TELOMERES AND PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR GAMMA IN THE DEVELOPMENT OF HUMAN BREAST CANCER
By
Fariborz Rashid-Kolvear
A Thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Laboratory Medicine and Pathobiology Faculty of Medicine University of Toronto
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While these forms may be included Bien que ces formulaires in the document page count, aient inclus dans la pagination, their removal does not represent il n'y aura aucun contenu manquant. any loss of content from the thesis. Canada Telomeres and peroxisome proliferator-activated receptor gamma in the development of human breast cancer
A Ph.D. thesis by Fariborz Rashid-Kolvear submitted in conformity with the requirements for the degree of Doctor of Philosophy, Graduate Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto, 2008
Abstract
Breast cancer is the most common internal malignancy afflicting North American women. The development of breast cancer involves a sequential progression through defined clinical and pathological stages beginning with normal epithelium and progressing to hyperplasia, ductal carcinoma in situ (DCIS), invasive ductal carcinoma
(IDC), and culminating in metastatic disease. This progression into metastatic disease is characterized by many molecular alterations, including the shortening of total telomere length. Cancer cells containing critically short telomeres activate telomerase to avoid cell death and it has been demonstrated to occur in 90% of breast carcinoma. Furthermore, telomerase activity has been shown to be modulated by ligands for peroxisome proliferator-activated receptor gamma (PPARy). The integral role of telomeres in cancer progression has motivated extensive research into targeting telomeres for diagnosis and treatment of breast cancer.
In the present study, the telomere shortening on chromosome 17q was analyzed in relation to average telomere length in normal epithelium, DCIS and IDC to determine if
ii the shortening of specific telomeres can be used as a molecular marker for breast cancer progression. Furthermore, the effect of the PPARy ligand troglitazone on telomerase
activity and gene expression was examined in MDA-MB-231 breast cancer cells to determine if PPARy ligands have an anti-telomerase effect.
Results indicated that telomere shortening on chromosome 17q, harboring several genes involved in breast cancer development, is higher than the average shortening of all telomeres. Furthermore, troglitazone reduced telomerase activity in MDA-MB-231 cells independently of PPARy activation. In addition, troglitazone inhibited cell proliferation and the cell cycle signaling pathway on MDA-MB-231 and MCF-7 breast cancer cell lines through different mechanisms. Results from microarray comparative genomic hybridization also indicated that the PPARy gene is lost or deleted in 58% of clinical breast cancers.
In conclusion, the high shortening of telomere on chromosome 17q suggests that telomere shortening is not a linear process and may serve as a marker for breast cancer progression. Furthermore, troglitazone demonstrated anti-telomerase and anti proliferative activity in breast cancer cells. However, these effects vary depending on the cell type and experimental models employed. Finally, the loss of the PPARy gene in over
50% of breast cancers indicates a potential tumor suppressor role for PPARy in the development of breast cancer.
m ACKNOWLEDGMENTS
I think this is the best time to express my deepest gratitude to all my teachers and
mentors from the time I began learning to now having a Ph.D. degree. I started learning
from a small family in a low middle neighborhood in Tehran and end up at one of the
most prestigious universities in the world: the University of Toronto. I owe this amazing
progression to many people. As a first teacher, my mother taught me the essence of life,
love, hope, humanity, and forgiveness. My wife was my excellent teacher as well. During
many years of marriage, she showed me true love in real life. Her lessons were part of my
success. And finally, my PhD supervisor, Dr. Susan Done, wrapped up my years of
training by teaching me how to think scientifically. I have been fortunate to have Susan
as a supervisor. Susan's outstanding energy, enthusiasm, and her excellent supports made
my learning experiences in her research laboratory very rewarding. I also wish to thank
my supervisory committee, Dr. Jeremy Squire and Dr. Ming-Sound Tsao for their
outstanding advice and input during my time as a graduate student. It was an excellent
opportunity for me to have Dr. Jeremy Squire's supports during my study. His great
supports opened a big door for my future. I will always remember this.
I would like to thank all the collaborators I have worked with, especially Michael
Taboski. I would also like to thank Johnny Nguyen. My experience as a graduate student would not have been the same without his outstanding assistance. I wish to express my gratitude to Melania Pintilie for her patience and continuous assistance with statistics. I also wish to acknowledge the members of Dr. Done's laboratory for their help and friendship: Dr. Dongyu, Dr. Danh Tran-thanh Wang, Vietty Wong, Dr. Chunjie Wang,
Yanxia Wen, Keisha Warren, and especially Dr. Vladimir Iakovlev. It was a pleasure to
IV have been friends and collaborated with Dr. Vladimir Iakovlev during my graduate studies. This acknowledgment would not be completed without special thanks to the coordinator of the Department of Laboratory Medicine and Pathobiology, Dr. Harry
Elsholts. I leaned a lot from his advices. I would also like to take this opportunity and thank all my friends as well as all people in the Department of Laboratory Medicine and
Pathobiology, the Ontario Cancer Institute, Princess Margaret Hospital, the Faculty of
Medicine, and the University of Toronto for their direct and indirect help and support.
Last but not least, I owe a little boy a lot. My son, Matin, will always be remembered in my heart for his amazing patience, support and unbelievable advice at the time that I was hopeless and lonely. I wish him my best for ever.
v To-
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Summer 2008 UvtiverbCty of Toronto- TABLE OF CONTENTS CHAPTER 1
Literature review 2
1.1 Breast cancer 3
1.1.1 Anatomy of human breast 3
1.1.2 Carcinoma of the breast 3
1.1.3 Duct carcinoma in situ 4
1.1.4 Invasive and metastatic breast carcinoma 5
1.1.5 Breast cancer treatment 5
1.2 Telomere 6
1.2.1 Telomere Structure 6
1.2.2 Telomere-associated proteins 7
1.2.3 Telomere Function 8
1.2.4 The length of telomere and chronic diseases 8
1.2.5 The length of telomeres in cancers 9
1.2.6 The length of telomeres in breast cancer 9
1.2.7 The length of telomeres in other types of cancer 12
1.2.8 Telomere maintenance 12
1.3 Telomerase 13
1.3.1 Regulation of hTERT 14
1.3.2 Regulation of hTR 14
1.3.3 Telomerase associated proteins 15
1.3.4. Human telomerase-associated protein 1 15
vii 1.3.5 Heat shock protein 90 chaperone complex 15
1.3.6 Dyskerin 16
1.3.7 hStau 17
1.3.8 L22 17
1.3.9 La autoantigen 17
1.3.10 Heterogeneous nuclear ribonucleoproteins (hnRNPs) 18
1.3.11 Telomerase structure 18
1.3.12 Telomerase in cancer 18
1.3.13 Alternative Lengthening of Telomeres (ALT) 19
1.3.14 ALT in cancer 19
1.3.15 Telomerase as a target for cancer treatment 20
1.4 Nuclear receptors 20
1.4.1 Peroxisome proliferator-activated receptors 21
1.4.2 Peroxisome proliferator-activated receptor a 21
1.4.3 Peroxisome proliferator-activated receptor (3/8 22
1.4.4 Peroxisome proliferator-activated receptor y 22
1.4.5 PPARy structure 23
1.4.6 Co-factors 24
1.4.7 Co-activators 24
1.4.8 Co-repressors 25
1.4.9 PPARy ligands 25
1.4.10 Natural ligands 25
1.4.11 Dual agonists 26 1.4.12 Synthetic ligands 26
1.4.13 PPARy function 27
1.4.14 The Role of PPARy in lipid metabolism 27
1.4.15 The Role of PPARy in type 2 diabetes 27
1.4.16 The Role of PPARy in cancers 29
1.4.17 PPARy and breast cancer 30
1.4.18 The effects of PPARy ligands on cell cycle regulation 31
1.4.19 The effects of PPARy ligands on apoptosis 33
1.5 Rationale 33
1.6 Hypothesis 37
1.7 Specific aims 37
CHAPTER 2
Telomere length on chromosome 17q shortens more than global telomere length in the development of breast cancer 39
2.1 Abstract 40
2.2 Introduction 41
2.3 Materials and Methods 43
2.3.1 Sample collection 43
2.3.2 Quantitative fluorescence in situ hybridization (Q-FISH) 43
2.3.3 Image capturing and analyzing 44
2.3.4 Immunohistochemical staining of paraffin sections for HER2 45
2.3.5 Data analysis 46
2.3.6 Statistical analysis 46
ix 2.4 Results 47
2.5 Discussion 48
CHAPTER 3
Troglitazone suppresses telomerase activity independently of PPARy transcriptional activity in breast cancer cells 61
3.1 Abstract 62
3.2 Introduction 63
3.3 Materials and Methods .65
3.3.1 Materials 65
3.3.2 Cell Culture 65
3.3.3 Cell toxicity and cell viability assay 66
3.3.4 Western blot analyzing 66
3.3.5 Real-time RT-PCR 68
3.3.6 Stable shRNA Mediated Repression of PPARyin MDA-MB-231 Cells 68
3.3.7 Telomeric repeat amplification protocol (TRAP) assay 69
3.3.8 The NKI microarray dataset analysis 70
3.3.9 Statistical analysis 70
3.4 Results 71
3.4.1 Evaluating the Expression of PPARy in different breast cancer cell lines 71
3.4.2 Determining the expression level of hTERT and telomerase activity in
MDA-MB-231 ells 71
3.4.3 The cell toxicity effect of troglitazone 71
3.4.4 Troglitazone reduces telomerase activity in a time and dose dependent
x manner 72
3.4.5 Troglitazone suppresses hTERT transcription 72
3.4.6 Reduction in telomerase activity is independent from the transcriptional role of
PPARy 73
3.4.7 Troglitazone does not induce apoptosis 74
3.4.8 Troglitazone does not induce the differentiation of MDA-MB-231 74
3.4.9 The expression of hTERT is not correlated with the expression of PPARy in
clinical samples 75
3.4.10 The level of hTERT expression is negatively correlated with ER status in
breast cancer samples 76
3.5 Discussion 76
CHAPTER 4
Genome-wide analysis of differential expression of troglitazone-mediated genes in the estrogen receptor negative breast cancer cell line, MDA-MB-231, and the role of
PPARy in the progression of breast carcinoma 103
4.1 Abstract 104
4.2 Introduction 105
4.3 Materials and Methods 106
4.3.1 Materials 106
4.3.2 Cell culture 106
4.3.3 Flow Cytometry analysis 107
4.3.4 Microarray analysis 108
4.3.5 Real-time RT-PCR 109
xi 4.3.6 Nuclear Extraction 109
4.3.7 Netherlands Cancer Institute (NKI) dataset microarray analysis 110
4.3.8 Array comparative genomic hybridization 110
4.3.9 Sample collection Ill
4.3.10 Statistical analysis Ill
4.4 Results Ill
4.4.1 Troglitazone reduces the number of MDA-MB-231 breast cancer cells by
suppressing Gl—>S cell cycle transition Ill
4.4.2 Troglitazone reduces the phosphorylation status of Rb 113
4.4.3 Troglitazone does not change the protein expression of cyclin Dl and D3 in the
MDA-MB-231 cells 113
4.4.4 mRNA expression profile of MDA-MB-231 cells in response to troglitazone
114
4.4.5 Troglitazone reduces the protein expression of cyclin E2 and CKD2 in MDA-
MB-231 cells 116
4.4.6 Real time RT-PCR confirmation of differentially up-regulated genes from
microarray analysis 117
4.4.7 The localization of PPARy remains unchanged in response to troglitazone in
breast cancer cell lines 117
4.4.8 DNA copy number of PPARy gene is altered in breast cancer patients 118
4.4.9 The level of PPARy expression correlates with good clinical prognostic
parameters in breast cancer patients 118
4.5 Discussion 119
xii CHAPTER 5
Discussion and future directions 157
5.1 Discussion 158
5.2 Future directions 167
REFERENCES LIST 171
Xlll LIST OF TABLES
Table 2.1 Comparing telomere shortening of chrl7q in DCIS and normal tissue with
total telomere shortening in the same tissues 56
Table 2.2 Association between telomere length and HER2 expression 59
Table 4.1 Down-regulated genes according to microarray analysis 146
Table 4.2 Up-regulated genes according to microarray analysis 150
Table 4.3 List of functional group from GO analysis 152
Table 4.4 List of functional group from KEGG analysis 153
Table 4.5 Genes in cell cycle group acquired from functional GO analysis 154
Table 4.6 Genes in cell cycle group acquired from functional KEGG analysis 155
xiv LIST OF FIGURES
Figure 2.1 Labeling strategy used to flag pantelomeres and subtelomeric region of
chromosome 17q 51
Figure 2.2 FISH analysis of breast FFPE sections 52
Figure 2.3 Comparison of the normalized intensities of telomere length between normal
breast epithelial tissue, DCIS and IDC 54
Figure 2.4 Distribution of telomere length between DCIS and DCIS associated with
IDC 55
Figure 2.5 FFPE tissue sections were used for immunohistochemistry using HER2
antibody 57
Figure 3.1 The expression of PAPRy mRNA, the level of PAPRy protein in various
breast cancer cell lines 82
Figure 3.2 The expression level of hTERT and telomerase activity in three different
breast cancer cell lines 83
Figure 3.3 IC50 of troglitazone was measured by MTS assay 85
Figure 3.4 Troglitazone suppresses the activity of telomerase in a dose dependent
manner 86
Figure 3.5 The effect of troglitazone on telomerase activity and hTERT expression is
time dependent 88
Figure 3.6 The suppression role of troglitazone on hTERT is independent from
PPARy 90
Figure 3.7 Suppression of the expression of PPARy by shRNA 91
xv Figure 3.8 Evaluation of hTERT mRNA expression and telomerase activity in PPARy
knocked-down cells 92
Figure 3.9 The effect of troglitazone on telomerase activity was measured in the
absence of PPARy 93
Figure 3.10 Troglitazone does not induce apoptosis in MDA-MB -231 95
Figure 3.11 Cell differentiation was not induced by troglitazone and troglitazone had no
effect on apoptosis 97
Figure 3.12 The expression of hTERT is independent from PPARy in clinical breast
cancer samples 98
Figure 3.13 Investigating the correlation between hTERT mRNA expression and clinical
prognostic parameters 100
Figure 4.1. Troglitazone reduces the number of cells 126
Figure 4.2. Troglitazone suppresses cell proliferation 128
Figure 4.3. Troglitazone inhibits Gl—>S cell cycle transition 129
Figure 4.4. Time course inhibitory effect of troglitazone on S phase cell cycle transition
131
Figure 4.5. The effect of troglitazone on phosphorylation status of Rb protein 132
Figure 4.6. The response to troglitazone is different in ER-negative and -positive breast
cancer cells 133
Figure 4.7. The effect of troglitazone on gene profiling of MDA-MB-231 cells 134
Figure 4.8. Common list of differentially expressed in cell cycle group identified by both
GO and KEGG functional annotation 135
Figure 4.9. Real time RT-PCR was used to analyze the expression of common genes
xvi identified by GO and KEGG annotation 136
Figure 4.10. The response to troglitazone is different in ER-negative and -positive breast
cancer cells 137
Figure 4.11. Real time RT-PCR confirmation of selected genes from the differentially up-
regulated list obtained from microarray analysis 138
Figure 4.12. Troglitazone does not change the localization of PPARy protein in MDA-
MB-231 and MCF-7 cell lines 140
Figure 4.13. Evaluating DNA copy number of PPARy gene in breast cancer patients... 141
Figure 4.14. Analyzing the expression of PPARy mRNA in NKI dataset 142
Figure 4.15. Investigating the correlation between PPARy mRNA expression and clinical
prognostic parameters 143
Figure 4.16.Troglitazone shows different effects on MDA-MB-132 and MCF-7 cell lines
145
xvii ABBREVATIONS
15d-PGJ2 15-deoxy-A ' prostaglandin J2
70GS 70-gene prognosis signature
aCGH Array comparative genomic hybridization
ACS acetyl-CoA synthetase
AF activation function
ALT alternative lengthening of telomeres
aP2 fat-specific adipocyte P2
APB ALT associated promyelocytic leukemia (PML) body
AR androgen receptor
BADGE Bisphenol A diglycidyl ether
CaP prostate cancer
CAP c-CBL-associated protein
CDK cyclin-dependent kinase
CDKI CDK inhibitor
CIN chromosomal instability
DBD DNA binding domain
DCIS duct carcinoma in situ
DKC dyskeratosis congenita
D-loop DNA displacement loop
DSBs double-stranded DNA breaks
ER estrogen receptor
FATP fatty-acid transport protein FBS fetal bovine serum
FCS fetal calf serum
FFA free fatty acid
FFPE formalin-fixed paraffin-embedded
FISH fluorescence in situ hybridization
FLOW-FISH flow cytometric fluorescence in situ hybridization assay
GR glucocorticoid receptor
GSK3p glycogen synthase kinase 3-beta
H&E hematoxylin and eosin
HAT histone acetyltransferase
HDAC histone deacetylase
HDL high density lipoprotein hnRNPs heterogeneous nuclear ribonucleoproteins
HPIN high-grade prostatic intraepithelial neoplasia
HRE hormone response elements hsp90 heat shock protein 90 hStau human staufen hTERT human telomerase reverse transcriptase hTR human telomerase RNA
IDC invasive carcinoma
EN intraepithelial neoplasia
IR insulin-stimulated receptor
IRS insulin receptor substrate
xix K19 keratin 19
LBD ligand binding domain
LN lymph node involvement
LPL lipoprotein lipase
MMTV mouse mammary tumor virus
MR mineralocorticoid receptor
MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-
sulfophenyl)-2H-tetrazolium, inner salt
Muc-1 mucin-1
NHR nuclear hormone receptor
NIH National Institutes of Health
NIMH National Institute of Mental Health
NKI Netherlands Cancer Institute
ONRs orphan nuclear receptors
PARP poly (ADP-ribose) polymerase
PML promyelocytic leukemia
PNA peptide nucleic acid
Pol RNA polymerase
POT1 protection of telomere
PPAR peroxisome proliferator-activated receptor
PPARy peroxisome proliferator-activated receptor gamma
PPRE peroxisome proliferator response element
PR progesterone receptor
xx pRb hyperphosphorylated Rb
FTP phosphotyrosine phosphatase
Q-FISH quantitative fluorescence in situ hybridization
RAP1 repressor activator protein 1
RAR all-trans retinoic acid receptor
Rb retinoblastoma
RNP ribonucleoprotein
R.XR 9-cis retinoic acid receptor
snoRNA classical small nucleolar RNA
TANK1 tankyrase 1
TC telomere DNA content
TDLU terminal duct lobular unit
TEP1 telomerase-associated protein 1
TIN TRF 1-interacting protein
T-loop telomere loop
TNFa tumor necrosis factor a
TR thyroid hormone receptor
TRAP telomeric repeat amplification protocol
TRF telomere repeat factor
TRF telomeric repeat fragment
TZD thiazolidinedione
UCP uncoupling proteins
VDR vitamin D3 receptor VSMC vascular smooth muscle cell
WS wound signature
xxu Chapter 1
i Literature Review
2 1.1 Breast cancer
Breast cancer is the second most common type of cancer after lung cancer and the most common malignancy affecting women worldwide, increasing significantly since early 1970s (1). It is estimated that there will be 22,400 new cases of breast cancer and more than 5,300 female deaths from this disease in 2008 in Canada (2). One in nine
Canadian women develop breast cancer during their lifetimes, and 1 in 28 women are expected to die from it (2) causing a big challenge in medicine. Despite numerous studies on breast cancer, the exact mechanisms underlying the initiation, development, and progression of this disease are not fully understood.
1.1.1 Anatomy of human breast
The mature female breast is composed primarily of glandular tissue, fat, and connective tissue centered over the pectoralis major muscle of the chest wall. The glandular tissue consists of ducts and lobules and is surrounded by connective tissue.
Each lobule is a cluster of several acini and terminal ducts called a terminal duct lobular unit (TDLU). In the TDLU, milk is produced in the acini and the terminal ducts transport milk from the acini to the nipple. Both the acini and ducts are made of two layers: the outer layer (basal membrane side) is composed of myoepithelial cells and the inner layer
(luminal side) lined with epithelial cells. The epithelial cells are responsible for synthesis and production of milk in the mammary glands. More importantly, carcinoma appears to arise from these epithelial cells (3).
1.1.2 Carcinoma of the breast
3 Familial predisposition to breast cancer has been the focus of intense studies
resulting in the identification of two susceptibility genes; BRCA1 and BRCA2 (4-6).
However, the majority of breast cancer cases is sporadic and occurs in women without a
strong family history. This implies that other genes must be involved. Despite the
heterogeneity, the natural history of breast cancer is thought to involve a sequential
progression through defined clinical and pathological stages beginning probably as early
as normal epithelium, progressing to hyperplasia, duct carcinoma in situ (DCIS) followed
by invasive carcinoma (IDC) and culminating in metastatic disease (7) . Carcinomas are
the most common breast malignancy and can be classified as in situ or invasive
carcinomas.
1.1.3 Duct carcinoma in situ
Duct carcinoma in situ is characterized by a proliferation of presumably malignant epithelial cells within the mammary ductal-lobular system without light- microscopic evidence of invasion into the surrounding stroma. However, DCIS has been found not to be a single disease. Rather, it is a heterogeneous group of lesions (8). While
DCIS itself presents no risk of metastasis, a proportion of patients with DCIS will develop invasive recurrences. Accurate identification of this latter group would allow increased surveillance and more aggressive treatment. Certain pathological features have already been associated with an increased risk of invasive breast cancer recurrence. These include size, the presence of comedo necrosis, and the distance between the DCIS lesion and the periphery of the surgical specimen (width of resection margin) (9-17). However, the development of histological grading schemes for DCIS has been hampered by poor
4 reproducibility of pathologic reporting of DCIS and limited follow-up data (18).
1.1.4 Invasive and metastatic breast carcinoma
Although the majority of early-stage breast cancers (lymph node negative) are not
life threatening, a small proportion of cases will invade adjacent tissue and progress to
metastatic breast cancer. Invasive carcinoma is defined as the extension of cancer cells
beyond the basement membrane into the adjacent tissue. On microscopic examination, it
is frequently observed to extend directly from ducts containing DCIS. It seems that the
invasive behavior of primary breast tumor cells and their potential to metastasize to other
organs such as bones, liver, brain, and lungs is the major cause of death in patients with
breast cancer.
1.1.5 Breast cancer treatment
Many standard chemotherapeutic agents currently used to treat breast cancer are
relatively non-specific and act on all rapidly dividing cells. There is a desperate need for
new therapies to treat breast cancer as many cases fail to respond to current
chemotherapeutic agents. Specifically targeted therapies such as Trastuzumab
(Herceptin) and tamoxifen in HER2 and estrogen receptor (ER)-positive breast cancers respectively have been developed (19). However, despite the introduction of these specifically targeted therapies, special agents have not yet been developed for the treatment of ER-negative breast cancers. This has motivated considerable effort toward finding novel therapeutic approaches for the treatment of this group of breast cancers.
Since cell immortalization is a critical and rate-limiting step in cancer progression
5 (20), it has been shown that agents inhibiting cell immortalization can be considered as a
novel therapeutic approach for cancers. It is known that telomerase, the enzyme that
protects the ends of chromosomes, is active in many type of cancers including breast
cancers but inactive in adjacent normal tissues (21-23)). If it were possible to target cells
with active telomerase then this strategy could be used to treat breast cancer.
1.2 Telomere
The ends of chromosomes are capped by specialized nucleoprotein complexes
called telomeres. A telomere is a specialized structure, which enables cells to recognize
the ends of chromosomes from intrachromosomal double-stranded DNA breaks (DSBs)
that result from radiation, mechanical damage or chemical mutagens. By preventing
fusion of chromosomal ends and degradation of DNA, telomeres inhibit a DNA damage
cellular response and provide genomic stability (24). Telomeres consist of double-
stranded DNA containing a highly conserved G-rich repetition of a (TTAGGG)n
sequence, ending in a 150 to 200 nucleotide single-stranded overhang at the 3' end (G-
strand overhang) (25, 26). The length of telomeres varies between chromosomes and species with an average of 10 to 15 kb and 25 to 40 kb in human and mice respectively
(27-29).
1.2.1 Telomere Structure
Using electron microscopy analysis, Griffith et al. found that the ends of telomeres form a lasso structure by invasion of the 3' telomeric overhang into the duplex telomeric repeat array (29). This invasion creates a telomere loop (T-loop) and a small
6 three-stranded DNA displacement loop (D-loop) (29, 30). This structure is dynamic and
depends on a number of proteins associated with the telomere (31). This model can
explain the general mechanism to protect the chromosome ends from recognition by the
cellular DNA repair processes. However, there are other models such as recruiting
specialized proteins or a loop structure in which the 3' overhang is sequestered in the
interior of the loop but not embedded in the adjacent duplex that can also explain this
protection (32).
1.2.2 Telomere-associated proteins
The lasso structure of the telomere end critically depends on the recruitment of
telomere-associated proteins (known as sheltering) (33). These proteins play important roles in regulating telomere length, integrity, and function that can be classified into three
groups. In the first group, proteins directly bind to the single-stranded 3' G-rich overhang.
This includes the protein Protection of Telomere (POT1) (34, 35). The second group is implicated in double-stranded DNA. This group includes Telomere Repeats Factor 1 and
2 (TRF1, TRF2) (36). TRF1 is a negative regulator of telomere length homeostasis (37) and TRF2 is involved in the t-loop establishment (29). The third group consists of interconnecting proteins including TRF1-interacting protein 2 (TIN2) (38), Repressor
Activator Protein 1 (hRAPl) (39), tankyrase 1 (TANK1) (40) and 2 (TANK2) (41), and
TPP1 (42). In addition, there are other proteins recruited by TRF1 and TRF2 to the telomere that are known to participate in DNA repair including the RAD50-MRE11-
NBS1 (RMN) complex, ATM (43), Ku (DNA end binding subunit) (44) and DNA-PKcs
(catalytic subunit) (45).
7 1.2.3 Telomere Function
In a remarkable publication, Hayflick and Moorhead found that normal human
fibroblasts have finite life-spans in vitro (46). The mechanism underlying this observation
was not clear until Olovnikov et al. suggested that the cause of cellular senescence is the
gradual shortening of telomeres (47, 48). This shortening is mainly due to the inability of
DNA polymerase to replicate the end of the chromosome during lagging strand synthesis
and is known as the "end-replication problem" (49). During each cell division, telomeres
shorten by 50-100 bp until they become critically short inducing senescence (50). It has
been found that critically short telomeres make the ends of chromsomes unprotected
inducing DNA damage responses (51-54).
In addition, telomere shortening leads to the disruption of telomere structure
because of dysfunction or loss of telomere associated protein activity such as TRF2
inducing cell cycle arrest and activation of the ATM-p53 DNA damage-response
pathway (55-57).
1.2.4 The length of telomeres and chronic diseases
In humans, telomeres play a crucial role in the life of the cells by providing a
protective cap on chromosome ends and their shortening is associated with aging (58). A
correlation between telomere length in peripheral blood cells and increased mortality has
been documented in people over 60 years old, suggesting that telomere length could represent a prognostic marker of human aging and age related diseases such as an ATM- like protein deficiency in ataxia telangiectasia (58). However, no correlation has been found with survival in the older individuals (humans over 85 years), indicating that other
8 factors become dominant at very late age (59). In addition, accelerated telomere
shortening has been seen in a variety of chronic diseases that elevate the rates of cell
turnover (60), atherosclerosis and associated cardiovascular disease (61), chronic liver
diseases (62), chronic inflammatory bowel disease (63), chronic HIV infection (64),
different forms of anemia (65, 66), and Alzheimer's disease (67). Some studies have
shown that telomere shortening is linked to disease progression and the development of
chromosomal instability. This often complicates the end-stage of chronic disease and
possibly contributes to the increased rate of malignancies in old age (63, 68-72).
1.2.5 The length of telomeres in cancers
Genomic instability is widely accepted as a hallmark of cancer cells, which
contributes to the evolution of cancers. A line of evidence suggests a significant relationship between telomere maintenance and genomic instability (73). The role of
telomere alteration in tumorigenesis and cancer development has been studied and
established at the molecular level (73). It has been shown that excessively shortened or
dysfunctional telomeres result in end fusions and the onset of chromosomal instability
(CIN) through break-fusion-bridge cycles.
The length of telomeres are generally shorter in human tumors compared to their surrounding normal tissue (74-80). However, a few reports found elongation of telomeres in tumors compared to the adjacent normal tissues in a subset of patients (81-83).
1.2.6 The length of telomeres in breast cancer
The length of telomeres in breast cancer has been studied by several investigators.
9 They found that the length of telomeres in breast carcinomas are shorter than their length
in normal breast epithelial tissues (76, 79, 81, 84-87). However, the role of telomere
length as a prognostic factor and its correlation with clinical parameters is controversial.
For the first time, telomere length in breast cancer was published by Odigari et al.
in 1994 (84). Using Southern blot analysis to measure the length of telomeres, they
observed significant erosion in telomeric length in all breast tissues compared to placental
DNA. They also found a significant correlation between telomere shortening and
histologic grade in breast carcinoma. However, no correlation was found between the
length of telomere and HER2 gene amplification, tumor size, clinical stage, and steroid
receptors, as prognostic parameters in this study. The length of telomeres in breast cancer
was also investigated by other groups (85, 86). Rogalla et al. studied the length of
telomeres in 85 breast cancer samples (85). They noticed that the mean of telomeric
repeat fragment (TRF) length varied between samples. They also found no correlation between TRF length and age of patients, tumor size, lymph node status, and steroid receptor status. This group concluded that TRF size can not be considered as a limiting or a promoting factor for growth of breast cancer (85). In agreement with this finding,
Takubo et al. found that the degree of telomere reduction is not associated with breast cancer grade or the age of patients (86). The same observation was also reported by Rha et al. (88). Interestingly, Griffith et al. found a significant correlation between the content of telomere DNA, metastasis, and chromosomal aneuploidy in human breast cancer using slot-blot titration to measure telomere DNA content (87). This group suggested that telomere DNA content may have prognostic value, although they found no correlation between telomere DNA content and tumor size, grade, stage, fraction of cells in S-phase,
10 and the age of patients. It is important to note that all these investigators studied the
length of telomeres in bulk tissue, which is contaminated with variable amounts of
normal breast epithelial tissue. To avoid this problem, Meeker et al. used fluorescence in
situ hybridization (FISH) to measure the length of telomeres directly in ducts and lobules
of breast intraepithelial neoplasia (JEN) (77). They used a specific peptide nucleic acid
(PNA) probe to label telomeres and a second centromere specific PNA probe as a control.
Interestingly, unlike other investigators instead of applying both probes on the same
tissue section (74, 75, 79), they hybridized the tissue sections in parallel with either the
centromere or the telomere PNA probe in their study. Using this approach, they found
that the majority of DCIS samples contain markedly or moderately shortened telomeres.
More recently, Iwasaki et al. examined the role of telomere length of lymphocytes
in breast cancer susceptibility and radiosensitivity (89). They determined the length of
telomeres using a flow cytometric fluorescence in situ hybridization assay (FLOW-
FISH). Using this approach, no significant reduction in telomere length in cases was
found compared to controls. This observation led the authors to conclude that telomere
length does not appear to be predictive of acute skin reactions to radiotherapy (89).
More importantly, all these groups measured the average length of all telomeres in their studies. Whereas, it has been suggested that the length of the shortest telomere rather than average telomere length is the critical factor determining cell fate because the loss of telomere function may occur preferentially on chromosomes with critically short telomeres (90). However, little is known about the length of the shortest telomere and its role in the progression of cancers.
11 1.2.7 The length of telomeres in other types of cancer
It has been shown that the length of telomeres is significantly shorter in most
types of cancer (74, 75, 78, 82, 83, 91-100). However, the role of telomere length as a
prognostic factor has been found to be controversial. It has been shown that telomere
shortening is correlated with death and disease recurrence in patients with prostate cancer
(96, 97). However, no significant association was found between telomere shortening and nodal status, pathological grades, and the age of patients at diagnosis (96, 97). Similarly, other groups noticed a correlation between telomere shortening and poor prognosis in several other cancer types (75, 77, 78, 95).
In contrast, there is some evidence indicating that the length of telomeres is negatively correlated to poor prognosis. Using the terminal restriction fragment method,
Shirotani et al. discovered that the length of telomeres is reduced in the majority of small cell lung cancer samples, although a few cases showed elongated telomeres compared to control normal lungs (83). Interestingly, they found that longer telomeres are correlated to poor prognosis in colorectal carcinoma patients. This observation was supported by other groups finding that telomere length negatively correlated to clinical prognosis in various types of cancers (82, 98-100).
In general, these studies indicate that telomere shortening is a general observation in tumors compared to normal tissues. However, as it has been stated by these studies, the role of telomere length as a prognostic factor in solid cancers is controversial and the mechanisms underlying these differences remained to be understood.
1.2.8 Telomere maintenance
12 Progressive shortening of telomeres predisposes cancer cells to chromosomal
instability and genomic alteration early in cancer development (32). When telomeres
shorten to a certain threshold, an irreversible growth arrest known as replicative
senescence occurs through activation of p53 and Rb as DNA damage response pathways
(101, 102). In the absence of active DNA damage-response pathways, cells can
temporarily bypass this growth arrest and continue cell division and further telomere
shortening causing multiple chromosome end fusions and severe chromosomal
instability. These cells eventually reach a second proliferation block referred to as
"crisis", which is characterized by telomere dysfunction and cell death (103-107). To
escape from this "crisis" and to continue cell proliferation, cancer cells need to stabilize
their telomere length during cancer progression. To overcome this problem, most human
cancer cells stabilize their telomeres through activation of telomerase (108). However, it
has been shown that a small fraction of malignant cells maintain their telomere length
using a mechanism independent from telomerase activity known as alternative
lengthening of telomeres (ALT) (109). Using either of these two mechanisms leads to
cellular immortalization as a hallmark of human cancer.
1.3 Telomerase
Telomerase is a very large and complex ribonucleoprotein enzyme with an estimated mass over 600 kDa (110). In vitro, two components are essential for telomerase activity: human telomerase reverse transcriptase (hTERT) and the RNA template known as human telomerase RNA (hTR) (111). In humans, hTERT acts as the catalytic subunit of telomerase and tightly regulates the activity of this enzyme. However, many other
13 proteins are present in the telomerase complex to regulate the activity of telomerase (112-
119).
1.3.1 Regulation of hTERT
The mRNA expression level of hTERT has been shown to be the rate-limiting
step in telomerase activation. Because of its important role in telomerase activity,
extensive studies have been done to investigate the regulation of hTERT expression. The
results show that many factors can be involved in the regulation of hTERT expression
including MYC (120-123), estrogen receptor (124, 125), Ets (126), USF1/2 (127-129),
Spl(122, 130), p53 (131), Madl(132, 133), Rb (134, 135), andE2Fl (135). However, the
exact mechanism underlying the regulation of hTERT expression is not fully understood.
1.3.2 Regulation of hTR
hTR was the first telomerase component cloned in 1995 (136). hTR acts as a
template for the addition of telomeric repeat sequences (111). In its 5' exterimty, hTR contains a template region of 11 nucleotides 5'-CUAACCCUAAC-3' (136), whereas its 3' end, hTR has a classical small nucleolar RNA (snoRNA) structure called H/ACA motif
(137). H/ACA motif contains a special structure including a hairpin stem, a hinge sequence, a second hairpin stem, and an ACA sequence required for activity of hTR
(138). Mitcell et al. showed that two motifs within the independently stable H/ACA domain of hTR are required for the accumulation of the mature RNA in vivo (138).
The expression of hTR is tightly regulated at multiple levels by various mechanisms. The transcriptional regulation seems to be the main mechanism controlling
14 the expression of the hTR gene. A number of transcription factors including glucocorticoid, progesterone and androgen steroid hormone receptors, API, Ets, Spl,
Sp3, and Nuclear Factor-Y (NF-Y) have been found to regulate hTR expression. In addition, it has been demonstrated that pRb, MDM2, MAPK proteins also affect the expression of hTR (139).
1.3.3 Telomerase associated proteins
Telomerase associated proteins interact with the telomerase holoenzyme and are required for telomerase accumulation, activity, and function. These proteins include:
1.3.4 Human telomerase-associated protein 1
Telomerase-associated protein 1 (TEP1) is a component of the telomerase ribonucleoprotein complex (112). TEP1 was shown to interact specifically with mammalian telomerase RNA and catalyzes the addition of new telomeres on the chromosome ends. Northern blot analysis of both mouse and human tissues showed widespread expression of the gene, indicating that this component is unlikely to be linked with telomerase re-activation (140,141).
1.3.5 Heat shock protein 90 chaperone complex
The heat shock protein 90 (hsp90) chaperone complex is composed of at least hsp90, p23, hsp70, hsp40, and p60 (142). These proteins act as a foldosome mediating the assembly of a biologically active telomerase complex (143). It has been suggested that for the proper assembly of hTERT and hTR components in active telomerase, all five
15 chaperone proteins (hsp90, p23, hsp70, p60, and hsp40) are needed (113). Holt et al.
showed that hsp90 and p23 interact specifically with hTERT and influence its proper
assembly with hTR (113). They demonstrated that the dissociation of hsp90 and p23 with
hTERT blocks the assembly of functional telomerase in vitro and in vivo. In addition to
hsp90 and p23, it has been found that hsp70 chaperone also associates with hTERT in the
absence of hTR (144). In contrast to hsp90 and p23, which remain associated with
functional telomerase, hsp70 transiently associates with hTERT and dissociates from
hTERT when telomerase is folded into its active state. Similar to hsp70, hsp40 has been
found to interact with hTERT and supports the correct assembly of hTERT protein to
hTR resulting in active telomerase complex (144).
1.3.6 Dyskerin
Identified by Heiss et al. (145), it has been shown that dyskerin (DKC1) causes the X-linked recessive condition dyskeratosis congenita (DKC) and it has been predicted to be involved in the cell cycle and nucleolar function. In addition, primary dermal fibroblasts cultured from a DKC patient showed premature senescence with the presence of short telomeres (114). Further experiments also confirmed that defects in DKC patient cells arise solely from reduced accumulation of hTR (114). Accumulating data indicates that mutations in components of telomerase impair telomere maintenance and cause related human diseases such as the autosomal dominant form of dyskeratosis congenita.
As a consequence of hTR mutation, these patients show accelerated telomere shortening and a reduced lifespan (115, 146). These studies showed that changes in telomerase expression could influence human aging and disease. Furthermore, high turnover organs
16 such as the hematopoietic system or the intestinal epithelium can be easily affected by
telomerase dysfunction (147).
1.3.7 hStau
Human Staufen (hStau) is a double-stranded RNA-binding protein with multiple
double-stranded RNA-binding domains (148), which play a role in RNA transportation
and localization (149, 150). Le et al. showed that hSatu interacts with hTR in vivo and
can be considered as a telomerase-associated protein (116).
1.3.8 L22
L22 is another RNA-binding protein (151, 152), which has been found to interact
with hTR as a telomerase-associated protein (116).
1.3.9 La autoantigen
La protein has been found to interact directly with hTR and telomerase
ribonucleoprotein (RNP) and its expression level affects telomere length in vivo (117). As
a conserved RNA-binding phosphoprotein, La autoantigen is implicated in several
cellular and viral RNA-associated processes. The most ubiquitous function of La is its interaction with newly synthesized RNA polymerase (Pol) III transcripts. However, this protein is involved in other cellular processes such as RNP maturation, transcription termination, mRNA stabilization, and internal ribosome entry site-mediated translation in mammalian cells (153).
17 1.3.10 Heterogeneous nuclear ribonucleoproteins (hnRNPs)
hnRNPs were initially found to be associated with nuclear pre-mRNA. The
mammalian hnRNP proteins Al, CI, and C2 have been proposed to link the telomerase
holoenzyme to telomeres, potentially by bridging hTR and single-stranded DNA (118,
119).
1.3.11 Telomerase structure
At least 32 distinct proteins have been proposed to interact with human
telomerase. Interestingly, the size of the human telomerase complex was found to be
-650 to 670 kD (110), which is bigger than one hTERT (127 kD) and one hTR (153 kD)
and much smaller than the complex of all proposed associated proteins (-2.6 MD) (154,
155). Using mass spectrometric sequencing, Cohen et al. demonstrated that active
telomerase is composed of two copies each of hTERT, hTR and dyskerin proteins (110).
They concluded that other proteins are not required for nucleotide addition, nor do they
constitute integral components of the catalytically active enzyme complex. Rather, they may be involved in telomerase synthesis, trafficking, and its recruitment to the telomere.
1.3.12 Telomerase in cancer
Although telomerase has been found to be expressed in embryonic cells and in adult male germline cells (25, 156), its expression in normal somatic cells is not detectable except in proliferating cells in renewal tissues (157-159). In contrast to normal somatic cells, the vast majority of tumor cells express high levels of telomerase activity.
It has been found that telomerase is active in over 85% of human tumors and more than
18 90% of breast carcinomas in contrast to normal tissues where telomerase is inactive (21-
23). However, the role of telomerase in tumorigenesis is paradoxical (160). Although telomere shortening is an early event, telomerase activation has been found to be a late event in cancer development (161). Based on these finding and other observations, it has been suggested that although telomerase is not oncogenic (161), its activity is necessary for tumor progression (162). However, not all of the cancer cells maintain their telomere length by telomerase activity (163)
1.3.13 Alternative Lengthening of Telomeres
Approximately 10-15% of human tumors maintain their telomere length using a recombination-mediated alternative lengthening of telomeres (ALT) mechanism (163).
There is some evidence indicating that the ALT mechanism relies on the DNA recombination machinery (164). The length of telomeres in ALT positive cells is highly heterogeneous from very small to very long, suggesting that ALT telomeres are maintained by the DNA recombination machinery (165). This finding has been supported by the observation that tagged telomeres move onto other telomeres in an ALT cell line
(109). Furthermore, ALT cells contain ALT associated promyelocytic leukemia (PML) bodies (APBs). The APB is a unique nuclear structure containing telomeric DNA, telomere- associated proteins (166), and proteins involved in recombination and repair.
1.3.14 ALT in cancer
The ALT mechanism has been found commonly activated in tumors of neural
(astrocytomas) or mesenchymal origin (osteosarcomas and liposarcomas). However, this
19 mechanism is not common in epithelial tumors (167, 168). Although there is some
speculation, the mechanism underlying these differences remains to be understood (164).
1.3.15 Telomerase as a target for cancer treatment
Telomerase activation in the great majority of cancers (~ 85%) and its association
with cell immortalization has made it a promising target for anti-cancer therapy (296).
Targeting telomerase activity in cancerous cells, several successful methods have been
introduce including oligonucleotide-based therapeutics, nucleoside analogs and non-
nucleoside small molecules (169), immunotherapy (vaccines targeting telomerase) (170),
gene therapy (telomerase oncolytic virus), chemoprevention using pharmacological
agents, combination therapy, and agents that interact with quadruplex DNA (171, 172).
More recently, some data indicates that nuclear receptors such as estrogen receptor, Retinoid acid receptor, and peroxisome proliferator-activated receptor gamma
(PPARy) inhibit the activity of telomerase in some in vivo and in vitro models (173-175)
and may potentially be used for treating cancer patients.
1.4 Nuclear receptors
The nuclear receptor (NR) superfamily contains ligand-inducible transcription factors that are structurally related but functionally diverse. This family contains both nuclear hormone receptors (NHRs) and orphan nuclear receptors (ONRs) (176). NHRs are those receptors with identified hormonal ligands, whereas ONRs do not have known ligands, at least at the time the receptor is identified. The members of this superfamily are involved in metabolism, development, and reproduction by regulating the expression of
20 target genes.
More than 100 nuclear receptors are known to exist including estrogen receptor
(ER), progesterone receptor (PR), androgen receptor (AR), glucocorticoid receptor (GR),
mineralocorticoid receptor (MR), thyroid hormone receptor (TR), all-trans retinoic acid
receptor (RAR), 9-cis retinoic acid receptor (RXR), vitamin D3 receptor (VDR), and
peroxisome proliferator-activated receptors (PPARs) (177). Together, these proteins
comprise the single largest family of metazoan transcription factors, the nuclear receptor
superfamily.
1.4.1 Peroxisome proliferator-activated receptors
Peroxisome proliferator-activated receptors are members of the nuclear hormone
receptor superfamily that includes several ligand-activated transcription factors involved
in a variety of physiological and pathological processes (178). PPARs were originally
named for their ability to induce hepatic peroxisome proliferation in mice in response to xenobiotic stimuli (179). There are three known subtypes of PPARs, PPARa (180),
PPARp/5 (181), and PPARy (182). The subfamily members of PPARs are encoded by separate genes located on different chromosomes and share 60%-80% homology in their ligand-and DNA-binding domains (183).
1.4.2 Peroxisome proliferator-activated receptor a
Murine PPARa was the first member of this nuclear receptor subclass to be cloned (184) followed by cloning of human PPARa in 1993 (180). Human PPARa was found to be highly expressed in organs that carry out significant catabolism of fatty acids
21 such as liver, kidney, heart, skeletal muscle, brown adipose tissue, and intestine (185-
187), in addition to its expression in monocytic (188), vascular endothelial (189), and
vascular smooth muscle cells (190). Activation of PPARa has been reported to improve
levels of triglycerides, high density lipoprotein (HDL), and the overall atherogenic
plasma lipid profile (191-198).
1.4.3 Peroxisome proliferator-activated receptor (3/8
Human PPAR.p/5 was identified by cDNA cloning from an oesteosarcoma cell
library (181) and it is widely and often abundantly expressed in brain, adipose tissue, and
skin (186, 187, 199, 200). PPARp78 has an important role in adipose tissue metabolism
and weight control by enhancing fatty acid catabolism and energy uncoupling in adipose
tissue and muscle. It also suppresses macrophage-derived inflammation (201). Compared
to the other subfamily members of PPARs, PPARJ3/8 has been studied the least mainly because of its wide tissue expression pattern, which raised early speculation that it may
serve for general housekeeping (202).
1.4.4 Peroxisome proliferator-activated receptor y
PPARy was cloned independently as a new member of the PPAR subgroup of nuclear hormone receptors and as a transcriptional regulator of fat-specific gene expression (182, 192, 203). The PPARy gene can be translated into four different PPARy proteins, PPARyl, PPARy2, PPARy3, and PPARy4 by alternative promoters and RNA splicing (185, 204). The PPARy gene spans more than 100 kb containing 9 exons (exon
Al, exon A2, exon B, and exons 1-6). PPARyl includes exon Al, A2, and exon 1-6.
22 PPARy2 contains exon B plus exon 1-6, whereas PPARy3 consists of exon A2 and exon
1-6. PPARy4 contains only exon 1-6. Since exons Al and A2 are untranslated, therefore
PPARyl, PPARy3, and PPARy4 genes code the same protein. In contrat, PPARy2 has an
additional 28 amino adics at its amino terminus as the result of exon B translation (182).
1.4.5 PPARy structure
As with other NRs, all four PPARys have similar structures consisting of the
(A/B) N-terminal region, the DNA binding domain (DBD), the flexible hinge region, the
ligand binding domain (LBD), the transactivation domain, and the C-terminal domain
(205). The DBD is the most conserved domain between all NRs and facilitates the interaction between PPARy protein and DNA. This domain contains two zinc finger motifs that allow NRs to bind to specific sequences of DNA known as hormone response elements (HRE) within the promoters of target genes. It has been shown that the precise sequence of the HRE, its orientation (direct repeat, palindromic, or inverted palindromic sequences), and the spacing between core recognition motifs are the features that regulate the specificity of DNA recognition by a particular set of NRs (206). Based on these observations and the interaction modes with HREs, NRs can be classified in three groups as monomer, homodimer, and heterodimer receptors. Monomer receptors such as NGFIb contain a single receptor motif. Homodimers such as ER and heterodimers such as TR,
VDR, RAR, and PPARs typically consist of two core recognition motifs within the promoters of target genes (207-214). PPARs have been found to form a heterodimer with the retinoic X receptor a (RXRa) as the DNA-binding partner and preferentially recognize the core of two direct hexanucleotide repeats (AGGTCA)2 with 1 bp spacing
(DR1). In addition, the transcriptional activation of PPARy depends on the activation
23 function (AF) domain, located at the N-terminal of PPARy. AF-1 can be phosphorylated
by kinases and activates PPARy in the absence of ligands. In contrast, AF-2 is a ligand-
dependent transcription domain located at the C-terminal of PPARy. AF-2 is a short
conserved helical sequence, which recruits co-activators to induce the transcriptional
activity of the ligand-bound PPARy/RXRoc heterodimer (215).
1.4.6 Co-factors
The rate of gene transcription is dependent on chromatin remodeling and hi stone
acetylation in eukaryotes. Histone acetyltransferases (HATs) give rise to hyperacetylated
regions with a loose chromatin structure that can be highly transcribed. In contrast, the
regions, which are hypoacetylated by histone deacetylases (HDACs) are highly
condensed and cannot be transcribed. It has been demonstrated that PPARy regulates the
expression of target genes by recruiting a large number of co-factors containing either
HAT or HDAC activity (216). These co-factors can be divided into two groups called co-
activators and co-repressors.
1.4.7 Co-activators
A diverse group of proteins have emerged as potential co-activators for PPARy such as PGC-la, the SWI/SNF chromatin remodeling complex,
TRAP220/DRIP205/PBP, PRIP/NRC/RAP250/TRBP, CBP/p300, and the members of the pl60 family (RC-1/NCoAl, TIF2/GRIP1/NCOA2/SRC-2, and pCIP/ACTR/AIBl/SRC-3) (216). These co-activators have different roles in induction of transcriptional activity of PPARy. Some of the well known co-activators of PPARy such
24 as CBP/p300, and SRC-1 have HAT activity, whereas the other group forms a bridge
between PPARy and the transcription initiation machinery. This group includes members
of the DRIP/TRAP complex such as PPAR binding protein (PBP)/TRAP220, (216).
1.4.8 Co-repressors
Published data suggests that PPARy can act as a repressor by recruiting co-
repressors such as RIP140 (217), SMRT, and NCoR (218) in the absence of ligand or in
the presence of certain antagonists. Compared to co-activators, little is known about co-
reppressors and their roles in the regulation of PPARy transcriptional activity due to the
weak repressor activity of PPARy (216).
1.4.9 PPARy ligands
PPARy receptor can be activated by several types of ligands. These ligands can be
divided in three classes; natural ligands, dual agonists, and synthetic ligands.
1.4.10 Natural ligands
Natural ligands are lipophilic agents that activate PPARy at physiologic levels.
This group of ligands include fatty acids and eicosanoids (219), components of oxidized low-density lipoproteins (220), and oxidized alkyl phospholipids including lysophosphatidic acid (221), and nitrolinoleic acid (222). Among natural ligands, cyclopentone prostaglandin (15-deoxy-A 12, 14-prostaglandin J2) is the most potent and commonly used endogenous PPARy agonist (223).
25 1.4.11 Dual agonists
Dual functional agonists are a new class of ligands that activate both PPARy and
PPARa simultaneously. Having synergetic effects, dual PPARy/PPARa ligands have
been designed to improve anti-diabetic and cardio-protective effects by reducing
triglycerides and raising HDL as the cardioprotective factor. This group includes
naveglitazar, netoglitazone, muraglitazar, ragaglitazar, tesaglitazar, and imiglitazar (224).
1.4.12 Synthetic ligands
The synthetic ligands are the members of the thiazolidinedione (TZD) family
including troglitazone, rosiglitazone, pioglitazone, ciglitazone (225). TZDs are also
known as insulin sensitizers used in the treatment of type 2 diabetes. This group was
originally developed without knowledge of their mechanisms of action. It is now
recognized that TZDs are pharmacological PPARy ligands (226). It also has been found
that TZDs promote the differentiation of various cell lines (227-231). Furthermore, it has
been shown that some TZDs, especially troglitazone and ciglitazone, demonstrate
antiproliferative activities in several cancer models including breast cancer (232).
However, there is some evidence indicating an increase in the frequency of tumors in mice treated with synthetic PPARy agonists (233). Interestingly, accumulating data
suggests that many biological effects of TZDs on cell processes are independent from
PPARy transcriptional activity (234-237). Therefore, it becomes very important to understand the clinical and/or biological roles of PPARy and its agonists in the progression or suppression of malignancies in order to assess a potential therapy for cancer patients.
26 1.4.13 PPARy function
PPARy plays an important role in regulation of dyslipidemia, type 2 diabetes,
inflammation, and various cancers.
1.4.14 The Role of PPARy in lipid metabolism
PPARy has been identified as a necessary and sufficient transcription factor for
the differentiation of adipocytes. Tontonoz et al. showed that the introduction of PPARy
into fibroblasts in the presence of PPARy ligands induces the differentiation of the cells
into adipocytes (238). Furthermore, it has been shown that ectopic expression of
dominant-negative PPARy mutants can inhibit the differentiation of 3T3-L1 cells into
adipocytes even in the presence of ligands (239, 240). Using animal models, the role of
PPARy in lipid metabolism has been further investigated. A line of evidence showed that
PPARy knocked out mice were extremely lipodystrophic (241), whereas PPARy
heterozygous null mice exhibited reduced amounts of adipose tissue (241-243). As a lipid
regulator, PPARy controls the expression of numerous genes involved in lipid
metabolism such as fat-specific adipocyte P2 (aP2), lipoprotein lipase (LPL), fatty-acid transport protein (FATP), acetyl-CoA synthetase (ACS), phosphoenolpyruvate carboxykinase, glycerol kinase, glycerol transporter aquaporin 7, and oxidized low density lipoprotein (LDL) receptor 1. In addition, PPARy plays an important role in the regulation of energy homeostasis by inducing the expression of uncoupling proteins 1, 2 and 3 (UCP1, UCP2 and UCP 3) genes as mediators of thermogenesis (244, 245).
1.4.15 The Role of PPARy in type 2 diabetes
27 PPARy is involved in the regulation of cellular glucose uptake by increasing
insulin sensitivity and decreasing insulin resistance in adipose tissue, skeletal muscle, and
liver. PPARy activation by TZDs improves insulin sensitivity and effectively reduces
hyperglycemia in patients with diabetes. TZDs predominantly decrease hemoglobin Aic
and fasting glucose concentrations by inhibiting lipotoxicity-induced insulin resistance.
This effect of PPARy especially in pancreatic P cells inhibits the apoptosis of p cells and
increases the mass of (3 cells, which in turn results in both diabetes prevention and
treatment (246, 247).
Using troglitazone in animal models, Miles et al. found a significant reduction of
free fatty acid (EFA) concentration in rat blood (248). They showed that stimulated
insulin receptor autophosphorylation in the animal muscles resulted in the elevation of
glucose uptake (248). In addition, they discovered that PPARy suppresses the expression
of tumor necrosis factor a (TNFa), a pro-inflammatory cytokine, in TNFoc-induced
insulin resistance (248). Activation of PPARy also stimulates the expression of c-CBL-
associated protein (CAP) in cultured adipocytes (249). CAP contains a functional PPRE within the 5' regulatory region of its gene and appears to play a positive role in the insulin
signaling pathway (250). Expression of insulin receptor substrate-2 (IRS-2), a protein with a proven role in insulin signal transduction in insulin-sensitive tissue, was also increased in differentiated 3T3-L1 adipocytes and cultured human adipose tissue treated with pioglitazone (251). Furthermore, it has been found that pioglitazone had no effects on IRS-1, PKB/Akt, or glucose transporter-4 (GLUT4) gene expression (251). In contrast, Zhang et al. observed a significant reduction in insulin sensitivity with dramatically decreased expression of IRS-1 in skeletal muscle, liver, and white adipose
28 tissue (WAT) and a significant decrease of GLUT4 in the skeletal muscle in male
PPARy2~'~ mice (252). Surprisingly, they found that the impairment of insulin sensitivity
was dramatically improved in PPARy2~'~ mice treated with rosiglitazone (252).
Recent data also showed that ciglitazone increased insulin-stimulated glucose
uptake in the cardiomyocytes of rats (253). This finding was associated with an increase
in Akt phosphorylation, an important protein in downstream insulin signaling regulating
glucose transportation. In agreement with these findings, it has been shown that PPARy
inhibition by siRNA in rat vascular smooth muscle cells (VSMCs) significantly
suppresses the phosphorylation of insulin-stimulated receptor (IR)-beta, Akt, and
glycogen synthase kinase 3-beta (GSK3|3) with an increase in the expression of
phosphotyrosine phosphatase (PTP)-IB (254).
1.4.16 The Role of PPARy in cancers
PPARy has been suggested to be a tumor suppressor gene based on several
observations (255). PPARy was mapped to chromosome 3p25 (256), a region with
detected abnormalities in a variety of human cancers (257-259), suggesting the genes
located in this region may function as tumor suppressor genes. In agreement with this
suggestion, Mueller et al. found that of 21 patients with prostate cancer, 8 (40%) of them had hemizygous deletions of the PPARy gene (260). Furthermore, it has been shown that colon cancer in humans is associated with loss-of-function mutations in PPARy (261).
In contrast, other studies identified PPARy as an oncogene. To study the role of
PPARy in breast cancer development, Saez et al. constructed a constitutively active
PPARy by fusing the activation domain of the herpes simplex virus Vpl6 protein to the
29 PPARyl isoform (262). Using this construct, they generated new transgenic mice
expressing the active form of PPARY under the control of the mouse mammary tumor
virus (MMTV) promoter. When these transgenic mice were bred to the MMTV-PyV
strain (a strain prone to mammary gland cancer), bigenic animals developed tumors with
greatly accelerated kinetics compared to their control matched groups. Based on these
observations, the authors concluded that increased activity of PPARy serves as a tumor
promoter in the mammary gland (262). In addition, it has been found that the treatment of
APC deficient mice with various TZDs unexpectedly increase the number of colon
tumors in these animals (263, 264).
Finally, there is some evidence indicating neither a tumor suppressor nor an
oncogenic role for PPARy in the progression of cancers. Using mutational analysis,
Ikezoe et at. showed that PPARy is expressed in most cancers, and its mutation is a very rare event in a variety of human cancer samples and human cancer cell lines (326 clinical cancer samples and 71 cancer cell lines) (265).
Taken together, these results indicate that the exact role of PPARy in cancer development is not clearly understood and needs more investigation.
1.4.17 PPARy and breast cancer
Human primary and metastatic breast adenocarcinomas express high levels of
PPARy (228, 265, 266). It is also found that many breast cancer cell lines including
MDA-MB-231, MDA-MB-436, MCF-7, and T47D express PPARy protein (265, 267).
However, no PPARy mutations or deletions were detected in clinical breast cancer samples and cell lines (265). For the first time in 1998, Mueller et al. showed that the
30 21PT breast cancer cell line can undergo significant morphologic changes following exposure to various types of PPARy ligands (228). They also found that these morphological changes are associated with the increased expression of maspin (a tumor suppressor gene and a marker of normal breast development). Furthermore, they showed that PPARy ligands suppress the expression of mucin-1 (Muc-1) and keratin 19 (K19) genes, two markers associated with a more malignant state of breast epithelial cells. It was also revealed that PPARy ligands arrest the growth of 21PT cells after long-term treatment. Based on these observations, the authors concluded that the cell growth arrest seems to be the consequence of cellular differentiation that occurs over several days.
However, not all of the breast cancer cell lines responded to the PPARy ligands in their study. Despite its high level expression of PPARy mRNA, 21MT breast cancer cells were not differentiated in response to ligand treatment (228). Further investigations by other groups showed that PPARy ligands especially TZD family members suppress the proliferation of not only breast cancer but also many other type of cancers (230, 231,
268-276).
Interestingly, it has been shown that that TZDs target genes involved in cell cycle regulation, apoptosis, and DNA damage response more than those regulating lipid metabolism and differentiation (268).
1.4.18 The effects of PPARy ligands on cell cycle regulation
The mammalian cell cycle consists of Gl, S, G2, and M phases. Cell cycle progression is regulated by members of the cyclin-dependent kinase (CDK) and cyclin families. Activated cyclin/CDK complexes phosphorylate and inactivate retinoblastoma
31 protein (Rb). Hyperphosphorylated Rb (pRb) induces the expression of E2F-regulated
genes such as cyclin E, and cyclin A leading to induction of cell proliferation (277, 278).
The activity of cyclin/CDK complexes can be inhibited by two families of CDK
inhibitors (CDKIs); the cyclin inhibitor protein/kinase inhibitor protein (CIP/KIP) family
and INK4 inhibitors (279). CIP/KIP family induces p21CIF1/WA]F\ p27Kn>1, and p57KIP2
inhibiting both Gi- and S-phase transitions (280). Whereas, the INK4 inhibitors suppress
the Gi-phase cyclin/Cdk complexes by induction of pie11™*, plS™ plS^40, and
pl9iNK4dproteins(281)
It has been shown that troglitazone inhibits MCF-7 cell proliferation by blocking
events critical for Gl—>S progression (282). Using the same breast cancer cell line, MCF-
7, Wang et al. found that progression of the cell cycle can be inhibited by 15d-PGJ2
through direct repression of cyclin Dl gene expression (283). To determine whether the regulation of cyclin Dl was PPARy receptor dependent, this group also showed that both
15d-PGJ2 natural and synthetic ligands (troglitazone and BRL49653) inhibit the cyclin
Dl promoter in PPARy-deficient HeLa cells expressing ectopic PPARy protein.
However, the involvement of PPARy in the inhibition of cyclin Dl was not shown directly in MCF-7 cells in this study (283).
Conversely, Huang et al. found that ablation of cyclin Dl in the MCF-7 cell line by troglitazone and ciglitazone is independent of PPARy transcriptional activity (234).
Interestingly, another group found that high doses of troglitazone (50/^M) selectively suppress cyclin D3 expression by a PPARy-independent mechanism in MDA-MB-231 cells without affecting other D-type cyclins and cell cycle regulators (cyclin E, p27, and p21) (284). In contrast, treatment of MCF-7 cells with rosiglitazone was found to induce
32 UM/WA1 1 mRNA and protein levels of the tumor suppressor p53 and p2i ' in a PPARy-
dependent mechanism (285). Taken together, this evidence indicates that the biological
effects of PPARy ligands on cell cycle regulation are controversial and can be dependent
or independent of PPARy activity.
1.4.19 The effects of PPARy ligands on apoptosis
TZDs induce apoptosis in various cancer cell lines by different mechanisms (286-
290). It was shown that high concentrations of troglitazone decrease the level of anti-
apoptotic bcl-2 protein in MCF-7 cells (290). Bae et al. found that troglitazone increases
the level of pro-apoptotic proteins such as Bad and Bax in HepG2 hepatoma cell lines
(291). It was also revealed that troglitazone is able to induce changes in Bcl-2 members in the prostate cancer PC3 cell line (288). Using human thyroid carcinoma cell line
BHP18-21, Ohta et al. demonstrated that 10/xM troglitazone can induce DNA fragmentation in BHP18-21 cultured in 0.1% fetal calf serum (FCS) (292). However, this group failed to detect any changes in the expression level of bcl-2 and bax genes in
BHP18-21 cells in response to troglitazone treatment.
In general, published data indicates an apoptosis response to high concentrations of troglitazone. However, this response was not observed at low concentrations of troglitazone. More importantly, regardless of the concentration of troglitazone used, all groups showed that induction of apoptosis was independent from PPARy activity (286-
290, 292).
1.5 Rationale
33 Breast cancer is one the highest causes of female death worldwide (1). The
development of breast cancer involves a sequential progression through defined clinical
and pathological stages beginning with normal epithelium and progressing to hyperplasia,
DCIS, IDC, and culminating in metastatic disease (7).
In humans, telomeres play a crucial role in the life of the cells by providing a
protective cap on chromosome ends (24). Furthermore, telomere alteration in
tumorigenesis and cancer development has been studied and established at the molecular
level (73). A line of evidence suggests a significant correlation between telomere length
and genomic instability in cancers (293). Excessively shortened or dysfunctional
telomeres have been shown to cause end fusions and onset of chromosomal instability
through break-fusion-bridge cycles (293). It has been shown that telomere lengths are
shorter in invasive breast cancer compared to normal breast epithelial tissue (76-79). In
addition, several other studies also identified telomere shortening as a common
observation in various types of cancers compared to their matched normal tissues (74, 75,
80, 91-94, 96, 97). However, the role of telomere length as a prognostic factor and its
correlation with clinical prognostic parameters is controversial. This can be partially
explained by the fact that all research groups measured the average length of total
telomeres in their studies, and no studies were conducted to investigate the lengths of individual telomeres and their correlation with prognostic parameters in cancer samples.
It has been shown that genetic alterations on certain chromosomes are more frequent compared to others (294-303). This suggests that the length of telomeres on these specific chromosomes may play an important role in the progression of cancers.
Furthermore, it has been suggested that the length of the shortest telomere is the critical
34 factor determining cell fate rather than the average of all telomere length (90), supporting
the idea that some individual telomere lengths may have specific roles in the
development of cancers. Numerous studies have demonstrated that genetic abnormalities
such as loss of heterozygosity (LOH) on chromosome 17 in DCIS is as high as 60% to
80% in some studies (297-303), suggesting that genes located on this chromosome may
be particularly important in the development of DCIS. In agreement with this idea, it has
been shown that a frequently amplified gene in IDC and DCIS is HER2 located on
chromosome 17q (19, 304). Additionally, since DCIS is believed to be an intermediate
stage between normal and invasive breast cancer, we were interested to study the length
of telomere on chromosome 17q in normal breast epithelium, DCIS and IDC samples.
Our aim was to determine whether shorter chromosome 17q telomeric lengths are
associated with an increased risk of invasive recurrence. The results of the present
investigation may allow us to predict which DCIS patients have a risk of future invasion.
Furthermore, the progressive shortening of telomeres with each cell division has been shown to limit replicative life span of human cells in culture (305). Most human cancer cells maintain their telomeres through activation of telomerase (108). In over 90% of breast carcinomas and 85% of other types of human tumors telomerase is active (21,
306, 307). In contrast, normal tissues have low or undetectable levels of telomerase activity. Telomerase is a very large complex ribonucleoprotein enzyme that maintains the length of the telomeres (110). It has been shown that inhibition of telomerase limits the growth of human cancer cells (108). Consequently, various anti-telomerase strategies are currently under investigation to limit tumor proliferation in cancer patients. This is even more important in breast cancer, since the current available therapy options are not
35 effective in all breast cancer patients. Standard chemotherapeutic agents currently used to
treat breast cancer are relatively non-specific and act on all rapidly dividing cells. In
recent years, more specifically targeted therapies have been introduced. While tamoxifen
and Herceptin have been successfully used to treat ER-positive and HER2 over-
expressing breast cancers respectively, few special cancer prevention and treatment
strategies are available for ER-negative breast carcinomas (308). This has motivated
considerable efforts toward finding novel therapeutic approaches for the treatment of ER-
negative breast cancer.
Resent evidence showed that PPARy can inhibit telomerase activity in some
primary and cancer cells (173-175). However, whether PPARy exerts anti-telomerase
activity in breast cancer models has not yet been studied. PPARy is a member of the
nuclear hormone receptor superfamily that includes several ligand-activated transcription
factors involved in a variety of physiological and pathological processes (178). Upon
activation by its ligands, PPARy regulates gene expression by binding to specific peroxisome proliferator response elements as a heterodimer with retinoid X receptor in the enhancer sites of target genes (309). Several studies confirmed an anti-tumor role for
PPARy in various cancers (228, 230, 231, 260, 270, 276, 310). It has been shown that synthetic PPARy ligands, especially troglitazone and ciglitazone, demonstrate antiproliferative activities, in addition to their role in the induction of cell differentiation, in several cancer models including breast cancer (227, 228, 230, 231, 311, 312).
However, the molecular mechanism(s) responsible for this antiproliferative effect is not well understood.
Because of the growing potential roles of PPARy ligands as novel anti-cancer
36 therapies, we investigated the effect of the classical PPARy ligand troglitazone on
telomerase activity and cell cycle regulation in an ER-negative breast cancer model. To
our knowledge, this is the first time that the effect of troglitazone on telomerase activity
in ER-negative breast cancer has been investigated. We believe that the PPARy ligand
plays a critical role in this process. The objective of our study is to investigate the effect
of drugs (which interact with PPARy) particularly on telomerase but also by using
microarray analysis to identify and better understand the other genes involved in this
pathway. These results may lead to novel therapeutic mechanisms that could be used to
treat ER-negative breast cancer.
1.6 Hypothesis
This study is based on the hypothesis that telomere length and its control by the nuclear hormone receptor PPARy plays an important role in the development and progression of breast cancer.
1.7 Specific aims
Specific amis are:
1. To compare the length of telomeres on chromosome 17q in pure DCIS with DCIS
associated with IDC.
2. To investigate the role of troglitazone in regulating telomerase activity in an in
vitro model.
3. To assess the gene expression profile of established cell lines in response to
troglitazone treatment using RNA expression microarray analysis.
37 Chapter 2
This chapter has been published as "Telomere length on chromosome 17q shortens more
than global telomere length in the development of breast cancer"
Rashid-Kolvear F, Pintilie M, and Done SJ., Neoplasia. 2007 Apr;9(4): 265-70.
Statistical analysis was performed Pintilie M.
38 Telomere length on chromosome 17q shortens
more than global telomere length in the
development of breast cancer
39 2.1 Abstract
It is known that total telomere length is shorter in invasive breast cancer than in
normal breast tissue but the status of individual telomere lengths has not been studied.
Part of the difficulty is that usually telomere length in interphase cells is measured on all
chromosomes together. In this study, we compared normal breast epithelium, duct
carcinoma in situ (DCIS), and invasive duct carcinoma (IDC) from 18 patients. Telomere
length was specifically measured on chromosome 17q and in DCIS and IDC was found to
be shorter than in normal breast epithelial cells with more heterogeneity in telomere
length in DCIS associated with IDC than in DCIS alone. More importantly, we found that
the shortening of the telomere on chromosome 17q is greater than the average shortening
of all telomeres. This finding indicates that telomere shortening is not simply the result of
the end replication problem; otherwise all telomeres should be subjected to the same rate
of telomere shortening. It seems there are mechanisms that preferentially erode some
telomeres more than others or preferentially protect some chromosome ends. Our results
suggest that the increased level of telomere shortening on chromosome 17q may be involved in chromosome instability and the progression of DCIS.
40 2.2 Introduction
Breast cancer is one of the most common cancers afflicting North American
women. Although the majority of early-stage breast cancers are not life threatening, a
small proportion of cases will progress to metastatic breast cancer. Invasive duct cancer is
frequently observed to extend directly from ducts containing duct carcinoma in situ.
However, not all cases of DCIS develop into invasive tumors. Molecular markers hold
the promise of becoming clinically useful diagnostic tools, particularly markers which
can be studied in formalin-fixed paraffin-embedded (FFPE) tissue samples as virtually all
DCIS is processed in this way for routine pathological diagnosis. As telomeres are
involved in maintenance of chromosomal stability they represent one group of markers of
particular interest.
The telomere is a specialized structure at the end of chromosomes consisting of a
highly conserved repetitive DNA sequence, (TTAGGG)n (25). Telomeres form caps on
the ends of chromosomes that prevent fusion of chromosomal ends and provide genomic
stability. In normal somatic cells, telomeres are progressively shortened with every cell
division. This shortening in normal human cells limits the number of cell divisions. For
human cells to proliferate beyond the senescence checkpoint, they need to stabilize
telomere length. This is accomplished mainly by reactivation of telomerase (313).
Telomerase expression is under the control of many factors (121, 122, 128, 314-319).
Expression of telomerase can lead to cell immortalization and it is activated during tumorigenesis (21).
Using various approaches, it has been shown that telomere length in DCIS is generally shorter than in normal breast epithelial cells with inconsistent results with
41 respect to associations between tumor telomere length and clinicopathological features
(76, 81, 84, 85). However, in other cancer types a correlation between longer telomeres
and more aggressive behavior of cancer cells has been found (82, 320). While all these
groups were measured the average length of pantelomeres in their studies, it has been
reported that it may not be the average but rather the shortest telomeres that constitute
telomere dysfunction and limit cellular survival in the absence of telomerase (90). It has
been suggested that loss of telomere function occurs preferentially on chromosomes with
critically short telomeres (90). This suggestion indicates that certain telomeres may have
important roles in the development of cancers.
Numerous studies have demonstrated genetic abnormalities on chromosome 17 in
DCIS with rates approaching 60 to 80% (85, 298, 303), suggesting that oncogenes and
tumor suppressor genes in these regions may be particularly important in the
development of DCIS. A frequently amplified gene in IDC and DCIS is the HER2
(human epidermal growth factor receptor-2) oncogene located on chromosome 17q.
HER2 is a member of a family of transmembrane receptor tyrosine kinases with no identified ligand. HER2 is overexpressed and amplified in 20-30% of IDC and up to 80% of DCIS. Many groups have reported a correlation between HER2 amplification and poor prognosis (19, 304). Despite much interest, the mechanism of HER2 amplification/overexpression in many cases is unclear. We hypothesized that telomere erosion on chromosome 17q could lead to instability of chromosome 17q and this may be associated with HER2 amplification/overexpression.
In this study, we developed a technique to allow measurement of specific telomeres in routinely processed clinical samples. This allowed us to measure telomere
42 length specifically on chromosome 17q in formalin-fixed paraffin-embedded samples.
We also investigated the relationship between HER2 expression and telomere shortening
on chromosome 17q.
2.3 Materials and Methods
2.3.1 Sample collection
Breast tissue was obtained from archival formalin-fixed paraffin-embedded
(FFPE) blocks that were stored in the Pathology Department of University Health
Network and had been obtained initially for diagnosis. Institutional Research Ethics
Board approved was obtained for the study.
2.3.2 Quantitative fluorescence in situ hybridization (Q-FISH)
To measure the relative changes in telomere length on chromosome 17q in breast
epithelium, Q-FISH was used. Metaphase slides prepared from normal peripheral blood
lymphocytes (Vysis Inc., Downers Grove, IL, USA) were used to optimize Q-FISH
conditions for two different probes, a PNA (CCCTAA)3 pan-telomere probe-FITC from
Applied Biosystems (Bedford, MA, USA) and a specific subtelomeric DNA probe for
chromosome 17q (TelVysion 17q SpectrumOrange) from Vysis Inc. (Downers Grove, IL,
USA) (Fig. 2.1 A). Since the (CCCTAA) 3 PNA probe is not able to distinguish the
parental telomeres from each other, all the telomere signals are the average of two
homologous chromosomes in our report. We also used the specific subtelomeric DNA probe for chromosome 17q (TelVysion 17q SpectrumOrange) as a guide to locate
43 chromosome 17q telomeres. Using these two probes, we were able to detect strong
signals from telomeres and the chromosome 17q subtelomeric region simultaneously
(Fig. 2.1B-D).
To measure the telomere length in our study, five-urn thick FFPE breast cancer
tissue samples on positively charged glass slides were deparaffinized with xylene,
washed with 100% ethanol and air dried followed by RNase A treatment. Slides were
then placed in 1M NaSCN at 80°C and washed in water at room temperature. After
pepsin digestion, slides were thoroughly washed with water followed by dehydration in
serial ethanol dilutions (70%, 90%, and 100%) at room temperature and air dried. The
sections and the PNA pan-telomere probe-FITC were then co-denatured at 80°C for 5 minutes in a Hybrite programmable heating block (Vysis Inc., Downers Grove, IL) followed by incubation in a dark humidified chamber for 90 minutes at 25°C. Then slides were hybridized with a denatured specific chromosome 17q probe mixture (TelVysion
17q SpectrumOrange) and incubated overnight at 33°C. Slides were washed in
2xSSC/0.3%NP-40 at 73°C, 2xSSC/0.3%NP-40, and 2xSSC at room temperature followed by dehydration in serial ethanol dilutions and air dried. Finally, the slides were counterstained with 4', 6 diamidino-2-phenylindole (DAPI) from Vector Laboratories,
Inc. (Burlingame, CA) and viewed with a fluorescence microscope (Leica DMRA2).
2.3.3 Image capturing and analyzing
Using Hematoxylin and Eosin (H&E) staining, the normal breast epithelium,
DCIS and invasive areas in each tissue section were identified (Fig. 2.2A). Then parallel sections were studied for the Q-FISH analysis (Fig. 2.2B). The telomeres (Fig. 2.2C) and
44 sub-telomeric chromosome 17q (Fig. 2.2D) signals were visualized as gray in the
outlined nuclei. In this study, we measured the length of telomere in normal breast
epithelium (n= 8), DCIS (n= 14), and IDC (n= 12) from 18 patients. For each type of
tissue (normal breast epithelium, DCIS and IDC) a minimum of 20 nuclei in five
different areas were scored for the following variables: the total telomere length
(expressed as intensity), the telomere length at the end of chromosome 17q (expressed as
intensity) and subtelomeric chromosome 17q (expressed as intensity).
Images were captured using Openlab 4.0.3d8 software (Improvision, Viscount
Centre II, University of Warwick Science Park, Coventry, England,
http://www.improvision.com). Finally the images were analyzed using Image J software
(developed at the Research Services Branch (RSB) of the National Institute of Mental
Health (NIMH), National Institutes of Health (NIH), USA, http://rsb.info.nih.gov/nih- image).
2.3.4 Immunohistochemical staining of paraffin sections for HER2
Adjacent FFPE tissue sections from the same blocks were dewaxed in xylene and blocked for endogenous peroxidase in 3% hydrogen peroxide for 10 minutes. Following microwave antigen retrieval, the sections were blocked for endogenous biotin using the
Vector Laboratories, Inc. (Burlingame, CA, USA) biotin blocking kit. The sections were incubated with 1:300 diluted primary antibody HER2 rabbit antibody from Dako
(Carpinteria, CA, USA) for 1 hour at room temperature and rinsed in PBS followed by incubation with biotin conjugated anti-rabbit IgG from Signet Laboratories, Inc.
(Dedham, MA, USA) for 30 minutes. After incubation with Streptavidin labeled
45 horseradish peroxidase (HRP) for 30 minutes (Signet Kit) and rinsing in PBS, the
sections were incubated in NovaRed substrate from Vector Laboratories, Inc.
(Burlingame, CA, USA) for another 5 minutes. Finally the nuclei were counterstained in
Mayer's Haematoxylin for 10 seconds and the sections were dehydrated, cleared and
permounted for microscopic evaluation. Samples having more than 10% cancer cells with
complete cell membrane staining of moderate to strong intensity were considered as
HER2 positive.
2.3.5 Data analysis
In this study, the following variables were used in the analysis: telomere length
(TelGen) = average of the telomere length intensity (per patient, per type of tissue, over
the minimum 20 nuclei), normalized telomere length (N-TelGen)= average telomere
length divided by the average chromosome 17q intensity, chromosome 17q telomere
length (TelChl7q) = average of the telomere length intensity of the chromosome 17q
PNA probe-FITC, normalized chromosome 17q telomere length (N-TelChl7q) =
chromosome 17q telomere length divided by the average chromosome 17q intensity, and immunohistochemical percentage positivity for HER2 overexpression. The intensity of the subtelomeric chromosome 17q signals was used to normalize the values for telomere length. Cells without 17q signals were not scored.
2.3.6 Statistical analysis
The exact Wilcoxon signed-rank test and the exact Wilcoxon rank-sum test were used to compare the telomere length between normal breast epithelium, DCIS and IDC
46 samples.
2.4 Results
In the present study, the variables (N-TelGen and N-TelChl7q) were compared
between normal, DCIS, and IDC tissues. Using the exact Wilcoxon signed-rank test, we
found that total telomere length in DCIS was significantly shorter than that measured in
normal breast epithelium (p= 0.023) (Fig. 2.3A). Likewise, the telomere length on
chromosome 17q was notably shorter than its counterpart in DCIS compared with normal
tissues (p= 0.0078) (Fig. 2.3B). In contrast, the differences in the length of total telomere
and telomere on chromosome 17q were minimal between DCIS versus IDC (Fig. 2.3A
and B). Upon investigating the variables, N-TelGen and N-TelChl7q, amongst DCIS
alone (n=6) and DCIS+IDC (n=8) no significant changes were apparent between these
two groups (Fig. 2.4A and B).
To compare the telomere shortening on chromosome 17q with total telomeres in
DCIS and normal tissue, we defined the telomere shortening as the fold decreases in
DCIS as compared to normal tissue for both chromosome 17q and for total telomeres in each sample. We found that the median telomere length on chromosome 17q in DCIS decreased by 89.8% as compared with normal tissue, while the median of telomere length on all the rest of the chromosomes decreased by 49% (Table 2.1).
To study the relationship between telomere shortening on chromosome 17q and
FTER2 expression, all samples were stained with HER2 antibody. We found that 18 samples including 8 normal breast epithelium (Fig. 2.5A), 4, DCIS (Fig. 2.5B), and 6
IDC (Fig. 2.5C) were negative and 16 samples containing 10 DCIS (Fig. 2.5D) and 6
47 IDC (Fig. 2.5E) were positive for HER2. We compared the intensities for TelGen, N-
TelGen, TelChl7q and N-TelChl7q between HER2 negative and HER2 positive
samples. Using the exact Wilcoxon rank-sum test, we found that total telomere length in
the HER2 positive group is not significantly different from telomere length in the HER2
negative group (Table 2.2). Likewise, no significant differences were found between the
telomere length on chromosome 17q in both HER2 positive and negative (Table 2.2),
indicating that the expression of HER2 is independent from telomere shortening for both
total chromosomes and chromosome 17q.
2.5 Discussion
Invasive breast cancer is frequently observed to extend directly from ducts
containing DCIS; however, not all cases of DCIS develop into invasive tumors. The
challenge is to identify molecular markers, which will allow prediction of which cases of
DCIS will progress to IDC. Telomere length is reduced in IDC. Hence, we chose to
investigate telomere length as a potential marker.
It has been reported that it may not be the average telomere length but rather the
shortest telomeres that constitute telomere dysfunction as loss of telomere function has
been shown to occur preferentially on chromosomes with critically short telomeres (245).
However, although the p arm of chromosome 17 has been reported to be the shortest telomere (321), it is the q arm of chromosome 17 not chromosome 17p that shows a signal-free end with high frequency in senescent cells (322). This means that telomere shortening does not occur at the same rate for all telomeres, otherwise chromosome 17p should be one of the signal-free end chromosomes. In agreement with these findings, our
48 data shows that telomere shortening on chromosome 17q is significantly greater than the
average erosion of total telomeres in DCIS compared to normal breast epithelium
supporting the idea that the erosion of telomeres can be varied for different
chromosomes.
While we observed this event in malignant cells, other groups have shown similar
results in different cell types (322, 323). Using BJ fibroblasts, Zou etal. found that there
are no signal-free ends in young BJ cells, whereas in near senescent BJ cells chromosome
17q exhibited one of the highest frequencies of signal-free ends (322). Their report
showed that chromosome 17p has no signal-free ends (0%), although this arm of
chromosome 17 has been reported to have the shortest telomere among human
chromosomes. Similarly, Martens et al. showed that out of 13 samples from 10 different
donors, three of them had significantly short chromosome 17q telomere lengths compared
to the median for each individual donor (323). Interestingly, all these three samples were
from haematopoietic bone marrow (BM) cells, which have a high proliferation capacity.
Based on these observations, it can be suggested that cells with higher proliferation rates
show greater telomere shortening on chromosome 17q compared to chromosome 17p and
the average of total telomeres. This suggests that telomere shortening may not be simply the result of the end replication problem and there are potential mechanisms that preferentially erode or protect specific telomeres more than others. Therefore, it is necessary to evaluate the length of each individual telomere and to study its role in the development of cancers. Based on these findings, we conclude that telomere length on chromosome 17q plays a role in the progression of DCIS, possibly via chromosome instability. However, further studies are required to validate the mechanism behind the
49 differential telomere shortening for different chromosomes as well as the role of this mechanism in chromosome instability and cancer.
50 Figure 2.1. Labeling strategy used to flag pan-telomeres and sub-telomeric region of
chromosome 17q.
A) A schematic figure of a pair of chromosome 17. In this study we used a pantelomeric probe to label all telomeres (green color), chromosome 17q specific subtelomeric DNA probe (orange color), and DAPI to stain whole DNA (blue color). B)
Metaphase slides prepared from normal peripheral blood lymphocytes were used to optimize Q-FISH conditions for two different probes, a PNA (CCCTAA)3 pan-telomere probe-FITC (green color) and a specific sub-telomeric DNA probe for chromosome 17q,
TelVysion 17q SpectrumOrange, (orange color). C-D) magnified figures of chromosome
17.
51 Figure 2.2. FISH analysis on breast FFPE tissue sections.
Comparable H&E sections shown in (A) were used to determine regions of interest. (B-D): FISH with subtelomeric DNA probe to label chromosome 17q (orange signals) and pantelomeric PNA probe (green signals) on FFPE sections with xlOO ((B-D) objective. Representative images of normal breast epithelial tissue, DCIS and IDC are shown in each row. DAPI was used as a counterstain. Identical images in black and white shown in (C-D) with nuclei outlined in gray and (C) telomere and (D) sub-telomeric chromosome 17q signals represented by the dark spots (indicated by arrows). Note reduction in telomere signal number, intensity and size in DCIS and IDC compared to normal tissue.
52 * .-si *"i •-.. . - M 1 t •*"" rji *«. *•> *«
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* k J« <*
,, •* . >" i* * lv* . * 3t «- * '* ~""V _ D '' ^' B ~ *
53 A \ Normal vs. DCIS: p=0.023 R \ Normal vs. DCIS: p=0.0078 1.5 - DCIS vs. IDC: p=0.25 cr DCIS vs. IDC: p=0.58 0.5 • .•#—c• D) C 0) <$ O -^ 0.4 •• C •D 1.0 - CD N o 0.3 • -ti CO omer e rmaliz e %'\ N N v (1) O N. ^<"'\ "* \ —$ = i 02 - 0.5 - ~ z «<" V\"» \-' J!> o CD ,s\\ 0.1 - To t \.—;—-=--^_„, E _o 0 0.0 - o 0.0 -
Normal DCIS IDC Normal DCIS IDC
cr r^ G Normal vs. DCIS: p=0.016 ts - DCIS vs. IDC: p=0.31 sz CO c T5 CI) 1.0 i \ N *-,. o **^<\ K . *s CD
E rm a "^§^\... ""w^x _o v o •^J^^^cr^^^- B *N ~t CD 0.5 • ,*-—-*•-""*** ^N ^*Si *-""*-...*. ^^ _____—• •*-> ^ o
0,0 ' •
Normal DCIS IDC
Figure 2.3. Comparison of the normalized intensities of telomere length between
normal breast epithelial tissue, DCIS and IDC.
A) normalized total telomere length, B) normalized chromosome 17q telomere
length. C) normalized total telomere length excluding chromosome 17q telomere length.
Note significant reduction in telomere intensity in DCIS and EDC in comparison with normal breast epithelial tissue. • A 0.8 - I • • -3 0.6 - Ks • • XS • 8 0.4 - •
er e 1 * • •
1•*3— "3 0.2 - » X •
0.0 - • OnlyDCIS DCIS+IDC
o.oo H OnlyDCIS DCIS+IDC
Figure 2.4. Distribution of telomere length between DCIS and DCIS associated with
IDC.
A) normalized total telomere lengths, B) normalized chromosome 17q telomere lengths. There are no significant differences in telomere length between DCIS and DCIS associated with IDC.
55 Table 2.1 Comparing telomere shortening of chromosome 17q in DCIS and normal
tissue with total telomere shortening in the same tissues.
Samples Group A* Group B**
1 82.517 59.4769
2 100.000 71.4190
3 97.081 77.3483
4 90.364 38.5657
5 89.248 64.1070
6 88.889 38.6999
7 72.074 -47.4690
8 100.000 39.3684
Median 89.8063 49.4227
StdDev 9.4823 39.5324
The telomere shortening in DCIS as compared to normal breast epithelium for both chromosome 17q (Group A), and for total telomeres (Group B). Samples 2 and 8 had a
100% erosion which indicated that there was no detection of telomere signal in these two samples.
* Group A: fold decreased of N-TelChl7q in DCIS as compared to normal tissue
** Group B: fold decreased of N-TelGen in DCIS as compared to normal tissue
56 Figure 2.5. FFPE tissue sections were used for immunohistochemistry using HER2 antibody.
Samples having more than 10% of complete cell membrane staining of moderate to strong intensity were considered as HER2 positive. A) negative HER2 normal epithelial breast, B) negative HER2 DCIS, C) negative HER2 IDC, D) positive HER2
DCIS, and E) positive HER2 IDC.
57 ""V *#>• - -'
o LU '1V1J ^ «.,
CQ
.••*«'>.
58 Table 2.2 Association between telomere length and HER2 expression.
HER2 negative HER2 positive Wilcoxon sum-rank (n=18) (n=16) (p-value) Median Median Total telomere lengths (Normalized) 0.34 0.45 0.82
Telomere length on Chl7q* (Normalized) 0.058 0.029 019
Wilcoxon rank-sum test was used to compare the intensities for the total telomere lengths and teleomere length on Chl7q between HER2 negative samples (n=18) and HER2 positive samples (n=16). * Chl7q= chromosome 17q Chapter 3
60 Troglitazone suppresses telomerase activity independently of PPARy transcriptional activity
in breast cancer cells
61 3.1 Abstract
Many standard chemotherapeutic agents currently used to treat breast cancer are
relatively non-specific and act on all rapidly dividing cells. In recent years, more
specifically targeted therapies have been introduced. It is known that telomerase, the
enzyme that protects the ends of chromosomes, is active in over 90% of breast cancers
but inactive in adjacent normal tissues. If it were possible to target cells with active
telomerase then this strategy could be used to treat breast cancer. Recent evidence
suggests that telomerase activity can be suppressed by peroxisome proliferator activated
receptor gamma (PPARy) ligands in primary cells. PPARy is a nuclear hormone receptor
that stimulates the terminal differentiation of a variety of cancers, including breast
carcinoma. In the present study, we investigated the effect of the PPARy ligand,
troglitazone, on telomerase activity in breast cancer cells. To our knowledge, this is the
first time that the effect of troglitazone on telomerase activity in breast cancer has been
investigated. We demonstrated that troglitazone reduced the mRNA expression of hTERT and telomerase activity in the MDA-MB-231 breast cancer cell line. Using
different approaches, we showed that troglitazone reduced telomerase activity even in the
absence of PPARy. In agreement with this result, we also found no correlation between
PPARy and hTERT transcript levels in breast cancer patients. In summary, given the important role of telomerase in breast cancer progression, our data suggest that troglitazone can be used as an anti-telomerase agent; however, the mechanism underlying this inhibitory effect remains to be determined.
62 3.2 Introduction
Breast cancer is the most common malignancy of North American women.
During their lifetime, 1 in 9 women are expected to develop breast cancer, and 1 in 28
women are expected to die from it. In Canada, it is estimated that there will be 22,400
new cases of breast cancer, and more than 5,300 women will die from this disease in
2008 (2). Current therapy for primary breast cancer includes surgical resection with or
without radiation and/or chemotherapy. Nevertheless, a large percentage of women with
early-stage disease will experience a distant relapse leading to death from recurrence-
related complications and many women will experience significant chemotherapy-
induced toxicity. This has motivated considerable efforts toward finding novel
therapeutic approaches for the treatment of breast cancer.
Immortalization is a necessary step toward malignant transformation of normal
human somatic cells, which have intrinsic mechanisms that monitor cell divisions and
limit their life span. Progressive shortening of telomeres with each cell division has been found to limit replicative life span of human cells in culture (305). Most human cancer cells maintain their telomeres through activation of telomerase (108). In over 85% of human tumors, and more than 90% of breast carcinomas, telomerase is active in contrast to normal tissues where telomerase is active at low levels or is undetectable (21, 306,
307). Telomerase is a very large complex ribonucleoprotein enzyme with an estimated mass of -650 to 670 kDa (110). In vitro, two components are minimally required for telomerase activity: human Telomerase Reverse Transcriptase (hTERT), the catalytic subunit of telomerase, and human telomerase RNA (hTR), the RNA template (111). It has been shown that inhibition of telomerase limits the growth of human cancer cells, and
63 as a consequence various anti-telomerase strategies are currently under investigation to limit tumor proliferation in cancer patients (108).
Peroxisome proliferator activated receptors are members of the nuclear hormone receptor super-family, regulating gene expression via their ligand-activated transcriptional activity. In addition to controlling the expression of many genes involved in lipid metabolism, and insulin sensitization, it has been revealed that PPARy functions as a tumor suppressor in a variety of malignancies such as breast cancer, colon cancer, liposarcoma, ovarian cancer, and prostate cancer (244).
There are three classes of ligands for PPARy; natural ligands, dual ligands, and synthetic ligands. Synthetic ligands are members of the thiazolidinedione (TZD) family including troglitazone, rosiglitazone, pioglitazone, and ciglitazone (268). TZDs are known as insulin sensitizers and are used in the treatment of type 2 diabetes. In addition, it has been shown that TZDs promote the differentiation of various cell lines (227, 228,
230, 231, 311), and some of them, especially troglitazone and ciglitazone, demonstrate antiproliferative activities in several cancer models including breast cancer (232).
PPARy regulates gene expression by forming a heterodimer with retinoid X receptor (RXR) and binding to peroxisome proliferator response elements on the target genes. Using the RXR ligand, Choi et al. demonstrated inhibition of cell growth and telomerase activity of breast cancer cells in vitro (324). Interestingly, the PPARy/RXR heterodimers can be activated by ligands for either PPARy or RXR (325). Interestingly, resent evidence showed that PPARy can inhibit telomerase activity in some experimental models (173-175), however, the same role for PPARy in breast cancer has not been studied. To determine if PPARy regulates telomerase activity in breast cancer, we tested
64 the effect of a PPARy ligand, troglitazone, on telomerase activity in breast cancer cell
lines.
3.3 Materials and Methods
3.3.1 Materials
The MDA-MB-231, MCF-7, and T47D cell lines were obtained from American
Type Culture Collection (ATCC) (Manassas, VA, USA). The PPARy agonist troglitazone
and its antagonist GW9662 were purchased from Sigma (Sigma-Aldrich, St Louis, MO,
USA). Bisphenol A diglycidyl ether (BADGE) was from Cayman (Cayman, Ann Arbor,
MI, USA). Antibodies against Maspin and PPARy were purchased from BD Biosciences
(BD Biosciences, Mississauga, ON, CA) and Santa Cruz Biotechnology (Santa Cruz
Biotechnology, Santa Cruz, CA, USA) respectively. TaqMan specific primers for PPARy, hTERT, K19, Muc-1, and GAPDH were obtained from Applied Biosystems (Branchburg,
New Jersey, USA). Cell culture media was from the Ontario Cancer Institute (OCI)
(Princess Margaret Hospital, Toronto, ON, CA).
3.3.2 Cell Culture
MDA-MB 231 cells were cultured in alpha MEM medium (aMEM) (Princess
Margaret Hospital, ON, CA) supplemented with 10% v/v fetal bovine serum (HyClone,
Logan, UT, USA) at 37°C in a humidified atmosphere with 5% C02 for 24 hours, then treated with either troglitazone or the same amount of DMSO (Sigma-Aldrich Life
Science, Saint Louis, MO, USA) and incubated for different periods of time as indicated
65 in the figure legends.
3.3.3 Cell toxicity and cell viability assay
The cell toxicity assay was measured using CellTiter96 nonradioactive proliferation assay kit (Promega, Madison, WI, USA). This assay can measure cell viability as well as cell toxicity using the dose-response curve to determine IC50 (50% inhibitory concentration at 50%), the concentration of the test substance required to reduce the light absorbance capacity of exposed cell cultures by 50%. To measure cell toxicity, cells were seeded in 96-well plates at a density of 7xl03 cell/well and treated with indicated concentrations of troglitazone. At the end of each time point, cells were incubated with 20 u,l MTS/PMS solution for a further 3 hours in a humidified environment. Finally, the toxicity effect of troglitazone was determined by measuring the formazan produced by proliferating cells at 490nm on a Tecan SpectraFluor Plus Plate
Reader (MTX Lab Systems, Inc, Vienna, VA, USA).
For the cell viability assay, cells were cultured in 6-well culture plates. The next day, cells were exposed to troglitazone or vehicle. After 24 hours inculabation in cell culture incubator, cells were trypsinized and washed with PBS and then resuspended in growth media. The cell viability was measured by automated Vi-CELL, which uses the trypan blue dye exclusion method (Beckman Coulter, Brussels, Belgium).
3.3.4 Western blot analysis
Total protein was extracted from cells using CytoBuster™ Protein Extraction
Reagent (Novagen, Darmstadt, Germany) containing an appropriate concentration of
66 complete protease inhibitor cocktail tablets (Roche Diagnostic GmH, Mannheim,
Germany) and Phosphatase Inhibitor Set II (Calbiochem, La Jolla, CA, USA). Protein concentrations were measured using the Bradford assay (Bio-Rad Laboratories, Inc.,
Hercules, CA, USA) according to the manufacturer's protocol. Proteins were mixed with
IX laemmli buffer (10% SDS, 0.5 M Tris, pH 6.8, (3-mercaptoethanol, glycerol, 0.1%
Bromophenol Blue) and boiled for 5 minutes, followed by separation on 4-12% gradient
NuPAGE® Novex Bis-Tris Gels (Invitrogen, Carlsbad, CA, USA). After electrophoresis, the proteins were transferred to Hybond™-C Extra nitrocellulose membrane (Amersham
BioSciences, Little Chalfont, UK). The transblotted membranes were blocked in 5% skim milk-TBST [lOx Tris Buffered Saline (TBS); 24.2 g Tris base, 80 g NaCl in one liter distilled H20; at pH to 7.6 (use at lx plus 0.1% Tween-20)] for 1 hour at room temperature and washed 3 times each time for 5 minutes with TBST. Blots were then incubated with the appropriate primary antibody overnight at 4°C. After washing three times in TBST, membranes were incubated with the appropriate secondary antibody conjugated to horseradish peroxidase (Amersham BioSciences, Buckinghamshire, UK)
(1:10,000 dilution in 5% skim milk) for 1 hour at room temperature and then washed three times in TBST each time for 10 minutes. The immunoblots were visualized using
ECL Advance™ Western Blotting Detection Kit (GE Healthcare, Buckinghamshire, UK) as described by the manufacturer. The resultant films were analyzed and quantified using
Image J software [Research Services Branch (RSB) of the National Institute of Mental
Health (NIMH), National Institutes of Health (NIH), USA, http://rsb.info.nih.gov/nih- imagel.
67 3.3.5 Real-time RT-PCR
Total RNA was extracted using RNeasy® plus kit (Qiagen, Mississauga, ON,
CA), and used for real-time reverse transcription (RT) in a two-step procedure. In the first step, an aliquot of 2 fig total RNA from each sample was reverse transcribed to cDNA using TaqMan® Reverse Transcription Reagents (Applied Biosystems, Branchburg, New
Jersey, USA). In the second step, 100 ng cDNA was used for PCR using TaqMan®
Universal PCR Master Mix (Applied Biosystems, Branchburg, New Jersey, USA) in a
396-well plate according to the manufacturer's instructions. We used TaqMan specific primers for hTERT, PPARy, Mucl, K19, and GAPDH in our experiments (Branchburg,
New Jersey, USA). The real-time quantitative PCR and analysis were carried out using the ABI Prism 7900HT Sequence Detection system (Foster City, CA, USA).
3.3.6 Stable shRNA mediated repression of PPARy in MDA-MB-231 cells
Human PPARy expression in wild type MDA-MB-231 breast cancer cells was silenced by the shRNA method. For this purpose, four lentiviral gene transfer vectors expressing shRNA against PPARy (NM_138712) were purchased from Open Biosystems
(Huntsville, AL, USA); shPPARy-71 (ClonelD: TRCN0000001671), shPPARy-72
(CloneE): TRCN0000001672), shPPARy-73 (ClonelD: TRCN0000001673), and shPPARy-74 (ClonelD: TRCN0000001674). An adopted Qiagen non-silencing control siRNA sequence (TTCTCCGAACGTGTCACGT) that was not complementary to any human gene was used as a control shRNA (generous gift from Dr. M.S. Tsao, University of Toronto). Lentiviruses were prepared by transfecting three plasmids (pMDLg/pRRE, the vesicular stomatitis virus (VSV-G) envelope plasmid pCMV-VSG, rev expressing
68 plasmid pRSV-Rev and gene transfer vectors containing the self-inactivating LTR) into
293T cells as described (326, 327). MDA-Mb-231 cells were transduced as previously
described (326, 327). Transduced cells were then selected for two weeks in growth media
supplemented with 0.5 u.g/ml puromycin. Selected cells were subjected to real-time RT-
PCR to test the expression level of PPARy in the transduced MDA-MB-231 cells.
3.3.7 Telomeric repeat amplification protocol (TRAP) assay
MDA-MB-231 cells were grown in 60-mm dishes in the presence and absence of
troglitazone at 37°C in a humid incubator. After 24 hours incubation, the medium was removed from each dish and the cells were washed two times with 2ml of ice-cold IX
PBS. A final 2 ml of PBS was added and the cells were detached using a cell scraper and transferred to a 2 ml Eppendorf tube. The cells were then centrifuged at 1 500 g for 5 min at 4°C. CHAPS lysis buffer [lOmM Tris HC1, pH7.5, 1 mM MgCl2, 5 mM beta- mercaptoethanol, 0.5% W/V CHAPS (Sigma-Aldrich, St Louis, MO, USA), and 10% v/v glycerol with 400 U RNase Inhibitor (Roche, Basel, Switzerland) and one tablet of complete (mini) EDTA free Protease Inhibitor Cocktail (Roche, Basel, Switzerland)] at a volume of 4-5 times the volume of the cell pellet was used to lyse the cells on ice for 30 minutes. The cells were then centrifuged at 13,000 x g for 30 mins to pellet the lysed cells. After centrifugation, the protein lysate was transferred to a new 1.5 ml Eppendorf tube. The concentration of the cleared whole cell lysate was determined by the Bradford assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA). TRAP assay reactions were performed, using the radioactive method of the TRAPeze kit (Millipore, Billerica,
MA,USA) with some modifications which follow. The telomerase extension step was
69 performed in the absence of Taq Polymerase in the reactions. After the telomerase
extension, the reactions were heated to 94°C for 2 minutes, followed by the addition of 2
units of Taq polymerase (NEB, Ipswich, MA, USA) and PCR amplification. PCR
conditions were 25 cycles of 94°C for 30 seconds, 50°C for 30 seconds, and 72°C for 90
seconds. Half of the 50/xl reaction was loaded on a 10% non-denaturing PAGE gel in
0.6X TBE buffer. The gel was dried and exposed to a phosphor-imager screen and
scanned using a Typhoon Trio Imager (GE Healthcare, UK). Protein titrations were performed to ensure TRAP reaction products exhibited a semi-linear response.
3.3.8 The NKI microarray dataset analysis
The Netherlands Cancer Institute (NKI) dataset was used to compare the expression of PPARy and hTERT (328). The NKI dataset is published genome-wide gene expression microarray data from 295 breast cancer samples collected between 1984 and
1995. Samples were selected from patients younger than 53 years with primary invasive breast cancers. All patients had stage I and II breast cancer and were either lymph-node- negative (151 patients) or lymph-node-positive (144 patients). These patients received modified radical mastectomy or breast conserving surgery as their treatments. The published expression dataset of PPARy (Probe ID: 17022) and hTERT (Probe ID: 1809) for the 295 patients were analyzed by unpaired Student's t test.
3.3.9 Statistical analysis
All numerical data were expressed as median values ± SD. Statistical significance was determined by performing a paired Student's t test.
70 3.4 Results
3.4.1 Evaluating the expression of PPARy in different breast cancer cell lines
Expression levels of PPARy gene and protein were evaluated in several breast
cancer cell lines by real-time RT-PCR and western blot analysis. Three different breast
cancer cell lines; MDA-MB-231, MCF-7, and T47D were tested for this purpose. Real
time RT-PCR showed that mRNA expression of PPARy is higher in MDA-MB-231 cells
compared to the other two cell lines (Fig. 3.1 A). In agreement with this result, we found
that although all three cell lines expressed PPARy protein, the expression of PPARy
protein was higher in MDA-MB-231 compared to the two other cell lines (Fig. 3.IB).
3.4.2 Determining the expression level of hTERT and telomerase activity in MDA-
MB-231 cells
We examined the expression level of hTERT mRNA, as well as telomerase
activity in these cell lines. Results from real-time RT-PCR indicated that all three cell lines analyzed expressed hTERT mRNA (Fig. 3.2A) and the TRAP assay confirmed that telomerase was active in these cells (Fig. 3.2B).
3.4.3 The cell toxicity effect of troglitazone
To determine the non-toxic concentration of troglitazone for cell treatment in our study, we measured IC50 for troglitazone using the CellTiter96 non-radioactive proliferation assay kit. This assay measures the activity of dehydrogenase found in metabolically active cells. Dehydrogenase, a mitochondrial enzyme, reduces MTS (3-
71 (4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-
tetrazolium, inner salt) chemically into formazan (329). Since the production of formazan
is proportional to the activation of this enzyme in viable cells, the intensity of the
produced color is an indicator of cell viability. The MTS result showed that the IC50 of
troglitazone after 24 (Fig. 3.3A) and 48 (Fig. 3.3B) hours exposure was 190 /iM.
3.4.4 Troglitazone reduces telomerase activity in a time and dose dependent manner
In order to examine whether troglitazone regulates telomerase activity, MDA-
MB-231 cells were cultured in growth medium enriched with 10% fetal bovine serum
(FBS) in the presence and absence of troglitazone. In the cells exposed to troglitazone, a
decrease in telomerase activity was observed at 24 hours in a dose dependent manner
(Fig. 3.4). MDA-MB-231 cells were also treated with 20 fiM troglitazone or the equal
volume of DMSO. Cells were then harvested at indicated time points and their telomerase
activity was measured by the TRAP assay. Our results showed that the reduction of telomerase activity by troglitazone is time dependent (Fig. 3.5A).
3.4.5 Troglitazone suppresses hTERT transcription
To examine whether the suppression of telomerase by troglitazone is at the mRNA level of hTERT, MDA-MB-231 cells were treated with different concentrations of troglitazone for 24 hours and the mRNA expression of hTERT was determined by real-time RT-PCR. Troglitazone exhibited a significant dose dependent reduction in the expression of hTERT mRNA compared to control (P < 0.05) (Fig. 3.5B), indicating that troglitazone suppressed telomerase activity by decreasing the expression of hTERT
72 mRNA. Time-chase experiments showed that this effect can be observed in 24 hours of
treatment starting with a minimum of 20uM troglitazone, whereas treating the cells with
the same concentration of troglitazone for 12 hours did not show significant changes in
hTERT mRNA compared to controls (Fig. 3.5C). Our results show that the minimum
concentration for the inhibitory effect of troglitazone is 20\iM for a minimum of 24
hours. Based on these observations, the effect of 20uM of troglitazone at 24 hours was
selected for further studies.
3.4.6 Reduction in telomerase activity is independent from the transcriptional role
ofPPARy
To assess the involvement of PPARy in the reduction of telomerase activity,
MDA-MB-231 cells were exposed to two different PPARy antagonists, 10 uM GW9662
(330) and lOOuM of BADGE (331), for 24 hours prior to troglitazone treatment. Results from real-time RT-PCR showed that addition of either GW9662 or BADGE did not recover the suppressive effect of troglitazone on hTERT gene expression (Fig. 3.6).
To confirm these results, we knocked down the expression of PPARy using lentivirus. Results from real-time RT-PCR showed that the expression of PPARy mRNA
(Fig. 3.7A) and its protein level (Fig. 3.7B) in knock-down cells has been significantly decreased compared to wild type and scrambled shRNA cells. Next, we studied the telomerase activity in the knocked-down PPARy cells. Using TRAP assay, we did not find a significant change in telomerase activity in knocked-down versus wild type MDA-
MB-231 cells (Fig. 3.8). Furthermore, to examine the effect of troglitazone in the absence of PPARy, wild type and knocked-down MDA-MB-231 cells were treated with
73 troglitazone for 24 hours and the activity of telomerase was measured. Our data show that
troglitazone was able to suppress telomerase activity in the absence of PPARy (Fig. 3.9).
These results indicate that the effect of troglitazone on telomerase activity does not rely
on PPARy transcriptional activity.
3.4.7 Troglitazone does not induce apoptosis
It has been shown that PPARy ligands induce programmed cell death (apoptosis)
(for review see (332)). To examine the effect of troglitazone on cell viability and
apoptosis in our study, MDA-MB-231 cells were treated with control or troglitazone for
24 hours and cell viability was measured using the trypan blue dye exclusion method.
Cells treated with troglitazone showed a reduction in cell viability compared to controls, however; this reduction was not statistically significant (Fig. 3.10A). Troglitazone treated cells were also evaluated for caspase-3, one of the key executioners of apoptosis, as an apoptosis marker. Caspase-3 did not show any differences in treated cells compared to control DMSO (Fig. 3.1 OB). Furthermore, we examined the protein levels of poly (ADP- ribose) polymerase (PARP), a protein which undergoes caspase-3 mediated cleavage during apoptosis and produces an 89 kDa fragment. In agreement with the caspase-3 result, we did not observe a reduction of PARP protein levels in treated cells compared to control cells (Fig. 3.10B). These results confirm that troglitazone at 20/xM does not induce apoptosis and is not toxic to the cells.
3.4.8 Troglitazone does not induce the differentiation of MDA-MB-231
Accumulating evidence indicates that PPARy promotes cell differentiation
74 following activation by its ligand (227, 228, 230, 231, 311) and since telomerase is not
active in differentiated cells (333, 334), we were interested to determine if the effect of
troglitazone in the reduction of telomerase activity is the result of cell differentiation.
MDA-MB-231 cells were treated with various concentrations of troglitazone for
different periods of time. We measured the expression of maspin as a marker for
differentiated breast epithelial cells (335). As shown in Fig. 3.11A, troglitazone did not
increase the protein level of maspin, indicating that treated cells are not more
differentiated. We also measured the expression of two genes associated with breast malignancy, Keratin 19 (K19) and mucin-1 (Muc-1) as described by Mueller et al. (228).
Real-time RT-PCR showed the expression of Muc-1 (Fig. 3.11B) and K19 (Fig- 3.11C) genes remained unchanged in troglitazone treated cells vs. vehicle treated cells.
3.4.9 The expression of hTERT is not correlated with the expression of PPARY in clinical samples
Published NKI data from 295 young patients with primary invasive breast cancers was used to compare the expression of PPARy and hTERT (328, 336). We found that the expression of PPARy and hTERT in this set of samples was 44% and 62% respectively.
Our results showed that there is no correlation between the level of PPARy expression and hTERT expression (R= -0.152) in these samples (Fig. 3.12A). Since MDA-MB-231 cells are estrogen receptor negative, we compared the expression of PPARy and hTERT genes in estrogen receptor-negative (N= 69) and estrogen receptor-positive (N= 225) tumor samples. We found no significant correlation between the two genes in estrogen receptor-positive (R=-0.156) (Fig. 3.12B) and estrogen receptor-negative (R = -0.08)
75 (Fig. 3.12C) tumors.
3.4.10 The level of hTERT expression is negatively correlated with ER status in
breast cancer samples
To assess the strength of the expression status of hTERT gene as a predictor of
the outcome of disease, we examined the correlation of hTERT gene expression with
different clinical prognostic parameters; tumor ER content, tumor differentiation (grade),
tumor size (Size), 70-gene prognosis signature (70GS) (337), and wound signature (WS)
(336) (Fig. 3.13A). Our results showed that in ER negative patients the expression of
hTERT is significantly higher than in ER positive patients (p<0.02) (Fig. 3.13B).
However, no significant correlation was found between hTERT expression and tumor
differentiation (Fig. 3.13C), WS (Fig. 3.13D), 70GS (Fig. 3.13E), and size (Fig. 3.13F).
3.5 Discussion
It has been shown that inhibition of telomerase limits the growth of human cancer cells (107), and as a consequence various anti-telomerase strategies are currently under investigation to limit tumor proliferation in cancer patients (108). There is some evidence indicating that PPARy may play a critical role in this process (173-175). The objective of this study was to investigate the effect of troglitazone on telomerase as a potential novel therapeutic approach that could be used to treat breast cancer. We studied three human breast cancer cell lines; MDA-MB-231, MCF-7, and T47D. MDA-MB-231 is estrogen receptor negative (338), EGF responsive and IGF-I non-responsive. MCF7 is estrogen receptor positive (338), EGF non-responsive and IGF-I responsive and T47D is estrogen
76 receptor positive, EGF responsive and IGF-I non-responsive (339). Real-time RT-PCR
data showed the expression of PPARy mRNA was higher in MDA-MB-231 cells
compared to MCF-7 and T47D. In agreement with this result, western blot analysis
confirmed that MDA-MB-231 cells produced more PPARy protein than the other two cell
lines. We also validated the expression of hTERT and telomerase activity in these cell
lines. Real-time RT-PCR showed that all three cell lines express hTERT mRNA. This
data has been confirmed by the TRAP assay, which showed that telomerase is active in
all three cell lines. To study the effect of PPARy on telomerase activity, the MDA-MB-
231 cell line was chosen as an ER-negative breast cancer in vitro model as this cell line
contains active telomerase and high PPARy protein. Using the TRAP assay, we showed
that troglitazone suppresses telomerase activity. The presence of a strong internal control
in the troglitazone treated samples indicates that the suppression of telomerase is not due
to PCR inhibition in the TRAP assay. We also found that this suppression is at the level
of gene transcription. We found that hTERT mRNA levels were significantly reduced by
troglitazone. Our results showed that 20jiiM of troglitazone is the minimum concentration
that can suppress telomerase activity in a minimum time of 24 hours. We also found that
troglitazone at this concentration is not toxic to the cells indicating that the inhibition of telomerase by troglitazone is not a consequence of cell toxicity.
There is accumulating published data indicating that troglitazone can act independently from PPARy (268). To study the role of PPARy in modulating the expression of hTERT and telomerase activity, we studied the effects of troglitazone on hTERT expression in the absence and presence of PPARy antagonists, BADGE and
GW9662. We found that neither BADGE nor GW9662 were able to prevent the
77 suppressive effect of troglitazone on hTERT expression. This finding was confirmed
using shRNA silencing of PPARy. We showed that troglitazone suppresses telomerase
even in the absence of PPARy expression. This result indicates that the effect of
troglitazone on telomerase activity is independent from PPARy. There is some evidence
showing that activated PPARy abolishes telomerase activity (173-175). It is noteworthy
that none of these groups used MDA-MB-231 cells as their models. Moreover, none of them used troglitazone as the ligand for PPARy. Recently, using pancreatic cancer cell
12 14 lines, Kondoh et al. showed that 15-deoxy-A ' prostaglandin J2 (15d-PGJ2), a natural ligand for PPARy, suppresses the expression of hTERT by blocking ER functions (340).
However, this group did not demonstrate whether this suppression is through PPARy activation. Although the underlying mechanism for these differences remains to be discovered, it may reflect differences in experimental models and approaches such as cell type, particular ligand, duration of treatment, and dosage.
A line of evidence indicates that PPARy ligands induce cell apoptosis (286-290).
To examine whether the suppression of telomerase is the result of apoptosis induction, we investigated the effect of troglitazone on cell viability and the protein level of caspase-3 and PARP as apoptosis markers. Although it has been shown that troglitazone induces apoptosis in different cancer cell lines by different mechanisms (286-289), our result showed that troglitazone does not induce apoptosis in MDA-MB-231 cells. It is noteworthy that these groups used a higher concentration of troglitazone. However, published data from other groups indicates that a low concentration of troglitazone cannot promote apoptosis. Elstner et al. showed that 10/xM troglitazone does not induce apoptosis in the MCF-7 cell line after 4 days incubation (290). In agreement with this
78 group, Ohta et al. demonstrated that 10/iM troglitazone was not able to change the
expression level of bcl-2 and box genes in BHP18-21, a thyroid papillary carcinoma cell
line (292).
More importantly, all groups showed that regardless of the concentration of
troglitazone used, the induction of apoptosis was independent from PPARy activity (286-
290, 292). Since the promotion of apoptosis was not observed in our cell model, it can be
postulated that the suppression of hTERT and its telomerase activity by troglitazone is not due to apoptosis activation.
It has been also shown that PPARy promotes cell differentiation (227, 228, 230,
231, 311). Since differentiated cells do not show telomerase activity (333, 334), we investigated whether telomerase suppression in our study is the result of cell differentiation. MDA-MB-231 cells were exposed to various concentrations of troglitazone for different time periods. We examined the expression of three different markers associated with breast cancer. It has been shown that the mRNA expression of maspin is decreased in malignant breast cells compared to normal epithelial breast cells
(335). In contrast, K19 and Muc-1 are associated with more malignant breast epithelial cells (228). Our results showed that troglitazone was not able to change the expression of these genes, indicating that treated cells with troglitazone had not been differentiated.
Furthermore, by increasing the concentration of troglitazone, we did not observe any signs of cell differentiation but we observed cell toxicity and apoptosis. This suggests that troglitazone inhibits telomerase activity independently from cell differentiation at low concentration, where no cell toxicity and apoptosis were observed.
To compare our findings with clinical samples, we analyzed the expression of
79 PPARy and hTERT from genome-wide gene expression microarray data for 295 patients
(328, 336). We found no correlation between these two genes (R= -0.152). Furthermore,
since the MDA-MB-231 cell line is an estrogen receptor negative cell line, we compared the expression of hTERT and PPARy in estrogen receptor (ER) negative patients. No significant correlation was found between the expression of hTERT and PPARy (R= -
0.08) in this group of patients. In agreement with our result from in vitro model, this finding suggests that telomerase inhibition by PPARy ligands is independent from PPARy transcriptional activity and conducted by an unknown mechanism.
Using the NKI dataset, we also analyzed the correlation of hTERT expression with clinical prognostic parameters. It has been hypothesized that reactivation of telomerase in breast epithelial tissue may be an important event in the progression to breast cancer (341). Published data reveal that the prognostic significance of telomerase activity in malignant disease is controversial. There is evidence indicating no correlation between telomerase activity and established prognostic factors such as tumor size and lymph node status (307, 342-345). Mokbel et al. showed that despite a significant association between telomerase activity with advanced histopathological grade and tumor type (ductal vs. lobular), there is no correlation between telomerase activity and nodal status or disease outcome in human breast cancer (346). However, the same group found an association between telomerase activity and nodal metastasis in another published report (347). Moreover, others found an association between telomerase activity and prognostic factors (348-350). Analyzing the NKI published dataset, we found no correlation between hTERT expression and tumor size, lymph-node status, 70-gene signature, wound signature, and proliferation status of tumors. However, the incidence of
80 hTERT expression was found to be negatively associated with ER content (p=0.019).
hTERT expression is higher in ER negative patients compared to ER positive patients.
This is in agreement with other published data (349) but in contradiction to the results
reported by Carey et al. (307). We also found that troglitazone significantly suppresses
telomerase activity at low concentration without involving PPARy transcriptional
activity, apoptosis, and cell differentiation. Furthermore, this result was confirmed by
comparing with clinical data, showing that there is no correlation between PPARy and hTERT gene expression in the NKI dataset.
To our knowledge this is the first time that the effect of troglitazone on telomerase
activity has been studied in human breast cancer. We showed that the expression of hTERT and PPARy are two independent events and troglitazone reduces the activity of telomerase by recruiting other pathway(s) rather than PPARy activity. Our study shows although the mechanism underlying this suppression remains unclear, and may be indirect, troglitazone can be considered as an anti-telomerase agent in estrogen-receptor negative breast cancer cells. In addition, based on data from our studies as well as others, we suggest that the role of troglitazone, and probably the other members of TZD family, should be revisited beyond their original role as PPARy ligands.
81 A
^ s 5 O 0.8
PL, >
P* 0.0 MDA-MB-231 MCF-7 T47D
B MDA-MB-231 MCF-7 T47D
MHH PPARY (protein) 1 L i •', i. >
GAPDH (protein)
Figure 3.1. The expression of PPARy mRNA and protein in various breast cancer cell lines.
A) Total RNA was prepared from MDA-MB-231, MCF-7, and T47D cells. Using real-time PCR, the mRNA expression of PPARy was measured. MDA-MB-231 cells express higher PPARy mRNA compared to the other two cell lines. B) Protein lysates were prepared from three different breast cancer cell lines and then analyzed via immunoblotting to examine the level of PPARy protein in these cell lines. Representative immunoblot suggests that MDA-MB-231 cells express more PPARy protein compared to
MCF-7 and T47D.
82 Figure 3.2. The expression level of hTERT and telomerase activity in three different breast cancer cell lines.
A) Using a Taqman probe, total RNA was used to examine the expression level of hTERT in MDA-MB-231, MCF-7, and T47D cells by real-time PCR. Values express the relative quantity of the genes to the level of mRNA expression of GAPDH. B)
Telomerase activity was measured in MDA-MB-231, MCF-7, and T47D cell lines. For each sample two different protein concentrations, 500 ng and 250 ng, were used for
TRAP assay. CHAPS buffer and protein lysate from HeLa cells were used as negative and positive controls respectively. The arrow head indicates a PCR product that serves as positive internal controls (IC) for the PCR reaction itself. TRAP assay showed that telomerase is active in all three cell lines.
83 A
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'w O CO •4-'
x 3
MDA-MB-231 MCF7 T47D
B
O -v er^ ^ ^
r ^'» *
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k ^t* ^.^ ic • ••
84 A
ON
N
o o B
•os CO 3 0 12 3 4 Logarithmic concentration of troglitazone (/JM) B
0 12 3 4 Logarithmic concentration of troglitazone ((iM)
Figure 3.3. IC50 of troglitazone was measured by MTS assay.
MDA-MB-231 cells were treated with various concentrations of troglitazone (0,
10, 20, 50, 100, 250, 500, 1000 /iM) for A) 24 or B) 48 hours and troglitazone IC50 was determined by MTS assay as described in Materials and Methods. All data were normalized against the control group (DMSO) in the respective treatment condition.
85 Figure 3.4. Troglitazone suppresses the activity of telomerase in a dose dependent manner.
Lysates from MDA-MB-231 cells with no treatment (wt), treated with various concentrations of troglitazone (Tro) or the equal volume of DMSO were examined for telomerase activity by TRAP assay. 500 ng and 250 ng of total protein were used for each sample, with a 25-cycle amplification step (see Materials and Methods). The arrow head indicates a PCR product that serves as positive internal controls (IC) for the PCR reaction itself. Triangles for each sample represent higher (500 ng) and lower amounts of protein
(250 ng).
86 %. <%>.
<2> >*• J, %\ V- £-~ t*b * % \
%, i I" ¥ ** <% ;•' -si* jr »•- % ^ %, °*. % *e_ feaaiwainmi
% V ^ V. % *s
J" d t
87 Figure 3.5. The effect of troglitazone on telomerase activity and hTERT expression is time dependent.
A) MDA-MB-231 cell lysate with no treatment (wt), treated with 20/iM troglitazone (Tro 20/xM) or the equal volume of DMSO for 24 hours or 48 hours were examined for telomerase by TRAP assay. 500 and 250 ng of total protein was used for each sample. The arrow head indicates a PCR product that serves as positive internal controls (IC) for the PCR reaction itself. Triangles for each sample represent higher (500 ng) and lower amounts of protein (250 ng). B-C) MDA-MB cells were treated with with
DMSO or 20 piM of troglitazone (Tro 2(fytM). Using real time RT-PCR, the expression of level of hTERT was analyzed after B) 24 hours or C) 12 hours of treatment. Values express the relative quantity of hTERT to GAPDH. The volume of DMSO was not more than 0.1%. Results shown are as mean ± SD and are representative of at least three independent experiments. * p < 0.05.
[HE DMSO (equal volume to 10/iM troglitazone) HD 10/xM troglitazone
ED DMSO (equal volume to IOJJM troglitazone) H 15/xM troglitazone
• DMSO (equal volume to 10/xM troglitazone) • 20/LiM troglitazone
88 mRNA expresson of hTERT DO (Relative to GAPDH) o o p o bo
r f ^ \ \ i • • -m - * X o i • ?L % I f % II 7 ** t M :•'-.'. -. . mRNA expresson of hTERT <•• i V (Relative to GAPDH) o o o p b bo t .t 'i t-Mfct-ttlt *.:•'> - 100
80 -^ Q 60 o U w O w *-> 40 ^
s P4 20
Tro20 GW9662 BADGE +Tro20 +Tro20
Figure 3.6. The suppressive role of troglitazone on hTERT is independent from
PPARy.
MDA-MB-231 cells were exposed to either 10 [iM GW9662 or lOOjuM BADGE for 24 hours. Cells then were treated with 20 /xM troglitazone in the presence of GW9662 or BADGE for another 24 hours. At the end of the incubation time, the expression level of hTERT was determined by real time PCR. Values represent the relative quantity of hTERT to GAPDH. The volume of DMSO was not more than 0.1%. Results shown are as mean ± SD and are representative of three independent experiments.
90 A
12
9 8 Jo oo O
x +3
•7 P5.
_^_ wt scrambled 71 72 73 74
B
wt scrambled 71 72 73 74
PPARy •tm>
GAPDH -'«&•* - nimiiir 4MHNNIW' 1 | Tfl^Wr ^^PF( **HUIP"i~ «.,
Figure 3.7. Suppression of the expression of PPARy by shRNA.
Using shRNA protocol, the expression of PPARy was blocked with four different
shRNA oligos (71, 72, 73, and 74) in the MDA-MB-231 cell line. To assess the
specificity of shRNA oligos against PPARy , MDA-MB-231 cells were transfected with
scrambled oligo and the extracted protein was used as a control for further experiments.
A) The mRNA level of PPARy was examined by real-time PCR. B) The protein level of
PPARy was subjected to western blot analysis.
91 o& * ^ ^
* / 0^ ^ ^ &
' ' i » ' , * * t 1 r "
i » J j * i
* 1 • - H . • •' ' ' • • -1. 11 ,4-' | "', i » * • ':•:,'.
•• 1 ,J A J ' 1 ' * ' -. • »^;..,A''
1 ' ! •' r
1
1 i
IC • iftiiirti«iFigure 3.8. Evaluation of hTERT mRNA expression and telomerase activity in
PPARy knocked-down cells.
TRAP assay was used to examine telomerase activity in MDA-MB-231 cells in the absence of PPARy (01igo71, 72, 73, 74) compared to non-transfected MDA-MB-231 cells (wt) and cells transfected with a scrambled oligo. IC represents positive internal controls.
92 Figure 3.9. The effect of troglitazone on telomerase activity was measured in the absence of PPARy.
MDA-MB-231 cells carrying silenced PPARy by shRNA were treated with 20 fjM troglitazone or the equal volume of DMSO for 24 hours. Two different concentrations of protein, 500 ng and 250 ng, from each sample were subjected to TRAP assay. CHAPS buffer and the extracted protein from HeLa cells were used as negative and positive controls respectively. The arrow head indicates a PCR product that serves as an internal positive control for the PCR reaction itself. Result shown is representative of two independent experiments.
93 "** i; VvJi'. J* "<*, Y, % J ; M * ' 9 I & •' • • -l *
\.- > / 1 1 A : iy. J