INVOLVEMENT OF SINGLE- AND DOUBLE-STRAND BREAK REPAIR
PROCESSES IN BETA-LAPACHONE-INDUCED CELL DEATH
by
MELISSA SROUGI BENTLE
Submitted in partial fulfillment of the requirements
For the degree of Doctor of Philosophy
Dissertation Adviser: Dr. David A. Boothman
Department of Pharmacology
CASE WESTERN RESERVE UNIVERSITY
August, 2007
CASE WESTERN RESERVE UNIVERSITY
SCHOOL OF GRADUATE STUDIES
We hereby approve the dissertation of
______
candidate for the Ph.D. degree *.
(signed)______(chair of the committee)
______
______
______
______
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(date) ______
*We also certify that written approval has been obtained for any proprietary material contained therein.
ii
For Noline, Mom and the memory of my Father
iii TABLE OF CONTENTS
Title Page i
Signature Sheet ii
Dedication iii
Table of Contents 1
List of Tables 4
List of Figures 5
Preface 7
Acknowledgements 8
List of Abbreviations 9
Abstract 13
CHAPTER 1: Introduction
New Tricks for Old Drugs: The Anticarcinogenic Potential of DNA Repair
Inhibitors
Introduction to DNA Repair 15
Targeting DNA DSB Response Pathways for the Prevention and
Treatment of Cancer 19
Mechanisms of SSB Response Pathways and Utility as Targets for
Chemotherapy 25
Targeting Direct Repair for Chemotherapy 27
Poly(ADP-ribosyl)ation, PARP-1, and DNA Repair 28
PARP-1 Intervention as a Means of Inhibiting Multiple DNA Repair
1 Pathways and Transformation 32
β-Lap: an Anticarcinogenic DNA Repair Inhibitor 35
Mechanism of β-Lap Toxicity in Cancer Cells 39
β-Lap and DNA Repair Inhibition 46
Concluding Remarks 47
CHAPTER 2: Calcium-Dependent Modulation of Poly(ADP-ribose) Polymerase-1
Alters Cellular Metabolism and DNA Repair
Abstract 49
Introduction 50
Experimental Procedures 52
Results 58
Discussion 89
CHAPTER 3: Non-Homologous End Joining is Essential for Cellular Resistance to
the Novel Antitumor Agent, β-Lapachone
Abstract 98
Introduction 99
Experimental Procedures 102
Results 105
Discussion 126
CHAPTER 4: Discussion and Future Directions 134
A.1 Conclusions: PARP-1 Mediates β-Lap-Induced Cell Death 135
A.2 Future Directions
β-Lap’s Cytotoxic Properties Resemble Ischemia-Reperfusion 137
2 Metabolic Implications of β-Lap Usage for Cancer Treatment 141
The Role of PARG in Modulating β-Lap-Induced Cell Death 142
B.1 Conclusions: Targeting DSB Repair to Enhance β-Lap-Induced
Lethality 144
B.2 Future Directions
LMDS as Potential Critical Lesions Formed After β-Lap
Exposure 145
Model of β-Lap-Induced DNA Damage Responses 147
Interplay Between PARP-1 and DNA-PK in Improving the Efficacy
of β-Lap 147
C. Summary 148
BIBLIOGRAPHY 150
3 LIST OF TABLES
Table 2.1. Addition of BAPTA-AM allows DNA repair after β-lap exposure 94
Table 4.1. Comparison of the mechanism(s) of cell death induced by ischemia-
reperfusion and β-lap 138
4 LIST OF FIGURES
Figure 1.1. Model of anticarcinogenic potential of DNA repair 17
inhibitors
Figure 1.2. Mechanism of β-lap-induced cell death 44
Figure 2.1. β-Lap-induced cell death is time- and Ca2+ -dependent 60
Figure 2.2. Ca2+ chelation prevents β-lap-induced nuclear morphological
changes and atypical PARP-1 and p53 proteolysis, but not STS-
induced PARP-1 cleavage 62
Figure 2.3. β-Lap induces NQO1- and Ca2+-dependent PARP-1
hyperactivation 66
Figure 2.4. NQO1- and Ca2+-dependent PARP-1 hyperactivation after
β-lap exposure is not cell type specific 68
Figure 2.5. PARP-1-dependent NAD+ and ATP pool depletion leads to cell
death after β-lap exposure in MCF-7 cells 72
Figure 2.6. PARP-1 plays an essential role in β-lap-induced apoptotic cell
death as monitored by TUNEL 75
Figure 2.7. β-Lap-induced γ-H2AX foci formation is abrogated by BAPTA-AM
pre-treatment 78
Figure 2.8. Ca2+ modulates DNA repair in β-lap-treated cells 81
Figure 2.9. Ca2+ chelation modulates DNA repair after β-lap treatment 84
2+ Figure 2.10. H2O2 causes Ca -dependent PARP-1 hyperactivation and cell
death 86
5 Figure 2.11. H2O2 causes time-dependent ATP depletion in NQO1 expressing
cells 87
2+ Figure 2.12. H2O2 causes Ca -dependent, presumably µ-calpain-mediated,
atypical PARP-1 and p53 proteolysis 88
Figure 3.1. The MRN complex is activated upon β-lap treatment 107
Figure 3.2. Perinuclear localization of DNA repair proteins following β-lap
treatment 108
Figure 3.3. Dose-dependent ATM and DNA-PK activation after β-lap
administration 111
Figure 3.4. Loss of DNA-PKcs activity potentiates β-lap-induced cell death 116
Figure 3.5. β-Lap-induced cell death is not dependent on ATM 119
Figure 3.6. β-Lap causes ATR activation and SSBs 123
Figure 3.7. β-Lap exposure induces DNA damage 125
Figure 3.8. Model of β-lap-induced cell death after lethal and sub-lethal doses 127
Figure 3.9. β-Lap-induced DNA damage is NQO1-mediated 129
6 PREFACE
As research intensifies on the search for agents that are effective against cancer, more attention has focused on the use of naturally occurring compounds. One such agent, β-lapachone, is currently under Phase I/Phase II clinical trials for the treatment of pancreatic and other cancers. β-Lapachone has a rich history stemming from its use as a folk medicine centuries past. It is a metabolite of lapachol, a main constituent of the inner bark of the Lapacho tree (Tabebuia heptaphylla, T. impetiginosa, or T. avellanedae). Natives would use the bark in a variety of preparations for the treatment of bacterial, viral, and fungal diseases to cancer. This thesis describes in more mechanistic detail the cancer chemotherapeutic properties of β-lapachone and its future as an effective and selective treatment for human cancers.
7 ACKNOWLEDGMENTS
The scientific world is an opened-door community. As such, this body of work is not solely the result of an individual effort; rather it is the melding of a number of ideas and perspectives. I would like to express my gratitude to my advisor, David A.
Boothman, Ph.D. and the members of my thesis committee Monica Montano, Ph.D.,
George Dubyak, Ph.D, Clark Distelhorst, M.D., Anna-Liisa Nieminen, Ph.D and Paul
MacDonald, Ph.D. I am especially indebted to the following individuals for their guidance, technical assistance and support: John J. Pink, Ph.D., Erik A. Bey, Ph.D.,
Kathryn E. Reinicke, Ph.D., Minh Lam, Ph.D., John-Paul Lavik, as well as the members of the Case Comprehensive Cancer Center on the 3rd floor of the Wolstein Research
Building. Thank you.
8 LIST OF ABBREVIATIONS
AGT O6-alkylguanine-DNA-alkyltransferase
AIF Apoptosis-inducing factor
AP Apurinic or apyrimidinic
AT Ataxia telangiectasia
ATM Ataxia telangiectasia mutated
ATP Adenosine 5’-triphosphate
ATR Ataxia telangiectasia and Rad3-related
ARE Antioxidant response element
BAPTA-AM 1,2-bis-(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid
tetra-(acetoxymethyl ester)
BER Base excision repair
β-Lap β-Lapachone
BRCA1 Breast cancer 1, early onset
Ca2+ Calcium
Chk2 Checkpoint 2
DCF 6-carboxy-2′7′-dichlorodihydrofluorescin diacetate,
di(acetoxymethyl ester)
DIC Dicoumarol
DNA Deoxyribonucleic acid
DNA-PK DNA-dependent protein kinase
DNA-PKcs DNA-dependent protein kinase catalytic subunit
9 DPQ (3,4-dihydro-5[4-(1-piperindinyl)butoxy]-1(2H)-
isoquinoline
DSB Double-strand break
ER Endoplasmic reticulum
γ-H2AX Phosphorylated histone H2AX
GGR Global genome repair
HR Homologous recombination
H2O2 Hydrogen peroxide
HQ Hydroquinone iCAD Inhibitor of caspase-activated DNAse
IR Ionizing radiation
KU-55933 2-morpholin-4-yl-6-thianthren-1-yl-pyran-4-one
LDH-A Lactate dehydrogenase A
MGMT O6-methyltransferase-DNA methyltransferase
MMP Mitochondrial membrane depolarization
MNNG N-methyl-N′-nitro-N-nitroguanidine
MOMP Mitochondrial outer membrane permeabilization
MMP Mitochondrial member permeabilization
MRN Mre11-Rad50-Nbs-1
NAD+ Nicotinamide adenine dinucleotide
NER Nucleotide excision repair
NHEJ Non-homologous end joining
NSCLC Non-small cell lung carcinoma
10 ns-shRNA Non-silencing short hairpin RNA
NQO1 NAD(P)H quinone oxidoreductase-1, DT-diaphorase
O6-BG O6-benzylguanine
OXPHOS Oxidative phosphorylation
PAR Poly(ADP-ribose)
PARG Poly(ADP-ribose) glycohydrolase
PARP-1 Poly(ADP-ribose) polymerase-1
PIKKs Phosphatidylinositol 3-kinase related kinases
PI3Ks Phosphoinositide-3 kinases
PLDR Potentially lethal DNA damage recovery
PMCA Plasma membrane Ca2+-ATPase
PP2A Protein phosphatase 2A
ROS Reactive oxygen species
RPA Replication protein A
UV Ultraviolet radiation
SERCA Sarcoplasmic/endoplasmic reticulum ATPase
SSB Single-strand break ss Single-stranded
STS Staurosporine
SQ Semiquinone
TCR Transcription coupled repair
TMZ Temozolomide
Topo Topoisomerase
11 TRPM Transient receptor potential-melastatin-like
TUNEL Terminal deoxynucleotidyl transferease-mediated dUTP
nick-end labeling
U1-Mel Human malignant melanoma cells
XRE Xenobiotic response element
3-AB 3-aminobenzamide
231 MDA-MB-231
231-NQ- MDA-MB-231-NQO1-negative
231-NQ+ MDA-MB-231-NQO1-positive
12 Involvement of Single- and Double-Strand Break Repair Processes in Beta-
Lapachone-Induced Cell Death
Abstract
by
MELISSA SROUGI BENTLE
β-Lapachone (β-Lap; a.k.a. ARQ 501) is a novel antitumor quinone currently in
Phase II clinical trials for the treatment of pancreatic and head/neck cancers. β-Lap has been shown to be an effective cancer chemotherapeutic agent both in vitro and in vivo against a number of human cancers that express NAD(P)H:quinone oxidoreductase-1
(NQO1). Bioactivation of β-lap by NQO1 caused a futile oxidoreduction, leading to reactive oxygen species generation (ROS). Unique to this compound was its ability to kill cancer cells regardless of abnormalities in commonly altered apoptosis-related proteins, such as p53, Bcl-2, and Bax/Bak.
This thesis describes in more mechanistic detail the cell death pathway activated after β-lap treatment, with particular focus on its involvement with, and resistance by,
DNA repair. We demonstrated that the NQO1-dependent reduction of β-lap caused ROS generation, DNA breaks, and triggered calcium (Ca2+)-dependent γ-H2AX formation and
PARP-1 hyperactivation (Chapter 2). PARP-1 hyperactivation was an integral part of cell death triggered by this compound, causing NAD+ and ATP losses that suppressed
DNA repair and caused cell death. PARP-1 inhibition or intracellular Ca2+ chelation
13 protected cells from β-lap-induced cell death. Similarly, hydrogen peroxide (H2O2), but not N-Methyl-N’-nitro-N-nitrosoguanidine (MNNG), caused Ca2+-mediated PARP-1 hyperactivation and death. Thus, Ca2+ appears to be an important co-factor in PARP-1 hyperactivation after ROS-induced DNA damage.
To explore DNA repair as a resistance factor(s) that might impede cell death, we explored the contribution of DNA double-strand break (DSB) repair following β-lap exposure. β-Lap treatment resulted in the NQO1-dependent activation of the MRE11-
Rad50-Nbs-1 (MRN) complex, as well as ATM Serine 1981, DNA-PKcs Threonine
2609, and Chk1 Serine 345 phosphorylation, indicative of ATR activation. These data suggested the simultaneous activation of both homologous recombination (HR) and non- homologous end joining (NHEJ) pathways. However, inhibition of NHEJ potentiated β- lap lethality.
These data provide insight into the mechanism by which β-lap kills cancer cells expressing endogenously elevated NQO1 levels. Furthermore, targeting NHEJ to enhance the therapeutic efficacy of β-lap alone, or in combination with other agents that are also potentiated by DSB repair inhibitors, is warranted.
14 Chapter 1: Introduction
New Tricks for Old Drugs: The Anticarcinogenic Potential of DNA Repair
Inhibitors
This work was published in the Journal of Molecular Histology
The Journal of Molecular Histology 2006 September; 37(5-7):203-18
INTRODUCTION
Mammalian cells are continuously bombarded with a wide variety of exogenous and endogenous genotoxic stresses that target DNA producing genetic alterations. DNA breaks may result from exposure to exogenous agents, such as ultraviolet radiation (UV), ionizing irradiation (IR), and many cancer chemotherapeutic agents (e.g. cisplatin, DNA
Topoisomerase (Topo) poisons). Spontaneous DNA breaks are primarily a consequence of cellular metabolism and the byproducts of oxidative phosphorylation which causes the generation of a number of electrophilic species that have the potential to damage DNA, lipids, and proteins within the cell. Inefficient or inaccurate repair can cause cell death or genomic instability that is fundamental to a number of human pathologies, including neurodegeneration, cardiovascular abnormalities and cancer (1). Therefore, a number of crucial defense mechanisms have evolved to combat the potential deleterious consequences of DNA damage in order to maintain genomic integrity and maintain normal cellular phenotypes. However, lack of proper DNA repair, or inaccurate repair of
DNA lesions, may result in cell death or survival of cells with mispaired DNA which
15 may then lead to tumorigenesis. Under these circumstances, it may be advantageous for organisms with severely damaged cells to convert these DNA lesions into lethal events, promoting cell death rather than transformation (Figure 1.1).
In this article, we review the basic mechanisms of DNA double- and single- strand break repair with particular focus on poly(ADP-ribose) polymerase-1 (PARP-1) and the potential for pharmacological intervention of these DNA repair mechanisms as a means of preventing carcinogenesis. We review recent data on the mechanism of action of a unique quinone, β-lapachone (β-lap), that holds promise for use as a dual anticancer and anticarcinogenic agent.
16
Figure 1.1. Model of anticarcinogenic potential of DNA repair inhibitors. Cells are continuously exposed to a wide-variety of both endogenous and exogenous DNA damaging agents. To protect the integrity of the genome, mammalian cells have evolved a number of distinct, yet overlapping, DNA repair mechanisms to correct various lesions that may be formed. In some instances, if DNA damage cannot be repaired completely and with high fidelity it is more advantageous for the cell to die rather than survive as a neoplastic transformant. It is at the point of carcinogenic exposure that DNA repair inhibitors can be utilized to either: (1) shift the repair process from a less accurate repair mechanism (e.g. NHEJ) to a more accurate repair pathway such as HR; or (2) commit the cell to death before initiating error-prone DNA repair, leading to large deletions and insertions, as well as increases in mutation rates that would cause a pre-malignant
17 phenotype. This idea was first proposed by Boothman, Shlegel, and Pardee in 1989
(Boothman et al. 1989), but is updated here due to the increase in knowledge of DNA repair mechanisms over the past 10 years.
18 Targeting DSB response pathways for the prevention and treatment of cancer
DSB repair mechanisms
Extensive studies using the yeast genetic systems, Saccharomyces cerevisiae and
Schizosaccharomyces pombe have revealed a number of important genes and proteins essential to checkpoint signaling mechanisms that compose the DNA damage response pathways. Nearly all of the identified yeast genes implicated in the DNA damage response have mammalian homologues illustrating the highly conserved nature of DNA damage signaling (2). Due to the large variety of genetic lesions that eukaryotic cells must deal with, a multitude of different and partly overlapping DNA damage repair pathways have evolved. The most deleterious lesion, the DSB, can arise from IR, free radicals, chemicals, or during replication through DNA single-strand breaks (SSBs) (3).
The signal transduction pathways activated by DSBs require the recruitment and activation of the phosphatidylinositol 3-kinase related kinases (PIKKs) (4) ataxia- telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR). There are two primary mechanisms of DSB repair: homologous recombination (HR) which is regulated in an ATR- or ATM-dependent manner (5-7), and non-homologous end joining
(NHEJ) regulated by the DNA-PK complex.
Conservative HR repairs breaks using genetic information provided by an undamaged sister chromatid or chromosomal homologue during S-G2 phase. ATM is the primary responder in HR, which is evident by the hereditary genetic disorder ataxia telangiectasisa (AT), a radiosensistivity and genome instability disorder that is
19 characterized by cerebellar degeneration and a predisposition to cancer. ATR may be activated later if stalled replication forks arise after replication is blocked, or after bulky lesions are caused by UV-induced DNA damage (8). In mammalian cells, it is believed that DSBs are recognized by the Mre11-Rad50-Nbs1 (MRN) complex that in turn, recruits ATM to the break site (9). ATM signaling mediates a number of pathways, including cell cycle arrest after IR-induced damage (10). Cell cycle arrest mediated by p53 at G1-S phase occurs through inhibition of checkpoint 2 (Chk2) which is mediated by p21 that provides time for DNA to be repaired prior to replication. A permanent G1-S arrest prevents the proliferation of severely damaged cells allowing time for repair or initiation of cell death (e.g. apoptosis). Furthermore, ATM can regulate a G2-M arrest for cells that are irradiated in G2. The substrates of ATR phosphorylation overlap with those of ATM, and ATR also causes similar cell-cycle-checkpoint arrest (4).
Another early event in DNA damage detection responses is the phosphorylation of histone H2AX (γ-H2AX) which can be performed redundantly by ATM, ATR or
DNA-dependent protein kinase (DNA-PK) (11, 12). γ-H2AX foci formation is necessary to recruit and maintain mediator proteins such as breast cancer 1 early onset (BRCA1), and the MRN complex at the site of the DSB (13). Free DNA ends are processed by the
MRN complex and results in the formation of 3’ single stranded (ss) DNA overhangs where replication protein A (RPA) can bind. Rad51 and Rad52 are then recruited to the
DSB. The complex of RPA-Rad51-Rad52 comlex then searches for regions of homology in the non-damaged sister chromatid. Upon finding a homologous region, this complex mediates strand invasion and DNA exchange occurs creating the DNA Holliday junction.
DNA polymerase synthesizes the genetic information required to seal the break, which is
20 followed by ligation and resolution of the Holliday junction resulting in error-free DNA repair.
NHEJ of DSB repair involves the DNA-PK complex, which is composed of the
DNA-dependent protein kinase catalytic subunit (DNA-PKcs, a member of the PIKK- related kinase family (14, 15)) and the targeting subunit, Ku70-Ku80 heterodimer.
Unlike HR, NHEJ takes place throughout the cell cycle. It involves the direct ligation of
DNA ends via the Ku70-Ku80 heterodimer and the recruitment of protein kinase, DNA-
PKcs (16, 17). In a process still poorly understood, Ku70-Ku80 holds the two DNA ends together and aids in the end-to-end ligation by DNA ligase IV and XRCC4. DNA repair mediated by this pathway is considered non-conservative, as it frequently leads to error- prone repair of DSBs by loss of a small number of terminal nucleotides or insertions at the repair joint (18).
HR pathway inhibitors: effects on DNA repair and carcinogenicity
In general, if the theory that inhibiting error-prone repair would prevent carcinogenesis, inhibition of HR would not be advised even if the methodology for enhancing anti-tumor activity could be devised. Many traditional inhibitors of HR that prevent ATM activation, such as wortmannin and LY294002, are non-specific and inhibit other phosphoinositide-3 kinases (PI3Ks) (19). These drugs are, therefore, restricted in their use clinically. Recent reports of the isolation of a novel small molecule ATM inhibitor
2-morpholin-4-yl-6-thianthren-1-yl-pyran-4-one (KU-55933), may provide the first true
21 test of its use as a DNA repair inhibitor to enhance radiosensitivity. KU-55933 is a competitive ATP inhibitor that has high selectivity for ATM and low cytoxicity (20).
In addition to ATM, RAD51 and BRCA1/2 have been studied as putative targets to block HR for chemotherapy against non-small cell lung carcinomas (NSCLC) and breast cancer (21, 22). Protein knock-down of RAD51 using anti-sense oligonucleotides was found to cause an increase radiosensitivity in NSCLC. To date, however, no compounds have been found to modulate the activities of RAD51 or BRCA1.
The foremost challenge in combating cancer as a disease is preventing its onset.
Thus far, most research has relied on ways to treat existing malignancies rather than prevent them. Of the few studies examining compounds to prevent cancer, much attention has been focused around natural products and foodstuffs (e.g. green tea, garlic, caffeine, etc.). Most of these compounds function to decrease initial DNA damage by serving as antioxidants and/or enhancing cellular DNA repair systems, thereby preventing carcinogenesis. The steps that lead to the transformation of normal to tumor cells are complex, making it difficult to pinpoint targets to prevent neoplastic initiation and possibly later, progression. Caffeine was one of the earliest DSB repair inhibitors examined for its anticarcinogenic properties. Administration of caffeine in vitro decreased the occurrence of skin tumors (23). It was hypothesized that this effect was due to the abrogation of G2 phase cell cycle checkpoint regulation caused by DNA damage.
It was theorized that this cell cycle checkpoint abrogation by caffeine left no time for potentially lethal DNA damage recovery (PLDR), and prevented the survival of possibly tumorigenic cells. Further studies in vivo demonstrated that unscheduled DNA synthesis
(USD) decreased approximately 70% in hepatocytes from caffeine treated mice. Taken
22 together, these studies suggested that the anticarcinogenic effect of caffeine might be caused by suppression of DNA repair (24). Although caffeine inhibits ATM it also affects many other cellular processes, making interpretation of its effects difficult at best.
Specific genetic manipulations of ATM in vitro and in vivo, through knock-out or knock- down technologies should be used to examine the role of HR in carcinogenesis following the administration of agents that induce HR or NHEJ.
Cell cycle checkpoints can be initiated by ATM and ATR in response to DNA damage, thereby allowing cells time to repair damage before duplicating their DNA or initiating cell death. Cell cycle checkpoint activation is one of the main causes of normal cell transformation after radio/chemotherapy, since the likelihood of initiating PLDR is greatly increased. In theory, targeting checkpoints under these circumstances, should prohibit the recovery of potentially carcinogenic cells. Checkpoint interference can be initiated in G1, S, or G2 phases of the cell cycle. In the past ten years, G2 checkpoint control has become considerably attractive to researchers in drug development. For example, targeting Chk1 with inhibitors, such as benzimidazole and quinolinone B, has been utilized to abrogate the G2 checkpoint and sensitize cells to most DNA therapeutic agents both in vitro and in vivo (25). The effects of these inhibitors on carcinogenesis need to be examined.
NHEJ pathway inhibitors: effects on DNA repair
Due to the predominant role of DNA-PK in NHEJ, a large number of comprehensive studies have focused on this protein complex for cancer therapy and prevention (26).
23 Resistance to chemo/radiotherapy has been demonstrated to result from an increased level of DNA-PK in tumor cells. A number of potent DNA-PK inhibitors have been created and are currently in Phase I clinical trials. The vast majority of these compounds inhibit DNA bound DNA-PKcs, and prevent autophosphorylation of the molecule. This prohibits DNA-PK release from free DSBs, thereby blocking end processing and inhibiting repair of the break.
In addition, these compounds sensitize tumor cells to DNA damaging agents. In particular, NU7026 increased the sensitivity of irradiated tumor cells to the PARP-1 inhibitor AG14361, thus decreasing its LD50 (27, 28). Other DNA-PK inhibitors,
IC87102, IC86621, and IC87361, increase the cytotoxic effects of radiation in both tumor cells and their surrounding microvasculture (29-31). Vanillin, a selective inhibitor of
DNA-PKcs, exhibits synergistic function with the anti-cancer drug, trichostatin A (TSA, a histone deacetylase inhibitor), but not radiation (32). SU11752 is another selective inhibitor of DNA-PK that does not interfere with the ATM kinase pathway or cell cycle progression (33). Salvicine, a diterpenoid quinone, was found to have anti-tumor properties via inhibition of DNA-PK-mediated NHEJ and Topo II activity (34, 35).
Salvicine treatment can overcome multidrug-resistance in several different tumor cell lines (36, 37). The newly developed DNA-PK inhibitor, arylmorpholine (AMA37), in concert with G2 checkpoint inhibitors, dramatically enhanced the selective killing of tumor cells treated with IR or DNA-damaging chemotherapeutic agents (38).
In addition to enhancing the effects of a multitude of DNA damaging agents, inhibition of DNA-PK has the potential to prevent cellular transformation. The splice variants of DNA-PKcs can influence which DSB repair pathways are activated following
24 DNA damage. In particular, DNA-PKcs isoform II can inhibit repair by HR in quiescent cells, thereby reducing the potential loss of genetic information or genetic rearrangements when no sister chromatids are available to serve as a template (39). This regulatory mechanism can be manipulated in situations of PLDR, when both HR and NHEJ are activated. Thus, in actively proliferating cells it is feasible that inhibition of DNA-PKcs isoform II after DNA damage would lift the inhibitory control over HR and shift the repair pathways from error-prone NHEJ to that of error-free HR. This would increase the fidelity of repair (39), and enhance the reversion of damaged cells to a normal phenotype, as well as prevent secondary carcinogenesis. However, the process may not radiosensitize cancer cells rendering the therapeutic rationale ineffective.
Mechanisms of SSB response pathways and utility as targets for chemotherapy
Basic mechanisms of mammalian base excision and nucleotide excision repair
Mammalian cells have two basic types of excision repair: nucleotide excision repair
(NER), and base excision repair (BER). NER is essential in mammals for the removal of bulky DNA lesions, such as UV-induced pyrimidine dimers. Defects in NER cause the autosomal recessive disorder xeroderma pigmentosum (XP), which leads to an increase in sun-induced skin cancer. There are two major types of NER: transcription coupled repair
(TCR) and global genome repair (GGR) (40, 41). TCR is specifically activated in response to damage-blocked RNA polymerase II, while GGR surveys the entire genome for lesions, relying on the XPC-hHR23B proteins to recognize damage located in non-
25 transcribed regions of DNA (42). Both mechanisms, however, serve to unwind condensed chromatin around the lesion allowing for the recruitment of repair-associated proteins to the break site (40, 43).
BER recognizes and repairs small base modifications mainly as a result of cellular metabolism, as well as alkylated and deaminated bases (44). A lesion-specific glycosylase removes the modified base, creating an apurinic or apyrimidinic (AP) site, or alkaline-labile sites. AP sites are recognized by AP endonuclease (APE1) that cleaves the sugar-phosphate backbones at the position of the missing bases. Finally, exonucleases are recruited to remove the additional nucleotides from around the site of the damage, and the newly created gap is filled and resealed by DNA polymerase β and ligase III, respectively (44).
Using the DNA SSB repair pathways for cancer treatment
Targeting BER is an alternative strategy to enhance the effects of DNA damaging agents.
Methoxyamine (MX) is an agent that can directly react with the aldyhydic C1 atom of AP sites exposed by glycosylases (45). These sites are resistant to binding and cleavage by
APE1, potentiating the effects of cytotoxic agents that mediate formation of AP sites.
Alternatively, blocking APE1 function can be used to directly target BER, and several small molecule inhibitors have been developed. E3330 is one such compound that prohibits APE1 by blocking its redox activity (46), while other compounds like
CRT0044876 have been shown to be effective in sensitizing cells to IR in preclinical studies (47).
26 DNA polymerase β plays an essential role in maintaining DNA strand integrity, and as such it has become a target to inhibit BER (48). Recently, the synthetic compound pamoic acid has been identified and can specifically block the polymerase activity of
DNA polymerase β in vitro (49). However, the degree to which it can interfere with normal DNA synthesis is still unclear and further studies need to be performed to better elucidate its mechanism of action. Modulating BER for the prevention of carcinogenesis will be discussed later in terms of PARP-1 inhibitors.
Targeting direct repair for chemotherapy
One of the challenges in treating cancers with DNA alkylating agents is overcoming direct repair by the O6-alkylguanine-DNA-alkyltransferase (AGT), and by O6- methylguanine-DNA-methyltransferase (MGMT), which remove O6-alkyl guanine lesions. Thus, combination treatment of alkylating agents with AGT inhibitors has been utilized to overcome methylating and chloroethylating agent resistance (50). Loss of
MGMT is related to increased carcinogenic risk and increased sensitivity to alkylating drugs. Temozolomide forms O6-methylguanine (O6mG), 7-methylguanine (N7mG) and 3- methyladenine (N3mA) DNA adducts that lead to genotoxic lesions and cytoxicity (51).
O6-benzylguanine (O6-BG) was the first pseudosubstrate identified and used to inactivate
MGMT by covalent transfer of the benzyl group to the cysteine residue in the active site
(52, 53). Subsequent O6-BG derivatives, such as O6-benzyl-2’-deoxyguanosine, 2- amino-O4-benzylpteridine and O6-4-bromothenylguanine, show higher efficacy and water solubility than O6-BG. These derivatives are now some of the most potent drugs in
27 clinical trials. Thus, future AGT inhibitors need to be administered in synergy with other
DNA damaging agents and their effects need to be evaluated both in vitro and in vivo.
The use of these inhibitors to prevent carcinogenesis has not, thus far, been examined in detail.
Poly(ADP-ribosyl)ation, PARP-1, and DNA repair
PARP-1: the basics
In 1963, Chambon and colleagues reported that exogenous addition of NAD+ to rat liver extracts resulted in the synthesis of a polyadenylic acid, later to be identified as poly(ADP-ribose) (PAR) (54). Later, this ADP-ribosyl transfer reaction was found to be primarily mediated by the ~116 kDa nuclear DNA damage sensor protein PARP-1 isoform.
PARP-1 is an abundant nuclear eukaryotic enzymes present at approximately 106 molecules/cell. PARP-1 serves as a DNA nick-sensor and signaling molecule that responds to DNA strand breaks, acting to facilitate DNA repair (55). The two terminal zinc fingers of PARP-1 recognize and bind with high affinity single- and double- stranded DNA breaks helping to facilitate BER, as well as HR DSB repair (56, 57).
PARP-1 is rapidly activated up to 100-fold after binding SSBs and subsequently forms homodimers that catalyze the conversion of β-nicotinamide adenine dinucleotide (β-
NAD+) to poly(ADP-ribose) branched or linear polymers of 50-200 units. Once formed,
PARP-1 catalyzes the covalent transfer of PAR onto glutamic acid, aspartic acid, or
28 lysine residues on a variety of nuclear acceptor proteins, including histones and PARP-1 itself as part of its autoregulation. PARP-1 is responsible for nearly 90% of DNA damaged-induced PAR synthesis (58). The biological effects of PARP activation rely mostly on the ADP(ribosyl)ation reaction: the liberation of free PAR molecules from
PAR-modified proteins confers a multitude of intracellular effects (e.g. cyclic (ADP- ribose) is a potent Ca2+ activator), covalent PAR-modification of nuclear target proteins affects their intracellular function, as well as reduction in NAD+ levels. Poly(ADP- ribosyl)ation reactions occur in a number of multicellular organisms ranging from plants to humans, but it is absent in yeast and prokaryotes. Since the discovery of PARP-1, an entire superfamily of PARPs have been characterized in humans, each encoded by a set of 18 separate genes with homologous catalytic domains (i.e. “the PARP signature sequence”), and distinct structures and functions (59). PARP-1 is constitutively expressed during the cell cycle. Therefore, its function is primarily regulated at the level of its catalytic activity through downregulation via auto(ADP-ribosyl)ation (60).
However, some reports have indicated augmented PARP-1 protein levels in a variety of human cancer cell lines (61, 62).
PARP-1-mediated poly(ADP-ribosyl)ation is a dynamic process with a short polymer half-life in the range of minutes. Two unique evolutionarily conserved enzymes poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribosyl protein lyase are responsible for the catabolism of PAR (63). PARG liberates both linear and branched PAR, whereas
ADP-ribosyl lysase removes the protein-proximal ADP-ribose monomer (63).
Targeting these proteins should accelerate caspase-independent cell death in tumors defective in caspase-mediated apoptosis after treatment with agents that kill via
29 PARP-1 hyperactivation (see mechanism of β-lapachone below). Targeting these processes should kill damaged cells rather than allowing them to survive with carcinogenic lesions. Therefore, this strategy should allow one to augment DNA damaging agents, while preventing carcinogenesis. Ideally, agents that hyperactivate
PARP-1 would be tumor selective.
PARP-1 and DNA repair
PARP-1 activation and subsequent PAR synthesis are immediate responses to DNA damage. A number of genetic studies have clearly established the importance of DNA- damage-induced PARP-1 activity to aid in the repair and recovery of cells following low to moderate levels of DNA damage (64). In addition, PARP-1 inhibitors can potentiate the action of a number DNA-damage-inducing cancer drugs (60). PARP-1 is crucial for the repair of SSBs by BER through its interactions with XRCC-1 and ligase III, which activates DNA polymerase β (65-67). It is postulated that PAR-modified histones facilitates relaxation of chromatin at the site of DNA damage, and recruits specific DNA repair factors to the sites of DNA breaks. PARP-1 appears to play a role in DSB repair independently of p53 function (68) or DNA-repair involving Cockayne syndrome-B protein (CSB) (69). Interestingly, genetic ablation or chemical inhibition of PARP-1 in
BRCA1- or BRCA2-deficient cells reduced clonogenic survival of these cells compared to PARP-1 deficient BRCA1- or BRCA2-proficient cells (22, 70). These and other studies implicated PARP-1/PAR formation as major players in a number of DNA repair
30 pathways. However, the exact mechanisms by which PARP-1 modulates these repair pathways remains unresolved.
PARP-1 hyperactivation
DNA strand-break-induced PARP-1 activation is a double-edged sword. On one hand, it has a role as a survival factor in the presence of low to moderate levels of DNA damage, promoting genomic integrity and DNA repair. On the other hand, since PARP-1 responds to DNA damage in a dose-dependent manner, it can be hyperactivated, leading to cellular depletion of NAD+ through (ADP)-ribosylation of nuclear substrates. The net result for the cell is a rapid and dramatic energy loss leading to cell death (71, 72).
PARP-1 hyperactivation is, therefore, an energetically expensive process. Rapidly declining NAD+ levels can reduce ATP levels since NAD+ synthesis is ATP-dependent.
Also, NAD+ loss directly affects a vast array of metabolic pathways such as glycolysis and the pentose phosphate pathway. The type of cell death (e.g. necrosis or apoptosis) elicited in response to PARP-1 hyperactivation is thought to be controlled by the type/duration of genotoxic stimulus in addition to the metabolic state of the cell at the time of damage. For example, cancer cells that are highly glycolytic and rapidly growing die more readily from PARP-1-mediated NAD+ and ATP depletion (e.g. programmed necrosis) than less actively growing normal cells that use oxidative phosphorylation and a variety of metabolic substrates (73).
PARP-1 activation in response to cellular trauma such as ischemia-reperfusion
(74, 75), myocardial infarction (76), and reactive oxygen species-induced injury (3, 72),
31 has been well documented. Under these conditions, genetic deletion or pharmacological inhibition of PARP-1 can protect cells from death. To date, few studies have attempted to exploit PARP-1 hyperactivation for enhanced radio/chemotherapies. Agents known to activate these processes such as H2O2 and N-Methyl-N’-nitro-N-nitroguanidine (MNNG) only do so at micro- and millimolar doses, respectively. Therefore, selective agents that can activate these processes are needed and theoretically have a multitude of benefits including: (a) killing independently of p53 and caspases; (b) selectively killing cancer cells that rely on glycolysis rather than oxidative phosphorylation; (c) augmenting anti- cancer agents that damage DNA, including agents that were not efficacious due to their formation of SSBs, instead of more lethal DSBs; and (d) killing cancer cells that have up- regulated levels of pro-survival factors that protect against caspase-mediated processes.
These processes include over-expressed Bcl-2, Bcl-XL, and loss of Bax expression.
Recent data indicate that one agent, β-lapachone, holds great promise in this regard.
PARP-1 intervention as a means of inhibiting multiple DNA repair pathways and transformation
Since PARP-1 has been implicated in a number of separate DNA repair pathways it is has come to the forefront of cancer therapy. In recent years, many small molecule PARP-1 inhibitors have been synthesized to enhance the susceptibility of tumor cells to DNA damaging agents (77, 78). Many first generation compounds are competitive NAD+ inhibitors. These compounds tend to be analogues of nicotinamide, such as 3- aminobenzamide, and have been used mainly for in vitro studies of PARP-1 activity (60).
32 The second generation of PARP-1 inhibitors from KuDOS Pharmaceuticals, such as
NU1025, NU1064 and NU1085, function mainly to enhance the cytotoxic effects of
DNA-alkylating agents and other agents, such as camptothecin (79, 80). Unfortunately, the lack of specificity of these drugs restricted their use in clinical trials. Agouron
Pharmaceuticals developed another line of inhibitors (e.g. AG14361) with improved specificity and significant chemo/radiosensitization in tumor xenografts (81).
Furthermore, the newly patented triciclic benzimidazoles exhibit great promise for effectively inhibiting PARP-1 in an animal tumor model (60).
The discovery that BRCA1/2 mutant cells are hypersensitive to PARP-1 inhibitors opened new avenues for treatment of tumors from BRCA2 deficient patients (70, 82). As mentioned previously, BRCA1/2-deficient cells are exquisitely sensitive to PARP-1 inhibitors. In these cells, it is postulated that PARP-1 inhibition causes an increase in
SSBs, and ultimately leads to replication fork collapse. Normally, under these circumstances HR would be activated, but the BRCA1/2 deficiencies prevent these cellular events from occurring. Small-molecule PARP-1 inhibitors, such as KU0058684 and KU0058, have been designed and exploited in treating BRCA-deficient cells (22).
An alternative strategy is to enzymatically inactivate PARP-1 bound to DNA since this would prevent other DNA repair proteins from having access to DNA lesions.
This is similar to the mechanism utilized by the cell when it is undergoing apoptosis (83).
PARP-1 cleavage by caspases to a 24 kDa fragment containing the DNA binding domain facilitates apoptosis by blocking access of other repair proteins to fragmented DNA (84,
85). AG14699 and the methylating agent temozolomide (TMZ), have been a successful combination through Phase I clinical trials. INO-1001, another promising PARP-1
33 inhibitor, can also increase the efficacy of TMZ for the treatment of malignant gliomas, and is currently in Phase II clinical trials (86). The number of inhibitors of the PARP family has grown exponentially over the last decade, however, caution must be taken as the side-effects of inhibiting DNA repair in undamaged normal cells can be detrimental.
Tumor-selectivity is at present missing in these proposed strategies.
Aside from cancer therapy, PARP-1 inhibition has been utilized to combat other diseases. One compound, 4-iodo-3-nitrobenzamide (INO2BA), represents a new class of therapeutic agents for the treatment of acquired immune deficiency syndrome (AIDS).
This work has now expanded for use as a cancer treatment and is ready for use in clinical trials. INO2BA selectively disrupts the F1 DNA binding zinc finger of PARP-1/2, thereby prohibiting PARP binding to DNA and becoming activate. This effect occurs independently of the type of DNA breaks (87).
The limited number of studies using PARP inhibitors to prevent carcinogenesis have been performed using the traditional benzamide family of PARP inhibitors. Most studies in vivo using these compounds and their derivatives have been shown to enhance carcinogenesis (88, 89). An increase in pancreatic islet cell tumors was seen in rats given a combination of alloxan and nicotinamide (90). Other studies have shown that methylazoxymethanol acetate added to water generated tumors in zebra fish, and this carcinogenic action was potentiated by the addition of 3-aminobenzamide (3-AB) (91).
However, other laboratories have found decreased transformation in human and rodent cells after treatment with DNA damaging agents in combination with PARP-1 inhibitors
(92, 93). X-ray- and UV-induced transformation were significantly decreased in murine and hamster cells treated with 3-AB or benzamide (92). In addition, transformation by
34 phorbol esters was inhibited by benzamides (88). These mixed reports using PARP inhibitors to prevent transformation need further investigation in order to determine the utility of these compounds for the prevention of carcinogenesis.
β-Lapachone: an anticarcinogenic DNA repair inhibitor
Brief history of β-lapachone
β-Lapachone (β-lap, 3,4-dihydro-2,2-dimethyl-2H-naptho[1,2-b] pyran-5,6-dione) is a naturally occurring quinone derived from the lapacho tree, (Tabeuia avellanedae) native to Central and South America. The synthesis and chemistry of β-lap and related compounds was initially investigated in the late 19th and early 20th centuries by the chemist, Samuel Hooker (94, 95). Following Hooker’s studies, investigations of quinones and their toxicities revolved around their antimalarial effects (96). Although some clinical studies of the bioactivity of napthoparaquinones were reported early in
1940, it was Decampo’s report of β-lap’s potential as an antitrypanosomal agent in 1977 that revitalized the study and characterization of this unique natural compound (97).
β-Lap: use as an antineoplastic agent
Decampo et. al., reported that β-lap inhibited the growth of sarcoma 180 (S-180) ascites tumors in mice (98). β-Lap was cytotoxic to leukemia cells in Balb/c mice infected with
Rauscher Murine Leukemia virus (99). In 1987, studies from the laboratory of Pardee
35 and colleagues reported that a 4 h β-lap treatment, following radiation, enhanced the radiosensitization of Hep-2 cells (100). Subsequent studies using human malignant melanoma cells (U1-Mel) confirmed the synergistic lethality of x-rays when β-lap was administered post-irradiation (101). These studies firmly established the ability of β-lap to act as a radiosensitizer. However, the mechanism of action of β-lap remained unknown, partially due to the focus of elucidating its mechanism in combination with IR exposure.
Our laboratory then adopted a strategy to better understand the lethal effects of β- lap alone in the absence of IR exposure. β-Lap was found to be cytotoxic to a variety of specific human cancers. These included breast, colon, prostate and lung (102-105).
Unfortunately, many initial studies in vitro with β-lap (excluding our own studies) were performed with continuous administration of this drug (106-109). The continuous treatment of cancer cells with β-lap greatly masked the therapeutic efficacy of this compound. The mechanism of this “masking” of the therapeutic window is related to its effects on normal cells and will be discussed later.
Further studies from our laboratory determined the principle determinant of β-lap- mediated cytotoxicity. Although other reports indicating the involvement of Topo poisons and E2F-1 appeared, our laboratory clearly established that the two-electron oxidoreductase, NAD(P)H quinone oxidoreductase-1 (NQO1) was the principle determinant for cytoxicity in human cells (110). Cells deficient in NQO1 were resistant to β-lap, and those same cells transfected with NQO1 were made sensitive. Furthermore,
NQO1-positive cells exposed to the NQO1 inhibitor, dicoumarol, were rendered resistant
(110). No other protein target in the cell has demonstrated as much importance for β-lap-
36 mediated cytoxicity as NQO1. This finding has led to many of our current studies that take advantage of the NQO1-dependent, antitumor therapeutic window, for the selective treatment of NQO1-positive cancers. NQO1-positive cancers account for >60% of human NSCLC, prostate, pancreatic, breast, and colon cancers (111, 112)
Anticarcinogenic potential of β-lap
NQO1 (DT-diaphorase) is a ubiquitously expressed flavoenzyme that catalyzes the two- electron reduction of a variety of substrates (113). NQO1 reduces toxic, reactive and unstable quinones. This one-step two-electron reduction bypasses the creation of toxic intermediates (e.g. a semiquinone radical), and spares the cell from the formation of reactive oxygen species (ROS) by producing a glutathione-conjugated non-toxic hydroquinone that can be excreted from the cell. However, in the case of β-lap, NQO1
“bioactivates” the compound, creating a futile cycle between the parent quinone and the hydroquinone form. As a result of NQO1-mediated futile cycling, ROS are generated that contribute to the toxicity associated with β-lap administration to cancer cells. The mechanism of NQO1-mediated β-lap-induced cell death will be discussed in more detail in the next section.
NQO1 is ubiquitously expressed at low levels in most murine and human tissues
(111). Interestingly, no detectable levels of NQO1 activity are found in the liver of humans (111). Importantly, many human tumors including those found in breast, colon, lung, prostate and pancrease, overexpress NQO1 more than 10-fold when compared to adjacent or nonadjacent normal tissues (111). This knowledge has led to a number of
37 clinical screens for compounds that are “bioactivated” by NQO1 (114-117). In addition to β-lap, compounds such as mitomycin-C, EO9 and streptonigrin have been reported to selectively kill NQO1-proficient cancer cells (113).
One critical factor for the development of such compounds as global chemotherapeutic agents is that there are NQO1 polymorphisms within the human population. Two polymorphic point mutations have been characterized: (a) C609T which accelerates proteasomal-mediated degradation of the protein; and (b) C465T which leads to increased alternative splicing, producing a truncated mRNA lacking exon 4 (118-121).
Tissues (including patient tumors) that carry these point mutations lack NQO1 activity.
Therefore, compounds “bioactivated” by NQO1 would not be efficacious against these cancers. Thus, rapid screening techniques to exclude such patients are crucial to develop prior to Phase I/II clinical trial development.
In addition to using β-lap as an antitumor agent, an added strategy for the simultaneous or separate use of β-lap may be to administer it prophylactically. This use of β-lap and other NQO1-activated quinones may prevent carcinogenesis prior to solid tumor formation with minimal toxicity in low NQO1- expressing normal tissues. Since the NQO1 gene is regulated by both the antioxidant response element (ARE) and the xenobiotic response element (XRE), one may envision that the elevated NQO1 levels found in many human tumors may occur as a survival advantage to highly metabolic cells. ARE-mediated NQO1 gene upregulation has been shown to be increased by a variety of antioxidants as well as tumor promoters, H2O2 and hypoxia (122-124). This over-expression may lead to malignant cellular phenotypes that utilize the upregulation of
38 NQO1 for their survival and protection from cytotoxic oxidative stress which occurs during tumor formation.
Earlier studies from our laboratory demonstrated that a major contribution of β- lap’s ability to synergize with IR was its ability to inhibit DNA repair. Inhibiting the repair of malignant cellular phenotypes by killing NQO1-over-expressing metastatic or neoplastically initiated cells may prevent solid tumors from forming. Thus, prophylactic administration of β-lap may halt the transformation of normal cells to cancer cells, as well as metastatic cells in adjuvant therapies. Importantly, the unique mechanism of β- lap-induced cell death is not inhibited by the loss of apoptotic factors such as Bax, Bak, p53 or alterations in cell cycle check-points. Therefore, all known resistant cancer variations would not escape the prophylactic administration of NQO1 bioactivated compounds such as β-lap.
Mechanism of β-lap toxicity in cancer cells
NQO1 metabolism of β-lap generates ROS
Due to the rapid and robust futile cycling of β-lap with NQO1, a large burst of ROS is produced in the cytoplasm as measured by increased oxygen consumption, accumulation of disulfide glutathione, and fluorescence of the redox sensitive dye dichlorodihydrofluorescein diacetate (DCF). The massive NQO1-dependent production of ROS results in concomitant DNA damage as observed by comet assays (125, 126).
39 ROS generation is a major catalyst of a multitude of downstream effects that will be described in more detail below.
β-Lap causes calcium release from the endoplasmic reticulum (ER)
Calcium (Ca2+) is a key cellular regulatory molecule that plays many essential roles in life-sustaining as well as life-ending processes. A sustained rise in Ca2+ levels has been associated with perturbations in cellular structures and functions. The release of Ca2+ induces oxidative stress, mitochondrial membrane depolarization and cessation of ATP synthesis (127-130). Therefore, drugs that alter Ca2+ homeostasis may have an advantage in killing cancer cells. Our studies with β-lap have shown that the mechanism of toxicity is intimately involved with alterations in Ca2+ levels. Early studies showed that a rise in cytosolic Ca2+ from the ER was observed within minutes of β-lap administration (131).
β-Lap-induced elevations of cytosolic Ca2+ levels were blocked when cells were pretreated with the intracellular calcium chelator, 1,2-bis-(2-aminophenoxy)ethane-
N,N,N’,N’-tetraacetic acid tetra-(acetoxymethyl ester) (BAPTA-AM). Cells that were co- treated with β-lap and dicoumarol were completely spared from intracellular calcium level elevations, strongly indicating that NQO1 was important in this process. In these early experiments, pretreatment with BAPTA-AM partially protected cells from downstream affects including mitochondrial membrane depolarization, ATP loss and the formation of apoptotic (TUNEL positive) cells (131). More recently, we noted that a critical time to death (CTD) following β-lap exposure was associated with Ca2+ release
(125). A 2 h exposure of β-lap was sufficient to induce cell death in NQO1-proficient
40 MCF-7 cells, as well as other cell types (132, 133). Pre-treatment of cells with BAPTA-
AM during this time completely suppressed β-lap-induced cell death. These studies indicated that early changes in intracellular Ca2+ levels were key mediators of β-lap- induced cell death.
Altered Ca2+levels and their downstream effects
Shortly after the administration of β-lap, an NQO1-expressing cell must deal with a variety of stresses. These include oxidative stress induced by the futile cycling of the parent quinone and alterations in intracellular Ca2+ levels resulting in DNA damage. The immediate focus of the cell is to repair the DNA damage induced by these potentially cytotoxic events and as a consequence, PARP-1 is activated, then hyperactivated once the damage becomes severe. This immediate response to β-lap has been detected in all the
NQO1-proficient cancer cells investigated by our laboratory. Our studies suggest that
DNA damage is not the only critical determinant in the death pathway activated by β-lap.
The release of intracellular Ca2+ is another major determinant in β-lap-induced lethality, which mediates PARP-1 hyperactivation. In neuronal cells, PARP-1 hyperactivation can be induced by changes in intracellular Ca2+ levels even in the absence of DNA damage (134, 135). In the case of β-lap, we discovered that hyperactivation of PARP-1 can be abrogated by dicoumarol, as well as BAPTA-AM.
Thus, β-lap induced PARP-1 hyperactivation is mediated in an NQO1- and Ca2+- dependent manner. Our studies revealed that PARP-1 mediates cell death after β-lap treatment via depletion of cellular nucleotides NAD+ and ATP. These drastic changes in
41 nucleotide levels were abrogated by inhibition of PARP-1 activity (125). In addition, when similar studies were performed with isogenically matched NQO1-proficient and
NQO1-deficient breast and lung cancer model systems, the results showed that NAD+ and ATP levels did not drop significantly in NQO1-deficient cells (125, 133). Therefore, regulation of ROS, Ca2+ levels, and PARP-1 hyperactivation after β-lap administration was exclusively dependent on elevated NQO1 levels in specific human breast, prostate, lung, pancreatic, and colon cancer cells. These early events after β-lap exposure (all occurring within 2 h) greatly determine the therapeutic use of this drug.
Ca2+-activated µ-calpain mediates PARP-1 cleavage, apoptosis and endonuclease activities
Once the cell has committed to death as a result of β-lap exposure, the dismantling of cellular structure can occur in an ordered and energy-independent fashion (136).
Apoptotic processes normally require ATP for the activation of executioner caspases (3,
6, or 7) (136, 137). Activated caspases catalyze the specific cleavage of key cellular proteins such as β-fodrin, epidermal growth factor receptor (EGFR), the inhibitor of caspase-activated DNAse (iCAD, which results in CAD activation) and PARP-1 (138).
The cleavage of these proteins results in membrane blebbing, fragmentation of DNA, chromatin shrinkage, and nuclear condensation. Apoptotic cell death protects the body from the unwanted inflammatory responses accompanied by necrotic processes. Necrotic cell death involves loss of cell membrane integrity and a “bursting” of the cell, with the liberation of cellular contents, which may stimulate an inflammatory reaction.
42 β-Lap-mediated cell death appears to have features of both necrosis (energy depletion) as well as apoptosis (early nuclear condensation, specific proteolytic cleavage, etc.) We have found that β-lap causes a 60 kDa atypical PARP-1 proteolytic cleavage fragment mediated by µ-calpain (104, 139). Purified µ-calpain cleaved PARP-1 into a 60 kDa fragment, unlike the 89 kDa cleavage fragment seen in typical caspase-mediated apoptosis (139). Furthermore, unlike caspase-mediated apoptosis, cell death caused by this agent was not prevented by the pancaspase inhibitor z-VAD (104, 125). β-Lap- stimulated, µ-calpain mediated atypical PARP-1 cleavage was blocked by co-treatment with dicoumarol or pretreatment with BAPTA-AM (104, 125). In addition to blocking
PARP-1 cleavage, β-lap-mediated endonuclease activity was also abrogated by pretreatment with BAPTA-AM or co-treatment with dicoumarol (125, 131). Thus, β-lap- induced µ-calpain-mediated activation appears to be the “executioner protease” that mediates a programmed termination of cancer cells (Figure 1.2).
43
Figure 1.2. Mechanism of β-lap-induced cell death. NQO1 performs a two-electron reduction of the parent β-lap molecule to its hydroquinone form (HQ), that avoids enzymes that perform one-electron reductions forming the semiquinone (SQ).
Unfortunately for the cell, the β-lap HQ is unstable and spontaneously reverts to its parent, oxidized form through two reactions with molecular oxygen. Reactive oxygen species (ROS) are formed. Up to 60 moles of NAD(P)H can be used per mole of β-lap in
10 min during this reaction (Pink et al. 2000), thereby rapidly depleting NAD(P)H levels.
ROS formation causes DNA damage concomitant with increases in cytosolic Ca2+
44 released from ER stores. ROS-dependent DNA damage and Ca2+ release collectively hyperactivate PARP-1. PARP-1 hyperactivation causes depletion of nucleotides, NAD+ and ATP leading to µ-calpain activation and ultimately cell death.
45 β-Lap cytotoxicity is independent of p53, Bax/Bak status and Bcl-2 over-expression
A major disadvantage of many chemotherapeutic agents is their dependence on apoptotic factors to elicit cell death, processes that are altered in many cancers allowing them to maintain a growth advantage. Consequently, many cancers are resistant to chemotherapy due to loss of p53 and Bax/Bak expression, or the over-expression of Bcl-2. We discovered that β-lap-mediated cell death is independent of p53 expression or Bax expression (105). β-Lap mediated cell death was also cell cycle independent (103) which is an advantage for killing tumors (such as prostate cancers) that grow extremely slowly.
β-Lap and DNA repair inhibition
Early studies by Boorstein and Pardee suggested that cotreatment of 3-AB or β-lap inhibited repair of damaged cells, blocking DNA replication and thymidylate synthase activity (140, 141). They proposed that β-lap inhibited a ligation step in the DNA repair process. Additional studies suggested that β-lap prevented the transformation of
CHEF/18A cells treated with IR through the compound’s inhibition of PLDR (101, 142,
143). In our current studies, we propose that β-lap inhibits DNA repair via PARP-1 hyperactivation. β-Lap-induced PARP-1 hyperactivation depletes NAD+ and ATP levels thereby inactivating energy-dependent DNA repair processes. The resulting accumulation of DNA damage caused by NQO1-dependent ROS formation and ER Ca2+ release leads to DNA repair inhibition and the initiation of cell death.
46 CONCLUDING REMARKS
Ideally, the best strategy for cancer therapy is cancer prevention. Fundamentally, it is easier to kill a few cells than a small tumor mass that can exceed >109 tumor cells. DNA repair inhibitors have the potential for use as anticarcinogenic agents, since they can prevent error-prone NHEJ and thereby prevent the cellular transformation of normal cells. Unfortunately, there is limited information regarding the utility of DNA repair inhibitors in preventing carcinogenesis. These compounds clearly could have effects on
DNA repair, tumorigenesis, mutagenesis, and cytoxicity. However, few recent studies on the anticarcinogenic potential of DNA repair inhibitors have been performed. Prior results are often difficult to interpret because they are not well controlled by genetic manipulation, rely on various cellular models, and rodent systems (which have naturally elevated mutation and transformation rates compared to human cells), as well as use non- relevant types of carcinogenic exposure. Therefore, more studies need to be performed to specifically examine the effects of these agents on DNA repair fidelity, mutation rates, and neoplastic transformation.
The use of quinones that target NQO1 that is over-expressed in many tumors are leading candidates for the treatment and prevention of a variety of human cancers. Since
NQO1 levels are elevated early in the neoplastic initiation process, then use of such
“bioactivated” compounds to kill metastatic cells is warranted (144). Thus, compounds such as β-lap would be prime candidates for the advancement of preventative cancer treatments that include prophylactic administration.
47 In summary, early work on the anti-neoplastic properties of β-lap demonstrated that the compound worked through a number of different mechanisms. Data from our lab and others, showed that a variety of pathways and proteins were thought to be important for β-lap’s cytotoxicity. These were, but not limited to, β-lap’s ability to: inhibit NFκB
(145), kill independently of caspases (139) and p53 (102), inhibit Topo I and Topo II-β
(146), and activate µ-calpain (139). Adding to the compounds unique attributes was its ability to kill cells independently of cell cycle status (103) and sensitize cells to IR (100).
Of all these mechanisms, our lab found that the key determinant for β-lap cytotoxicity was NQO1 activity (110). However, β-lap was not shown to cause DNA damage (101).
Despite all these data, there has been no unifying theme to explain all of β-lap’s purported effects. This thesis will seek to describe one such unifying mechanism. We demonstrated that the NQO1-mediated metabolism of β-lap generated ROS, γ-H2AX foci formation, and DNA breaks that hyperactivated PARP-1, which was required for cell death. PARP-1-mediated NAD+/ATP depletion can account for many of the previously reported effects of the drug. Furthermore, we showed that β-lap activated DSB repair, and inhibition of NHEJ could enhance the potency of β-lap.
48 CHAPTER 2: Calcium-Dependent Modulation of Poly(ADP-ribose) Polymerase-1
Alters Cellular Metabolism and DNA Repair
This work was published in the Journal of Biological Chemistry
The Journal of Biological Chemistry 2006 November 3; 281(44):33684-33696
After genotoxic stress poly(ADP-ribose) polymerase-1 (PARP-1) can be hyperactivated, causing (ADP-ribosyl)ation of nuclear proteins (including itself), resulting in NAD+ and ATP depletion and cell death. Mechanisms of PARP-1- mediated cell death and downstream proteolysis remain enigmatic. β-Lapachone
(β-Lap) is the first chemotherapeutic agent to elicit a Ca2+- mediated cell death by
PARP-1 hyperactivation in cancer cells expressing elevated NAD(P)H:quinone oxidoreductase 1 (NQO1) levels. β-Lap induces the generation of NQO1-dependent reactive oxygen species (ROS), DNA breaks, and triggers Ca2+-dependent γ-H2AX formation and PARP-1 hyperactivation. NAD+ and ATP losses suppress DNA repair and cause cell death. Reduction of PARP-1 activity or Ca2+ chelation protects
2+ cells. Interestingly, Ca chelation abrogates hydrogen peroxide (H2O2), but not N-
Methyl-N’-nitro-N-nitrosoguanidine (MNNG)-induced PARP-1 hyperactivation and cell death. Thus, Ca2+ is an important co-factor in PARP-1 hyperactivation after
ROS-induced DNA damage, which alters cellular metabolism and DNA repair.
49 INTRODUCTION
Alterations in the initiation and regulation of caspase-mediated apoptosis are associated with an array of pathological disease states, including chemotherapy-resistance in cancer (147). Therefore, elucidating mechanisms that initiate non-caspase-mediated cell death are crucial for the development and use of novel anticancer agents.
A growing number of chemotherapeutic approaches focus on targeting specific
DNA repair enzymes. In particular, inhibitors of poly(ADP-ribose) polymerase-1 (PARP-
1) that sensitize cells to DNA damaging agents are under extensive investigation (81).
PARP-1 functions as a DNA damage sensor that responds to both single- and/or double- strand DNA breaks (SSBs, DSBs), facilitating DNA repair and cell survival. After binding to DNA breaks, PARP-1 converts β-NAD+ into polymers of branched or linear poly(ADP-ribose) units (PAR) and attaches them to various nuclear acceptor proteins, including XRCC1, histones, and PARP-1 for its autoregulation (148). However, in response to extensive DNA damage, PARP-1 can be hyperactivated, eliciting rapid cellular NAD+ and ATP pool depletion. PARP-1-mediated NAD+ and ATP losses have affects on mitochondrial function by decreasing the levels of pyruvate and NADH. Loss of mitochondrial membrane potential (MMP) ensues, causing caspase-independent cell death by as yet unknown mechanisms (148). PARP-1 hyperactivation was documented in the cellular response to trauma, such as ischemia-reperfusion, myocardial infarction, and reactive oxygen species (ROS)-induced injury (148). In each case, inhibition of
PARP-1 was necessary for the long-term survival of damaged cells (149).
50 β-Lapachone (β-Lap) elicits a unique cell death process in various human breast, lung, and prostate cancers that have elevated levels of the two-electron oxidoreductase,
NAD(P)H:quinone oxidoreductase 1 (NQO1) (E.C. 1.6.99.2) (110). β-Lap induces an
NQO1-dependent form of cell death wherein PARP-1 and p53 proteolytic cleavage fragments were noted (104), concomitant with µ-calpain activation (139). β-Lap-induced lethality and proteolysis were abrogated by dicoumarol (an NQO1 inhibitor), and were muted in cells deficient in NQO1 enzymatic activity (110). Restoration of NQO1 caused increases in drug sensitivity (110). In contrast to staurosporine (STS), global caspase inhibitors had little effect on β-lap lethality (110). β-Lap-mediated cell death exhibited classical features of apoptosis (e.g. DNA condensation, trypan blue exclusion, sub-G0-G1 cells, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling
(TUNEL)-positive cells). β-Lap cell death was not, however, dependent on typical apoptotic mediators, such as p53 or caspases (103). To date, the mechanisms responsible for the initiation of this unique cell death have not been delineated.
We report that β-lap induces an NQO1-dependent, PARP-1-mediated cell death pathway involving changes in cellular metabolism leading to cell death. NQO1- dependent reduction of β-lap results in a futile redox cycle between the parent β-lap molecule and its hydroquinone form (110) wherein ROS generation causes extensive
DNA damage, H2AX phosphorylation (γ-H2AX) and PARP-1 hyperactivation. Drastic decreases in NAD+ and ATP pools, in turn inhibit DNA repair and accelerate cell death.
In addition, chelation of intracellular Ca2+ by 1,2-bis-(2-aminophenoxy)ethane-
N,N,N’,N’-tetraacetic acid tetra-acetoxymethyl ester (BAPTA-AM) abrogates β-lap- induced: (i) γ-H2AX formation, (ii) PARP-1 hyperactivation, (iii) atypical PARP-1 and
51 p53 proteolysis, and (iv) cytotoxicity without affecting NQO1-dependent ROS production. A similar Ca2+ sensitive cell death is observed after hydrogen peroxide
(H2O2) exposure. Interestingly, N-Methyl-N’-nitro-N-nitrosoguanidine (MNNG)-induced
PARP-1 hyperactivation is not sensitive to BAPTA-AM. These data support a critical role for Ca2+ as a regulator of cellular metabolism in response to ROS-induced DNA damage.
EXPERIMENTAL PROCEDURES
Reagents- β-Lap (3,4-dihydro-2,2-dimethyl-2H-naphthol[1,2b]pyran-5,6-dione) was synthesized by Dr. William G. Bornmann (MD Anderson, Texas), dissolved in dimethyl sulfoxide (DMSO) at 40 mM, and the concentration verified by spectrophotometric analyses (103). β-Lap stocks were stored at -80°C. Hoechst 33258, 3-aminobenzamide
(3-AB), dicoumarol (DIC), H2O2, STS, and MNNG were obtained from Sigma Chemical
Company (St. Louis, MO). BAPTA-AM was dissolved in DMSO and used at 5 µM unless otherwise stated. DPQ (3,4-dihydro-5[4-(1-piperindinyl)butoxy]-1(2H)- isoquinoline) was dissolved in DMSO and used at 20 µM. DPQ and BAPTA-AM were obtained from Calbiochem (La Jolla, CA). Z-VAD-fmk was obtained from Enzyme
Systems Products (Dublin, CA), diluted in DMSO and used at 50 µM. 6-carboxy-2’,7’- dichlorodihydrofluorescin diacetate, di(acetoxymethyl ester) (DCF) was used at 5 µM and dissolved in DMSO and used to monitor ROS formation. DCF was obtained from
Molecular Probes (Eugene, OR).
52 Cell Culture- Human MCF-7 and MDA-MB-468-NQ+ breast cancer cells were maintained and used as described (110). Human MDA-MB-231 (231) breast cancer cells that contain a 609C>T polymorphism in NQO1 (144) and are deficient in enzyme activity, were obtained from the American Type Culture Collection (Manassas, VA).
Cells were stably transfected with a CMV-driven NQO1 cDNA or the pcDNA3 vector alone (110). All cells were grown in high-glucose-containing RPMI 1640 tissue culture medium containing 5% fetal bovine serum (FBS), 2 mM L-glutamine, 100 units/ml penicillin, and 100 mg/ml streptomycin at 37°C in a 5% CO2,-95% air humidified atmosphere (104). 231-NQO1+ (NQ+) and –NQO1- (NQ-) cells were maintained in medium containing 400 µg/ml geneticin (103), but all experiments were performed without selection. All tissue culture components were purchased from Invitrogen
(Carlsbad, CA) unless otherwise stated. All cells were routinely tested and found free of mycoplasma contamination.
PARP-1 knockdown- A puromycin-selectable- pSHAG-MAGIC2 retroviral vector containing a short hairpin small interfering RNA against PARP-1 (PARP-1-shRNA) and a non-silencing sequence (ns-shRNA) control were used to infect both NQ+ and NQ- 231 cells (Open Biosystems (Huntsville, AL)). Cells were then selected and grown in 0.5
µg/ml puromycin and screened for PARP-1 protein expression and NQO1 enzymatic activity.
Relative survival assays- Relative survival assays were performed as previously described (110). MCF-7 cells were pretreated or not with BAPTA-AM (5 µM, 30 min) then treated with or without 2 h pulses of β-lap at the doses indicated, in the presence or absence of 40 µM dicoumarol. In some experiments, cells were exposed to 5 µM β-lap
53 followed by delayed (t=0-2 h) addition of 5 µM BAPTA-AM. After drug addition, media were removed and drug-free media added. Cells were then allowed to grow for an additional six days and relative survival, based on DNA content (Hoechst 33258 staining), was determined (110). Prior studies using β-lap showed that relative survival assays correlated directly with colony forming ability assays (110). Data were expressed as treated/control (T/C) from separate triplicate experiments (means, +SE), and comparisons analyzed using a two-tailed Student’s t-test for paired samples.
Immunoblotting and confocal microscopy- Western blots were prepared as previously described (103). α-PARP (sc-8007) and α-p53 (DO-1) antibodies were utilized at dilutions of 1:1000 (Santa Cruz, Santa Cruz, CA). The α-PAR antibody was used at
1:2000 dilution (BD-Pharmingen, San Jose, CA). Antibodies to total H2AX or γ-H2AX were used at dilutions of 1:100-1:500 and purchased from Bethyl Laboratories
(Montgomery, TX) and Upstate (Charlottesville, VA), respectively. An NQO1 antibody was generated and used directly for immunoblot analyses in medium containing 10%
FBS, 1X PBS, and 0.2% Tween 20 (150).
Confocal microscopy was performed as previously described (139). Cells were fixed in methanol/acetone (1:1) and incubated with α-PAR (10H; Alexis, San Diego, CA) or α-γ-H2AX (Trevigen, Gaithersburg, MD) for 2 h at rm. temp. Nuclei were visualized by Hoechst 33258 (10 mg/mL) staining at a 1:3000 dilution. All confocal images were acquired using a Zeiss LSM 510 inverted laser-scanning confocal microscope (Jena,
Germany) with a 63X numerical aperture of 1.4 oil immersion planapochromat objective.
Images of Alexa Fluor 488 dye were collected using a 488-nm excitation light from an argon laser, a 488-nm dichroic mirror and 500-550-nm band pass barrier filter. All
54 Hoechst 33258 stained nuclear images were collected using a Coherent Mira-F-V5-XW-
220 (Verdi 5W) Ti-Sapphire laser tuned at 750-nm, a 700-nm dichroic mirror and a 390-
465 nm band pass barrier filter. Images were representative of experiments performed at least four times. The number of PAR positive cells and γ-H2AX foci/cell were quantified from counting 60 or more cells from four independent experiments (means, + SE).
Formation of ROS was monitored by the conversion of non-fluorescent 6- carboxy-2’7’-dichlorodihydrofluorescin diacetate, di(acetoxymethyl ester) to fluorescent
6-carboxy-2’,7’-dichlorofluorescein diacetate di(acetoxymethyl ester) (DCF) as previously described (151, 152). Briefly, MCF-7 cells were seeded at 2-3 x 105 cells per
35-mm glass bottom Petri dishes (MatTek Corp., Ashland, MA) and allowed to attach overnight. Cells were loaded with 5 µM DCF in media for 30 min at 37 °C. After loading, cells were washed twice with PBS, and incubated for an additional 20 min at 37
°C to allow for dye de-esterification. Confocal images of DCF fluorescence were collected using the same filter settings as for the Alexa Fluor 488 dye on the Zeiss LSM
510 system. Three basal images were collected before drug addition (5-8 µM β-lap, + 5
µM BAPTA-AM or 40 µM dicoumarol). Subsequent images were taken after the indicated treatments at 15-s intervals and similar results were found at 37 °C or rm. temp.
BAPTA-AM was co-loaded with DCF where indicated. Mean pixel intensities were determined in regions of interest for at least 40 individual cells at each time point.
Shown are representative traces of at least three independent experiments (means, + SE).
Single-Cell Gel Electrophoresis (Alkaline Comet) Assays- DNA damage was assessed after different drug treatments by evaluating DNA “comet” tail shape and migration distance (153). MCF-7 cells were pretreated with BAPTA-AM (5 µM 30 min) or DMSO
55 (1:1000 dilution), and then exposed to H2O2 (500 µM, 1 h), β-lap (4 µM, various times), or vehicle alone, and harvested at various times. Cell suspensions (3 x 105/ml cold PBS) were mixed with 1% low melting temperature agarose (1:10 (v/v)) at 37°C and immediately transferred onto a CometSlideTM (Trevigen, Gaithersburg, MD). After solidifying (30 min at 4oC), slides were submerged in prechilled lysis buffer [2.5 M
NaCl, 100 mM EDTA pH 10, 10 mM Tris Base, 1% sodium lauryl sarcosinate, and 1%
Triton X-100] at 4°C for 45 min., incubated in alkaline unwinding solution [300 mM
NaOH, and 1 mM EDTA] for 45 min at rm. temp. and washed twice (5 min) in neutral 1
X TBE [89.2 mM Tris Base, 89 mM Boric Acid, and 2.5 mM EDTA disodium salt].
Damaged and undamaged nuclear DNA was then separated by electrophoresis in 1 X
TBE for 10 min at 1 V/cm, fixed in 70% ethanol, and stained using SYBR-green
(Trevigen). Comets were visualized using an Olympus fluorescence microscope
(Melville, NY), and images captured using a digital camera. Images were analyzed using
ImageJ software (154, 155) and comet tail length was calculated as the distance between the end nuclei heads and the end of each tail. Tail moments were defined as the product of the %DNA in each tail, and the distance between the mean of the head and tail distributions: % of DNA in the tail = tail area (TA) x tail average intensity (TAI) x
100/(TA x TAI) + [head area (HA) x head area intensity (HAI)]. Importantly, tail moment and tail area calculations yielded similar experimental results. Each datum point represented the average of 100 cells +SE, and data are representative of experiments performed three times.
56 Determination of NAD+ and ATP levels-
Intracellular NAD+ levels were measured as described (156) with modification. Briefly, cells were seeded at 1 x 106 and allowed to attach overnight. Cells were pretreated for 2 h with 3-AB (25 mM), DPQ (20 µM), BAPTA-AM (pretreatment 30 min, 5 µM), or
DMSO and then exposed to β-lap (5-6 µM) for the indicated times. Cell extracts were prepared in 0.5 M perchloric acid, neutralized (1 M KOH, 0.33 M KH2PO4/K2HPO4 (pH
+ 7.5)), and centrifuged to remove KClO4 precipitates. Supernatants or NAD standards were incubated 4:1 (v/v) for 20 min at 37°C with NAD+ reaction mixture as described
(73). Measurements from extracts were taken at an O.D. of 570 nm and intracellular
NAD+ levels were normalized to 1 x 106 cells. Data were expressed as %NAD+ +SE, for
T/C samples from nine individual experiments.
ATP levels were analyzed using a luciferase-based bioluminescence assay as described
(157). Data were graphed as means +SE of experiments performed at least three times.
Results were compared using the two-tailed Student’s t-test for paired samples.
NQO1 enzyme activity assays- NQO1 enzymatic assays were performed as described
(158) using cytochrome c (practical grade, Sigma Chemical) in Tris-HCl buffer (50 mM, pH 7.5). NADH (200 µM) was the immediate electron donor, and menadione (10 µM) the electron acceptor. Changes in absorbance were monitored using a Beckman DU 640 spectrophotometer (Beckman Coulter, Fullerton, CA). Dicoumarol (10 µM) inhibitable
NQO1 levels were calculated as nmol cytochrome c reduced/min/µg protein based on initial rate of change in absorbance at 550 nm and an extinction coefficient for cytochrome c of 21.1 nmol/L/cm (159). Results were expressed as means +SE of three or more separate experiments.
57 Flow cytometry and apoptotic measurements- Flow cytometric analyses of TUNEL positive cells were performed as described using APO-DIRECTTM (BD Pharmingen)
(110). Samples were analyzed in an EPICS Elite ESP flow cytometer using an air-cooled argon laser at 488 nm, 15 mW (Beckman Coulter Electronics, Miami, FL) and Elite acquisition software. Experiments were performed a minimum of five times and data expressed as means +SE. Statistical analyses were performed using a two-tailed
Student’s t-test for paired samples.
Glutathione measurements- Glutathione disulfide and total glutathione (GSH + GSSG) levels were determined using a spectrophotometric recycling assay (160, 161). Following indicated treatments, pellets were thawed and whole cell homogenates prepared as described (160, 161). Data were expressed as the %GSSG normalized to protein content, as measured using the method of Lowry et al. (162). Shown are averages +SE for experiments performed at least three times.
RESULTS
Time- and Ca2+-dependence of β-lap-induced cell death- To elucidate the signaling events required for β-lap-induced cell death, log-phase human MCF-7 breast cancer cells, with high levels of endogenous NQO1 activity, were tested for their sensitivities to various concentrations of β-lap at various times. The purpose of these experiments was to determine the minimal time of β-lap exposure required to kill the entire cell population. Cells exposed to doses of ≤3 µM β-lap required ≥4 h to elicit cell death, whereas 2 h exposures of β-lap at ≥4 µM killed all MCF-7 cells (Figure 2.1A).
58 Prior data from our laboratory demonstrated that Ca2+ was released within 2-5 min from endoplasmic reticulum (ER) Ca2+ stores after β-lap treatment (131), suggesting that this was a required initiating factor in β-lap-induced cell death (131). To test this,
MCF-7 cells were pretreated with the intracellular Ca2+ chelator BAPTA-AM (5 µM, 30 min), then exposed to 4 µM β-lap for various times (Figure 2.1B). Under these conditions we previously demonstrated that BAPTA-AM pretreatment was sufficient to block the rise in cytosolic Ca2+ caused by β-lap treatment (131). BAPTA-AM abrogated β-lap- induced cytotoxicity and nuclear condensation (Figure 2.1B and Figure 2.2A). To determine the kinetics of Ca2+-dependent cell death, MCF-7 cells were treated with 5 µM
β-lap, and 5 µM BAPTA-AM was added at various times thereafter, up to 2 h. A time- dependent decrease in survival was observed with delayed addition of BAPTA-AM
(Figure 2.1C), indicating that Ca2+ release was a necessary event, occurring before cells were committed to death. Abrogation of β-lap cytotoxicity by BAPTA-AM was equivalent to that noted with NQO1 inhibition by dicoumarol (Figure 2.1B). Addition of
BAPTA-AM also prevented β-lap-induced, atypical proteolysis (e.g. ~60 kDa PARP-1 and p53 cleavage fragments), in a manner as effective as dicoumarol (Figure 2.2B, lanes
3, 4). Interestingly, Z-VAD-fmk (50 µM), a pan-caspase inhibitor, did not block atypical
PARP-1 and p53 proteolysis in β-lap-treated MCF-7 cells (lane 7). As expected, Z-VAD- fmk inhibited STS, (1 µM)-induced caspase-mediated proteolysis (163). However,
BAPTA-AM did not affect STS-induced apoptotic proteolysis (Figure 2.2C). These data, in conjunction with our prior data showing β-lap-induced ER Ca2+ release (131), support a role for Ca2+ in the initiation of cell death induced by this drug.
59
Figure 2.1. β-Lap-induced cell death is time- and Ca2+-dependent.
A-C, Cell death was examined using relative survival assays in NQO1+ MCF-7 cells. A,
Cells were exposed to various doses of β-lap for different lengths of time to determine the minimal exposure time required for cell death. After drug exposure, media were
60 removed and drug-free media added. Cells were then allowed to grow for an additional six days and relative survival, based on DNA content was determined by Hoechst 33258 staining as described in “Experimental Procedures”. Student’s t-test for paired samples, experimental group compared with 3 µM β-lap (*p ≤ 0.001; **p ≤ 0.005). Prior studies using β-lap showed that relative survival assays directly correlated with colony forming ability. B, Relative survival assays using MCF-7 cells treated with 4 µM β-lap alone, or in combination with 40 µM dicoumarol or 5 µM BAPTA-AM. C, MCF-7 cells were treated with 5 µM β-lap at t=0. BAPTA-AM (5 µM) was then added to β-lap-treated cells at various times post-β-lap exposure as indicated.
61
Figure 2.2. Ca2+ chelation prevents β-lap-induced nuclear morphological changes and atypical PARP-1 and p53 proteolyses, but not STS-induced PARP-1 cleavage.
A, Nuclear morphology changes after β-lap treatment alone with or without 5 µM
BAPTA-AM in Hoechst 33258-stained cells using fluorescence microscopy. B,
Immunoblots of PARP-1 and p53 apoptotic proteolytic cleavage 24 h after exposure to β- lap alone (5 µM, 2 h), in combination with 40 µM dicoumarol or the broad-spectrum
62 caspase inhibitor, 20 µM Z-VAD-fmk. Some cells were pretreated for 30 min with 5 µM
BAPTA-AM, then exposed to β-lap (5 µM, 2 h). MCF-7 cells were also treated with 1
µM STS overnight in the presence or absence of 20 µM Z-VAD-fmk. Equal protein was confirmed by α-tubulin expression. (*denotes typical 89 kDa PARP-1 fragment, and
**denotes atypical, µ-calpain-mediated 60 kDa PARP-1 proteolysis). C, STS-induced, caspase-mediated cleavage of PARP-1 is not blocked by BAPTA-AM. MCF-7 cells treated with 5 µM β-lap or 1 µM STS alone. Other MCF-7 cells were pretreated with 5
µM BAPTA-AM for 30 min prior to 5 µM β-lap or 1 µM STS exposure. Representative immunoblots of PARP-1 and α-tubulin proteins from whole cell extracts taken 24 h after drug exposures are shown. (*denotes typical, caspase-mediated 89 kDa PARP-1 fragment, and **denotes atypical, µ-calpain-mediated 60 kDa PARP-1 fragment).
63 PARP-1 hyperactivation after β-lap treatment is NQO1-dependent and BAPTA-AM- sensitive- Since β-lap-induced cell death was accompanied by a ≥80% loss of ATP within 1 h (131), we suspected PARP-1 hyperactivation played a role in the mode of action for this drug. To investigate this, we generated 231 NQO1-proficient (231-NQ+) cells that are sensitive to β-lap (LD50: ~1.5 µM), after a 2 h pulse, and compared these cells to vector alone, 231 NQO1-deficient (231-NQ-) cells, that are resistant to the drug
(LD50: 17.5 µM). Only β-lap-treated, 231-NQ+ cells exhibited an increase in PAR- modified proteins, mostly PARP-1, consistent with the role of PARP-1 as the predominant ADP(ribosyl)ated species. This response peaked ~30 min after β-lap exposure (Figure 2.3A). In contrast, treatment of 231-NQ- cells with equal or significantly higher doses of β-lap did not induce PAR accumulation (data not shown).
In contrast, both 231-NQ+ and 231-NQ- cells hyperactivated PARP-1 in response to
H2O2. The NQO1-dependence of PARP-1 hyperactivation after β-lap exposure was confirmed in a number of other cell lines (e.g. breast, prostate, and lung cancers) that have elevated NQO1 activity demonstrating that the responses to β-lap were not cell type specific. In all cases, dicoumarol suppressed β-lap-induced PAR formation (Figure 2.4A-
C).
We then examined a possible connection between the involvement of Ca2+ in lethality and PARP-1 hyperactivation. MCF-7 cells were pretreated with 5 µM BAPTA-
AM, then exposed to 5 µM β-lap for the indicated times (Figure 2.3B). The kinetics of
PAR accumulation were faster in MCF-7 cells than in 231-NQ+ cells, (10 min v. 20 min
Figure 2.3A and 2.3B respectively), consistent with higher NQO1 levels in MCF-7 cells.
64 PAR levels decreased after 90 min, corresponding to auto-(ADP-ribosyl)ation of PARP-
1, and efficient PAR hydrolysis by poly(ADP-ribose) glycohydrolase (PARG) (164).
Interestingly, BAPTA-AM pretreatment abrogated PARP-1 hyperactivation induced by β-lap (Figure 2.3B) as confirmed by confocal microscopy (Figure 2.3C).
Robust and extensive poly(ADP-ribosyl)ation occurred within 30 min (87% + 17 PAR positive cells) after drug exposure and dissipated between 60-90 min (Figure 2.3C and
2.3D). However, in the presence of BAPTA-AM PAR accumulation in β-lap-treated
MCF-7 cells was prevented (Figure 2.3C and 2.3D). To determine the global nature of this response, other cancer cell lines such as NQO1+ PC-3 human prostate cancer cells were examined and similar responses noted (Figure 2.4D). Collectively, these data suggested a role for Ca2+ in the modulation of PARP-1 hyperactivation after β-lap exposure.
65
Figure 2.3. β-Lap induces NQO1- and Ca2+-dependent PARP-1 hyperactivation.
A, Immunoblots of PAR formation as a measure of PARP-1 hyperactivation, and steady- state PARP-1 protein levels from whole cell extracts of isogenic 231-NQ+ and 231-NQ- cells treated with 6 µM β-lap for 10-90 min. Relative PAR levels were calculated by densitometric analyses by NIH ImageJ using PARP loading controls wherein controls
66 were set to 1.0. B, Immunoblots of PAR formation and steady state α-tubulin expression in extracts from MCF-7 cells treated with β-lap or H2O2 (2 mM, control for PARP-1 hyperactivation). Other cells were pretreated with BAPTA-AM with or without β-lap (5
µM). Samples were harvested at the indicated times. Relative PAR levels were calculated by densitometric analyses by NIH ImageJ using α-tubulin loading controls wherein controls were set to 1.0. C, Assessment of PARP-1 hyperactivation, measured by PAR formation, in MCF-7 cells treated with β-lap alone or in cells pretreated with
BAPTA-AM for 30 min prior to β-lap exposure. PAR formation was visualized using confocal microscopy. D, Quantified percentages of PAR-positive cells from confocal microscopy analyses of at least 60 cells from four independent experiments (means, +
SE).
67
Figure 2.4 NQO1- and Ca2+-dependent PARP-1 hyperactivation after β-lap exposure is not cell- type specific. A-C, NQO1-dependent PARP-1 hyperactivation
(PAR formation) in human cancer cell lines that have endogenously elevated NQO1 enzyme levels in response to β-lap exposure. Immunoblots of PAR and α-tubulin proteins from whole cell extracts of cells exposed to β-lap alone, or β-lap in combination
68 with 40 µM dicoumarol. Cells used were: A, human MCF-7 breast cancer cells; B, human PC-3 prostate cancer cells; and C, human A549 non-small cell lung cancer cells.
D, Ca2+-dependent PARP-1 hyperactivation (PAR formation) after β-lap treatment is not cell-type specific. Immunoblots of PAR and α-tubulin protein levels from whole cell extracts of PC-3 cells exposed to various doses of β-lap alone or with a 30 min pretreatment of 5 µM BAPTA-AM prior to β-lap exposure. Immunoblots shown are representative of experiments performed three or more times.
69 β-Lap-induced PARP-1 hyperactivation alters cellular energy dynamics causing cell death- PARP-1 hyperactivation can elicit depletion of cellular NAD+ levels and cause cell death in situations of extreme DNA damage or ischemia-reperfusion (72, 165).
Treatment of MCF-7 cells with doses of β-lap ≥5 µM resulted in decreases (>80%) in
NAD+ and ATP levels 1 h after treatment (Figure 2.5A-C). To determine if NAD+ and
ATP losses were attributable to PARP-1 hyperactivation, MCF-7 cells were pretreated for
2 h with PARP inhibitors (i.e. 3-AB or DPQ), prior to 5 µM β-lap exposure. Similar to pretreatment with BAPTA-AM, NAD+ and ATP losses in β-lap-treated MCF-7 cells were abrogated by 3-AB or DPQ (Figure 2.5B and C). 3-AB was more effective at preventing nucleotide loss than DPQ presumably because it has two distinct modes of PARP-1 inhibition, preventing NAD+ binding to the catalytic site and competing with the PARP-1
DNA binding domain (166), whereas DPQ is a competitive inhibitor of NAD+ (78).
Similar effects of DPQ on NAD+ and ATP losses after DNA damage have been reported
(167). Neither 3-AB nor DPQ (used at >2-fold higher doses than in the above experiments) altered NQO1 activity in enzyme assays in vitro. Finally, 3-AB did not affect β-lap-induced ROS formation (data not shown).
To confirm that the energetic consequences of PARP-1 hyperactivation (e.g.
NAD+ and ATP losses) were necessary and sufficient for β-lap-induced cell death, the effects of 3-AB or DPQ, on apoptosis, was measured by TUNEL assay. Pretreatment with either inhibitor resulted in a reduction in apoptosis (2% and 15% total apoptosis respectively for 3-AB and DPQ) compared to 55% in β-lap-treated cells alone (Figure
2.5D). Thus, PARP-1 inhibition by 3-AB or DPQ spared β-lap-induced apoptosis in
70 NQO1+ MCF-7 cells consistent with the effects of these inhibitors to prevent NAD+ and
ATP losses.
Cumulatively, these data strongly suggest that Ca2+-dependent PARP-1 hyperactivation caused NAD+ and ATP loss in NQO1+ human cancer cell lines after β- lap treatment.
71
Figure 2.5. PARP-1-dependent NAD+ and ATP pool depletion leads to cell death after β-lap exposure in MCF-7 cells. A and B, NAD+ pool depletion occurs immediately after β-lap treatment in MCF-7 cells. A, MCF-7 cells were exposed to varying concentrations of β-lap for 1 h, or B, 5 µM β-lap alone with or without pre- and co-treatment of PARP inhibitors, (20 µM DPQ or 25 mM 3-AB) or 5 µM BAPTA-AM for various times and harvested for NAD+ content. Student’s t-test for paired samples, comparing experimental groups containing β-lap + 3-AB or DPQ versus β-lap alone are indicated (*p ≤ 0.001; ** p ≤ 0.05, respectively). C, Intracellular ATP levels were
72 monitored using a luciferase-based bioluminescence assay in MCF-7 cells treated with β- lap, or in cells pre- and co-treated with 20 µM DPQ, or 25 mM 3-AB 2 h prior to β-lap addition. Differences were compared using two-tailed Student’s t-test. Groups having *p
≤ 0.05 values compared with β-lap alone are indicated. D, Apoptotic DNA fragmentation was assessed using TUNEL assays in β-lap-exposed, log-phase MCF-7 cells with or without pre- and co-treatment with DPQ or 3-AB.
73 PARP-1 hyperactivation is required for β-lap cytotoxicity- Since the use of PARP inhibitors was only partially effective at preventing nucleotide loss after β-lap treatment, we confirmed the above findings using a genetic system. 231 breast cancer cells were chosen since they have lower NQO1 levels than MCF-7 cells. PARP-1 protein in 231-
NQ+ and 231-NQ- cells was suppressed by stable PARP-1-shRNA expression. Both
231-NQ+ and 231-NQ- cells infected with PARP-1-shRNA showed ~3- to 4-fold knockdown of PARP-1 protein levels compared to cells containing a non-silencing shRNA sequence (ns-shRNA) (Figure 2.6A). Poly(ADP-ribosyl)ation was decreased in
231-NQ+ PARP-1-shRNA cells after β-lap exposure compared to 231-NQ+ ns-shRNA cells (Figure 2.6B). The remaining PAR-modified proteins noted in cell extracts from β- lap treated 231-NQ+ PARP-1-shRNA cells were due to residual PARP-1 activity since
NQO1 activity was not altered after viral knock-down (Figure 2.6C). PARP-1 protein knock-down spared 231-NQ+ cells from β-lap-induced NAD+ and ATP losses compared to 231-NQ+ ns-shRNA cells (Figure 2.6D and E respectively). Importantly, PARP-1 protein knock-down in 231-NQ+ PARP-1-shRNA cells was sufficient to abrogate β-lap- induced apoptosis at concentrations that killed all 231-NQ+ ns-shRNA cells (Figure
2.6F). In contrast, without NQO1 activity, nominal PAR accumulation, NAD+ or ATP losses were observed in 6 µM β-lap-treated 231-NQ- cells containing PARP-1-shRNA or ns-shRNA (data not shown). These cells remained resistant to β-lap independent of altered PARP-1 levels.
74
Figure 2.6. PARP-1 plays an essential role in β-lap-induced apoptotic cell death as monitored by TUNEL. A, Immunoblots of steady state PARP-1 and α-tubulin protein levels from whole cell extracts of 231-NQ+ ns-shRNA, 231-NQ+ PARP-1-shRNA, 231-
NQ- ns-shRNA, and 231-NQ- PARP-1-shRNA cell lines. Relative PARP-1 protein levels were calculated by densitometry analyses using α-tubulin loading controls by NIH
ImageJ wherein controls were set to 1.0. B, Immunoblots of PAR, PARP-1, and α- tubulin protein levels from control and β-lap (6 µM, 10-90 min)-exposed 231-NQ+ ns-
75 shRNA or PARP-1-shRNA cells. Relative PAR levels were calculated by densitometry analysis using α-tubulin loading controls by NIH ImageJ. C, PARP-1 protein knock- down does not alter NQO1 enzymatic activity. NQO1 enzyme activities were determined for 231-NQ+ ns-shRNA, 231-NQ+ PARP-1-shRNA, 231-NQ- ns-shRNA, 231-NQ-
PARP-1-shRNA cells. NQO1 enzyme activities were calculated as nmol cytochrome c reduced/min/µg protein. D, PARP-1 is necessary for NAD+ loss after β-lap exposure.
231-NQ+ ns-shRNA and 231-NQ+ PARP-1-shRNA cells were treated with 6 µM β-lap for 0-120 min at which times NAD+ content was determined as previously described. E,
β-Lap-induced ATP loss is PARP-1-dependent. ATP levels were monitored in 231-NQ+ ns-shRNA or PARP-1-shRNA cells after exposure to β-lap (6 µM, 0-120 min). Student’s t-test for paired samples, comparing experimental groups 231-NQ+ ns-shRNA versus
231-NQ+ PARP-1-shRNA are indicated (*p≤ 0.05; **p≤ 0.001). F, Knock-down of
PARP-1 protein protects cells from β-lap-induced cell death. 231-NQ+ ns-shRNA, and
231-NQ+ PARP-1-shRNA cells were exposed to a 2 h pulse of β-lap and monitored for apoptosis 24 h later. Students’s t-test for paired samples, experimental group compared with vehicle control (*p≤ 0.001).
76 NQO1-dependent DNA damage after β-lap treatment- To date, there is little direct evidence that β-lap treatment causes DNA damage as assessed by alkaline or neutral filter elution, p53 induction, or covalent complex protein-DNA formation (101, 103,
168). However, many of these prior studies were performed in cells expressing little to no NQO1 (169). Since PARP-1 activation typically requires DNA damage, we examined
NQO1-positive cells exposed to β-lap for DNA breaks, by measuring γ-H2AX. H2AX contains a highly conserved serine residue (ser139), that is rapidly phosphorylated upon
DNA damage (170). Significant γ-H2AX foci formation was observed in 30 min, similar to that seen 15 min following 5 Gy (Figure 2.7A). In contrast, total H2AX and α-tubulin levels remained unchanged. These results were confirmed by confocal microscopy
(Figure 2.7B).
Since Ca2+ chelation blocked both β-lap-induced PARP-1 hyperactivation and cell death, we tested the effects of BAPTA-AM on γ-H2AX foci formation. Similar to the immunoblot analyses and the PAR formation kinetics (Figure 2.8C), β-lap-treated MCF-7 cells showed γ-H2AX foci in 10 min (~17 foci/cell) of exposure, with peak levels at 60 min (~40 foci/cell) (Figure 2.7C). Importantly, β-lap-induced γ-H2AX foci formation was partially abrogated by BAPTA-AM addition, with fewer γ-H2AX foci noted in 30-90 min (~5-25 foci/cell respectively) (Figure 2.7C). These results were confirmed by immunoblot blot analyses (Fig. 2.7D).
77
Figure 2.7. β-Lap-induced γ-H2AX foci formation is abrogated by BAPTA-AM pretreatment. A, Immunoblot analyses of γ-H2AX, total H2AX, and α-tubulin protein levels in whole cell extracts from MCF-7 cells exposed to β-lap for various times, or IR
(5 Gy) harvested after 15 min. B, Visualization of γ-H2AX foci in MCF-7 cells at various times after treatment with 5 µM β-lap or 15 min post-IR (5 Gy) by confocal
78 microscopy. C, BAPTA-AM pretreatment followed by β-lap exposure abrogates γ-
H2AX foci formation in MCF-7 cells as visualized by confocal microscopy. The number of γ-H2AX foci per cell was determined by eye from at least 60 cells for each treatment group from four independent confocal experiments (means, +SE). D,
Immunoblot of γ-H2AX, total H2AX, and α-tubulin protein levels in whole cell extracts from MCF-7 cells exposed to β-lap for various times with or without BAPTA-AM (5
µM) pretreatment, or IR (5 Gy) harvested after 15 min.
79 Ca2+ chelation modulates DNA repair after β-lap treatment- We postulated that metabolism of β-lap by NQO1 would generate superoxide, peroxide, and other ROS
(171). We directly monitored intracellular ROS formation, using the conversion of non- fluorescent 6-carboxy-2’,7’-dichlorodihydrofluorescin to fluorescent 6-carboxy-2’,7’- dichlorodihydrofluorescein (DCF). Indeed, exposure of MCF-7 cells with 5 or 8 µM β- lap treatment, caused an increase in fluorescence within 5 min compared to DMSO- treated cells (Figure 2.8A, left panel). Region-of-interest analyses showed a ~2000 fold increase in fluorescence with β-lap alone over control cells which could be abrogated by inhibiting NQO1 activity with dicoumarol (Figure 2.8A, right panel). Since BAPTA-AM has moderate affinity for divalent cations other than Ca2+, we explored the possibility that
BAPTA-AM may protect cells from DNA damage and subsequent cell death by interfering with Fenton chemistry. Cells pretreated with 5 µM BAPTA-AM and then exposed to 5 or 8 µM β-lap exhibited no significant difference in the rate or extent of
ROS formation compared with β-lap alone treated cells (Figure 2.8A). These results were confirmed by examining the oxidative state of MDA-MB-468-NQ+ cells after treatment with 4 µM β-lap in the presence or absence of 5 µM BAPTA-AM. β-Lap treatment caused an ~65% rise in disulfide glutathione (GSSG) levels, that persisted during drug exposure (Figure 2.8B). Addition of BAPTA-AM did not alter the kinetics or levels of GSSG formation after β-lap exposure (Figure 2.8B). These data suggest that the protective effects of BAPTA-AM on β-lap-treated NQO1+ cells were not due to interference with β-lap-induced ROS formation. Similar results were found in 231-NQ+ cells (data not shown).
80
Figure 2.8. Ca2+ modulates DNA repair in β-lap treated cells. A, β-lap treatment causes the formation of ROS that is not blocked by BAPTA-AM pretreatment. MCF-7 cells were loaded with DCF as described under “Experimental Procedures”. Images were collected before drug treatment (0 min) and 15 s after treatment up to 5 min. Cells were treated with β-lap (5 or 8 µM) with or without BAPTA-AM pretreatment or cotreatment with dicoumoral. Values represent the average of total cellular DCF fluorescence expressed as arbitrary units (AU) from 0-5 min (left panel). X-fold changes in
81 fluorescence were calculated as the difference in fluorescence at 5 min minus the initial fluorescence obtained at the time of drug addition (right panel). Results are expressed as means + SE from 20-30 cells per treatment group. Shown are representative data from three independent experiments. B, BAPTA-AM does not block β-lap-induced ROS production in NQO1+ MDA-MB-468 cells as monitored by changes in levels of disulfide glutathione (% GSSG) following treatment with 4 µM β-lap alone, or in cells pretreated for 30 min with 5 µM BAPTA-AM and then exposed to 4 µM β-lap. C, DNA damage in
MCF-7 cells during (left) or following (right) vehicle (DMSO) alone, 500 µM H2O2 (),
4 µM β-lap, 5 µM BAPTA-AM () or in MCF-7 cells pretreated with 5 µM BAPTA-
AM prior to β-lap (4 µM) exposure. DNA damage was assessed by alkaline comet assays at the indicated times. The comet tail area of 100 cells (means +SE) for each time and condition were quantified with NIH image software and normalized to untreated cells. Similar results were obtained by analyzing comet tail length.
82 To assess the effects of Ca2+ on DNA damage and repair, β-lap-treated MCF-7 cells were analyzed by alkaline comet assays to monitor total DNA strand breaks with or without
BAPTA-AM addition. β-Lap-treated cells exhibited significant DNA strand breakage by
10 min, resembling the positive control (H2O2), and after 30 min, β-lap-induced DNA damage exceeded those levels (Figure 2.8C and Figure 2.9). Cells pretreated with
BAPTA-AM exhibited less DNA damage compared to β-lap alone and their repair of
DNA damage correlated well with their ability to survive (Figure 2.8C and 2.1B).
We then examined the kinetics of repair in MCF-7 cells following 2 h β-lap exposures with or without BAPTA-AM pretreatment. After β-lap exposure, DNA damage persisted and gradually increased over time (Figure 2.8C), indicative of inhibition of DNA repair and consistent with the drop in NAD+ and ATP levels (Figure
2.5B and C). Although cells treated with β-lap and BAPTA-AM at 2 h exhibited equivalent damage to 10 min of β-lap exposure alone (4.6 + 0.2 v 4.7 + 0.4, p>0.5 comet tail areas respectively), BAPTA-AM pretreated cells were protected from PARP-1 hyperactivation (Figure 2.3B) as well as decrements in NAD+ levels (Figure 2.5B).
BAPTA-AM pretreated cells showed a time-dependent recovery from DNA damage
(Figure 2.8C and Figure 2.9). In contrast, β-lap exposed cells resulted in extensive DNA damage with no signs of DNA repair. Collectively, these data suggest that NQO1- mediated metabolism of β-lap leads to the generation of ROS and subsequent DNA damage that hyperactivates PARP-1.
83
Figure 2.9. Ca2+chelation modulates DNA repair after β-lap treatment.
Log-phase MCF-7 cells were treated with vehicle alone or BAPTA-AM and then exposed to β-lap as indicated. Cells were then assessed for total DNA strand breaks by alkaline comet assays as described in “Experimental Procedures”. Shown are representative images of separate experiments performed three or more times. The arrow indicates the direction of electrophoresis.
84 2+ H2O2 causes Ca -dependent PARP-1 hyperactivation- To examine the universality of
Ca2+-modulated PARP-1 function in response to other DNA damaging agents, we examined responses to H2O2 or MNNG. Unlike β-lap, H2O2 treatment caused PARP-1 hyperactivation in both 231-NQ+ and 231-NQ- cells (Figure 2.10A). However, expression of NQO1 spared PAR formation in 231 cells in a dose-dependent manner
(Figure 2.10A). These data suggest that NQO1 has a broader antioxidant role by protecting against ROS-induced damage as previously proposed (172-174). Consistent with β-lap, however, was the abrogation of PAR formation by BAPTA-AM in 231 cells independent of NQO1 activity (Figure 2.10B).
H2O2 treatment also caused a dose-dependent increase in apoptosis in both 231-
NQ- and 231-NQ+ cells that was blocked by BAPTA-AM (Figure 2.10C). However,
231-NQ+ cells were much less sensitive to H2O2 than 231-NQ- cells. ATP loss was seen within minutes of H2O2 exposure in 231-NQ-, but not in the NQO1-positive cell line 231-
NQ+ (Figure 2.11). In addition, PARP-1 hyperactivation and cell death in response to equivalent doses of H2O2 in 231-NQ- cells was much more robust than in the 231-NQ+ cells (Figure 2.10B and C). Interestingly, as noted with β-lap exposure, treatment of
MCF-7 cells with ≥200 µM H2O2 for 2 h resulted in formation of a 60 kDa PARP-1 and
40 kDa p53 fragments. This atypical proteolysis was effectively inhibited by BAPTA-
AM pretreatment (Figure 2.12).
Finally, BAPTA-AM had no effect on PARP-1 hyperactivity or cytotoxicity caused by treatment with the monofunctional DNA alkylating agent, MNNG (Figure
2+ 2.10E). Since MNNG does not cause Ca release like β-lap or H2O2, these data suggest that Ca2+ modulation of PARP-1 hyperactivation is unique to ROS-producing agents.
85
2+ Figure 2.10. H2O2 causes Ca -dependent PARP-1 hyperactivation and cell death.
A, PARP-1 hyperactivation after H2O2 exposure occurs regardless of NQO1 status.
Immunoblot analyses of PAR, NQO1, and α-tubulin protein levels in whole cell extracts from 231-NQ- and 231-NQ+ cells after exposure to varying doses of H2O2 for 15 min. B,
2+ PARP-1 hyperactivation after H2O2 treatment is Ca -dependent. 231-NQ- (left) and
231-NQ+ cells (right) were pretreated with BAPTA-AM or vehicle alone for 30 min and then treated with varying doses of H2O2 and harvested after 15 min. Immunoblots of
PAR, and α-tubulin protein levels from whole cell extracts were then analyzed. C, Ca2+
86 chelation protects 231-NQ- and 231-NQ+ cells from H2O2-induced apoptosis. TUNEL assays were performed in H2O2-exposed, log-phase 231-NQ- and 231-NQ+ cells, with or without pretreatment with 5 µM BAPTA-AM. D, MNNG-induced PARP-1 hyperactivation is not blocked by Ca2+ chelation. Immunoblots of PAR and α-tubulin protein levels from whole cell extracts of MCF-7 cells treated with MNNG or in cells pretreated for 30 min with BAPTA-AM and then exposed to MNNG. Cells were treated for the indicated times and immediately harvested.
Figure 2.11. H2O2 causes time-dependent ATP depletion in NQO1 deficient cells.
Human breast cancer 231-NQ- and 231-NQ+ cells were treated with 500 µM H2O2 and harvested for ATP content at various times during drug exposure. Data are means, +SE of experiments performed three times in triplicate.
87
2+ Figure 2.12. H2O2 causes Ca -dependent, presumably µ-calpain-mediated, atypical
PARP-1 and p53 proteolysis. Shown are representative immunoblots of PARP-1, p53, and α-tubulin in MCF-7 cells exposed to 5 µM BAPTA-AM or vehicle alone for 30 min prior to varying doses of H2O2 or to 5 µM β-lap for 2 h. Samples were harvested 24 h later (**denotes atypical 60 kDa PARP-1 fragment). Immunoblots shown are representative of experiments performed three or more times.
88 DISCUSSION
The regulatory mechanisms controlling PARP-1 function to either promote cell survival or cell death in response to DNA damage remain enigmatic. PARP-1 facilitates
DNA repair and cell survival in response to a variety of DNA damaging agents.
However, it also mediates programmed necrosis (73) as well as caspase-independent apoptotic cell death following severe levels of DNA damage (175). The downstream pathways essential for the execution of cell death in response to PARP-1-mediated metabolic alterations remain poorly understood.
In elucidating the cell death pathway after exposure to β-lap, we uncovered a novel mechanism of PARP-1-mediated cell death. Our data suggest that this mechanism occurred selectively in response to ROS-generating agents. We demonstrated, for the first time, that Ca2+-mediated PARP-1 hyperactivation commits cells to death as a consequence of metabolic starvation without the involvement of caspases.
PARP-1 hyperactivation in response to β-lap treatment was not cell type specific and has been observed in all cells that express elevated NQO1 levels (Figure 2.3A, and
Figure 2.4A-C). As a result, cells exposed to β-lap exhibited depletion of NAD+ and
ATP, occurring 30-60 min after drug exposure. NAD+ and ATP losses were, in part,
PARP-1-mediated since PARP inhibitors (e.g. 3-AB and DPQ) partially abrogated nucleotide loss (Figure 2.5B and C). Chemical inhibition of PARP-1, or PARP-1 protein knock-down, not only prevented NAD+ and ATP losses, but also abrogated β-lap-induced apoptosis (Figure 2.5D and 2.6D-F). These data established PARP-1-mediated NAD+ and ATP losses as crucial upstream events in β-lap-mediated cell death.
89 Alterations in cellular metabolism caused by β-lap explains many of its purported effects in vitro and in vivo. These include, but are not limited to: (i) inhibition of NFκB activation via inhibition of IKK-α (145), (ii) lack of caspase activation (104) and p53 stabilization (103), and (iii) inhibition of Topoisomerase (Topo) I and Topo II-β (176).
Furthermore, β-lap can initiate cell death independently from Bax and/or Bak activation as changes in mitochondrial outer membrane permeabilization (MOMP) can occur via
PARP-1-mediated NAD+ loss1.
A unique feature of PARP-1-mediated cell death stimulated by β-lap was that administration of BAPTA-AM abrogated PARP-1 hyperactivation (Figure 2.3B and
Figure 2.4D), nucleotide pool loss (Figure 2.5B), atypical proteolysis (assessed by p53 and 60 kDa PARP-1 cleavage) (Figure 2.2B), and cell death (Figure 2.1B and 2.1C).
When BAPTA-AM was added >20 min after β-lap treatment, cells were not protected from cell death (Figure 2.1C), suggesting that events occurring within the first 20 min of drug exposure committed cells to death. BAPTA (free acid form) did not alter NQO1 activity in vitro (131). This appears to be confirmed by the inability of BAPTA-AM to prevent ROS generation, which arises from NQO1-mediated metabolism of β-lap.
Instead, our data suggest that the ability of BAPTA-AM to prevent β-lap-induced lethality in NQO1+ cancer cells was due to the specific prevention of PARP-1 hyperactivation. The observed differences in the amount of DNA damage and γ-H2AX foci formation between β-lap alone and that of β-lap co-administered with BAPTA-AM suggest that preventing PARP-1 hyperactivation and subsequent changes in cellular metabolism can allow for cell recovery, noted by more rapid and extensive DNA damage
1 Zong, W.X., Bey, E.A., and Boothman, D.A., unpublished data
90 repair (Figure 2.7C and Figure 2.8C). Recent data suggest that protein phosphatase 2A
(PP2A) dephosphorylates γ-H2AX and is required for DSB repair (177). It is possible that Ca2+ chelation not only prevents PARP-1 hyperactivation, but also augments γ-
H2AX dephosphorylation through PP2A activity. ROS-induced ER Ca2+ release may poison PP2A. However, we favor the theory that NQO1-mediated β-lap-induced ER
Ca2+ release has its predominant affect on PARP-1 hyperactivation, thereby inhibiting
DNA repair and cell recovery.
The mechanism of cell death induced by β-lap could be recapitulated by treatment with high doses of ROS-generating agents such as H2O2 (Figure 2.10A-D). Notable
2+ similarities included, H2O2-mediated PARP-1 hyperactivation, Ca -dependent proteolytic cleavage of PARP-1 and p53, and apoptotic DNA fragmentation.
Furthermore, H2O2-induced lethality was abrogated by BAPTA-AM (Figure 2.10A-D and
Figure 2.12). Although β-lap and H2O2 initiate a similar downstream death pathway, the compounds differed in their lethality in cells with respect to NQO1 expression. β-Lap lethality was enhanced in cells that express NQO1, whereas H2O2 caused greater cytotoxicity in NQO1-deficient cells (Figure 2.10C). We noted striking similarities between β-lap- or H2O2-induced cell death and the caspase-independent cell death induced by ischemia-reperfusion. ROS produced during ischemia-reperfusion induces
DNA strand breaks beyond a normal threshold that lead to PARP-1 hyperactivation, metabolic catastrophe, and an increase in intracellular Ca2+ levels leading to µ-calpain activation (178). These data suggest that programmed PARP-1-mediated cell death is a global response to these types of cellular insults.
91 PARP-1 hyperactivation was also observed following high doses of MNNG, however, this response was not affected by BAPTA-AM (Figure 2.10D). These data highlight two separate PARP-1 regulatory mechanisms. First, ROS-induced, PARP-1- mediated cell death appears to require Ca2+ as a co-factor, whereas alkylation-mediated
PARP-1-induced cell death does not. We propose that Ca2+ release following ROS- induced stress directly influences PARP-1 and PARG function. Both Mg2+ and Ca2+ exert significant (≥3 fold increases) allosteric activation of PARP-1 auto(ADP- ribosyl)ation in vitro, which is inhibited by EDTA addition (179). We, therefore, speculate that Ca2+ chelation modulates PAR synthesis by dampening PARP-1 auto(poly-
ADP)-ribosylation. Furthermore, since increases in [Ca2+] can inhibit PARG function by up to 50% in vitro, maintenance of homeostatic Ca2+ levels after drug treatment would thereby restore the normal turnover of PAR by PARG, lifting PARP-1 self-inhibition
(180). Our data are consistent with the hypothesis that both PAR synthesis and PAR degradation can be modulated by BAPTA-AM to spare the cell from metabolic catastrophe via Ca2+-mediated NAD+ and ATP losses (Figure 2.3B, and Figure 2.5B,C)
(181). The remaining PARP-1 activity would be necessary for DNA break repair, ultimately providing a survival advantage to damaged cells (Figure 2.1B, 2.7C, 2.8C and
2.10C). We are currently exploring the mechanism by which Ca2+ modulates PARP-1 hyperactivation and subsequent DNA repair after H2O2 or β-lap treatments versus
MNNG.
There appears to be some disagreement as to the role of Ca2+ in PARP-1- dependent cell death. Ca2+ can hyperactivate PARP-1 in the absence of DNA breaks
(135). In neuronal cells, glutamate caused Ca2+-mediated ROS production through
92 mitochondrial dysfunction, leading to DNA damage, PARP-1 hyperactivation, and cell death. Furthermore, Ca2+ chelators, such as BAPTA-AM, EGTA-AM, and Quin-2-AM, protected against other oxidative stress-induced apoptotic and necrotic cell death mechanisms (182, 183). In these studies, Ca2+ chelation did not directly inhibit PARP-1 activity, but rather prevented DNA damage by inhibiting ROS. Contrary to these observations, in our system BAPTA-AM did not alter the direct production of ROS or oxidative stress in H2O2- or β-lap-exposed cells (Figure 2.8A). Therefore, there does not appear to be an interference with transition metal-mediated oxidant production by
BAPTA-AM (e.g. Fenton reaction) as previously suggested after H2O2 treatment (184,
185). In fact, our data demonstrate that β-lap caused equivalent ROS production in both
β-lap alone and β-lap + BAPTA-AM treated cells. In contrast to β-lap alone treated cells, cells pretreated with BAPTA-AM did not exhibit notable PARP-1 hyperactivation, associated NAD+ and ATP losses, and showed a decrease in DNA damage over time
(Table 2.1). While these data are suggestive of ongoing DNA repair in the presence of
Ca2+ chelators, we cannot discount that these observations could also be the result of a decrease in the initial amount of DNA damage created in NQO1+ cells in response to β- lap. Initial DNA damage and active DNA repair would be indistinguishable in these experiments. Thus, although we believe it is unlikely, the Ca2+-dependence of PARP-1 hyperactivation could be an indirect consequence of a BAPTA-AM-mediated decrease
(i.e., protection) in the initial amount of DNA lesions created in response to β-lap. Future studies will address this issue by utilizing DNA repair compromised NQO1+ cells.
93
End points examined β-Lapa β-Lap+BAPTA-AMa p valuesc Relative PAR levels 0.6 0 N/A % NAD+ loss 92% + 6 3% + 3 ≤0.001 Number of γ-H2AX foci/cell 17 + 1 16 + 2 ≥0.2 ROS formation (fold change) 1999 + 147 2171 + 148 ≥0.2 Comet tail area 4.7 + 0.4 4.6 + 0.2b ≥0.2 aComparisons were taken using NQO1 positive MCF-7 cells treated with 5 µM β-lap for 10 min vs. cells treated with 5 µM β-lap + 5 µM BAPTA-AM for 90 min unless otherwise noted bComparisons were taken using 5 µM β-lap for 10 min vs. 5 µM β-lap + 5 µM BAPTA- AM for 120 min cp values were performed using the two-tailed Student’s t-test for paired samples 5 µM β- lap for 10 min vs. cells treated with 5 µM β-lap + 5 µM BAPTA-AM for 90 min unless otherwise noted; p values ≥0.2 were deemed not significant.
Table 2.1. Addition of BAPTA-AM allows DNA repair after β-lap exposure.
Comparison of PARP-1 hyperactivation (noted as PAR formation), NAD+ loss, γ-H2AX foci formation, ROS formation, and total DNA damage monitored by comet assays in
MCF-7 cells treated with β-lap alone or in cells pretreated with BAPTA-AM prior to β- lap treatment. MCF-7 cells exposed to β-lap alone for 10 min exhibited an average comet tail area of 4.7 +0.4 and 17 +1 γ-H2AX foci/cell, concomitant with a 60% increase in relative PAR levels, resulting in a 92% loss of NAD+. In contrast, cells pretreated with
BAPTA-AM prior to β-lap exposure exhibited an equivalent amount of DNA damage at
90 min post-treatment to that of β-lap alone (i.e., cells exposed to 10 min with β-lap, see above) indicated by an average comet tail area of 4.6 + 0.2 and 16 + 2 γ-H2AX foci/cell.
However, exposure of NQO1+ MCF-7 cells to β-lap + BAPTA-AM did not show a subsequent increase in PARP-1 hyperactivation (observed as an increase in PAR-
94 modified proteins, or a substantial drop in NAD+ levels; NAD+ levels dropped only 3% under these conditions). Importantly, ROS formation in NQO1+ MCF-7 cells in response to β-lap alone compared to β-lap + BAPTA-AM was equivalent (1999 +147 versus 2171 +148, respectively). Since β-lap alone and β-lap + BAPTA-AM have equivalent amounts of ROS production and DNA damage but differ in the amount of
PAR accumulation and NAD+ loss, these data suggest that the decrease in DNA damage after treatment with β-lap + BAPTA-AM is due to ongoing DNA repair, allowed for by the inhibition of PARP-1 hyperactivation by calcium chelation, rather than an alternative mechanism whereby BAPTA-AM prevented initial DNA damage. The results of Table
2.1 are a summary of experiments performed at least three times in triplicate.
95 Collectively, our data suggest that PARP-1 is necessary for the initiation of cell death caused by β-lap. To date, however, the endonuclease responsible for the execution of cell death in response to β-lap treatment remains unknown. We therefore propose, that
PARP-1-mediated NAD+ and ATP losses, in addition to PARG-liberated ADP-ribose, causes an influx of Ca2+ from extracellular and intracellular sources. Impairment of ATP- dependent membrane/organelle transporters (e.g. plasma membrane Ca2+ ATPases
(PMCA) and sarcoplasmic/endoplasmic reticulum Ca2+ ATPases (SERCA)) by ATP loss, and activation of plasma membrane cation channels (e.g. transient receptor potential- melastatin-like (TRPM)) by ADP-ribose, leads to high intracellular Ca2+ levels (186) sufficient to activate the Ca2+-dependent protease µ-calpain and commit the cell to death.
Previous studies from our laboratory have demonstrated that β-lap causes the downstream activation of µ-calpain resulting in its translocation to the nucleus concomitant with nuclear proteolytic cleavage of p53 and PARP-1 (139). Studies from our laboratory indicate that β-lap treatment causes apoptosis-inducing factor (AIF) translocation from the mitochondria to the nucleus, leading to nuclear condensation following µ-calpain activation2. To date, the mechanism responsible for PARP-1- mediated AIF release remains unclear. We speculate that AIF release under conditions of
DNA damage may be mediated through a concerted effort of both µ-calpain and PARP-1.
Disruption of the mitochondrial membrane potential through PARP-1-dependent NAD+ and ATP losses, in conjunction with µ-calpain mediated cleavage of Bid or of AIF itself, may mediate its release from the mitochondria (187, 188).
2Bey, E.A., and Boothman, D.A., unpublished observations
96 In conclusion, our studies offer new insights into the signal transduction pathways necessary for PARP-1-mediated cell death, providing a connection between PARP-1 hyperactivation and cell death via fluctuations in Ca2+ homeostasis. Knowledge of this pathway may be used to understand, and effectively treat, a large number of human pathologies (e.g. ischemia-reperfusion during heart attacks and stroke, and diabetes), as well as to enhance current cancer chemotherapeutic agents through modulation of PARP-
1 hyperactivation.
97 CHAPTER 3: Non-Homologous End Joining is Essential for Cellular Resistance to
the Novel Anitumor Agent, β-Lapachone
This work was submitted to Cancer Research
Commonly used antitumor agents, such as DNA Toposiomerase I/II poisons, kill cancer cells by creating non-repairable DNA double-strand breaks (DSBs). To repair DSBs, error-free homologous recombination (HR) and/or error-prone non- homologous end joining (NHEJ) are activated. These processes involve the phosphatidylinositol 3′-kinase related kinase (PI3K) family of Serine/Threonine protein kinases: ataxia telangiectasia mutated (ATM), ATM- and Rad3-related
(ATR) for HR, and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) for NHEJ. Alterations in these repair processes can cause drug/radiation resistance and increase genomic instability.
β-Lapachone (a.k.a. ARQ 501), currently in Phase II clinical trials for the treatment of pancreatic cancer, causes a novel caspase- and p53-independent cell death in cancer cells expressing NAD(P)H:quinone oxidoreductase-1 (NQO1).
NQO1 catalyzes a futile oxidoreduction of β-lapachone leading to reactive oxygen species (ROS) generation, DNA breaks, γ-H2AX foci formation, and the hyperactivation of poly(ADP-ribose) polymerase-1 (PARP-1), which is required for cell death. Here, we report that β-lapachone exposure results in the NQO1- dependent activation of the MRE11-Rad50-Nbs-1 (MRN) complex. In addition, phosphorylation of ATM Serine 1981, DNA-PKcs Threonine 2609, and Chk1 Serine
98 345 were noted; indicative of simultaneous HR and NHEJ activation. However, inhibition of NHEJ, but not HR, by genetic or chemical means potentiated β- lapachone lethality. These studies give insight into the mechanism by which β- lapachone radiosensitizes cancer cells and suggests that NHEJ is a potent target for enhancing the therapeutic efficacy of β-lapachone alone or in combination with other agents in cancer cells that express elevated NQO1 levels.
INTRODUCTION
Many cancer chemotherapeutic agents, such as ionizing radiation (IR) and DNA- damaging chemotherapeutic compounds cause cell death by creating DNA double-strand breaks (DSBs) (189, 190). DSBs can occur from endogenously produced reactive oxygen species (ROS) or conversion of single-strand breaks (SSBs) to DSBs by advancing replication forks (191). Although cells maintain the capability to survive low levels of DNA damage, as little as one non-repaired DSB can be lethal (192).
Homologous recombination (HR) and non-homologous end joining (NHEJ) are two distinct, yet complementary, mechanisms for mammalian DSB repair that can interact simultaneously at DSB sites (1, 3, 193). Essential to both HR and NHEJ, is the activation of one or all three related phosphatidylinositol 3′-kinase-like kinases (PI3Ks) in response to DNA damage (3). Ataxia telangiectasia mutated (ATM), and ATM- and
Rad3-related (ATR) are associated with HR, and are typically activated during S/G2 phase by DNA breaks (e.g. ATM) or after replication fork arrest (e.g. ATR activation).
In contrast, DNA-protein kinase catalytic subunit (DNA-PKcs; part of the DNA-PK
99 complex including the Ku70/Ku80 heterodimer) is involved in NHEJ that operates throughout the cell cycle in response to DSBs. These kinases operate as transducer proteins, relaying and amplifying damage signals to mediator proteins. A common substrate of all PI3Ks is histone variant, H2AX. Formation of phosphorylated histone
H2AX is a sensitive and early marker of DSBs (170, 194). Shortly after DSB detection and PI3K activation, H2AX becomes phosphorylated on Serine 139 (γ-H2AX) in a 2-Mb region surrounding the break. Microscopically, this phosphorylation event occurs on a multitude of H2AX molecules leading to foci that are visible when labeled with an antibody specific for γ-H2AX. γ-H2AX foci facilitate recruitment of DNA-damage regulating protein complexes to the sites of damage (13), while γ-H2AX de- phosphorylation assists repair (177). The MRE11/Rad50/Nbs-1 (MRN) complex serves as the initial protein complex to participate in both NHEJ and HR pathways (4). In
NHEJ, the MRN complex modifies DSB ends by its endo- and exo-nuclease activities
(195). In HR, the complex acts as an exonuclease to produce 3′ single-strand overhangs bound by Rad52 (4).
β-Lapachone (β-lap; a.k.a. ARQ501) is currently in Phase II clinical trials for the treatment of pancreatic adenocarcinoma in combination with gemcitabine3. β-Lap is a novel antitumor agent that is bio-activated by the two-electron oxidoreductase, NAD(P)H quinone oxidoreductase-1 (NQO1) (E.C. 1.6.99.2). Since NQO1 is expressed at high levels in many human cancers (e.g. pancreatic, breast, lung, and prostate cancers) it is an attractive target for selective cancer chemotherapy using β-lap alone or in combination with IR (101, 110, 196). β-Lap-induced cell death is triggered by the NQO1-dependent
3 http://clinicaltrials.gov/ct/show/NCT00102700?order=4
100 oxidoreduction of β-lap (110), resulting in a futile cycling, wherein β-lap is reduced to an unstable hydroquinone that spontaneously reverts to its parent structure using two oxygen molecules (146). As a result, ROS are generated causing DNA damage, γ-H2AX foci formation, poly(ADP-ribose) polymerase-1 (PARP-1) hyperactivation, and subsequent loss of ATP and NAD+ (197). β-Lap-induced cell death is unique in that PARP-1 and p53 proteolysis occurred concomitant with µ-calpain activation (146). β-Lap-mediated cell death exhibits classical features of apoptosis (e.g. DNA condensation, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells), but is not dependent on typical apoptotic mediators such as p53, Bax/Bak, or caspases
(103).
To date, few studies have explored the contribution of DSB repair in β-lap- induced cell death. We previously demonstrated that β-lap caused DNA damage, γ-
H2AX focus formation, and PARP-1 hyperactivation selectively in NQO1-expressing
(NQO1+) cells. We investigated whether β-lap exposure of NQO1+ cancer cells activated
HR and/or NHEJ, and explored the extent to which these repair systems influenced cellular sensitivity. Various model cell systems with altered ATM, ATR, and DNA-PKcs functions, as well as selective kinase inhibitors, were utilized. β-Lap caused delayed, compared with IR (10-15 min), dose-dependent activation of the MRN complex, as well as ATM, DNA-PKcs and ATR. Importantly, only inhibition of DNA-PKcs enhanced β- lap potency, indicating a predominant role for NHEJ in DSB repair, and resistance of cancer cells to this agent. These results suggest the combinatorial use of NHEJ inhibitors to enhance β-lap cytotoxicity for the treatment of human cancers that express elevated
NQO1 levels.
101 EXPERTIMENTAL PROCEDURES
Reagents- β-Lap was synthesized by Dr. William G. Bornmann (MD Anderson), dissolved in dimethyl sulfoxide (DMSO) at 40 mM, and the concentration verified by spectrophotometry (197). Hoechst 33258, etoposide (ETO), and dicoumarol (DIC) were obtained from Sigma (St. Louis, MO). The DNA-PKcs inhibitor, Nu7026 (2-
(Morpholin-4-yl)-benzo[h]chromen-4-one), and ATM/ATR kinase inhibitor (AAI) were obtained from Calbiochem (La Jolla, CA). They were dissolved in DMSO and used at 10
µM unless otherwise stated. 2-Morpholin-4-yl-6-thianthren-1-yl-pyran-4-one (KU-
55933) was synthesized by KuDOS Pharmaceuticals Ltd. (Cambridge, UK), dissolved in
DMSO and used at 10 µM.
Cell culture- MCF-7 breast cancer cells were maintained and used as described (110).
Human MO59K and MO59J cells, proficient and deficient in both DNA-PK activity and p350 protein, respectively (198) were obtained from the American Type Culture
Collection (Manassas, VA). U2OS-derived stable cell lines that conditionally regulate wild-type (WT) or kinase-dead ATR (KD-ATR) levels by doxycylin (dox) were generously provided by Dr. Paul Nghiem/Dr. Stuart L. Schreiber (Harvard University)
(199). Recombinant ATM was stably expressed in immortalized human A-T cells using an episomal expression vector (200) and designated: A-T cells (ATM-/-) and A-T cells ectopically expressing ATM (ATM+/+). MO59K, MO59J, ATM-/-, and ATM+/+ cells were stably infected using a puromycin-selectable pLPCX retroviral vector alone or one containing the NQO1 cDNA packaged in Phoenix-Ampho cells (201). Uninfected cells were removed by puromycin selection (0.5 µg/mL). However, all experiments were
102 performed without selection. NQO1 expression was evaluated in all cells as described
(110). All cells were grown in high glucose-containing DMEM containing 10% fetal bovine serum (FBS) or tetracycline-free FBS (U2OS cells), 2 mM L-glutamine, penicillin
(100 units/mL), and streptomycin (100 mg/mL) at 37°C in a 5% CO2, 95% air humidified atmosphere (104). All tissue culture components were purchased from Invitrogen
(Carlsbad, CA). Cells were free of mycoplasma contamination.
Relative Survival Assays- Relative survival assays were performed as described (110).
MCF-7, MO59K, and MO59J cells were pretreated for 1 h with 10 µM Nu7026 or KU-
55933 prior to co-treatment with β-lap at the indicated doses for 2-4 h. After β-lap treatment, medium containing Nu7026 or KU-55933 alone was added and then removed after 16 h. Drug-free medium was added and survival was assessed after 6 days. U2OS
WT or KD-ATR cells were treated with 1 µg/mL dox 48 h prior to treatment with various
β-lap doses, with or without DIC (40 µM) for 2-4 h, then replaced with drug-free media.
Prior studies using β-lap showed that relative survival assays correlated directly with colony forming ability (110). Data were expressed as means, + S.E. for treated/control
(T/C) from separate triplicate experiments, and comparisons analyzed using a two-tailed
Student’s t test for paired samples.
Cell irradiation- For ultraviolet light C (UVC) exposures, U2OS WT and KD-ATR culture medium were removed, and then placed uncovered under a UV lamp emitting primarily 254 nm radiation (fluency rate of 2.2 J/m2/s). After exposure, medium was replaced and cultures incubated for various times. Other cells were treated with 10 µM
β-lap or 500 µM H2O2 and harvested for immunoblotting 2 h after treatment. For IR,
103 cells were irradiated using a 137Cs source as described and analyzed for foci formation
(see below) (169).
Immunoblotting- Western blots were prepared as described (103) with modifications. To examine Chk1, whole cell extracts were prepared using lysis buffer (50 mM Tris, pH 8.0,
150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% NP-40, 10 µl/ml protease inhibitor mixture, and 1 mM sodium metavanadate). Total α-Chk-1 and the α-phospho-Ser345
Chk-1 (Chk1-pSer345) antibody were used at a dilution of 1:100 (Cell Signaling
Technology, Danvars, MA), α-tubulin was used at a dilution of 1:5000 (Calbiochem), and α-PAR was used at 1:2000 (BD Pharmingen). An NQO1 antibody was generously provided to us by Dr. David Ross (Univ. of Colorado Health Science Center) and used at a 1:2000 dilution (150).
Confocal microscopy- MCF-7 cells were treated with 1-5 µM β-lap for various times with or without pre- and co-treatment with Nu7026, KU-55933 or DMSO. As a positive control for DSB damage responses, cells were irradiated with 5 Gy, and fixed 15 minutes later. After treatment, cells were fixed in methanol/acetone (1:1) and incubated with primary antibodies: α-MRE11, α-Rad50 (GeneTex, San Antonio, TX), α-Nbs-1 phospho-Ser343 (Nbs-1-p) (Abcam, Cambridge, MA), α-ATM phospho-Ser1981, α-
DNA-PKcs phospho-Thr2609 (Rockland, Gilbertsville, PA), or α-γ-H2AX (Trevigen,
Gaithersburg, MD; Upstate, Charlottesville, VA) overnight at 4°C at 1:100-500 dilutions.
Alexa Fluor fluorescent secondary antibodies (Molecular Probes, Eugene, OR) were added for 2 h at room temperature. Nuclei were visualized by Hoechst 33258 (10 mg/mL) staining at 1:3000 dilution. All confocal images were acquired using a 63X numerical aperture of 1.4 oil immersion planapochromat objective using a Zeiss LSM
104 510 inverted laser-scanning confocal microscope (Jena, Germany). Images of Alexa
Fluor 488 dye were collected using a 488-nm excitation light from an argon laser, a 488- nm dichroic mirror and 500-550-nm band pass barrier filter. Images of Alexa Fluor 594- nm dye were collected using a 543-nm excitation light from HeNe1 laser using a 543-nm dichroic mirror and 560-nm long pass filter. All Hoechst 33258 stained nuclear images were collected using a Coherent Mira-F-V5-XW-220 (Verdi 5W) Ti-Sapphire laser tuned at 750-nm, a 700-nm dichroic mirror and a 390-465 nm band pass barrier filter. Images shown were representative of experiments performed at least three times. The average number of foci/1 µM slice was calculated as means, + S.E. by counting ≥30 cells from three independent experiments. Student’s t tests were used for comparison.
Alkaline and Neutral Comet Assays- Single cell gel electrophoretic comet assays were performed under alkaline or neutral conditions (197). MCF-7 cells were treated with 5
µM β-lap, 100 µM ETO, 500 µM H2O2, or vehicle alone and harvested at various times.
For neutral comet assays, after cellular lysis, slides were immersed in neutral buffer (1 X
TBE, pH 7.0) for 60 min at room temperature in the dark. Each datum point represents the average of 100 cells + S.E., and data are representative of experiments performed in duplicate.
RESULTS
MRE11/Rad50/Nbs-1 (MRN) complex activation by β-Lap- Previously, we showed that exposure of NQO1-expressing cancer cells to β-lap caused ROS and DNA damage, measured by alkaline comet assays and γ-H2AX formation (197). Furthermore, activation
105 of the MRN complex following β-lap exposure was recently reported in Saccharomyces cerevisiae (202). We wanted to determine if, and which, DSB repair pathways were activated in response to β-lap by examining MRN complex recruitment. Although the true nature of damage-induced foci has not been elucidated, these protein complexes are suggested to be a visual indication of DNA repair centers (13). Since the MRN complex is central to both HR and NHEJ, we examined foci formation in MCF-7 cells after various times of β-lap exposure. Mock- or β-lap-treated cells were stained with antibodies complementary to MRE-11, Rad50, and phosphorylated Ser343 Nbs-1 (Nbs-1- p). A marked increase in the organized localization of MRE-11 and Rad50 were observed in the nuclei of β-lap treated cells at 15 min, with foci persisting >60 min
(Figure 3.1A and Figure 3.1B). Similarly, Nbs-1-p nuclear foci were visible beginning 15 minutes after drug exposure, with similar kinetics as γ-H2AX (Figure 3.1A and Figure
3.1B); both proteins are downstream targets of ATM activation (203). MRN foci were not visible prior to 15 min (data not shown). Foci formation was delayed compared to foci observed after IR exposure, where prior studies have demonstrated γ-H2AX foci formation within 1-5 mins post-IR (204). Interestingly, with IR-treated cells MRN foci,
γ-H2AX, and phosphorylated DNA-PKcs randomly appeared throughout the nucleus, whereas with β-lap-treated cells all these proteins formed foci that were predominantly perinuclear (Figure 3.2 and data not shown). The appearance of perinuclear foci is consistent with the fact that NQO1 is largely cytoplasmic (205) and where ROS would diffuse into the nucleus, leading to SSBs. SSBs may then be converted to DSBs at a later time, thereby explaining the delayed activation of the MRN complex.
106
Figure 3.1. The MRN complex is activated upon β-lap treatment. β-Lap causes the punctate nuclear localization of MRE11, Rad50, and the phosphorylation of Nbs-1 (Nbs-
1-p). A, Visualization of MRE11, Rad50, Nbs-1-p and γ-H2AX in MCF-7 cells after treatment for 30 min with 5 µM β-lap by confocal microscopy. B, Average number of
MRE11, Rad50, Nbs-1-p, and γ-H2AX foci per cell was determined from ≥60 cells for
107 each treatment group from three independent confocal experiments (means + S.E.). Scale bar = 20 µm
Figure 3.2. Perinuclear localization of DNA repair proteins following β-lap treatment. MCF-7 cells were treated with 5 µM β-lap for 30 min or 10 Gy. Samples were fixed and probed with antibodies to γ-H2AX and phosphorylated DNA-PKcs at
Thr2609 (DNA-Pkcs-pThr2609). Nuclei were stained with Hoechst 33258 and cells were visualized by confocal microscopy. Scale bar = 20 µm
108 Dose-dependent activation of ATM and DNA-PK following β-lap treatment-
Since the MRN complex was recruited to form discrete foci after exposure to β-lap, we examined cells for activation of ATM, an HR-associated PI3K. After interacting with a
DSB, ATM undergoes autophosphorylation at Serine 1981, causing dissociation of the
ATM homodimer (206). Activated ATM monomers phosphorylate numerous downstream proteins, including Nbs-1 (203). To test for ATM activation, MCF-7 cells were mock-treated or exposed to various β-lap doses. Fixed cells were stained with a
Serine 1981 phospho-specific antibody to ATM (ATM-pSer1981). ATM activation occurred in a dose-dependent manner after β-lap treatment. β-Lap doses (0-3 µM) resulted in few activated ATM foci, consistent with low levels of DNA damage detected
(Figure 3.3A and Figure 3.7). In contrast, lethal doses of β-lap (≥4 µM) caused considerable ATM activation with ~8-fold increase in the number of foci/cell, corresponding to the net increase in total damage (Figure 3.3C and Figure 3.7). These data suggest that the accumulation of large numbers of β-lap-induced SSBs leads to
DSBs that, in turn, activate the canonical HR DSB repair pathway involving MRN and
ATM.
Due to the cell-cycle independence of NQO1-mediated bioactivation of β-lap
(104) and generation of DNA damage, we examined DNA-PK activation, since it functions in a cell-cycle independent manner (103). Prior work indicated that DNA-PKcs was autophosphorylated at Thr2609 (DNA-PKcs-pThr2609) in vivo in response to IR, and DNA-PKcs-pThr2609 co-localizes with γ-H2AX after damage (207, 208). MCF-7 cells were mock-treated or exposed to 1-5 µM β-lap for 30 min. Similar to ATM activation, DNA-PKcs-pThr2609 foci formed in a dose-dependent manner. Non-toxic β-
109 lap doses caused ≤ 2 foci/cell DNA-PKcs-pThr2609 foci over background, whereas lethal doses led to increases in DNA-PKcs-pThr2609 foci that co-localized with γ-H2AX (207)
(Figure 3.3B). Importantly, at 3 µM β-lap (~LD50 for MCF-7 cells), DNA-PKcs was significantly activated (9 + 0.2 foci/cell), while ATM and γ-H2AX were not as robustly activated (3.9 foci/cell + 1.98 and 3.0 foci/cell + 0.12, respectively). These data suggested that both HR and NHEJ were activated after β-lap exposure, but the predominant DNA repair pathway activated was NHEJ (Figure 3.2C).
110
Figure 3.3. Dose-dependent ATM and DNA-PK activation after β-lap administration. MCF-7 cells were treated with 0-5 µM β-Lap for 30 min at which time samples were fixed and probed with antibodies to A, phosphorylated ATM at Ser1981
(ATM-pSer1981) or B, phosphorylated DNA-PKcs at Thr2609 (DNA-Pkcs-pThr2609) and visualized by confocal microscopy. C, MCF-7 cells were treated with 0-5 µM β-lap for 30 min, fixed and probed for ATM-pSer1981, DNA-PKcs-pThr2609, and Nbs-1-p
111 after 30 min. Shown is the quanitation of the average number of foci/cell per dose of β- lap treated cells for ≥60 cells per treatment (means + S.E.). Student’s t-test for paired samples, comparing Nbs-1-p or γ-H2AX foci/cell versus the number of DNA-PKcs- pThr2609 foci/cell at various doses of β-lap are indicated (*, p<0.05). Scale bar = 20 µm
112 NHEJ is necessary for β-lap-induced cell death- Since DNA-PK appeared to be the most activated in response to β-lap, we examined the consequence of its inhibition on β- lap-induced lethality. Glioblastoma cell lines, MO59K (containing DNA-PKcs) and
MO59J (lacking DNA-PKcs) were used (209). These cells lacked NQO1 and were resistant to β-lap. After isolating NQO1-expressing pooled variants, MO59K (MO59K-
NQ+) and MO59J (MO59J-NQ+) cells had NQO1 enzymatic activities of 790 + 20 and
690 + 10 µmols/cyt c reduced/µg protein, respectively. MO59J-NQ+ and MO59K-NQ+ were mock-treated or exposed to various β-lap doses for 2 h, with or without DIC cotreatment (a selective NQO1 inhibitor). DNA-PKcs-deficient MO59J-NQ+ cells were significantly more sensitive to β-lap than their DNA-PKcs-proficient counterparts,
MO59K-NQ+ cells (Figure 3.4A). MO59J-NQ+ cells required doses of ~5 µM β-lap to elicit cell death, whereas MO59K-NQ+ cells were resistant, with only 30% cytotoxicity by 12 µM β-lap. In both cell lines, cytotoxicity was abrogated by inhibiting NQO1 activity with 40 µM DIC (Figure 3.4A). To assess DNA-PKcs functionality, we pretreated both MO59J-NQ+ and MO59K-NQ+ with a DNA-PKcs selective inhibitor
Nu7026 prior to β-lap exposure. Nu7026 is a potent radiosensitizer in both proliferating and quiescent cells (210). As anticipated, Nu7026 had little effect on β-lap-induced cell death in MO59J-NQ+ cells, since they are devoid of DNA-PKcs activity. In contrast,
Nu7026 significantly sensitized MO59K-NQ+ cells to β-lap (Figure 3.4B). Treatment with Nu7026 and 12 µM β-lap resulted in an ~80 + 3% reduction in survival versus ~5 +
1% loss of survival in MO59K-NQ+ cells that were pretreated with vehicle (Figure 3.4B).
To confirm that DNA-PK was essential in resistance to β-lap-induced cell death,
MCF-7 cells were treated with sub-lethal doses of β-lap with or without pre- and co-
113 treatment with 30 µM Nu7026 for 2 h and relative survival determined. β-Lap doses ≤ 2
µM had little to no cytotoxicity alone, whereas β-lap doses ≥ 2.5 µM caused significant lethality. Furthermore, MCF-7 cells treated with Nu7026 and β-lap potentiated β-lap- induced lethality (Figure 3.4C). Significant differences in cell death were noted at 2-2.5
µM β-lap, where co-treatment with otherwise non-toxic doses of Nu7026 and β-lap resulted in an >80% reduction in survival compared to cells treated with β-lap alone
(Figure 3.4C). To confirm that DNA-PKcs and not ATM was important in β-lap-induced cell death, MCF-7 cells were treated with 5 µM β-lap alone with or without pre- and co- treatment with 30 µM Nu7026. Effects on DNA-PKcs versus ATM foci formation were then assessed. After 30 min of β-lap exposure, cells were fixed and stained with antibodies to both phosphorylated forms of DNA-PKcs-pThr2609 and ATM-pSer1981 and the average number of foci/cell were examined by confocal microscopy. β-Lap- treated MCF-7 cells resulted in 14 + 0.1 DNA-PKcs-p-Thr2609 foci/cell, that was reduced to 3 + 1 foci/cell after Nu7026 coadministration (Figure 3.4D). ATM-pSer1981 foci were not altered after Nu7026 and β-lap treatment versus β-lap alone (5 + 3 v. 5 +
0.7, respectively) indicating that DNA-PKcs was the predominate PI3K inhibited after treatment with Nu7026 (Figure 3.4D).
We previously demonstrated that β-lap-induced cell death was mediated by
PARP-1 (197). Since a deficiency in DNA-PKcs potentiated β-lap-induced cell death, we examined whether NHEJ inhibition was accompanied by PARP-1 hyperactivation at sub-lethal doses of β-lap. PARP-1 is associated with both SSB and DSB repair. After binding to DNA breaks, PARP-1 converts β-NAD+ into polymers of branched or linear poly(ADP-ribose) (PAR) units and attaches them to various nuclear proteins, including
114 PARP-1 itself as part of its autoregulation (148). MO59J-NQ+ and MO59K-NQ+ cells were treated with various doses of β-lap and cell extracts prepared at 20 min post- treatment. Lethal doses of β-lap in MO59J-NQ+ cells resulted in PAR accumulation, while the same dose that was non-toxic to MO59K-NQ+ cells resulted in little or no PAR accumulation (Figure 3.4E). Increasing levels of PAR polymers were noted in the
MO59K-NQ+ cells with increasing (≥5 µM) β-lap doses (Figure 3.4E). β-Lap treatment of MCF-7 cells in combination with Nu7026, but not with the ATM and ATR inhibitors
(KU55933 and AAI) resulted in PAR formation (data not shown).
115
Figure 3.4. Loss of DNA-PKcs activity potentiates β-lap-induced cell death. A-C, cell death was examined using relative survival assays in NQO1 containing cells. A,
MO59K-NQ+ (DNA-PKcs positive) and MO59J-NQ+ (DNA-PKcs negative) cells were treated with varying doses of β-lap alone or in combination with 40 µM DIC for 2 h.
After drug exposure, media were removed and drug-free media added. Cells were then allowed to grow for an additional 6 days and relative survival, based on DNA content was determined by Hoechst 33258 staining as described under “Experimental
116 Procedures.” B, Loss of DNA-PKcs kinase activity sensitizes MO59K-NQ+ cells to β-lap.
Relative survival assays using MO59K-NQ+ and MO59J-NQ+ pre- and co-treated with the DNA-PKcs inhibitor, Nu7026 (10 µM) for 1 h, prior to treatment with varying doses of β-lap for 2 h. C, Inhibition of DNA-PKcs with Nu7026 potentiates β-lap-induced cell death in NQO1+ MCF-7 cells. MCF-7 cells were treated with sub-lethal to lethal doses of
β-lap alone with or without pre- and co-treatment with 30 µM Nu7026 for 2 h.
Differences were compared using two-tailed Student’s t-test. Groups having *, p≤0.05;
**p≤0.001 values compared with β-lap alone are indicated. D, Nu7026 inhibits phosphorylation of DNA-PKcs-pThr2609 but not ATM-pSer1981. MCF-7 cells were treated with 5 µM β-lap with or without pre- and co-treatment with 30 µM Nu7026 for 30 min. After treatment, cells were fixed and probed with antibodies to DNA-PKcs- pThr2609 and ATM-pSer1981 and foci were visualized by confocal microscopy. Shown is the quanitation of the average number of foci/cell for at least 60 cells per treatment group (means + S.E.). Student’s t-test for paired samples, experimental groups containing β-lap + KU55933 (KU) or Nu7026 (NU) versus β-lap alone are indicated
*p<0.001. E, Lack of DNA-PKcs causes PARP-1 hyperactivation after β-lap treatment.
Immunoblot analyses of PAR and α-tubulin protein levels from whole cell extracts from
MO59J-NQ+ and MO59K-NQ+ cells that were mock treated or treated with 5 or 12 µM
β-lap and harvested after 30 min. Relative PAR levels were determined by densitometry analyses using α-tubulin loading controls by NIH ImageJ wherein controls were set to
1.0 (means + S.E.; arbitrary units (AU)).
117 HR-associated PI3K ATM is not necessary for β-lap-induced cell death- Since ATM autophosphorylation was observed after β-lap treatment in NQO1-proficient cancer cells, we investigated whether loss of ATM would alter β-lap-mediated lethality. Isogenic
NQO1+ human immortalized fibroblasts from A-T patients deficient in ATM (ATM-/-) or proficient via ectopic ATM expression (ATM+/+) were used (200). ATM+/+ and ATM-/- cells were mock-treated or exposed to various β-lap doses with or without DIC for 4 h.
There was no observable difference in β-lap-induced lethality between NQO1-proficient
ATM+/+ or ATM-/- cells, and both cells were protected from lethality by DIC (Figure
3.5A).
To corroborate these findings, MCF-7 cells were mock-treated or exposed to sub- lethal-to-lethal doses of β-lap in the presence or absence of the ATM kinase inhibitor
KU55933, or the general ATM/ATR inhibitor, AAI, for 2 or 4 h (20). KU55933 inhibited ATM-p-Ser1981 after β-lap or IR treatments, but did not inhibit NQO1 (Figure
3.4D and data not shown). Although weak ATM activation was observed after sub- lethal doses of β-lap, inhibition of ATM by KU55933 or AAI had little affect on β-lap- induced lethality (Figure 3.3C and Figure 3.5B-C).
118
Figure 3.5. β-Lap-induced cell death is not dependent on ATM. A-C, Cell death was monitored using relative survival assays in NQO1+ cells. A, Loss of ATM does not potentiate β-lap-induced cell death. NQO1-proficient ATM-/- and ATM+/+ cells were treated with varying doses of β-lap alone or in combination with 40 µM DIC for 4 h. B,
MCF-7 cells were treated with 0-4 µM β-lap alone or with pre- and co-treatment with the
119 ATM kinase inhibitor, KU55933 (10 µM), for 2 h or 4 h or C, the ATM and ATR kinase inhibitor, AAI (10 µM), for 2 h.
120 ATR activation after β-lap exposure- HR can also be mediated by ATR, which is recruited to single-stranded DNA regions, arising due to replication fork arrest or during the processing of bulky lesions such as UV photoproducts (211). We reasoned that β-lap could generate ROS-induced SSBs (197), causing replication fork stalling. Thus, we examined whether ATR activation occurred in response to β-lap by utilizing a set of stable, NQO1+ cell lines derived from U2OS cells (human osteosarcoma). These cells are wild-type for p53, have an intact G1 DNA-damage checkpoint, and allow doxycycline- inducible expression of either wild-type ATR or a dominant-negative (kinase-dead) ATR point mutant (199). ATR activation was confirmed by monitoring Chk1-pSer345 levels in
WT U2OS cells after exposure to UVC or β-lap (Figure 3.6A). Chk1-pSer345 was muted in U2OS KD-ATR cells after either UVC or β-lap exposures (Figure 3.6A). Importantly, neither expression of WT ATR or inhibition of ATR by KD-ATR affected the survival of
NQO1+ U2OS cells after β-lap exposure (Figure 3.6B). Administration of DIC abrogated
β-lap-induced lethality in both cell lines (Figure 3.6B).
To confirm our findings that HR was not necessary for β-lap-induced cell death, we treated MCF-7 cells with AAI, an inhibitor of both ATM and ATR, prior to β-lap exposure. Inhibition of both enzymes was not sufficient to enhance β-lap-mediated cytotoxicity compared to the 80% enhancement observed after inhibition of DNA-PKcs
(Figure 3.4C and Figure 3.5C). These data indicate that ATR signaling is activated after
β-lap exposure, but that it is not a predominant factor required for survival.
β-Lap-induced ATR activation and PARP-1 hyperactivation suggested that this compound caused extensive SSBs, while MRN, ATM, and DNA-PK foci formation indicated delayed DSB formation. Neutral and alkaline comet assays were performed to
121 elucidate the type(s) of breaks created in NQO1-proficient human cancer cells after β-lap exposure. Sub-lethal doses of β-lap resulted in no detectable strand breakage over time, consistent with their survival (Figure 3.7) (197). In contrast, lethal doses of β-lap resulted in significant total DNA breaks, occurring immediately after drug exposure at levels surpassing breaks created after 500 µM H2O2 (Figure 3.6C). Prior data show that
ROS formation and DNA damage were detected within ≤ 5 min after β-lap addition
(197). The amount of DNA strand breaks increased over time after 5 µM β-lap treatment, suggesting that the lethal event may be related to the total amount of DNA breaks generated4. Interestingly, when cells were analyzed using neutral conditions, little to no
DSBs were detected, in contrast to ETO treatment (Figure 3.6C). As expected, H2O2 treatment caused few DSBs, similar to damage observed after β-lap exposure (Figure
3.6C). The formation of DNA breaks after β-lap treatment was NQO1-dependent since
DIC prevented damage (197). These studies indicate that the majority of DNA damage caused by the NQO1-mediated metabolism of β-lap was SSBs, consistent with the genesis of “long-lived” ROS precursors (e.g. H2O2).
4 Reinicke, K.E., Bentle, M.S., Bey, E.A., Dong, Y. Bornmann, W., Spitz, D.R., and Boothman, D.A. NAD(P)H:quinone oxidoreductase 1-dependent reactive oxygen species are necessary but not sufficient for β-lapachone-mediated cell death.
122
Figure 3.6. β-Lap causes ATR activation and SSBs. A, Chk1-pSer345 occurs after β- lap treatment. Immunoblots of Chk1-pSer345 and α-tubulin protein levels from whole cell extracts of U2OS KD-ATR treated with or without 1 µg/mL of dox for 48 h prior to treatment with 10 µM β-lap or 15 J/m2 UVC. Cells were harvested at the indicated times after treatment. Relative Chk1-pSer345 levels were calculated by densitometric analyses by NIH ImageJ using α-tubulin, wherein controls were set to 1.0 (means + S.E.; arbitrary units (AU)). B, KD-ATR does not alter cell death caused by β-lap. U2OS KD-ATR cells were treated with or without 1 µg/mL of dox for 48 h to induce expression of the dox-
123 inducible kinase-dead ATR prior to treatment with varying doses of β-lap alone or in combination with 40 µM DIC for 2 h. C, β-Lap causes the formation of DNA SSBs as shown by comet assay under alkaline and neutral conditions. MCF-7 cells were treated with 5 µM β-lap for 0-120 min. At the indicated times during drug treatment, cells were harvested for comet tail formation under either alkaline or neutral conditions. Shown are quantified comet tail lengths of 100 cells (means + S.E.) for each time and condition calculated using NIH ImageJ software. The arrow indicates the direction of electrophoresis. Not shown: 100 µM ETO for 60 h under neutral and alkaline conditions had a comet tail length of roughly 81 + 4 microns v. 64 + 1 microns, respectively.
124
Figure 3.7. β-Lap exposure induces DNA damage. NQO1+ MCF-7 cells were treated with various doses of β-lap (1, 2, or 5 µM), or H2O2 (500 µM, as a positive control) for the indicated times. Medium was removed, cells harvested and analyzed by comet assays using ImageJ software as described in “Experimental Procedures.” Each point represents the mean ± SEM of 100 individually analyzed cells from three independent experiments performed in triplicate.
125 DISCUSSION
β-Lap, a natural product-based antitumor agent, elicits a unique cell death pathway selectively in cancer cells that express elevated NQO1 levels. The drug is currently in phase I/II clinical trials for the treatment of pancreatic, as well as other cancers5. We recently demonstrated that β-lap-induced cell death was dependent upon
PARP-1 hyperactivation, but not typical apoptotic mediators, such as p53, Bax/Bak or caspases (146). Data from others suggested that β-lap does not cause DNA damage (176).
However, we recently showed that β-lap-induced cell death was initiated by the NQO1- dependent generation of ROS, subsequent formation of DNA damage, and calcium- dependent PARP-1 hyperactivation. Once stimulated, PARP-1 hyperactivation depletes
ATP/NAD+ pools inhibiting DNA repair. Therefore, once a threshold level of DNA breaks are formed, PARP-1 hyperactivation appears to be the dominant factor, dictating downstream events leading to cell death (Figure 3.8A) (197). Since reaching the threshold level of DNA breaks required to hyperactivate PARP-1 is critical for the lethality of this drug, understanding the mechanism(s) by which cells resist this threshold
(e.g. amplified DNA repair) is important for improving its efficacy.
To determine the repair pathways activated by β-lap, we examined a number of proteins involved in DSB repair, due to their very rapid and specific localization, and modification at double-stranded breaks (13).
5 http://clinicaltrials.gov/ct/show/NCT00102700?order=4 and http://clinicaltrials.gov/ct/show/NCT00358930?order=5
126 A. Lethal B. Sub-Lethal dose dose
Figure 3.8. Model of β-lap-induced cell death after lethal and sub-lethal doses. A, β-
Lap-induced cell death at lethal doses is PARP-1-dominated. β-Lap-mediated metabolism by NQO1 generates ROS that cause DNA SSBs. Extensive SSBs cause the hyperactivation of PARP-1, subsequent NAD+ and ATP loss, which inhibits DNA repair and causes cell death. B, Non-lethal doses of β-lap are converted to lethal events via
DNA-PK inhibition. At sub-lethal doses of compound, repairable amounts of SSBs are generated, some of which may be converted to DSBs possibly via replication fork arrest.
This causes the activation of members of the PI3K family of kinases, ATM, ATR, and
DNA-PKcs. However, NHEJ appears to be the primary repair pathway needed to repair
DSBs after drug treatment as only inhibition of DNA-PKcs by either chemical (Nu7026) or genetic (DNA-PKcs-/-) means potentiated the toxicity of sub-lethal doses of β-lap leading to PARP-1-mediated cell death.
127 The appearance of β-lap-induced DNA damage and MRN complex activation was delayed (e.g. 15 min) with respect to IR-mediated damage and MRN activation (e.g. < 2 min) (Figure 3.1A-B and Figure 3.6). Simultaneously, we noted the activation of ATM and DNA-PK as monitored by their autophosphorylation products, which only occurred in NQO1 expressing cells (Figure 3.3A-C and Figure 3.9). We did, however, detect low levels of γ-H2AX and DNA-PKcs-pThr2609 foci in NQO1-deficient cells, but we hypothesize that this may be due to two one-electron reductions of β-lap by cytochrome
P-450 reductase and cytochrome b5 reductase (212). Overall, however, these data support a role for NQO1 in amplifying the lethal effects of β-lap via its two-electron oxidoreduction (Figure 3.9). Activation of ATM, DNA-PK, and the MRN complex were dose-dependent (Figure 3.1 and Figure 3.3). Evidence from our laboratory suggests that the dose-dependent nature of DSB repair activation may be due to a minimal threshold of
DNA damage created after β-lap treatment.
DNA-PKcs plays a direct role in DSB repair by acting as a key component of
NHEJ, because defects in kinase activity result in radiosensitivity (209). Since NHEJ is the primary mechanism by which mammalian cells repair DSBs, we tested the hypothesis that NHEJ was not only activated after β-lap treatment, but was necessary to protect cells from β-lap-induced lethality at sub-lethal doses. We noted a ~2-fold increase in the number of DNA-PKcs-pThr2609 versus ATM-pSer1981-induced foci after β-lap exposure (Figure 3.3C). Further examination of MO59K-NQ+ and MO59J-NQ+ cells revealed that NHEJ was essential for the survival of cells after β-lap treatment (Figure
3.4A).
128
Figure 3.9. β-Lap-induced DNA damage is NQO1-mediated. MDA-MB-231 NQO1- positive and NQO1-negative (231-NQ+ and 231-NQ- respectively) cells were treated with
6 µM β-lap for 60 min and fixed and probed with antibodies to DNA damage-associated proteins; γ-H2AX and DNA-PKcs-pThr2609. Cells were visualized by confocal microscopy. Scale bar = 20 µm
129 Importantly, co-treatment of cells with β-lap + Nu7026, a DNA-PKcs inhibitor, sensitized MO59K-NQ+ cells as well as MCF-7 cells (Figure 3.4B-C), indicating a major role for NHEJ in repair of β-lap-induced DNA damage.
Furthermore, Nu7026 cotreatment with β-lap shifted the LD50 from 2.5 µM to less than 1.5 µM (Figure 3.4C). While this shift may appear modest, the dose response curve for β-lap alone was steep. At 2 µM β-lap, the drug was relatively non-toxic, whereas ≥
50% of the cells survived 2 µM β-lap and Nu7026 exposure. Nu7026 + β-lap eliminated the shoulder of the dose-response curve caused by β-lap alone, but the overall survival curve was parallel to that of β-lap alone (Figure 3.4C). These data indicate that the shoulder, representing a DNA repair threshold, was inhibited by Nu7026. Interestingly, unlike in MCF-7 cells treated with Nu7026 and β-lap, the overall survival curves between the MO59J-NQ+ and MO59K-NQ+ cells did not parallel one another (Figure 3.4A-C).
The relative discrepancies between these two cells lines may be explained by the DNA repair deficiency in the MO59J-NQ+ cells. Since these cells lack DNA-PKcs, and thus
NHEJ ability, they may have accumulated other genetic abnormalities over repeated passages, that further sensitized them to the toxic effects of β-lap.
To determine if NHEJ inhibition caused PARP-1-mediated cell death at otherwise sub-lethal β-lap doses, we examined PAR accumulation in MO59J-NQ+ and MO59K-
NQ+ cells. In DNA-PKcs-deficient MO59J-NQ+ cells, doses of β-lap ≥ 5 µM alone caused significant PAR-modification (Figure 3.4E). However, proficiency of NHEJ in
MO59K-NQ+ cells muted PAR accumulation, consistent with its role as a cellular resistance factor in β-lap-induced lethality (Figure 3.4E). Co-administration of Nu7026 and β-lap in MCF-7 cells converted non-lethal events to a cytotoxic event accompanied
130 by the accumulation of PAR polymers not seen in the presence of ATM or ATR inhibitors (Figure 3.4C, Figure 3.5B-C, and data not shown). These data indicate that interruption of NHEJ during β-lap treatment commits cells to PARP-1-mediated cell death (Figure 3.8). A relationship between DNA-PK and PARP-1 has been established, and inhibition of both proteins increased net DSB over time after chemically-induced damage (213, 214). Thus, combining β-lap and NHEJ inhibitors would be an ideal “two- hit” cancer therapy, by inhibiting mechanistically diverse DNA repair enzymes and thereby retarding DSB rejoining.
In addition to phosphorylation of DNA-PKcs-pThr2609, ATM-pSer1981 was also noted after β-lap treatment (Figure 3.3A). While there is some discrepancy as to the exact role ATM plays in HR, it does play a pivotal role in general DNA damage signaling, cell cycle regulation, and the slow component of DSB repair (5, 215, 216).
After IR, A-T cells fail to show recovery from damage, and experiments using reverse genetics failed to show potentiation in cell death arising from the conditional loss of
RAD54 in ATM-/- cells, suggesting an inherent HR defect in ATM-/- cells (217). An interplay between ATM and DNA-PKcs may be important for the proper initiation of
DSB signaling and processing of DSBs to ensure repair and maintenance of genomic integrity after damage (218). Using cells deficient in ATM, we tested whether the loss of
ATM and therefore HR-mediated repair, would alter β-lap-mediated cell death to result in a form of cell death similar to that observed with NHEJ loss. Data from our studies revealed no significant difference between ATM+/+ and ATM-/- cells in terms of β-lap sensitivity (Figure 3.5A). Studies using KU55933 corroborated these findings, as inhibition of ATM did not augment lethality in β-lap-treated MCF-7 cells (Figure 3.5B).
131 The dose of KU55933 used in these studies was sufficient to inhibit phosphorylation of
ATM-pSer1981 after IR or β-lap-induced DNA damage (Figure 3.4D). Interestingly, inhibition of phosphorylation of ATM-pSer1981 by this inhibitor also abrogated phosphorylation of DNA-PKcs-pThr2609, as recently noted by Chen et. al. (218) supporting a role for ATM in the phosphorylation of DNA-PKcs-pThr2609 in response to
DNA damage (Figure 3.4D). Inhibition of DNA-PKcs with Nu7026 blocked ATM- mediated phosphorylation of DNA-PKcs-pThr2609 (Figure 3.4D). These data suggested that ATM-mediated phosphorylation of DNA-PKcs-pThr2609 was an added component of an amplification scheme whereby autophosphorylation of DNA-PKcs at Thr2609 was required prior to signal amplification via ATM.
ATR is recruited by ATR-interacting protein (ATRIP) to replication protein A
(RPA)-coated single-stranded DNA that accumulates at stalled DNA replication forks or is generated by processing of the initial DNA damage (203). Thus, ATR activation in response to β-lap exposure may be consistent with stalled replication forks and could indicate the formation of DSBs from SSBs in response to β-lap exposure (219, 220). The primary lesions generated after β-lap exposure were SSBs (Figure 3.6C). The lack of detectable DSBs could be the result of low sensitivity of neutral comet assays compared to γ-H2AX-induced foci formation to detect the few DSB populations. Importantly, formation of γ-H2AX, as well as the activation of MRN, ATM, DNA-PK, and ATR appeared in a delayed manner with respect to initial SSBs (< 5 min versus 10-15 min respectively) (197). These data support the hypothesis that DSBs are formed as a secondary DNA lesion after initial SSBs were generated, likely resulting from stalled
132 replication forks (Figure 3.8B) and such lesions appear not to be detected by the neutral comet assays.
Nevertheless, ATR signaling does not appear to be a major factor in β-lap resistance. Using U2OS KD-ATR and WT-ATR, we showed an increase in phosphorylation of Chk1-pSer345 after UVC, as well as after β-lap treatment. This response was muted in cells with low ATR expression (Figure 3.6A). Total levels of
Chk1 and NQO1 were relatively unchanged under dox treatment (data not shown).
U2OS KD-ATR cells treated with or without dox showed no enhanced β-lap toxicity
(Figure 3.6B). Furthermore, addition of AAI had no apparent effect on β-lap-induced cytotoxicity in MCF-7 cells (Figure 3.5C). Thus, neither ATM nor ATR appear to play major roles as resistance factors in β-lap lethality.
Many currently used cancer chemotherapeutic agents function primarily as non- selective inducers of DSBs in highly proliferative cells. A major problem with many of these agents is their lack of selectivity, in which both normal and cancerous tissues are targeted. In contrast, β-lap, which is currently under investigation in PhaseI/II clinical trials, selectively targets cancer cells that express elevated NQO1 levels, resulting in
DNA damage and cell death mediated by PARP-1 (197). Here, we demonstrate that in addition to SSB-induced DNA repair pathways, β-lap activates various DSB repair pathways. In particular, NHEJ was a key factor in the survival of cells exposed to β-lap, since inhibition of NHEJ causes PARP-1-mediated cell death after treatment with sub- lethal doses of β-lap. In summary, these data warrant the combinatorial use of β-lap with inhibitors of NHEJ thereby increasing the therapeutic efficacy of this compound by targeting two distinct repair mechanisms: NHEJ and PARP-1.
133 CHAPTER 4: Discussion and Future Directions
Chemical chemotherapeutic agents are used to treat many types of cancers, and most act as non-selective inducers of DNA damage in highly proliferative cells.
However, a major pitfall of these drugs is their lack of selectivity, whereby both cancerous and normal tissues are affected. As we understand more about the biological characteristics and mechanisms that make cancer cells distinct from non-cancerous cells, we are more apt to engineer targeted therapies, thereby reducing many of the deleterious side-effects associated with these chemotherapeutic agents.
β-Lap (a.k.a. ARQ501), a unique antitumor quinone, is currently in Phase II clinical trials for the treatment of pancreatic and head and neck cancers6. Our laboratory discovered that the key determinant for cell death mediated by β-lap was the activity of the two-electron oxidoreductase NQO1 (110). NQO1 is over-expressed in many human cancers of epithelial origin, such as breast, non-small cell lung, prostate and pancreatic cancers. Exploiting this fact, β-lap can be utilized to target these types of tumors in patients that overexpress NQO1. Understanding the mechanisms of β-lap-induced cell death will allow us to increase the therapeutic efficacy of this compound alone, or in combination with other chemotherapeutic agents and/or IR. In addition, details about the mechanism of action of this agent, will be a springboard for the rationale design of other agents that are bioactivated by NQO1 or other reductases overexpressed in cancer.
The studies detailed in this thesis build upon work started by our laboratory in
1989 and further characterized the molecular mechanisms of β-lap-induced cell death. In particular, we demonstrated that β-lap caused NQO1-dependent, Ca2+-mediated PARP-1
6 http://clinicaltrials.gov/ct/show/NCT00102700?order=4 and http://clinicaltrials.gov/ct/show/NCT00358930?order=5
134 hyperactivation (Chapter 2). Furthermore, we showed that the NQO1-mediated metabolism of β-lap generated ROS which, caused activation of the DNA DSB damage response pathways, and that NHEJ is the primary pathway responsible for the repair of
DSBs incurred after β-lap exposure (Chapter 3).
A.1 Conclusions
PARP-1-mediates β-lap-induced cell death
We found that the NQO1-dependent reduction of β-lap results in a futile redox cycle between the β-lap hydroquinone form and the parent compound (110). As a result, extensive ROS were generated causing DNA damage, H2AX phosphorylation and
PARP-1 hyperactivation (Chapter 2). PARP-1 hyperactivation results in the utilization and ultimate decline in NAD+ and ATP levels, causing inhibition of DNA repair and acceleration of non-caspase-mediated cell death. Interestingly, Ca2+ chelation could abrogate β-lap-induced: (i) γ-H2AX formation, (ii) PARP-1 hyperactivation, (iii) atypical PARP-1 and p53 proteolysis, and (iv) cytotoxicity. The mechanism of β-lap- induced cell death mimicked that observed after H2O2 exposure, but not MNNG.
Therefore, these data suggest a role for Ca2+ in the regulation of cellular metabolism after
ROS-induced DNA damage.
NQO1-mediated metabolism of β-lap resulted in ROS production concomitant with DNA damage and γ-H2AX (Figures 2.7A,B and 2.8). PARP-1 hyperactivation was observed ~10 min after β-lap treatment in an NQO1-dependent, but not cell type specific manner (Figures 2.3 and 2.4). Furthermore, NAD+ and ATP losses were observed within
30-60 min. PARP-1 inhibition or knock-down abrogated both nucleotide loss and cell
135 death (Figures 2.5, and 2.6D-F). Interestingly, chelation of intracellular Ca2+ with
BAPTA-AM efficiently suppressed all events occurring after β-lap treatment, thereby protecting the cells from death (Table 2.1). Notably, BAPTA-AM was able to block
PARP-1 hyperactivation, nucleotide pool depletion, atypical proteolysis, and cell death while having no measurable effect on NQO1 enzymatic activity, or ROS production.
However, BAPTA-AM pre-treatment did not completely block all DNA damage, as measured by both comet assay and γ-H2AX formation (Table 2.1). We, therefore, concluded that the observed decrease in DNA damage after β-lap + BAPTA-AM treatment was due to Ca2+ directly modulating and preventing PARP-1 hyperactivation.
Inhibition of PARP-1 hyperactivation spares cellular nucleotide levels allowing for repair of β-lap-induced DNA damage, promoting cell survival. Although Ca2+ was shown to affect PARP-1 function, we still cannot discount the possibility that Ca2+ chelation was dampening the initial damage created. The observed differences in damage levels may be due to the initial amount of damage created, rather than ongoing DNA repair, as these two processes would be indistinguishable in these experiments.
β-Lap-mediated cell death was similar to cell death caused by H2O2, an ROS
2+ generating agent. H2O2 treatment caused Ca -mediated PARP-1 hyperactivation (Figure
2.10A), atypical PARP-1 and p53 cleavage (Figure 2.12), apoptotic fragmentation (Figure
2.10C), and cell death. Interestingly, both β-lap and H2O2 elicited similar downstream pathways, but β-Lap was lethal in cells that expressed NQO1, whereas H2O2 was more cytotoxic in cells that were NQO1-deficient (Figures 2.3B versus 2.10A).
136 A.2 Future Directions
β-Lap’s cytotoxic properties are similar to that of ischemia-reperfusion
Globally, there are striking similarities between cell death caused by β-lap and ischemia-reperfusion. One of the hallmarks of ischemia is the failure to maintain normal
ATP levels due to decreases in mitochondrial energy production (e.g. oxidative phosphorylation, (OXPHOS)) (221). As a direct or indirect result of ATP loss, ion transporters such as the Na+/K+ ATPase, and the plasma-membrane Ca2+-ATPase
(PMCA) as well as ion exchangers including the Na+/Ca2+ exchanger, cease to function, altering cellular ion homeostasis. The resultant ion imbalance causes activation of a number of hydrolases including µ-calpain, and an increase in cellular membrane permeability (222). Concomitantly, dysregulation of ion transport systems from ongoing
2+ ATP loss and pH imbalances results in a rapid (within minutes) release of Ca from IP3 receptors located in the sarco/endoplasmic reticulum stores leading to a rise in cytosolic
Ca2+ (222). In cells still viable after ischemia, a number of enzymes integral to the mitochondrial electron transport chain become damaged. During ischemia, damage to the iron-sulfur peptide within Complex III, acts as the primary site for the net increase in
ROS generation. Complex III enhances ROS production because it directs the ROS formed away from the antioxidant defenses present in the mitchondrial matrix favoring its release from the mitochondria (223). ROS produced in ischemia-reperfusion include
• -• the hydroxyl radical (OH ), the superoxide anion radical (O2 ), H2O2 and peroxynitrite
- (ONOO ) (98, 224-226). During the reperfusion stage, H2O2, formed by the SOD-
-• 2+ mediated conversion of O2 , is converted to the hydroxyl radical catalyzed by free Fe .
137 Ischemia-Reperfusion β-Lap1 2+ 2+ IP3-mediated Ca release from ER stores, Ca release from ER stores, within 5 within 5 minutes of insult (227) minutes of drug treatment, receptor(s) unknown (131) • -• • -• Formation of ROS: H2O2, OH , O2 , Formation of ROS: H2O2, OH , O2 , ONOO- (221, 224) ONOO- (98, 225, 226)
ROS-mediated DNA damage (72) ROS-mediated DNA damage (197)
PARP-1-mediated depletion of NAD+ (72) PARP-1-mediated depletion of NAD+ (197, 228) Depletion of ATP due to impaired Depletion of ATP correlated with NAD+ OXPHOS and NAD+ consumption (222) consumption (197, 228) MMP due to Ca2+ overload from increased MMP due to Ca2+ overload from increased cytosolic Ca2+ (229) cytosolic Ca2+ (131) Extracellular Ca2+ influx from Na+/K+ Extracellular Ca2+ influx, receptor(s) ATPase, PMCA and SOC as a result of ER unknown (131) Ca2+ release (230, 231) µ-calpain activation (232) from µ-calpain activation from extracellular Ca2+ extracellular Ca2+ influx (233) influx (104, 139) Caspase-independent cell death (178) Caspase-independent cell death (104, 139)
p53-independent cell death2 (234, 235) p53-independent cell death3 (103)
1Studies performed in NQO1+ human cancer cells 2Studies performed in vivo 3Bey, E.A. and Boothman, D.A., unpublished data
Table 4.1. Comparison of the mechanism(s) of cell death induced by ischemia- reperfusion and β-lap.
138 These reactive species react directly with lipid membranes, proteins, and DNA thereby altering the ROS-generating and ROS-eliminating systems, causing further formation of
ROS. ROS produced during ischemia-reperfusion induces DNA strand breaks, leading to
PARP-1 hyperactivation, and NAD+/ATP loss (44). Furthermore, buffering of excess cytosolic Ca2+ by the mitochondria causes mitochondrial membrane depolarization
(MMP), leading to caspase-independent, µ-calpain-induced cell death (236).
The mechanism of β-lap-induced cell death stimulates a similar cell death pathway as seen in ischemia-reperfusion (Table 4.1). β-Lap administration in NQO1+ cells, causes a rise in cytosolic Ca2+ from ER stores within 3-5 minutes of drug exposure
(131). The NQO1-mediated reduction of β-Lap generates the same ROS species as in
• -• - ischemia-reperfusion that include: OH , O2 , H2O2, and ONOO (98, 225, 226). The
ROS formed cause DNA damage, leading to PARP-1-mediated NAD+ and ATP depletion
(197). After NAD+ and ATP are completely utilized, MMP ensues due to the increase in cytosolic Ca2+ (131). Presumably as a result of ion transporter inhibition due to ATP loss, a secondary influx of extracellular Ca2+ also occurs, activating the cysteine protease, µ- calpain and causing cell death (139).
These parallels between β-lap and ischemia-reperfusion have important implications for the future use of β-lap. Prevention of β-lap’s futile cycle under low oxygen conditions could decrease the effectiveness of the drug. Therefore, tumors with hypoxic regions may be less susceptible to β-lap-mediated cell death. Alternatively, targeting β-lap to the more vascularized tumor periphery, by localized delivery methods, could starve the tumor cells on the edge of the hypoxic region(s) due to the drug’s anti- angiogenic properties (237).
139 Information gleaned from the comparison of ROS generation agents (e.g. β-lap) and ROS-mediated pathologies (e.g. ischemia-reperfusion) suggests that PARP-1- mediated cell death is a global response to ROS-mediated cellular insults. For example,
PARP-1 inhibition during a number of ischemia-reperfusion conditions, especially in in vivo and in vitro models of myocardial infarction, was effective at decreasing cellular injury (72, 238). PARP-1 inhibitors, such as PJ34 attenuated PARP-1 activity, NAD+ and ATP depletion, as well as mitochondrial depolarization inhibiting both apoptotic and necrotic forms of cell death in ischemia-reperfused cadiomyoblasts (239). In vivo,
PARP-1 inhibition, or genetic deletion, has also been able to significantly rescue neurologic function in mice and pigs with aortic-occlusion induced spinal cord injury
(240). PARP-1 inhibition in these circumstances may prevent the aggravation of tissue damage, by switching necrotic cell death to apoptotic cell death (197, 241). This would thereby prevent the leakage of cellular content from necrotic cells, which is pro- inflammatory. Likewise, modulating PARP-1 activity after H2O2 or β-lap treatment was able to prevent nucleotide loss and cell death (197). These findings indicate that PARP-
1 plays an important role in the recognition and signaling of ROS-mediated DNA damage. Understanding the mechanisms of how these injuries contribute to human pathologies, as well as agents that can mimic these processes are useful for the development of models for future pharmacological intervention.
140 Metabolic implications of β-lap usage for cancer treatment
In addition to NQO1-mediated bioactivation of the compound, activating PARP-
1-mediated cell death is another key determinant in the effectiveness of β-lap as a viable antitumor agent. Thompson and colleagues demonstrated that, relative to cells that catabolize non-glucose substrates for oxidative phosphorylation, cells that use aerobic glycolysis are more susceptible to damage-induced PARP-1-mediated cytosolic NAD+ consumption, leading to cell death (73). The conversion of tumor cells from oxidative phosphorylation to aerobic glycolysis, even in the presence of adequate oxygen supply, for the production of ATP is important not only for tumorigenicity, but will also confer sensitivity to agents that manipulate this property, such as β-lap. This phenomenon, first reported by Warburg in the 1920s (242) explains the sensitivity of tumor cells to DNA- damaging agents, as cancer cells produce the majority of their ATP through aerobic glycolysis (73, 243).
In contrast, PARP-1 hyperactivation in cells that can maintain non-glucose dependent oxidative phosphorylation, are resistant to DNA damage-induced cell death since PARP-1 activation does not compromise the cell’s bioenergetic state (73). This would provide an innate protective mechanism for normal endothelial and epithelial cells, that commonly express NQO1, after systemic β-lap treatment (244). Since healthy cells would rely almost exclusively on OXPHOS, they should be resistant to β-lap-induced
PARP-1 mediated cell death, even in the presence of NQO1.
However, a caveat to solely targeting metabolic alterations in tumor cells, was demonstrated in neu-initiated mammary tumor cells. Knock-down of lactate dehydrogenase A (LDH-A) resulted in an increase in oxygen consumption and OXPHOS
141 activity, thereby preventing these cells from growing under hypoxic conditions (245).
These data demonstrate that cancer cells maintain the ability to re-engage in OXPHOS even when they preferentially use glycolysis as their primary metabolic mechanism
(246). The high rate of glycolysis maintained by most tumor cells is required to support cell growth rather than compensate for alterations in mitochondrial function (246). Many of β-lap’s purported effects can be explained by PARP-1-mediated metabolic alterations, which can: (A) inhibit NFκB, (B) inhibit caspase-activation, and stabilize p53, (C) inhibit
Topo I and Topo II-β, (D) and cause MOMP via NAD+ loss, (E) and result in cell death not dependent on typical apoptotic mediators such as Bcl-2 and Bax/Bak7. These data have important implications for the effectiveness of β-lap in overcoming drug resistance in glycolytic cells because two separate pathways; metabolism and quinone detoxification are targeted.
The role of PARG in modulating β-lap-induced cell death
We established the importance of PARP-1 in β-lap-induced cell death (Chapter
2). Since PARP-1-mediated cell death also requires the concomitant action of PARG
(247) it would be interesting to examine the latter enzyme’s function after β-lap treatment. PAR has a quick turnover rate, with a half-life of approximately 1 min, due to the degradation by endo-exoglycosidase PARG (164, 248). Future endeavors should focus on the dynamic of PAR signaling and PARG function, which are currently under investigation.
7 Bey, E.A., and Boothman, D.A. unpublished data
142 PARG is a essential enzyme in the catabolism of PAR that acts by hydrolyzing both terminal ADP-ribose units from poly(ADP-ribose) polymers via exoglycosidic activity and removes larger oligo(ADP-ribose) fragments via endoglycosidic cleavage
(247, 249). PARG has not been as intensely researched as PARP-1 for a number of technical reasons, mainly due to its low cellular abundance and its sensitivity to
-/- -/- proteolytic degradation during purification (250, 251). Similar to parp animals, parg110 animals (ablation of 110 kDa PARG isoform) have decreased injury, dysfunction and inflammation caused by renal ischemia-reperfusion (252). Interestingly, PARG inhibitors gallotannin and nobotannin reduced astrocyte cell death induced by H2O2 treatment, N-methyl-N9-nitro-N-nitroguanidine or N-methyl-D-aspartate (253, 254).
PARG inhibition attenuated NAD+ depletion, but increased PAR accumulation, thus retarding the rate of PAR turnover and PARP-1 activity. Therefore, one would predict that PARG inhibition after β-lap treatment would act in an analogous manner to PARP-1 inhibition, by sparing NAD+ depletion, and thus cell death. However, it would not prevent PAR polymer synthesis as would PARP-1 inhibition or protein knock-down, which could affect the repair of DNA damage.
PAR is a signaling molecule implicated in a number of normal cellular processes including transcription and initiation of cell death (255). It would be interesting to examine the DNA repair kinetics and dynamics after β-lap exposure in the presence of
PARG inhibitors. Furthermore, PARG inhibition may decrease the possible side-effects associated with PAR loss (255). These studies will provide valuable insight on the role of
PARG and PAR in β-lap-induced cell death. Furthermore, these findings will shed light
143 on the mechanism of PAR degradation, as well as the biological functions of poly(ADP- ribosylation).
B.1 Conclusions
Targeting DSB repair to enhance β-lap-induced lethality
In Chapter 2, we clearly demonstrated that β-lap caused NQO1-dependent ROS generation leading to DNA damage (Figures 2.7 and 2.8). However few studies have explored the contribution of DNA repair, in particular DSB repair, in cell death mediated by β-lap. We, therefore, investigated whether β-lap exposure in NQO1+ cells activated
HR and/or NHEJ, and explored the extent to which these pathways influenced cellular sensitivity to the drug (Chapter 3).
To first elucidate the repair pathway(s) activated after β-lap treatment, we examined a number of DNA repair proteins known to specifically co-localize with DNA
DSBs (10). β-Lap caused the time-dependent localization and activation of the MRN complex (Figure 3.1), concomitantly with ATM/DNA-PK autophosphorylation (Figure
3.3) and Chk1-pSer345 phosphorylation (indicative of ATR activation) (Figure 3.6). The activation of these DSB damage proteins was delayed compared to IR exposure.
To test what DSB damage proteins were involved in response to β-lap, we utilized a number of cellular model systems with altered DNA-PKcs, ATM, and ATR functions, as well as selective inhibitors. We found that although components of both HR and
NHEJ were activated, only NHEJ was a key factor in the survival of cells exposed to β- lap. Furthermore, inhibition of NHEJ or DNA-PKcs-deficiency caused PARP-1- mediated cell death after treatment with sub-lethal doses of β-lap. Interestingly, despite
144 the activation of DSB repair pathways, β-lap caused primarily SSBs as measured by comet assay. Our inability to observe DSBs could be due to a sensitivity issue with regard to the comet assay. However, we were able to observe γ-H2AX foci formation, which has been directly correlated with DSBs (256).
In addition, activation of the MRN complex, ATM, DNA-PK, and ATR all occurred within 10-15 min of β-lap exposure, however SSBs were detected within ≤ 5 min. (197). β-Lap-induced ATR activation, suggests that SSBs are generated immediately from the NQO1-mediated reduction of β-lap, and DSBs are formed as a secondary insult likely resulting from stalled replication forks (Figure 3.8).
B.2 Future Directions
LMDS as potential critical lesions formed after β-lap exposure
Studies examining ionizing radiation have revealed another type of DNA insult that results in locally multiply damaged sites (LMDS, a.k.a. clustered lesions) (257).
LMDS are defined by two or more closely spaced damages (strand breaks, abasic sites, or oxidized bases) within a few helical turns on the DNA. This observation was initially noted for radiation induced damage because non-uniform energy deposits follow the passage of secondary electrons through matter. This produces ROS that attack DNA within the surrounding volume, which supersedes the limited number of radical scavengers. This energy deposition creates multiples of radicals in a localized area, and result in multiple damage sites on a small portion of the DNA (258). These types of complex damage sites pose huge repair problems for the cell, since they result in dramatically lowered rates of repair. LMDS frequently caused stalled replication (259,
145 260), DSBs and loss of sequence information, giving rise to deletions and/or chromosomal aberrations. LMDS has also been observed after successive reactions of oxidative species with a bound metal ion such as Fe2+ that could cause multiple radical damages at one site producing a site-specific DSB (261, 262). Since NQO1 is highly expressed in the cytosol, and at lower levels in the nucleus, futile-cycling of β-lap would produce a very high local concentration of ROS in direct contact with the DNA and free iron/copper (262). It is therefore plausible, that β-lap, in a fashion similar to IR, can produce DSBs via processing of LMDS. Methods have been designed to test for LMDS by treating DNA with specific endonucleases to induce single-strand cleavage at an oxidized base or abasic site. If there are two closely spaced damages on opposite strands, such cleavage will reduce the size of the DNA on a nondenaturing gel (263). Future studies should investigate LMDS after β-lap treatment, as the mechanism of cellular repair of such clusters is virtually unknown.
In addition, localized clusters of multiple base damage attract PARP-1 and BER repair proteins prior to formation of DSBs. It has been demonstrated that repair of this type of damage by BER gives rise to DSBs both in vitro (264, 265) and in vivo (266).
Some data even indicate that PARP-1 could participate in the repair of DSBs at stalled replication forks (267). It would, therefore, be interesting to determine if agents such as methoxyamine, that inhibit BER by binding to AP sites (51), could attenuate the DSB response during/after β-lap treatment.
146 Model of β-lap-induced DNA damage responses
Based on data presented in this thesis, we propose two models for β-lap-induced- cell death. At lethal doses, β-lap is reduced by NQO1 causing the generation of extensive ROS that cause SSBs sufficient to hyperactivate PARP-1. PARP-1 hyperactivation results in an increasing amount of PAR, consuming cellular NAD+ and
ATP. In highly glycolytic cancer cells, nucleotide depletion prevents DNA repair and results in cell death. In this scenario, PARP-1 dominates the pathway since it directly regulates the metabolic state (NAD+/ATP levels) of the cell (Figure 3.8A). However, at non-lethal doses of β-lap, repairable amounts of SSBs are generated, and some of these lesions will be converted to DSBs possibly via replication fork collision and subsequent arrest. DSBs cause the recruitment and activation of members of the PI3K family of kinases, ATM, ATR, and DNA-PKcs. Although all three PI3Ks are activated, only
NHEJ inhibition potentiated the toxicity of sub-lethal doses of β-lap by retarding repair, which causes PARP-1-mediated cell death (Figure 3.8B).
Interplay between PARP-1 and DNA-PK in improving the efficacy of β-lap
DSBs are preferentially repaired by NHEJ/DNA-PK. However, other mechanisms also promote end-joining, such as BER (XRCC1/DNA ligase III complex
(XL)) in association with PARP-1 (268, 269). This information adds to the extensive and diverse relationship between DNA-PK and PARP-1. Genetic ablation of both PARP-1 and Ku80 in mice results in embryonic lethality (270, 271) and inhibition of both proteins increases net DSBs over time after chemically-induced DNA damage.
147 Inhibition of DNA-PK and/or PARP-1 is an interesting therapeutic strategy.
Combining both β-lap and NHEJ inhibitors should therefore target two distinct, mechanistically diverse, yet overlapping DNA repair enzymes. As a result, such therapy would potentiate DNA damage and cell death, as well reducing the potential for drug resistance. Many commonly used cancer agents such as IR, alkylating agents, and Topo
II poisons frequently cause tumor relapse several years after initial treatment. These new secondary cancers can arise as a result of potentially mutagenic damage to normal tissues.
To circumvent this scenario, combined treatment of the “targeting agent” (β-lap) coupled with a NHEJ inhibitor (Nu7026) may reduce the occurrence of secondary cancers by eliminating those cells with potentially mutagenic damage as a result of chemotherapy
(Chapter 1). Furthermore, both β-lap (101, 142, 169) and DNA-PKcs inhibitors (272) are potent radiosensitizers. Attention should, therefore, be focused on what the effects of this type of drug combination would have on selectively enhancing the effects of radiation therapy, especially on hard to treat malignancies.
C. Summary
It is imperative to establish the links between specific tumor cell phenotypes and sensitivity to cancer chemotherapeutic agents to allow the tailoring of individual treatment options to the tumors that will respond to it most effectively. As we move forward into an era of more personalized medicine, it will be necessary to efficiently and accurately model tumor protein expression profiles with appropriate treatment regimes.
This should result in future diagnoses of “cancer” that will not be so closely associated with mortality. In the case of β-lap, screening tumors for NQO1 protein levels and
148 activities would be a key diagnostic test, as some individuals have polymorphisms in the
NQO1 gene that renders cells NQO1-deficient and resistant to β-lap (110, 119, 120, 144,
273). Diagnostic screens coupled with optimized local delivery methods, such as poly(D,L-lactide-co-glycolide) (PLGA) polymer millirods, will further increase tumor targeting and decrease healthy tissue damage. Our laboratory is currently working in collaboration with others to develop these specialized delivery tools (274).
In conclusion, the data and ideas within this thesis describe the molecular mechanisms of β-lap-induced cell death, with particular focus on the DNA damage response. Knowledge gained from these studies will contribute to the understanding of this promising agent in hopes of optimizing its effects alone and/or in combination with
IR/other drugs for more selective cancer chemotherapy.
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