MULTIPLE GENOTOXIC AGENTS ACTIVATE ATR KINASE SIGNALLING IN

QUIESCENT HUMAN CELLS

A thesis submitted in partial fulfillment of the

requirements for the degree of

Master of Science

By

MARIYYAH AHMED O MADKHALI

B.Pharm., King Saud University, Saudi Arabia 2016

2020

Wright State University

WRIGHT STATE UNIVERSITY

GRADUATE SCHOOL April 17th, 2020

I HEREBY RECOMMEND THAT THE THESIS PREPARED UNDER MY SUPERVISION BY MARIYYAH AHMED O MADKHALI ENTITLED MULTIPLE GENOTOXIC AGENTS ACTIVATE ATR KINASE SIGNALLING IN QUIESCENT HUMAN CELLS.BE ACCEPTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science.

______Michael G Kemp, Ph.D. Thesis Director ______Jeffrey B. Travers, M.D., Ph.D. Chair, Department of Pharmacology and Toxicology Committee on Final Examination:

______Jeffrey B. Travers, M.D., PhD. ______Michael Kemp, Ph. D. ______Yong-jie Xu, M.D., Ph.D. ______Barry Milligan, Ph.D. Interim Dean of the Graduate School

ABSTRACT

Madkhali, Mariyyah Ahmed O., M.S., Department of Pharmacology and Toxicology, Wright State University, 2020. Multiple genotoxic agents activate ATR kinase signaling in quiescent human cells.

The ATR protein kinase is activated in response to DNA damage and other forms of genotoxic stress caused by both environmental carcinogens and anti-cancer drugs.

However, much of our understanding of ATR kinase function is limited to proliferating cells in which DNA replication stress is the primary signal for ATR activation and where the major regulatory targets of ATR signaling are proteins involved in DNA synthesis and progression. Here we have used HaCaT keratinocytes maintained in a non- replicating, quiescent state in vitro to examine how cell killing by different genotoxic agents is impacted by cell growth status and by treatment with small molecule ATR kinase inhibitors. The genotoxins we examined included drugs from several classes of anti-cancer agents, including topoisomerase inhibitors (, ), alkylating agents

(, , and ), and compounds that interfere with RNA polymerase movement ((5-6-dichlorobenzimidazole 1-beta-D-ribofuranoside), actinomycin D). As expected, we find that quiescent cells are more resistant to the acute, lethal effects of these genotoxins than replicating cells. However, though we find that nearly all of these compounds led to the activation of ATR kinase signaling in the quiescent state, little-to-no effect of ATR kinase inhibitors was observed on quiescent cell viability.

These results indicate that ATR can be activated in the absence of canonical replication stress and that its function does not significantly impact acute cell survival. To examine

potential alternative functions for ATR signaling in quiescent cells, we then examined how

ATR kinase inhibition impacted the activation of the translesion synthesis (TLS) pathway of DNA synthesis, which involves the use of specialized, potentially mutagenic DNA polymerases to fill in DNA repair gaps and complete DNA repair. Interestingly, we found that ATR kinase inhibition potentiated the activation of this pathway in response to mitomycin C and cisplatin but not following treatment with other genotoxins. Both mitomycin C and cisplatin induce the formation of DNA adducts that are repaired by the nucleotide excision repair system, and thus these results suggest that the ATR kinase may be required to limit the dependence on mutagenic TLS DNA polymerases in quiescent cells. Because ATR kinase inhibitors are currently being tested in clinical trials for use in anti-cancer therapies, this work provides valuable information on the positive and negative consequences of ATR kinase inhibition in quiescent or slowly growing cancer cells.

Keywords: DNA damage response, ATR kinase inhibition, DNA damaging agents,

Translesion DNA synthesis, PCNA mono-ubiquitination.

iv TABLE OF CONTENTS

INTRODUCTION……………………………………………………………………1-31

1. DNA damage and DNA repair mechanisms………………………………...1-9

a. DNA damage……………………………………………...…………...1

b. DNA repair mechanisms…………………………………….……….2-9

2. DNA damage response vs. DNA damage tolerance………………………..9-14

a. DNA damage response……………………………………………..9-12

b. DNA damage tolerance……………………………...…………….12-14

3. Ataxia telangiectasia and Rad3-related activation and inhibition in replicating

cells vs. non-replicating cells...... 15-20

a. ATR activation and inhibition in replicating cells.……...... …….15-18

b. ATR activation and inhibition in non-replicating cells……...…….18-20

4. ATR inhibitors………………………………………………..……………21-24

5. Genotoxin compounds……………………………………………………..24-31

a. Alkylating agents…………………………………………………..26-28

b. Topoisomerase inhibitors……………………………………………...29

c. Transcriptional stressors…………………………………………...29-30

d. Translational stressors…………………………………..…………30-31

v Materials and methods………………………………………………………….…..32-35

1. Cell culture………………………………………..……………………….32-33

2. BrdU dot blot………………………………………………………………….33

3. MTT assay…………………………………………………………………33-34

4. Immunoblotting/chromatin fractionation…………………………...……..34-35

5. Clonogenic Survival assay……………………...……………………………..35

Results……………………………………………………………………………..…36-61

1. Validation of the non-replicating status of quiescent HaCaT keratinocytes by

using BrdU labeling………………………….………………………………36-37

2. Quiescent cells are less sensitive to the acute effects of genotoxic agents than

proliferating cells……………………………………………….…………….38-40

3. ATR kinase inhibition in genotoxin-treated quiescent cells dose not impact acute

survival………………………………………………………………...... 41-45

4. Many genotoxins induce ATR kinase activation in quiescent cells……….46-48

5. ATR kinase inhibition generally results in an elevation in PCNA mono-

ubiquitination in genotoxin-treated quiescent cells…………………………..49-54

6. The effect of NER inhibitors on TLS pathway activation and ATR kinase

signaling in genotoxin-treated quiescent cells………………………………..55-60

7. Effect of ATR kinase inhibition on the clonogenic survival of quiescent cells

following their induction of the proliferative state……………………………61-63

vi Discussion………………………………………………………………………….64-69

References………………………………………………………………………….70-78

vii LIST OF FIGURES

Figure 1: DNA damage and repair mechanisms………………………………………...2

Figure 2: DNA damage response protein kinases...……………………………...….....10

Figure 3: DNA damage checkpoints in human cells …………………………….…….11

Figure 4: DNA damage response vs. DNA damage tolerance………………………....14

Figure 5: ATR-CHK1 activation and inhibition in replicating cells…………………...17

Figure 6: ATR activation and inhibition in replicating cells vs. non-replicating cells....20

Figure 7: Genotoxic agents in the cellular level…………..……………………………25

Figure 8: Validation of the non-replicating status of quiescent HaCaT keratinocytes by using BrdU labeling……………………...……………………………………….……..37

Figure 9: Comparison of cell survival in replicating and non-replicating HaCaT cells treated with different DNA damaging agents and genotoxic stressors………………39-40

Figure 10: ATR kinase inhibition has differing effects on acute cell survival following treatment of quiescent and proliferating cells with different genotoxic compounds...42-45

Figure 11: ATR kinase signaling is activated in quiescent cells in response to a variety of different genotoxic compounds…………………………………………………………..48

Figure 12: Examination of the effects of ATR kinase inhibition on TLS pathway activation in genotoxin-treated quiescent cells in shorter time. …………………………………….52

Figure 13: The effect of ATR inhibition in elevation in PCNA mono-ubiquitination in genotoxin-treated in quiescent cells in longer time treatment…………………………….53

viii Figure 14: Mitomycin C and temozolomide dose responses different two times points…………………………….………………………………………………….……54

Figure 15: Diagram of the effect of PCNA mono-ubiquitination and ATR kinase signaling with NER inhibitors……………………………………………………………………...58

Figure 16: The effect of PCNA mono-ubiquitination by using NER inhibitors

(spironolactone and triptolide) followed by mitomycin C or temozolomide for 4 hr or 24 hr treatment………………………………………………………………………….…...59

Figure 17: The effect of ATR kinase signaling and NER inhibitors exposed to mitomycin

C or temozolomide at different times points…………………………………………...... 60

Figure 18: Clonogenic survival assay diagram………………………………………....62

Figure 19: Effect of ATR kinase inhibition on the clonogenic survival assay of quiescent

HaCaT cells exposed to different genotoxic agents. …………………………………….63

Figure 20: Schematic summarizing the effect of the effect of ATR kinase inhibition in temozolomide, mitomycin C, cisplatin, and UVB treated quiescent cells……………….67

ix

LIST OF TABLES

Table 1: Summary of DNA damage, DNA repair and examples of human disease with defect in DNA repair……………………..……………………………………………..8-9

Table 2: Some examples of ATR inhibitors…………………………………..…...... 22-24

Table 3: Examples of DNA damaging agents- Alkylating agents……………...……….28

x ACKNOWLEDGEMENTS

I would thank Dr. Michael Kemp (Thesis Advisor) for helping and explaining the project. He has been very patient with me to teach me skills that I should need them throughout my project. Also, I appreciate him teaching the DNA damaging agents’ class that help us to understand the background of our thesis project and help us to write the introduction of our thesis. He always explains to me the basics of information and skills that I did not know about them or I forgot them.

I would thank my committee members, Dr. Jeffrey Travers (Department Chair) and

Dr. Yong-jie Xu for their valuable advices and suggestions to improve my thesis project.

As well as I would thank Dr. Travers’s lab for using a CO2 incubator for cell culture and for thesis meeting every week and month, which helps me to read, analyze, correct and even conclude our data.

Also, I appreciate Wright State University Proteome Analysis Laboratory and

Center for Genomics Research for the use of equipment, Dr. Yong-jie Xu and Dr. Ravi

Sahu (Mentors for thesis proposal class), Dr. Terry Oroszi (The Program Director),

Catherine Winslow (the Assistant to the Chair), my lab’s classmates and my classmates,

Pharmacology and toxicology department. Wright State University. My government and

SACAM to give me an opportunity to complete my study. The last and not the least, I would thank my husband and my entire family for supporting and encouragements. This work was supported by a grant (GM130583) from the NIH.

xi INTRODUCTION

1- DNA damage and DNA repair mechanisms.

a. DNA damage:

Human cells are continually exposed to many compounds that cause damage to deoxyribonucleic acid (DNA) (1). DNA damage changes the structure of DNA either physically or chemically to disrupt the function and the genomic integrity of DNA (2). Thus, the damage of DNA can be exogenous ways or from external environments, such as ultraviolet light, ionizing radiation, and . Besides, the damage of DNA can be endogenous ways or from internal metabolic ways, such as reactive oxygen species or replication stress (3,4).

If the DNA of cells has been damaged, they leave lesions on the site of the damaged area. The different types of genotoxic agents cause a variety of types of

DNA damages (3). These lesions have many types such as lesions to nucleotides, single-strand break, double-strand break, inter-and intra- strand DNA crosslinks, or

DNA adduct (2). Then, recognition of DNA damage to begin DNA damage responses by specific DNA repair proteins to initiate repair of DNA lesions, which is known as DNA repair mechanisms (4).

1 b. DNA repair mechanisms:

Every lesion has a specific type of DNA repair mechanisms. These include mismatch repair, base excision repair, nucleotide excision repair, single and double-strand break repair mechanisms (figure 1) (3). DNA repair mechanisms are essential because deficiency in repair leads to increased human pathogenesis, such as cancers (table 1) (4,5).

Figure 1: DNA damage and DNA repair mechanisms.

2 Mismatch repair is fixing the damage of deletion or insertion of nitrogenous bases through replication on the newly synthesized DNA by slipping polymerases (1-3). These

DNA polymerases interact with proliferating cell nuclear antigen (PCNA), which is a loading clamp for DNA polymerases to fill gaps. Then, DNA ligase seals nick of the DNA

(6). If there is defect of human genes that effect to mismatch repair, these lead to increase mutation rate and linked to inherited colon cancer in humans (3,6,7,30).

Base excision repair (BER) is the most common pathway that cuts out a single nucleotide base by removing the damaged base from the DNA backbone. This occurs when single bases are damaged by alkylated or oxidizing agents that modified the nitrogenous base (3,6). It is started by a DNA glycosylase enzyme that detects and removes the damaged base. Then, an apurinic-apyrimidinic (AP) endonuclease and a phosphodiesterase recognize and remove the sugar phosphate that have a missing base by leaving a single base gap (6). After that, DNA polymerase and DNA ligase add the correct nucleotide as a cytosine and seals nick in the DNA helix (1-3). Examples of DNA damaging agents that cause this type of damage are X-rays, hydrogen peroxide, and alkylating agents, such as cisplatin, mitomycin C and temozolomide. The defective of BER leads to an increase in mutation rates, ataxia and neurodegeneration, which is genetic neurodegenerative disorder that effect the coordination of voluntary movement and immune system (7,30).

Nucleotide excision repair (NER) is the second major DNA repair which is correcting the damage of bulky nucleotides caused by UV radiation and carcinogen benzopyrene which is found in tobacco smoke or coal tar (1-3,7). Aziz Sancar characterized

3 the mechanism of NER and won a Nobel prize in chemistry in 2015 (3). This led bulky lesions to pyrimidine dimers as cytosine to cytosine, thymine to thymine, or thymine to cytosine causes thymine bases to react with each other to cause dual incision (6,31,32).

Then, excision nuclease is a complex enzyme which detects and scans the damaged nucleotides in the double helix instead of a single nucleotide base as in base excision repair

(3). After that, it comes DNA helicase that cuts around 25-30 nucleotides in humans and leave gap (31,32). Then, DNA polymerase and DNA ligase add new and correct nucleotides and seals gap in the DNA helix (7). The example of human disease that linked with defect in this repair is xeroderma pigmentosum (XP) which is affecting humans who have high sensitivity to ultraviolet radiation that lead to increase mutation rate and skin cancer. There are two pathways of NER are transcription-coupled NER repair and global genomic repair to recognize DNA damage (30-32).

Transcription-coupled NER repair (TC-NER) targets DNA damage in transcribed

DNA (3,6). This pathway involves RNA polymerase which is an enzyme that transcribes

DNA to RNA that repair the damaged nucleotides and using coupling proteins at stalls of

RNA polymerase (31, 32). An example of a defect of this repair is Cockayne syndrome which has symptoms of high sensitivity of sunlight, growth abnormality (growth, skeletal and progressive neural retardation) but it is not susceptible to cause skin cancer (3,6).

However, global genomic repair (GG-NER) targeted the both transcribed and not- transcribed DNA (29-30). It occurs when xeroderma pigmentosum C (XPC) recruited the downstream proteins as xeroderma pigmentosum A (XPA), xeroderma pigmentosum

4 D(XPD), and Human transcription factor IIH subunit 5. Then, XPA recruits replication protein A (RPA) to recognize DNA damage and fill gaps by DNA polymerases. The last,

DNA ligase comes to seal the nick of DNA (6,31,32). An example of a defect of this repair is Trichothiodystrophy is neurodegeneration disorder which characterized with brittle hair, extremely UV light sensitivity, growth and mental retardation (6).

Double strand DNA break is the most dangerous type of DNA damage because it involves break of the both strands of the double helix and can cause rearrangement of chromosomes leading to increased mutations rates and cancer development (3,6). The examples of DNA damaging agents that cause this damage are X-rays, ionizing radiation and chemotherapies, such as cisplatin and mitomycin C (3,6). Ataxia telangiectasia (AT) is a rare inherited disease that characterized of acute sensitivity to radiation and X-rays, genomic instability, immunodeficiency, and susceptible to cancers. AT is a defect in double strand repair and loss in ATM protein kinase which activated by double strand break (3,29-

32). Correcting the double strands breaks occurs by either homologous recombination (HR) or non-homologous end-joining (NHEJ) pathways (3).

HR uses a normal sister chromatid as a template to repair dsDNA breaks and to allow an accurate error-free repair of the DNA, which occur in S and G2 phases of the cell cycle (1-3,6). Thus, it is essential for proliferating cells or replication cells because it cause stalled in DNA replication forks that arising from UV light and alkylating agents (1,29). In addition, HR is playing an important role in sexually reproducing organisms, especially in sperms and eggs production during in gametes step (30). Thus, defective HR repair

5 is linked to ovarian, breast, and prostate cancers (1). It is linked to Seckel’s syndrome which is rare genetic disorder of dwarfism that characterized with microcephaly, growth, mental retardation (6). In addition, it correlates to Fanconi anemia (FA) which is an inherited genetic disease that is characterized with defect in birth, bone marrow failure which leads to anemia (1,3,6). HR occurs when the daughter duplex DNA helix close to the broken double DNA stands in replication of DNA. After nuclease enzyme cuts out the ends of broken double strands of DNA, the strand exchange by complementary base pairing through DNA polymerase from damaged strands that is using the correct repairing with the undamaged daughter as template. The last step is DNA ligase to seal the gaps between the two strands of DNA (7,30).

In contrast, NHEJ does not required another sister chromatid to fix that repair by direct ligation, and it occurs in all phases of the cell cycle (3,6). It is a prone-error process, which can induce mutation in the DNA. The initiation and recognition of NHEJ repair mechanism by Ku heterodimer protein which is a protein to hold the double strands break or the broken ends of chromosomes. Then, the additional proteins come to hold the broken ends and processing of DNA ends which the KU heterodimer recruit DNA-dependent protein kinase, catalytic subunit (DNA-PKcs) (1-3,6). Eventually DNA ligation to seal the

DNA nick with deleted of nucleotides which lead to mutation and changes in DNA sequence. There is relation between deficiency of NHEJ repair and deficiency of adaptive immune system. That is because in NHEJ repair mechanism involves the Ku protein which

6 is essential in recombination of V, D, and J joining segments antibodies and T cells receptors (1,30).

Inter-strand cross link repair is cross link between two strands to form break in double strand of DNA which lead chromosomal rearrangement (3,6). It is considered toxic because it blocks replication and transcription of DNA that leads to arrest of the cell cycle and cell death (6). In non-replicating cells, which are in G0-G1 phase, DNA adducts may be repair occurs via NER (6). However, in replicating cells, which are in , crosslink repair occurs by two repair mechanisms. First, when crosslink occurs in replication forks stall to recruit endonuclease to cut it to form double stand DNA break to repair by HR and translesion synthesis polymerase (TLS). Second, After TLS polymerase filled the gaps followed NER to end repair. In replicating cells is more toxic than in non-replicating cells because has robust effect to replication (6,33,37)

However, DNA repair mechanisms are the one of the responses to sense and repair

DNA damage, and to maintain genomic stability. These responses are known as DNA damage responses that has four ways to detect and fix DNA lesions. DNA repair mechanisms are important because some human’s disease that linked to defect in DNA repair mechanisms as shown in (table 1) (3-6,31,32).

7

Table 1: Summary of DNA damage, DNA repair and examples of human disease with defect in DNA repair.

Type of DNA damage DNA repair Some disease in defects in

mechanisms DNA repair mechanisms

1- Single strand break-insert or Mismatch Colon cancer

delete base during replication. repair(MMR)

2- Single strand break- Base excision Ataxia and

deamination from cytosine to repair(BBR) neurodegeneration

uracil base.

3- Single strand break-intra-strand Nucleotide Xeroderma pigmentosum

crosslinks excision (XP),

repair(NER) Cockayne syndrome,

Trichothiodystrophy

4- Double strand break-inter-strand Double strand Ataxia telangiectasia (AT),

crosslinks break repair Fanconi anemia (FA),

Seckel’s syndrome,

ovarian, breast, and prostate

cancers.

8 5- Translesion DNA synthesis DNA damage Xeroderma pigmentosa (XP)

tolerance variant

2- DNA damage response vs. DNA damage tolerance.

a) DNA damage response (DDR):

The DNA damage response (DDR) is a network of protein kinase signaling pathways that detect and repair DNA lesions (21). DDR recognizes theses different types of genotoxic agents by four responses (2,3). These are cell cycle checkpoints activation,

DNA repair mechanisms, transcriptional response, and cell death or senescence if damage cannot repair (4, 16, 24). Thus, DDR has crucial role in maintaining genomic stability and cell viability (5, 21).

After DNA is damaged, DNA damage sensors are activated, and checkpoints activated. DNA damage response is a complex signaling pathways that activate checkpoints through a family of phosphoinositide 3 kinase-related kinases (PIKKs) (4,5).

There are three subgroup protein kinases in human cells, which is controlled by the DNA damage response (3,6,7,25,26). These are DNA-dependent protein kinase (DNA-PK), ataxia telangiectasia mutated (ATM), and ataxia telangiectasia and rad3-related (ATR).

ATM and DNA-PK have known to be recruited when DNA damage in double strand DNA break while ATR protein kinase have known be recruited after DNA damage in single strand DNA break. (16-19) Thus, these proteins sense DNA damage to repair DNA, cell

9 cycle regulation, DNA replication, transcription regulation, RNA processing, and inducing cell death (figure 2) (7,8,9).

Figure 2: DNA damage responses protein kinases.

10 ATM and ATR have common phosphorylation substrate proteins which are involved in regulation and activation which consider overlap between these protein kinases through checkpoint kinase 1(CHK1) and checkpoint kinase 2(CHK2) (6,16-17). The components of DNA damage checkpoints are sensors, signal transducers, and effectors

(figure 3) (2). Sensor proteins are the first to recognize the damaged DNA to repair and stop the progression of the cell cycle. These are ATR, ATM, the RFC/PCNA (clamps loader/polymerase clamp)-related Rad 17-RFC/9-1-1 complex, and RPA. Then comes mediators along with sensors to help signal transducers and effectors to affect cellular processes in the cell cycle (figure 3) (2,7).

Figure 3: DNA damage checkpoints in human cells.

11 However, ATM protein kinase is not essential protein because loss of ATM gene cause (A-T) ataxia telangiectasia whereas ATR is essential protein because loss of ATR gene cause death of embryonic in early stage of life (6). Thus, ATR-checkpoint kinase signaling is more common signaling pathway because it involves single strand breaks, which is the most common DNA damage that occur around 10,000 lesions per cell per day

(6). and resected single strand break which involve checkpoints kinases (16-19).

b) DNA damage tolerance (DDT):

In DNA damage tolerance (DDT), some DNA lesions escape from repair mechanisms and have longer time to add mismatch nucleotides opposite to damaged DNA template to help bypass DNA lesions via specific polymerases. Lesion bypass by

(Ubiquitination PCNA) has two ways: 1- PCNA poly-ubiquitination; Error-free template switching (TS). 2- PCNA mono ubiquitination; Error-prone translesion synthesis (TLS), which leads to increase mutation (figure 4) (41-43,48).

Translesion synthesis (TLS) is one of DNA damage tolerance that bypass lesions through translesion DNA polymerases in DNA replication and escape from DNA repair mechanisms such as base excision repair, nucleotides excision repair or mismatch repair

(1,30). However, DNA polymerase is correcting the wrong nucleotides by adding the correct nucleotides on the undamaged complementary strand and it is not fixing the damaged nucleotides on template strand of DNA. Thus, the TLS pathway can increase , genomic instability and apoptosis because TLS is error prone that recruited

PCNA-mono-ubiquitination which the ubiquitination of PCNA is an essential role in

12 regulating from template switching (TS) to translesion DNA synthesis (TLS), which is a damage avoidance pathway and it is error free (1,27-28,30-32).

TLS involves two type of DNA polymerases. These are polymerase switching and

DNA extension past the lesion. In polymerase switching, which involve the high-fidelity

DNA polymerase such as Pol delta to come along with PCNA in replication fork and to assist leading and lagging DNA strand synthesis (31,32,41-43,48).Then, these polymerases are replaced with the low fidelity DNA polymerase such as in Y-family (TLS) polymerase;

Ubiquitin-binding Zinc finger (POL eta, and POL kapa) and helical ubiquitin-binding motifs (Rev1) to fill gaps via putting mismatch nucleotide across from lesions to help bypass lesions (31,32,). Hence, translesion synthesis (TLS) DNA polymerases will interact with PCNA only if PCNA is mono-ubiquitinated (41-43,48). The example of some inherited disease that effect humans who have defect in translation DNA synthesis, especially in polymerase eta is Xeroderma pigmentosa variant which effecting humans who have high sensitivity to UV light and they are susceptible to have skin cancer (1,30) (table

1).

However, TLS is most recognized for its function in S phase in replicating cells as described in previous studies (1,30,41-43,48). However, TLS polymerase recruitment to sites of DNA damage has recently been shown to occur in non-replicating G0-G1 phase as well (27,28,51,52). In non-replicating cells, TLS pathway has been shown that polymerase specialized DNA synthesis as pol kapa, which is responsible to fill gaps by NER to prevent the single strand DNA break from conversion to double strand DNA break, which is the

13 most dangerous and toxic DNA lesion (51, 54). Thus, the TLS pathway is dependent on mono-ub-PCNA and specialized polymerase DNA synthesis as pol eta, which recruited after the HR in replicating cells in S phase (52, 54).

Figure 4: DNA damage response vs. DNA damage tolerance. ATR-Checkpoint kinase, which is the key regulator of DNA damage response, and ubiquitination of PCNA, which is the key regulator of DNA damage tolerance. They activated after replication protein A recruited at replication forks stall in a single strand DNA. Ubiquitination of PCNA at lysine residue at lysine 146 has two major lesion bypasses. These are Poly-ubiquitination -PCNA, which is error-free template switching (TS) bypass and mono-ubiquitination-PCNA, which is error-prone translesion DNA synthesis (TLS) leading to increase mutagenic cell survival.

14 3- Ataxia telangiectasia and Rad3-related activation and inhibition in

replicating cells vs. non-replicating cells.

a. ATR kinase in replicating cells.

ATR is ataxia telangiectasia and Rad3-related is activated after cells are exposed to genotoxic agents to sense and activate DNA damage response kinase pathway (21). It is one of the phosphatidylinositol-3 kinase-related kinase (PIKKs), which is a member of the phosphatidylinositol3- kinase family (PI3K) (13,14). These are serine/ threonine kinase, which are upregulated in a different type of cancer development and function in the ATR-

CHK1 pathway. (16-19). The activation of ATR plays a crucial role in DNA repair, inhibiting replication fork stabilization, decreasing in replication origin firing, and cell cycle inhibition by activation of intra-S and G1/M checkpoints (10,11-15).

There are three-ways to induce ATR-CHK1 pathway activation and downstream signaling (21-24). First, induction of ATR by increasing replication stress that causes a stalled in replication fork by breaking in a single strand of DNA in S phase in replication cells through topoisomerase 1 inhibitor (camptothecin), alkylating agents, or hydroxyurea

(10-13,21-24). Second, UV and cross-linking agents that causes bulky DNA adducts by nucleotide excision repair (NER) at stalled replication forks in single strand DNA break through alkylating agents (10-13,21). Moreover, an additional pathway that may activate

ATR is transcription stress caused by RNA polymerase stalling, which recruited transcriptional coupled nucleotide excision repair (TC-NER) through phosphorylation of transcription factor p53 at ser-15 site. Examples of transcriptional stressors that

15 phosphorylate p53 is actinomycin D and 5,6-Dichlorobenzimidazole 1-beta-D- ribofuranoside (DRB) (25,26,34). Third, the activation of ATR-check point by breaking in a double strand of DNA through topoisomerase II inhibitors (Etoposide) or ionizing radiation which they cause resected in double-strand DNA break to be involved in the process of HR (10-13,21-24).

Therefore, ATR steps happen once it is triggered by single-stranded DNA gaps

(ssDNA), which are primarily responsible for signaling of DNA damage (21-24). Then,

Replication protein A (RPA) coats ssDNA, which is localization in the site of DNA damage. RPA recruits the ATR-interacting protein complex, which is phosphorylated by

ATR to recruited DNA topoisomerase II-binding protein 1 (TOPBP1) and claspin/9-1-

1which are protein adaptors to activate ATR via phosphorylate checkpoint (CHK1) (11-

15). Finally, it has essential roles in DNA repair, cell survival, DNA replication, and cell cycle progression. Most of the known activation mechanisms and functions of ATR kinase involve DNA replication and cell cycle progression through S/M stage in the cell cycle

(10-15,25-28).

ATR kinase inhibition prevents ATR-checkpoint kinase signaling pathway, which leads to inhibit of DNA repair and cell cycle arrest and induce cell death through using small molecule inhibitors, such as VE-821, VE-822, and/or AZD678 to decrease tumors activity and DNA damage (11,21). Theses inhibitors are blocking ATR-CHK1 signaling pathways which leads to prevent checkpoint activation, DNA repair, DNA replication

(origin firing), fork stabilizing, and deoxynucleotides pools (dNTPs) (figure 5) (12).

16

Figure 5: ATR-CHK1 activation and inhibition in replicating cells.

17 However, ATR kinase activation and inhibition have primarily been studied in cells that actively synthesizing DNA, such as during the S phase of cell cycle. Thus, ATR kinase protein can protect cells from lethal effects in response of replication stress. In contrast,

ATR kinase inhibition can promote cells to lethal effects when exposed to genotoxins (25-

28). Most of researches have been shown that ATR has only functions and activated only in the replication stage (S phase) of cell cycle (figure 6) (25.26,33,34).

b. ATR kinase in non-replicating cells.

The most of ATR kinase activation and inhibition in non-replicating quiescent cells are undiscovered yet or not completely understood. Thus, a few articles have been studied that ATR can be activated in non-replicating quiescent cells, which in G0-G1phase in the cell cycle (figure 6) (25,26,33,34). However, quiescent cells are essential to study because of the majority of human cells and cancer stem cells in the G0-G1 cell cycle in the non- replication stage, which DNA not actively proliferating through cell cycle progression. In contrast, a few human cells are in the replicating stage (25,26,33,34). Thus, Dr. Kemp had found that ATR kinase inhibition partially protects quiescent HaCaT cells treated with N- acetoxy-2-acetylaminofluorene (NA-AAF), which generates bulky adducts on in

DNA. He had shown that ATR kinase inhibition has opposite effects on the viability of replicating and non-replicating cells (25,26).

A few previous studies have focused on agents that induce DNA lesions repaired via NER and have suggested a possible reliance on the TLS pathway that promote mutagenesis in quiescent cells (27,28,33,34). Dr. Kemp’s lab has been found recently that

18 ATR kinase inhibition sensitizes quiescent cells to the lethal effects of cisplatin or UVB, but ATR inhibition caused an increase in PCNA mono-Ub, which it is a marker of activation of the translesion synthesis (TLS) pathway, which is potentially mutagenic after

UVB and cisplatin (27, 28).

19

Figure 6: ATR activation and inhibition in replicating cells vs. non-replicating cells. ATR kinase can activate after it exposed to DNA damage either in proliferating cells in S phase or in non-replicating quiescent cells. In proliferating cells, ATR activated after replication stress to lead to protect cell from lethal effects when it exposed to genotoxins but in inhibition of ATR kinase can promote cells to apoptosis or cell death when exposed to genotoxic compound. However, in quiescent cells, ATR kinase activation and inhibition not accurately known or discovered.

20 4- ATR inhibitors.

ATR inhibitors are inhibiting ATR-checkpoint signaling pathway in response of

DNA damage with or without DNA damaging agents to decrease cancers development (21-

24). ATR kinase inhibitors are currently ongoing in clinical trials with different types of chemotherapies to treat human cancers patients (20,25-28).

The first ATR inhibitor was 3-amino-6-arylpyrazines, which known as VE-821 that developed by Vertex Pharmaceuticals in 2011 and it has antitumor activity (21). It has selectivity and potency to inhibit ATR protein kinase and ATR-CHK1 signaling pathway.

It has synergistic effects and sensitization effect to inhibit ATR which targeted cancer cells but not to normal cells. VE-821 had been demonstrated in preclinical studies with radiation and chemotherapies such as cisplatin, , , , and camptothecin (table1) (11-23).

The second ATR inhibitor was VE-822; M6629 which developed by Vertex

Pharmaceutical and it was the first ATR inhibitor to undergo clinical trials as VX-970. It is an analogue of VE-821 and it has more selectivity and potency of ATR inhibitor. It sensitizes and synergizes cancer cells with or without radiation and chemotherapies such as cisplatin (for gastric cancer, lung cancer, and head and neck squamous cell carcinoma),

Camptothecin, topotecan, , (for colorectal cancer patients) gemcitabine, etoposide (for lung cancer patients) (table1) (11-24).

The third ATR inhibitor was AZD6738 that was developed by AstraZeneca and it is an analogue of AZ20. It has the highest selectivity and potency to inhibit ATR kinase

21 and ATR-CHK1 signaling pathways. It is the first drug which is administered orally. It is the second ATR inhibitor which undergoes clinical trials in combination with chemotherapies or alone. It is sensitizing and synergizes tumor cells with or without radiation and chemotherapies, such as , , , or durvaumab

(table1) (11-24).

Table 2: Some examples of ATR inhibitors

ATR VE-821 VE-822 AZD6738

inhibitors (VX-970; M6620)

Structure

Description It is the first selective It is an analogue of It is an analogue of

ATR inhibitor and VE-821 and it has AZ20 and it is having

potent. It was more selectivity and the highest potency

discovered by vertex potency of ATR and the highest ATR

pharmaceutical inhibitor. It is the first inhibitor selectivity.

company in 2011. It ATR inhibitors that It is the first drug

sensitizes cancer cells undergo clinical trials which is

rather than normal with combination administered orally.

22 cells with with chemotherapies It is the second ATR

chemotherapies and and ionization inhibitor which

ionization radiation. radiation. It is undergoes clinical

sensitizing cancer trials in combination

cells rather than with chemotherapies

normal cells with or alone. It is also

chemotherapies and sensitizing cancer

ionization radiation. cells rather than

normal cells with

chemotherapies and

ionization radiation.

Clinical Not undergoes in Ongoing ongoing trials clinical trials

Chemothera cisplatin, gemcitabine cisplatin, topotecan, paclitaxel, pies with topotecan, irinotecan, gemcitabine,

ATR doxorubicin, and gemcitabine, carboplatin, inhibitors camptothecin. etoposide. olaparib, durvaumab.

23 Target and All three ATR inhibitors prevent DNA damage checkpoint signalling function activity of ATR/Chk1.Thus, ATR inhibitors inhibit DNA damage

checkpoint activation, DNA damage repair, and increases cancer cells

death.

References (11-24)

1- Genotoxic compounds.

Genotoxic agents are classified as four major classes according to the

cellular level that we have been tested them at the Dr. Kemp’s lab with ATR kinase

inhibitors. They are alkylating agents, topoisomerase inhibitors, transcription stress

inducing compounds, and translational inhibitor (figure 7). Thus, from the central

dogma of cells’ life, there are two main process to replicate and transcribes DNA

to RNA, and to translate RNA. In the first step is transcription which begun in

nucleolus in DNA to be replicated and transcribed to messenger RNA (mRNA).

Then, in the translation processes, mRNA transfer to cytoplasm at a ribosome to

synthesize protein (49,50).

24

Figure 7: Genotoxic agents in the cellular level. Alkylating agents (cisplatin, mitomycin c, temozolomide) inhibited replication of DNA at the cellular level as well as topoisomerase inhibitors (camptothecin, etoposide). Transcriptional stressors (actinomycin D, 5,6-Dichlorobenzimidazole 1-beta-D-ribofuranoside) prevented transcription of messenger RNA to transfer to cytoplasm whereas translational stressor(cycloheximide) inhibited all process at cellular level.

25 As in a diagram of different classes of genotoxic agents which effect in the cellular level. Alkylating agents and topoisomerase inhibitors inhibit replication of DNA at the cellular level. Then, transcriptional stressors prevent transcription of the mRNA. The last class is translational stressor which inhibit mRNA to transfer to protein as shown in (figure

7) (25,26).

a. Alkylating agents:

Alkylating agents have an alkyl group to attach to DNA or RNA and result in the inhibition of DNA, RNA, or protein synthesis. Thus, inhibiting replication on S phase and replication in G1 phase. The example of alkylating agents as mitomycin C, temozolomide, and cisplatin. (1,45) (1,29,30,45). Mitomycin C and cisplatin are considered as crosslink

DNA that prevents DNA replication and transcription by the formation of DNA adducts within or between DNA strands (44). In contrast, temozolomide is used to treat and considered as the methylating agent that generate damages which repaired via base excision repair (BER) (36,44). Besides, Mitomycin C and cisplatin are produced lesions that fixed by nucleotide excision repair (NER) and translation synthesis pathway (TLS) on quiescent cells in G0/G1 phase. Whereas in replicating cells in S phase are repaired by homologous recombination (HR) through ATR signaling activation (table3) (50,51,54,58).

26 Most alkylating agents which cause killing of tumors cells are bifunctional alkylating agents. These agents have two alkylating groups to form inter-stand cross links and intra-strand cross links, such as cisplatin (29,30) (table 3). Cisplatin has been shown inhibiting in replication S phase through breaking double stand break that repaired by non- homologous end-joining (NHEJ) or homologous repair (HR) which are dependent on recruitment of specialized DNA polymerase pol eta to help to fill gaps and protect cells from the toxic effect of double strand break (52,54).

However, cisplatin has shown inhibiting ATR kinase signaling through G1 phase on quiescent cells treated with ATR kinase inhibitors which are ongoing in clinical trials to promotes cell killing more than cisplatin alone and decreasing resistance of cisplatin

(20,27-30). Suggesting that ATR kinase inhibitor and cisplatin have promising result to increase sensitivity of cisplatin to treat patients. The possible repair mechanisms are nucleotide excision repair (NER) or translesian DNA synthesis on quiescent cells cisplatin with ATR inhibitors have been found that increase mutagenicity through increase induction of mono-ubiquitination PCNA that depend on translesian DNA synthesis (TLS) (26-27,51).

27 Table 3: Examples of DNA damaging agents- Alkylating agents

Agents Sub Mechanism Type of DNA The possible Refe classificati of action damage DNA repair renc on mechanisms es

1- Inter-strand Prevent DNA Transcriptional NER 1, Mitomycin cross links and RNA stress through 29, C synthesis and single strand 30, through break. 35, alkylating 44 DNA to Double strand produce inter- break. HR strand cross TLS links. 2- Triazane Single strand break BER 1, Temozolo- of through 29, mide base at O6 Methylation 30, position to MMR 36, insert 44 thymidine instead of cytosine 3- Inter-strand Prevent DNA Double strand HR 1, Cisplatin cross links synthesis breaks 27- through cross 30, Intra-strand links two Single strand NER 37- cross links guanine to break 40, form inter- TLS 44, stand or intra- 45 strand cross links.

28 b. Topoisomerase inhibitors:

Topoisomerase inhibitors are chemotherapies that inhibit topoisomerase enzymes that are responsible to regulate supercoiled DNA to relax lead to break in single strand

DNA or double strand DNA break (1,29,30). They are classified as Topoisomerase I or II inhibitors. Camptothecin is topoisomerase I inhibitor which break single strand. Dr.

Michael G Kemp had found that ATR inhibition partially protects quiescent cells from

NAAF which is N-acetoxy-2-acetylaminoflurene (25,26). Camptothecin with ATR kinase inhibitor had been shown that inhibit replication in S and G2 phase in proliferating cells which is being tested in clinical trials (20,33). However, the second class is topoisomerase

II inhibitor is Etoposide which is breaking double strand of DNA. Inhibit replication S and

G2 phase on cell cycle on proliferating cells with ATR kinase inhibitors which is ongoing in clinical trials. (20,38,44). However, the process of transcription also generates transcriptional stress in DNA that must be relaxed by topoisomerases to allow for efficient transcription by RNA polymerases. Thus, topoisomerase inhibitors may also affect the function of non-replicating cells.

c. Transcriptional stressors:

Actinomycin D (ActD) and 5,6-Dichlorobenzimidazole 1-beta-D-ribofuranoside

(DRB) inhibit RNA synthesis by impeding the movement of RNA polymerase II during transcription (25,26). Actinomycin D is transcription inhibitor that inhibits transcription and RNA polymerase through DNA intercalation, which binds to guanine and cytosine

29 base pairs (44). DRB inhibits RNA polymerase transcription and messenger RNA transcription (44). Thus, ActD and DRB will all induce transcription stress by interfering with RNA polymerase movement (25,26). ActD and DRB have been shown that with ATR kinase inhibitors on quiescent cells inhibited ATR weakly compared to (NA-AAF)

(25,26,34).

d. Translational stressor:

Translational inhibitor is Cycloheximide (CHX) which inhibits protein synthesis through elongation phase of protein synthesis in messenger RNA and translesion RNA in eukaryotic cells (44). CHX was used as a control for a general stress that is not known to generate genotoxic stress (25,26,53). In addition, CHX has been shown that the budding yeast homolog of ATR was essential in protein homeostasis. Thus, there may be other signals that can activate ATR besides canonical genotoxic stress (53).

However, ATR kinase activation and function in genotoxin-treated quiescent cells or G1 stage on cell cycle are not accurately known. ATR inhibitors are being tested in clinical trials for human cancer patients (20). Thus, a few studies have indicated that ATR can be activated in non-replicating quiescent cells, which is important because only a minority of cells in the human body are undergoing replication at any given time. In contrast, most of the cells in the human body are in non-replicating quiescent or differentiated state. In addition, most tumor cells are in G1 phase in non-replicating state

(25-28,33,34). A few previous studies have focused on agents that induce DNA lesions repaired by NER and have suggested a possible reliance on the TLS pathway that promote

30 mutagenesis in quiescent cells. Recently, Dr. Kemp’s lab had found that ATR inhibition with (NA-AAF) was partially protected cells from lethal effect when exposed on quiescent cells (25,26). However, more recent data from Dr. Kemp’s lab has been found that ATR inhibition UVB and cisplatin sensitize cells to apoptotic or cell death but increased in mutagenesis to examine long-term effect of ATR kinase inhibition on cell proliferative potential and mutagenesis in quiescent cells exposed to UVB and cisplatin (27,28).

Based on our previous data, we hypothesize ATR inhibition in quiescent cells have different functions when exposed to different type of genotoxins that generate variety of

DNA lesions. This hypothesis will be tested by following three specific aims. The first aim is to determine how ATR kinase inhibition impacts acute cell survival following the treatment of quiescent cells with different genotoxic agents. The second aim is to characterize the effects of different genotoxic compounds on ATR kinase activation in non- replicating cells. The last aim is to examine the long-term consequences of ATR kinase inhibition on cell proliferative potential and mutagenesis in quiescent cells exposed genotoxins.

31 MATERIALS AND METHODS

1. Cell culture:

The immortalized human HaCaT keratinocytes were cultured in DMEM/high glucose supplemented with 10% FBS clone III (Hyclone), 6 mM L-glutamine, 100 units/ml

O of penicillin, and 100 ug/ml of streptomycin at 37 C in a 5% CO2 humidified incubator.

In proliferating HaCaT cells were subconfluent after it cultured with normal complete media 10% FBS while quiescent HaCaT cells were change the media to low serum 0.5%

FBS clone III (Hyclone) DMEM/high glucose after it reach confluency two or three days.

Next two days HaCaT cell were treated with DMSO or ATR inhibitor VE-821, VE-822, or AZD6738 (Selleckchem) for 30 min prior to use different DNA damaging agents. These are cisplatin (Sigma) was dissolved in PBS at a 3 mM stock concentration, camptothecin

(Sigma) was diluted with DMSO at a 10 mM and a 1 mM stock concentration, actinomycin

D (Sigma) was diluted from 1 mg/ml concentrated stock with DMSO to make 100ug/ml and 10 ug/ml stock concentration, etoposide (Sigma) was diluted with DMSO at a 50 uM stock concentration, cycloheximide (Sigma) was dissolved in sterile water to be 20 mg/ml stock concentration, mitomycin C (Sigma) of 2 mg was dissolved in 4 ml of sterile water to be 0.5 mg/ml at a 1.5 mM stock concentration, 5,6-Dichlorobenzimidazole 1-beta-D- ribofuranoside (DRB) (Sigma) was dissolved in 1.57 ml DMSO to be a 100 mM stock concentration, temozolomide (Sigma) was dissolved in 1.29 ml of DMSO at a 100 mM stock concentration. Quiescent HaCaT cells were exposed to DMSO as a (vehicle),

32 spironolactone (APExBio) was dissolved in DMSO at 10 uM stock concentration, and triptolide (Sigma) was dissolved in DMSO at 1 mM stock concentration. Cells remained in the incubator for 4 hr or 24 hr to BrdU dot blot, MTT assay, immunoblotting/chromatin fractionation, and clonogenic survival assay.

2. BrdU dot blot:

Bromodeoxyuridine (BrdU) was added 10 ug/ml to a final concentration to proliferating HaCaT cells and quiescent HaCaT cells for 30 min prior harvesting the cells.

The genomic DNA was purified by using the GenElute Mammalian Genomic DNA

Miniprep kit (Sigma). Then, quantifying the genomic DNA with PicoGreen florescence

(Invitrogen) on a Synergy H1 Hybrid Multi-Mode microplate reader (BioRad). After that,

DNA was immobilized on nitrocellulose, dried, and immunodot blotting with antibodies against BrdU (Sigma) or ssDNA (Millipore) at 1:5000 dilution in 10 ml TBST as previous described (25-28).

3. MTT assay:

Acute cell survival was determined by using (3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide, a tetrazole) staining which was performed using 96 well

plates and survival determined after two days following treatment as mentioned before.

Discard media from 96 well plates and add 1.25 ml of 5 mg/ml MTT solution to 23.75

ml of low serum medium at a 0.25mg/ml MTT final concentration. Pour solution of 50

ml tube to the boat and use multichannel pipettor of 100uL to each well. Incubate 96 well

o plate at 37 C CO2 incubator for 1 hr. Then, remove or aspirate MTT-reagent containing

33 medium and add 100 ul of DMSO to each well to solubilize the MTT dye, then put it on the shaker to ensure it solubilize completely. Measure at 570 nm absorbance from reader plate (Bio-Rad) and calculate relative survival and standard deviation by comparing treated samples with DNA damaging agents to the untreated DMSO control group and each of the ATR inhibitor-treated samples.

4. Immunoblotting/chromatin fractionation:

After quiescent HaCaT cells treated for 4 hr or 24 hr, cells were washed with cold

PBS and scrape cells by cell scraper. Then, transfer cells to 1.5 ml microfuge tubes on the ice and centrifuge the samples for 4 hr at 4000 rpm. Aspirate the PBS without disturbing the cell pellet. Then, cells were lysed for 20 min on ice in 20 mM Tris-HCl

(PH 7.4), 150 mM NaCl, 1mM EDTA, 1mM EGTA, and 1% Triton X-100. After that, cells were centrifuged in a cold centrifuge at maximum speed for 15 min at 4oC and soluble lysates were transferred to new tubes. While chromatin- associated proteins were obtained from cells by two times extractions with cytoskeletal buffer (10 mM Tris-HCL pH 7.4, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA, 1 mM NaVO3, 10 mM NaF,0.1 % Triton X-100). Then, sonicate them for 10 pulse per two times by sonicator. Soluble and chromatin lysates were separated by using SDS-PAGE in equal amounts. Then, transferred to nitrocellulose membrane, and stained with Ponceau S.

After that, washed 2-3 times with TBST (Tris-buffered saline containing 0.1 %Tween-

20) and blocked in 5% milk in TBST. Probe them with primary antibodies with diluted with 1:2000 of Actin (BETHYL, A300-485A), phosphor-p53(Ser15) (Cell signaling

34 9284S), phosphor-ATR(Thr1989) (Gene Tex, GTX 128145), PCNA (Santa Cruz

Biotechnology, sc-56), or Ubiquityl-PCNA (Lys146) (Cell signaling,13439S) in overnight with TBST. After washing five times for five min, secondary antibodies were probe them in 5% milk in 1xTBST in 1:2000 dilution. Secondary antibodies included horseradish peroxidase (HRP)-linked goat anti-Rabbit IgG (Invitrogen) and goat anti-

Mouse IgG (Invitrogen) for Ubiquity-PCNA. Then visualized by using clarity Western

ECL substrate (Bio-Rad) or Supersignal West Femto substrate (Thermo Scientific) to detect chemilunescence signals by using a Molecular Imager Chemi-Doc XRS+ imaging system. Signals or bands were detected by using Image Lab (Bio-Rad) (25-28).

5. Clonogenic Survival assay:

Quiescent HaCaT cells were replaced drug-containing medium with fresh

0.5% FBS low serum media, after it treated for two days in 6 well plates. Then, incubate cells for another three days and trypsinize cells for re-plating at 100 cells per 10 cm plate containing 10 ml of complete 10% FBS media. After that, incubate cells for two weeks and change the media with fresh complete 10% FBS media after one week. Finally, cells were stained with crystal violet after cells washed by PBS and fixed them by cold methanol for 20 min. After plate dried, cells were counted the number of colonies and determined the relative survival of the treatment plates and normalized with untreated control plates DMSO or ATR inhibitor (VE-821) (27,28).

35 RESULTS

1. Validation of the non-replicating status of quiescent HaCaT keratinocytes by

using BrdU labeling.

To validate that our cell culture conditions can generate non-replicating

quiescent HaCaT cells and proliferating HaCaT cells, BrdU labeling and

quantification was carried out on the two populations of cells. Proliferating cells

were grown to be sub-confluent about 50-60% confluency with 10% FBS serum

media. In contrast, quiescent cells were grown to be confluent (about 95%

confluency) then changed media with 0.5% FBS media (figure 8A). We confirmed

the cellular proliferation to validate differences between quiescent cells and

proliferating cells by adding 10 µg/ml of BrdU for 30 min. Then, the purification

of the genomic DNA was quantified by PicoGreen kit and visualized by conducting

immunoblotting BrdU-labeled genomic DNA with reprobing with anti ssDNA

antibody as a control and anti-BrdU antibody. Relative DNA synthesis of

proliferating HaCaT cells was higher than quiescent HaCaT cells. Thus, BrdU

incorporated into proliferating HaCaT cell while in quiescent HaCaT cells have a

few enough spaces to be incorporated less than in replicating cells (figure 8B).

Thus, DNA synthesis in quiescent cells was decreased around 10 times less than in

proliferating cells.

36

Figure 8: Validation of the non-replicating status of quiescent HaCaT keratinocytes by using the BrdU labelling. (A) HaCaT cells in replication state with 10% FBS serum media while non- replication state it become confluent with 0.5% FBS media. (B) replication and non-replication cells were added 10 µg/ml BrdU for 30 min to purify the genomic DNA, quantify genomic DNA with PicoGreen kit and immunodot blotting BrdU-labeled genomic DNA. Blots were re-probed with anti-ssDNA antibody as control or anti-BrdU antibody. Relative incorporation of BrdU from two separate experiments was quantified (Mean +/-SE of N=2) and graphed.

37 2. Quiescent cells are less sensitive to the acute effects of genotoxic agents than

proliferating cells.

To determine how the different genotoxic agents impact acute cell survival,

quiescent and replicating cells were treated with increasing concentrations of

various genotoxic agents and then cell viability was measured. First, non-

replicating quiescent HaCaT cells or replicating HaCaT cells were grown to be

confluent with low serum media or sub-confluent with high serum media,

respectively, as (in figure 8A).

After that, non-replicating quiescent HaCaT cells or replicating HaCaT cells

were treated with increasing concentrations of different classes of genotoxic

compounds. These are alkylating agent: mitomycin C (figure 9A), temozolomide

(figure 9B), and cisplatin (figure 9C); topoisomerase inhibitors: camptothecin,

which inhibit topoisomerase I (figure 9D), and etoposide, which inhibit

topoisomerase II (figure 9E); transcriptional stressors: actinomycin D (figure 9F),

and 5,6-Dichlorobenzimidazole 1-beta-D-ribofuranoside (DRB) (figure 9G), and

the translational stressor: cycloheximide (figure 9H). Finally, after two days of

treatment, non-replicating quiescent HaCaT cells or replicating HaCaT cells were

stained by MTT. Relative survival and standard deviation were quantified and

graphed. In general, most of the genotoxic compounds are significantly different

between quiescent and proliferating HaCaT cells. Therefore, quiescent HaCaT cells

are more resistant to be lethal than in replicating HaCaT cells, as shown in Dr.

38 Kemp’s articles, except temozolomide has no difference between quiescent or proliferating HaCaT cells (25-26).

39

Figure 9: Comparison of cell survival in replicating and non-replicating HaCaT cells treated with different DNA damaging agents and genotoxic stressors. Proliferating (replicating) and quiescent (non-replicating) HaCaT cells were treated with the increasing concentrations of the indicated compounds. Then cell survival was assessed 2 days later by using an MTT assay. (A) Mitomycin C, (B) Temozolomide (TMZ), (C) Cisplatin, (D) Camptothecin, (E) Etoposide, (F) Actinomycin D, (G) 5,6- Dichlorobenzimidazole 1-beta-D-ribofuranoside (DRB), and (H)Cycloheximide. Relative survival was performed 2 times in replicating HaCaT cells and 4 times in quiescent HaCaT cells with (Mean +/- SE of N=2 or 4), respectively.

40 3. ATR kinase inhibition in genotoxin-treated quiescent cells does not impact

acute survival.

To determine how ATR kinase inhibition impacts acute cell survival following the treatment of quiescent cells with different genotoxic agents. As in figure 9, quiescent

HaCaT cells and proliferating HaCaT cells were treated with vehicle (DMSO) or different

ATR kinase inhibitors (VE-821, VE-822, and AZD6738) for 30 min followed by the different genotoxic agent in a dose-dependent manner. Quiescent cells were then treated with the various genotoxic compounds as described above and analyzed by MTT assay.

ATR kinase inhibition has varying effects on acute survival in replication cells treated with different genotoxins, as shown on the right side (figure 10). However, ATR kinase inhibition generally has no significant effect on acute survival in quiescent cells, as shown on the left side (figure 10). The rationale for measuring the survival is to test the response to DNA damage and genotoxic stress with ATR kinase inhibitors. Thus, proliferating HaCaT cells are more sensitive to lethal effect than quiescent HaCaT cells, which treated with ATR inhibitors and exposed to a different type of genotoxins, except

DRB is having the opposite impact than others genotoxins because it may not inhibit replicating HaCaT cells (25,26,44).

41

42

43

44

Figure 10: ATR kinase inhibition has differing effects on acute cell survival following treatment of quiescent and proliferating cells with different genotoxic compounds. Proliferating and quiescent HaCaT cells were pre-treated with vehicle (DMSO) or the ATR inhibitor (ATRi) VE-821, VE-822 or AZD6738 for 30 min before treatment with the indicated concentrations of genotoxic compounds. MTT assays were used to measure cell survival 2 days later. (A) Mitomycin C, (B) Temozolomide (TMZ), (C) Cisplatin, (D) Camptothecin, (E) Etoposide, (F) Actinomycin D, (G) 5,6-Dichlorobenzimidazole 1-beta-D-ribofuranoside (DRB), and (H)Cycloheximide. Relative survival was performed 2 times in replicating HaCaT cells and 4 times in quiescent HaCaT cells with the average and standard error.

45 4. Many genotoxins induce ATR kinase activation in quiescent cells.

The data presented in Figure 10 indicated that ATR kinase inhibition did not affect acute cell survival in the quiescent cells. To determine if ATR was activated in these cells, we next carried out experiments in which we treated quiescent HaCaT cells with the genotoxin at concentrations that were shown to inhibit survival in the proliferating state. Quiescent cells were treated with DMSO or ATR kinase inhibitor

(VE-821) for 30 min. They were then exposed to different genotoxins in concentrations for 24 hr that show effect in quiescent HaCaT cells in acute survival as in (figure11). These are A) Mitomycin C (20 µM), (B) Temozolomide (TMZ) in

(500 µM), (C) Cisplatin (40 µM), (D) Camptothecin (20 µM), (E) Etoposide (250

µM), (F) Actinomycin D (100 ng/ml), (G) 5,6-Dichlorobenzimidazole 1-beta-D- ribofuranoside (DRB) (100 µM), and (H) cycloheximide (50 µg/ml).

After that, cells were harvested, and lysates were probed for p53 and/or ATR phosphorylation by immunoblotting. Because p53 gave the strongest and most consistent results among the different genotoxins, we focused our analyses on p53 phosphorylation. In quiescent HaCaT cells, p53 phosphorylation was increased in response to all of the different genotoxic compounds except DRB. Moreover, treatment with the ATR inhibitor VE-821 significantly blocked ATR and/or p53

46 phosphorylation in response to most of the genotoxins. Residual p53 phosphorylation in the presence of the ATR inhibitor is likely due to the action of the related kinase

ATM, which is known to be activated in response to DNA double-strand breaks

(25,26,33,34). Nonetheless, these results demonstrate that ATR kinase signaling is activated in quiescent HaCaT cells in response to a variety of different types of genotoxic compounds. Thus, replication stress during S phase is not the only signal for ATR activation (25,26).

47

Figure 11. ATR kinase signaling is activated in quiescent cells in response to a variety of different genotoxic compounds. Quiescent HaCaT cells were treated with vehicle (DMSO) or the ATR kinase inhibitor (VE-821; 10 µM) for 30 min before exposure to the indicated genotoxins. Cell lysates were prepared 24 hr later and were analyzed by western blotting with antibodies against phosphorylated ATR (Thr1989), phosphorylated p53 (Ser15) and Actin. The following concentrations of genotoxins were used(A) Mitomycin C (20 µM), (B) Temozolomide (TMZ) in (500 µM), (C) Cisplatin (40 µM), (D) Camptothecin (20 µM), (E) Etoposide (250 µM), (F) Actinomycin D (100 ng/ml), (G) 5,6-Dichlorobenzimidazole 1-beta-D- ribofuranoside (DRB) (100 µM), and (H) Cycloheximide (50 µg/ml).

48 5. Effects of ATR kinase inhibition on TLS pathway activation in genotoxin-

treated quiescent cells.

To examine possible functions for ATR kinase signaling in quiescent cells

unrelated to acute viability, we next examined a potential role in mutagenesis by

monitoring activation of the translesion synthesis (TLS) pathway. The TLS

pathway is potentially error-prone and may increase the risk of mutation and cell

death (27,28,41-43,48). Quiescent HaCaT cells were treated with DMSO or ATR

kinase inhibitor (VE-821) for 30 min and then were exposed to different genotoxins

for 4 hr (figure 12), 24 hr (figure 13). After two days, quiescent HaCaT cells were

harvested and extracted to enrich for chromatin-associated proteins. PCNA

becomes loaded onto DNA during DNA repair synthesis and becomes mono-

ubiquitinated on Lys164 upon activation of the TLS pathway.

As shown in figure 12, there is an induction of PCNA mono-ubiquitination

at lysine 164 after 4 hr of treatment with mitomycin C (figure 12 A), temozolomide

(figure 12 B), and cisplatin (figure 12 C). All three of these compounds induce the

formation of adducts on DNA that are repaired by either NER or BER and thus

require PCNA to complete DNA repair synthesis. Though some of the agents

induced an increase in chromatin-associated PCNA, they did not stimulate

49 significant PCNA mono-ubiquitination and thus do not seem to induce the TLS pathway. However, ATR kinase inhibition did not cause any difference in PCNA mono-ub after treated with these drugs. We then repeated the experiments but treated for a longer period of time (24 hr.). As previously reported (27), ATR kinase inhibition stimulated PCNA mono-ubiquitination after cisplatin treatment (figure

13 C). Similarly, we observed that treatment mitomycin C resulted in PCNA mono- ubiquitination that was stimulated by ATR kinase inhibition (figure 13 A). In contrast, after 24 hr treatment of temozolomide (figure 13 B) with ATR kinase inhibition did not cause any increase in PCNA mono-ub.

Then, dose response of mitomycin C and temozolomide were performed

(figure 14) because mitomycin C and temozolomide treatment resulted in the most robust activation of the TLS pathway, we performed dose response experiments monitoring PCNA mono-ubiquitination after 4 hr and 24 in the presence and absence of the ATR inhibitor. As shown in figure 14, we observed that that ATR kinase inhibition caused an increase in PCNA mono-ub after treated for 24 hr with different doses of mitomycin C but not with temozolomide.

In conclusion, ATR kinase inhibition caused an increase in PCNA mono- ub after treatment with mitomycin C and cisplatin in quiescent cells. Both of these

50 agents are bifunctional alkylating agent that induce DNA lesions that are repaired by NER (1,29,30,35) and thus ATR may function in this process to limit the dependence on TLS polymerases. In contrast, ATR kinase signaling does not appear to be involved in the DNA repair synthesis that takes place in response to the monofunctional methylation agent temozolomide, which induces methyl adducts repaired by BER. Thus, ATR kinase inhibitors with some chemotherapies as cisplatin and mitomycin C may increase the risk of mutation and secondary cancer to the non-replicating cells of the human’s cells (27,35). ATR kinase inhibition had been studied in replicating cells with anticancer drugs that decreased significantly in cell survival, but not in quiescent cells which needed to be monitored (27,28).

51

Figure 12: Examination of the effects of ATR kinase inhibition on TLS pathway activation in genotoxin-treated quiescent cells in shorter time. Quiescent HaCaT cells were treated with vehicle (DMSO) or the ATR kinase inhibitor (VE-821; 10 µM) for 30 min before exposure to the indicated genotoxins. Cell lysates were prepared 4 hr later and were analyzed by western blotting with antibodies against PCNA mono-ub, total PCNA and Ponceau staining as a control. The following concentrations of genotoxins were used: (A) Mitomycin C (20 µM), (B) Temozolomide (TMZ) in (500 µM), (C) Cisplatin (40 µM), (D) Camptothecin (20 µM), (E) Etoposide (250 µM), (F) Actinomycin D (100 ng/ml), (G) 5,6-Dichlorobenzimidazole 1-beta-D-ribofuranoside (DRB) (100 µM), and (H) Cycloheximide (50 µg/ml).

52

Figure 13: The effect of ATR inhibition in elevation in PCNA mono-ubiquitination in genotoxin-treated in quiescent cells in longer time treatment. Quiescent HaCaT cells were treated with vehicle (DMSO) or the ATR kinase inhibitor (VE-821; 10 µM) for 30 min before exposure to the indicated genotoxins. Cell lysates were prepared 24 hr later and were analyzed by western blotting with antibodies against PCNA mono-ub, total PCNA and Ponceau staining as a control. The following concentrations of genotoxins were used: (A) Mitomycin C (2.5 µM), (B) Temozolomide (TMZ) in (500 µM), (C) Cisplatin (40 µM), (D) Camptothecin (20 µM), (E) Etoposide (250 µM), (F) Actinomycin D (100 ng/ml), (G) 5,6- Dichlorobenzimidazole 1-beta-D-ribofuranoside (DRB) (100 µM), and (H) Cycloheximide (50 µg/ml).

53

Figure 14: Mitomycin C and temozolomide dose response in different two times points. Quiescent HaCaT cells were treated with vehicle (DMSO) or the ATR kinase inhibitor (VE-821; 10 µM) for 30 min before exposure to the indicated genotoxins. Cell lysates were prepared 4 hr., 24hr later and were analyzed by western blotting with antibodies against PCNA mono-ub, total PCNA and Ponceau staining as a control. The following concentrations of genotoxins were used: (A) Mitomycin C dose response (2.5 uM, 2uM,10 uM, 20uM, and 30 uM), (B) Temozolomide (TMZ) dose response (50uM, 100uM, 200uM,500 µM, and 1000 uM).

54 6. The effect of NER inhibitors on TLS pathway activation and ATR kinase

signaling in genotoxin-treated quiescent cells.

To see the effect of PCNA mono-ubiquitination and ATR kinase signaling with

using NER inhibitors. After 2 days of quiescent HaCaT cells were confluent with low

serum media to be confluent from proliferating in S phase to non-replication in

G0/G1stage. Then after 2 days, cells were pre-treated 2 hr or 30 min with spironolactone,

triptolide, or DMSO, respectively. Cells were followed with mitomycin C (20 uM) or

temozolomide (500 uM) for 4 hr or 24 hr treatment. After that, cells were harvested and

cell lysates either by fractionation buffer to extract chromatin-fractionation three times

to see the effect of PCNA mono-ubiquitination or by lysate buffer in soluble lysate to see

the effect of ATR signaling pathway activation after 4hr or 24hr, respectively. Finally,

western blot assays were performed (figure 15).

The effect of PCNA mono-ubiquitination by using NER inhibitors (spironolactone and triptolide) followed with mitomycin C or temozolomide for 4 hr or 24 hr treatment

(figure 16). We can see from figure 16, when quiescent HaCaT cells treated with mitomycin C in the top left side the treatment decreased the induction of PCNA mono-ub with spironolactone or triptolide for 4 hr treatment. Therefore, the use of NER inhibitors

(spironolactone and triptolide) inhibits the induction of PCNA mono-ub. Similarly, Dr.

55 Kemp’s lab previously has been shown that inhibiting NER by (spironolactone or triptolide) blocked PCNA mono-ub with cisplatin and UVB, which TLS pathway may be required to fill in NER gaps (27,28). This suggests that the TLS pathway may be needed to fill gaps in NER during quiescent cells to inhibit the single-strand DNA break from converting to double-strand DNA break, which is the one of the most dangerous and toxic

DNA lesions (51,52,54). However, mitomycin C with NER inhibitors showed toxicity to the quiescent cells, especially when cells were treated with triptolide for 24 hr. Thus, we cannot see the effect of NER inhibitors with PCNA mono-Ub because of the toxicity of these drugs on cells treated with either mitomycin C or temozolomide for 24 hr.

Temozolomide induces BER that has been considered as not toxic as mitomycin C which likely to generate of sufficient single strand to activate TLS pathway. Also, the temozolomide result is suggesting that DNA polymerase on the BER pathway prevented

RPA and PCNA on single-strand intermediates on quiescent cells to be error-prone TLS, which is the opposite of lesions (52,54).

The effect of ATR kinase signaling pathway and using NER inhibitors

(spironolactone and triptolide) followed by treatment with mitomycin C or temozolomide was examined at 4 hr or 24 hr time points (figure 17). We can see with mitomycin C treatment for 24 hr is more dependent on ATR signaling pathway because there is

56 increasing phosphorylation of p-53(Ser15) and ATR (Thr1989) when exposed to NER inhibitors or alone. Similarly, previous studies have shown that pre-treatment with NER inhibitors blocked cisplatin and UVB dependent ATR kinase signaling through phosphorylation of ATR and p53. Thus, they conclude that ATR kinase signaling on quiescent cells are dependent on NER pathway when exposed to UVB or cisplatin

(27,28,46,52).

57

Figure 15: Diagram of the effect of PCNA mono-ubiquitination and ATR kinase signaling with NER inhibitors. HaCaT quiescent cells were confluent and changed media to low serum 0.5% serum after 2 days from S phase in proliferating cells to be confluence in G0/G1 phase in quiescent cells. Then, after 2 days, cells were treated with spironolactone, triptolide, or DMSO before treated with mitomycin C (20 uM) or temozolomide (500 uM) by 2 hr or 30 min, respectively. After that, cells were harvested and cell lysates either by fractionation buffer to extract chromatin-fractionation three times or by lysate buffer in soluble lysate after 4hr or 24hr, respectively. Finally, cells were performed immunoblotting assay.

58

Figure 16: The effect of PCNA mono-ubiquitination by using NER inhibitors (spironolactone and triptolide) followed with mitomycin C or temozolomide for 4 hr or 24 hr treatment. Quiescent HaCaT cells were pre-treated with spironolactone, triptolide, or DMSO as vehicle for 2hr or 30 min, respectively. Then, cells were followed with mitomycin C (20 uM) or temozolomide (500 uM). Finally, cells were harvested and lysated with fractionation buffer for three times and cells were performed immunoblotting assay.

59

Figure 17: The effect of ATR kinase signaling and NER inhibitors exposed to mitomycin C or temozolomide in different times points. Quiescent HaCaT were treated with spironolactone, triptolide, or DMSO as a vehicle for 2 hr or 30 min, respectively. Then, cells were followed with mitomycin C or Temozolomide for 4hr or 24 hr treatment. Finally, cells were harvested and cell lysates with lysate buffer in soluble lysate and then cells were performed immunoblotting assay.

60 7. Effect of ATR kinase inhibition on the clonogenic survival of quiescent cells

following their induction of the proliferative state.

Because the different genotoxins induced ATR kinase signalling in the

quiescent cells (figure 11) but ATR kinase inhibition had little effect on acute

viability in quiescent cells (figure 10), we next wanted to determine whether ATR

kinase inhibition impacts the ability of quiescent cells to proliferate and form colonies

upon withdrawal of the different genotoxins. We therefore carried out clonogenic

survival assays with after exposure of quiescent cells to the different genotoxic

agents. Quiescent HaCaT cells were treated with DMSO or ATR inhibitor (VE-821)

for 30 min followed by genotoxic compound treatment for two days. After two days,

the culture medium was changed media to low serum media and the cells were

cultured for an additional 3 days. The cells were then trypsizined and re-plateed at

low density to monitor colony formation. Cells were incubated for two weeks and

were then stained with crystal violet to visualize and count the colonies (27,28). A

schematic of this experimental methodology is shown in figure 18.

From figure 19, ATR kinase inhibition with different genotoxic compounds

have not consistent results, but may be decreased clonogenic generally. Dr. Kemp’s

lab previously showed that ATR kinase inhibition with UVB radiation have

significantly decreased clonogenic survival but not with cisplatin which have not

decreased significantly (27,28).

61

Figure 18: Clonogenic survival assay diagram. After two days of quiescent HaCaT cells were confluent with low serum media and then treated with DMSO or ATR inhibitors (VE-821) for 30 min followed by using genotoxic agents. It changed media with 0.5% FBS serum media for three days and then re-plate cells and trypsinize it with two weeks incubated. It changed media with 10% FBS serum after 7 days incubated. Finally, cells were stained with crystal violet and counted colonies and graphed.

62

Figure 19: Effect of ATR kinase inhibition on the clonogenic survival assay of quiescent HaCaT cells exposed to different genotoxic agents. Quiescent HaCaT cells were treated with DMSO or ATR inhibitor (VE-821) for 30 min followed with different genotoxic compounds in concentration were used: (A) Mitomycin C (20 µM), (B) Temozolomide (TMZ) in (500 µM), (C) Cisplatin (40 µM), (D) Camptothecin (20 µM), (E) Etoposide (250 µM), (F) Actinomycin D (100 ng/ml), (G) 5,6-Dichlorobenzimidazole 1-beta-D-ribofuranoside (DRB) (100 µM), and (H) Cycloheximide (50 µg/ml). Relative survival cells were quantified (Mean +/- SE of N=2) and graphed by using crystal violet staining.

63 DISCUSSION

There are different types of DNA damaging agents that cause a variety of DNA lesions which employ specific types of DNA repair mechanisms. Hence, many of these defects in DNA repair mechanisms have been linked to human disease and increase the risk of cancers and mutations. Many DNA damaging agents also induce the activation of

ATR kinase signaling. However, most of our knowledge on ATR kinase activation and function is limited to replicating cells, where ATR regulates replication origin firing, replication fork stabilization, and cell cycle progression. Because most cells in the body are in a non-replicating quiescent or differentiated state, it is important to understand how

DNA damaging agents impact non-replicating cell function and how ATR modulates these effects. As shown in figure 11, ATR kinase is activated under normal conditions after quiescent cells are exposed to different types of genotoxins. The mechanisms by which each genotoxin activates ATR signaling remain to be determined. However, as both DNA repair intermediates and RNA polymerase stalling potentially involve the generation of long stretches of single-stranded DNA, ATR activation in quiescent cells may exhibit similarities to canonical activation by replication fork stalling. The functions of ATR in non-replicating cells is also important to determine. ATR kinase inhibition in proliferating cells generally sensitizes the cells to undergo cell death in response to treatment with different genotoxins. In contrast, as shown in figure 10, ATR kinase inhibition generally has no significant impact on acute survival in quiescent cells. However, the observation that ATR kinase signaling is activated in quiescent cells treated with a variety of different

64 genotoxins (figure 11) implies that ATR kinase may have other functions in quiescent cells that do not directly impact acute survival. These functions may include the regulation of

RNA splicing (59) or translesion DNA synthesis (TLS) (25-28,37).

Most DNA repair mechanisms involve some level of DNA synthesis to replace damaged nucleotides removed during the repair process. For example, in cells exposed to

UV radiation or cisplatin, the nucleotide excision repair system removes the damaged nucleotides in the form of 30-nt-long DNA oligonucleotide. This process leaves a 30-nt single-stranded DNA gap in the DNA that must be filled by DNA synthesis to restore the duplex to its native double-stranded state. Though the replicative polymerases pol delta and epsilon have long been assumed to be involved in this gap filling synthesis, more recent data show that TLS polymerases may be required for this process (51, 60, 61). The recruitment of TLS polymerases to sites of DNA damage and repair requires the mono- ubiquitination of the polymerase clamp protein PCNA. In the case of UVB radiation and cisplatin, which induce lesions removed by nucleotide excision repair, ATR kinase inhibition results in an increased level of PCNA mono-ubiquitination (27, 28). These results indicate that DNA repair synthesis is more reliant on TLS polymerases when ATR is inactive in quiescent cells. The alkylating agent mitomycin C (MMC) is similar to cisplatin in that it induces monoadduct and inter-strand crosslinks that require the nucleotide excision repair machinery for damage removal. As shown in figure 13 and 14,

ATR kinase inhibition in MMC-treated quiescent cells results in a higher level of PCNA mono-ubiquitination. Moreover, treatment with the nucleotide excision repair inhibitor

65 spironolactone partially abrogates both MMC-induced ATR signaling (figure 17) and

PCNA mono-ubiquitination (figure 16), which indicates that both DNA damage response processes are dependent on nucleotide excision repair in quiescent cells. Thus, ATR kinase inhibitors may induce mutagenesis and elevate the risk of carcinogenesis in humans undergoing chemotherapies regimens that involve both ATR kinase inhibitors and a DNA damaging drug.

In contrast, temozolomide (TMZ) treatment generates smaller methylated purine lesions that can be removed from DNA by the base excision repair system. During BER,

PCNA becomes loaded onto DNA to allow DNA polymerase delta or epsilon to carry out

DNA repair synthesis, in a manner similar to nucleotide excision repair. Moreover, the data in figure 13 demonstrate that PCNA becomes mono-ubiquitinated after TMZ treatment in quiescent cells, which suggests that some BER DNA synthesis involves TLS polymerases.

However, the observation that ATR inhibition does not further exacerbate this PCNA mono-ubiquitination (figure 13, 14) indicates that ATR does not affect repair synthesis during long-patch BER, unlike the situation with UVB, cisplatin, and MMC (figure 20). A possibly explanation for this difference is the extent of single-stranded DNA generation during NER and BER. NER gaps are much larger than BER gaps, and thus only NER gaps may provide sufficient amounts of single-stranded DNA for RPA binding and subsequent

ATR recruitment to the repair gap to regulate gap filling synthesis. However, future studies will be necessary to test this hypothesis.

66

Figure 20: Schematic summarizing the effect of the effect of ATR kinase inhibition in temozolomide, mitomycin C, cisplatin, and UVB treated quiescent cells. Most genotoxins under normal conditions are repaired by different DNA repair mechanisms to fill gaps by DNA synthesis after ATR becomes activated. However, when ATR inhibited, the gap filling process depend on the translesien synthesis TLS pathway because it is increased in PCNA monoubiquitination mitomycin C, cisplatin, or UVB-treated quiescent cells. These leads to DNA adducts to be repaired by the NER. These genotoxins can result in increasing mutagenesis or cell death. In contrast, temozolomide is generated single methyl group on guanine which it is repaired by BER machinery. Thus, after ATR inhibited, the gap filling process becomes more dependent on PCNA polyubiquitination to fill gaps without PCNA and RPA, which can result in increasing cell survival because it is error-prone lesion bypass.

67

To summarize the results in three points: 1) Most of the genotoxins lead to the activation of ATR kinase signaling in quiescent cells. 2) Acute cell viability for genotoxins were not significantly affected by ATR kinase inhibition, except for cisplatin and modestly for MMC. 3) None of these genotoxic compounds induced much PCNA mono- ubiquitination except MMC and TMZ. These findings provide the potential to understand the role of the ATR kinase for the treatment and prevention of human cancers. These provide new insight on how modulation of ATR dependent on DNA damage signaling, which has therapeutic effects of mitigating or limiting the toxicity of certain genotoxic compounds. Hence, as we know that ATR inhibitors are currently in clinical afflictions as anti-cancer treatment with or without chemotherapies or radiotherapies, thus, it is essential to mitigate or limit the toxicity or adverse effects of DNA damaging anti-cancer agents.

ATR kinase inhibition may have positive or negative results with different types of genotoxins. Thus, mitomycin C, cisplatin, UVB with ATR inhibitors should be monitored because it increases TLS, which leads to increase mutation and secondary cancers.

For future studies, we need determine how ATR impacts TLS activation

(mechanism) and whether ATR affects recruitment of specific TLS polymerase to damaged

DNA. In addition, we need to identify relevant substrates for ATR in genotoxin-treated quiescent cells. Moreover, are these effects seen in other non-replicating cell types including differentiated cells and senescent cells that compose most tissues in vivo. Finally, we need to confirm these results on three dimensional cultured microenvironments, which

68 mimic the in vivo experiments as in human’s bodies because 3D cultured cells have extracellular matrix that interacts with cells together (37).

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