNA binding motifs. The C-terminal portion of the VDR contains a ligand binding domain (LBD) that associates with 1,25(OH)2D3 (Jurutka et al., 2001). The binding of
1,25(OH)2D3 to VDR initiates a conformational change in the VDR that enhances its interaction with one of the several isoforms of retinoid X receptor (RXR) to form a heterodimer that can bind with high affinity to vitamin D-responsive elements (VDREs) on the promoter of 1,25(OH)2D3-responsive genes (Thompson et al., 1998). The VDREs are located in the up-stream promoter region of genes regulated by 1,25(OH)2D3
(Ohyama et al., 1996).
22 Like other steroid hormone receptors, VDR is phosphorylated (Hsieh et al., 1991;
Hilliard et al., 1994; Jurutka et al., 1996). Low levels of phosphorylation are seen in the absence of 1,25(OH)2D3 whereas, 1,25(OH)2D3 treatment can increase phosphorylation of VDR. The functional role of phosphorylation remains unknown. However, it has been suggested that it might be important for nuclear localization, hormone binding and transcriptional activation or repression of the receptor (Whitfield et al., 1995; Jurutka et al., 2002).
1.4.2 Plasma membrane VDR
Beside genomic actions via the nuclear VDR and transcriptional activation, 1,25(OH)2D3 can initiate a non-genomic response, a more rapid response which occurs within minutes.
The non-genomic response includes the opening of chloride channels, activation of protein kinase C (PKC) and mitogen-activated protein (MAP) kinases (Norman et al.,
2001; Fleet, 2004). It has been suggested that the non-genomic response is initiated through binding of 1,25(OH)2D3 to a putative plasma membrane VDR that is distinct from the classical nuclear VDR (Fleet, 1999; Fleet, 2004). Norman et al., (2001) demonstrated that 6-s-trans bowl-shaped 1,25(OH)2D3 binds nuclear VDR whereas planar 6-s-cis ligand shaped 1,25(OH)2D3 binds to plasma membrane VDR. Another study by Nemere et al., (1994) demonstrated that the membrane VDR has different ligand binding affinity and molecular weight from nuclear VDR. The selective binding of
1,25(OH)2D3 and the different binding affinity of plasma membrane VDR suggest the presence of a different type of membrane VDR compared to the classical nuclear VDR.
23 However, it has been suggested that the plasma membrane VDR is derive from nuclear
VDR and that the altered selectivity and ligand specificity are due to interaction of the nuclear VDR with unknown cytoplasmic or plasma membrane protein co-factors (Erben et al., 2002). Erben et al., (2002) showed that both the genomic and non-genomic response to 1,25(OH)2D3 were abrogated in osteoblasts of mice expressing VDR with an intact hormone binding domain, but lacking the first zinc finger necessary for DNA binding. This finding demonstrates that the plasma membrane VDR is derived from the nuclear VDR. Consistent with the data by Erben et al., (2002), Capiati et al., (2002) showed that 1,25(OH)2D3 induce the translocation of VDR from the nuclear to the plasma membrane. This process of relocation involves the transport of nuclear VDR by microtubules and tyrosine kinases.
1.4.3 Regulation of VDR expression
Up-regulation of VDR expression in the presence of 1,25(OH)2D3 has been shown in
various systems, including rat kidney and intestine, pig kidney, human leukemic cells and
mouse fibroblast cells (Healy et al., 2005; Costa et al., 1985; Mahonen et al., 1990).
Beside 1,25(OH)2D3, forskolin, PTH, genistein and 17beta-estradiol can also up-regulate
VDR expression (Song, 1996; Armbrecht et al., 1998; Lechner and Cross, 2003). Studies
by Barletta et al., (2003) have shown that up-regulation of VDR protein expression by
1,25(OH)2D3 in LLCPK-1 porcine kidney cells can be enhance by phorbol 12-myristate
13-acetate (PMA). They also showed that over expression of PKCα increased the
induction of the –1,500/+60 hVDR promoter construct which was further stimulated by
24 PMA (Barletta et al., 2003). This suggests that PKC plays a role in regulating of VDR expression. Other studies that support transcriptional regulation of VDR by PKC activation include a recent study by Qi X et al., (2002) who identified a functional AP-1 site in the mouse VDR promoter.
However, there are also reports that show that the increase in VDR protein levels by
1,25(OH)2D3 does not involve an increase in transcriptional activation (an increase in
VDR mRNA) but are due to increased stability of hormone occupied VDR (Wiese et al.,
1992; Arbour et al., 1993).
1.4.4 Non-classical VDRs
1.4.4.1 Annexin II
Recent studies have shown that 1,25(OH)2D3 can initiate rapid non-genomic actions by binding to annexin II membrane receptor (Baran et al., 2000). Using an antibody against annexin II, it was shown that annexin II played an important role in mediating the effect of 1,25(OH)2D3 on the activation of MAP kinases by the hormone (Johansen et al.,
2003). However, Mizwicki et al., (2004) reported that annexin II did not specifically bind
1,25(OH)2D3 and thus was not consider a putative receptor for 1,25(OH)2D3. Mizwicki et al. (2004) also suggested that the inhibition of 1,25(OH)2D3-mediated rapid non- genomic effects after addition of anti-annexin II antibody were likely to have been due to a blockade of the ability of annexin II to form complexes with itself and other proteins,
25 and not the inability to specifically bind 1,25(OH)2D3 in the presence of the antibody.
Thus, this issue remains to be resolved.
1.4.4.2 1,25D3-MARRS
1,25(OH)2D3-Membrane associated, rapid response, steroid-binding (1,25D3-MARRS) protein, a 66 kDa protein, was first purified from basolateral membranes of chick intestinal epithelial cells and latter identified as a candidate receptor for 1,25(OH)2D3
(Nemere et al., 1994). 1,25D3-MARRS protein was shown to bind 1,25(OH)2D3 more readily than 25(OH)D3 or 24,25(OH)2D3 (Nemere et al., 1994). At present, the membrane receptor can be detected or block by using a 1,25D3-MARRS antibody
(Ab099), an antibody that recognize the N-terminus of the MARRS protein (Nemere et al, 2004; Nemere et al., 1998). The membrane receptor has been found in intestinal epithelial cells, ameloblasts, odontoblasts, osteoblast and osteoclasts of chicks, mice, rat, human embryos and fetuses and has been shown to play an important role in; phosphate and calcium adsorption, cartilage formation and bone mineralization (Rohe et al., 2005;
Nemere et al., 2004; Berdal et al., 2003; Mesbah et al., 2002; Zhao and Nemere, 2002).
Activation of 1,25D3-MARRS protein by 1,25(OH)2D3 has been shown to stimulate
PKC. A study by Boyan et al, 2003 showed that 1,25(OH)2D3 can stimulate PKC with in minutes in the prehypertrophic and hypertrophic zone of costochondral cartilage of VDR knockout mice, suggesting the presence of a new responsive 1,25(OH)2D3 receptor other than the classical VDR. Furthermore, Boyan et al, 2003 showed that anti-1,25D3-
26 MARRS antibody can attenuate the stimulatory effect of 1,25(OH)2D3 on PKC ativity.
Similar studies perform on isolated intestinal epithelial cells of chicken using ribozyme
MARRS6, previously shown to actively cleave 1,25D3-MARRS sequence, also demonstrated the role played by 1,25D3-MARRS in 1,25(OH)2D3 medated PKC activation (Nemere et al, 2004). At presence, there is no literature on the activation of
MAPK signalling pathway by 1,25D3-MARRS.
1,25D3-MARRS has been shown to play a role in 1,25(OH)2D3 mediated phosphate uptake as the used of MARRS6 and anti-1,25D3-MARRS antibody inhibited
1,25(OH)2D3 mediated phosphate uptake. Similar studies have also shown that PKC is involved in regulating phosphate uptake. PMA has been shown to stimulate phosphate uptake while PKC inhibitor has been shown to inhibit 1,25(OH)2D3 mediated phosphate uptake (Sterling and Nemere, 2005). These suggest that the activation of PKC by 1,25D3-
MARRS protein plays an important role in regulating 1,25(OH)2D3 mediated phosphate uptake.
Unlike the classical VDR promoter, analysis of the 1,25D3-MARRS promoter region in mouse, rat and human failed to find any conventional VDRE sequence within the 1000 bp region immediately upstream of the transcriptional start site. Consistant with this,
1,25(OH)2D3 treatment did not increase mRNA or protein expression of 1,25D3-
MARRS. However, the hormone treatment triggers a rapid nuclear translocation of
1,25D3-MARRS. Interestingly, TGFβ1 was found to stimulate mRNA and protein expression of 1,25D3-MARRS (Rohe B et al., 2005).
27 The evidence for new classes of VDRs is overwhelming. Currently, it is important to characterise the functionality of these receptors. If each of these VDRs has unique functions, there would be potentials for the design of specific inhibitors to target these
VDRs. For example, if 1,25D3-MARRS protein has a unique role in Ca2+ transport, specific inhibitors of 1,25D3-MARRS might diminish the side effect of hypercalcemia when 1,25(OH)2D3 is used to treat cancer.
1.5 Non-genomic action of vitamin D
In addition to the classical manner in which the nuclear VDR, in partnership with RXR, mediate the genomic actions of 1,25(OH)2D3, it is now well accepted that 1,25(OH)2D3 can also exert non-genomic actions as alluded to in the above sections. The stimulation of the non-genomic response by 1,25(OH)D3 plays an important role in regulating cell differentiation, growth, apoptosis and many other cell functions (McGuire et al., 2001;
Morales et al., 2004; Fleet, 2004; Wang et al., 2005). The precise mechanism of how
1,25(OH)D3 mediates the non-genomic response is currently of great interest. This hormone has been shown to activate different cell signaling pathways in a range of cell- types. Examples of the signaling molecules activated by 1,25(OH)D3 in different cells types are: 1) protein kinase C (PKC) in keratinocytes (Bikle et al., 2001), bone cells
(Boyan and Schwartz, 2004), muscle cells (Buitrago et al., 2003), HL60 cells
(Marcinkowska et al., 1997) and macrophages (Higashimoto et al., 1995), 2) MAP kinases in bone cells (Boyan et al., 2002; Chae et al., 2002), muscle cells (Buitrago et al.,
2003), duodenal mucosae (Balogh et al., 2000) and HL60 cells (Marcinkowska et al.,
28 1997) and 3) protein kinase A (PKA) in intestinal cells (Phadnis and Nemere, 2003),
bone cells (Boyan et al., 2002), cardiac muscle (Santillan and Boland, 1998) and
pancreatic islet cells (Bourlon et al., 1997). Using pharmacological inhibitors, a number
of studies have suggested that 1,25(OH)2D3 also stimulates the activity of
phosphatidylinositol 3-kinase (PI3K) in bone cells (Vertino et al., 2005), keratinocytes
(Johansen et al., 2003), muscle cells (Buitrago et al., 2002), vascular smooth muscle
(Rebsamen et al., 2002), macrophages (Hmama et al., 2004), T-lymphocytes (Belkowski
et al., 1999), promonocytic U937 cell line (Hmama et al., 1999) and HL60 cells
(Marcinkowska et al., 1998). It seems that different signaling molecules play different
functional roles in different cell-types. Thus, it is difficult to predict whether the same
signaling molecule is involved in mediating the actions of 1,25(OH)2D3 in a given cell-
type (Nutchey et al., 2005).
1.6 Vitamin D and intracellular signaling
1.6.1 Mitogen-activated protein kinases
MAP kinases play an important role in regulating cell growth, differentiation, immune
responses, stress responses and apoptosis (Cobb and Goldsmith, 1995). At least four
distinct MAP kinases pathways are found in mammalian cells (Figure 1.4). These are the
extracellular signal-regulated kinase (ERK)1/ERK2, c-Jun NH2-terminal kinases (JNK), p38 MAP kinase and Big MAP kinase 1 (BMK1) or (ERK5). MAP kinases can be activated by different membrane receptors such as receptor tyrosine kinases, cytokine
29 receptors, antigen receptors, G protein-coupled receptors, integrins and stressors such as radiation, hyperosmotic shock and anisomycin (Pearson et al., 2001; Chang and Karin,
2001).
1.6.1.1 ERK1/ERK2
ERK1/ERK2 have been shown to play an important role in 1,25(OH)2D3 mediated non- genomic action. ERK1/ERK2 was also found to regulate cellular responses to
1,25(OH)2D3 in different cell types. Examples include intestinal permeability (Gentili et al., 2004), HL60 cell differentiation (Marcinkowska, 2001), regulation of Ca2+ levels in skeletal muscle cell, cell contractility, proliferation and differentiation (Morelli et al.,
2001), and stimulation of CYP24 induction in kidney cells (Dwivedi et al., 2002).
Receptor-mediated stimulation of ERK1/ERK2 activity requires the activation of the small GTP binding protein, ras, PKC or PI3K. These in turn act on raf-1, a serine/threonine kinase which phosphorylates and activates the dual specificity MAP kinase/ERK kinase (MEK)1 and MEK2 (Figure 1.4) (Pearson et al., 2001). The MEKs phosphorylate the threonine and tyrosine residues on the TEY amino acid sequence in the activation motif of ERK1/ERK2 (Pearson et al., 2001). Activated ERK1/ERK2 then translocate to the nucleus and directly phosphorylate the transcriptional regulatory proteins. The amino acid sequence for ERK1/ERK2 are 90% identical (Boulton et al.,
1990). Since ERK1/ERK2 can regulate cytoplasmic as well as nuclear events, substrates for ERK1/ERK2 include cytoplasmic proteins such as the cytosolic phospholipase A2
30 (Pearson et al., 2001) and transcription factors such as TCF/ElK-1, NF-IL6 (Pearson et al., 2001) and 9-cis retinoid X receptor alpha (RXR alpha) (Dwivedi et al., 2002).
31
Figure 1.4 Mitogen-activated protein kinase (MAPK) signal transduction
pathway. Abbreviations: ASK, apoptosis signal-regulating kinase; MLK, mixed lineage
kinases, MEK, MAP kinase/ERK kinase; MKK; MAP kinase kinase; MAP, mitogen-
activated protein; c-Jun N-terminal protein kinases (JNK); Cx43, connexion 43;
MAPKAP kinase, MAP kinase-activated protein kinase; PRAK, p38-regulated and - activated kinase; SGK, serum- and glucorcoticoid-stimulated kinase; COT, cancer osaka thyroid; Elk-1, E twenty six (ets)-like transcription factor-1; CREB, cAMP response element binding protein, MEF, myocyte enhancing factor; ATF2, activated transcription factor (Hayashi and Lee, 2004)
32 1.6.1.2 BMK1/ERK5
Extracellular signal-regulated kinase 5 (ERK5) is also known as Big MAP kinase 1
(BMK1) because of its large protein size (~ 90 kDa) compared to other MAP kinases
Like other MAP kinases, ERK5 plays an important role in regulating cell survival, cell proliferation and differentiation and cell function (Hayashi and Lee, 2004). ERK5 plays a critical role in regulating embryo development. In vivo studies showed that ERK5 knockout mice die between embryonic days 9.5 and 11.5 due to defects in angiogenesis and cardiovascular development (Regan et al., 2002; Sohn et al., 2002). ERK5 activity has been found to be up-regulated in prostate and breast cancer cells (Weldon et al.,
2002; Mehta et al., 2003). ERK5 has also been shown to regulate CYP24 promoter induction by 1,25(OH)2D3 (Dwivedi et al., 2002). Similar to other MAP kinases, the
ERK5 pathway is activated by a wide variety of mitogens and stress stimuli. Agents that activate ERK5 include serum, epithelial growth factor (EGF), brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2) and phorbol ester (Hayashi and Lee, 2004). The stress stimuli that activate ERK5 include hyperosmotic shock, oxidative stress, laminar flow shear stress and UV irradiation (Hayashi and Lee, 2004). This signaling molecule is exclusively activated by MEK5, which can be phosphorylated by two direct upstream kinases, MEKK2 and MEKK3 (Chao et al., 1999; Sun et al., 2001) (Figure 1.4). These findings were supported by studies using ERK5, MEKK3 and MEKK5 knockout mice.
Consistent with biochemical studies on the ERK5 pathway, the phenotype of ERK5-
33 deficient embryos resembles that of knockout embryos of MEKK3 and MEK5 (Yang et
al., 2000; Wang et al., 2005).
A number of molecules have been identified as substrates of the ERK5 pathway. These
include the MEF2 (myocyte enhancer factor 2) family of transcriptional factors (MEF2A,
MEF2C and MEF2D), Sap1a (Ets-domain transcription factor), SGK (serum- and glucocorticoid-inducible kinase), Cx43 (connexin 43; a gap junction protein), and Bad (a pro-apoptotic member of Bcl-2 family) (Hayashi and Lee, 2004). In 1,25(OH)2D3- stimulated kidney cells, ERK5 was reported to phosphorylate the transcription factor Ets-
1 (E26-1).
1.6.1.3 JNK
The c-Jun N-terminal protein kinases (JNKs) are stress-activated protein kinases
(SAPKs) since they are activated in response to a variety of environmental stress such as
radiation, heat shock, osmotic imbalance, DNA damage and inflammatory cytokines
(Kyriakis and Avruch, 2001). In addition to environmental stressors, JNKs are also
activated in some cell types by agonists such as growth factors, ligands which act via
heterotrimeric G-protein-coupled receptors, phorbol esters and 1,25(OH)2D3 (Ichijo,
1999; Cuenda, 2000; Nutchey et al., 2005). JNK is also activated in T lymphocytes by a
number of ligands (Singh and Zhang, 2004).
Activation of JNK has been associated with cell apoptosis. Studies have shown that
persistent activation of JNK either through over-expression of upstream activators or
34 following exposure to external stimuli can result in apoptosis. Furthermore, inhibition of
JNK signaling by over-expression of the dominant negative mutant of MKK4 (an
upstream regulator of JNK, see Figure 1.4) was shown to enhance cell survival
(Bogoyevitch and Court, 2004). However, there are also studies showing that under
certain circumstances, JNK activation can enhance cell survival (Roulston et al., 1998;
Andreka et al., 2001). Besides regulating cell survival, JNK also has a role in regulating
cell function. JNK was shown to play a role in 1,25(OH)2D3 mediated cell differentiation
in keratinocytes and HL60 cells (Johansen et al., 2003; Wang et al., 2003). A recent
study from our laboratory shows that JNK plays an important role in 1,25(OH)2D3
induction of the CYP24 promoter and its synergistic action with PMA in HEK293T cells
(Nutchey et al., 2005). In T lymphocytes, JNK1 and JNK2 have been proposed to play
essential roles in TH1 and TH2 differentiation (Dong et al., 2002).
At least 10 JNK isoforms have been identified. These include four isoforms of JNK1
(α1, α2, β1, β2) and JNK2 (α1, α2, β1, β2) and two isoforms of JNK3 (α1, α2). These
originate from alternative splicing of three mammalian genes, Jnk1, Jnk2, Jnk3 (Kyriakis and Avruch, 2001). Each of the three JNK genes is expressed on different chromosomes
(Manning et al., 2002). Jnk1 and Jnk2 are ubiquitously expressed, whereas Jnk3 expression is restricted predominantly to the brain and testes (Kyriakis and Avruch,
2001). The JNK protein is preferentially phosphorylated at Tyr and Thr by MKK4 and
MKK7, respectively (Lawler et al., 1998). MKK4 and MKK7 are in turn activated by
MAP kinase/ERK kinase kinases (MEKKs) which include MEKK1, MEKK2, MEKK3
and MEKK4 (Figure 1.4) (Kyriakis and Avruch, 2001). However, some of these MEKKs
are not specific for the JNK pathway. For example, MEKK2 and MEKK3 are also up-
35 stream activators of p38 and ERK5 (Deacon and Blank, 1997; Deacon and Blank, 1999).
Other activators of MEK4 and MKK7 include tumor progression locus (Tpl)-2 (Salmeron et al., 1996), apoptosis signal-regulating kinase1 (ASK1) (Matsukawa et al., 2004) and mixed lineage kinases (MLK) family (Hirai et al., 1997). Like the MEKKs, these activators can also activate p38 and ERK1/ERK2. PI3K and PKC have also been shown to play a role in regulating JNK signaling. Stimulation of PI3K by 1,25(OH)2D3 was shown to activate JNK in human keratinocytes (Johansen et al., 2003). A study by
Brandlin et al., (2002) has demonstrated that over-expression of PKCε can stimulate JNK activity whereas PKCµ plays an inhibitory role in JNK activation.
A number of molecules have been identified as substrates of the JNK pathway. They include the transcription factor c-Jun, ATF2 (that heterodimerises with c-Jun and stimulates expression of the c-jun gene) (van Dam et al., 1995) and Elk-1 (which is involved in the induction of the c-fos gene, whose product forms the AP-1 heterodimer with c-Jun) (Cavigelli et al., 1995; Shaulian and Karin, 2001). In addition to the transcription factors, other substrates of JNK include Bcl-x(L), an anti-apoptotic protein which is found in the mitochondria (Ito et al., 2001).
1.6.2 Phosphathdylinositol 3-kinase
Phosphathidylinositol 3-kinase (PI3K) plays an important role in regulating a variety of cellular responses, including organization of the cytoskeleton, cell motility, cell
36 differentiation and growth, transformation, cell survival, and insulin action (Fry, 1994;
Toker and Cantley, 1997; Toker and Newton, 2000).
PI3K is a family of lipid kinases that catalyze the phosphorylation of the inositol ring of
phosphoinositides (PI) at the 3′ position. The sugar moiety can be phosphorylated at
different positions to produce different types of phosphatidylinositol (PtdIns)
(Vanhaesebroeck and Alessi, 2000). To date, nine members of the PI3K family,
belonging to 3 classes of PI3K, have been isolated from mammalian cells
(Vanhaesebroeck and Alessi, 2000). Three different lipid products can be generated by
the different PI3K members. They consist of the singly phosphorylated form PtdIns-3-P,
the doubly phosphorylated forms, PtdIns-3, 4-P2 and finally the triply phosphorylated
form PtdIns-3, 4, 5-P3 (Rameh and Cantley, 1999).
Three classes of PI3K are Class I, Class II and Class III PI3K. The Class I PI3K consists
of two subunits, a catalytic subunit and a regulatory adapter subunit. There are four
isoforms of the subunit, p110α, p110β, p110γ and p110δ and three mammalian genes
encode the adapter subunits, p85α, p85β and p85γ (Fruman et al., 1998; Vanhaesebroeck
and Alessi, 2000). Class I PI3K is composed of Class 1a and Class 1b kinases
(Vanhaesebroeck and Alessi, 2000). Whereas Class 1a PI3K appears to be expressed
ubiquitously, Class 1b appears to be restricted to leukocytes. Class II PI3K have
characteristic C-terminal C2 homology domains whereas, Class III PI3K are homologous
to Saccharomyces cervisiae VPS34 (Vacuolar protein sorting mutant) (Schu et al., 1993).
Currently, little is known about Class II and III PI3K.
37
The activation of Class 1a PI3K involves the interaction of a p85 adapter subunit with an
activated receptor tyrosine kinase which in turn recruits the p110 catalytic subunit of
PI3K to the plasma membrane where it can phosphorylate its main substrate PtdIns-4, 5-
P2 (Cantrell, 2001). Besides receptor tyrosine kinase, other non-receptor tyrosine kinases such as Janus kinases in leucocytes can activate PI3K. Binding of p85 to signaling scaffold from by three adapter proteins Shc, Grb2 and Gab2 can also recruit the p110 catalytic subunit to the plasma membrane (Cantrell, 2001). In contrast to Class1a PI3K,
Class 1b kinase is activated by guanine-nucleotide-binding proteins (G proteins)
(Cantrell, 2001). For example, PI3Kγ, the sole member of Class 1b PI3K is activated solely by G-protein coupled receptors via Gβγ. PI3Kγ is composed of a p110γ catalytic subunit and a 101-kDa regulatory subunit (Vanhaesebroeck and Alessi, 2000). Class 1b
PI3K plays an essential role in regulating neutrophil and dendritic cell migration (Del
Prete et al., 2004; Puri et al., 2005).
PI3K has been shown to play a role in the activation of MAP kinase signaling pathways
(Baier et al., 1999; Pandey et al., 1999). A study by (Pandey et al., 1999) using Chinese
hamster ovary cells show that PI3K inhibitors, wortmannin and LY294002, can inhibit
the activation of ras, c-Raf-1, MEK and ERK1/ERK2 by vanadyl sulfate and insulin.
Another study by Baier et al. (1999), using similar PI3K inhibitors, show that retinoic
acid could induce PI3Kγ expression resulting in increased ERK2 activation in the
myelomonocytic cell-line U937. Similar results were also observed in a study using
human keratinocytes which also show that both wortmannin and LY294002 can
completely block the 1,25(OH)2D3 induced phosphorylation of ERK1/ERK2 and JNK1
38 (Johansen et al., 2003). These findings demonstrate that the PI3K is required for the activation of ras/c-Raf1/MEK/ERK or JNK signaling cascade induced by vanadyl sulfate, insulin, retinoic acid and 1,25(OH)2D3 (see Figure 1.5).
Several studies have demonstrated that the activation of certain PKC isozymes is
regulated by the PI3K pathway (Toker et al., 1994; Chou et al., 1998). The inhibition of
PI3K activity by wortmannin and LY294002 which results in inhibition of PKCζ in
HL60 cells, further demonstrate that PKCζ is a downstream target of PI3K (Liu et al.,
1998). This regulation is facilitated by the phosphorylation of PKCζ by phosphoinositide-
dependent kinase (PDK)-1 which is activated by PtdIns-3, 4-P2 and PtdIns-3, 4, 5-P3
(Vanhaesebroeck and Alessi, 2000).
39
Insulin
1,25(OH)2D3
Receptor
Nucleus
Gene Expression
Figure 1.5 Insulin and 1,25(OH)2D3 stimulates gene expression by activating
PI3K/Ras and down stream MEK/ERK1/ERK2 and JNK1 (Modified figure of
Johansen et al., 2003).
40 1,25(OH)2D3 can activate PI3K in different cell types (Hmama et al., 1999; Rebsamen et al., 2002). Studies have shown that PI3K plays a crucial role in 1,25(OH)2D3-induced differentiation of both myeloid cells and HL-60 cells (Marcinkowska et al., 1998;
Hmama et al., 1999). 1,25(OH)2D3 can also activate PI3K to promote vascular smooth muscle cell growth and calcification (Rebsamen et al., 2002). Beside this 1,25(OH)2D3 can also stimulate PI3K in human keratinocytes to promote cell differentiation (Johansen et al., 2003). Currently there are no reports regarding the role PI3K has on the regulation of CYP24 by 1,25(OH)2D3.
1.6.3 Protein Kinase C
Protein Kinase C (PKC) plays an important role in modulating a wide variety of cellular processes such as cell growth, differentiation, secretion, apoptosis and tumor development (Gschwendt, 1999). The activity of PKC is stimulated by a diverse range of agonists which act via G-protein coupled receptors (e.g: bombesin, vascular endothelial growth factor, neurotransmitters and formylated peptides), receptor tyrosine kinases (e.g receptors for epidermal growth factor and platelet derived growth factor) and other agonists such as fatty acids (Nishizuka, 1995; Hii et al., 1995; Clerk et al., 2005). With the exception of the fatty acids, receptor-mediated activation of PKC results from the hydrolysis of membrane phosphatidylinositol (4,5) bisphosphate by phospholipase C.
This results in the production and accumulation of diacylglycerol (DAG), and inositol trisphosphate (IP3). While DAG activates PKC (see below), IP3 mobilizes stored Ca2+
(Nishizuka, 1995)
41
The PKC family is subdivided into three groups: conventional, novel and atypical isozymes, based on their requirements for Ca2+ and diacylglycerol (DAG) for activation
(Parker and Murray-Rust 2004). The conventional PKCs (PKC α, βI, βII, γ) are
dependent on calcium and are activated by DAG or PMA. The novel PKC isozymes
(PKC ε, η, δ, θ) are also activated by DAG or PMA but are calcium-independent. The
atypical PKC isozymes (PKC ζ, ι, λ) are calcium-independent and do not respond to
DAG or PMA (Gschwendt, 1999; Parker and Murray-Rust 2004). The difference in
response to DAG and requirement for Ca2+ between the three PKC subfamilies is due to
differences in their molecular structures. The structure of PKC isozymes consists of
conserved domains (C1 –C4) and in between these conserved domains are variable
regions (V1 to V5A). The C1 regions of the conventional and novel PKCs contain two
cysteine-rich sequences (Liu and Heckman, 1998). Ono et al, (1989) showed that
phospholipid and diglyceride/phorbol ester interact with the cysteine-rich sequences of
the conventional PKCs and novel PKCs. Deletion of the C1 region containing the two
cysteine-rich sequences completely abolishes phorbol ester binding (Burns and Bell,
1991). Furthermore, the lack of one of these cysteine-rich sequences in atypical PKC
renders it non-responsive to DAG or PMA (Ono et al., 1989). These findings suggest that
the two cysteine-rich sequences are DAG/phorbol ester binding domains that are
important for PKC activation. Unlike the conventional PKC subfamily, the novel PKCs
lack the C2 region that mediates Ca2+-binding (Ono et al., 1989).
42 1,25(OH)2D3 has been shown to activate PKC in a variety of cell types (Wali et al.,
2003; Shimizu et al., 2002). Although the precise mechanism through which this is
achieved is not known. Activation of PKC by 1,25(OH)2D3 was shown to play a part in
regulating the growth of chondrocytes (Schwartz et al., 2002), reducing insulin-induced
glucose uptake in rat adipocytes (Huang et al., 2002), promoting keratinocytes
differentiation (Bollinger Bollag and Bollag, 2001), increasing calcium influx into
skeletal muscle cells (Capiati et al., 2001) and reducing proliferation, but increasing
apoptosis and differentiation in human colon cancer cell lines (Chen et al., 1999). PKC
has also been shown to play an important role in 1,25(OH)2D3 induction of the CYP24
promoter and its synergistic action with PMA in HEK293T and COS-7 cells (Barletta et
al., 2004; Nutchey et al., 2005). Using dominant negative PKC isozyme mutants,
Nutchey et al., (2005) demonstrated that PKCα, in particular, is crucial for the synergistic action of the CYP24 promoter by 1,25(OH)2D3 and PMA.
1.6.4 Ras
The Ras signaling pathway is an essential signal transduction cascade that controls cell
survival, growth, differentiation and transformation. Three human Ras genes encode the
four isoforms of ras, H-ras, N-ras, K-ras4A and K-ras4B that are the founding members
of a large superfamily of Ras-related proteins (Repasky et al., 2004; Wennerberg et al.,
2005). Ras proteins are small GTP-binding and hydrolyzing proteins (GTPases) that are
targeted to the plasma membrane and exist in two distinct structural and functional
confirmations (Reuther and Der, 2000). Ras is activated when it is bound to GTP and
43 inactivated when bound to GDP. The level of signaling response induced by Ras is
dependent on the effector molecules that bind Ras, such as the MAP kinase kinase kinase,
Raf-1, Ral guanine nucleotide dissociation stimulator, PI 3K, phospholipase C, Ras
inhibitor, RIN1, and polarity protein AF6/Canoe (Ory and Morrison, 2004). The best- characterized Ras effectors are the three Raf serine/threonine kinases, A-Raf, B-Raf and
Raf-1 which stimulate MEK1/MEK2, the immediate upstream regulators of ERK1/ERK2
(Alberola-Ila and Hernandez-Hoyos, 2003; Vojtek and Der, 1998). The process of
ERK1/ERK2 activation by Ras involves the binding of Raf-1 to activated Ras (Ras -
GTP). This process then brings Raf to the plasma membrane where it becomes activated.
The activated Raf then phosphorylates and activates MEK1/MEK2. Various stimuli such
as hormones, cytokines and various drugs have been shown to activate Ras. Studies in
our laboratory have shown that Ras plays a role in the activation of ERK1/ERK2 and
ERK5 by 1,25(OH)2D3 in kidney cells (Dwivedi et al., 2002). Furthermore, using
dominant negative mutant of Ras, we showed that Ras plays a role in regulating the
ability of transcriptional factor, Ets-1, to transactivate the CYP24 promoter (Dwivedi et
al., 2002). Other studies also showed that Ras is involved in the activation of Raf-1 by
1,25(OH)2D3 (Solomon et al., 1999; Buitrago et al., 2003). It was suggested that the activation of Ras and downstream Raf-1 by 1,25(OH)2D3 was due to the hormone inhibiting the Ras -GTP hydrolysis in skeletal muscle cells (Buitrago et al., 2003). The activation of the transcription factor, activator protein 1 (AP1), in keratinocytes by
1,25(OH)2D3 was shown to be dependent on Ras signaling pathway (Johansen et al.,
2003).
44 1.7 Enzymes involved in vitamin D Metabolism
The biosynthesis and biodegradation of 1,25(OH)2D3 have been introduced above
(Figure 1.1), and these processes will be discussed in greater detail in the following sections. The discussion will include a description of the enzymes which are involved in the bio-activation and inactivation of 1,25(OH)2D3, their tissue distribution and how these enzymes are regulated.
1.7.1 Hepatic 25-hydroxylase enzyme.
The main circulatory form of vitamin D, 25-hydroxyvitamin D3 [25(OH)D3] is synthesized by the 25-hydroxylase. Until now, it is still unclear which CYP enzymes catalyse the 25 hydroxylation reaction. Recent studies have identified five CYP enzymes from various species that are capable of vitamin D 25-hydroxylation. These enzymes include the CYP2D11, CYP2D25, CYP2J2/3, CYP3A4 and CYP2R1. However, only the latter three seem to match the enzymatic properties and distribution pattern of the physiologically relevant human liver microsomal enzyme (Prosser and Jones, 2004).
Although the main site for 25-hydroxylation of vitamin D3 occurs in the hepatocytes in the liver, 25-hydroxylase activity has also been found in other tissues such as bone, intestine, kidney and skin (Ichikawa et al., 1995; Hosseinpour et al., 2000; Gascon-Barre et al., 2001). Furthermore, although both hepatocytes and nonparenchymal cells of the rat liver can efficiently take up vitamin D3, only the hepatocytes can carry out the 25- hydroxylation of vitamin D3 (Dueland, 1981).
45
Two types of hepatic 25-hydroxylase enzyme have been identified, the microsomal and
the mitochondrial forms (Dahlback and Wikvall, 1987). Since the microsomal 25-
hydroxylase enzyme has a higher affinity for 25(OH)D3, hence lower Km value than the
mitochondrial enzyme, it is thought that the microsomal 25-hydroxylase is responsible
for maintaining physiological levels of 25(OH)D3 whereas, the mitochondrial enzyme is
used only when very high levels of circulating 25(OH)D3 are required (Holick et al.,
1994; Omdahl et al., 2002). The microsomal 25-hydroxylase uses vitamin D3,
1α(OH)D3, vitamin D2 and 1α(OH)D2 as substrates (Wikvall, 2001). Between
1α(OH)D3 and vitamin D3, the former is a much better substrate for 25-hydroxylase
enzymes than vitamin D3 (Dahlback and Wikvall, 1988). The mitochondrial 25-
hydroxylase has a broad substrate specific and is apparently identical with CYP27, an
obligatory enzyme in bile acid biosynthesis (Andersson et al., 1989). The 25-hydroxylase
is less regulated compared to 1α-hydroxylase and 24-hydroxylase enzyme.
1.7.2 Kidney 1α-hydroxylase enzyme
1.7.2.1 Role of 1α-hydroxylase.
The 1α-hydroxylase is a member of the cytochrome P450 family and is a heme- containing protein designated as CYP27B1 by the Cytochrome P450 gene superfamily
Nomenclature Committee. 1α-hydroxylase plays an important role in the synthesis of
1,25(OH)2D3. 1α-hydroxylase knockout mice has no 1,25(OH)2D3 in the circulation.
46 These mice develop hypocalcemia, secondary hyperparathyroidism, retarded growth,
skeletal abnormalities, and are defective in the reproductive system and immune function
(Panda et al., 2001; Dardenne et al.,2003). Examination of the bone structure reveals alterations to non-collagenous matrix protein expression, reduced numbers of osteoclasts and the presence of rachitic bone lesions. The defects in these 1α-hydroxylase knockout
mice are similar to patients with the genetic disorder, vitamin D-dependent rickets type 1
(Panda et al., 2001).
1α-hydroxylase can catalyze both the C-1 hydroxylation of 25(OH)D3 and
24,25(OH)2D3. 24,25(OH)2D3 is the preferred substrate for 1α-hydroxylase when
compared to 25(OH)D3. However, because of the higher concentration of 25(OH)D3 in
normal physiological conditions (approximately 10 times higher than 24,25(OH)2D3),
more 1,25(OH)2D3 is synthesized than 1,24,25(OH)3D3 (Omdahl et al., 2002).
Subcellular analysis of enzyme activity indicates that the 1α-hydroxylase enzyme is
located in the mitochondria rather than in the microsomal fraction (Henry, 1997). The
biochemical reduction catalysed by 1α-hydroxylase is dependent on NADPH as a co-
factor, as well as flavoprotein NADPH-ferrodoxin reductase and ferrodoxin (Zehnder and
Hewison, 1999).
The process of hydroxylating 25(OH)D3 to 1,25(OH)2D3 occurs mainly in the kidney
which is the main organ for maintaining circulatory levels of 1,25(OH)2D3. 1α-
hydroxylase enzymes are express throughout the kidney such as the proximal tubules,
distal tubules, collecting ducts, cortical and medullary renal tissue (Figure 1.6). It is
47 thought that the production of 1,25(OH)2D3 in the proximal tubules function to support circulating levels of 1,25(OH)2D3, whereas more distal parts of the nephron fulfill an autocrine or paracrine function, i.e. to regulate calcium reabsorption via the calcium- sensing receptor (Hewison et al., 2000).
Extrarenal 1α -hydroxylase is found in a wide variety of tissue. Immunohistochemical staining for 1α-hydroxylase reveals the presence of this enzyme in skin, bone cells, macrophages, colon, ganglia of mesenteric plexus, adrenal gland, pancreas, purkinje cells of the brain, prostate and placental tissue. It is suggested that this enzyme functions in an autocrine or paracrine fashion and may provide 1,25(OH)2D3 to localized area for cell growth and differentiation (Zehnder et al., (2001).
48
Bowman’s Distal Glomerulus capsule tubule Proximal tubule Distal tubule
Proximal tubule
Corte x
Medulla Descending Loop of Limb of loop Henle of Henle
Collecting
duct Ascending Limb of loop of Henle
To renal pelvis
Figure 1.6 Diagram of Nephron
The 1α-hydroxylase enzymes are express in the proximal tubules, distal tubules,
collecting ducts of the cortical and medullary renal tissue.
49 1.7.2.2 Regulation of the 1α-hydroxylase
The renal 1α-hydroxylase is tightly regulated by a wide range of substances such as circulating parathyroid hormone (PTH), insulin-like growth factor I, calcium, phosphorus and 1,25(OH)2D3 (Portale and Miller, 2000; Brenza and DeLuca, 2000; Zoidis et al.,
2002; Zhang et al., 2002). Two of the main regulators of 1α-hydroxylase are PTH and
1,25(OH)2D3. Studies done on avian and mammalian species showed that parathyroidectomy diminishes, whereas PTH administration increases, circulating levels of 1,25(OH)2D3 (Henry, 1997). Studies by Kong et al.,(1999) identified the presence of
PTH responsive regions in the promoter of the 1α-hydroxylase gene and further demonstrated that PTH could stimulate the 1α-hydroxylase promoter activity in a kidney cell line.
In vivo studies using VDR knockout mice showed that mice lacking the VDR develop abnormally high levels of 1-hydroxylase expression and serum concentrations of
1,25(OH)2D3, indicating that 1,25(OH)2D3 inhibits 1α-hydroxylase activity (Takeyama et al., 1997; Murayama et al., 1999). Besides this, 1,25(OH)2D3 was also shown to suppress the 1α-hydroxylase promoter activity in opossum kidney cells (Armbrecht et al.,
2003). The above studies show that PTH has a stimulatory effect on 1α-hydroxylase whereas the effect of 1,25(OH)2D3 is inhibitory. In the physiological system both PTH and 1,25(OH)2D3 work side by side to regulate calcium homeostasis. Low calcium and
1,25(OH)2D3 stimulate the release of PTH which in turn stimulates the production of 1α-
50 hydroxylase. As sufficient 1,25(OH)2D3 is produced, 1,25(OH)2D3 act as a negative
feedback to inhibit the expression of 1α-hydroxylase.
1.7.3 24-hydroxylase enzymes (CYP24)
While the above two enzymes are involved in the bioactivation of vitamin D, CYP24
catalyses the bioinactivation of vitamin D metabolites. CYP24 is mainly found in the
kidney. The major site for renal 24-hydroxylase activities is found in the proximal
convoluted tubule (Iida et al., 1993). However, CYP24 is also found in nearly all cells, or
cells with VDR such as in the immune cells, bone, intestines, skin, brain, muscle and
reproductive organs (Henry and Norman, 1984). The widespread distribution of CYP24
suggests that 24-hydroxylase enzyme play a crucial role in regulating circulating and
local concentration of 25(OH)D3 and 1,25(OH)2D3 by metabolizing them to inactive
forms. CYP24 plays an important role in preventing hypervitaminosis. There are also
findings indicating that the by-product of CYP24, 24,25(OH)2D3 is essential for bone fracture healing (Seo et al., 1997)
In vitro studies on Escherichia coli expressed human and rat CYP24 have demonstrated that human CYP24 was able to catalyze both C-23 and C-24 hydroxylation whereas rat
CYP24 can only catalyze C-24 hydroxylation (Sakaki et al., 2000). Human CYP24 not only catalyzes the 24R-hydroxylation of 25(OH)D3 and 1,25(OH)2D3, but also the subsequent oxidation of the hydroxyl group to a ketone group, (hydroxylation at the C-23 or C-24 position and cleavage of the C23-C24 bond). Studies by Taniguchi et al., (2001)
51 have demonstrated that 1,25(OH)2D3 is the preferred substrate for CYP24. However, a
recent study by Masuda et al., (2004) showed that both 1,25(OH)2D3 and 25(OH)D3 are metabolized at a similar rate by the CYP24 enzyme.
CYP24 knockout mice were found to have high circulating levels of 1,25(OH)2D3
(Masuda et al., 2004 and 2005). These mice also have impaired intramembranous bone formation. Since CYP24 converts 25(OH)D3 to 24,25(OH)2D3, it was suggested that the deficiency in 24,25(OH)2D3 may have contributed to the defect in bone growth and repair
(St-Arnaud, 1999). However, later experiments to rescue the bone phenotype, by cross breeding CYP24 knockout mice with VDR knockout mice revealed that the impaired bone formation in CYP24 knockout mice was not the result of 24,25(OH)2D3 deficiency but because of high levels of 1,25(OH)2D3 (St-Arnaud et al., 2000), acting via as yet uncharacterized mechanisms.
In vivo studies using transgenic rats that constitutively express CYP24 showed a significant reduction in circulating 25(OH)D3 and 24,25(OH)2D3 levels in these rats.
These transgenic animals also developed albuminuria and hyperlipidemia shortly after
weaning (Kasuga et al., 2002; Hosogane et al., 2003). It was suggested that the reduced
24,25(OH)2D3 levels were due to an excessive amount of albumin being excreted from
the glomeruli because of nephritis, therefore causing a decrease in the reuptake of
25(OH)D3 from the proximal tubule of the kidney. The reason for how constitutive
CYP24 expression induces nephritis in rats remains to be elucidated (Hosogane et al.,
2003).
52 1.7.3.1 Regulation of the CYP24 promoter by vitamin D
Of all the compounds that have been found to stimulate CYP24 transcription,
1,25(OH)2D3 is the most studied. The promoter of the CYP24 gene is known as the most transcriptionally responsive vitamin D-inducible gene identified to date. Sequencing of the human CYP24 promoter has mapped out two functional vitamin D response elements
(VDREs) located about 100 bp apart within the –300-bp promoter region (see Figure 1.7)
(Chen and DeLuca 1995; Carlberg, and Polly 1998) whereas, the rat CYP24 promoter contains three VDREs within its promoter region (Zierold et al., 1995). Studies in our laboratory suggest that the proximal VDRE (VDRE-1) is crucial for 1,25(OH)2D3 activation of the CYP24 promoter whereas distal VDRE (VDRE-2) is recruited if further activation is required. For example, in the presence of high concentrations of
1,25(OH)2D3, both VDREs are utilized in a synergistic manner to ensure high levels of
CYP24 expression (Kerry et al., 1996). Activation of the CYP24 promoter by
1,25(OH)2D3 requires the binding of 1,25(OH)2D3 to the VDR, as shown in VDR knock out mice (Takeyama et al., 1997). VDR interacts with RXR to form VDR-RXR heterodimer that then binds to the VDRE in the CYP24 promoter (Zou et al., 1997). In the absence of 1,25(OH)2D3, the heterodimer VDR/RXR binds to one or both VDREs to recruit a corepressor and lower basal expression (Dwivedi et al., 1998; Polly et al., 2000).
Upon 1,25(OH)2D3 binding to the VDR, the corepressor is replaced by a coactivator complex.
53 -2 9 8 G Illus F i C g -2 , u CG CG 58
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54 Besides this, recent studies in our laboratory on VDREs of the CYP24 promoter have
identified an E26 (Ets)-1 binding site (EBS) (-128/-119) located downstream of VDRE-1
(-136/-150) that is important for maximal transcriptional activation (see Figure 1.7)
(Dwivedi et al., 2000). Over expressed Ets-1 enhanced 1,25(OH)2D3 induction of
CYP24 expression by 34% whereas, over expression of a mutant Ets-1 (Ets-1 T38A)
resulted in a substantially lowered (42% reduction) CYP24 promoter induction. This
level of reduction is comparable to that observed when the Ets-1 binding site was mutated
(Dwivedi et al., 2002). In addition to the EBS, further analysis by our laboratory of the
CYP24 promoter sequence using a computer program MatInspector (V2.2)
(http://transfac.gbf.de/), has identified a putative GC box (-114/-101) and a putative
CCAAT box (-62/-51) located downstream from the EBS which plays an important role in CYP24 promoter induction (see Figure 1.7) (Dwivedi, Tan, Ferrante, Morris, May, and
Hii, manuscript in preparation). Mutation to GC box reduced basal and 1,25(OH)2D3 induction of the CYP24 promoter by approximately 50% in HEK293T and UMR106 bone cells. Mutation to CCAAT box also reduced the basal and 1,25(OH)2D3 induction of the CYP24 promoter in HEK293T cells by 66% and 75%, respectively (Dwivedi, Tan,
Ferrante, Morris, May, and Hii, manuscript in preparation). Mutation to both GC and
CCAAT box further reduced basal promoter activity and 1,25(OH)2D3 induction of the
CYP24 promoter, (75% and 90% reduction, respectively). In these studies, the transcription factor, Sp1, was shown to interact with the GC box and over expression of
Sp1 increased CYP24 promoter induction, which was then abrogated when the GC box was mutated. Transcription factor nuclear factor Y (NF-Y) was also shown to interact with CCAAT box while over expression of NF-Y increases CYP24 promoter activity that
55 was abrogated when CCAAT box was mutated (Dwivedi, Tan, Ferrante, Morris, May, and Hii, manuscript in preparation). Dwivedi, Tan, Ferrante, Morris, May, and Hii
(manuscript in preparation) using mammalian two-hybrid system showed that Sp1 interacts with Ets-1 efficiently and may play a role in transcriptional cooperation. Studies in our laboratory by Nutchey et al., (2005) identified a vitamin D stimulatory element
(VSE), a sequence of 5’-TGTCGGTCA-3’ which is located about 30bp upstream of
VDRE-1 at –171/-163 in the CYP24 proximal promoter (see Figure 1.7). Mutation to this
VSE site reduced 1,25(OH)2D3 induction of the CYP24 promoter by 60% and completely abolished the synergistic response to PMA and 1,25(OH)2D3 (Nutchey et al.,
2005). This finding shows that the VSE site is not only crucial for 1,25(OH)2D3 induction of the CYP24 promoter but is also required for the synergistic response to PMA and 1,25(OH)2D3.
1.7.3.2 Signaling pathways that regulate the CYP24 promoter in response to vitamin D.
As described earlier (see section 1.7.3.1), the CYP24 promoter contains the VDREs,
EBS, GC box, CCAAT box and VSE site. The stimulation of the CYP24 promoter by
1,25(OH)2D3 involves both genomic and the non-genomic action. The genomic action involves direct interaction of 1,25(OH)2D3 with nuclear VDR which in turn acts on the
VDRE to initiate transcriptional activation of the CYP24 promoter. The non-genomic action of 1,25(OH)2D3 involves the binding of 1,25(OH)2D3 to a putative membrane
VDR which results in the activation of various signaling molecules such as MAP kinases,
PKC and PI3K. These signaling molecules in turn phosphorylate a number of
56 transcription factors (e.g RXRα, Ets-1, Sp1 and NF-Y proteins) which play a crucial role in the CYP24 promoter induction (Dwivedi et al., 2002; Gao et al., 2004). 1,25(OH)2D3 is known to activate different signaling molecules in different cell types (see section 1.5).
Studies from our laboratory and those of others have shown that the MAP Kinases play an important role in the induction of the CYP24 promoter (Dwivedi et al., 2000; Dwivedi et al., 2002; Nutchey et al., 2005). 1,25(OH)2D3 has been shown to activate
ERK1/ERK2 and ERK5 and these MAP kinases play an important role in the induction of the CYP24 promoter in COS-1 cells (Dwivedi et al., 2002; Pearson et al., 2001).
Further more, the dominant negative Ras mutant has been shown to inhibit 1,25(OH)2D3 induction of the CYP24 promoter in COS-1 cells (Dwivedi et al., 2000; Dwivedi et al.,
2002) as well as the activation of down stream ERK1/ERK2 and ERK5 (Dwivedi et al.,
2002). However, the signaling molecules needed to induce the CYP24 promoter can vary between different cell types. For example, a study by Nutchey et al. (2005) showed that in HEK293T cells, the dominant negative ERK1 mutant did not inhibit the CYP24 promoter induction by 1,25(OH)2D3, whereas the dominant negative MKK4 (an upstream activator of JNK) mutant caused a significant reduction in the ability of
1,25(OH)2D3 to stimulate CYP24 promoter activity. Consistent with this, Nutchey et al.
(2005) demonstrated that 1,25(OH)2D3 could stimulate JNK activity and not
ERK1/ERK2 in HEK293T cells. These results seem to differ from the results obtained by
Dwivedi et al. (2002) who showed that in COS-1 cells, 1,25(OH)2D3 could stimulate
ERK1/ERK2 activity and that these kinases were required for 1,25(OH)2D3 induction of the CYP24 promoter. Dwivedi et al. (2002) also found that JNK did not play any role in the CYP24 promoter induction by 1,25(OH)2D3 in COS-1 cells. Differences in the
57 requirement for the type of different cell signaling molecules for CYP24 promoter induction can also be observed in different bone cell lines. Thus, overexpression of Raf-1
(an upstream activator of ERK) led to the stimulation of CYP24 mRNA induction by
1,25(OH)2D3 in MG-63 bone cells whereas overexpression of Raf-1 in MC3T3-E1 bone cells resulted in suppression of CYP24 mRNA (Narayanan et al., 2004). Furthermore,
CYP24 mRNA expression was enhanced when MC3T3-E1 cells were treated with a
MEK inhibitor (U0126) (Narayanan et al., 2004). These findings suggest that ERK can have different effects on CYP24 expression depending on the cell-type involved
(stimulation in MG-63 cells or inhibitory in MC3T3-E1). Narayanan et al. (2004) suggested that this differences in the regulation of the CYP24 promoter induction by
ERK between the different cell-types is dependent on the type of RXR expressed in the cells. There are three different isoforms of RXR: RXRα, RXRβ and RXR γ. MC3T3-E1 cells have low levels of RXRβ and RXRγ whereas MG-63 cells have low levels of
RXRα. These workers suggested that phosphorylation of RXRα by ERK resulted in the
inability of RXRα to heterodimerise to VDR to form VDR/RXR heterodimer and
therefore prevent the transcriptional activation of the CYP24 (Narayanan et al., 2004).
Besides the MAP kinases, PKC has also been shown to play an important role in the
induction of the CYP24 promoter by 1,25(OH)2D3 (Partridge et al., 1998; Armbrecht et
al., 2001). PMA a direct activator of PKC, was shown to enhance the 1,25(OH)2D3
induction of CYP24 mRNA expression in rat intestinal epithelial cells (Koyama et al.,
1994; Armbrecht et al., 2001). In the presence of 1,25(OH)2D3, PMA was shown to
synergistically increase CYP24 promoter activity in kidney cells (Barletta et al., 2003;
58 Chen et al., 1993). In addition, a recent study by Nutchey et al. (2005) show that PKC is also required for the synergistic increase in CYP24 promoter activity caused by PMA and
1,25(OH)2D3 in HEK293T cells. These workers also investigated the roles played by the different isoforms of PKC (PKCα, PKCβ1, PKCδ, and PKCε) on CYP24 induction by
PMA and 1,25(OH)2D3. Results from these studies demonstrate that PKCα plays an important role in the synergistic induction of the CYP24 promoter by PMA. In contrast,
PKCβ1 was found to be required for 1,25(OH)2D3 to stimulate CYP24 promoter activity
(Nutchey et al., 2005). Thus, there is overwhelming evidence that show that PKC, ERK and JNK are involve in stimulating CYP24 induction and the signaling molecules that are required depend on the cell-type being studied.
1.8 Regulation of CYP24 by compounds other than vitaminD.
Besides 1,25(OH)2D3, the CYP24 promoter has been shown to be regulated by a variety of substances including parathyroid hormone (PTH), retinoid X receptor (RXR) ligand
(LG100268), genistein, forskolin and PMA (Armbrecht et al., 2003; Allegretto et al.,
1995; Kallay et al., 2002; Christakos et al., 1996; Barletta et al., 2003). PTH was shown to decrease both CYP24 mRNA and enzyme activity in kidney cells by reducing the stability of the CYP24 mRNA (Zierold et al., 2003). The effect of PTH on CYP24 in bone cells is different from kidney cells. PTH by itself has no effect on the CYP24 levels in bone cells but acts synergistically with 1,25(OH)2D3 to increase the CYP24 mRNA and protein (Armbrecht et al., 1998). The synergistic increase in CYP24 mRNA caused by 1,25(OH)2D3 and PTH was shown to be mediated through the cAMP signaling
59 pathway (Armbrecht et al., 1998). Consistent with the role of cAMP, a study using HL60
cells has demonstrated that the activation of cAMP by forskolin resulted in an increase in the CYP24 enzyme activity (Smith et al., 1999). In contrast, forskolin was shown to inhibit CYP24 enzyme activity in a dose dependent manner in mouse renal tubule
(Mandla and Tenenhouse, 1992). The basis for this difference in action for forskolin is not known.
Genistein, a plant derived isoflavone has been shown to reduce CYP24 mRNA in tumor
cells by down regulating CYP24 promoter induction (Farhan et al., 2003). Currently, the
mechanism for the reduction in CYP24 promoter induction by genistein is unknown.
The RXR ligand (LG100268) was shown to increase CYP24 promoter activity by binding
to RXR which in turn act through the VDRE within the CYP24 promoter to stimulate its
induction (Zou et al., 1997).
There has been quite a number of studies that investigated the action of PMA on CYP24
induction, either in the presence or absence of 1,25(OH)2D3. PMA was shown to
enhance 1,25(OH)2D3 induction of CYP24 promoter activity in COS-7 and LLCPK-1
cells by acting through PKC and MAP kinase to increase VDR expression and
phosphorylation (Barletta et al., 2004). However, there are also studies that suggest that
the increase in CYP24 induction by PMA is independent of VDR expression but through
a direct action on the CYP24 promoter (Koyama et al., 1994). Similarly, PMA was also
shown to increase CYP24 enzyme activity and in the presence of 1,25(OH)2D3,
synergistically increase CYP24 promoter activity in kidney cells. PKC, ERK and JNK
60 have all been shown to play a role in up-regulating the expression of CYP24 (Mandla et
al., 1990; Nutchey et al., 2005)
1.9 Chemotherapy and plasma vitamin D
Cancer is the most common cause of disease-related death in children in industrialized
societies (Parker et. al., 1997). With the improvement in the efficacy of anticancer drugs,
surgical techniques, radiation therapy and the development of intricate imaging
technologies for earlier detection and monitoring of the disease, the survival rate of
childhood cancer patients has approached 60-70% (Green, 2002; Parisi et al., 1999,
Meadows et al., 1986 and Dreyer et al., 2002) and is projected to reach 85% by the year
2010 (Harras, 1996). With the improved survival rate, therapy-associated complications have become more evident and thus have become a major management challenge. These patients can develop skeletal defects, reduced bone mineral mass and density, osteoporosis and fractures during and at the end of the treatment period. Many of these skeletal defects, such as scoliosis, craniofacial dysplasia, and limb-length discrepancy, are readily apparent. However, other side effects such as osteoporosis and osteonecrosis are silent until they reach advanced stages where they may compromise quality and duration of survival and be more difficult to ameliorate (Kaste, 2004). The basis for the reduction in bone mass and density are likely to be multi-factorial. Cancer treatments such as corticosteroids, antineoplastic drugs and radiotherapy have been reported to reduce bone mass and density (Abrams et al., 2004 and van Leeuwen et al., 2000)
Besides this, endocrinopathies (e.g growth hormone deficiency and hypogonadism),
nutritional deficits and relatively sedentary lifestyle during and even after therapy can
61 interfere with bone-mineral accretion and skeletal development (Tillmann et al., 2002,
Arikoski et al., 1999, Warner et al., 1998). Another factor could involve a disturbance in the metabolism of 1,25(OH)2D3 which is an essential regulator of calcium homeostasis
(Glorieux, 1990).
Studies by Arikoski and colleagues have demonstrated that children undergoing chemotherapy for leukemia as well as solid tumours had reduced plasma levels of
25(OH)D3 and 1,25(OH)2D3 and that the vitamin D metabolites remain low even after the completion of chemotherapy (Arikoski et al., 1999a, 1999b). Halton and colleagues reported that more than 70% of paediatric patients with acute lymphoblastic leukemia showed reduced levels of plasma 1,25(OH)2D3 following chemotherapy (Halton et al.,
1996; Atkinson et al., 1998). Although most of the reports for the reduction of serum levels of 1,25(OH)2D3 were found in children, there is some evidence that chemotherapy can also reduce plasma 1,25(OH)2D3 in adults patients with gynaecological malignancies
(Gao et al., 1993). Given the importance of 1,25(OH)2D3 in the maintenance of calcium levels and bone density, chemotherapy-induced disturbance in 1,25(OH)2D3 metabolism could be a major contributing factor to the reduction in bone mass during and after treatment.
The mechanism for the reduction in 1,25(OH)2D3 is not known. Halton et al., (1996) speculated that the reduction in 1,25(OH)2D3 was due to receptor-mediated binding of
1,25(OH)2D3 to leukemic cells. Other possible mechanisms suggested by Halton and colleagues included the down-regulation of 1α-hydroxylase which is required for the
62 biosynthesis of 1,25(OH)2D3 (Halton et al., 1996) or up-regulation of metabolism to inactive products. Arikoski et al., (1999b) suggested that the reduction in 1,25(OH)2D3 was due to factors such as malnutrition and diminished sunlight exposure, vitamin D malabsorption due to intestinal mucositis and deficiency in 25(OH)2D3. This is because
Arikoski et al., (1999a and b) found that the levels of plasma 25(OH)D3 and
1,25(OH)2D3 were reduced in patients with leukemia as well as with solid tumor even at completion of their chemotherapy treatment. Other proposed mechanisms for the reduction in serum 1,25(OH)2D3 during chemotherapy include impairment of mitochondrial function in the proximal tubules of the kidneys by chemotherapeutic drugs such as cisplatin (Gao et al., 1993). However, none of these possibilities have been proven and identifying the causes remains a major challenge.
1.10 Chemotherapeutic drugs
The realization that a large number of children who are undergoing or have completed chemotherapy have reduced levels of 25(OH)D3 and 1,25(OH)2D3 metabolites, suggests to us that chemotherapeutic drugs may have a direct effect on the metabolism of vitamin
D. The following sections briefly review the major classes of drugs and examples, their effects and associated mechanisms of action, including the signaling molecules which they have been reported to stimulate.
63 1.10.1 Categories of chemotherapeutic drugs
Alkylating agents, include mustargen-nitrogen mustard, cyclophosphamide (cytoxan), melphalan (alkeran), chlorambucil (leukeran), cis-platinum ("non-classical" alkylating agent), carbo-platinum ("non-classical" alkylating agent), carmustine (BCNU), thiotepa and busulfan (myleran).
Vinca alkaloids and related substances, include vincristine, vinblastine and VP-16.
Anthracycline antibiotics, include doxorubicin (adriamycin), actinomycin D, daunorubicin (daunomycin), bleomycin, idarubicin and mitoxantrone.
Glucocorticoids, include prednisone/prednisolone, dexamethasone and triamcinolone
(vetalog).
Inhibitors of protein/DNA/RNA synthesis, include Methotrexate, 6-thioguanine, 5- fluorouracil (5-FU), cytosine arabinoside (ara-C, cytosar), L-asparaginase (Elspar), dacarbazine (DTIC), hydroxyurea (hydrea) and procarbazine (matulane).
Miscellaneous agents, includes paclitaxel.
64 1.10.2 Principal targets of Chemotherapeutic drugs: the cell cycle and
apoptosis.
The ultimate aims of anti-cancer therapy are to disrupt cancer cell cycling and kill the
cancer cells. Cancer cells have the advantage over normal cells of disregulated growth
due, predominantly, to their resistance to the normal regulation of apoptosis
(programmed cell death in which the cell commits suicide by a highly regulated
biochemical process). Consequently, anti-cancer drugs are designed to target the cell
cycle and to increase the sensitivity of cancer cells to death inducers and apoptotic
effectors. The cell cycle can be divided into: Mitosis (M) phase (cell division phase,
duration 0.5-1 hr), growth 1 (G1) postmitotic phase (duration depending on G0. A phase
for RNA and protein synthesis in preparation for S phase), growth zero (G0) phase
(variable duration time and is temporary or permanent if the cell has reached the end of
its cycling capability), DNA synthesis (S) phase (duration 10-20 hrs, DNA replication occurs during this phase), growth 2 (G2) premitotic phase (RNA and protein synthesis).
The anti-cancer drugs can have actions either on a specific phase (e.g vincristine which has a specificity on the M phase but can work in S or G1 phase at high concentrations) or be non-specific (e.g. daunorubicin and doxorubicin, but most effective in S phase).
Apoptosis comes from the Greek words apo = from and ptosis = falling. This is a key
process in the body which responds to cell damage, infection, stress or DNA damage.
Apoptosis is required for homeostasis, development and immune cell regulation. It can
occur, for instance, when a cell is damaged beyond repair, or infected with a virus. The
65 "decision" for apoptosis can come from the cell itself, from its surrounding tissue or from
a cell that is part of the immune system such as the monocyte/macrophage-derived cytokine, tumour necrosis factor α, and CD8+ cytototic T lymphocytes. If the ability of the cell to undergo apoptosis is impaired (e.g. by mutation), or if the initiation of apoptosis is blocked (by a virus), the damaged cell can continue to divide without restrictions and develops into cancer. For example, as part of the hijacking of the cell's
genetic system carried out by human papillomaviruses (HPV), the E6 gene is expressed and this protein causes the degradation of p53 protein, a vital protein which regulates the
decision for apoptosis. This interference of the apoptotic capability of cells is believed to be a major cause for the development of cervical cancer in individuals with persistent oncogenic HPV infection. Owing to its crucial role in cancer development and progression, many of the above listed anti-cancer drugs have also been developed due to their ability to not only block the cell cycle but also to cause apoptosis or sensitize cancer cells to apoptosis-inducing agents, some of which will be described in greater detail below.
1.10.3 Actions of anticancer drugs
For this discussion, we will focus on four commonly used and better characterized anti-
cancer drugs which we have selected from the above-listed classes of drugs. These four
drugs are also the subject of investigation in this thesis.
66 1.10.3.1 Daunorubicin hydrochloride
Daunorubicin hydrochloride is a member of the anthracycline family of antibiotics that is
produced from a strain of Streptomyces coeruleorubidus. Its molecular structure is shown
in Figure 1.8 A. It is one of the major anti-tumour agents which are widely used in the
treatment of acute myeloid leukemias and acute lymphocytic leukemia (Tallman et al.,
2005). The drug can significantly increase the rate of complete remission compared to
others such as vincristine, prednisone, and L-asparaginase alone.
Daunorubicin hydrochloride is rapidly taken up by different tissues. Typically, it has been
found that the highest concentrations of daunorubicin hydrochloride are found in the
spleen, kidney, liver, lung and heart. Daunorubicin hydrochloride and its hydroxylated
derivative, doxorubicin hydrochloride, are both anti-mitotic and cytotoxic by virtue of
their ability to cause DNA damage by intercalating between base pairs and to generate
free radicals which can cause DNA damage (Minotti et al., 2004; Gervasoni et al., 2004).
The drugs also inhibit topoisomerase II activity by stabilizing the DNA topoisomerase II
complex (Minotti et al., 2004). Topoisomerase II is a ubiquitous enzyme which is
essential for the survival of all eukaryotic organisms and plays critical roles in virtually
every aspect of DNA metabolism. The enzyme unknots and untangles DNA by passing
an intact helix through a transient double-stranded break that it generates in a separate
helix. Owing to its central role in DNA replication, topoisomerase II is the target for some of the most active and widely prescribed anticancer drugs currently utilized for the treatment of human cancers (Wang, 2002). The list of potential toxic effects includes
67 A: Daunorubicin Hydrochloride
B: Etoposide
C: Vincristine Sulfate
D: Cisplatin
Figure 1.8 Molecular structures of 4 selected chemotherapeutics drugs
68 persistent and severe myelosuppression, and cardiac toxicity which can lead to heart
failure, fetal abnormality, secondary leukemias and tissue necrosis.
Daunorubicin hydrochloride can activate a number of cell signaling molecules, including
MAP Kinases, PI3K and PKC which regulate cell survival and apoptosis (Mas et al.,
2003). Indeed, numerous reports have demonstrated that this drug can trigger apoptosis of
cancer cells in vitro (Masquelier et al., 2004; Gervasoni et al., 2004; Dimanche-Boitrel et al., 2005). This will be discussed further below.
1.10.3.2 Etoposide
Etoposide (also known as VP-16) is a semisynthetic derivative of podophyllotoxin. The
molecular structure of etoposide is shown in Figure 1.8 B. Etoposide is used to treat a
variety of malignancies and shows considerable activity in treating leukemias,
neuroblastomas and many other malignancies such as refractory testicular tumors and
small cell lung cancer (Frappaz et al., 1992; Hijiya et al., 2004; Tsuchiya et al., 2005).
The cytotoxicity of etoposide is due, in part, to its ability to inhibit topoisomerase II,
resulting in the inhibition of DNA synthesis. Etoposide can also activate apoptosis
signaling via activation of the MAP kinases, PKC and p53 signalling molecules (Shimada
et al., 2003; DeVries et al., 2002). Toxic side effects of etoposide include
myelosuppression, hypotension, fetal malformation, gastrointestinal toxicities and
alopecia. Toxicity studies in non-cancer cells such as hepatocytes, seem to show that non-
cancer cells are less sensitive to etoposide than cancer cells such as HL-60 and liver
69 cancer cells. For example, Zhuo et al. (2004) showed that 40 µM of etoposide was not
cytotoxic to normal human hepatocytes. Furthermore, a study using normal rat
hepatocytes showed that etoposide at concentrations below 176 µM did not induce
apoptosis (Gomez-Lechon et al., 2002). The concentration of etoposide used in these
studies were higher than those needed to kill cancer cells such as human hepatocellular
carcinoma cell line (less than 17 µM of etoposide) (Miao et al., 2003) and HL60 (30µM
of etoposide) (Bjorling-Poulsen and Issinger, 2003). Thus, like most anti-cancer drugs,
etoposide shows selectivity towards cancer cells.
1.10.3.3 Vincristine Sulfate
Vincristine Sulfate is derived from a common flowering plant, the periwinkle plant. The
molecular structure of vincristine sulfate is shown in Figure 1.8 C. The mechanism of
anti-cancer action of vincristine sulfate is via the inhibition of microtubule formation in
the mitotic spindle. In addition, vincristine sulfate can promote apoptosis by regulating
different signaling molecules such as ras, Raf, MAP Kinases, PKA and PKC (Wang et
al., 1999; Stone and Chambers, 2000). Furthermore, the PI3K inhibitor, LY294002, has been demonstrated to synergistically enhance the cytotoxicity of vincristine sulfate towards human malignant glioma cell lines, suggesting that the PI3K pathway promotes resistance towards vincristine sulfate (Shingu et al., 2003). Indeed, some types of cancers have developed resistance to apoptosis by upregulating their expression of Acute
Transforming (Akt)/protein kinase B (PKB) which is an effector of PI3K (Siddik, 2003;
Cenni et al., 2004).
70 Vincristine sulfate is normally used together with other chemotherapeutic drugs to treat acute leukemia, Hodgkin’s disease, non-Hodgkin’s malignant lymphomas, rhabdomyosarcoma, neuroblastoma and Wilms’ tumor (Zoubek et al., 1994; Zinzani et al., 2002; Bisogno et al., 2005; Mori et al., 2005). Toxic side effects of vincristine sulfate include acute uric acid nephropathy, acute shortness of breath and severe bronchospasm, impairment of fertility, hair loss, hypertension or hypotension, sensory impairment, neuritic pain and motor difficulties.
1.10.3.4 Cisplatin
Cisplatin is a heavy metal complex containing a central atom of platinum surrounded by two chloride atoms and two ammonia molecules in the cis position (see Figure 1.8 D for molecular structure). The anti-cancer effect of cisplatin is due to the drug reacting with the DNA, causing DNA damage. Cisplatin reacts with DNA to produce mostly intra- strand crosslinks between adjacent guanine base pairs or adjacent guanine and adenine base pairs (Nowosielska and Marinus 2005). This formation of intra-strand crosslinks between DNA blocks the action of DNA polymerases, resulting in the inhibition of DNA synthesis (Pinto and Lippard, 1985). Cisplatin can also promote the formation of crosslinks between DNA and proteins, thus shielding the cisplatin-modified DNA from being repaired. The damaged DNA triggers cell-cycle arrest and apoptosis (Jordan and
Carmo-Fonseca, 2000). Cisplatin can also activate signaling molecules such as the MAP kinases and PKC to initiate apoptosis (Wang et al., 2004; Sedletska et al., 2005; Iioka et al.,2005).
71
Cisplatin has been used together with other approved chemotherapeutic drugs to treat a variety of cancers, including metastatic testicular tumor, metastatic ovarian tumor and advanced bladder cancer (Einhorn, 2002; Rosenberg et al., 2005). Toxic side effects of cisplatin include low levels of white blood cell counts which can lead to increased risk of infection, low levels of red blood cell (anemia), low platelet levels which can result in increased risk of bleeding, nausea and vomiting, kidney damage, hearing loss and infertility.
1.11 Activation of cell signaling molecules by chemotherapeutic drugs.
A number of studies have shown that chemotherapeutic drugs such as those mentioned above can activate PKC and MAP Kinases, ERK1/ERK2 and JNK (Nowak, 2002; Boldt et al., 2002; Hershberger et al., 2002; Hayakawa et al., 2003; Mas et al., 2003). For example, Nowak (2002) found that 50 µM of cisplatin caused the activation PKCα in renal proximal tubular cells. A detectable increase in kinase was observed at 30 min and kinase activation peaked at around 1 h. Cisplatin also stimulates the activity of
ERK1/ERK2 MAP kinases (Nowak, 2002). Increases in ERK1/ERK2 activity could be observed at 30 min after the addition of the drug and kinase activity peaked at around
6hrs and this was maintained for up to 24hrs (Nowak, 2002). The effect of cisplatin on
ERK1/ERK2 activity was shown to be PKC-dependent since the PKC inhibitor, Go 6976, inhibited the stimulation of ERK1/ERK2 activity by cisplatin.
72 Etoposide, as mentioned above, has also been reported to stimulate the activity of
ERK1/ERK2. Thus, Tang et al. (2002) reported that etoposide (100µM) caused the activation of ERK1/ERK2 in mouse embryonic fibroblasts. This effect was evident within 1 h of treatment and was maintain for at least 8 h. Studies by Boldt et al. (2002)
showed that the chemotherapeutic drugs, taxol and etoposide, could stimulate the
activities of ERK1/ERK2, JNK and/or p38 MAP kinases in three human cancer cell lines:
human squamous carcinoma (A431), human cervix carcinoma (HeLa) and human breast
epithelial carcinoma (MCF7). At 68 µM, etoposide was found to stimulate the activity of
ERK1/ERK2 in A431, HeLa and MCF7 cells at 3 h, 15 min and 3 h, respectively (Boldt et al., 2002). Increases in JNK activity following treatment with etoposide was observed at 3, 1 and 3 h in A431, HeLa and MCF7, respectively. These authors also investigated the effect of taxol on the activities of the MAP kinases. In A431 and HeLa cells, the actions of 50 nM taxol was found to be somewhat similar to those of etoposide, with the exception that in HeLa and MCF7 cells, stimulation of ERK1/ERK2 activity was biphasic. The second phase of activation occurred between 12-18 h after treatment. JNK activation in MCF7 cells the presence of taxol was biphasic, with the second phase commencing at 9-12 h. Etoposide also stimulated the activation of p38 in these cell lines, assessed by measuring MAPKAP-2 activity. Kinetics studies in HeLa and MCF7 cells demonstrate that etoposide increased p38 activity at 1 h after the commencement of treatment. In A431 cells, the increase occurred at 6 h (Boldt et al., 2002). In contrast, taxol did not have much of an effect on p38 activity in the three cell lines. Despite these increases, it was found that suppression of the drug-induced activation of the MAP kinases did not have a consistent effect of cancer cell survival or death. These results
73 prompted Boldt et al. (2002) to propose that the original view of ERK promoting cell
survival, and JNK and p38 furthering apoptosis is too simplistic, and in this form, cannot
be exploited as a general tool to enhance the effectiveness of chemotherapy. There is also
a functional dependency on which cancer cell-type and drug are being investigated.
Daunorubicin hydrochloride has been reported to stimulate the activities of ERK1/ERK2,
the MAP kinase kinase kinase, raf-1, and PI3K in U937 cells (Mas et al., 2003). Increases
in ERK1/ERK2 and raf-1 activities were detectable within 10-30 min of the addition of
the drug. Daunorubicin hydrochloride also stimulated the hydrolysis of
phosphatidylcholine (PC) by a PC-specific phospholipase C and the activation of PKCζ in these cells (Mas et al., 2003). The stimulatory effect of the drug on ERK1/ERK2 activity required PKCζ and PI3K since a kinase-dead mutant of PKCζ blocked the
activation of ERK1/ERK2 and cytoxicity. Wortmannin, an inhibitor of PI3K, also
blocked ERK1/ERK2 activation by daunorubicin hydrochloride (Mas et al., 2003). A role
for reactive oxygen species (ROS) in the activation of the MAP kinase and PI3K by
daunorubicin hydrochloride was also demonstrated. Thus, Mas et al. (2003) not only
showed that daunorubicin hydrochloride stimulated H2O2 generation but activation of
ERK1/ERK2 and PI3K was blocked by the antioxidant, N-acetyl cysteine. This is
consistent with ROS being generated by and involved in the actions of chemotherapeutic
agents (Sinha and Mimnaugh, 1990). Studies in human malignant testicular germ cells by
Schweyer et al. (2004a) have also shown that cisplatin can stimulate the production of
ROS which in turn activates Raf-1 and its downstream MEK and ERK signaling
pathway.
74 Currently, there is much interest in the roles MAP kinases play in regulating cell survival and apoptosis. The ERK signaling pathway is normally regarded as having a protective role during stress condition (Rosseland et al., 2005) and it promotes cell survival (Boldt et al., 2002). It is known that some tumour cell-types such as melanoma cells can constitutively exhibit high levels of ERK activity (Smalley, 2003). Consequently, there has been much interest in the use of selective inhibitors of the MAP kinase in conjunction with chemotherapeutic drugs to increase the efficacy of the anti-cancer drugs (Kohno and
Pouyssegur, 2003). Consistent with this view, the inhibition of MEK1/MEK2 or
ERK1/ERK2 has been shown to increase the sensitivity of tumour cells to chemotherapeutic drugs. At the same time, over expression of ERK1/ERK2 was shown to increase the survival of tumour cells when they are exposed to chemotherapeutic drugs
(Brantley-Finley et al., 2003; Mas et al., 2003). Studies by Brantly-Finley et al. (2003) showed that inhibition of the ERK1/ERK2 pathway by U0126 in KB-3 carcinoma cells strongly enhanced the cytotoxicity of vinblastine and doxorubicin but had no effect on etoposide-treated cells. Similarly, studies by Mas et al. (2003) showed that the MEK inhibitor, PD98059, increased the sensitivity of U937 cells to killing by daunorubicin hydrochloride. These studies support the view that ERK1/ERK2 plays an important role in mediating cell survival and promoting resistance against chemotherapeutic drugs.
In contrast, there are also studies which show that ERK1/ERK2 plays a role in initiating apoptosis of tumour cells and enhancing the cytotoxicity of chemotherapeutic drugs (Yeh et al., 2002; Tang et al., 2002; Schweyer et al., 2004b). Studies by Schweyer et al.
(2004b) in human malignant germ cells have demonstrated that cisplatin mediates
75 apoptosis by stimulating MEK and its downstream ERK1/ERK2 to activate caspase-3
(executing caspase). Consistent with this observation, Nowak (2002) using MEK and
PKC inhibitors have also shown that PKCα and ERK1/ERK2 play a role in mediating
cisplatin-induced renal cell apoptosis by causing a sustained decrease in mitochondrial
function and by inhibiting Na+/K+-ATPase activity. Tang et al. (2002) reported that
ERK1/ERK2 could play a role in promoting NIH3T3 cell apoptosis. These authors showed that etoposide and adriamycin not only stimulated the activity of ERK1/ERK2 but that the MEK inhibitor, PD98059, significantly reduced the degree of apoptosis caused by etoposide and adriamycin. Furthermore, the expression of constitutively active
ERKs could considerably increase the sensitivity of immortalized NIH3T3 cells to etoposide-induced cell-death (Tang et al., 2002). As mentioned above, Boldt et al.
(2002), using a clonogenic assay to determine the ability of cancer cells to form colonies after taxol treatment, showed that in HeLa cells, ERK1/ERK2 has a pro-apoptotic role whereas in A431 and to a lesser degree in MCF7 cells, both ERK and p38 play a pro- survival role. These results suggest that the role played by the ERKs is dependent on the
cell type in question as well as the chemotherapeutic drug being used. This stresses the
need to investigate cell signalling in each cell-type of interest. What occurs in one cell-
type may not occur in another.
Besides ERK1/ERK2, it has also been demonstrated that chemotherapeutic drugs can
activate JNK and that the JNK signaling pathway may play an important role in
regulating drug-induced cell death. The activation of JNK is normally linked to cell death
under stressful stimuli. Thus, Brantley-Finley et al. (2003) showed that the JNK inhibitor,
76 SP600125, protected KB-3 carcinoma cells against the cytotoxic effects of vinblastine and doxorubicin hydrochloride and almost abrogated etoposide-induced cell death. The notion that JNK plays a destructive role in the mechanism of action of chemotherapeutic drugs is further supported by observations in various other tumour cell-types, including prostate cancer cells and breast cancer cells (Shimada et al., 2003; Kim et al., 2003).
Furthermore, the ability of 1,25(OH)2D3 to enhance the killing of murine squamous cell carcinoma by cisplatin has been reported to be mediated by the JNK pathway
(Hershberger et al., 2002). These data are consistent with JNK playing a role as a pro- apoptotic signalling molecule.
However, there are also some studies which showed that JNK could play a role in promoting cell survival (Nishina et al., 2004). NH2-terminal phosphorylation of c-Jun by
JNK was shown to play a role in DNA repair and promoting cell survival after cisplatin treatment in human cancer cell lines such as glioblastoma, ovarian, breast, and prostate carcinoma cells (Gjerset et al., 1999; Hayakawa et al., 1999; Potapova et al., 2001). A study by Hayakawa et al., (2003) showed that inhibition of JNK increased the sensitivity of tumour cells to cisplatin and etoposide and that JNK played a role in drug resistance by enhancing DNA repair in the human breast cancer BT474 cell line. Recent studies have suggested that the different JNK isotypes may play opposing roles in regulating cell survival and death (Dietrich et al., 2004; Ventura et al., 2004; She et al., 2002). Thus, the above results could be due, at least in part, to which JNK isoform was activated in a particular cell-type by a particular drug.
77 From the above discussion, it is therefore clear that many of the chemotherapeutic drugs are able to stimulate the activities of signalling molecules such as PKC, PI3K and the
ERK1/ERK2, JNK and p38 MAP kinases, to mention a few. The roles these signalling molecules play in the cytotoxic action of the drugs are dependent, to a large degree, on the cell-type and the drug under investigation. Given that the MAP kinases have moved into the limelight of drug discovery, modulation of the MAPK pathways to enhance the efficacy of chemotherapeutic drugs is likely to require some tailoring to suit the specific combination of drug and cancer.
Currently, there are no reports on whether chemotherapeutic drugs can regulate the
CYP24 promoter. However, given the roles PKC and the MAP kinases play in mediating the effect of 1,25(OH)2D3 on the CYP24 promoter, and the ability of chemotherapeutic drugs to stimulate the activities of these signalling molecules, it is possible that the drugs could alter the expression of CYP24 and hence the rate of 1,25(OH)2D3 metabolism.
1.12 Hypotheses
Based on the above information, I hypothesise that chemotherapeutic drugs cause a reduction in the circulating levels of 1,25(OH)2D3 by up-regulating the expression of cytochrome p450C24 (CYP24) in kidney cells. This is due to their ability to stimulate the activities of the MAP kinases. Blocking the activities of these kinases inhibits the ability of the drugs to stimulate the transcriptional activation of the CYP24 promoter.
78 1.13 Aims
The aims of my project are to investigate whether:
chemotherapeutic drugs such as daunorubicin hydrochloride, etoposide, cisplatin and vincristine sulfate are able to stimulate CYP24 promoter activation in kidney cell-lines. the above drugs alter the ability of 1,25(OH)2D3 to stimulate the CYP24 promoter. the chemotherapeutic drugs are able to stimulate the activities of ERK1/ERK2, ERK5 and JNK in the kidney cells.
inhibition of ERK1/ERK2/ERK5 and JNK activities blocks the ability of the chemotherapeutic drugs per se to stimulate CYP24 promoter activity and whether this is modulated by the presence of 1,25(OH)2D3.
79
Chapter 2
Materials and Methods
80 2.1 Materials
2.1.1 General chemicals, drugs and reagents
Trizma base (tris- (Hydroxymethyl) aminomethane), EGTA, EDTA, Triton X-100,
Ponceau S, Nonidet-P40, D-glucose, phorbol 12-myristate 13-acetate (PMA), Folin and
Ciocalteu’s Phenol Reagent, sodium diatrizoate were from Sigma-Aldrich Pty. Ltd,
(Sydney, NSW, Australia). Tween-20, sucrose, NaCl, Na2CO3 were from Asia Pacific
Specialty Chemical Limited, (Seven Hills, NSW, Australia). β-mercaptoethanol, acetic acid, Na/K-tartrate, sodium hydroxide (NaOH), diethyl ether, hexane, acetic acid, and acetone were from AJAX Finechem, (Seven Hills, NSW, Australia). CuSO4 was from
BDH Chemicals, (Port Fairy, VIC, Australia). Dimethyl sulphoxide (DMSO) was from
Merck, Germany. Trypsin-EDTA was from Gibco, (Grand Island, NY, USA). HEPES was from Paton Scientific, (Stepney, SA, Australia). Bovine Serum Albumin was from
Boehringer Manheim, (Mannheim, Germany). Trypan Blue was from Sigma, (St Louis,
MO, USA).
2.1.2 Reagents for Western blots and Kinase assays
Acrylamide (N, N’-Methylbisacrylamide 30%), ammonium persulfate (APS), sodium dodecyl sulfate (SDS), N,N,N’,N’,-Tetramethylethylenediamine (TEMED), Bio-Rad
Minigel Apparatus, and low range molecular markers were from Bio-Rad Laboratories,
(Sydney, NSW, Australia). Chemiluminescence reagent was from Perkin Elmer Life
Sciences, (Boston, MA, USA). Western Blot recycling kit was from Alpha Diagnostic
International, (San Antonio, TX, USA). Nitrocellulose membrane (0.2 mm thickness
Optitran BA-S83, reinforced NC) was from Schleicher & Schuell, (Dassel, Germany). γ- 81 32P ATP (specific activity 4000 Ci/mM) was from Geneworks (Adelaide, SA, Australia).
X-ray film (Curix Ortho HT-G 100 NIF) was obtained from AGFA, (Mortsel, Belgium).
2.1.3 Chemotherapeutic drugs
Daunorubicin hydrochloride, doxorubicin hydrochloride, vincristine sulfate salt, etoposide and cis-platinum ІІ diammine diachloride (cisplatin) were from Sigma, (St
Louis, MO, USA).
2.1.4 Reagents for lysis, washing and assay buffer,
Leupeptin, pepstatin A, benzamidine hydrochloride, DL-dithiothreitol (DTT), PMSF
(phenylmethylsulfonyl fluoride) and Sigma 104 (phosphatase substrate) were from
Sigma-Aldrich Pty. Ltd, (Sydney, NSW, Australia). Aprotinin (bovine lung, 100,000 U/5 ml) was from Calbiochem, (La Jolla, CA, USA).
82 2.1.5 Reagents for transfection
DOTAP Liposomal Transfection Reagent was from Roche, (Indianapolis, IN, USA).
2.1.6 Reagents for Luciferase Assay
Dual luciferase assay kit was from Promega (Madison, WI, USA)
2.1.7 Antibodies
Anti-mouse IgG, (HRP conjugated) was from DAKO Corporation, (Carpinteria, CA,
USA). Anti-rabbit IgG (HRP conjugated) was from Chemicon, (Boronia, VIC, Australia).
Anti-ERK2, anti-active ERK and anti-β actin antibodies were from Santa Cruz
Biotechnology, (Santa Cruz, CA USA). Anti-ERK5 antibody was from Sigma-Aldrich
Pty. Ltd., (Sydney, NSW, Australia). Anti-VDR and anti-rat IgG antibodies were kind gifts from Dr EM Gardiner (Garvan Institute of Medical Research, NSW, Australia).
2.1.8 Serum, Culture media and buffers
Fetal calf serum, RPMI-1640 and L-glutamine (200 mM) was from JRH biosciences,
(Lenexa, KS, USA). Dulbecco’s Minimal Essential Media (DMEM), RPMI-1640 without phenol red and Trypsin EDTA were from Gibco BRL, (Carlsbad, California, USA).
Penicillin/streptomycin (5000 U/ml) was from Commonwealth Serum Laboratories
83 (CSL), (Parkville, Victoria, Australia). Phosphate buffered saline (PBS) was from Cell
Image, (Adelaide, SA, Australia).
2.1.9 Flasks and plates for culture
Culture flasks (75 cm2, 125 cm2) were from Corning, (Corning, NY, USA) and Becton
Dickinson Biosciences, (Franklin Lakes, NJ USA). Tissue culture plates (100x20 mm) were from TPP, (Trasadingen, Switzerland). Multi-well plates (24 wells) were from
Becton Dickinson Labware, (Franklin Lakes, NJ, USA).
2.1.20 Cell lines
Human primary embryonic kidney cell line (HEK-293T) was from J Health, Oxford
University, (Oxford, UK, England). Monkey kidney fibroblast cell line (COS-1) and human promyelocytic leukemic (HL60) cells were obtained from American Type Culture
Collection (ATCC), (Rockville, MD, USA). Isolated primary rat hepatocytes were from
Dr Roland Gregory, Department of Medical Biochemistry (Flinders University, SA,
Australia). They were prepared fresh on the day of use.
2.1.21 Plasmids/Vectors
The rat CYP24 promoter luciferase constructs pCYP24WT(-1.4kb)-Luc) and pCYP24WT(-298bp)-Luc contain the promoter sequence from either -1.4 kb or -298 bp to the transcription start site as well as 74 bp of 5´-untranslated region cloned upstream of
84 the firefly luciferase reporter gene in the pGL3 basic vector, as described previously
(Hahn et al., 1994; Dwivedi et al., 2000) (Figure 2.1 A and B).
The pCYP24mVDRE1&2(-298bp)-Luc (where Luc stands for luciferase) containing mutations to VDRE1 and VDRE2 has been described previously (Dwivedi et al., 2000).
(Figure 2.1 C).
The pCYP24(mEBS)(-298bp)-Luc reporter construct containing a mutation in the EBS has been described previously (Dwivedi et al., 2000) (Figure 2.1 D).
The pCYP24mGC(-298bp)-Luc containing a mutation to the GC box at -114/-101 has been described previously (Gao et al., 2004) (Figure 2.1 E).
85
VDRE-2 VDRE-1 EBS GC-Box Luciferase A +1 -1.4kb pCYP24WT(-1.4kb)-Luc +74
B VDRE-2 VDRE-1 EBS GC-Box Luciferase +1 -298bp pCYP24WT(-298bp)-Luc +74
C VDRE-2 VDRE-1 EBS GC-Box Luciferase +1 pCYP24mVDRE1&2-Luc +74 -298bp
D VDRE-2 VDRE-1 EBS GC-Box Luciferase +1 -298bp pCYP24mEBS-Luc +74
E VDRE-2 VDRE-1 EBS GC-Box Luciferase +1 -298bp pCYP24mGC-Luc +74
Figure 2.1 Diagram showing the CYP24WT(-1.4kb)-Luc, CYP24WT(-298bp)-Luc
and the site-directed mutagenesis for the VDRE1&2, EBS and putative GC site of
the CYP24WT(-298bp)-Luc.
86 The pCYP24WT(-298bp)-Luc, pCYP24WT(-1.4kb)-Luc, pCYP24mVDRE1&2-Luc, pCYP24mEBS-Luc, pRSV-hVDR, pRSV-hRXR S260A, VP16-RXR and pCYP24mGC-
Luc were provided by Dr. P Dwivedi (School of Molecular Sciences, University of
Adelaide, SA, Australia) (Hahn et al., 1994; Dwivedi et al., 2000; Dwivedi et al., 2002).
The promoterless pRL-null Renilla luciferase vector (pRL-Null-Luc) and HSV-thymidine kinase promoter pRL-TK Renilla luciferase vector (pRL-TK-Luc) was purchased from
Promega (Madison, WI, USA).
Dominant negative PKC-ζ, PKCζK281M, was generously provided by Dr. JW Soh
(Columbia University, USA) (Soh et al., 1999).
Dominant negative Ras, Ras17N, and dominant negative ERK1, ERK1K71R were kindly provided by Prof. C. J. Der (University of North Carolina at Chapel Hill, NC, USA)
(Quilliam et al., 1994; Westwick et al., 1994)
Dominant negative MEK5, MEK5A, was kindly provided by Prof. E. Nishida (Kyoto
University, Kyoto, Japan) (Kamakura et al., 1999).
Dominant negative MKK4, MKK4K116R, was provided by Prof. D. Riches (National
Jewish Medical and Research Center, Denver, CO, U.S.A.) (Chan et al., 1999)
GST-Ets-1 was kindly provided by Professor Ismail Kola (Institute of Reproduction and
Development, Monash University, Victoria, Australia) (Dwivedi et al., 2002)
87 His-Ets-1 and GST Ets-1T38A mutant were provided by Dr. P Dwivedi (School of
Molecular Sciences, University of Adelaide, SA, Australia) (Dwivedi et al., 2002).
2.1.22 Buffers and Solutions
The following buffers were used in this thesis:
Lysis buffer – 20 mM HEPES, pH 7.2, NP-40 0.5% (v/v), 100 mM NaCl, 2 mM Sigma
104, 1 mM DTT, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin A and 10 µg/ml benzamidine hydrochloride.
Washing buffer – 10 mM HEPES, pH 7, 100 mM NaCl, 2 mM DTT, 2 mM Sigma 104,
1 mM PMSF and 10 µg/ml each of aprotinin, leupeptin, pepstatin A and benzamidine.
Assay buffer – 20 mM HEPES, pH 7.2, 20 mM β-glycerophosphate, 10 mM Sigma 104,
10 mM MgCl2, 1 mM DTT, 50 µM orthovanadate, 20 µM ATP.
Laemmli buffer – 20 mM Tris-HCl, pH 6.8, 40% sucrose, 6% SDS and 10 mM β- mercaptoethanol.
Blocking buffer for western blots - 5% skim milk, 25 mM Tris/HCl pH 7.4, 100 mM
NaCl.
Washing buffer for western blots – blocking buffer + 0.1% Tween-20.
GTE buffer - 50 mM glucose, 25 mM Tris pH 8.0, 10 mM EDTA pH 8.0.
88
1x Tris-EDTA buffer (1xTE buffer) – 10 mM Tris-HCl, 1.0 mM EDTA pH 8.0.
Lowry’s Solution - 2% Na2CO3, 1% SDS, 0.4% NaOH, 0.16% Na/K-tartrate.
Solution II for plasmid DNA extraction - 0.2 M NaOH, 1% SDS.
2.1.23 Reagents for RNA extraction and real time PCR
Trizol Reagent and SuperScript III reverse transcriptase was from Invitrogen (Mount
Waverley, Vic, Australia). Random hexamer primers, dNTPs, and PCR primers were purchased from Geneworks (Adelaide, SA, Australia). Primers designed to amplify cDNA for CYP24 (F: 5’-cctgctgccagattctctggaa-3’; R: 5’-atacttcttgtggtactccacca-3’) and
GAPDH (F: 5’-acccagaagactgtggatgg-3’; R: 5’-cagtgagc ttcccgttcag-3’) were based on
Genebank sequences (CYP24 Acc: NM000782; GAPDH Acc: NM002046). The iQ™
SYBR® Green Supermix was purchased from Bio-Rad Laboratories (Regents Park, NSW,
Australia).
89 2.2 Methods
2.2.1 Cell Culture
2.2.1.1 Culture of HEK293T and COS-1 cells
HEK293T and COS-1 cells were maintained in DMEM supplemented with 10% heat- inactivated FCS, 100 U/ml penicillin/streptomycin, 4 mM L-glutamine. They were incubated in a humidified atmosphere of 5% CO2 in air at 37°C.
2.2.1.2 Cell Culture for kinase assay and for western blotting.
HEK293T and COS-1 cells were seeded at 2.5 x 106 cells per 10 cm2 culture plate in 6 ml of DMEM supplemented with 10% heat inactivated FCS, 100 U/ml penicillin/streptomycin, 4 mM L-glutamine. The cells were incubated at 37°C in a humidified atmosphere of 5% CO2 in air overnight before being washed with 4 ml serum- free RPMI-1640. The cells were then incubated with 6 ml of serum-free RPMI-1640 overnight before being treated with chemotherapeutic drugs and/or 1,25(OH)2D3.
90 2.2.1.3 Culture of HL 60 cells
HL-60 cells were maintained in RPMI-1640 supplemented with 10% heat-inactivated
FCS, 100 U/ml penicillin/streptomycin, 4 mM L-glutamine. They were incubated in a
6 humidified atmosphere of 5% CO2 in air at 37°C, and kept at a density of <1x10 cells/ml.
2.2.2 Cell Viability Assay using trypan blue.
HEK293T cells, COS-1 cells, isolated hepatocytes and HL60 cells were seeded at 75,000 cells in 400 µl of DMEM containing 10% FCS in a 24 well plates. The cells were incubated overnight before being treated with one of the following chemotherapeutic drugs: etoposide, cis-platinum, vincristin sulfate or daunorubicin hydrochloride at concentrations indicated in the Results section. After 24 hours, the cells were harvested and 44 µl of Trypan blue (1% w/v) was added. The total number of cells and the trypan blue-positive cells were counted using a haemocytometer under a light microscope. The percentage of dead cells was calculated based on the number of cells that had taken up trypan blue relative to the total number of cells counted.
91 2.2.3 Plasmid DNA Preparation and Transfection
2.2.3.1 Midi-preparation using midi-plasmid DNA preparation kit (Qiagen)
Isolation of up to 100 µg plasmid DNA was performed using the midi-preparation kit according to the manufacture’s instruction.
2.2.3.2 Large scale preparation by cesium-chloride (CsCl) density gradient procedure
High concentrations of plasmid DNA can be obtain using a modified version of
Sambrook et al. (1989). The desired clone culture e.g.20 ml was inoculated into 250 ml of
LB medium with appropriate antibiotics (100 µg/ml Ampicillin) and incubated at 37oC with shaking. The bacterial cells were then pelleted at 4oC in a SS-34 rotor at 6000 rpm for 5 min. Plasmid DNA was extracted using the alkaline lysis procedure: 10 ml of freshly prepared GTE buffer with lysozyme (2 mg/ml) was used to lyse the cells on ice for 20 min; 20 ml of freshly prepared solution II was added and mixed with the cell lysate by inverting the tubes and then 15 ml of NaAc (3M, pH 4.6) was added, mixed and left on ice for 10 min. The supernatant was collected and passed through a funnel containing cloth bandage and the volume recorded in a 50 ml measuring cylinder. Plasmid DNA was precipitated by the addition of 0.6 volume of isopropanol and incubated on ice for 10 min.
The plasmid DNA was pelleted at 9000 rpm for 20 min and the supernatant discarded.
The pelleted plasmid DNA was allowed to air dry and resuspended in 4 ml of 1xTE buffer. To separate plasmid DNA from RNA and genomic DNA, the plasmid was further purified using the CsCl/ethidium bromide density gradient procedure. CsCl (4.2 g) was mixed with the dissolved plasmid DNA by vortexing thoroughly. Ethidium bromide (100
µl of 10 mg/ml) was added as the marker of plasmid DNA. The mixture was then
92 centrifuged in a Beckman TL-100 bench top ultracentrifuge and TLA-100.2 rotor at
80,000 rpm at 20oC overnight. Plasmid DNA with ethidium bromide (the lower most central red band) was havested using syringe and needles. The ethidium bromide was removed by butanol extraction. The plasmid DNA was then precipitated with 2.5 volumes of 100% ethanol, rinsed with 70% ethanol, resuspended in 1xTE buffer, and quantified by spectrophotometry (260 nm) and analysed by agarose gel electrophoresis to confirm concentration and supercoiling.
2.2.3.3 Transient transfection of mammalian cells and treatment with 1,25(OH)2D3 and/or chemotherapeutic drugs.
Transfection of cells was performed with the liposomal transfection reagent, DOTAP, according to Dwivedi et al., (2002). In preparation for transfection, approximately 5x104 cells were seeded into 24 well plates in 400 µl of DMEM containing 10% FCS to achieve
60-70% confluence. Following incubation for 4-5 h to allow the cells to attach, the wells were washed with 250 µl of serum-free RPMI-1640 and the medium replaced with 400 µl of serum-free RPMI-1640. Transfection was performed in triplicate using 200 ng of the
CYP24 promoter luciferase construct and 50 ng of pRL-Null-Renilla luciferase vector
(pRL-Null-Luc) as control for transfection efficiency. Whenever indicated, 200 ng of dominant negative mutants or blank plasmids were also co-transfected. All plasmid DNA was diluted in HEPES buffer (20 mM, pH 7.4) to a final volume of 5 µl and mixed with
1.5 µl of DOTAP diluted to 3.5 µl with HEPES buffer. DNA-DOTAP complex formation was achieved by incubating the mixture for 15-20 min at room temperature. A total of 10
µl DNA-DOTAP complex was transfected into the cells. After an overnight incubation, the media was replace with 400 µl serum-free medium RPMI-1640. The cells were treated with different chemotherapeutic drugs, 1,25(OH)2D3 or vehicle (H2O, ethanol or
93 DMSO). The maximum amount of vehicle was 0.1% (v/v), which did not affect promoter activity. After 24 h, the medium was aspirated and the cells were lysed using 50 µl of 1X passive lysis buffer (Promega) for 20 min at 25oC.
2.2.3.4 Dual Luciferase assay
Dual luciferase activity was measured using a TD-20/20 luminometer. The first luminescence from the firefly luciferase (representing CYP24 promoter activity) was recorded by adding 25 µl of LARII substrate into the 2 µl of cell lysate in an eppendorf tube. The second luminescence for Renilla luciferase (representing reference luminerscence) was quantified by the addition of 25 µl of Stop and Glo substrate to quench the first reaction and simultaneously initiate the Renilla luciferase reaction. Data were recorded as relative luciferase activity, the ratio of the first luminescence over the second luminescence. Results were then presented as fold induction over vehicle (EtOH or DMSO)-treated control cells. Analysis of renilla luciferase activity from experiments in which chemotherapeutic drugs were used revealed that the drugs did not significantly alter this activity.
2.2.4 Preparation of cell lysates
After treatment the plates were placed on ice and the medium was removed using vacuum suction. The cells were harvested using a rubber policeman in 300 µL of lysis buffer. The samples were rocked for 2 h at 4o C, centrifuged (14,000 g x 5 min, 4o C) and the supernatants were collected and stored at –70o C until assayed. An aliquot was taken for protein estimation.
94 2.2.5 Lowry’s Protein determination
Protein content of samples was determined using the Lowry method (Harrington, 1990).
Protein standards were obtained using serial dilutions of BSA (1 mg/ml) with H2O, resulting in 6 standards, 0 µg, 3.125 µg, 6.25 µg, 12.5 µg, 25 µg and 50 µg. Each unknown sample (5 µl) was diluted in 45 µl of H2O, then 150 µl of CuSO4/Lowry’s solution (1:100) was added to all tubes and these were left to stand for 10 min at room temperature. Folin and Ciocalteu’s Phenol Reagent/H2O (1:1), 15 µl, was then added and the tubes were left to stand for 30 min. 180 µl of each sample was transferred to a 96 well plate and the absorbance at 570 nm was determined in a plate reader (Dynatech MR5000,
Guernsey, Channel Islands, UK). The absorbance of the protein standards was plotted on a linear regression nomogram (Instat, GraphPad Software Incorporated, San Diego, CA,
USA) and the protein content of the test samples was calculated.
2.2.6 Kinase Assay and Western Blotting
2.2.6.1 Purification of GST-jun (1-79)
A plasmid encoding the 1-79 amino acid sequence of c-jun was kindly provided by Prof.
C.J. Der (University of North Carolina, Chapel Hill, USA). After transformation, a single colony was inoculated into 100 ml of Luria-Bertani (LB) broth supplemented with 100
µg/ml ampicillin (CSL, Victoria, Australia) and shaken at 200 rpm for 16 h at 37oC in a
G24 Environmental Incubator shaker (New Brunswick Scientific, Edison, USA). After 16 h the entire inoculum was transferred into 400 ml of fresh LB broth supplemented with
100 µg/mL ampicilin and shaken at 200 rpm for 1 h at 37oC. The expression of GST c-jun
95 (1-79) fusion protein was induced by the addition of 1 mM Isopropyl-β-D- thiogactopyranoside (Boehringer Mannheim, Castle Hill, Australia) to the culture. The flasks were shaken for another 3 h at 200 rpm at 37oC. The cells were then collected by centrifugation (10 min x 12,500 g) at 4oC. The pellets were resuspended in 20 ml of ice cold phosphate buffer (22 mM, pH 7.0) and the cells disrupted using a French Pressure
Cell (SLM Instruments). The lysate was cleared by centrifugation (30 min x 48,000 g) at
4oC. The clarified lysate was then added to glutathione-sepharose CL4B (Pharmacia
Biotech, Uppsala, Sweden) and incubated overnight with constant mixing at 4oC. The
GST-c-jun (1-79)-bound glutathione sepharose was then washed with 50 ml of PBS (3 x
5 min, 600 g) and finally resuspended in PBS (1:1, PBS:beads). The GST-c-jun (1-79)- bound glutathione sepharose was stored at –70o C.
2.2.6.2 JNK kinase assay
A solid phase assay (Hii et al., 1998) was employed to determine the activity of JNK.
Equal amounts of lysate protein (0.8 mg to 1.2 mg) were added to 50 µl of GST-c-jun (1-
79) glutathione sepharose and MgCl2 and ATP added to a final concentration of 15 mM and 10 µM, respectively, and the samples rocked for 2 h or overnight at 4o C. The samples were washed once each with 400 µl with lysis buffer, washing buffer and assay buffer. The assay was initiated by adding 30 µl of reaction mixture (assay buffer, containing 33 mM of DTT, 3.8 mM of Sigma 104 and 10 µCi of [γ-32P] ATP) to the tubes, which were then incubated at 30o C for 20 min. The reaction was terminated by the addition of Laemmli buffer (ratio of 1:2 of laemmli buffer: assay mixture) and the samples heated at 100o C for 5 min. Phosphorylated GST c-jun (1-79) was fractionated on a 12% SDS-PAGE gel (BioRad, NSW, Australia) (175 volts for 90 min). An aliquot of
Low Range molecular weight markers (Bio-Rad, Sydney, NSW, Australia) was loaded in
96 a separate lane to provide a range of molecular weight markers. The radioactivity incorporated in the GST-c-jun (1-79) peptide was quantified using a Packard Instant
Imager (Packard, Canberra, Australia) and integrated using the Imager software (version
2.0.5).
2.2.6.3 ERK1/ERK2/ERK5 kinase assays
Equal amounts of lysate protein (0.8 mg to 1.2 mg) were added to 15 µl of protein A
Sepharose (Sigma Chemical Company, St Louis, USA) and the samples rocked for 30 min at 4oC and centrifuged (15,000 g x 10 s, 4oC) to pre-clear the samples. The supernatants were removed and incubated with 15 µl of protein A Sepharose and 3 µg of anti-ERK2 or anti-ERK5 antibody. After rocking for 2 h or overnight at 4oC, the samples were centrifuged (15,000 g x10 s, 4 o C) and the supernatants were discarded. The pellets were washed as described above for JNK assay. The assay was initiated by adding 30 µl of reaction mixture (assay buffer containing, 33 mM of DTT, 3.8 mM of Sigma 104, 0.03 mg/ml MBP and 10 µCi of [γ-32P] ATP). Samples were incubated at 30o C for 20 min.
The reaction was terminated by the addition of Laemmli buffer (15 µl) followed by heating at 100oC for 5 min. Phosphorylated MBP was separated from unincorporated 32P
ATP on a 16% SDS-PAGE gel run at 175 volts for approximately 1.5 h. Phosphorylated
MBP was visualised in a Packard Instant Imager (Packard, Canberra, Australia) and the amount of radiolabel was quantified using the Imager software (version 2.0.5).
2.2.6.4 Western Blotting
Western blotting was used to determine the levels of ERK1/ERK2, ERK5, active ERK and VDR protein. We used a Bio-Rad Mini gel apparatus or CBS Scientific MGV-201
97 mini-gel unit to fractionate ERK5 (10% SDS-PAGE gel), ERK1/ERK2 and VDR (12%
SDS-PAGE gel). Lysates were mixed with Laemmli buffer, boiled for 5 min at 100° C and between 20 to 40 µg of proteins were loaded per lane, depending on the experiment.
Within each experiment, the same amount of lysate proteins were loaded for each lane.
The gel was run at 175 volts for approximately 1 h or until the dye front had migrated out of the gel. The separated proteins were subsequently transferred onto nitrocellulose membrane in a Bio-Rad Mini Trans-Blot cell (Bio-Rad, Sydney, NSW, Australia) at 100
V for 1.5 h. The blots were stained with Ponceau S (0.1% in 5% acetic acid) to visualise the transferred proteins and confirm even transfer of proteins to the membrane. After destaining, the membrane was blocked for 1 h at 37° C or overnight at 4° C in blocking buffer, then incubated with primary antibody (Table 2.1) diluted in Tris-Tween (pH 7.4) for 1 h. The membrane was washed (3 x 10 min) in washing buffer and then incubated for
1 h with horseradish peroxidase-conjugated secondary antibody (Table 2.1), diluted in blocking buffer. The membrane was washed again (3 x 10 min) with washing buffer. The target protein was visualized by enhanced chemiluminescence (Hii et al., 1995). The relative density of each band was determined using Image Quant software, version 3.3
(Molecular Dynamics, Charlottesville, VA, USA).
Table 2.1: Antibodies types and dilution for primary and secondary antibodies.
Primary Antibodies Secondary Antibodies
Anti ERK5 1:10,000 Anti Rabbit 1:2000
Anti ERK2 1: 2000 Anti Goat 1: 2000
Anti-active ERK 1:10,000 Anti Rabbit 1:2000
Anti VDR 1:1000 Anti Rat 1:50000
Anti β-actin 1:10,000 Anti Mouse 1:2000
98 2.2.6.5 Western Blot recycling
Following Western blotting, nitrocellulose membranes were stored at -20° C. When required the blots were stripped using a Western blot recycling kit. The membrane was incubated in stripping solution (1:10) for 10 min, then blocked using the supplied blocking solution (1:20) (2x5 min). The membrane was then re-probed with primary and secondary antibodies as previously described. Stripping and re-probing was possible up to
3 times. The bands were again subjected to densitometric analysis. Densitometry permits the correction of unequal protein loading and the results are expressed as a ratio of a target protein: reference protein.
2.2.7 Quantification of mRNA expression using real-time PCR
2.2.7.1 RNA Extraction
Following treatment, cells were lysed in 1 ml of Trizol Reagent. RNA was than partitioned into the aqueous phase by adding chloroform (0.2 ml), mixed and centrifuged
(15min x 12,000 g). The aqueous phase was than transferred to a clean eppendolf tube.
RNA was precipitated by adding 0.5 ml of isopropanol and 10 µg of RNase-free glycogen. After incubating overnight at -70° C, the sample was centrifuged (15min x
12,000 g). Supernatent was discared and RNA pellet washed with 1 ml of 75% EtOH.
The RNA pellet was than air dry and resuspended in 15 µl of H2O. Total amount of RNA was quantified using a spectrophotometer (by reading the absorption at 260nm).
2.2.7.2 Generation of cDNA cDNA was generated by reverse PCR. Briefly, 1 µg of RNA was mixed with Reaction
Mixture I which consisted of 2 µl of Random Hexamer, 2 µl of 10mM dNTPs and topped
99 up to a final volume of 23 µl with H2O. After incubating for 5 min at 65° C, the mixture was place on ice for 1 min. Mixture II consisting of 8 µl of 5x first strand Buffer, 2 µl of
0.1 M DTT and 2 µl of Superscript III Reverse Transcriptase was than added and the mixture incubated at 50° C for 60 min. Following completion of reaction, the enzyme was than inactivated by heating to 70° C for 15 min.
2.2.7.3 Real-time PCR
The cDNA samples were amplified by mixing 1 µl of cDNA together with 5 µl of iQ
SYBR Green Supermix (2x), 0.5 µl of CYP24 or GAPDH forward primer (5µM), 0.5 µl of reverse primer (5µM) and 3 µl of H2O. The reaction mixture was than placed into a
Rotor-Gene Real-Time PCR thermo cycler and the following programme was run. 15 min at 94° C to melt the cDNA, 35 cycle of 30 sec at 94° C, 30 sec at 60° C, 30 sec at 72° C to amplify cDNA and 4 min at 72° C to deactivate enzyme. To check purity a melt curve was generated by heating for 15 sec at 72° C and 90° C for 5 sec. Relative expression between samples was calculated using the comparative cycle threshold (CT) method
(∆CT) (Livak and Schmittgen, 2001).
2.2.8 Statistics
ANOVA, Tukey-Kramer multiple comparisons test and Dunnett’s multiple comparisons test and Student’s t-test were conducted using Graph Pad InStat V2.02 (Graph Pad
Software, San Diego, CA, USA) and SPSS 11 student version (SPSS, Chicago, IL, USA).
Results were considered statistically significant when p<0.05.
100
Chapter 3
Effects of chemotherapeutic drugs on the induction of the CYP24 promoter in kidney
cells.
101 3.1 Introduction
As discussed in Chapter 1.11, chemotherapeutics drugs have been reported to stimulate the activities of MAP kinases which we have been previously reported to regulate the activity of the CYP24 promoter by 1,25(OH)2D3. This raises the possibility that the drugs could induce CYP24 promoter activity by themselves. The aim of this chapter is to investigate whether chemotherapeutic drugs such as daunorubicin hydrochloride, etoposide, cisplatin, vincristine sulfate, doxorubicin hydrochloride and dexamethasone can stimulate the CYP24 promoter activity in kidney cells. The effect of the drugs were tested in two different CYP24 promoter constructs, the -1.4kb and the -298bp promoter.
Unlike the -1.4kb promoter, the -298bp promoter has been studied extensively and has been very well characterized. However, we chose to investigate the -1.4 kb promoter construct first because it resembles more of the endogenous promoter in length.
COS-1 and HEK293T cells were used in this study. Both kidney cell lines have been previously validated for studying the regulation of CYP24 promoter activity (Dwivedi et al., 2000 and Nutchey et al., 2005). The concentrations of drugs used in this study are in the range of levels found in the serum of patients receiving chemotherapy. List et al.
(2001) found that the peak serum concentration of daunorubicin hydrochloride in patients with acute myeloid leukemia to be around 0.9 µM – 1.1 µM. Kato et al. (2003) found that etoposide levels in Japanese children and adolescents were around 16 µM – 54 µM.
Marina et al. (2002) found that the peak serum concentration of doxorubicin hydrochloride in children with refractory tumours to be around 63 µM – 83 µM. Peng et al. (1997) reported that the peak concentration of cisplatin in pediatric patients was around 13 µM – 20 µM. These concentrations were also used in a study to investigate the
102 effects of the drugs on apoptosis in osteoblasts (10 µM etoposide, 0.1 µM vincristine sulfate and 0.1 µM daunorubicin hydrochloride) (Davies et al., 2003).
Prior to undertaking the promoter induction studies, the effects of the drugs on cell viability were investigated to exclude the possibility that these drugs are cytotoxic to cells which either synthesise or metabolise 1,25(OH)2D3. Using the trypan blue exclusion test to determine viability, the data demonstrate that whereas daunorubicin hydrochloride, etoposide, cisplatin and vincristine sulfate were effective at killing HL60 human promyelocytic leukaemic cells at the above-listed dose ranges, these drugs did not affect the viability of COS-1, HEK293T and primary hepatocyte cells, even at concentrations higher than those found in the serum of patients receiving chemotherapy (data not shown). Thus, it is unlikely that the reduction in serum 25(OH)2D3 and 1,25(OH)2D3 levels seen in children undergoing chemotherapy is the result of the cytotoxic effects of the drugs on hepatocytes and kidney cells which produce the vitamin D metabolites.
3.2 Methods for investigating CYP24 promoter induction
COS-1 and HEK293T kidney cells were transiently transfected with a construct containing the –298bp or the –1.4kb CYP24 promoter sequence fused to the firefly luceferase reporter gene (pCYP24WT (-298bp)-Luc and pCYP24WT (-1.4kb)-Luc). A recombinant vector pRL-Null (0) expressing renilla luciferase was also co-transfected as an internal control for transfection efficiency. Cells were then treated with daunorubicin hydrochloride, etoposide, cisplatin or vincristine sulfate alone or in combination with
1,25(OH)2D3. Data obtain (ratio of firefly:renilla luciferase activity) were expressed as fold induction, the ratio of drug treated cells over ethanol treated cells (control group)
103 3.3 Results
3.3.1 Effects of chemotherapeutic drugs on the activity of the -1.4kb CYP24 promoter in COS-1 and HEK293T kidney cells.
COS-1 and HEK293T cells were transiently cotransfected with a construct containing the
-1.4 kb CYP24 promoter-luciferase gene (pCYP24WT (-1.4 kb-Luc) and the pRL-0 vector expressing Renilla luciferase. Cells were treated with daunorubicin hydrochloride, etoposide, cisplatin or vincristine sulfate alone or in combination with 1,25(OH)2D3. The data obtain were expressed as fold induction, the ratio of drug treated cells over ethanol treated cells (control group).
The data show that daunorubicin hydrochloride by itself increase the activity of the -1.4 kb promoter in COS-1 and HEK293T cells by 2 ± 0.5 and 2.4 ± 0.4 fold, respectively over ethanol treated cells (Figure 3.1a). As expected, 1,25(OH)2D3 stimulated the activity of the CYP24 promoter in both cell lines. The induction by 1,25(OH)2D3 in
HEK293T cells was substantially higher than in COS-1 cells. Treatment with daunorubicin hydrochloride and 1,25(OH)2D3 together caused an additive effect on
CYP24 promoter activity in COS-1 cells but no further increase in CYP24 promoter activity was observed in HEK293T cells under this condition (Figure 3.1a).
The effect of etoposide was investigated next. This drug increased the -1.4 kb CYP24 promoter activity in COS-1 and HEK293T cells by 3 ± 0.6 and 2.8 ± 0.5 fold, respectively (Figure 3.1b). Interestingly, a synergistic increase in CYP24 promoter activity was observed when COS-1 and HEK293T cells were treated with both etoposide and 1,25(OH)2D3, giving approximately 7.4 and 4.3 fold, respectively, higher induction than that caused by 1,25(OH)2D3 (Figure 3.1b).
104
Vincristine sulfate alone increase the -1.4 kb CYP24 promoter activity by 2.9 ± 0.5 fold in COS-1 cells (Figure 3.1c). However, the drug did not affect the activity of the CYP24 promoter in HEK293T cells. A synergistic increase in CYP24 promoter activity was observed when COS-1 and HEK293T cells were treated with both vincristine sulfate and
1,25(OH)2D3, approximately 4.2 and 2 fold respectively higher than induction cause by
1,25(OH)2D3 (Figure 3.1c).
Cisplatin alone did not stimulate the -1.4 kb CYP24 promoter activity in both cell lines
(Figure 3.1d). However, the treatment of the cells with cisplatin and 1,25(OH)2D3 resulted in a synergistic increase in CYP24 promoter activity in COS-1 cells, approximately a 6.3 fold higher than induction cause by 1,25(OH)2D3. No further increase in promoter activity was observed in HEK293T cells (Figure 3.1d).
105 a b
25 ### 80 ) n on) i o i t ct c
u 20 ***
d COS-1 du n I n
COS-1 HEK293T I 60 d l old HEK293T
(F 15 n n (Fo o i t io t c c u 40 u d d n I n I
10 r ## e t oter o m
om *** r # 20
5 pro
* 4 * * 2 P *** CYP24 p CY 0 0 Daunorubicin - + - + Etoposide - + - + 1,2 5(OH)2D3 - - + + 1,25(OH)2D3 - - + +
c d
40 25
) ### ### ) n ction io t u
c d 20 *** n du I 30 COS-1 COS-1 n I old
HEK293T d HEK293T l F ( o F 15 on i t n ( c io
*** t c du 20 u n d I r n
### I
10
r ote e t o om r m p 10 o * pr 24 5 4 * * 2 P CYP
CY 0 0 Vincr istine - + - + Cisplatin - + - + 1,2 5(OH)2D3 - - + + 1,25(OH)2D3 - - + +
106 Figure 3.1 Effects of chemotherapeutic drugs on the -1.4kb CYP24 promoter in
COS-1 and HEK293T cells. Cells were transiently transfected with 200 ng of pCYP24WT (-1.4kb)-Luc and incubated with daunorubicin hydrochloride (2 µΜ), etoposide (100 µΜ), vincristine sulfate (10 µΜ), cisplatin (100 µΜ) and/or 1,25(OH)2D3
(10 nM) for 24 h. Control cells received ethanol (0.1%, v/v). The results are presented as fold induction over ethanol-treated control cells. Data are mean ± SEM of three independent experiments performed in triplicate. Significance of difference between control and drug or 1,25(OH)2D3: *p<0.05, **p<0.01, ***p<0.001; Significance of difference between 1,25(OH)2D3 and 1,25(OH)2D3 + drug: #p<0.05, ##p<0.01,
###p<0.001 (ANOVA and Tukey-Kramer multiple comparisons test). The response observed with HEK293T cells in the presence of 1,25(OH)2D3 either alone or in the presence of a drug was generally greater than that seen with COS-1.
107
3.3.2 Effects of chemotherapeutic drugs on the activity of the -298 bp CYP24 promoter in COS-1 kidney cells.
In this study, we compared the effects of daunorubicin hydrochloride, etoposide, vincristine sulfate and cisplatin on the -298bp and -1.4kb CYP24 promoter in COS-1 cells. Treatment of COS-1 cells with daunorubicin hydrochloride resulted in an increased the activity of the -298 bp CYP24 promoter activity by 2.7 ± 0.5 fold. This result is similar to the results seen in the -1.4 kb promoter. As expected, 1,25(OH)2D3 up- regulated the -298bp CYP24 promoter activity. Unlike the -1.4 kb CYP24 promoter, treatment with both daunorubicin hydrochloride and 1,25(OH)2D3 resulted in a substantial increase in the -298bp CYP24 promoter activity, (a 13.3 ± 1.1 fold higher than ethanol treated cells, and approximately 7 fold higher than the induction caused by
1,25(OH)2D3) (Figure 3.2a).
Likewise to the data obtain from the -1.4 kb promoter, etoposide was also able to increase the -298bp CYP24 promoter induction by 2.1 ± 0.2 fold over ethanol treated cells. When combined with 1,25(OH)2D3, etoposide treatment caused a synergistic increase in -298bp
CYP24 promoter activity (a 26.3 ± 3.9 fold increase over ethanol treated cells, and approximately 2.9 fold higher than CYP24 induction by 1,25(OH)2D3) (Figure 3.2b).
This result is similar to the result obtained in the -1.4 kb promoter.
108
a b
16 35 ### ) ### n o n)
cti 28 ## ctio du u n
12 d 298bp n d I
I
l 298bp
o 1.4KB F
1.4KB old (
n 21 (F io n t c u
ctio
d 8 u n d I r In
e 14 t
o # ** m oter o m o pr
4 ** * r 4
2 7 * 4 p P * * * * CY
CYP2 0 0
Daunorubicin - + - + Etoposide - + - + 1 ,25(OH)2D3 - - + + 1,25(OH)2D3 - - + +
c d
20 24 ###
ion) t on) c i t u 298bp 20 c
d 16 u n 298bp I 1.4KB ###
Ind 1.4KB old
(F 16 old
n (F
12 o i n t ** o c i t c du 12 ###
du n I er In 8 ot er ot 8 om r p om r * * 4 * * 4 P24 p * CYP24 Y C
0 0 Vincristine - + - + Cisplatin - + - + 1,25(OH)2D3 - - + + 1,25(OH)2D3 - - + +
109 Figure 3.2 Comparison of the effects of chemotherapeutics drugs on the -298bp and
-1.4kb CYP24 promoter constructs in COS-1 cells. Cells were transiently transfected with 200 ng of pCYP24WT (-298bp)-Luc or pCYP24WT (-1.4kb)-Luc and incubated with daunorubicin hydrochloride (2 µΜ), etoposide (100 µΜ), vincristine sulfate (10 µΜ), cisplatin (100 µΜ) and/or 1,25(OH)2D3 (10 nM) for 24 h. Control cells received ethanol
(0.1%, v/v). The results are presented as fold induction over control cells. Data are mean
± SEM of three independent experiments performed in triplicate. Significance of difference between control and drug or 1,25(OH)2D3: *p<0.05, **p<0.01, ***p<0.001; significance of difference between 1,25(OH)2D3 and 1,25(OH)2D3 + drug: #p<0.05,
##p<0.01, ###p<0.001 (ANOVA and Tukey-Kramer multiple comparisons test).
110 Vincristine sulphate can also increased the -298bp CYP24 promoter activity in COS-1 cells by 1.9 ± 0.3 fold over ethanol treated cells (Figure 3.2c). However, the addition of vincristine sulphate and 1,25(OH)2D3 did not further increase the -298bp CYP24 promoter activity, which is in contrast to data obtain from the -1.4 kb promoter which showed a synergistic increase in promoter activity.
In contrast to the other drug, cisplatin alone did not affect the activity of either the -298bp or the -1.4 kb CYP24 promoter. However, when incubated together with 1,25(OH)2D3, a synergistic increase in the -298bp promoter activity was observed. This amounted to a 9.2
± 1.5 fold increase over ethanol treated cells which is approximately 4.9 fold higher than
CYP24 induction by 1,25(OH)2D3 alone (Figure 3.2d).
We also tested cisplatin at a lower concentration (10 µM). The results show that cisplatin alone did not stimulate the -298bp CYP24 promoter but in the presence of 1,25(OH)2D3 the drug synergistically increased the CYP24 promoter activity which was 3 fold higher than induced by 1,25(OH)2D3 alone (data not shown). These results demonstrate that while there are subtle differences between the two promoter constructs, there is a general consensus between the two constructs.
111 3.3.3 Daunorubicin hydrochloride stimulated the –1.4kb CYP24 promoter activity in a dose-dependent manner in COS-1 kidney cells.
COS-1 cells were transiently transfected with pCYP24WT (-1.4 kb)-Luc and the pRL-0 vector and treated with 0.2, 0.5, 1 and 2 µΜ of daunorubicin hydrochloride ±
1,25(OH)2D3. Results from these experiments (Figure 3.3) show that daunorubicin hydrochloride per se can increase the –1.4 kb CYP24 promoter activity in a dose dependent manner. Increases in CYP24 promoter activity by 3.6 ± 0.8 fold and 5.3 ± 1.5 fold were observed at 1 µΜ and 2 µΜ of daunorubicin hydrochloride respectively. In the presence of 1,25(OH)2D3, the drug demonstrated an enhancing effect on the promoter activity especially at 1 and 2 µΜ of daunorubicin hydrochloride. However, the effect was not synergistic. In addition, lower doses of daunorubicin hydrochloride (0.2 µΜ and 0.5
µΜ) did not alter the ability of 1,25(OH)2D3 to stimulate the activity of the -1.4 kb
CYP24 promoter (Figure 3.3).
These results demonstrate that daunorubicin hydrochloride can stimulate the -1.4 kb
CYP24 promoter in a dose dependent manner and can enhance the induction by
1,25(OH)2D3 in COS-1 cells.
112
12 #
#
n 1,25(OH)2D3 o i
ct 9 ) du n n o I i
t c * du oter
n
I 6 om r Vehicle * (Fold
* CYP24 p 3
0
00.511.52
Daunorubicin Hydrochloride (µM)
Figure 3.3 Dose-response profile of the effects of daunorubicin hydrochloride on the activity of the –1.4kb CYP24 promoter in COS-1 cells. Cells were transiently transfected with 200 ng of pCYP24WT (-1.4kb)-Luc and incubated with daunorubicin hydrochloride (0.2 µΜ, 0.5 µΜ, 1 µΜ and 2 µΜ) ± 1,25(OH)2D3 (10 nM) for 24 h. Data shown are mean ± SEM of three independent experiments performed in triplicate.
Significance of difference between untreated cells and drug- or 1,25(OH)2D3-treated cells: *p<0.05. Significance of difference between 1,25(OH)2D3 and drug +
1,25(OH)2D3: #p<0.05 (ANOVA and Tukey-Kramer multiple comparisons test).
113 3.3.4 Dose-dependent effect of daunorubicin hydrochloride on the -298bp
CYP24 promoter in COS-1 cells and HEK293T cells.
The dose-response profile of the effect of daunorubicin hydrochloride was also investigated using the -298bp promoter construct. The results in Figure 3.4 show a dose dependent increase in the CYP24 promoter activity in COS-1 cells incubated with daunorubicin hydrochloride. At 1 µΜ of daunorubicin hydrochloride, CYP24 promoter induction was increased by 3.7 ± 0.4 fold when compared to untreated cells. The threshold of stimulation was around 0.5 µΜ. Synergistic increases in CYP24 promoter activity were observed in cells treated with 1,25(OH)2D3 and low concentrations (0.13 and 0.25 µΜ) of daunorubicin hydrochloride. However, the enhancing effect of daunorubicin hydrochloride on the effect of 1,25(OH)2D3 diminished as the concentrations of daunorubicin hydrochloride were increased.
In contrast to data obtain from COS-1 cells, daunorubicin hydrochloride alone did not stimulate the activity of the -298bp promoter in HEK293T cells in the dose range tested.
However, treatment of HEK293T cells with both 1,25(OH)2D3 and daunorubicin hydrochloride produced a synergistic increase in CYP24 promoter activity, which amounted to approximately 2.7, 3.0, 3.7 and 2.7 fold induction at 0.13, 0.25, 0.5 and 1
µΜ daunorubicin hydrochloride, respectively, over the effect of 1,25(OH)2D3 alone.
These data demonstrate that in this cell line, the predominant effect of daunorubicin hydrochloride was observed in the presence of 1,25(OH)2D3, thereby confirming that
114 COS-1 8 ##
n o 6 i # t c
n) ** o i Indu
duct 1,25(OH)2D3 oter 4 n I Vehicle (Fold * 2 CYP24 prom
0 0 0.2 0.4 0.6 0.8 1
Daunorubicin Hydrochloride (µM)
18 HEK293T ##
15 ## n ## 1,25(OH)2D3 o i t ## duc n) 12 n o I i
t c du oter
n 9 I om r
(Fold 6 ***
CYP24 p 3 Vehicle
0 0 0.2 0.4 0.6 0.8 1 Daunorubicin Hydrochloride (µM)
115 Figure 3.4 Dose-response profile of the effect of daunorubicin hydrochloride on the
-298bp CYP24 promoter in the presence or absence of 1,25(OH)2D3 in COS-1 and
HEK293T cells. COS-1 and HEK293T cells were transfected with 200 ng of vector containing pCYP24WT (-298bp)-Luc promoter. Daunorubicin hydrochloride and/or
1,25(OH)2D3 were added and promoter activity was assayed 24 h later as described under Materials and Methods. Results shown are mean + SEM of 3 experiments conducted in triplicate determinations. Significance of difference between control and drug or 1,25(OH)2D3 treatment: *p<0.05, **p<0.01, ***p<0.001; significance of difference between 1,25(OH)2D3 and 1,25(OH)2D3 + drug: #p<0.05, ##p<0.01
(ANOVA followed by Tukey-Kramer multiple comparisons test).
116 daunorubicin hydrochloride could enhance CYP24 promoter induction under our experimental conditions.
3.3.5 Etoposide stimulated the -1.4kb CYP24 promoter activity in a dose- dependent manner in COS-1 kidney cells.
Etoposide had the greatest effect amongst the other three drugs on the induction of the -
1.4 kb promoter (Figure 3.1 and 3.2). Thus, it was of interest to also investigate the relationship between varying dosage of etoposide and CYP24 induction in COS-1 cells using the -1.4 kb promoter. Cells were co-transfected with pCYP24WT (-1.4kb)-Luc and the pRL-0 vector and were then treated with 12.5 and 100 µΜ of etoposide and/or 10 nM of 1,25(OH)2D3. The data in Figure 3.5 demonstrate that 12.5 and 100 µΜ of etoposide increased the transcriptional activity of the CYP24 promoter by 2.7 ± 0.7 and 1.8 ± 0.4 fold respectively over untreated cells. Treatment of the cells with both etoposide and
1,25(OH)2D3 produced a synergistic increase in CYP24 promoter activity. These data show that etoposide per se could stimulate CYP24 promoter induction, whereas in the presence of 1,25(OH)2D3 could enhance CYP24 promoter induction.
117
36 ## ##
30 1,25(OH)2D3 n o i
ct ) du n 24 n o I i
ct er du ot n
I 18
om r old
(F P24 p 12 Y ** C
6 * Vehicle *
0 0 20 40 60 80 100
Etoposide (µM)
Figure 3.5 Effects of different concentrations of etoposide on the activity of the -
1.4kb CYP24 promoter in COS-1 cells in the presence or absence of 1,25(OH)2D3.
Cells were transiently transfected with 200 ng of pCYP24WT (-1.4kb)-Luc and incubated with etoposide (12.5 µΜ and 100 µΜ) and/or 1,25(OH)2D3 (10 nM). The results are presented as fold induction. Data shown are mean ± SEM of three independent experiments performed in triplicate. Significance of difference between control and drug or 1,25(OH)2D3: *p<0.05 and **p<0.001; significance of difference between
1,25(OH)2D3 and 1,25(OH)2D3 + drug: #p<0.05 (ANOVA followed by Tukey-Kramer multiple comparisons test).
118 3.3.6 Doxorubicin hydrochloride did not stimulate the activity of the -298bp
CYP24 promoter or enhance CYP24 induction by 1,25(OH)2D3 in COS-1 cells.
Doxorubicin hydrochloride (adriamycin), the hydroxylated derivative of daunorubicin hydrochloride, is another anthracycline anti-cancer drug that is commonly used to treat various form of cancers including leukemia. Since the above data show that daunorubicin hydrochloride increased both basal and 1,25(OH)2D3 induction of CYP24 promoter in
COS-1 cells, this experiment was conducted to determine whether doxorubicin hydrochloride could increase the transcriptional activation of the CYP24 promoter in
COS-1 kidney cells. Results from this study (Figure 3.6) show that doxorubicin hydrochloride at all concentrations tested did not increase CYP24 promoter activity or enhanced CYP24 induction by 1,25(OH)2D3 in COS-1 cells. Thus, not all drugs are able to stimulate CYP24 promoter activity. In this situation, hydroxylation of the daunorubicin structure resulted in the loss of promoter-inducing activity of the drug.
119
5
4 ion t
c u ) d n n o I i t
c 3 1,25(OH)2D3 du oter * In
om r old p 2 4
(F Vehicle 2
CYP 1
0 00.511.52 Doxorubicin Hydrochloride (µM)
Figure 3.6 Lack of effect of doxorubicin hydrochloride on the -298bp CYP24 promoter in COS-1 cells. Cells were transiently transfected with 200 ng of pCYP24WT
(-298bp)-Luc and incubated with doxorubicin hydrochloride at the indicated concentrations and/or 1,25(OH)2D3 (10 nM). Data are mean ± SEM of three independent experiments, performed in triplicate. Significance of difference between untreated and
1,25(OH)2D3 treated cells: * p<0.05. No significant differences were detected between untreated and drug treated cells or between 1,25(OH)2D3 and 1,25(OH)2D3 + drug
(ANOVA followed by Tukey-Kramer multiple comparisons test).
120
3.4 Summary
The data demonstrate that in COS-1 cells, daunorubicin hydrochloride, etoposide and vincristine sulfate per se could stimulate the -1.4kb promoter. In contrast, cisplatin did not. Similarly, in HEK293T cells daunorubicin hydrochloride and etoposide per se were both able to stimulate the -1.4kb CYP24 promoter. However, both cisplatin and vincristine sulfate did not stimulate the -1.4kb promoter in HEK293T cells.
The data with the -1.4kb promoter construct also showed that in COS-1 cells, daunorubicin hydrochloride was only able to produce an additive effect in the presence of
1,25(OH)2D3. On the other hand, etoposide, vicristine sulfate and cisplatin were able to synergistically up-regulate the -1.4kb CYP24 promoter in the presence of 1,25(OH)2D3.
However in HEK293T cells, unlike daunorubicin hydrochloride and cisplatin, both etoposide and vincristine sulfate were able to synergistically up-regulate the CYP24 promoter induction by 1,25(OH)2D3.
Results from the -298bp CYP24 promoter demonstrate that daunorubicin hydrochloride, etoposide and vincristine sulfate can also stimulate the promoter activity by itself in COS-
1 cells. In contrast, cisplatin was not able to stimulate the -298bp CYP24 promoter. These data are consistent with the data from the -1.4kb promoter.
Furthermore, the data also show that daunorubicin hydrochloride, cisplatin and etoposide in the presence of 1,25(OH)2D3, synergistically up-regulated the -298bp CYP24 promoter activity in COS-1 cells. On the other hand, vincristine sulfate was only able to cause an additive effect on the CYP24 promoter activity in the presence of 1,25(OH)2D3.
121 The effect of daunorubicin hydrochloride per se on both the -298bp and the -1.4kb promoter activity in COS-1 cells was dose dependent. In the presence of 1,25(OH)2D3, no synergistic increase in -1.4kb promoter activity was observed at all different concentrations of daunorubicin hydrochloride. In contrast, synergistic response were only observed at lower concentrations of daunorubicin hydrochloride in the -298bp promoter.
The dose response profile for daunorubicin hydrochloride was some what different between COS-1 and HEK293T cells. Unlike in COS-1 cells, daunorubicin hydrochloride alone did not affect the -298bp promoter activity in HEK293T cells. However, in the presence of 1,25(OH)2D3, a synergistic increase in CYP24 promoter activity was detected.
Although etoposide per se was less able to stimulate the -1.4kb CYP24 promoter in COS-
1 cells over the concentration range tested, etoposide was found to synergistically stimulate the -1.4kb promoter at higher concentrations (12.5 µM and 100 µM) in the presence of 1,25(OH)2D3.
The investigations with doxorubicin hydrochloride (a hydroxylated derivative of daunorubicin hydrochloride) showed that the drug did not stimulate the CYP24 promoter alone or in the presence of 1,25(OH)2D3. Similarly, investigation with dexamethasone also showed that the drug neither stimulate the CYP24 promoter alone or in the presence of 1,25(OH)2D3 (data not shown). These results demonstrate that some chemotherapeutic drugs do not stimulate CYP24 induction.
Overall, these results demonstrate that a number of chemotherapeutic drugs were able to activate the CYP24 promoter and have the potential to further stimulate CYP24 induction in the presence of 1,25(OH)2D3. Furthermore, the effects of the chemotherapeutic drugs
122 on CYP24 promoter varied somewhat between two different kidney cell-lines and different promoter lengths.
123
Chapter 4 Mechanism I
Mechanism of action of daunorubicin hydrochloride and etoposide on CYP24 promoter activity: Role of reactive oxygen species (ROS),
VDR, VDRE and Sp1
124 4.1 ROS are not required for CYP24 promoter induction by daunorubicin hydrochloride and etoposide in COS-1 cells.
As discussed in Chapter 1.10, anticancer drugs such as daunorubicin hydrochloride, etoposide and cisplatin can activate MAP kinases and PKC through the production of reactive oxygen species (ROS). It was therefore possible that these drugs could act via
ROS in the induction of CYP24 promoter. To determine if the stimulation of CYP24 promoter activity by daunorubicin hydrochloride and etoposide involved ROS, COS-1 cells were transiently co-transfected with the -298bp CYP24 promoter-Luc construct and a vector containing renilla luciferase. The cells were then treated with the anti-oxidant, N- acetyl L-cysteine (10 mM), for one hour before being treated with ethanol (EtOH),
1,25(OH)2D3 (10 –8 M), daunorubicin hydrochloride (2 µM) or etoposide (100 µM) for
24 h. This concentration of N-acetyl L-cysteine has previously been reported to be effective in inhibiting the production of oxygen radicals in cells (Ravid et al., 1999;
Gouaze et al., 2001; Kurz et al., 2004; Corna et al., 2004). The data in Figure 4.1 demonstrate that N-acetyl L-cysteine did not reduce CYP24 promoter induction by daunorubicin hydrochloride or etoposide, suggesting that ROS did not play a role in mediating the effects of the drugs on CYP24 promoter activity in the kidney cells.
However, N-acetyl L-cysteine enhanced CYP24 promoter induction by 1,25(OH)2D3, demonstrating that the anti-oxidant was active, Although the precise reason for the increase CYP24 promoter activity in
125
20 * RPMI -1640 16 N-acetyN-acetyl lL-cystein L- Cysteinee y y it it tiv tiv c c n) n) 12 oter a oter a ductio ductio m m d In d In l l 4 pro 4 pro (Fo (Fo 8 P2 P2 Y Y C C
4
0
n e 5D ci tOH 2 i id E 1, ub os or top un E Da
Figure 4.1 Effect of N-acetyl L-cysteine on CYP24 promoter induction by daunorubicin hydrochloride, etoposide and 1,25(OH)2D3 in COS-1 cells. Cells were transiently transfected with 200ng of pCYP24-WT (298bp)-Luc and treated with N-acetyl
L-cysteine (10 mM) for 1hr prior to incubation with daunorubicin hydrochloride (2 µΜ), etoposide (100 µΜ) or 1,25(OH)2D3 (10-8 M) for 24 h. Results are presented as fold induction over EtOH treated cells. Data are representative of three independent experiments performed in triplicates. Significance of difference between RPMI-1640 and
N-acetyl L-cysteine treatment. * p<0.01 (Student’s t-test)
126 the presence of N-acetyl L-cysteine remains unclear, it is possible that, ROS production in the presence of 1,25(OH)2D3 could inhibit CYP24 promoter induction by the hormone and this was relieved by N-acetyl L-cysteine. Consistent with this possibility, there are reports that 1,25(OH)2D3 can deplete cancer cells of reduced glutathione and this has been proposed as a mechanism for the anti-cancer action of 1,25(OH)2D3. 1,25(OH)2D3 has also been shown to suppress the expression of the antioxidant enzyme, Cu 2+/Zn 2+ superoxide dismutase, an enzyme that is responsible for cellular defense against ROS and this results in a built up of ROS within cells (Ravid A et al., 1999; Ravid A & Koren R
2003). Our data demonstrate that treatment of the cells with N-acetyl L-cysteine resulted in a higher induction of the CYP24 promoter activity by 1,25(OH)2D3, suggesting the generation of ROS by the hormone in the kidney cells.
4.2 Over expression of VDR did not enhance CYP24 promoter induction by daunorubicin hydrochloride in COS-1 and HEK293T cells.
In this set of experiments, only daunorubicin hydrochloride was tested. Since ectopic expression of the nuclear VDR greatly enhances the CYP24 promoter induction by
1,25(OH)2D3 (Dwivedi et al., 2002), we investigated whether the VDR was required for
CYP24 promoter induction by daunorubicin hydrochloride. COS-1 cells were co- transfected with a plasmid carrying the nuclear VDR or empty vector, pcDNA3, the -
298bp CYP24 promoter sequence fused to the firefly luceferase reporter gene
(pCYP24WT (298)-Luc) and a vector (pRL-0) expressing Renilla luciferase. The cells were treated with either daunorubicin hydrochloride, 1,25(OH)2D3 or both. The data obtained were expressed as fold induction over control. As reported previously (Dwivedi
127 et al.,2002), CYP24 promoter activation by 1,25(OH)2D3 was greatly increased in VDR- transfected cells compared to pcDNA3-transfected cells (Figure 4.2). In contrast, VDR overexpression inhibited the ability of daunorubicin hydrochloride per se to stimulate the induction of CYP24 promoter.
Similar results were observed in HEK293T cells. Thus cells transfected with the VDR exhibited a greater induction of CYP24 promoter activity than control cells when treated with 1,25(OH)2D3 (Figure 4.3) as was observed in COS-1 cells (Figure 4.2).
Interestingly, VDR overexpression did not affect promoter activity when the cells were treated with both daunorubicin hydrochloride and 1,25(OH)2D3. The synergistic effect of
1,25(OH)2D3 and daunorubicin hydrochloride was still evident in the VDR overexpressing cells.
These results suggest that unlike 1,25(OH)2D3, daunorubicin hydrochloride did not require the nuclear VDR to stimulate the CYP24 promoter. The lack of a further increase in the VDR overexpressing cells treated with 1,25(OH)2D3 and daunorubicin hydrochloride suggest that maximum induction had been reached in these cells.
128
30 **
n o i t
c u
d 20 n on) i I t
er ot
om r P
4 10 (Fold Induc 2 P * Y C
0
VDR (200ng) --++
Daunorubicin (2µM) ++ --
1,25D (10-8 M) -- ++
Figure 4.2 Effect of overexpression of VDR on CYP24 promoter induction by daunorubicin hydrochloride or 1,25(OH)2D3 in COS-1 cells. Cells were co- transfected with 200ng of pCYP24-WT (298bp)-Luc and 200ng of VDR or empty vector pcDNA3 and treated with daunorubicin hydrochloride or 1,25(OH)2D3 for 24 h. The results are presented as fold induction over that seen with EtOH-treated cells. Data are representative of three independent experiments performed in triplicates. Significance of difference between pcDNA3 and VDR transfected cells. * p < 0.05 and ** p < 0.01
(Student’s t-test).
129
50
40 ppCDNcDNA3A n
o i t
c VDR
u d n on) i I t 30 * er
ot om r
P 20 (Fold Induc 24 P Y
C 10
0
EEtOHtOH 1,25(OH)2D3 Daunorubicin Daunorubicin + 1,25(OH)2D3
Figure 4.3 Effect of over expression of VDR on CYP24 induction by daunorubicin and/or 1,25(OH)2D3 in HEK293T cells. Cells were co-transfected with 200 ng of pCYP24-WT (298bp)-Luc and 200 ng of VDR or empty vector and treated with daunorubicin hydrochloride (0.25 µM) and/or 1,25(OH)2D3 (10-8 M) for 24 h. The results are presented as fold induction over the response seen in EtOH-treated cells. Data are representative of three independent experiments performed in triplicates. Significance of difference between pcDNA3 and VDR transfected cells. * p<0.01 (Student’s t-test)
130 4.3 VDRE and GC box are not involved in CYP24 promoter induction by daunorubicin hydrochloride in COS-1 cells.
Again, only the effect of daunorubicin hydrochloride was tested in this set of experiments. The vitamin D responsive element (VDRE) and GC box element (see
Chapter 1.7.3.1) play an important role in the activation of the CYP24 promoter by
1,25(OH)2D3. The transactivation of the CYP24 promoter by 1,25(OH)2D3 requires the binding of both VDR/RXR heterodimers and specific protein 1 (Sp1) transcription factor to the two VDREs and GC box, respectively, within the CYP24 promoter. Studies in our laboratory (Dwivedi et al., 2000); Dwivedi et al., manuscript in preparation) have demonstrated that mutation of the VDREs abolishes CYP24 promoter induction by
1,25(OH)2D3 and mutation of the GC box reduces basal as well as 1,25(OH)2D3- stimulated promoter activity. We therefore investigated whether these promoter regions were required for promoter induction by daunorubicin hydrochloride.
Cells were transiently transfected with wild type CYP24 (pCYP24WT-Luc), mutant
VDRE1 and VDRE2 (pCYP24mVDRE1+2–Luc) or CYP24 promoter with mutated GC box (pCYP24mGC-Luc) and a vector expressing Renilla luciferase. Cells were treated with 1,25(OH)2D3 or daunorubicin hydrochloride for 24 h. As expected, CYP24 promoter induction by 1,25(OH)2D3 was significantly reduced when VDRE1+2 or the
GC box was mutated (Figure 4.4). However, the ability of daunorubicin hydrochloride to stimulate CYP24 promoter activity was not affected by mutation of VDRE1+2 or the GC box. These results demonstrate that unlike 1,25(OH)2D3, daunorubicin hydrochloride did not require the VDRE or GC box to stimulate CYP24 promoter activity.
131
18 pCYP24WT-Luc
pCYP24mVDRE-Luc 15 pCYP24mGC-Luc
on
ti c 12 u d n) In io
t er
ot 9 om d Induc r l 4 P (Fo 2 6 P
Y C
3 **
0 EtOH 1,25(OH)2D3 Daunorubicin
Figure 4.4 Effects of mutant VDRE and mutant GC-box on CYP24 induction by daunorubicin hydrochloride and 1,25(OH)2D3 in COS-1 cells. COS-1 cells were co- transfected with wild type pCYP24WT (-298bp)-Luc, a construct carrying mutant
VDRE1+2 (pCYP24mVDRE-Luc) or a vector carrying mutant GC box (pCYP24mGC-
Luc). Cells were treated with 1,25(OH)2D3 (10-8 M) or daunorubicin hydrochloride (2
µM) for 24 h and luciferase activity determined. Results are presented as fold induction, over EtOH-treated cells. Significance of difference between CYP24WT, CYP24mVDRE and CYP24mGC-box. * p <0.001 (ANOVA and Dunnet multiple comparisons test).
132
4.4 Daunorubicin hydrochloride did not alter the activity of the VDR in COS-1 cells
To confirm that daunorubicin hydrochloride did not have a direct effect on the activity of
VDR itself, COS-1 cells were transiently transfected with a vector which contained the consensus vitamin D response element (VDRE) of the DR3-type (two AGGTCA motifs separated by 3bp) cloned upstream of the TK promoter driving the firefly luciferase, and pRL-0 carrying Renilla luciferase. The data in Figure 4.5 demonstrate that daunorubicin hydrochloride did not affect VDR activity because the drug did not affect reporter expression when added alone or in combination with 1,25(OH)2D3. As expected,
1,25(OH)2D3 stimulated the activity of the VDR. The smaller response seen in the presence of the hormone was due to the absence of accessory elements such as the Ets binding site (please see Chapter 5), GC-Box and the VSE (Nutchey et al, 2005) in this construct. The data imply that daunorubicin hydrochloride does not act via the VDREs and that elements other than the VDREs are required for synergism.
133
3
ion * 2 * duct
In er t
old Induction)
(F 1
DR3 promo
0 Daunorubicin - - + +
1,25(OH)2D3 - + - +
Figure 4.5 Effect of daunorubicin hydrochloride on the activity of VDR in COS-1 cells. COS-1 cells were transiently transfected with a vector which contained the consensus vitamin D response element (VDRE) of the DR3-type and pRL-0. Cells were treated with 1,25(OH)2D3 (10-8 M) ± daunorubicin hydrochloride (2 µM) for 24 h and luciferase activity determined. Results are presented as fold induction, over EtOH-treated cells. Significance of difference between control and 1,25(OH)2D3-, or 1,25(OH)2D3 ± daunorubicin hydrochloride-treated cells p<0.01 (ANOVA followed by Dunnet’s multiple comparisons test).
134 4.5 Daunorubicin hydrochloride and etoposide increased the expression of VDR in COS-1 and HEK293T cells.
The data in Figure 4.3 demonstrate that the ability of the cells to respond to 1,25(OH)2D3 was enhanced when the level of the VDR was increased. It is therefore possible that the drugs could enhance CYP24 promoter induction by 1,25(OH)2D3 by increasing the level of VDR in the kidney cells. We therefore investigated whether daunorubicin hydrochloride and etoposide could increase the expression of VDR in these cells. COS-1 cells were incubated with daunorubicin hydrochloride or etoposide for 24 h, lysed and the expression of VDR was determined by Western blotting using an anti-VDR antibody. The data in Figure 4.6 demonstrate that COS-1 cells treated with daunorubicin hydrochloride or etoposide had increased VDR expression. Equal protein loading was also demonstrated by reprobing with anti β-actin antibody (Figure 4.6).
Similar results were obtained in HEK293T cells treated with daunorubicin hydrochloride or etoposide, where both drugs caused an increase in VDR expression compared to ethanol-treated cells (Figure 4.7). These results could explain, at least in part, the enhancing effects of daunorubicin hydrochloride and etoposide on CYP24 promoter induction by 1,25(OH)2D3.
135
EtOH Daunorubicin Etoposide
Figure 4.6 Effects of daunorubicin hydrochloride and etoposide on vitamin D receptor (VDR) expression in COS-1 cells. COS-1 cells were treated with ethanol
(EtOH), daunorubicin hydrochloride (2 µΜ) and etoposide (100 µM) for 24 h. The cells were lysed and samples were Western blotted using an anti-VDR antibody (upper panel).
The blots were stripped and reprobed with anti-β-actin antibody to assess loading (lower panel). The data above are representative of 3 experiments with similar results.
136
EtOH Daunorubicin Etoposide
Figure 4.7 Effects of daunorubicin hydrochloride and etoposide on vitamin D receptor (VDR) expression in HEK293T cells. HEK293T cells were treated with ethanol (EtOH), daunorubicin hydrochloride (0.25 µΜ) and etoposide (100 µM) for 24 h.
The cells were lysed and samples were Western blotted using an anti-VDR antibody
(upper panel). The blots were stripped and reprobed with anti-β-actin antibody to assess loading (lower panel). The data above are representative of 3 experiments with similar results.
137 4.6 Summary
Overall, the above results demonstrate that the drugs tested did not act via ROS,
VDRE1/VDRE2 or the GC Box to stimulate the CYP24 promoter activity. However, an increase in VDR expression caused by daunorubicin hydrochloride and etoposide could mediate some of the enhancing effects of the drugs on promoter induction by
1,25(OH)2D3.
138
Chapter 5 Mechanism II
Role of MAP Kinases in 1,25(OH)2D3 mediated transactivation regulation of the CYP24 promoter in
COS-1 cells
139 5.1 Introduction
The data in Chapter 3 show that a selection of chemotherapeutic drugs could stimulate
CYP24 promoter activity and in the presence of 1,25(OH)2D3 cause a synergistic up- regulation of the CYP24 promoter activity. To understand how these chemotherapeutic drugs up-regulated the CYP24 promoter, it is important to first understand how
1,25(OH)2D3 regulates the CYP24 promoter.
The classical mode for the induction of CYP24 by 1,25(OH)2D3 involves the binding of
1,25(OH)2D3 to VDR-RXR heterodimer, which in turn binds to vitamin D response elements (VDRE) to mediate the activation of the CYP24 promoter. The rat CYP24 promoter has two VDRE (Hahn et al 1994), VDRE1 at –150/-136 and VDRE2 at –258/-
244. The two VDREs can synergise with each other in response to 1,25(OH)2D3 (Kerry et al., 1996). Although VDRE1 has a lower binding affinity than VDRE2 towards the
VDR-RXR complex, it is able to mediate 1,25(OH)2D3 induction of the CYP24 promoter to a greater extent than VDRE2. Computer analysis of the promoter region proximal to
VDRE1 led to the identification of an Ets-binding site (EBS) (-128/-119) located downstream of VDRE1. Mutation to the EBS resulted in a significant reduction in CYP24 promoter activity, indicating that the EBS plays an important role in the transcriptional activation of the CYP24 promoter especially at lower concentrations of 1,25(OH)2D3
(Dwivedi et al., 2000). Studies by Dwivedi et al. (2000) also showed that over expression of Ets-1 could enhance 1,25(OH)2D3 induction of a truncated CYP24 (-186bp) promoter containing just VDRE1 and the adjacent EBS. In our studies conducted in collaboration with Dwivedi et al. (2002), we showed that transfection of kidney cells with the Ets-1
140 mutant, Ets-1 T38A, or mutant EBS reduced 1,25(OH)2D3-mediated induction of the
CYP24 promoter by similar extents. Mutation to both VDRE1 and VDRE2 reduced promoter activity to near basal levels. These data show that, unlike the EBS, VDRE1 and
VDRE2 are crucial for the transcriptional activation of the CYP24 promoter. Over expression of exogenous RXR did not further increase CYP24 promoter activity whereas mutation of RXR (RXRS260A) significantly reduced promoter activity. These data suggest that, although RXR plays an important role in the induction of the CYP24 promoter, unlike Ets-1, RXR is not limiting in these cells.
1,25(OH)2D3 has been shown to activate the mitogen-activated protein (MAP) kinases and protein kinase C (PKC) (Chapter 1; Lissoos et al., 1993; Beno et al., 1995; Schwartz et al., 2002). However, the roles these MAP kinases play in the activation of the CYP24 promoter by 1,25(OH)2D3 are poorly understood. Furthermore, the targets of the MAP kinases within the context of CYP24 promoter activation by 1,25(OH)2D3 are not known. In addition to ERK1/ERK2, recent studies have demonstrated that the recently discovered MAP kinase, ERK5, is expressed in many cell-types and many ligands which stimulate ERK1/ERK2 also stimulate ERK5 activity. However, this has not been reported for 1,25(OH)2D3. These issues will be addressed in this Chapter in collaboration with Dr
Prem Dwivedi and Prof Brian May (School of Molecular Bioscience, Adelaide
University). These data have been presented by Dwivedi et al. (2002).
141 5.1.1 Vectors used:
A number of vectors are described in this Chapter. (See also Dwivedi et al. (2002) for further description) These include:
pCYP24WT (-298bp)-Luc: Wild type CYP24 promoter–reporter construct. pRL-TK: Renilla luciferase control reporter vector pRSVhVDR: Human VDR construct pRSVhRXRα: Human RXRα construct pRSVhRXRα S206A: Human RXR mutant construct pCYP24mEBS (-298bp)-Luc: CYP24 promoter-reporter construct with mutated Ets binding site. pCMVERK1K71R: Dominant negative ERK1 mutant pSRαMEK5A: Dominant negative MEK5 mutant. pZIP-Ras(17N): Dominant negative Ras mutant.
142 5.2 Results:
5.2.1 1,25(OH)2D3 stimulated phosphorylation of ERK1/ERK2 in COS-1 cells.
We examined whether 1,25(OH)2D3 could stimulate the activation / phosphorylation of
ERK1/ERK2 in COS-1 cells. We first determined the optimum time for ERK1/ERK2 stimulation by 1,25(OH)2D3. COS-1 cells were incubated with 1,25(OH)2D3 (10-7 M) for 0, 1, 5, 15 and 30 min. Cells were also incubated for 5 min with PMA (10-7 M), a
-3 standard stimulator for ERK1/ERK2 (positive control), and H2O2 (10 M), which can stimulate ERK1/ERK2 activity in some but not all cell-types. However, H2O2 is also a standard activator of ERK5 (see below). Cells were lysed and lysates were western blotted for phosphorylated ERK1/ERK2. As expected, results from cells treated with
PMA showed increased phosphorylation of ERK1/ERK2 whereas no change in the phosphorylation of ERK1/ERK2 was detected in cells treated with H2O2 (Figure 5.1).
Cells treated with 1,25(OH)2D3 showed increased ERK1/ERK2 phosphorylation at 1, 5,
15 and 30 min treatment time. Maximum ERK1/ERK2 phosphorylation was observed at
5 min. Equal amounts of ERK1/ERK2 were present in each lane, (confirmed by stripping and reprobing with anti-ERK2 antibody (Figure 5.1, lower panel). This shows that the increase in the level of phosphorylated ERK1/ERK2 was not due to a greater amount of
ERK1/ERK2 being loaded.
143
Figure 5.1 Effects of different 1,25(OH)2D3 treatment times on the phosphorylation of ERK1/ERK2 in COS-1 cells. COS-1 cells were treated with 1,25(OH)2D3 (10-7 M) for 0, 1, 5, 15, 30 min. Cells were also stimulated with PMA (10-7 M) (positive control) and H2O2 (10-3 M) (negative control) for 5 min. Cells were lysed and samples western blotted using an anti-active ERK antibody to determine the amount of phosphorylated
ERK1/ERK2 (upper panel). The blot was stripped and reprobed with anti-ERK2 antibody to assess loading (lower panel). The experiment was repeated twice and similar results were observed.
144
We then tested whether phosphorylation of ERK1/ERK2 was dose-related. COS-1 cells were treated with the biologically inactive 25(OH)D3 (10-7 M), ethanol and 1,25(OH)2D3
(10 –10 M, 10 –9 M and 10 -7 M) for 5 min. Cells were lysed and lysates western blotted for phosphorylated ERK. The data in Figure 5.2 show that 25(OH)D3 did not affect the phosphorylation of ERK1/ERK2. Significant increases in ERK1/ERK2 phosphorylation were detected at 10 –9 M and 10 -7 M of 1,25(OH)2D3. However, no increase in
ERK1/ERK2 phosphorylation was observed in cells treated with 10 –10 M of
1,25(OH)2D3. These results demonstrate that 1,25(OH)2D3 stimulated the phosphorylation of ERK1/ERK2 in COS-1 cells at concentrations greater than 10–10 M and that maximum ERK1/ERK2 phosphorylation occurs at 5 min.
5.2.2 1,25(OH)2D3 stimulated the activity of ERK5 activity in COS-1 cells
We also investigated whether 1,25(OH)2D3 could stimulate ERK5 activity in COS-1 cells. We first investigated the kinetics of ERK5 activation by 1,25(OH)2D3. COS-1 cells were transfected with HA-ERK5 and treated with H2O2, a well known stimulator of
ERK5 (positive control), ethanol (control) or 1,25(OH)2D3 (10-7 M) for 2, 10, 15 and 30 min. After lysis, HA-ERK5 was immunoprecipitated with anti-HA antibody and kinase assay was performed using MBP as a substrate. ERK5 activity was quantified using an
Instant Imager. As expected, ERK5 activity was strongly stimulated in H2O2 treated cells
(Figure 6.3). Cells incubated with 1,25(OH)2D3 for 2 min did not show an increase in
145
pERK1 pERK2
ERK2 WB: αERK2
C M M
D3 M) M
0 -7 -9 -7 -1 0 10 10 10 (1 25(OH) 1,25(OH)2D3
Figure 5.2 Effects of different concentrations of 1,25(OH)2D3 on the phosphorylation of ERK1/ERK2 in COS-1 cells. COS-1 cells were treated with vehicle
Control, 25(OH)D3 (10 –7 M) or 1,25(OH)2D3 (10 –10 M, 10 -9 M and 10 -7 M). Cells were lysed and samples western blotted using an anti-ERK antibody (upper panel). The blot was stripped and reprobed with anti-ERK2 antibody (lower panel) to assess loading.
The experiment was repeated twice and similar results were observed.
146
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i
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) 8000
A
m 5
p
c
K
(
R E
- 4000 A
H
2000
0 H2O2 C 2 10 15 30
1,25(OH)2D3 (minutes)
Figure 5.3 Kinetics of 1,25(OH)2D3-stimulated activation of ERK5 in COS-1 cells.
COS-1 cells were transfected with HA-ERK5 and stimulated with H2O2 (1 mM) for 15 min or with 1,25(OH)2D3 (10–7 M) for 2, 10, 15 and 30 min. Cells were lysed and HA-
ERK5 immunoprecipitated. Kinase activity was assayed as described in Materials and
Methods. Results are representative of three experiments.
147 ERK5 activity compared to control. However, significant increases in ERK5 activity were observed at 10, 15 and 30 min. Peak ERK5 activity was detected at 15 and 30 min (Figure
5.3).
We then investigated whether the stimulation of ERK5 activity by 1,25(OH)2D3 was dependent on the concentration of the hormone. COS-1 cells were transfected with HA-
ERK5 and treated with ethanol, 10-7 M of 25(OH)D3 or 10-10 M, 10-9 M and 10-7 M of
1,25(OH)2D3 for 15 min. As observed for ERK2 activity, ERK5 activity was not affected by 25(OH)D3. However, 1,25(OH)2D3 caused a dose-dependent increase in ERK5 activity (Figure 5.4), such that 10-7 M > 10-9 M > 10-10 M.
5.2.3 Dominant negative ERK1 and MEK5 inhibited 1,25(OH)2D3 mediated induction of the CYP24 promoter.
To investigate whether the ERK1 and ERK5 modules were involved in regulating the
1,25(OH)2D3-induction of the CYP24 promoter, COS-1 cells were transiently transfected with pRSVh VDR, pCYP24WT(-298bp)-Luc, dominant negative (DN)ERK1 (ERK1
K71R), DN MEK5 (MEK5A) or an empty vector. MEK5 is the immediate upstream activator of ERK5 (Sun et al., 2001). Cells were treated with 1,25(OH)2D3 (10-7 M) for
24 h. Results from these experiments (Figure 5.5), show that DN ERK1 significantly reduced the 1,25(OH)2D3 induction of the CYP24 promoter activity from approximately
26 fold to 6 fold. Note that the level of induction is greater than that seen in the results
148
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y y
t t
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v v i
i 8000
t t
c c
) )
A A
m m
5 5
p p
c c
K K
( (
R R
E E
- - A
A 4000
H H
0 E 2 1 1 1 5 0 tO 0 0 ( – – – H O 7 9 1 0 H M M ) M D 3
1,25(OH)2D3
Figure 5.4 Effects of varying concentrations of 1,25(OH)2D3 on ERK5 activity in
COS-1 cells. COS-1 cells were transfected with HA-ERK5 and treated with ethanol,
25(OH)D3 (10-7 M), or different concentrations of 1,25(OH)2D3 (10-7 M, 10–8 M, and 10-
10 M) for 15 min. Cells were lysed and HA-ERK5 immunoprecipitated. Kinase activity was assayed as described in Materials and Methods. Results are representative of three experiments.
149
30
25
n
tio )
n 20 duc io t
In * r c
e t 15
d Indu
ol F 4 promo ( 10
P2 ** **
CY 5
0 pCYP24WT(-298bp)-Luc + + + + ERK1 K71R - + - + MEK5A - - + +
Figure 5.5 Effects of dominant negative ERK1 and MEK5 mutants on 1,25(OH)2D3 mediated induction of the CYP24 promoter. COS-1 cells were co-transfected with 600 ng of pCYP24WT(-298bp)-Luc, 150 ng of pRL-TK and 500 ng of dominant negative
(DN) ERK1 (pCMVERK1 K71R), DN MEK5 (pSRαMEK5A) or a blank vector (-).
Cells were treated with 1,25(OH)2D3 (10-7 M) for 24 h. The results presented here are the mean ± S.D of three independent experiments. Significance of difference between control and (DN) ERK1 or/and DN MEK5. *p<0.05, **p<0.001 (ANOVA and Tukey’s multiple comparison test)
150 presented in the other chapters. This is because these cells were co-transfected with VDR.
DN MEK5 also suppressed CYP24 promoter activity from 26 fold to 14 fold. No further reduction in CYP24 promoter activity was observed in cells transfected with both DN
ERK1 and DN MEK5 compared to cells transfected with just DN ERK1 or DN MEK5.
These data demonstrate that both ERK1 and MEK5 played a role in 1,25(OH)2D3- mediated induction of the CYP24 promoter. Preliminary dose response studies have indicated that 500 ng of DN vectors produced the maximum degree of inhibition. The greater degree of inhibition by DN ERK1 than DN MEK5 suggests that the ERK1/ERK2 module may be more important than the MEK5-ERK5 module in the regulation of the
CYP24 promoter activity by 1,25(OH)2D3.
5.2.4 Dominant negative Ras inhibited the stimulation of ERK1/ERK2 activity by 1,25(OH)2D3 in COS-1 cells.
Ras is one of the upstream regulators of the ERK1/ERK2 modules (Wennerberg et al.,
2005) and it is not known whether 1,25(OH)2D3 utilizes Ras or other upstream regulators of the ERK1/ERK2 module, such as protein kinase C, to activate this module. We therefore investigated the effect of DN Ras (Ras 17N) on ERK1/ERK2 activity stimulated by 1,25(OH)2D3. COS-1 cells were cotransfected with HA-ERK2 ± DN Ras or empty vector. Cells were treated with 1,25(OH)2D3 (10-7 M) for 5 min, lysed and HA-ERK2 immunoprecipitated. Kinase assay was performed using myelin basic protein (MBP) as a
151 substrate and activity measured and quantified using an Instant Imager. Results from this set of experiments (Figure 5.6), show that 1,25(OH)2D3 increased HA-ERK2 activity when compared to untreated cells and that HA-ERK2 activity diminished in cells co- transfected with DN Ras. These results demonstrate that 1,25(OH)2D3 acted via Ras to stimulate the activity of ERK1/ERK2 in COS-1 cells.
5.2.5 Dominant negative Ras inhibited the stimulation of ERK5 activity by
1,25(OH)2D3 in COS-1 cells.
The data show that Ras was required for 1,25(OH)2D3 to stimulate ERK1/ERK2 activity
(Figure 5.6). We also investigated whether the stimulation of ERK5 activity by
1,25(OH)2D3 was dependent on Ras. COS-1 cells were co-transfected with HA-ERK5 ±
DN Ras (Ras17N) or empty vector and treated with ethanol or 1,25(OH)2D3 (10-7 M) for
15 min. The data in Figure 5.7 show that 1,25(OH)2D3 significantly increased ERK5 activity compared to ethanol-treated cells. However, 1,25(OH)2D3 did not cause an increase in ERK5 activity in cells transfected with DN Ras (Figure 5.7). This result demonstrates that Ras was required for the stimulation of ERK5 activity by 1,25(OH)2D3 in COS-1 cells.
152
MBP
10000
y 8000
t i
v
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t
c )
A
m 6000
2
p
c K
(
R E -
A 4000
H
2000
0
1,25(OH)2D3 - + +
Ras17N - + -
Figure 5.6 Effect of dominant negative Ras on the stimulation of ERK1/ERK2 activity by 1,25(OH)2D3 in COS-1 cells. COS-1 cells were transfected with HA-ERK2
± DN Ras (Ras17N). After 24 h, the cells were treated with 1,25(OH)2D3 (10 –7 M) for 5 min, lysed and HA-ERK2 immunoprecipitated using an anti-HA antibody. Kinase activity was assayed as described in Materials and Methods. Results are representative of three experiments.
153
MBP
16000
y y
t t
i i
v v
i i t
t 12000
c c
) )
A A
m m
5 5
p p
c c
K K
( (
R R
E E -
- 8000
A A
H H
4000
0 1,25(OH)2D3 - + + Ras17N - + -
Figure 5.7 Effect of dominant negative Ras on 1,25(OH)2D3 stimulation of ERK5 activity in COS-1 cells. COS-1 cells were transfected with HA-ERK5 ± DN Ras
(Ras17N) or empty vector. Cells were treated with 1,25(OH)2D3 (10 –7 M) for 15 min, lysed and HA-ERK5 immunoprecipitated. Kinase activity was assayed as described in
Materials and Methods. Results are representative of three experiments.
154 5.2.6 Dominant negative Ras inhibited CYP24 promoter induction by
1,25(OH)2D3
Since dominant negative Ras blocked the activation of ERK1/ERK2 and ERK5 by
1,25(OH)2D3, we investigate whether Ras also regulate CYP24 promoter activation by
1,25(OH)2D3. To investigate the possible involvement of endogenous Ras activity, we have overexpressed an inhibitory dominant-negative Ras construct, Ras17N. The results show that expression of Ras17N strongly reduced 1,25(OH)2D3 induction of the CYP24 promoter activity from 26- to 3.5-fold at the highest concentration tested (Figure 5.8).
This results demonstrates that Ras plays an important role in 1,25(OH)2D3-dependent transactivation of CYP24 promoter in COS-1 cells.
5.2.7 ERK2 interacted with RXRα independently of 1,25(OH)2D3.
Protein-protein interaction plays a crucial role in cell biology, especially for enzyme- substrate relationships. We therefore investigated whether an interaction could be detected between the MAP kinases and their effectors such as transcription factors. An interaction between the ERKs and RXRα has not been reported before. COS-1 cells were transfected with HA-RXRα and treated with ethanol (control) or 1,25(OH)2D3 (10-7 M) for 0.5, 2 and 24 h. After cell lysis, HA-RXRα was immunoprecipitated using anti-HA antibody and western blot for the possible presence of co-immunoprecipitated ERK2 using anti-ERK2 antibody. The results show the presence of ERK2 in all lanes (Figure
5.9), demonstrating that ERK2 interacted with RXR independently of 1,25(OH)2D3 and
155
35
n 30
io t
) 25 n duc
In r ctio 20
e t 15 d Indu
ol F
( 10
5 CYP24 promo
0
Ras 17N (ng) 050 100 200 50000
Figure 5.8 Effect of dominant negative Ras on 1,25(OH)2D3 induction of CYP24 promoter in COS-1 cells. pCYP24WT(-298bp)-Luc were co-transfected into COS-1 cells together with increasing doses of Ras 17N expression clone (100-500 ng). Cells were treated with 10-7 M of 1,25(OH)2D3 for 24 h. The results presented here are the mean ± S.D of three independent experiments.
156
EtOH 0.5 2 24 (Hours) 1,25(OH)2D3
Figure 5.9 Effect of 1,25(OH)2D3 on the interaction between RXRα and ERK2 or
ERK5 in COS-1 cells. COS-1 cells were transfected with HA-RXRα and treated with ethanol or 1,25(OH)2D3 (10-7 M) for 0.5, 2 and 24 h. Cells were lysed, immunoprecipitated with anti-HA antibody and western blotted with anti-ERK2 antibody.
Blots were strip and reprobed for ERK5 using anti-ERK5 antibody. To assess protein loading, the blots were stripped and reprobed with anti-HA antibody. Results are representative of two experiments.
157 incubation time. To determine whether ERK5 interacted with HA-RXRα, the blots were stripped and reprobed with anti-ERK5 antibody. The western blots did not show the presence of ERK5 in the immunoprecipitates. This demonstrates that ERK5 did not interact with HA-RXRα. To confirm that similar amounts of HA-RXRα were present in all lanes, the blots were stripped and reprobed with anti-HA antibody. Results show that similar amounts of HA-RXRα were loaded in all lanes. These data imply that the docking domain of RXR is specific for ERK2.
5.2.8 Phosphorylation of RXRα was dependent on the interaction between activated ERK1/ERK2 and RXRα.
It has been previously reported that RXRα becames phosphorylated when keratinocytes are treated with 1,25(OH)2D3 (Solomon et al., 1999). To investigate whether
1,25(OH)2D3 also stimulates RXRα phosphorylation and whether phosphorylation of
RXR is dependent on ERK1/ERK2, COS-1 cells were cotransfected with HA-RXRα ±
DN ERK1 (ERK1K71R). Cells were treated with ethanol (control) or 1,25(OH)2D3 (10-7
M) for 2 h. Phosphorylation of RXRα was determined by western blotting with an anti- phosphoserine antibody. The data in Figure 5.10 show that 1,25(OH)2D3-treated cells contained higher levels of phosphorylated RXR when compared to control (lower panel, duplicate samples shown for 1,25(OH)2D3-treated cells). However, in cells cotransfected with DN ERK1, no difference in RXR phosphorylation was detected when compared to
158
Figure 5.10 Effects of 1,25(OH)2D3, dominant negative ERK1 and mutated RXRα on serine phosphorylation of RXRα in COS-1 cells. COS-1 cells were co-transfected with HA-RXRα and ERK1K71R or HA-RXRS260A before being stimulated with
1,25(OH)2D3 (10-7 M) for 2 h. After lysis, cell lysates were immunoprecipitated with anti-HA antibody and probed for RXR phosphorylation by western blotting with an anti- phosphoserine antibody. To assess the level of protein loading, blots were stripped and reprobed with anti-HA antibody. Results are representative of three experiments.
159 control. These results show that phosphorylation of RXR by 1,25(OH)2D3 required activated ERK1. We also examined whether mutation of RXRα at S260, by replacing the serine residue with alanine, (S260A), affected phosphorylation of RXR. The data show that in cells transfected with HA-RXRα S260A, 1,25(OH)2D3 did not increase the phosphorylation of RXR when compared to cells treated with ethanol (Figure 5.10). The above data demonstrate that ERK1/ERK2 mediated the phosphorylation of RXRα at
S260 in cells incubated with 1,25(OH)2D3. The data also suggest that 1,25(OH)2D3- induced activation of ERK1/ERK2 and phosphorylation of RXRα on S260 might be important for the activation of the CYP24 promoter by the hormone.
5.2.9 RXR phosphorylation played an important role in CYP24 induction by
1,25(OH)2D3.
To investigate the importance of RXR phosphorylation in 1,25(OH)2D3 induction of the
CYP24 promoter, COS-1 cells were transfected with pCYP24WT(-298bp)-Luc, pRSVhVDR, pRSVhRXRα, or pRSVhRXRα S206A. Cells were treated with
1,25(OH)2D3 (10-7 M) for 24 h. Results from these experiments (Figure 5.11) show that overexpression of wild type RXRα did not influence the CYP24 induction by
1,25(OH)2D3. However, the 1,25(OH)2D3 induction was inhibited in cells transfected with mutant RXRα S260A. These results demonstrate that phosphorylation of RXRα at
160 difference betweenhRXRandS260A results presentedherearem each ofpCYP24W promoter inCOS-1cells Figure 5.11EffectofmutantRXRS260A pRSVhRXR
α CYP24 promoter Induction RX S206A.Thecellsweretr
R S260A (Fold Induction) T RXR ( - 298bp)-Luc ande . Cellswereco-transfected ean ± S. - - - - D ofthreeindependent eated with1,25(OH)2D3(10 m : *p<0.01(Student’sunpairedt-test) pty vector,pRSVhVDR,pRSVhRXR + + - - on1,25(OH)2D3inductionofC with 150ngofpRL-TK,600 * + + - - experim e -7 nts. Significanceof
M ) for24h.The Y P24 16 α 1 ,
serine 260 by ERK1/ERK2 is required for RXRα functionality during 1,25(OH)2D3 induction of the CYP24 promoter.
5.2.10 Interaction between ERK5 and Ets-1 was 1,25(OH)2D3-dependent.
Since Ets-1 plays an important role in enhancing 1,25(OH)2D3 induction of the CYP24 promoter (Dwivedi et al., 2000), the following experiments were performed to examine whether ERK1/ERK2 or ERK5 interacted with the Ets-1 protein in the presence of
1,25(OH)2D3. To test this, COS-1 cells were transfected with HA-Ets-1 and treated with
1,25(OH)2D3 (10-7 M) for 2 and 24 h. The cells were lysed and HA-Ets-1 immunoprecipitated using the anti-HA antibody. Western blotting was performed to determine the presence of co-immunoprecipitated ERK2 and ERK5. The data in Figure
5.12 show that there was no interaction between ERK5 and Ets-1 in untreated cells.
However, a faint ERK5 band was detected in cells treated with 1,25(OH)2D3 for 2 h.
Strong interaction between ERK5 and Ets-1 was clearly observed in cells incubated with
1,25(OH)2D3 for 24 h. A weak interaction between ERK2 and Ets-1 was detected but this interaction was independent of 1,25(OH)2D3 treatment. To confirm that the lanes contained similar amounts of Ets-1 protein, the blots were stripped and reprobed with anti-Ets-1 antibody. Results show that there were no differences in Ets-1 protein loading between the lanes. The data from this set of experiments demonstrate that the interaction between ERK5 and Ets-1 transcription factor was regulated by 1,25(OH)2D3. In contrast,
162 EtOH 2 24 (Hours) 1,25(OH)2D3
Figure 5.12 Effects of 1,25(OH)2D3 on the interaction between Ets-1 and ERK2 or
ERK5 in COS-1 cells. COS-1 cells were transfected with HA-Ets-1 and treated with
1,25(OH)2D3 (10-7 M) for 2 or 24 h. Cells lysates were prepared and HA-Ets-1 immunoprecipitated with anti-HA-antibody and probed for the presence of ERK5 by western blotting using an anti-ERK5 antibody. Blots were stripped and reprobed for
ERK2 using an anti-ERK2 antibody. The levels of Ets-1 protein in the lanes were determined by stripping and reprobing with an anti-Ets-1 antibody. Results were representative of three experiments.
163 the weak interaction between ERK2 and Ets-1 was constitutive and not regulated by
1,25(OH)2D3.
5.2.11 1,25(OH)2D3 stimulated the phosphorylation of Ets-1 protein.
Analysis of the sequence of Ets-1 revealed that the transcription factor contains the MAP kinase docking site motif, LXL, where X is any amino acid. This docking site can be found both upstream and downstream of the consensus Ser/Thr-Pro (S/TP) MAP kinase phosphorylation site. Thus it is possible that 1,25(OH)2D3 could stimulate Ets-1 phosphorylation via ERK5. To investigate whether 1,25(OH)2D3 increased the phosphorylation of Ets-1, COS-1 cells were transfected with HA-Ets-1 and treated with
1,25(OH)2D3 (10-7 M) for 2, 4 and 24 h. Results based on western blotting using an anti- phosphothreonine antibody show a very low level of threonine phosphorylation of Ets-1 in ethanol treated cells. However, a gradual increase in the level of threonine phosphorylation was evident in cells incubated with 1,25(OH)2D3, with an increase being detectable at 2 h and this increased over a 24 hour period (Figure 5.13). No difference in
Ets-1 protein loading was detected in the lanes, demonstrating that the increase in threonine phosphorylation in 1,25(OH)2D3 was not due to higher levels of Ets-1 being loaded.
164
EtOH 2 4 24 (Hours) 1,25(OH)2D3
Figure 5.13 1,25(OH)2D3 stimulated the threonine phosphorylation of Ets-1 in
COS-1 cells. COS-1 cells were transfected with HA-Ets-1 and treated with ethanol or
1,25(OH)2D3 (10 -7 M) for 2, 4 and 24 h. After cell lysis, HA-Ets-1 was immunoprecipitated with anti-HA antibody and assessed for equal Ets-1 protein loading by western blotting with an anti-Ets-1 antibody. To determine the level of Ets-1 phosphorylation, blots were stripped and reprobed with anti-phosphothreonine antibody.
Results were representative of three experiments.
165 5.2.12 Phosphorylation of Ets-1 requires the activation of HA-ERK5 by
MEK5 and Ras.
In Figure 5.12, we demonstrated that unlike ERK2, ERK5 interacted with Ets-1 in a
1,25(OH)2D3-dependent manner. To determine whether Ets-1 is a substrate for ERK5,
COS-1 cells were co-transfected with HA-ERK5 or HA-ERK2 ± Ras17N or MEK5(A).
The cells were then treated with 1,25(OH)2D3 or ethanol (control) for 5 min (HA-ERK2) or 15min (HA-ERK5). After lysis, HA-tagged kinase was immunoprecipitated, and kinase activity was determined using His-Ets-1 as a substrate. Figure 5.14 shows that HA-
ERK5 from 1,25(OH)2D3-treated cells phosphorylated Ets-1. Dominant negative MEK5 and dominant negative Ras inhibited the phosphorylation of Ets-1 by HA-ERK5. In contrast, HA-ERK2 did not phosphorylate Ets-1 even in the presence of 1,25(OH)2D3.
Thus, the data show that ERK5 and its upstream activators play an important role in mediating the phosphorylation of Ets-1 whereas Ets-1 is not a target of ERK1/ERK2.
Ets-1 contains a putative ERK phosphorylating site at threonine 38. To investigate whether this is indeed an ERK5 phosphorylating residue, we mutated threonine 38 of Ets-
1 to alanine (GST-Ets-1T38A). Figure 5.15 show that ERK5 was not able to phosphorylate Ets-1 when threonine 38 of Ets-1 was substituted with alanine. This study demonstrates that threonine 38 of Ets-1 is an important target for ERK5.
166
HA-ERK5 HA-ERK2
His-Ets-1
4000
) 3000
(cpm y vit
ti c a 2000
se na i
K
1000
1,25(OH)2D3 + - + + - + MEK5(A) - - + - - -
Ras 17N - - - + - -
Figure 5.14 Phosphorylation of Ets-1 by ERK5 but not by ERK2 and the role of
MEK5 and Ras. COS-1 cells were co-transfected with HA- ERK5 or HA-ERK2 ± dominant negative MEK5 (MEK5A) or Ras (Ras17N) and treated with 1,25(OH)2D3 or ethanol for 5 (HA-ERK2) or 15 min (HA-ERK5). After cell lysis, HA-tagged kinase was immunoprecipitated, and kinase activity was assayed using His-Ets-1 as a substrate.
Results are representative of three experiments.
167
3
2 ion) tivity t * c ula
5 a m
RK E - old sti A F 1 H (
0 Ets-1 Ets-1T38A
5.15 Substituting threonine 38 for alanine in Ets-1T38A diminished the phosphorylation of Ets-1 by ERK5. COS-1 cells were transfected with HA-ERK5 and treated with 1,25(OH)2D3 for 15 min. After lysis, HA-ERK5 was immunoprecipitated with anti-HA antibody and the activity of ERK5 was assayed using GST-Ets-1 or GST-
Ets-1T38A as a substrate. The results presented here are mean ± S.D of three independent experiments. Significance of difference between Ets-1 and Ets-1T38A: *p<0.01
(Student’s unpaired t-test).
168 5.2.13 CYP24 promoter induction by 1,25(OH)2D3 required the Ets binding site (EBS) and the ERK5 module
To investigate the role played by the EBS in 1,25(OH)2D3-mediated induction of the
CYP24 promoter and its functional relationship with ERK1/ERK2 and ERK5, COS-1 cells were co-transfected with pCYP24WT (-298bp)-Luc, pCYP24mEBS (-298bp)-Luc, pRL-TK, empty vector, ERK1 K71R and/or MEK5A. After 24 h, the cells were treated with 1,25(OH)2D3 (10-7 M) for 24 h and the luciferase activities assayed. The data in
Figure 5.15 show that 1,25(OH)2D3-stimulated CYP24 promoter activity was reduced by approximately 42% when the EBS was mutated. Promoter activity was reduced by a similar degree by the overexpression of the dominant negative MEK5. Co-transfection of the mutant EBS-Luc construct with dominant negative MEK5 did not produce a greater degree of suppression than that seen with one mutant alone. In contrast, expression of the dominant negative ERK1 reduced CYP24 promoter activity by approximately 80%
(Figure 5.16). This degree of inhibition was not enhanced by co-transfection of dominant negative ERK1 with dominant negative MEK5, mutant EBS or the combination of dominant negative MEK5 and mutant EBS. These results, together with the data on the phosphorylation of Ets-1 by ERK5, suggest that unlike ERK1/ERK2, the ERK5 module is specifically required for the functionality of the EBS binding protein in COS-1 cells. In contrast, ERK1/ERK2, functioning via RXRα, are essential for the induction of the
CYP24 promoter by 1,25(OH)2D3 via sites such as VDRE1 and VDRE2 which represent the principal signaling pathway in the induction process.
169 c b
a 30
25 n
tio d )
n 20 duc d In
r ctio e u t 15
d Ind ol F
( 10
CYP24 promo 5
0 pCYP24WT(-298bp)-Luc + + + + - - - -
pCYP24mEBS (-298bp)-Luc - - - - + + + + ERK1 K71R - + - + - + - + MEK5A - - + + - - + +
Figure 5.16 Roles of the EBS, ERK1/ERK2 and ERK5 in 1,25(OH)2D3-stimulated activation of the CYP24 promoter. COS-1 cells were transiently co-transfected with pCYP24WT (-298bp)-Luc (600 ng), pCYP24mEBS (-298bp)-Luc (600 ng), empty vector
(500 ng), ERK1 K71R (500 ng) and/or MEK5A (500 ng) as indicated, and pRL-TK (150 ng). After transfection, the cells were treated with 1,25(OH)2D3 (10-7 M) for 24 h, and the relative luciferase activity determined. Results are mean ± S.D of three independent experiments. Significance of difference between control and dominant negative
ERK1K71R: ap<0.001; between control and MEK5A: bp<0.01; between control and mEBS: cp<0.01; between ERK1K71R and a combination of ERK1K71R and MEK5A or mEBS + ERK1K71R + MEK5A: dp>0.05 (ANOVA and Tukey multiple comparison test).
170 5.3 Summary
Using western blotting and kinase activity assays, the data show that unlike 25(OH)D3,
1,25(OH)2D stimulated the activities /phosphorylation of ERK1/ERK2 and ERK5 and that the up-stream signaling molecule, Ras, played a crucial role in activating these MAP kinases in COS-1 cells. The data also established that both ERK1/ERK2 and MEK5 played a role in 1,25(OH)2D3-mediated induction of the CYP24 promoter. The
ERK1/ERK2 module was shown to play a greater role than the MEK5-ERK5 module in regulating CYP24 promoter induction by 1,25(OH)2D3.
Interestingly we found that ERK1/ERK2 but not ERK5 interacted with RXRα and that activation of ERK1 was required for the phosphorylation of RXRα by 1,25(OH)2D3.
Furthermore, phosphorylation of RXRα was required for CYP24 promoter induction by
1,25(OH)2D3, suggesting that ERK1/ERK2 acted via RXRα.
With respect to Ets-1, the data show that 1,25(OH)2D3 stimulated the binding of ERK5 to Ets-1. However, a weak interaction was observed between Ets-1 and ERK2. This interaction was constitutive and was not regulated by 1,25(OH)2D3. 1,25(OH)2D3 also stimulated the phosphorylation of Ets-1 on T38 and this was shown to be by ERK5.
Binding of Ets-1 to the EBS played a role in CYP24 promoter induction by
1,25(OH)2D3. These data provide strong evidence that the ERK5 module acted via Ets-1 and the EBS. Based on the data from this study, the following model is proposed (see
Figure 5.16). We proposed that 1,25(OH)2D3, acting on the plasma membrane VDR,
171 activates ERK1/ERK2 and ERK5 via up-stream Ras. Activated ERK1/ERK2 and ERK5 phosphorylate RXRα and Ets-1 which binds VDRE and EBS respectively. In addition,
1,25(OH)2D3 can also bind directly to the nuclear VDR to activate the CYP24 promoter.
RXR VDR P Ets-1 P
VDRE EBS CYP24
Figure 5.16 Regulation of the CYP24 promoter induction by 1,25(OH)2D3 via non- genomic and genomic action. (Dwivedi et al., 2002)
172
Chapter 6 Mechanism III
Role of MAP kinases, PI3K and PKC in mediating
CYP24 promoter induction by daunorubicin
hydrochloride and etoposide.
173 6.1 Introduction
Chemotherapeutics drugs such as daunorubicin hydrochloride, doxorubicin hydrochloride, etoposide, cisplatin and vincristine sulfate have been reported to stimulate the activity of PKCζ, PI3K and the MAP Kinases, ERK1/ERK2 and JNK, in cancer cells
(Mas et al., 2003; Brantley-Finley et al., 2003; Ohtsuka et al., 2003). Thus, chemotherapeutic drugs may stimulate CYP24 promoter activity through the activation of these signaling molecules.
6.2 Results
6.2.1 Dominant negative ERK1 and MEK5 prevent the induction of CYP24 promoter activity by daunorubicin hydrochloride in COS-1 cells.
In Chapter 5, we establish that ERK1/ERK2 and ERK5 plays an important role in
1,25(OH)2D3 induction of the CYP24 promoter in COS-1 cells. In this Chapter, we investigate whether ERK1/ERK2 and ERK5 are involved in the induction of the CYP24 promoter by daunorubicin hydrochloride and etoposide. Using kinase assay, we later confirm that the drugs can activate these signalling molecules.
174 COS-1 cells were co-transfected with pCYP24WT(-298bp)-Luc, pRL-Null-Luc and dominant negative (DN) ERK1 (ERK1K71R) or DN MEK5 (MEK5A) or a control vector using DOTAP. The cells were then treated with daunorubicin hydrochloride (2
µΜ) for 24 h. The relative luciferase activity of daunorubicin hydrochloride-treated cells was expressed as fold induction over ethanol-treated cells. The results (Figure 6.1) show that daunorubicin hydrochloride treatment increased CYP24 promoter activity to 9.6 ±
1.2 fold. However, in cells co-transfected with DN ERK1 or DN MEK5, stimulation of
CYP24 promoter activity by the drug was 4.5 ± 0.8 fold (53% reduction) and 4.0 ± 0.8 fold (59% reduction), respectively (Figure 6.1). These results demonstrate that daunorubicin hydrochloride-mediated induction of the CYP24 promoter in COS-1 cells required ERK1/ ERK2 and ERK5.
6.2.2 Daunorubicin hydrochloride stimulated the activities of ERK1/ERK2 and ERK5 in COS-1 cells.
To further confirm the above results that ERK1/ERK2 and ERK5 played a role in the daunorubicin hydrochloride-mediated induction of the CYP24 promoter, the activation of endogenous ERK1/ERK2 and ERK5 by daunorubicin hydrochloride was investigated.
175 12
10 on i 8 nduct I tion) 6 *
oter * prom
(Fold Induc 4 P24 CY 2
0 Control ERK1K71R MEKMEK5A
Figure 6.1 Dominant negative ERK1 and MEK5 inhibited CYP24 promoter induction by daunorubicin hydrochloride in COS-1 cells. COS-1 cells were co- transfected with pCYP24 WT (-298bp)-Luc, pRL-Null-Luc and dominant negative (DN)
ERK1 (ERK1K71R), DN MEK5 (MEK5A) or a control vector and treated with daunorubicin hydrochloride (2 µΜ) for 24 h. The results shown are mean ± SEM of three experiments, each conducted in triplicate determinations. Significance of difference between control and ERK1K71R or MEK5A: *p<0.01 (ANOVA followed by Dunnett multiple comparisons test).
176 COS-1 cells were treated with daunorubicin hydrochloride for 5 and 30 minutes for
ERK2 and ERK5 kinase assays, respectively. Results from preliminary data suggest that these were the optimum times for the stimulation of ERK1/ERK2 and ERK5 activities.
The data in Figure 6.2 demonstrate that daunorubicin hydrochloride increased
ERK1/ERK2 activity by 93% compared to EtOH treated cells. Daunorubicin hydrochloride also modestly increased ERK5 activity (21%) compared to EtOH treated cells (Figure 6.3). These results suggest that ERK1/ERK2 and ERK5 may be involved in mediating the stimulatory effect of daunorubicin hydrochloride on the CYP24 promoter.
6.2.3 Dominant negative MKK4 inhibited daunorubicin hydrochloride induction of CYP24 promoter activity in COS-1 cells.
Previous studies have shown that chemotherapeutic drugs could stimulate JNK activity in tumor cells (Mansat-de Mas et al., 1999; Hayakawa et al., 2003). JNK has also been shown to play a role in 1,25(OH)2D3 induction of the CYP24 promoter in HEK293T cells (Nutchy et al., 2005). We therefore investigated whether JNK was required for daunorubicin hydrochloride induction of the CYP24 promoter activity. To achieve this,
COS-1 cells were co-transfected with pCYP24WT(-298bp)-Luc, pRL-Null-Luc and dominant negative (DN) MKK4 (MKK4 K116R) or a control vector. MKK4 is one of the two direct upstream activators of JNK.
177 250 *
200 y )
l 150 o r ont
of c 100 % ( ERK1/ERK2 activit
50
0 EtOH DDauaunnororuubbiicincin
Treatment
Figure 6.2 Daunorubicin hydrochloride stimulated ERK1/ERK2 activity in COS-1 cells. COS-1 cells were incubated with daunorubicin hydrochloride (2 µΜ) for 5 minutes, lysed and the activities of ERK1/ERK2 assayed as described in Materials and Methods.
Results shown are mean ± SEM of three experiments. Significance of difference between
EtOH and daunorubicin hydrochloride treated cells: *p<0.01 (Student’s unpaired t-test).
178 150 *
100 ) y of control K5 activit R (% E 50
0 EtOH Daunorubicin
Treatment
Figure 6.3 Daunorubicin hydrochloride caused a modest increase in ERK5 activity in COS-1 cells. COS-1 cells were incubated with daunorubicin hydrochloride (2 µΜ) for
30 minutes, lysed and the activity of ERK5 assayed as described in Materials and
Methods. Results shown are mean ± SEM of three experiments. Significance of difference between EtOH and daunorubicin hydrochloride treated cells: *p<0.05
(Student’s unpaired t-test).
179 Cells were treated with daunorubicin hydrochloride (2 µΜ) for 24 h. The result from these experiments shows that DN MKK4 significantly inhibited the drug-induced CYP24 promoter activity (Figure 6.4). The data therefore show that the JNK pathway plays an important role in daunorubicin hydrochloride induction of the CYP24 promoter. This is in direct contrast to the data that showed that the JNK signaling pathway was not involved in 1,25(OH)2D3 induction of the CYP24 promoter in this cell-line (Dwivedi et al., 2002).
6.2.4 Daunorubicin hydrochloride stimulated JNK activity in COS-1 cells.
To further confirm that the JNK module was involved in mediating the effect of daunorubicin hydrochloride on CYP24 promoter activity in COS-1 cells, we also investigated whether daunorubicin hydrochloride could stimulate JNK activity. We first determined the optimal incubation time required for daunorubicin hydrochloride to activate JNK. COS-1 cells were incubated with daunorubicin hydrochloride for 5 minutes, 30 minutes, 1 h, 2 h and 6 h. The results show that daunorubicin hydrochloride only caused JNK activation at 6 h (Figure 6.5).
To study the dose-response relationship between daunorubicin hydrochloride and JNK activity, COS-1 cells were treated with 0.125 µΜ, 0.5 µΜ, 2 µΜ and 4 µΜ of daunorubicin hydrochloride for 6 h. A Significant increase in JNK activity was observed at 2 and 4 µΜ of daunorubicin hydrochloride. Stimulation of JNK activity was 1.5 fold
180 12
n 9 io ct ) Indu duction
n 6 omoter d I l r p 4 (Fo * 2 P
CY 3
0 Contrrolol MMKKK4K4 K116R
Figure 6.4 Effect of dominant negative MKK4 on CYP24 promoter induction by daunorubicin hydrochloride in COS-1 cells. COS-1 cells were co-transfected with pCYP24WT(-298bp)-Luc, pRL-Null-Luc and dominant negative MKK4 (MKK4 K116R) or a control vector and treated with daunorubicin hydrochloride (2 µΜ) for 24 h. The results shown are mean ± SEM of three experiments, each conducted in triplicate determinations. Significance of difference between control and MKK4: *p<0.001
(Student’s unpaired t-test).
181
Duration of treatment
6 hrs 5 min 30 min 1 hr 2 hrs 6 hrs
Phosphorylated GST-jun (1-79)
Control Daunorubicin hydrochloride (2µM)
Figure 6.5 Daunorubicin hydrochloride caused the activation of JNK in COS-1 cells. COS-1 cells were incubated with vehicle (control) or daunorubicin hydrochloride for 5 minutes, 30 minutes, 1 h, 2 h and 6 h, lysed and the activity of JNK was assayed as described in Materials and Methods. The experiment was repeated twice and similar results were obtained.
182 greater at 4 µΜ compared to 2 µΜ (Figure 6.6). These results demonstrated that the drug stimulated the activity of JNK in a time- and dose-dependent manner.
6.2.5 Dominant negative PKCζ inhibited daunorubicin hydrochloride induction of CYP24 promoter activity in COS-1 cells.
Previous studies have demonstrated that chemotherapeutic drugs can stimulate PKCζ activity in leukaemic cells (Mas et al., 2003) and recent studies from our laboratory have shown that PKCζ is involved in mediating the effect of 1,25(OH)2D3 on CYP24 promoter activity in HEK293T cells (Dwivedi et al., manuscript in preparation). We therefore investigated whether PKCζ was required for daunorubicin hydrochloride induction of the CYP24 promoter. To test this, COS-1 cells were co-transfected with pCYP24WT (-298bp)-Luc, pRL-Null-Luc and dominant negative (DN) PKCζ or a control vector. After transfection, the cells were treated with daunorubicin hydrochloride
(2 µΜ) for 24 h. Results from this experiment show that DN PKCζ significantly reduced the CYP24 promoter activity when compared to cells with control vector (Figure 6.7).
The data therefore show that PKCζ was involved in mediating the effect of daunorubicin hydrochloride on CYP24 promoter activity.
183
300 **
250 * 200 of control) 150 y (% t
100
JNK activi 50
0 012345 Daunorubicin (µΜ)
Figure 6.6 Effects of different concentrations of daunorubicin hydrochloride on
JNK activity in COS-1 cells. COS-1 cells were treated with 0, 0.125, 0.5, 2 or 4 µΜ of daunorubicin hydrochloride for 6 h, lysed and the activity of JNK was assayed as described in Materials and Methods. The data, expressed as % of control, are the mean ±
SEM of three experiments. Significance of difference between control cells and cells treated with daunorubicin hydrochloride: *p<0.05, **p<0.01 (ANOVA followed by
Dunnett multiple comparisons test).
184 12
10 on i 8 on) i Induct er
ot 6 om
old Induct * F ( 4 P24 pr CY 2
0 Control PKCζK281M
Figure 6.7 Dominant negative PKCζ inhibited CYP24 promoter induction by daunorubicin hydrochloride in COS-1 cells. COS-1 cells were co-transfected with pCYP24WT(-298bp)-Luc, pRL-Null-Luc and dominant negative PKCζ (PKCζ K281M) or a control vector and treated with daunorubicin hydrochloride (2 µΜ) for 24 h. The results shown are mean ± SEM of three experiments, each conducted in triplicate determinations. Significance of difference between control and PKCζ K281M: *p<0.05
(Student’s unpaired t-test).
185 6.2.6 Inhibitor of PI3K (LY294002) suppressed daunorubicin hydrochloride induction of CYP24 promoter activity in COS-1 cells.
PI3K had been shown to play an important role in daunorubicin hydrochloride stimulation of ERK1 and PKCζ (Mas et. al., 2003). Inhibition of PI3K by wortmannin was shown to partially inhibit ERK1 activation but significantly inhibit PKCζ in U937 cells (Mas et. al., 2003). We therefore investigated whether the specific pharmacological inhibitor of PI3K, LY294002, could inhibit CYP24 promoter activity. COS-1 cells were transiently co-transfected with pCYP24WT(-298bp)-Luc, pRL-Null-Luc and treated with
LY294002 (5 µΜ) or DMSO. The concentration of LY294002 used in this study was based on the concentration used by Versteeg et al. (2000) and Benini et al. (2004) to inhibit PI3K. Following 30 minutes treatment with LY294002, the cells were treated with daunorubicin hydrochloride (2 µΜ) for 24 h. The data in Figure 6.8 show that cells treated with LY294002 had a 71% reduction in CYP24 promoter activity compared to
DMSO-treated cells. These results demonstrate that PI3K played an important role in the induction of the CYP24 promoter by 1,25(OH)2D3 in COS-1 cells, most probably acting as an upstream regulator of the ERK1/ERK2 module and PKCζ.
186 10
8
6 ion) Induction
4 old Induct F ( * P24 promoter Y
C 2
0 LY 294002 - +
Daunorubicin + +
Figure 6.8 LY294002 inhibited CYP24 promoter induction by daunorubicin hydrochloride in COS-1 cells. COS-1 cell were transiently co-transfected with pCYP24WT(-298bp)-Luc, pRL-Null-Luc and treated with LY294002 (5 µΜ) for 30 minutes before being incubated with daunorubicin hydrochloride (2 µΜ) for 24 h. Results shown are mean ± SEM of three experiments, each conducted in triplicate determinations. Significance of difference between LY294002 treated and untreated:
*p<0.01. (Student’s unpaired t -test).
187 6.2.7 Inhibition of daunorubicin hydrochloride-mediated transcriptional activation of the CYP24 promoter activity by dominant negative ERK1 and
MEK5 in HEK293T cells.
The data in Chapter 3 demonstrate that daunorubicin hydrochloride alone was able to induce CYP24 promoter in a dose-dependent manner in COS-1 cells but not in HEK293T cells. However, the drug caused a greater enhancing effect on the stimulation of CYP24 promoter activity by 1,25(OH)2D3 in HEK293T cells than in COS-1 cells. Given these differences in the induction of the CYP24 promoter activity by daunorubicin hydrochloride between COS-1 and HEK293T cells, we also investigated the roles played by the ERK1/ERK2, ERK5 and JNK modules, and PKCζ in the induction of the CYP24 promoter in HEK293T cells. HEK293T cells were co-transfected with pCYP24WT(-
298bp)-Luc, pRL-Null-Luc and DN ERK1 (ERK1K71R), DN MEK5 (MEK5A) or a control vector. The cells were then incubated with daunorubicin hydrochloride (0.5 µΜ)
+ 1,25(OH)2D3 (10 nM) for 24 h. Co-treatment with 1,25(OH)2D3 and daunorubicin hydrochloride caused a synergistic activation of the CYP24 promoter in HEK293T cells
(Chapter 3). Results from these experiments (Figure 6.9) show that DN ERK1 or DN
MEK5 per se did not suppress the induction of CYP24 promoter activity by daunorubicin hydrochloride + 1,25(OH)2D3. However, when DN ERK1 and DN MEK5 were co- transfected together, a significant reduction in CYP24 promoter activity was observed
(11.4 ± 0.8 fold to 1.2 ± 0.3 fold). These data imply that unlike in COS-1 cells, the
ERK1/ERK2 and ERK5 signaling pathways in HEK293T cells were able to compensate
188 each other and thus one pathway was still able to transmit signals to the CYP24 promoter even when the other was inhibited. These results are consistent with recent data from our laboratory (Nutchey et al., 2005) which show that inhibition of ERK1 or MEK5 alone in
HEK293T cells did not suppress the 1,25(OH)2D3-mediated induction of the CYP24 promoter. However, Nutchey et al. (unpublished) demonstrated that dominant negative
Ras (Ras17N) inhibited the induction of the CYP24 promoter by 1,25(OH)2D3 in
HEK293T cells. Ras is located up-stream of ERK1/ERK2 and ERK5 and plays an important role in activating the ERK1/ERK2 and ERK5 modules and the CYP24 promoter (Chapter 5, Dwivedi et al., 2002)
189 16
12 n io ct ) on ndu i I
r e t
duct 8 n I omo 4 pr (Fold P2
CY 4 *
0 CCoontrol ERK1K71R MEK5A ERK1K71RK1K71R + MEK5A
Figure 6.9 Effects of dominant negative ERK1 and MEK5 on CYP24 promoter induction by daunorubicin hydrochloride and 1,25(OH)2D3 in HEK293T cells.
HEK293T cells were transiently co-transfected with pCYP24 WT (-298bp)-Luc, pRL-
Null-Luc and dominant negative (DN) ERK1 (ERK1K71R) and/or DN MEK5 (MEK5A) or a control vector and treated with daunorubicin hydrochloride (0.5 µΜ) + 1,25(OH)2D3
(10 nM) for 24 h. The results shown are mean ± SEM of three experiments, each conducted in triplicate determinations. Significance of difference between control and
ERK1K71R+MEK5A: *p<0.001 (ANOVA followed by Dunnett multiple comparisons test).
190 6.2.8 Daunorubicin hydrochloride stimulated ERK1/ERK2 and ERK5 activities in HEK293T cells.
The effects of daunorubicin hydrochloride on ERK1/ERK2 and ERK5 activities were investigated next. HEK293T cells were treated with daunorubicin hydrochloride (0.5
µΜ) for 5, 10 and 30 minutes (ERK2) and 6 h (ERK5) respectively. The data show that daunorubicin hydrochloride increased ERK2 activity by 1.4 fold compared to untreated cells (control) (Figure 6.10). In contrast, although 1,25(OH)2D3 showed a tendency to increase ERK1/ERK2 activity in HEK293T cells, this effect did not reach a statistically significant level (data not shown). This is consistent with the data of Nutchey et al.
(2005) who also found that 1,25(OH)2D3 did not significantly increase ERK1/ERK2 activity in HEK293T cells. These results are in direct contrast to the data in COS-1 cells in which 1,25(OH)2D3 stimulated the activity of ERK1/ERK2. (Dwivedi et al., 2002).
Similarly, in cells treated with daunorubicin hydrochloride alone and together with
1,25(OH)2D3, ERK5 activity was increased by 1.5 fold and 1.6 fold, respectively, compared to untreated cells (Figure 6.11). In these cells, 1,25(OH)2D3 per se did not stimulate or enhance the activation of ERK5 by daunorubicin hydrochloride although there was also a tendency for 1,25(OH)2D3 per se to cause a small increase in ERK5 activity which did not reach statistically significant levels.
191
160
* l)
140
(% of contro
y
it 120
100
ERK1/ERK2 activ
80
0102030
Time (minutes)
Figure 6.10 Daunorubicin hydrochloride stimulated ERK2 activity in HEK293T cells. HEK293T cells were incubated with daunorubicin hydrochloride (0.5 µΜ) for 5, 10 and 30 minutes, lysed and the activity of ERK1/ERK2 was assayed as described in
Materials and Methods. Results shown are mean ± SEM of three experiments.
Significance of difference between vehicle-treated (control) and daunorubicin hydrochloride-treated cells: *p<0.05 (ANOVA followed by Dunnett multiple comparisons test).
192 180 ** ) 120 of control
% ( y Activit 60 K 5 ER
0 1,25(OH)2D3 - + - + Daunorubicin - - + +
Figure 6.11 Daunorubicin hydrochloride stimulated ERK5 activity in HEK293T cells. HEK293T cells were incubated with daunorubicin hydrochloride (0.5 µΜ) for 6 h, lysed and the activity of ERK5 assayed as described in Materials and Methods. Results shown are mean ± SEM of three experiments. Significance of difference between cells treated with daunorubicin hydrochloride ± 1,25(OH)2D3 and untreated cells: *p<0.05
(ANOVA followed by Dunnett multiple comparisons test).
193 This is also in direct contrast to the ability of 1,25(OH)2D3 to stimulate ERK5 activity in
COS-1 cells (Chapter 5; Dwivedi et al., 2002). The above results show that daunorubicin hydrochloride could stimulate ERK1/ERK2 and ERK5 in COS-1 cells as well as in
HEK293T cells. In contrast, 1,25(OH)2D3 stimulated ERK1/ERK2 and ERK5 activities in COS-1 cells but not in HEK293T cells, thereby revealing subtle differences between the two different kidney cell lines which were derived from two different regions in the kidney and from two different species.
6.2.9 Effect of dominant negative MKK4 on daunorubicin hydrochloride- and 1,25(OH)2D3-mediated induction of CYP24 promoter activity in
HEK293T cells.
HEK293T cells were co-transfected with pCYP24WT(-298bp)-Luc, pRL-Null-Luc and
DN MKK4 (MKK4 K116R) or a control vector. After transfection, the cells were treated with daunorubicin hydrochloride (0.5 µΜ) + 1,25(OH)2D3 (10 nM) for 24 h.
The data in Figure 6.12 show that the ability of daunorubicin hydrochloride +
1,25(OH)2D3 to stimulate CYP24 promoter activity in cells co-transfected with DN
MKK4 was suppressed by approximately 54% compared to cells co-transfected with empty vector (DN MKK4: 3.8 ± 1.0 fold compared to empty vector: 8.2 ± 1.6 fold).
194 12
10
ion 8 duct n ion) I
er
duct 6 n * omot old I F ( 4 P24 pr CY 2
0 Control MKK4K116R
Figure 6.12 Effect of dominant negative MKK4 on CYP24 promoter induction by daunorubicin hydrochloride and 1,25(OH)2D3 in HEK293T cells. HEK293T cells were co-transfected with pCYP24 WT (-298bp)-Luc, pRL-Null-Luc and dominant negative (DN) MKK4 (MKK4 K116R) or a control vector and treated with daunorubicin hydrochloride (0.5 µΜ) + 1,25(OH)2D3 (10 nM) for 24 h. The results shown are mean ±
SEM of three experiments, each conducted in triplicate determinations. Significance of difference between control and MKK4 K116R: *p<0.05 (Student’s unpaired t-test).
195 6.2.10 Daunorubicin hydrochloride stimulated JNK activity in HEK293T cells.
To determine whether daunorubicin hydrochloride could stimulate JNK activity in
HEK293T cells, the cells were treated with daunorubicin hydrochloride (0.5 µΜ) for 6 h
(see Figure 6.5). The results show that daunorubicin hydrochloride caused a modest increase (1.2 fold) in JNK activity compared to vehicle-treated cells (Figure 6.13).
Neither 1,25(OH)2D3 nor daunorubicin hydrochloride alone significantly increased JNK activity (data not shown). These results show that the JNK signaling pathway also played a role in mediating the induction of the CYP24 promoter by daunorubicin hydrochloride and 1,25(OH)2D3 in HEK293T cells.
6.2.11 PKCζ was not required for the induction of the CYP24 promoter by daunorubicin hydrochloride and 1,25(OH)2D3 in HEK293T cells.
To investigate the role of PKCζ in the induction of the CYP24 promoter by daunorubicin hydrochloride and 1,25(OH)2D3 in HEK293T cells, the cells were co-transfected with pCYP24WT(-298bp)-Luc, pRL-Null-Luc and DN PKCζ (PKCζ K281M) or control
196 Phosphorylated GST-jun (1-79)
150 *
100
50 JNK activity (% of control)
0 EtOH Daunorubicin + 1,25(OH)2D3
Figure 6.13 Effect of daunorubicin hydrochloride and 1,25(OH)2D3 on JNK activity in HEK293T cells. HEK293T cells were treated with daunorubicin hydrochloride (0.5 µΜ) and 1,25(OH)2D3 (10 nM) for 6 h. Cells were lysed and the activity of JNK assayed as described in Materials and Methods. Results shown represent mean ± SEM of three experiments conducted in triplicate determinations. Significance of difference between EtOH-treated cells and daunorubicin hydrochloride + 1,25(OH)2D3- treated cells: *p<0.05 (Student’s unpaired t-test).
197 vector. Cells were then treated with daunorubicin hydrochloride (0.5 µΜ) +
1,25(OH)2D3 (10 nM) for 24 h. The data in Figure 6.14 show that DN PKCζ did not inhibit CYP24 promoter induction by daunorubicin hydrochloride + 1,25(OH)2D3. These data imply that PKCζ was not required for the induction of the CYP24 promoter by daunorubicin hydrochloride + 1,25(OH)2D3, unlike its role in COS-1 cells (Figure 6.7).
6.2.12 Role of MAP Kinases and PKCζ in the induction of CYP24 promoter by etoposide in COS-1 cells.
As with daunorubicin hydrochloride, etoposide has been shown to stimulate the activities of MAP Kinases in tumor cells in order to induce apoptosis. Besides this, etoposide also stimulated the transcriptional activation of the CYP24 promoter in COS-1 cells (Chapter
3). The following experiments were therefore conducted to investigate whether etoposide induction of the CYP24 promoter activity in COS-1 cells required the ERK1/ERK2,
ERK5, JNK and PKCζ signaling pathways.
198 12
10
8 duction n
6
(Fold Induction) 4 P24 promoter I CY 2
0 Control PKCζK281M
Figure 6.14 Dominant negative PKCζ did not affact CYP24 promoter induction by daunorubicin hydrochloride and 1,25(OH)2D3 in HEK293T cells. HEK293T cells were co-transfected with pCYP24 WT (-298bp)-Luc, pRL-Null-Luc and dominant negative (DN) PKCζ (PKCζ K281M) or a control vector and treated with daunorubicin hydrochloride (0.5 µΜ) + 1,25(OH)2D3 (10 nM) for 24 h. The results shown are mean ±
SEM of three experiments, each conducted in triplicate determinations. Significance difference between control and PKCζ K281M: *p>0.05 (Student’s unpaired t-test).
199 6.2.13 Dominant negative ERK1 and not MEK5 inhibited etoposide mediated induction of the CYP24 promoter activity in COS-1 cells.
Since CYP24 promoter induction by etoposide was greater in the -1.4Kb promoter than in the -298bp promoter (Chapter 3), the -1.4Kb CYP24 promoter was therefore used in these studies. COS-1 cells were co-transfected with pCYP24WT(-1.4Kb)-Luc, pRL-Null-
Luc and DN MEK5 (MEK5A) or DN ERK2 (ERK1K71R) or a control vector. Cells were then treated with etoposide (100 µΜ) for 24 h. Results from these experiments (Figure
6.15) show that DN MEK5 did not inhibit etoposide mediated induction of the CYP24 promoter. On the other hand, DN ERK1 severely suppressed CYP24 promoter activity. In fact, promoter activity was reduced by 94% below basal CYP24 promoter activity.
However, this was likely to be an overestimation of the effect because etoposide selectively caused an increase in renilla luciferase activity when the cells were transfected with DN ERK1. This effect of etoposide on the activity of renilla luciferase was not seen with the other DN mutants. Nevertheless, there was also a decrease in firefly luciferase activity in the presence of DN ERK1 compared to when an empty vector was transfected.
Thus, the ratio of firefly luciferase: renilla luciferase was exceptionally low when compared to control cells (please see Chapter 2.2.3.4 for data presentation). These results showed that ERK1/ERK2 but not ERK5 was involved in the induction of the CYP24 promoter by etoposide.
200 3 on 2 on) nducti i er I ot m
(Fold Induct 1 CYP24 Pro *
0 Control MEK5A ERKRK11KK771R1R
Figure 6.15 Effects of dominant negative MEK5 and ERK1 on CYP24 promoter induction by etoposide in COS-1 cells. COS-1 cells were co-transfected with pCYP24
WT (-1.4Kb)-Luc, pRL-null-Luc and dominant negative (DN) ERK1 (ERK1K71R), DN
MEK5 (MEK5A) or a control vector and treated with etoposide (100 µΜ) for 24 h.
Results shown are mean ± SEM of three experiments, each conducted in triplicate determinations. Significance of difference between control and ERK1K71R: *p<0.0001
(ANOVA followed by Dunnett’s multiple comparisons test).
201 6.2.14 Etoposide stimulated ERK1/ERK2 activity in COS-1 cells.
Since DN ERK1 inhibited etoposide-mediated induction of the CYP24 promoter activity, we investigated whether etoposide-stimulated the activity of ERK1/ERK2 in COS-1 cells. To test this, the cells were incubated with etoposide (100 µΜ) for 5 minutes. The results show that etoposide increased ERK1/ERK2 activity by 2.2 fold compared to untreated cells (Figure 6.16). Thus, these results demonstrate that the activation of
ERK1/ERK2 by etoposide played a role in the induction of the CYP24 promoter in COS-
1 cells.
6.2.15 Dominant negative MKK4 but not PKCζ inhibited etoposide-mediated induction of the CYP24 promoter activity in COS-1 cells.
To determine whether PKCζ and the JNK module played a role in the etoposide- mediated induction of the CYP24 promoter, COS-1 cells were co-transfected with DN
PKCζ (PKCζ K281M), or DN MKK4 (MKK4 K116R), pRL-Null-Luc and pCYP24WT(-1.4kb)-Luc. After transfection, the cells were treated with etoposide (100
µΜ) for 24 h. Results from this set of experiments (Figure 6.17) show that DN PKCζ did not suppress etoposide-mediated induction of the CYP24 promoter. However, DN MKK4 caused a significant reduction in CYP24 promoter induction (55% reduction compared to cells transfected with a empty vector). These results demonstrate that JNK
202 300
*
l) ro t
con 200 f
(% o ity
100
/ERK2 activ
ERK1
0 Control Etoposidetoposide Treatment
Figure 6.16 Effect of etoposide on ERK1/ERK2 activity in COS-1 cells. COS-1 cells were incubated with etoposide (100 µΜ) for 5 minutes, lysed and the activity of
ERK1/ERK2 was assayed as described in Materials and Methods. Results shown are mean ± SEM of three experiments. Significance of difference between vehicle-treated cells (control) and etoposide-treated cells: *p<0.05 (Student’s unpaired t-test).
203 4
3 n o nducti er I
ot 2 m
old Induction) * (F
CYP24 Pro 1
0 Control PKCζK281M MMKK4K116RKK4K116R
Figure 6.17 Effects of dominant negative PKCζ and MKK4 on CYP24 promoter induction by etoposide in COS-1 cells. COS-1 cells were co-transfected with pCYP24
WT (-1.4Kb)-Luc, pRL-Null-Luc and dominant negative (DN) PKCζ (PKCζ K281M) or
DN MKK4 (MKK4 K116R) or a control vector and treated with etoposide (100 µΜ) for
24 h. Results shown are mean ± SEM of three experiments, each conducted in triplicate determinations. Significance of difference between control and MKK4 K116R: *p<0.05
(ANOVA followed by Dunnett’s multiple comparisons test).
204 and not PKCζ was involved in the induction of the CYP24 promoter by etoposide. This contrasts with a role for PKCζ in the action of daunorubicin hydrochloride (Figure 6.7).
6.2.16 Etoposide stimulated JNK activity in COS-1 cells.
To further confirm the role of the JNK module in the action of etoposide, we examined whether etoposide could stimulate JNK activity in COS-1 cells. First we investigated the optimal incubation time for JNK activation in response to etoposide treatment. COS-1 cells were incubated with etoposide for 0.5, 2 and 6 h. The data show that etoposide significantly increased JNK activity at 6 hr although a small increase was also evident at
2 h (Figure 6.18). We then investigated whether the stimulation of JNK activity by etoposide was concentration-dependent. COS-1 cells were treated with 4, 20 and 100 µΜ of etoposide for 6 h. The results show that at all three concentrations of etoposide tested,
JNK activity was significantly increased (Figure 6.19). This is consistent with the data on
CYP24 promoter activity in that the lowest concentration of etoposide tested, 6.25 µΜ was also effective at stimulating CYP24 promoter activity (Figure 3.5).
205
Duration of Treatment (Hours) 6 hr 0.5 hr 2 hr 6 hr
Phosphorylated GST-Jun (1-79)
Control Etoposide
Figure 6.18 Effects of different etoposide exposure times on JNK activity in COS-1 cells. COS-1 cells were incubated with etoposide for 0.5, 2 and 6 h. Cells were lysed and the activity of JNK assayed as described in Materials and Methods. The experiment was repeated twice and similar results were obtained.
206 * 300 * * 250
200
150
100 JNK activity (% of control) 50
0 0014 20 10000 Etoposide (µΜ)
Figure 6.19 Effects of different concentrations of etoposide on JNK activity in COS-
1 cells. COS-1 cells were treated with 0, 4, 20, 100 µΜ of etoposide for 6 h. Cells were lysed and the activities of JNK was assayed as described in Materials and Methods.
Results shown represent mean ± SEM of three experiments, each conducted in triplicate determinations. Significance of difference between control and cells treated with etoposide: *p<0.05 (ANOVA followed by Dunnett’s multiple comparisons test).
207 6.3 Summary
In summary, the above data demonstrate that ERK1, ERK2, ERK5, JNK, PI3K or PKCζ were involved in regulating CYP24 promoter induction by daunorubicin hydrochloride or etoposide. The extent to which each of these signaling molecules was involved in mediating the action of the drugs depended on the kidney cell-line used as well as the drug involved.
208
Chapter 7
Effects of chemotherapeutic drugs on endogenous CYP24 mRNA expression in
kidney cells
209 7.1 Introduction
In the preceding chapters, we demonstrated that chemotherapeutic drugs can up-regulate the activity of the CYP24 promoter in kidney cells. We therefore aimed to investigate the effects of these drugs on endogenous CYP24 expression. Since it is not possible to measure the levels of CYP24 protein due to the unavailability of commercial anti-CYP24 antibody at present, we therefore investigated the effects of daunorubicin hydrochloride and etoposide on endogenous CYP24 mRNA expression in COS-1 cells.
7.2 Methods
COS-1 cells were treated with daunorubicin hydrochloride or etoposide in the presence or absence of 1,25(OH)2D3 for 24 h. Cells were lysed using Trizol reagent and RNA extracted according to manufacturer’s instructions. RNA (1µg) was reverse transcribed using Random hexamers and SuperScript III reverse transcriptase. All real-time PCR reactions were carried out in duplicate in a final volume of 10 µl. The cDNA samples were amplified using iQ SYBR Green Supermix primers (250 nM) specific for CYP24 and GAPDH cDNA. Relative expression between samples was calculated using the comparative cycle threshold (CT) method (∆CT) (24).
210 7.3 Results
7.3.1 Daunorubicin hydrochloride and etoposide inhibited the expression of endogenous CYP24 by 1,25(OH)2D3.
As expected, the results from this study showed that 1,25(OH)2D3 increased the levels of
CYP24 mRNA in COS-1 cells. Unexpectedly, daunorubicin hydrochloride and etoposide abrogated the 1,25(OH)2D3 induction of CYP24 mRNA expression whereas daunorubicin hydrochloride and etoposide per se did not increase the CYP24 mRNA level in COS-1 cells (Figure 7.1).
7.3.2 Pre-treatment of 1,25(OH)2D3 prevented etoposide from suppressing the induction of CYP24 mRNA expression by 1,25(OH)2D3.
Daunorubicin Hydrochloride and etoposide has been shown to inhibit topoisomerase II enzyme which has an important role in DNA replication and transcription (Mao et al.,
2001). It is possible that the inhibition of the endogenous CYP24 induction by
1,25(OH)2D3 is due to the inhibition of the uncoiling of DNA by both daunorubicin hydrochloride and etoposide. To test this hypothesis, COS-1 cells were incubated with
1,25(OH)2D3 for 6 h before adding etoposide which had a stronger synergistic action than daunorubicin hydrochloride. The level of CYP24 mRNA was determined 12 h later.
As shown in Figure 7.2, delaying the exposure of the cells to etoposide permitted the unhindered stimulation of CYP24 expression by 1,25(OH)2D3. However, there was no enhancement of the effect of 1,25(OH)2D3 by the drug. This result suggests that etoposide inhibited the initiation of CYP24 mRNA transcription by 1,25(OH)2D3.
211
0.05
*
0.04
0.03 : GAPDH mRNA
0.02
P24 mRNA *#* *#* CY 0.01
0 1,25(OH)2D3 - + - - + +
Daunorubicin - - + - + - Etoposide - - - + - +
Figure 7.1 Effects of daunorubicin hydrochloride and etoposide on endogenous
CYP24 mRNA expression in COS-1 cells. COS-1 cells were incubated with 2 µM of daunorubicin hydrochloride or 100 µM of etoposide ± 1,25(OH)2D3 for 24 h. Total RNA was extracted and the level of CYP24 mRNA was determined by quantitative real time
PCR. Significance of difference between control and drug or 1,25(OH)2D3 treatment:
*p<0.001; significance of difference between 1,25(OH)2D3 and 1,25(OH)2D3 + drug:
#p<0.001 (ANOVA followed by Tukey-Kramer multiple comparisons test).
212
0.016 * ***
0.012
mRNA
0.008 : GAPDH
P24 mRNA 0.004 CY
0 EtOH + + - - Me SO + - + - 2 1,25(OH)2D3 - - + + Etoposide - + - +
Figure 7.2 Etoposide did not suppress the ability of 1,25(OH)2D3 to stimulate
CYP24mRNA expression in cells which had been preincubated with 1,25(OH)2D3.
COS-1 cells were incubated with vehicle or 1,25(OH)2D3 for 6 h. Etoposide or vehicle was then added and the cells were incubated for a further 12 h. RNA was extracted and the level of CYP24 mRNA was determined. The results are expressed as a ratio of
CYP24 mRNA:GAPDH mRNA. Significance of difference between vehicle and
1,25(OH)2D3: *p<0.05.
213
3.3.3 Etoposide stimulated the degradation of CYP24 mRNA
The above data also suggest that etoposide might have another action in addition to its role as a topoisomerase inhibitor because it did not enhance the effect of 1,25(OH)2D3
(Fig 7.2) or cause the which would have been expected from the CYP24 promoter assays in the preceeding chapters. accumulation of CYP24 mRNA on its own (Figs 7.1 and 7.2),
We therefore investigated whether the drug could also cause the degradation of CYP24 mRNA. To investigate this possibility, COS-1 cells were incubated with 1,25(OH)2D3 for 6 h, washed and actinomycin D was added to stop transcription. The data in Figure
7.3 demonstrate that etoposide accelerated the decline in CYP24 mRNA over the period of study.
214
Figure 7.3 Etoposide accelerated CYP24 mRNA degradation
COS-1 cells in serum deficient media were incubated with 1,25(OH)2D3 (10 nM) for 6 h, washed and the cells were then incubated with actinomycin D (5 µg/ml) and etoposide (
■ ) or Me2SO ( ♦ ). At the indicated times, triplicate wells from each group were removed, total RNA extracted and CYP24 mRNA levels were determined by qRT-PCR.
Significance of difference between etoposide and Me2SO: *p<0.05 (Student’s unpaired t- test). Similar results were obtained in a replicate experiment.
215 7.4 Summary
The results from this study show that daunorubicin hydrochloride and etoposide alone did not cause an increase in CYP24 mRNA in kidney cells as would have been expected from the promoter studies in the preceeding chapters. Incubation of daunorubicin hydrochloride or etoposide together with 1,25(OH)2D3 resulted in the inhibition of
1,25(OH)2D3 induction of CYP24 mRNA expression. This inhibition could be abrogated with 1,25(OH)2D3 pre incubation suggesting the chemotherapeutic drugs inhibited the transcriptional machinery of the endogenous CYP24 promoter. This is most likely to be at the level of topoisomerase II as both drugs inhibit this enzyme. In addition, we demonstrated that etoposide increased the degradation of CYP24 mRNA. These results therefore demonstrate disparate effects of the drugs on transfected CYP24 promoter activity and endogenous CYP24 mRNA expression. The reason for this discrepancy and implications of these finding are discussed in the next chapter.
216
Chapter 8
Discussion
217 8.1 Discussion
8.1.1 Anti-cancer drugs and CYP24 promoter activity
The data demonstrate that a number of chemotherapeutic drugs representing several classes of the drugs were able to stimulate the transcriptional activity of the CYP24 promoter and/or amplify the effect of 1,25(OH)2D3 on the promoter in two kidney cell- lines which had been transiently transfected with one of two constructs that carried the
CYP24 promoter fused to the firefly luciferase gene. One promoter-reporter construct contained a -298 bp promoter sequence (relative to the transcription initiation site) whereas the other contained a -1.4 kb sequence which encompasses the 298 bp sequence.
These promoter sequences contained VDRE1 and VDRE2 (Kerry et al., 1996), an EBS
(Ets-binding site, Dwivedi et al., 2000), a VSE (vitamin D response element, Nutchey et al., 2005), a GC box (Dwivedi et al., manuscript in preparation) and perhaps other as yet unidentified elements. The drugs which were tested included the anthracycline, daunorubicin hydrochloride, the vinca alkaloid, vincristine sulphate, the alkylating agent, cisplatin, and etoposide, a podophyllotoxin. Between the two kidney cell-lines examined, some subtle differences in drug response were detected. Thus, when the -298 bp promoter was used, daunorubicin hydrochloride per se stimulated promoter activity in COS-1 but not HEK293T cells despite causing a synergistic activation of the promoter with
1,25(OH)2D3 in both cell-lines. The synergistic activation of the -298 bp promoter was greater in the HEK293T than in the COS-1 cells. The difference in drug response between the two kidney cell lines could be attributed to the difference in their origin.
218 While HEK293T is a human cell-line, derived from embryonic kidney and has characteristics of proximal tubular cells, COS-1 came from the kidney of an African green monkey. Despite this difference, both cell-lines responded to 1,25(OH)2D3 with enhanced CYP24 promoter activity and both have been used previously as models to investigate the regulation of CYP24 promoter activity by 1,25(OH2D3 (Dwivedi et al.,
2000, Nutchey et al., 2005). When the -1.4 kb sequence was used, daunorubicin hydrochloride, vincristine sulphate and etoposide per se also stimulated the transcriptional activation of the construct. In contrast, cisplatin alone did not stimulate
CYP24 promoter activity in COS-1 or HEK293T cells, either with the -298 bp or the -
1.4kb promoter construct. All of the four above listed drugs caused a synergistic enhancement in promoter activity when the cells were treated concurrently with a drug and 1,25(OH)2D3 although this effect was smaller with the -1.4 kb than the -298 bp construct in case of daunorubicin hydrochloride. Comparing the data obtained from the two promoter constructs, it can be concluded that the actions of the drugs were predominantly channelled through sequences in the -298 bp promoter.
In contrast to daunorubicin hydrochloride, the hydroxylated derivative, doxorubicin hydrochloride, was inactive on its own and did not amplify the action of 1,25(OH)2D3 on promoter induction, suggesting that not all chemotherapeutic drugs will affect vitamin D metabolism. Consistent with this view, ifosamide, a drug that is used to treat solid tumors in children, has been found not to affect the plasma levels of 1,25(OH)2D3 (de Schepper et al., 2004). In addition, we have also examined whether dexamethasone, a glucocorticoid which is frequently given to patients to prevent chemotherapy-induced
219 nausea and vomiting (Jordan et al., 2005) as well as having direct anti-tumour activity for some cancer-types (Child et al., 2005), could affect CYP24 promoter activity since glucocorticoids are known to cause bone loss (Sivagurunathan et al., 2005). Our data (not shown) demonstrate that dexamethasone neither stimulated nor enhanced the effect of
1,25(OH)2D3 on CYP24 promoter activity. Thus, these data exclude an effect of dexamethasone on CYP24 promoter activity and reinforce the view that not all chemotherapeutic drugs will affect vitamin D metabolism.
Very few agents, apart from 1,25(OH)2D3 and calcitonin, have consistently been demonstrated to induce CYP24 expression (Omdahl et al., 2002). However, agents such as insulin (Armbrecht et al., 1996) and PMA (Chen et al., 1993; Nutchey et al., 2005), although they do not increase CYP24 expression by themselves, enhance the action of
1,25(OH)2D3 on CYP24 induction. Thus, our findings place chemotherapeutic drugs in a class of agents that can both stimulate the transcriptional activation of the CYP24 promoter as well as amplify the effects of 1,25(OH) 2D3, as has previously been reported for calcitonin (Gao et al., 2004).
8.1.2 Cytotoxic effects of the drugs in HL60 cells but not in kidney cells
The tested drugs were found to readily kill the human promyelocytic leukaemic HL60 cells at relatively low doses but the drugs did not affect the viability of the kidney cell- lines, even at relatively high but pharmacologically achievable doses. Primary rat
220 hepatocytes also showed substantial resistance to the cytotoxic actions of the drugs (data not shown). Thus, it is unlikely that toxicity to kidney or liver cells and hence blockade of the bioactivation of 1,25(OH)2D3 could contribute in any significant manner to the reduction in plasma level 25(OH)D3 and 1,25(OH)2D3 levels in the patients.
8.1.3 Lack of a role for the VDR and VDREs in the actions of the drugs
The actions of 1,25(OH)2D3 are mediated via the VDR and overexpression of the nuclear
VDR in COS-1 cells greatly facilitates the ability of the hormone to stimulate CYP24 promoter activity (Dwivedi et al., 2002) and this was confirmed in the studies undertaken in this thesis. To investigate whether the actions of the drugs required the VDR, the cells were transiently transfected with the nuclear VDR and transcriptional activation of the
CYP24 promoter was investigated. The data suggest that the stimulatory effect of daunorubicin hydrochloride per se on CYP24 promoter activity was unlikely to have involved the VDR because VDR overexpression not only did not augment the action of the drug but it suppressed promoter induction by the drug. Consistent with this conclusion, mutating VDRE1 and VDRE2 within the CYP24 promoter did not affect promoter induction by daunorubicin hydrochloride. In contrast, overexpression of the
VDR augmented the effect of 1,25(OH)2D3 whereas mutating the VDREs greatly diminished the stimulatory effect of 1,25(OH)2D3. To demonstrate that the drugs did not directly affect VDR activity, we used a construct which contained a consensus VDRE of the DR3-type (two AGGTCA motifs separated by 3 bp), cloned immediately upstream of
221 the thymidine kinase promoter. When transfected in COS-1 cells, our data show that
1,25(OH)2D3 but not daunorubicin hydrochloride stimulated the activity of this reporter construct. The drug also did not alter the response to 1,25(OH)2D3. These data clearly demonstrate that the VDR and VDREs do not mediate the actions of drugs such as daunorubicin hydrochloride.
8.1.4 Daunorubicin and etoposide increased the expression of the VDR
Despite the VDR-independent action of daunorubicin hydrochloride per se on promoter activity, the VDR could be involved in mediating the synergistic effect of the drug and
1,25(OH)2D3. One likely scenario was that daunorubicin hydrochloride could increase the expression of the VDR. By analogy to the VDR-overexpression experiments in which transient transfection with the nuclear VDR augmented the response to 1,25(OH)2D3, a drug-induced enhancement in the expression of the VDR could have a similar effect.
Indeed, the data demonstrate that treatment of the cells with daunorubicin hydrochloride increased the expression of the VDR. This effect was also seen with etoposide. It has previously been demonstrated that etoposide could increase VDR mRNA and protein expression in leukaemic cells. Studies by Torres et al. (2000) have demonstrated that treating HL-60 and U-937 cells with 0.15 mM of etoposide for 72 h significantly increased VDR protein expression and 1,25(OH)2D3 binding activity. These authors showed that the increase in VDR mRNA expression by 0.15 mM etoposide was time dependent. Together, these data demonstrate that etoposide and daunorubicin can up- regulate the expression of the VDR in some cell-types, due primary to an increase in
222 VDR transcription (Torres et al., 2000). Although the effects of the other drugs on VDR expression were not investigated, the data strongly suggest that while the actions of the drugs per se did not require the VDR, drug-induced increase in VDR expression could be involved, at least in part, in mediating the synergistic effects of the drugs and
1,25(OH)2D3.
8.1.5 Reactive oxygen species and the promoter inducing effect of daunorubicin and etoposide
The anti-cancer actions of several anti-cancer drugs are dependent on the generation of reactive oxygen species (Gervasoni et al., 2004; Huang et al., 2003; Wu et al., 2002).
Thus, it was of interest to investigate whether the promoter-activating effects of daunorubicin and etoposide required the generation of reactive oxygen species. We used
N-acetylcysteine which is a precursor in the formation of the antioxidant glutathione in the body. The thiol (sulfhydryl) group confers antioxidant effects and is able to reduce free radicals. Contrary to the requirements for reactive oxygen species in the cytotoxic action of anthracyclines (Wu et al., 2002), the data demonstrate that reactive oxygen species were not required for daunorubicin hydrochloride to stimulate the activity of the
CYP24 promoter. Thus, N-acetylcysteine did not affect the action of daunorubicin hydrochloride. Similarly, N-acetylcysteine also did not affect the action of etoposide on the CYP24 promoter activity. Interestingly, treatment of the cells with N-acetylcysteine increased CYP24 promoter activity in response to 1,25(OH)2D3. Oxygen is a co-
223 substrate for CYP24 enzyme activity. The observation that the anti-oxidant enhanced the ability of 1,25(OH)2D3 to induce CYP24 promoter activity is most likely due to decreased activity of endogenous CYP24 activity with a concomitant increase in intracellular 1,25(OH)2D3. The redox state of the cell is likely to be altered by
1,25(OH)2D3 since the hormone has previously been reported to deplete cellular reduced glutathione. This has been suggested as a mechanism by which the hormone exerts its anti-cancer action (Ravid and Koren, 2003). Thus, our data with N-acetylcysteine are not only consistent with 1,25(OH)2D3 altering the redox state of the cells but also reveal that oxidative processes initiated by 1,25(OH)2D3 exert a negative impact on the induction of
CYP24 promoter by the hormone.
8.1.6 Non-genomic actions of 1,25(OH)2D3 involving intracellular signalling molecules
Whereas the genomic actions of 1,25(OH)2D3 are well established, recent studies have strongly suggested that the hormone also has non-genomic actions. The best examples are the observations that some of the actions of 1,25(OH)2D3 occur at kinetics which are incompatible with a genomic action and these include the activation of intracellular signalling molecules. Thus, we and others have demonstrated that 1,25(OH)2D3 not only caused the activation of PKC and the MAP kinases (Nutchey et al., 2005; Dwivedi et al.,
2002; Chae et al., 2002; Bikle et al., 2001) but the action of 1,25(OH)2D3 on CYP24 promoter induction was also shown to be mediated by the ERK1/ERK2 and ERK5
224 modules in COS-1 cells (Dwivedi et al., 2002). A recent study in HEK293T cells has demonstrated that PKCβ and JNK were also required for promoter activation by the hormone (Nutchey et al., 2005). In this study, PMA and 1,25(OH)2D3 were found to cause a synergistic activation of the CYP24 promoter. This well known phenomenon was found to be protein kinase C-dependent. Whereas ERK1/ERK2 was not activated by
1,25(OH)2D3 alone, its activation by PMA was potentiated by 1,25(OH)2D3 also. The importance of ERK1/2 for transcriptional synergy was demonstrated by transfection of the dominant-negative ERK1(K71R) mutant, which resulted in a reduced level of synergy on CYP24 promoter activity. JNK, in addition to being necessary for promoter activity incuced by 1,25(OH)2D3, was also shown to be required for synergy (Nutchey et al.,
2005). Whereas the likely physiological target of ERK1/ERK2 in COS-1 cells was
RXRα, ERK5 targeted Ets-1. We were able to demonstrate a direct interaction between
ERK1/ERK2 and RXRα, ERK1/ERK2 and Ets-1, and ERK5 and Ets-1. Whereas the interaction between ERK1/ERK2 and RXRα and between ERK1/ERK2 and Ets-1 was constitutive, the interaction between ERK5 and Ets-1 was induced by 1,25(OH)2D3.
Interestingly, Waas and Dalby (2001) reported that a His-tagged Ets-1 (1-138) was a good substrate for ERK2 in cell-free assays. Although we could not demonstrate such phosphorylation by ERK1 in cell-free assays using full length GST-Ets-1, our finding that Ets-1 showed constitutive binding to ERK1/ERK2 suggests a possible functional relationship between the MAP kinase and Ets-1 in intact cells. The precise reason for the difference between our data and those of Waas and Dalby (2001) is not known but could be due to different assay conditions. We used full length Ets-1 whereas Waas and Dalby
225 (2001, 2002, 2003) used a truncated Ets-1; we used 10 mM Mg2+ in our buffers whereas
Waas and Dalby used 20 mM Mg2+ and 100 mM K+.
As described in Chapter 1, RXRα and VDR form a complex which binds to the VDREs.
This is responsible for mediating the genomic action of the hormone. Our data demonstrate that S260 on RXRα is the ERK1/ERK2 phosphorylation site. On the other hand, Ets-1 binds to the EBS (5'-AGAGGATGGA-3' containing the core 5'-GGAT-3' consensus sequence) at -128/-119 (Dwivedi et al., 2000). The likely residue on Ets-1 which was phosphorylated by ERK5 was shown to be T38 since a T→A mutation at this residue rendered the protein non-phosphorylatable by ERK5. The function of Ets-1 was also lost when this mutation was introduced into the transcription factor. The studies by
Nutchey et al, (2005) demonstrate that JNK targets an unidentified transcription factor which interacts with the VSE (vitamin D stimulatory element) at -132/-126. Data obtained in HEK293T cells by Dwivedi et al., (manuscript in preparation) have also demonstrated that PI3 kinase and PKCζ target the transcription factor, Sp1, which binds to a GC box (5’-GGGGCGGGT-3’) at -114/-101. In this case, Sp1, acting via the GC box
(Gao et al., 2004), was demonstrated to regulate basal as well as 1,25(OH)2D3-mediated induction of the CYP24 promoter activity. These data demonstrate that a number of intracellular signalling molecules are involved in regulating the transcriptional activation of the CYP24 promoter by 1,25(OH)2D3 with Sp1 regulating the basal transcriptional activity.
226 8.1.7 Anti-cancer drugs and intracellular signalling molecules in CYP24 promoter activation
Many chemotherapeutic drugs, including daunorubicin hydrochloride and etoposide, have been found to stimulate the activities of the above signalling molecules. This is confirmed in this thesis. We have now presented data which demonstrate that the signalling molecules, ERK1/ERK2, ERK5, PI3 kinase and JNK were involved in mediating the effects of daunorubicin hydrochloride and etoposide per se on promoter activity in COS-
1 cells. Thus, promoter induction by the drugs was inhibited when the cells were transiently transfected with the dominant negative mutant of a target kinase or its upstream kinase, or incubated with an inhibitor of the target kinase. Consistent with a role for the MAP kinases in promoter induction, the drugs also stimulated the activities of
ERK1/ERK2, ERK5 and JNK in COS-1 cells. The lower than expected fold stimulation for some of the kinases was probably due to a high baseline kinase activity in the kidney cell-line compared to other cell-lines. The MAP kinases were also required for the synergistic activation of the CYP24 promoter in HEK293T cells by the drugs and
1,25(OH)2D3. Although previous studies have reported that anthracyclines and etoposide can stimulate the activities of ERK1/ERK2 and JNK (Boldt et al., 2002; Tang et al.,
2002; Brantley-Finley et al., 2003; Mas et al., 2003), their effects on ERK5 have not been investigated. Our data therefore extends the number of MAP kinases that these chemotherapeutic drugs can activate. The precise targets of the kinases within the -298 promoter remain to be identified but we can exclude the VDREs and GC-box as targets.
227 Our recent studies in HEK293T cells have demonstrated that 1,25(OH)2D3 can also stimulate the activity of PKCζ (Dwivedi et al, manuscript in preparation). With respect to this hormone, PKCζ appears to target Sp1 and the GC box. In contrast to the MAP kinases, our data suggest that PKCζ played a more selective role, depending on the drug and cell-line tested. Thus, while the dominant negative PKCζ mutant blocked the action of daunorubicin hydrochloride in COS-1 cells, it did not affect the response to etoposide in this cell-line. The PKCζ mutant did not affect drug-induced promoter activation in
HEK293T cells, suggesting that this PKC isozyme did not play any roles in mediating
CYP24 promoter induction by the drugs in HEK293T cells. These results again highlight the subtle differences between COS-1 and HEK293T kidney cells.
An interesting finding was that the hydroxylated form, doxorubicin hydrochloride, does not induce CYP24 promoter activity, despite being reported to stimulate the activity of
ERK1/ERK2 in a variety of cell cancer lines (Brantley-Finley et al., 2003; Yeh et al.,
2004). Although doxorubicin hydrochloride has been shown to be more active against solid tumour compared to daunorubicin hydrochloride (Weiss., 1992), overall, there are very little differences between doxorubicin hydrochloride and daunorubicin hydrochloride in terms of toxicity, pharmacokinetics and stability (Dine et al.,1992;
Maniez-Devos et al., 1986; Heijn et al., 1999). Like daunorubicin hydrochloride, doxorubicin hydrochloride has been shown to kill cancer cell by binding to DNA and causing the DNA to break (Swift et al., 2006). Furthermore, doxorubicin has been shown to inhibit topoisomerase II enzyme thus disrupting transcription and replication (Swift et al., 2006). Doxorubicin can also bind to cell membranes and sarcoplasmic reticulum and
228 interrupts the ion transport (Earm et al., 1994). It also cause the generation of oxygen free radicals (Tsang et al., 2003). Our data therefore suggest that activation of ERK1/ERK2 per se is insufficient for the induction of the CYP24 promoter activity.
Overall our findings are consistent with a model for daunorubicin hydrochloride or etoposide induction that involves the phosphorylation and activation by ERK1/ERK2,
ERK5, JNK and/or PKCζ/PI3 kinase of specific transcription factors associated with the first –298bp of the CYP24 promoter. Transcription factors known to enhance basal activity of the first –298bp of promoter and thus possible targets for the ERKs include
Ets-1, Sp1, and a VSE and CCAATT box binding proteins (Gao et al., 2004; Nutchey et al., 2005, Dwivedi et al., in preparation). A key issue is the question of how the chemotherapeutic drugs activate the kinases. Mas et al, (2003) reported that the ability of daunorubicin hydrochloride to stimulate the activity of ERK1/ERK2 in U937 cells was blocked by kinase dead DN PKCζ, demonstrating that this PKC isozyme is involved in regulating the activity of ERK1/ERK2. In cardiac myocytes, the stimulation of
ERK1/ERK2 activity by the anthracyclines was demonstrated to involve the small G protein, p21ras (Zhu et al., 1999). It is possible that p21ras could also be involved in the activation of not just ERK1/ERK2 but also ERK5 by the drugs in COS-1 cells since we and others have previously demonstrated this p21ras-ERK relationship under different conditions (English et al., 1999; Dwivedi et al., 2002). Unlike ERK activation by
1,25(OH)2D3 that most likely involves binding of the hormone to VDR located at the cytoplasmic membrane, our work eliminates the involvement of VDR and VDRE in the induction of the CYP24 promoter by daunorubicin hydrochloride or etoposide per se
229 although its ability to up-regulate the expression of endogenous VDR could be responsible, at least in part, for enhancing the effect of 1,25(OH)2D3 on promoter induction.
8.1.8 Suppression of CYP24 mRNA induction by daunorubicin and etoposide
Lastly, we also measured the levels of endogenous CYP24 mRNA in COS-1 cells using real time quantitative PCR. Unexpectectly, we found that daunorubicin hydrochloride and etoposide did not increase CYP24 mRNA expression. Furthermore, the drugs inhibited
1,25(OH)2D3 induction of CYP24 mRNA expression. Pre-incubating the cells with
1,25(OH)2D3 for 6 h before etoposide treatment resulted in the unhindered stimulation of
CYP24 expression by 1,25(OH)2D3. These results suggest that etoposide may be inhibiting the initiation of the transcriptional activation by 1,25(OH)2D3. One well- characterised action of daunorubicin hydrochloride and etoposide as anti-cancer drugs is the inhibition of topoisomerase II activity, thereby inhibiting DNA transcription (Minotti et al., 2004; Shimada et al., 2003). Topoisomerase enzyme plays a crucial role in gene transcription by helping DNA to uncoil (Mao et al., 2001). The inhibition of topoisomerase II by etoposide may explain the attenuation of 1,25(OH)2D3 induction of
CYP24 mRNA expression in the presence of etoposide. Since plasmid DNA do not require uncoiling for transcription to take place, daunorubicin hydrochloride and etoposide were able to stimulate the CYP24 promoter and hence resulting in an increase
230 in luciferase activity. However, the lack of an enhancing effect of etoposide also suggests that the drug may be increasing the degradation of the CYP24 mRNA. Using actinomycin
D to stop transcription, we were able to confirm that etoposide facilitates the degradation of CYP24 mRNA. This unique ability of etoposide to up-regulate CYP24 promoter while enhancing degradation of its mRNA has also been observed previously with parathyroid hormone (PTH). Earlier studies by Zierold et al, 2000 have shown that PTH enhanced the action of 1,25(OH)2D3 induction of the CYP24 promoter in AOK-B50 cells. However, further studies by Zierold et al, 2002 demonstrated that PTH reduces the half-life of
CYP24 mRNA by 4.2-fold. Whether the increase in degradation of the CYP24 mRNA caused by the drugs translates to a decrease in 24-hydroxylase enzyme and therefore a reduction in the degradation of 1,25(OH)2D3 is yet to be determine.
8.1.9 Clinical implications of this work and future directions
Apart from the classical role of 1,25(OH)2D3 in Ca2+ homeostasis, 1,25(OH)2D3 and analogues of 1,25(OH)2D3 have attracted immense interest as a novel therapy to treat malignancies. 1,25(OH)2D3 and analogues have anti-proliferative actions and they sensitize cancer cells to chemotherapeutic drugs (Trump et al., 2004). The idea that chemotherapeutic drugs could used in conjunction with 1,25(OH)2D3 to treat advanced tumours have already attracted immense interest. In novel approaches to treat advanced prostate cancers, several groups have focussed on combination therapy using
1,25(OH)2D3 and conventional drugs. This is based on the observations that
231 1,25(OH)2D3 and analogues can enhance, either synergistically or additively, the anti- tumor activities of several classes of anti-neoplastic agents, including platinum compounds, docetaxel, etoposide and paclitaxel (Beer, 2005; Moffatt et al., 1999).
Sevaral clinical trials are currently underway to evaluate the efficacy of combining
1,25(OH)2D3 and drugs such as docetaxel (taxotere) in the treatment of androgen- independent prostate cancer (Beer, 2005; Vijayakumar et al., 2005). How such combinations enhance the anti-tumor activity of each other is poorly understood.
Nevertheless, our data demonstrate that some anti-neoplastic agents cause CYP24 mRNA instability and prevent 1,25(OH)2D3 from increasing the level of CYP24, despite stimulating the promoter activity in transfection assays. There are possible important medical repercussions for anti-cancer therapy. In combination chemotherapy involving
1,25(OH)2D3 and an anti-neoplastic agent, relatively high doses of 1,25(OH)2D3 are generally required with the concomitant increase in risk of adverse side effects. Indeed, a major hurdle to achieving a favourable clinical response is the need for doses that are just below those which cause hypercalcaemia and hypercaluria (Beer, 2005). Moreover,
CYP24 is regarded as an oncogene and in some cancers, its expression is elevated compared to normal tissues (Albertson et al., 2000). This is likely to provide an escape mechanism for these cancer cells to avoid the cytotoxic and anti-proliferative effects of
1,25(OH)2D3. Our findings that anti-cancer drugs increased the expression of the VDR and prevented the expression of CYP24 suggest that it is possible to reduce the dose of
1,25(OH)2D3 to achieve a favorable clinical response because increased VDR expression would increase the sensitivity as well as responsiveness of cancer cells to 1,25(OH)2D3
232 while the inability of 1,25(OH)2D3 to induce CYP24 expression would maintain the bioavailability of circulating 1,25(OH)2D3 over a longer period.
While our data on CYP24 mRNA expression have provided some novel insights into how it may be possible to increase the efficacy of 1,25(OH)2D3 in anti-cancer treatments, more studies are required to expand this field and elucidate the mechanism by which the drugs promote CYP24 mRNA degradation. Future studies could investigate whether other anti-cancer agents can also promote CYP24 mRNA degradation and stimulate VDR expression. These studies could also explore the effect of chemotherapeutic drugs on the rate of 1,25(OH)2D3 degradation, either alone or in combination with other agents. Other studies could explore the effects of anti-cancer drugs on CYP24 expression in cancer cells especially those that express high levels of CYP24 e.g. DU145 prostate cancer cells
(Krishnan et al., 2003). In addition to kidney and cancer cells, other cell-types such as bone cells and monocytes are also known to express CYP24. Thus, it would be important to conduct similar studies using these cell-types. Finally, animal models with established tumours could be used to investigate the effects of chemotherapeutic drugs on circulating levels of 1,25(OH)2D3 and the levels of 1,25(OH)2D3 within tumour cells. These can be investigated in the nude mice model which have been transplanted with human tumours or the Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model which spontaneously develops prostate cancer.
233 8.2 Summary
In summary, the data in the present study demonstrate, for the first time, that certain anti- cancer drugs could stimulate the activity of the CYP24 promoter and enhance the induction of promoter activity by 1,25(OH)2D3. In addition, this study also demonstrate that ERK1/ERK2, ERK5, PKCζ and PI3K played an important role in the promoter induction by the anti-cancer drugs. These results are summarised in Table 8.1 and Table
8.2. However, the increase in promoter activity by chemotherapeutic drugs did not translate to an increase in CYP24 mRNA expression. Furthermore, daunorubicin hydrochloride and etoposide inhibited the expression of CYP24 mRNA by 1,25(OH)2D3.
The inhibitory effects were attributed to the inhibition of transcriptional activation and enhanced degradation of CYP24 mRNA by etoposide. Thus, although our data cannot explain why children undergoing chemotherapy had reduce levels of 1,25(OH)2D3, these results, nevertheless, open new avenues for the use of chemotherapy drugs in conjunction with 1,25(OH)2D3 to treat cancer and provide further justification for such use.
234 Table 8.1 Summary of the effects of chemotherapeutic drugs on the -298bp and -
1.4kb CYP24 promoter constructs in the presence or absence of 1,25(OH)2D3 in kidney cell lines
Cell Type COS-1 HEK293T
CYP24 298 bp 1.4 Kbp 298 bp 1.4 Kbp promoter Treatment Type EtOH 1,25D EtOH 1,25D EtOH 1,25D EtOH 1,25D
Daunorubicin + +++ + ++ + +++ + - Hydrochloride
Etoposide + +++ + +++ ND ND + +++
Vincristine + - + +++ ND ND - +++ Sulfate
Cisplatin - +++ - +++ ND ND - -
Doxorubicin - - ND ND ND ND ND ND Hydrochloride
+ = Increase in CYP24 promoter activity ++ = Enhance CYP24 promoter activity with 1,25(OH)2D3 +++ = Synergistic increase in CYP24 promoter activity with 1,25(OH)2D3 ND = Not determined
235 Table 8.2 Summary of the effects of dominant negative ERK1, MEK5, MEK4,
PKCζ mutants and inhibitor of PI3K on CYP24 promoter induction by daunorubicin hydrochloride and etoposide in kidney cell lines.
Cell Line COS-1 HEK293T
Promoter 298 bp 1.4 Kb 298 bp
Treatment Daunorubicin Etoposide Daunorubicin Hydrochloride Hydrochloride
ERK1K71R - MEK5A - -
ERK1K71R + ND ND MEK5A
MKK4K116R
PKCζK281M - -
LY294002 ND ND
= Inhibition of CYP24 promoter activity
= No changes in CYP24 promoter activity
ND = Not Determined
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