University of Cincinnati

UNIVERSITY OF CINCINNATI

Date: 5/8/06

I, Emily E. Bosco

Hereby submit this work as part of the requirements for the degree of:

Doctor of Philosophy in: Cell and Molecular Biology

It is entitled: RB Modifies the Therapeutic Response of Breast Cancer

This work and its defense approved by:

Chair: Erik Knudsen

Sue Heffelfinger

Kathy Hepner-Goss

Sohaib Khan

Yolanda Sanchez

The Retinoblastoma Tumor Suppressor Modifies

the Therapeutic Response of Breast Cancer

A dissertation submitted to the Division of Research and Advanced Studies

of the University of Cincinnati

in partial fulfillment of the requirements for the degree of

Doctorate of Philosophy (Ph.D.)

in the Department of Cell Biology, Neurobiology, and Anatomy

of the College of Medicine

5/8/06

Emily Elizabeth Bosco

B.S. University of Notre Dame, 2001

Committee Chairman: Erik S. Knudsen Ph.D. Abstract:

The retinoblastoma tumor suppressor (RB) is functionally inactivated in the majority of human cancers, and nearly half of all breast cancers. Here, we investigate the consequence of RB loss on the response to DNA damage and anti-estrogenic therapies used in the treatment of breast cancer. Initially, we demonstrate that downstream RB targets are severely mis-regulated following acute deletion in adult primary cells causing abrogation of the DNA damage checkpoint and consequently, accumulation of secondary DNA lesions upon treatment with chemotherapeutics. Additionally, we found that RB modifies the

DNA repair response in adult primary fibroblasts, such that RB-deficient cells are able to repair UV-induced lesions at an accelerated rate. These initial studies reveal that RB loss in primary cells modifies the response to DNA damage by promoting aberrant replication and inappropriately accelerating repair, both of which may ultimately sensitize cells to DNA damaging therapies. To specifically recapitulate RB loss in breast cancer, we directed shRNA against RB in MCF7 cells. RB-deficiency resulted in RB/E2F target gene deregulation and accelerated tumorigenic proliferation, thereby demonstrating that even in the context of a complex tumor cell genome, RB status exerts significant control over proliferation. Furthermore, the loss of RB compromised the short-term cell cycle inhibition following anti-estrogen, cisplatin, and ionizing radiation therapies. In the context of DNA damaging agents this bypass resulted in increased sensitivity to these agents in cell culture and xenograft models. In contrast, the bypass of anti- estrogen signaling resulted in continued proliferation and xenograft tumor growth in the presence of tamoxifen. These effects of RB loss were reiterated by ectopic

E2F expression, indicating that control of downstream target genes was

important for the observed responses. Specific analyses of the RB/E2F gene expression signature in 60 human patients indicated that deregulation of this pathway was associated with early recurrence following tamoxifen monotherapy.

Thus, because the RB-pathway is a determinant of tumorigenic proliferation and differential therapeutic response, it may represent a critical basis for informing

therapy in the treatment of breast cancer.

Acknowledgements:

I am extremely grateful to my advisor, Dr. Erik Knudsen, whose guidance,

creativity, and enthusiasm made four years feel much more like four days. Both

Erik and Karen Knudsen have been tremendous role models and I am thankful

for all of their advice and support. All members of the Knudsen laboratories, past

and present, have made my graduate work possible and my time in the lab more

enjoyable. I am especially appreciative of all of the time and assistance given to

me by the members of my thesis committee; Dr. Sue Heffelfinger, Dr. Yolanda

Sanchez, Dr. Kathy Hepner-Goss, and Dr. Sohaib Khan. Additionally, this work

could not have been accomplished without the generosity and efforts of many

collaborators and co-authors: Dr. Tyler Jacks, Dr. Julien Sage, Dr. Bob

Hennigan, Dr. Scott Lowe, Dr. Jack Zilfou, Dr. Christopher Mayhew, Sejal Fox,

Dr. Bruce Aronow, Huan Xu, Sandy Schwemberger, and Dr. George Babcock.

I would like to thank my family for their constant encouragement and for

giving me something to aspire to, both in life and in my career. Lastly, I must

thank my husband, Chris, not only for bringing me to Cincinnati, but for his patience and love, neither of which I could be here without.

TABLE OF CONTENTS

Page

List of Tables and Figures……………………………………………………………..2

Chapter I: Introduction…………………………………………………………………4

A. The Retinoblastoma Tumor Suppressor and Cancer……………………...4 B. RB and Cell Cycle Control…………………………………………………….5 C. RB and Breast Cancer Therapeutic Response…………………………….10 D. References……………………………………………………………………..13

Chapter II-IV: Results……………………………………………………………18-139

Chapter II: RB Signaling Prevents Replication-Dependent DNA Double-Strand

Breaks Following Genotoxic Insult…………………………………………………..18

A. Abstract………………………………………………………………………...19 B. Introduction…………………………………………………………………….20 C. Materials and Methods……………………………………………………….23 D. Results…………………………………………………………………………27 E. Discussion……………………………………………………………………..34 F. References…………………………………………………………………….38

Chapter III: Differential Role of RB in Response to UV and IR Damage……….56

A. Abstract………………………………………………………………………..57 B. Introduction……………………………………………………………………58 C. Materials and Methods………………………………………………………61 D. Results………………………………………………………………………...65 E. Discussion…………………………………………………………………….76 F. References……………………………………………………………………81

Chapter IV: RB Modifies the Therapeutic Response of Breast Cancer……….103

A. Abstract……………………………………………………………………….104 B. Introduction…………………………………………………………………...105 C. Materials and Methods………………………………………………………108 D. Results………………………………………………………………………...112 E. Discussion…………………………………………………………………….119 F. References……………………………………………………………………123

Chapter V: Summary and Conclusions…………………………………………….140 A. RB-mediated transcriptional repression and maintenance of cell cycle control………………………………………………………………………….140 B. Role of RB in Therapeutic Response……………………………………...143 C. Can RB Status Inform Therapy?...... 145 D. Interesting Questions and Future Investigations………………………….146 E. References…………………………………………………………………....149

LIST OF TABLES AND FIGURES Figure Page

I-1 Two-Hit Hypothesis of Retinoblastoma Heritability……………………….....5 I-2 RB is Targeted in Cancer………………………………………………………6 I-3 Regulation of Cell Cycle by RB…………………………………………….….8 I-4 RB and Breast Cancer Therapy………………………………………………11 II-1 Infection of Primary RbloxP/loxP MAFs with Adenoviral Cre Yields Acute Downregulation of RB Proteinand Deregulation of RB Repression Targets……46 II-2 RB Loss in Primary Adult Fibroblasts Impairs Replication in Response to DNA Damage……………………………………………………………………….48 II-3 Acute RB Loss Influences the Accumulation of DNA DSBs……………….50 II-4 Replication-Dependent DSB Accumulation………………………………….52 II-5 BrdU and γH2AX Foci Co-localization………………………………………..54 III-1 Acute Downregulation of RB Protein Abrogates the DNA Damage Checkpoint Response to UV and IR………………………………………………...90 III-2 Acute RB Loss Compromises the Rapid Checkpoint Response to UV and IR…………………………………………………………………………………..92 III-3 Kinetics of RB Loss is Concomitant with Target Gene Deregulation to promote Abrogation of the DNA Damage Checkpoint Response…………….94 III-4 Differential Effects of Acute RB Loss on UV and IR Induced DNA Lesion Removal……………………………………………………………………….96 III-5 Acute RB Loss Accelerates UV Induced DNA Damage Repair…………..99 III-6 RB Modifies Repair Factor Dynamics Following DNA Damage………….101 IV-1 Efficient RB Knockdown in MCF7 Cells Causes Deregulation of

2 RB/E2F Target Genes and Increased Proliferation Kinetics……………………129

IV-2 Tumor Growth in Nude Mouse Xenografts is Accelerated in RB Knockdown Cells…………………………………………………………………….130 IV-3 RB-deficiency Enables Bypass of the DNA Damage Checkpoint Resulting in Increased Sensitivity………………………………………………….132 IV-4 RB is Necessary for Sensitivity to Anti-Estrogen Therapy and Long Term Growth Arrest…………………………………………………………..134 IV-5 E2F3 Overexpression in MCF7 Cells Allows Bypass of Anti- Mitogenic Checkpoints……………………………………………………………...135 IV-6 E2F Downstream Target Deregulation Correlates with Poor Prognosis in Human Breast Cancers Treated with Tamoxifen Monotherapy…………………136 IV- Supplemental 1. Stable RB Knockdown in Several MCF7 Clones………...138 IV- Supplemental 2. RB-Deficiency Enables Accelerated Cell Cycle Progression………...... 138 IV- Supplemental 3. Bypass of Anti-Estrogen Checkpoint is Evident in Several RB-Deficient MCF7 Clones……………………………………………….138

3 Chapter I: Introduction

The Retinoblastoma Tumor Suppressor and Cancer

Cancer is a disease that develops in multiple steps, and each of these

steps is a genetic alteration that fuels the transformation of a normal cell into a

malignant one. Most genetic mutations that drive tumorigenesis disrupt proteins involved in signaling networks controlling cell division. These genetic mutations allow for uncontrolled cell division via a variety of mechanisms, for example, cells may become able to evade apoptosis, become self-sufficient for growth, or become insensitive to anti-growth signals (1). In 1971, the “two-hit hypothesis” was proposed to explain a mechanism by which cells are able to achieve such genetic changes during tumorigenesis. Through a detailed statistical analysis of two forms of a pediatric eye tumor, retinoblastoma, occurring in either a heritable or sporadic fashion, Alfred Knudson proposed that this disease arose from two mutations. Patients with the heritable form of the disease carried a germline mutation of one copy of a tumor suppressor in all of their cells. This predisposed these patients to somatic mutation of the second copy of the tumor suppressor gene early in their lifetime initiating the development of one or more tumors (2)

(Figure I-1). Following Knudson’s work, many laboratories began trying to understand the mechanism by which tumor suppressor genes could prevent aberrant proliferation. Therefore, it was not long before the retinoblastoma tumor suppressor (Rb) became the first identified tumor suppressor and was mapped to chromosomal locus 13q14 (3). Loss of heterozygosity at this locus specifically in retinoblastomas, lead to the conclusion that bi-allelic loss of a tumor suppressor

4 Sporadic:

1st “hit” 2nd “hit” in the same cell

Inherited:

One inherited “hit” in One sporadic hit Retinoblastoma: every cell in any cell biallelic loss of Rb gene

Figure I-1. Two-Hit Hypothesis of Retinoblastoma Heritability. The RB protein was

the first identified tumor suppressor based on its biallelic inactivation in rare childhood

eye tumors. Alfred Knudson proposed that two “hits” are necessary to inactivate a

tumor suppressor and induce retinoblastoma development. in fact induced tumor development (4). In addition to retinoblastomas, individuals who inherited a mutant copy of RB were predisposed to other forms of cancer later in life, namely sarcomas, brain tumors, and melanomas (5) (6), highlighting the importance of RB function in controlling cell division. RB is now known to be functionally inactivated through a variety of mechanisms in the majority of human cancers (7) (Figure I-2).

RB and Cell Cycle Control

RB function was further elucidated by the discovery that RB is bound and inactivated by the oncoproteins produced by DNA tumor viruses, including SV40 large T-Antigen, human papilloma virus E7, and adenovirus E1A (8-10).

Common among these oncoproteins is the LxCxE motif which was found to bind

5 >80% Pituitary >80% Glioma >90% Head and Neck >50% Breast >90% Lymphoma >80% Lung >20% Melanoma >80% Pancreas >90% Liver >80% Gastrointestinal >70% Prostate >80% Endometrium >90% Testes/ Ovary >70% Bladder >80% Osteosarcoma >80% Leukemias > Other sarcomas

Figure I-2. RB is targeted in cancer. RB inactivation is evident in

over 90% of all of cancers and occurs through many mechanisms:

transforming tumor viruses (T antigen, E1A, E7), mutation/ loss, or

aberrant phosphorylation. Adapted from Malumbres and Barbacid,

2001

directly to the matching sequence within RB, termed the “A/B pocket”.

Importantly, many other proteins were found to interact with RB in or around this same location, which would suggest that these viral oncoproteins may induce tumorigenesis by disrupting the ability of RB to bind with its endogenous targets.

The most comprehensively studied family of proteins that bind RB are the

E2F family of transcription factors, which are critical regulators of S-phase entry

(11). A large body of published studies exists which reveals that RB facilitates transcriptional repression of E2F-responsive genes. It accomplishes this by binding to the transactivation domain of E2F and assembling co-repressor complexes which include histone deacetylases, DNA methyltransferases,

6 SWI/SNF chromatin remodeling complex, and polycomb group proteins (12).

This interaction prevents the E2F-mediated expression of genes required for S-

phase entry and holds cells in G1 (13, 14). Together, these data suggest that a

critical facet of tumor suppression by RB may be via inhibition of S-phase entry

through interaction with E2F.

Extracellular signals modify RB function via cell-cycle dependent

phosphorylation. In quiescent cells, RB is hypophosphorylated and assembles in

transcriptional repressor complexes to maintain cell cycle arrest. In response to

mitogenic factors in G1, RB is inactivated through hyperphosphorylation

catalyzed by the combined action of cyclin D-cdk4 and cyclin E-cdk2 complexes.

These modifications are sufficient to disrupt RB-mediated transcriptional

repression and correspondingly allow for cell cycle progression (15). The hyperphosphorylation of RB is maintained throughout the rest of the cell cycle, where upon exit from M phase RB becomes dephosphorylated through the actions of serine/ threonine phosphatases (16). Once again in its hypophosphorylated form, RB is able to recruit transcriptional repressor complexes to target promoters to prevent cell cycle progression (Figure I-3).

In contrast, when growth conditions are not advantageous, anti-mitogenic factors such as DNA-damage or growth factor deprivation, inhibit activity of

Cdk4/cyclin D and cdk2/cyclin E complexes. This is accomplished through a variety of mechanisms including, regulation of protein expression, stability, or phosphorylation or through the influence of cdk inhibitors, such as p16ink4a,

7 HDAC/BRG-1 Mitogens Anti-mitogens Phosphatase RB X E2F DP CycD p16ink4a Cdk4/6 P CycE p21Cip1 RB Cdk2 p27Kip1

G0/G1 S-phase E2F DP genes M S P P RB G CycA 2 RB Cdk2 CycB Cdc2 CycA Cdc2

Figure I-3. Regulation of Cell Cycle By RB. Hypophosphorylated RB assembles in transcriptional repressor complexes in early G1. RB binds and inhibits E2F/DP heterodimers in addition to recruiting co-repressor molecules and chromatin modifying enzymes such as histone deacetylases (HDAC) and brahma related gene product-1 (BRG-1) respectively. Upon mitogenic stimulation in proliferating cells,

RB phosphorylation/ inactivation by cyclin/ cdk complexes releases E2F, thereby inducing the expression of critical S-phase genes, and S phases progresion. RB remains hyperphosphorylated throughout the cell cycle until mitotic exit when it is dephosphorylated by phosphatase activity. Anti-mitogenic signals, such as DNA damage or limited growth factors, upregulate the expression of cdk inhibitors

(p16ink4a, p21Cip1, p27Kip1) which prevent the activation of cdk/cyclin complexes maintaining hypophosphorylated RB and inducing cell cycle arrest.

8 p21Cip1, or p27Kip1. The effect of inhibition of these cyclin/cdk complexes in G1

is maintainance of RB in its hypophosphorylated form, which halts cell cycle

progression. This cell cycle arrest is known as a cell cycle checkpoint, or a period

during which cells ensure proper completion of one cell cycle stage before

entering the next. Cell cycle checkpoints are known to be critical for tumor

suppression because they ensure the maintenance of genomic integrity by

limiting the propagation of DNA mutations to daughter cells (17). DNA mutations arise naturally due to cell metabolism or exposure to environmental mutagens.

Following checkpoint activation, normal cells either repair the damaged DNA or activate apoptotic pathways if the damage is too extensive. Underscoring the

importance of cell cycle checkpoints, key components of checkpoint signaling

networks are targeted for inactivation at high frequency in tumorigenesis.

Critically, DNA damaging agents are the basis for many cancer

therapeutic regimens and function to specifically impart DNA lesions and induce

cell cycle arrest or cell death in proliferating cells. Therefore, because RB is

required for the appropriate cellular response to DNA damage, many studies

have focused upon the impact of RB loss on the response to DNA damaging

cancer therapies. These studies have revealed that cells deficient in RB exhibit

checkpoint deregulation following DNA damage, thereby allowing them to

progress through the cell cycle despite the presence of DNA lesions (18-20).

Understanding the extent and consequence of this genetic damage in RB

deficient cells can help us to more fully understand both cancer etiology and

cancer therapy.

9 RB and Breast Cancer Therapeutic Response

Breast cancer is the leading non-cutaneous cancer diagnosis in American

women, impacting over 240,000 new patients per year. RB is functionally

inactivated in approximately 30% of all breast cancers (21), largely through the

overexpression of cyclin D1 or cyclin E (7), both of which inactivate RB by

phosphorylation. RB inactivation in breast cancer is known to be associated with

tumor progression, poor response to therapeutics, and poor prognosis (22, 23).

While RB has been shown to be important in tumorigenesis and is a

modifier of the anti-mitogenic response, exploration of the specific function of RB

in breast cancer therapy has been limited. Treatment of breast cancer relies

predominantly upon the estrogen dependence of the tumor cells. Two-thirds of all

breast cancers are ER-positive, and ER serves as a molecular target for

endocrine based therapies (24). Anti-estrogens are the first line therapy for

these ER-positive tumors and efficiently elicit a G0/G1 phase arrest and hypo-

phosphorylate RB in hormone-dependent cancers (25, 26). This class of drugs is

initially effective in curbing the growth of ER-positive tumors. Unfortunately, many patients develop cellular resistance to anti-estrogen therapy while maintaining

ER-positive disease (27-29). Functional inactivation of RB and the deregulation of RB/E2F target genes have been implicated in the bypass of this hormone responsive pathway in vitro (30).

Second line therapies for tumors that exhibit resistance to anti-estrogens have traditionally included DNA damaging chemotherapies (Figure I-4). As previously discussed, RB is critical for cell cycle inhibition in response to DNA-

10 E2 4-OHT st Arrest Resistance 1 line 4-OHT

therapy 4-OHT (involves RB activation & target genes) ??????

IR nd P 2 line DD (involves RB activation C therapy & target genes)

Figure I-4. RB and Breast Cancer Therapy. ER serves as a molecular target for first

line therapy for ER+ tumors (anti-estrogens: 4-OHT, ICI). These therapies induce RB

activation and result in cell cycle arrest. Many ER+ tumors eventually develop cellular

resistance to anti-estrogen therapy through unknown mechanisms and are then treated

with DNA damaging agents, such as ionizing radiation (IR) or chemotherapies, such as

cisplatin (CDDP). RB is critical for proper activation of the DNA damage checkpoint.

Therefore, RB loss in ER+ breast cancer could impact the response to the two major

breast cancer therapeutic modalities.

damaging agents. However, upon RB functional inactivation as occurs in many

breast cancers, cells are able to bypass the G1/S DNA damage checkpoint

despite the presence of deleterious DNA lesions. As RB is known to regulate both apoptotic and DNA repair factors in addition to the cell cycle response (31,

32), it can be envisioned that RB loss has multiple effects upon the cellular response to breast cancer therapy.

The lack of effective treatment in human breast cancer is one of the main reasons for the high level of patient death associated with the disease. We believe that the analysis of RB function and its effect upon downstream targets in

modifying the response to therapeutic agents will contribute to the development

11 of efficacious treatments that specifically exploit the genetic composition of the tumor. Here, we describe our studies to address the consequence of RB loss on the two major facets of breast cancer therapy: DNA damage and anti-estrogenic checkpoint responses.

REFERENCES

1. Hanahan, D., and Weinberg, R.A. 2000. The hallmarks of cancer. Cell

100:57-70.

2. Knudson, A.G., Jr. 1971. Mutation and cancer: statistical study of

retinoblastoma. Proc Natl Acad Sci U S A 68:820-823.

3. Friend, S.H., Bernards, R., Rogelj, S., Weinberg, R.A., Rapaport, J.M.,

Albert, D.M., and Dryja, T.P. 1986. A human DNA segment with properties

of the gene that predisposes to retinoblastoma and osteosarcoma. Nature

323:643-646.

4. Dunn, J.M., Phillips, R.A., Becker, A.J., and Gallie, B.L. 1988.

Identification of germline and somatic mutations affecting the

retinoblastoma gene. Science 241:1797-1800.

5. Moll, A.C., Imhof, S.M., Bouter, L.M., and Tan, K.E. 1997. Second primary

tumors in patients with retinoblastoma. A review of the literature.

Ophthalmic Genet 18:27-34.

6. Fletcher, O., Easton, D., Anderson, K., Gilham, C., Jay, M., and Peto, J.

2004. Lifetime risks of common cancers among retinoblastoma survivors.

J Natl Cancer Inst 96:357-363.

7. Malumbres, M., and Barbacid, M. 2001. To cycle or not to cycle: a critical

decision in cancer. Nat Rev Cancer 1:222-231.

8. Munger, K., Werness, B.A., Dyson, N., Phelps, W.C., Harlow, E., and

Howley, P.M. 1989. Complex formation of human papillomavirus E7

13 proteins with the retinoblastoma tumor suppressor gene product. Embo J

8:4099-4105.

9. Whyte, P., Buchkovich, K.J., Horowitz, J.M., Friend, S.H., Raybuck, M.,

Weinberg, R.A., and Harlow, E. 1988. Association between an oncogene

and an anti-oncogene: the adenovirus E1A proteins bind to the

retinoblastoma gene product. Nature 334:124-129.

10. Zalvide, J., Stubdal, H., and DeCaprio, J.A. 1998. The J domain of simian

virus 40 large T antigen is required to functionally inactivate RB family

proteins. Mol Cell Biol 18:1408-1415.

11. Dimova, D.K., and Dyson, N.J. 2005. The E2F transcriptional network: old

acquaintances with new faces. Oncogene 24:2810-2826.

12. Dahiya, A., Wong, S., Gonzalo, S., Gavin, M., and Dean, D.C. 2001.

Linking the Rb and polycomb pathways. Mol Cell 8:557-569.

13. Flemington, E.K., Speck, S.H., and Kaelin, W.G., Jr. 1993. E2F-1-

mediated transactivation is inhibited by complex formation with the

retinoblastoma susceptibility gene product. Proc Natl Acad Sci U S A

90:6914-6918.

14. Helin, K., and Ed, H. 1993. The retinoblastoma protein as a transcriptional

repressor. Trends Cell Biol 3:43-46.

15. Jacks, T., and Weinberg, R.A. 1998. The expanding role of cell cycle

regulators. Science 280:1035-1036.

14 16. Ludlow, J.W., Glendening, C.L., Livingston, D.M., and DeCarprio, J.A.

1993. Specific enzymatic dephosphorylation of the retinoblastoma protein.

Mol Cell Biol 13:367-372.

17. Bartek, J., and Lukas, J. 2001. Pathways governing G1/S transition and

their response to DNA damage. FEBS Lett 490:117-122.

18. Harrington, E.A., Bruce, J.L., Harlow, E., and Dyson, N. 1998. pRB plays

an essential role in cell cycle arrest induced by DNA damage. Proc Natl

Acad Sci U S A 95:11945-11950.

19. Knudsen, K.E., Booth, D., Naderi, S., Sever-Chroneos, Z., Fribourg, A.F.,

Hunton, I.C., Feramisco, J.R., Wang, J.Y., and Knudsen, E.S. 2000. RB-

dependent S-phase response to DNA damage. Mol Cell Biol 20:7751-

7763.

20. Mayhew, C.N., Perkin, L.M., Zhang, X., Sage, J., Jacks, T., and Knudsen,

E.S. 2004. Discrete signaling pathways participate in RB-dependent

responses to chemotherapeutic agents. Oncogene 23:4107-4120.

21. Borg, A., Zhang, Q.X., Alm, P., Olsson, H., and Sellberg, G. 1992. The

retinoblastoma gene in breast cancer: allele loss is not correlated with loss

of gene protein expression. Cancer Res 52:2991-2994.

22. Ceccarelli, C., Santini, D., Chieco, P., Taffurelli, M., Gamberini, M., Pileri,

S.A., and Marrano, D. 1998. Retinoblastoma (RB1) gene product

expression in breast carcinoma. Correlation with Ki-67 growth fraction and

biopathological profile. J Clin Pathol 51:818-824.

15 23. Simin, K., Wu, H., Lu, L., Pinkel, D., Albertson, D., Cardiff, R.D., and Van

Dyke, T. 2004. pRb inactivation in mammary cells reveals common

mechanisms for tumor initiation and progression in divergent epithelia.

PLoS Biol 2:E22.

24. Jensen, E.V., and Jordan, V.C. 2003. The estrogen receptor: a model for

molecular medicine. Clin Cancer Res 9:1980-1989.

25. Osborne, C.K., Boldt, D.H., Clark, G.M., and Trent, J.M. 1983. Effects of

tamoxifen on human breast cancer cell cycle kinetics: accumulation of

cells in early G1 phase. Cancer Res 43:3583-3585.

26. Sutherland, R.L., Green, M.D., Hall, R.E., Reddel, R.R., and Taylor, I.W.

1983. Tamoxifen induces accumulation of MCF 7 human mammary

carcinoma cells in the G0/G1 phase of the cell cycle. Eur J Cancer Clin

Oncol 19:615-621.

27. Robertson, J.F. 1996. Oestrogen receptor: a stable phenotype in breast

cancer. Br J Cancer 73:5-12.

28. Howell, A., and Robertson, J. 1995. Response to a specific antioestrogen

(ICI 182780) in tamoxifen-resistant breast cancer. Lancet 345:989-990.

29. Encarnacion, C.A., Ciocca, D.R., McGuire, W.L., Clark, G.M., Fuqua, S.A.,

and Osborne, C.K. 1993. Measurement of steroid hormone receptors in

breast cancer patients on tamoxifen. Breast Cancer Res Treat 26:237-

246.

16 30. Varma, H., and Conrad, S.E. 2000. Reversal of an antiestrogen-mediated

cell cycle arrest of MCF-7 cells by viral tumor antigens requires the

retinoblastoma protein-binding domain. Oncogene 19:4746-4753.

31. Polager, S., Kalma, Y., Berkovich, E., and Ginsberg, D. 2002. E2Fs up-

regulate expression of genes involved in DNA replication, DNA repair and

mitosis. Oncogene 21:437-446.

32. Ishida, S., Huang, E., Zuzan, H., Spang, R., Leone, G., West, M., and

Nevins, J.R. 2001. Role for E2F in control of both DNA replication and

mitotic functions as revealed from DNA microarray analysis. Mol Cell Biol

21:4684-4699.

Chapter II: RB Signaling Prevents Replication-Dependent DNA Double-Strand Breaks Following Genotoxic Insult Emily E Bosco†, Christopher N Mayhew†, Robert F Hennigan†, Julien Sage±,

ψ Tyler Jacks± , and Erik S Knudsen†*

†Department of Cell Biology, Vontz Center for Molecular Studies, University of

Cincinnati College of Medicine, Cincinnati, Ohio and ±Department of Biology and

ψ Center for Cancer Research, Massachusetts Institute of Technology, Howard

Hughes Medical Institute, Cambridge, Massachusetts

* Corresponding author, Tel: (513) 558-8885; Fax: (513) 558-4454;

E-mail: [email protected]

Permission to reprint this work has been granted by Oxford University Press as it was published in Nucleic Acids Research, Vol 32 (1) 2004 pp 25-34.

18 Abstract:

Cell cycle checkpoints induced by DNA damage play an integral role in preservation of genomic stability by allowing cells to limit the propagation of deleterious mutations. The retinoblastoma tumor suppressor (RB) is crucial for the maintenance of DNA damage checkpoint function because it elicits cell cycle arrest in response to a variety of genotoxic stresses. Although sporadic loss of

RB is characteristic of most cancers and results in the bypass of the DNA damage checkpoint, the consequence of RB loss upon chemotherapeutic responsiveness has been largely uninvestigated. Here, we employed a conditional knockout approach to ablate RB in adult fibroblasts. This system enabled us to examine the DNA damage response of adult cells following acute

RB deletion. Using this system, we demonstrated that loss of RB disrupted the

DNA damage checkpoint elicited by either cisplatin (CDDP) or camptothecin

(CPT) exposure. Strikingly, this bypass was not associated with enhanced repair, but rather the accumulation of phosphorylated H2AX (γH2AX) foci, which indicate

DNA double-strand breaks. The formation of γH2AX foci was due to ongoing replication following chemotherapeutic treatment in the RB-/- cells. Additionally, peak γH2AX accumulation occurred in S-phase cells undergoing DNA-replication in the presence of damage, and these γH2AX foci colocalized with replication foci. These results demonstrate that acute RB loss abrogates DNA damage- induced cell cycle arrest to induce γH2AX foci formation. Thus, secondary genetic lesions induced by RB loss have implications for the chemotherapeutic response and the development of genetic instability.

19 Introduction:

DNA damage checkpoints are elicited to ensure the maintenance of

genomic stability following genotoxic insult (1,2). First observed in unicellular

eukaryotes, it has subsequently become clear that highly conserved checkpoint

pathways are present in virtually all organisms (3). It is believed that these

signaling pathways lead to transient inhibition of cell cycle progression thereby

ensuring repair of damaged DNA prior to DNA replication and cellular division.

Alternatively, severe damage may lead to permanent cell cycle withdrawal or

apoptosis to prevent the proliferation of cells harboring irreparable genetic

lesions. As such, loss of critical checkpoint pathways represents a means for

cells to accumulate deleterious mutations and is implicated in tumorigenesis (4-

6).

Several tumor suppressor proteins play key roles in the maintenance of

appropriate DNA damage response. For example, the p53 tumor suppressor was observed to accumulate in cells treated with UV or ionizing radiation and is

required for G1/S cell cycle inhibition (7). Cells mutant for p53 are defective in

delaying cell cycle progression in response to DNA damage (8). Similarly, ATM

(ataxia telangiectasia mutated) activity is stimulated immediately following

recognition of ionizing radiation-induced DNA damage. ATM-deficient cells have

been found to undergo radioresistant DNA synthesis, erroneous entry into

mitosis, and to exhibit extensive loss of the G1/S cell cycle checkpoint (9-11). In both the case of ATM and p53, loss of checkpoint function following DNA damage is associated with genomic instability and tumor predisposition.

20 While the retinoblastoma tumor suppressor, RB, is a central regulator of cell cycle progression, its role in checkpoint processes and genomic stability are less well understood. Initially, described as the gene mutated in retinoblastoma, it has subsequently become clear that RB is inactivated at high frequency in human cancers (5,12-17). RB functions to inhibit cell cycle progression by assembling repressor complexes that inhibit the expression of genes required for progression through S-phase (18-20). Following mitogenic signaling, RB is phosphorylated through the combined activities of CDK4 and CDK2-associated complexes (21-25). These phosphorylation events disrupt RB-mediated transcriptional repression and enable progression through the cell cycle (26-29).

It has recently been shown that DNA damage acts through RB to inhibit cell cycle progression (20). Specifically, it was observed that DNA damage signals to prevent RB phosphorylation, thus activating RB-signaling pathways (43). Cells deficient in RB exhibit checkpoint deregulation in the presence of DNA damaging agents, however, the extent and consequence of this genetic damage has not been specifically investigated.

A high proportion of tumors that are treated with DNA damaging chemotherapeutic agents happen to be RB-deficient (30-32). As such, understanding the interaction between RB loss and the chemotherapeutic response to these drugs is important for the improvement of existing therapeutics and the development of novel treatments. In principal, RB loss could have various effects on the processing of DNA lesions induced by chemotherapeutics and several non-exclusive possibilities exist. First, it has recently been

21 demonstrated that RB negatively regulates the expression of a wide range of

DNA repair factors (33-35); thus, in RB-deficient cells these lesions may be readily repaired due to the elevated levels of repair factors. Second, loss of RB in

cells could deregulate cell cycle checkpoints permitting the propagation of

deleterious mutations as is postulated to promote tumor progression. Third, RB

loss and subsequent checkpoint bypass could compromise viability in a manner

analogous to that observed in checkpoint-deficient yeast, wherein a failure to

elicit checkpoints leads to cell cycle catastrophe and death (36-39). In order to

probe these responses in RB mutant cells, here we investigated the development of double strand breaks (DSBs) as a secondary event from cisplatin (CDDP) or

camptothecin (CPT) treatment.

22 Materials and Methods:

Isolation of primary RbloxP/loxP murine adult fibroblasts:

Floxed Rb mice (RbloxP/loxP) of mixed 129/FVBN background (40), at least

5 weeks of age, were sacrificed by CO2 anaesthetization followed by cervical

dislocation. Fibroblasts were isolated from the peritoneal fascia by excision and

mincing of the peritoneum followed by dissociation by constant agitation for 40

min at 37°C in 0.2 mg/mL collagenase (Type I, Sigma) supplemented with 100 U

Dnase I (Roche). The dissociated tissue was washed with PBS and subsequently

incubated for 20 min at 37°C in 0.25% trypsin (Gibco) with constant agitation.

After washing twice, the isolated cells were plated in tissue culture dishes.

Cell culture, recombinant adenoviral infections:

RbloxP/loxP MAFs were subcultured in DMEM containing 10% FBS

supplemented with 100 U/mL penicillin/streptomycin and 2 mM L-glutamine at

37°C in air containing 5% CO2. Primary cells used in this study were between

passages 3 and 5. Replication defective recombinant adenovirus expressing

green fluorescent protein (Ad-GFP) or GFP in addition to Cre recombinase (Ad-

GFP-Cre) were obtained from G. Leone (Department of Molecular Genetics,

Ohio State University). The conditional RB knockout in primary RbloxP/loxP MAFs

was attained by infecting cells with adenovirus at approximately 2x107 virus particles per dish to achieve an infection efficiency of 90-95% as determined by

GFP immunofluoresence. Cells were cultured for at least 4 days post-adenoviral

23 infection before use. The same passage number and length of time post-infection

of the Ad-GFP and Ad-GFP-Cre infected MAFs were used in all experiments.

Immunoblotting:

Cells infected with Ad-GFP or Ad-GFP-Cre were harvested by

trypsinization and lysed in RIPA buffer and equal amounts of protein were

resolved by SDS-PAGE. Specific proteins were detected by standard immunoblotting procedures using the following primary antibodies: (Santa Cruz,

1:500 dilution) PCNA (pc10), Cyclin E (HE12), Cyclin A (C19), β-tubulin (D10), and anti-RB (G3-245, Becton Dickson, 1:100 dilution).

RT-PCR analysis of recombination:

RT-PCR analysis was performed to verify adenoviral-Cre-mediated recombination in primary MAFs. Total RNA was extracted using Trizol (Gibco) and cDNA was synthesized from 1µg of RNA with the SuperScript RT-PCR system (Gibco) according to the manufacturers protocol. cDNAs were amplified using PCR and the following primers: (sense) 5’-

CTGGCCAGGCTTGAGTTTGAAG-3’ and (antisense) 5’-

CAGTAGATAACGCACTGCTG-3’. PCR conditions consisted of initial

denaturation for 2 min at 94°C, followed by 30 cycles of 30 s at 94°C, 30 s at

51°C and 1 min at 72°C, followed by a final extension for 5 min at 72°C. 10 µl of

PCR product was run on a 2% agarose gel and visualized by Ethidium Bromide

staining.

24 DNA damage, Bromodeoxyuridine (BrdU) labeling, γH2AX immunoflourescence:

Primary MAFs infected with Ad-GFP or Ad-GFP-Cre were seeded on coverslips in 6-well dishes and allowed to attach. Cells were then incubated with chemotherapeutic drugs at the indicated concentrations for 16 h. Drugs were then removed by washing cells 3 x 5 min with tissue culture media and labeled with BrdU (Amersham Pharmacia Biotech) to detect DNA synthesis. Cells were labeled for 8 h, then washed, fixed in 3.7% formaldehyde, and processed to detect BrdU incorporation as previously described (41). Camptothecin (CPT) was purchased from Sigma while clinical grade cis-diamminedichloroplatinum II

(CDDP) was from Bristol Oncology.

γH2AX staining was performed as described in Paull, T. et. al. (2000) using anti-phospho-H2AX ser 139 mouse primary antibody (Upstate

Biotechnology).

BrdU and γH2AX colocalization studies were performed by BrdU labeling cells for 20 min followed by methanol fixation. Cells were stained for γH2AX as described above using Alexa 488 secondary 1:100 (Molecular Probes). Cells were then fixed in 3.7% formaldehyde for 20 min followed by 0.5% NP-40 for 5 min and washed with water. 1.5N HCl was used to depurinate the cell nuclei for

30 min followed by washing 3 x 5 min with water, permeabilization with 0.3%

Triton-X 100 for 20 min, and BrdU staining as above using 1:100 rat anti-BrdU primary antibody (McAb). Donkey anti-rat IgG rhodamine-red X-conjugated

25 secondary antibody (Jackson Immunoresearch) was used 1:100 and counterstained with DAPI (Sigma).

Flow Cytometry for γH2AX

Cells (5x105) were harvested and fixed in 70% ethanol. On the day of analysis, cells were prepared and analyzed as previously described (42). The cell cycle distribution was determined using Mod-Fit software.

26 Results:

Efficient deletion of RB from adult cells

During the genesis of human cancer RB is acutely lost in adult cells. As

most prior studies have used mouse embryo fibroblasts (MEFs) harboring loss of

RB throughout development or tumor lines which have been extensively cultured,

a specific role for RB loss has been difficult to ascertain. To eliminate these

complications, we utilized a novel system involving mice harboring a conditional

Rb allele (RbloxP/loxP mice) in which loxP sites flank Rb exon 3. Through

adenoviral expression of Cre recombinase, acute RB loss in cells that contain a

genetically stable primary cell background have recently been reported (40).

To examine the role of RB in adult cells, we employed murine adult fibroblasts (MAFs) infected with recombinant adenoviruses expressing both GFP and Cre recombinase (Ad-GFP-Cre) or GFP alone (Ad-GFP) as a control.

Efficient infection was demonstrated under previously described conditions such that greater than 90% Ad-GFP-Cre infected cells expressed high levels of GFP fluorescence 16-24h post-infection (data not shown). To confirm Cre-mediated recombination, primers in exon 2 and exon 4 of the Rb gene were utilized as described in Materials and Methods. RNA was prepared from uninfected MAFs

(0 h) or those infected with Ad-GFP-Cre at 48 h post-infection. Analysis of the

Rb transcript using RT-PCR showed the accumulation of the ∆exon3-PCR product in the infected cells relative to the control confirming efficient recombination at this locus (Fig. 1A). Western blotting with anti-RB monoclonal antibody revealed that the Cre-mediated recombination resulted in acute

27 downregulation of RB protein in Ad-GFP-Cre infected MAFs five days post-

infection (Fig.1B).

To determine the biochemical effect of acute RB loss, we evaluated the

expression of downstream targets in the RB/E2F signaling axis (34,43,44). MAFs

infected with either Ad-GFP or Ad-GFP-Cre were harvested five days post-

infection and levels of known RB target proteins were analyzed by

immunoblotting. Relative to control (lane 1), the Ad-GFP-Cre infected MAFs exhibited increased levels of proteins downstream of RB signaling including,

PCNA, cyclin E, and cyclin A (Fig. 1C). No changes were detected in tubulin

protein levels, which served as a loading control. Thus, RB can be conditionally

ablated in primary adult cells and leads to specific target gene deregulation.

RB loss in primary adult fibroblasts impairs the cell cycle response to cisplatin

and camptothecin damage

In order to evaluate the role of RB in the DNA damage response of adult

fibroblasts, asynchronously proliferating Ad-GFP or Ad-GFP-Cre infected MAFs

were treated with various concentrations of CDDP for 16 h. Initially, we used an

anti-platinum (Pt) antibody that detects Pt-DNA adducts to determine that both

Ad-GFP and Ad-GFP-Cre infected MAFs treated with CDDP produced similar

amounts of Pt-DNA adducts based on equal staining intensities (Fig. 2A, left

panel) (45). To quantify the amounts of anti-Pt staining present in these MAFs,

the average pixel intensity was compared using Metamorph software and

displayed graphically to confirm that there is no significant difference in the levels

28 of initial Pt-adduct damage (Fig. 2A, right panel). In parallel experiments, cells were treated with CDDP for 16 h, washed of drug, and then processed in one of the three following ways: 1. pulsed with BrdU for 8 h and subsequently fixed, 2. allowed to proliferate to 40 h before BrdU labeling and fixing, 3. allowed to proliferate to 64 h before BrdU labeling and fixing (Fig. 2B). The replicative fraction of treated cells was determined with respect to untreated control cells by detecting BrdU incorporation. MAFs containing functional RB exhibited a dose- dependent cell cycle inhibition, whereas cells lacking RB exhibited significantly reduced levels of arrest at each timepoint (Fig. 2C). Analogous RB-mediated dose-dependent checkpoint results were also observed when treating infected

MAFs with CPT (Fig 2D). Together, these data indicate that RB plays an instrumental role in the cell cycle response to DNA damage, in that acute loss of

RB in adult cells uncouples this checkpoint.

Acute RB knockout influences the accumulation of DNA double-strand breaks

Since neither CDDP nor CPT act directly to elicit DNA double-strand breaks (DSBs) (46,47), we studied the impact of RB loss upon the accumulation of these lesions. H2AX, a submember of the histone H2A family is rapidly phosphorylated at its C-terminus (γH2AX) in response to DSBs, and serves as an efficient measure of DSB accumulation (46-48). Ad-GFP and Ad-GFP-Cre infected primary MAFs were treated with CDDP for 16 h and examined for γH2AX focus formation using immunofluorescence. Capturing images of these drug treated cells at equal exposures revealed that the number and intensities of

29 γH2AX foci increased with CDDP treatment in both types of infected MAFs (Fig

3A). Interestingly, cells lacking RB in fact have a significantly increased quantity

and intensity of γH2AX foci as compared to the Ad-GFP infected MAFs after 16h of treatment (Fig 3A, top panel). Analogous results were evident 48 h post-

treatment (T=64 h) (Fig 3A, bottom panel). In order to quantitate the observed

intensity differences between the γH2AX foci in Ad-GFP and Ad-GFP-Cre

infected MAFs, cells were imaged by confocal microscopy and analyzed for

signal intensity using Metamorph software (Fig 3B). These data show that the

cells containing functional RB accumulate lower levels of DSBs that increase in

proportion to CDDP concentrations immediately after and 48 h post-treatment

(T=64 h) as compared to MAFs lacking RB. RB-deficient MAFs exhibit a much higher intensity level of γH2AX staining that increases with CDDP dose such that

the staining level appeared to saturate at the 4 µM dose (Fig. 3B). Cells

containing wild-type RB treated for 16 h with CDDP, washed free of drug, and

maintained in medium to 64 h contained similar levels of staining as compared to

the cells stained after 16 h of treatment. We confirmed these results by

evaluating the percentage of cells in the populations staining positive for γH2AX

(Fig. 3C). Similar results were evident after CPT treatment for 16 h (Fig 3D).

These data demonstrate the significant differences in amount and intensity of

nuclear γH2AX foci present in cells with and without RB and attests to the

importance of RB in preventing the accumulation of DSBs in response to

chemotherapeutic treatment.

30 Replication- dependent DNA double-strand break accumulation

There are two possible explanations for the observed increase in

accumulation of DNA damage in response to chemotherapeutic drugs in RB

mutant cells. First, loss of RB may contribute to DSBs due to downstream target

deregulation that promotes breaks (e.g. enhanced repair or nuclease expression). Second, replication across the primary lesion promotes additional

DSBs. We were able to address these possibilities by uncoupling RB-loss from

checkpoint deregulation. As shown in Fig. 4A, serum starvation for three days

inhibits both RB-proficient and deficient cells from undergoing replication. Under

these conditions no significant difference in the percentage of γH2AX positive foci

per cell was evident between the Ad-GFP and Ad-GFP-Cre infected MAFs

following 16 h CDDP treatment (Fig. 4B). These data indicate that most of the

DSB damage acquired by the RB-deficient cells occurred during cell cycle

progression, as is demonstrated by comparing Fig. 3C with Fig. 4B.

To specifically consider the possibility that DSB damage is replication-

dependent, flow cytometric analysis was utilized to examine cell cycle phase-

specific accumulation of γH2AX positive foci. Asynchronous Ad-GFP-Cre infected

MAFs were analyzed following 16 h CDDP treatment. By separating cells stained for γH2AX and DNA content, peak γH2AX staining was, in fact, specifically observed in cells with an S-phase DNA content (Fig. 4C). This profile is

consistent with the idea that continued replication in the CDDP-treated RB-

deficient cells drives the accumulation of γH2AX.

31 To directly associate γH2AX with ongoing replication, co-labeling

experiments were performed where DNA replication and γH2AX foci were coordinately analyzed. Initially, as a control to validate the specificity of the antibodies utilized we demonstrated that the γH2AX background staining observed in untreated cells did not colocalize with BrdU incorporation (Fig. 5A).

Ad-GFP and Ad-GFP-Cre infected MAFs treated with CDDP for 16 h, were washed of drug and pulsed with BrdU for 20 min, at which time they were fixed.

Immunofluoresence was used to observe sites of BrdU incorporation and γH2AX foci localization in untreated control and CDDP treated cells. No colocalization was evident in cells containing functional RB due to lower accumulation of γH2AX foci and a functional checkpoint yielding inhibition of BrdU incorporation following

16 h 4 µM CDDP treatment. Conversely, in RB-deficient cells with the same

CDDP exposure, γH2AX foci colocalized with the sites of ongoing BrdU incorporation (Fig. 5B). The BrdU and γH2AX stained cells were analyzed in greater detail using morphometric analysis to identify individual foci overlap

throughout the entire nucleus (Fig. 5C). As a control for background staining, the

pixel intensity thresholds for both the red and green channels in these 8-bit

images were held constant at 133 and 50, respectively. Sites of BrdU

incorporation were found to colocalize with approximately 7% of the γH2AX

background stained sites in untreated Ad-GFP infected MAFs (not shown).

However, in Ad-GFP-Cre infected MAFs that were treated with 4 µM CDDP for

16 h, sites of threshold BrdU incorporation staining were found to colocalize with

69% +/- 8% of threshold γH2AX foci staining present in the nuclei. These data

32 demonstrate that sites of ongoing replication and DSBs colocalize in RB-deficient adult cells following DNA damage.

33 Discussion:

Since RB is functionally inactivated in the majority of human cancers,

understanding the role of RB in modifying the cellular response to

chemotherapeutics is critical for the development of efficacious therapeutics. We

and others have shown that chronic RB loss in MEFs is sufficient to inactivate the

DNA damage checkpoint and yield deregulated growth (20,41). Here, we

observed that primary murine adult fibroblasts (MAFs) with functional RB were

able to successfully inhibit replication following treatment with cisplatin (CDDP),

which forms platinum-DNA adducts, and with camptothecin (CPT), which is an

inhibitor of topoisomerase I. However, acute RB loss in these same primary cells

was sufficient to impair cell cycle arrest after exposure to both chemotherapeutic

agents. These results indicate that RB is also required to mediate cell cycle

checkpoints in adult cells, consistent with what has been observed in MEFs

(41,48).

It has become increasingly clear that DNA damage functions to elicit a cell

cycle checkpoint via signaling pathways. One of the critical determinants of the

signaling from DNA double-strand breaks (DSBs) is the rapid phosphorylation of

histone H2AX at the site of the lesion (γH2AX) (49). In this study we utilize

antibodies specific for γH2AX to probe for the induction of DSBs (as described

below). However, the data from this study indicate that loss of RB does not compromise the signaling to phosphorylate H2AX. These results are consistent

with the observation that RB function after DNA damage signaling demonstrates

delayed kinetics relative to the rapid phosphorylation of H2AX by ATM that can

34 occur within minutes of a formed DSB. As such, these results position RB downstream from the signaling pathways giving rise to γH2AX.

Among the most damaging lesions caused by chemotherapeutic drugs are

DSBs. Several potent anti-cancer agents induce this lesion as the primary form of damage (e.g. ionizing radiation or doxorubicin). However, a number of chemotherapeutics do not generate DSBs as the primary lesion. For example,

CDDP or alkylating agents form DNA-adducts that do not spontaneously give rise to DSBs. Similarly, CPT and other topoisomerase I inhibitors cause only single strand breaks. However, the processing of these types of lesions can lead to the formation of DSBs (50-54). Here we specifically focused on determining the role of RB loss on the development of DSBs following damage with CDDP and CPT.

Presumably, the loss of RB could have diverse effects on the processing of these forms of damage. First, it has been found that RB regulates the expression of a large number of repair factors that are involved in the repair of adducts and DSBs

(e.g. RAD51, BRCA1, FEN1, RPA, MSH2) (33-35). The upregulation of such factors could actually limit the amount of damage and therefore lead to less

DSBs. Alternatively, loss of RB and the subsequent failure to halt DNA replication (43) could facilitate replication across the lesion and give rise to DSBs

(50-53). Analysis of these lesions, arising both after CPT and CDDP damage, indicated that loss of RB does in fact lead to additional DSBs (Fig. 3). This occurred on a per cell basis with low dose CDDP and CPT (not shown) damage capable of eliciting significantly more breaks in RB deficient cells. Additionally, this was valid for the entire population of cultured cells, wherein RB-deficient

35 cells acquired increased amounts of γH2AX foci at a given concentration of DNA damaging agent. This effect could be explained by the ability of the cells lacking

RB to replicate despite the presence of DNA damage as we observed through

BrdU incorporation assays so that failure to inhibit replication leads to secondary lesions.

Because functional inactivation of the RB pathway is characteristic of 90% of all tumors, understanding the relationship between chemotherapeutic response and the genetic status of a tumor has high clinical relevance and is crucial for the development of effective treatments. Our data suggest that CDDP and CPT may be particularly effective in the treatment of RB-deficient neoplasms because DSBs represent a difficult lesion to repair and can fuel the subsequent cell death elicited by the agents. Loss of RB seems to synergize with the drug to make it more effective in inducing damage. This observation may indicate why certain tumors characterized by loss of RB have good response rates to chemotherapeutics and why RB-deficient cells are generally more sensitive to cell death by such agents (41,55,56). However, the generation of additional genetic lesions through the loss of RB (in this case DSBs) can also have a deleterious consequence as such lesions could fuel tumor progression. Such an idea is not without precedent as it has been reported that 30% of heritable retinoblastoma patients present with secondary primary nonocular neoplasms outside of the field of their initial radiation therapy by age 40 (57).

Our results provide a framework for elucidating the consequence of RB- loss on chemotherapeutic response. We demonstrate that acute RB loss

36 abrogates the DNA damage checkpoint in adult cells. These cells acquired increased amounts of DSBs as compared to RB-proficient cells following CDDP or CPT treatment. Lastly, the accumulation of DSBs in RB-deficient cells was shown to be replication-dependent. Thus, loss of RB gives rise to secondary genetic lesions that will impact the response to therapeutic agents.

Acknowledgements: We are grateful to Dr. Karen Knudsen and Dr. Peter

Stambrook for their helpful comments on the manuscript and all members of the

Knudsen laboratories for insightful discussions. We thank Sandy Schwemberger and Dr. George Babcock for providing their expert assistance with the flow cytometry. This work was supported by ACS grant RSG-01-254-01-CCG to ESK.

EEB is supported by National Institute of Environmental Health Sciences Training

Grant 5T32 ES 07250-16. CNM is supported by National Cancer Institute

Training Grant T32 CA 59268. TJ is an investigator of Howard Hughes Medical

Institute.

37 References

1. Hartwell, L.H. and Weinert, T.A. (1989) Checkpoints: controls that ensure

the order of cell cycle events. Science, 246, 629-634.

2. Nyberg, K.A., Michelson, R.J., Putnam, C.W. and Weinert, T.A. (2002)

Toward maintaining the genome: DNA damage and replication

checkpoints. Annu Rev Genet, 36, 617-656.

3. Elledge, S.J. (1996) Cell cycle checkpoints: preventing an identity crisis.

Science, 274, 1664-1672.

4. Bartek, J. and Lukas, J. (2001) Mammalian G1- and S-phase checkpoints

in response to DNA damage. Curr Opin Cell Biol, 13, 738-747.

5. Bartkova, J., Lukas, J. and Bartek, J. (1997) Aberrations of the G1- and

G1/S-regulating genes in human cancer. Prog Cell Cycle Res, 3, 211-220.

6. Flatt, P.M. and Pietenpol, J.A. (2000) Mechanisms of cell-cycle

checkpoints: at the crossroads of carcinogenesis and drug discovery.

Drug Metab Rev, 32, 283-305.

7. Kastan, M.B., Zhan, Q., el-Deiry, W.S., Carrier, F., Jacks, T., Walsh, W.V.,

Plunkett, B.S., Vogelstein, B. and Fornace, A.J., Jr. (1992) A mammalian

cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in

ataxia-telangiectasia. Cell, 71, 587-597.

8. Lu, X. and Lane, D.P. (1993) Differential induction of transcriptionally

active p53 following UV or ionizing radiation: defects in chromosome

instability syndromes? Cell, 75, 765-778.

38 9. Savitsky, K., Bar-Shira, A., Gilad, S., Rotman, G., Ziv, Y., Vanagaite, L.,

Tagle, D.A., Smith, S., Uziel, T., Sfez, S. et al. (1995) A single ataxia

telangiectasia gene with a product similar to PI-3 kinase. Science, 268,

1749-1753.

10. Canman, C.E., Lim, D.S., Cimprich, K.A., Taya, Y., Tamai, K., Sakaguchi,

K., Appella, E., Kastan, M.B. and Siliciano, J.D. (1998) Activation of the

ATM kinase by ionizing radiation and phosphorylation of p53. Science,

281, 1677-1679.

11. Lavin, M.F. and Shiloh, Y. (1997) The genetic defect in ataxia-

telangiectasia. Annu Rev Immunol, 15, 177-202.

12. Wang, J.Y., Knudsen, E.S. and Welch, P.J. (1994) The retinoblastoma

tumor suppressor protein. Adv Cancer Res, 64, 25-85.

13. Weinberg, R.A. (1995) The retinoblastoma protein and cell cycle control.

Cell, 81, 323-330.

14. Sherr, C.J. (1996) Cancer cell cycles. Science, 274, 1672-1677.

15. Reed, S.I. (1997) Control of the G1/S transition. Cancer Surv, 29, 7-23.

16. Bartek, J., Bartkova, J. and Lukas, J. (1997) The retinoblastoma protein

pathway in cell cycle control and cancer. Exp Cell Res, 237, 1-6.

17. Kaelin, W.G., Jr. (1997) Recent insights into the functions of the

retinoblastoma susceptibility gene product. Cancer Invest, 15, 243-254.

18. Harbour, J.W. and Dean, D.C. (2000) The Rb/E2F pathway: expanding

roles and emerging paradigms. Genes Dev, 14, 2393-2409.

39 19. Dyson, N. (1998) The regulation of E2F by pRB-family proteins. Genes

Dev, 12, 2245-2262.

20. Harrington, E.A., Bruce, J.L., Harlow, E. and Dyson, N. (1998) pRB plays

an essential role in cell cycle arrest induced by DNA damage. Proc Natl

Acad Sci U S A, 95, 11945-11950.

21. Brugarolas, J., Chandrasekaran, C., Gordon, J.I., Beach, D., Jacks, T. and

Hannon, G.J. (1995) Radiation-induced cell cycle arrest compromised by

p21 deficiency. Nature, 377, 552-557.

22. Lundberg, A.S. and Weinberg, R.A. (1998) Functional inactivation of the

retinoblastoma protein requires sequential modification by at least two

distinct cyclin-cdk complexes. Mol Cell Biol, 18, 753-761.

23. Mittnacht, S. (1998) Control of pRB phosphorylation. Curr Opin Genet

Dev, 8, 21-27.

24. Zarkowska, T. and Mittnacht, S. (1997) Differential phosphorylation of the

retinoblastoma protein by G1/S cyclin-dependent kinases. J Biol Chem,

272, 12738-12746.

25. Resnitzky, D., Hengst, L. and Reed, S.I. (1995) Cyclin A-associated

kinase activity is rate limiting for entrance into S phase and is negatively

regulated in G1 by p27Kip1. Mol Cell Biol, 15, 4347-4352.

26. Bremner, R., Cohen, B.L., Sopta, M., Hamel, P.A., Ingles, C.J., Gallie, B.L.

and Phillips, R.A. (1995) Direct transcriptional repression by pRB and its

reversal by specific cyclins. Mol Cell Biol, 15, 3256-3265.

40 27. Weintraub, S.J., Prater, C.A. and Dean, D.C. (1992) Retinoblastoma

protein switches the E2F site from positive to negative element. Nature,

358, 259-261.

28. Sellers, W.R., Rodgers, J.W. and Kaelin, W.G., Jr. (1995) A potent

transrepression domain in the retinoblastoma protein induces a cell cycle

arrest when bound to E2F sites. Proc Natl Acad Sci U S A, 92, 11544-

11548.

29. Weintraub, S.J., Chow, K.N., Luo, R.X., Zhang, S.H., He, S. and Dean,

D.C. (1995) Mechanism of active transcriptional repression by the

retinoblastoma protein. Nature, 375, 812-815.

30. Bornfeld, N., Schuler, A., Bechrakis, N., Henze, G. and Havers, W. (1997)

Preliminary results of primary chemotherapy in retinoblastoma. Klin

Padiatr, 209, 216-221.

31. Zheng, L. and Lee, W.H. (2002) Retinoblastoma tumor suppressor and

genome stability. Adv Cancer Res, 85, 13-50.

32. Yamasaki, L. (2003) Role of the RB tumor suppressor in cancer. Cancer

Treat Res, 115, 209-239.

33. Polager, S., Kalma, Y., Berkovich, E. and Ginsberg, D. (2002) E2Fs up-

regulate expression of genes involved in DNA replication, DNA repair and

mitosis. Oncogene, 21, 437-446.

34. Markey, M.P., Angus, S.P., Strobeck, M.W., Williams, S.L., Gunawardena,

R.W., Aronow, B.J. and Knudsen, E.S. (2002) Unbiased analysis of RB-

41 mediated transcriptional repression identifies novel targets and distinctions

from E2F action. Cancer Res, 62, 6587-6597.

35. Ishida, S., Huang, E., Zuzan, H., Spang, R., Leone, G., West, M. and

Nevins, J.R. (2001) Role for E2F in control of both DNA replication and

mitotic functions as revealed from DNA microarray analysis. Mol Cell Biol,

21, 4684-4699.

36. Tercero, J.A. and Diffley, J.F. (2001) Regulation of DNA replication fork

progression through damaged DNA by the Mec1/Rad53 checkpoint.

Nature, 412, 553-557.

37. Takada, S., Kelkar, A. and Theurkauf, W.E. (2003) Drosophila checkpoint

kinase 2 couples centrosome function and spindle assembly to genomic

integrity. Cell, 113, 87-99.

38. Andreassen, P.R., Lacroix, F.B., Lohez, O.D. and Margolis, R.L. (2001)

Neither p21WAF1 nor 14-3-3sigma prevents G2 progression to mitotic

catastrophe in human colon carcinoma cells after DNA damage, but

p21WAF1 induces stable G1 arrest in resulting tetraploid cells. Cancer

Res, 61, 7660-7668.

39. Arroyo, M.P. and Wang, T.S. (1998) Mutant PCNA alleles are associated

with cdc phenotypes and sensitivity to DNA damage in fission yeast. Mol

Gen Genet, 257, 505-518.

40. Sage, J., Miller, A.L., Perez-Mancera, P.A., Wysocki, J.M. and Jacks, T.

(2003) Acute mutation of retinoblastoma gene function is sufficient for cell

cycle re-entry. Nature, 424, 223-228.

42 41. Knudsen, K.E., Booth, D., Naderi, S., Sever-Chroneos, Z., Fribourg, A.F.,

Hunton, I.C., Feramisco, J.R., Wang, J.Y. and Knudsen, E.S. (2000) RB-

dependent S-phase response to DNA damage. Mol Cell Biol, 20, 7751-

7763.

42. MacPhail, S.H., Banath, J.P., Yu, Y., Chu, E. and Olive, P.L. (2003) Cell

cycle-dependent expression of phosphorylated histone H2AX: reduced

expression in unirradiated but not X-irradiated G1-phase cells. Radiat Res,

159, 759-767.

43. DeGregori, J., Kowalik, T. and Nevins, J.R. (1995) Cellular targets for

activation by the E2F1 transcription factor include DNA synthesis- and

G1/S-regulatory genes. Mol Cell Biol, 15, 4215-4224.

44. Sever-Chroneos, Z., Angus, S.P., Fribourg, A.F., Wan, H., Todorov, I.,

Knudsen, K.E. and Knudsen, E.S. (2001) Retinoblastoma tumor

suppressor protein signals through inhibition of cyclin-dependent kinase 2

activity to disrupt PCNA function in S phase. Mol Cell Biol, 21, 4032-4045.

45. Ghazal-Aswad, S., Tilby, M.J., Lind, M., Baily, N., Sinha, D.P., Calvert,

A.H. and Newell, D.R. (1999) Pharmacokinetically guided dose escalation

of carboplatin in epithelial ovarian cancer: effect on drug-plasma AUC and

peripheral blood drug-DNA adduct levels. Ann Oncol, 10, 329-334.

46. Fuertesa, M.A., Castillab, J., Alonsoa, C. and Perez, J.M. (2003) Cisplatin

biochemical mechanism of action: from cytotoxicity to induction of cell

death through interconnections between apoptotic and necrotic pathways.

Curr Med Chem, 10, 257-266.

43 47. Streltsov, S.A. (2002) Action models for the antitumor drug camptothecin:

formation of alkali-labile complex with DNA and inhibition of human DNA

topoisomerase I. J Biomol Struct Dyn, 20, 447-454.

48. Lan, Z., Sever-Chroneos, Z., Strobeck, M.W., Park, C.H., Baskaran, R.,

Edelmann, W., Leone, G. and Knudsen, E.S. (2002) DNA damage invokes

mismatch repair-dependent cyclin D1 attenuation and retinoblastoma

signaling pathways to inhibit CDK2. J Biol Chem, 277, 8372-8381.

49. Rogakou, E.P., Pilch, D.R., Orr, A.H., Ivanova, V.S. and Bonner, W.M.

(1998) DNA double-stranded breaks induce histone H2AX

phosphorylation on serine 139. J Biol Chem, 273, 5858-5868.

50. Vaisman, A., Lim, S.E., Patrick, S.M., Copeland, W.C., Hinkle, D.C.,

Turchi, J.J. and Chaney, S.G. (1999) Effect of DNA polymerases and high

mobility group protein 1 on the carrier ligand specificity for translesion

synthesis past platinum-DNA adducts. Biochemistry, 38, 11026-11039.

51. Vaisman, A. and Chaney, S.G. (2000) The efficiency and fidelity of

translesion synthesis past cisplatin and oxaliplatin GpG adducts by human

DNA polymerase beta. J Biol Chem, 275, 13017-13025.

52. Vaisman, A., Masutani, C., Hanaoka, F. and Chaney, S.G. (2000) Efficient

translesion replication past oxaliplatin and cisplatin GpG adducts by

human DNA polymerase eta. Biochemistry, 39, 4575-4580.

53. Strumberg, D., Pilon, A.A., Smith, M., Hickey, R., Malkas, L. and Pommier,

Y. (2000) Conversion of topoisomerase I cleavage complexes on the

44 leading strand of ribosomal DNA into 5'-phosphorylated DNA double-

strand breaks by replication runoff. Mol Cell Biol, 20, 3977-3987.

54. Furuta, T., Takemura, H., Liao, Z.Y., Aune, G.J., Redon, C., Sedelnikova,

O.A., Pilch, D.R., Rogakou, E.P., Celeste, A., Chen, H.T. et al. (2003)

Phosphorylation of histone H2AX and activation of Mre11, Rad50, and

Nbs1 in response to replication-dependent DNA double-strand breaks

induced by mammalian DNA topoisomerase I cleavage complexes. J Biol

Chem, 278, 20303-20312.

55. Samuelson, A.V. and Lowe, S.W. (1997) Selective induction of p53 and

chemosensitivity in RB-deficient cells by E1A mutants unable to bind the

RB-related proteins. Proc Natl Acad Sci U S A, 94, 12094-12099.

56. Nahle, Z., Polakoff, J., Davuluri, R.V., McCurrach, M.E., Jacobson, M.D.,

Narita, M., Zhang, M.Q., Lazebnik, Y., Bar-Sagi, D. and Lowe, S.W.

(2002) Direct coupling of the cell cycle and cell death machinery by E2F.

Nat Cell Biol, 4, 859-864.

57. Mohney, B.G., Robertson, D.M., Schomberg, P.J. and Hodge, D.O. (1998)

Second nonocular tumors in survivors of heritable retinoblastoma and

prior radiation therapy. Am J Ophthalmol, 126, 269-277.

45 Figure 1 Ad-GFP Ad-GFP-Cre A. B. RB Ad-GFP-Cre infected MAFs 0 48 (h) 1 2 RbLoxP/LoxP Rb exon 3 1 2 Ad-GFP Ad-GFP-Cre C. PCNA Cyclin E

Cyclin A

Tubulin

1 2

Figure 1. Infection of primary RbloxP/loxP MAFs with adenoviral Cre yields acute downregulation of RB protein and deregulation of RB repression targets. (A)

Asynchronously proliferating primary RbloxP/loxP MAFs were infected with Ad-GFP

or Ad-GFP-Cre adenovirus. RNA was isolated at 0 and 48 h post-infection and

RT-PCR was performed employing primers specific for regions flanking the loxP

sites in the murine RB gene. Recombination at the floxed RB locus is evident by

the appearance of the smaller transcript post-infection. (B) Asynchronously

proliferating primary MAFs were infected with Ad-GFP or Ad-GFP-Cre and

harvested in RIPA buffer. Protein concentrations were normalized by BioRad DC

assay. Equal amounts of protein were separated by electrophoresis and

immunoblotted with anti-RB monoclonal antibody. (C) Asynchronously

proliferating MAFs were infected with adenovirus, harvested, and protein concentrations were quantified as in Fig. 1B. The effect of acute RB loss upon

46 downstream targets was determined by immunoblotting for PCNA, Cyclin E, and

Cyclin A. Lysates were immunoblotted for β-tubulin to confirm equal loading.

Figure 2 A. MAF p3 MAF p3 16 h CDDP: 0 µM 8 µM 32 µM

300

Ad-GFP 250 Ad-GFP Pt Ad-GFP-Cre 200 150 Ad-GFP-Cre 100

Pt IntensityAverage 50

0 0 123µM 8 µM 32 µM B. 16 h CDDP treatment

1. Harvest

Treat cells 2. Wash & grow to 40 h Harvest with CDDP for 16 h

3. Wash & grow to 64 h Harvest

C. T= 16 h T= 40 h T=64 h

120 100 80 Ad-GFP 60 Ad-GFP-Cre 40

BrdU positive BrdU 20 0 percent untreated control 00uM µM 4 µM 8uM8 µM 0 0uM µM 4 µM 8uM8 µM 0 0uM µM 4 µM 8uM 8 µM CDDP treatment

D. T=16 h 120

100 Ad-GFP 80 Ad-GFP-Cre 60

40 BrdU positive positive BrdU 20

percent untreated control untreated percent 0 0uM0 µM 5uM5 µM 10 10uM µM CPT treatment

48 Figure 2. RB loss in primary adult fibroblasts impairs replication in response to

DNA damage. (A) Left Panel: Immunofluorescence using an anti-platinum antibody was performed on Ad-GFP and Ad-GFP-Cre infected RbloxP/loxP MAFs in order to confirm that the cells incorporated similar amounts of DNA-Pt adducts after 16 h CDDP treatment. Right Panel: The average pixel intensities from the anti-Pt staining shown in the left hand panel of Fig. 2A. are represented graphically to verify similar DNA-Pt adduct incorporation after CDDP treatment.

(B) The following studies incorporate the experimental format as outlined, such that asynchronously proliferating primary RbloxP/loxP MAFs infected with Ad-GFP or Ad-GFP-Cre were treated with 0, 4, and 8 µM CDDP for 16 h. The drug was washed away and a subset of the cells were allowed to proliferate to 40 and 64 h

(C) CDDP treated cells from 2B were washed of drug and pulsed with BrdU for 8 h. The proliferative fraction of treated cells was determined with respect to untreated control through immunofluorescence. (D) The adenovirus infected primary cells from 2B were treated with 0, 5, and 10 µM CPT for 16 h and the proliferative fraction of cells was determined by BrdU incorporation to show an

RB-mediated role in the dose-dependent cell cycle response to DNA damage.

49 Figure 3 MAF p3 A. 16 h CDDP: 0 µM 4 µM 8 µM

Ad-GFP γH2AX

Ad-GFP-Cre γH2AX

MAF p3

64 h CDDP: 0 µM4 µM8 µM

γH2AX Ad-GFP

γH2AX Ad-GFP-Cre

B.T=16 h T=64 h C. T=16 h T=64 h

7 70

X Ad-GFP v 60 i Ad-GFP

A 6

Ad-GFP-Cre s

o γ 5 50 Ad-GFP-Cre

t X s

a 40

4 A

e H

t 3 γ 30

n i

e v 2 20

e 1 P 10

R 0 0 0uM0 µM 4 4uM µM 8 µ 8uMM 0 0uM µM 4 4uM µM 8 8uM µM 0uM1uM4uM8uM0µM 1 µM 4 µM 8 µM 0uM1uM4uM8uM0 µM 1 µM 4 µM 8 µM CDDP treatment CDDP treatment

D. T=16 h

i 60

o 50 Ad-GFP

X 40 Ad-GFP-Cre

H2AX positive H2AX H 30

e 20

e 10

PPercent 0 0 0uMµM 5 5uM µM 10uM10 µM CPT treatment 50 Figure 3. Acute RB loss influences the accumulation of DNA double-strand breaks. (A) Top panel: Asynchronously proliferating primary RbloxP/loxP MAFs infected with Ad-GFP or Ad-GFP-Cre were treated with 0, 4, and 8 µM CDDP for

16 h. MAFs were analyzed for γH2AX foci formation by immunofluorescence using an anti-γH2AX monoclonal antibody. Bottom panel: A subset of the CDDP treated cells from 3A were washed of drug and allowed to proliferate to 64 h. The

γH2AX positive foci were analyzed as above to show the effect of RB on the levels of DNA double-strand breaks at longer periods after chemotherapeutic treatment. (B) The difference in γH2AX staining intensity between the Ad-GFP

and Ad-GFP-Cre infected MAFs treated with 0, 4, and 8 µM CDDP for 16 h from

3A was further quantified using Metamorph software to analyze average pixel intensities of each stained nucleus. This data is represented graphically as the

relative increase in γH2AX intensity. (C) In order to quantify the effect of RB loss

upon levels of DNA damage, the Ad-GFP and Ad-GFP-Cre infected MAFs from

from 3A were treated with 0, 1, 4, and 8 µM CDDP for 16 h and either fixed or

maintained in media to 64 h. These cells were then scored as being positive or

negative for γH2AX foci and the data is represented graphically. (D) Ad-GFP or

Ad-GFP-Cre infected MAFs were treated with 0, 5, and 10 µM CPT for 16 h and

immunofluorescence using an anti-γH2AX monoclonal antibody is performed. As

in 3B, the cells were scored for the presence of γH2AX positive foci and the data

was represented graphically.

51 Figure 4

A. Serum Starved Cells 50 45 40 35 Ad-GFP 30 Ad-GFP-Cre 25 20 15 10

Percent BrdU positive BrdU Percent 5 0 00uM µM 4 4uM µM 8uM8 µM CDDP treatment

Serum Starved Cells B. 70

i 60

i s Ad-GFP o 50

X 40 Ad-GFP-Cre

2 30

t 20

c 10 r

P 0 0uM0µM 4 4uMµM 8 8uM µM CDDP treatment

16 h CDDP: 0 µM 4 µM

Ad-GFP-Cre

1000 1000

100 100

10 10 H2AX intensity H2AX intensity γ γ FL1 LOG: LOG 1 FL1 LOG: FL1 LOG 1 Log Log Log Log

0.1 0.1 0 200 400 600 800 0 200 400 600 800 PIFL3: PI FL3:PI PI

52 Figure 4. Replication-dependent DSB accumulation. (A) Primary RbloxP/loxP MAFs

infected with Ad-GFP or Ad-GFP-Cre were serum starved for 3 days and treated with 0, 4, and 8 µM CDDP for 16 h. The cells were washed of drug, labeled with

BrdU for 8 h to confirm that none of the cells were able to enter S-phase. (B)

CDDP treated MAFs from 4A were analyzed for γH2AX foci formation by immunofluorescence using an anti-γH2AX monoclonal antibody. (C)

Asynchronously proliferating primary RbloxP/loxP MAFs infected with Ad-GFP-Cre

were treated with 0 and 4 µM CDDP for 16 h. The cells were harvested and

processed for flow cytometry by staining with γH2AX-FITC and propidium iodide.

These data were analyzed using ModFit software.

53 Figure 5 A.

0 µM CDDP: DAPI γH2AX BrdU Merge

Ad-GFP

Ad-GFP-Cre

B. 4 µM CDDP: Ad-GFP Ad-GFP-Cre

DAPI γH2AX BrdU Merge DAPI γH2AX BrdU Merge

C. Ad-GFP-Cre 4 µM CDDP

BrdU Threshold γH2AX Threshold

BrdU and γH2AX Threshold Colocalization

69% +/- 8% BrdU and γH2AX Colocalization (n=5) 54 Figure 5. BrdU and γH2AX foci colocalization. (A) Immunofluorescence analysis of BrdU and γH2AX foci co-labeling in untreated primary RbloxP/loxP MAFs infected

with Ad-GFP or Ad-GFP-Cre. (B) Cells from 5A were treated for 16 h with 4 µM

CDDP and immunofluorescence was used to analyze BrdU and γH2AX foci

localization. (C) Co-labeled Ad-GFP-Cre infected cells treated with CDDP from

5B were imaged by confocal micoroscopy and examined using morphometric

analysis. As a control for background staining, the pixel intensity thresholds for

both the red (BrdU) and green (γH2AX) channels in these 8-bit images were held

constant at 133 and 50, respectively. Throughout the entire nucleus, sites of

BrdU incorporation were found to colocalize with 69 +/- 8% of γH2AX foci (n=5).

55 Chapter III: Differential Role of RB in Response to UV and IR

Damage

Emily E Bosco† and Erik S Knudsen†*

†Department of Cell Biology, Vontz Center for Molecular Studies, University of

Cincinnati College of Medicine, Cincinnati, Ohio

* Corresponding author, Tel: (513) 558-8885; Fax: (513) 558-4454;

E-mail: [email protected]

Permission to reprint this work has been granted by Oxford University Press as it was published in Nucleic Acids Research, Vol 33 (5) 2005 pp 1581-1592.

56 Abstract:

The retinoblastoma tumor suppressor (RB) is functionally inactivated in the majority of cancers and is a critical mediator of DNA damage checkpoints.

Despite the critical importance of RB function in tumor suppression, the coordinate impact of RB loss on the response to environmental and therapeutic sources of damage has remained largely unexplored. Here, we utilized a conditional knockout system to ablate RB in adult fibroblasts. This model system enabled us to investigate the temporal role of RB loss on cell cycle checkpoints and DNA damage repair following ultraviolet (UV) and ionizing radiation (IR) damage. We demonstrate that RB loss compromises rapid cell cycle arrest following UV and IR exposure in adult primary cells. Detailed kinetic analysis of the checkpoint response revealed that disruption of the checkpoint is concomitant with RB target gene deregulation, and is not simply a manifestation of chronic RB loss. RB loss had a differential effect upon repair of the major DNA lesions induced by IR and UV. Whereas RB did not affect resolution of DNA double-strand breaks, RB-deficient cells exhibited accelerated repair of pyrimidine pyrimidone photoproducts. In parallel, this repair was coupled with enhanced expression of specific factors and the behavior of proliferating cell nuclear antigen (PCNA) recruitment to replication and repair foci. Thus, RB loss and target gene deregulation hastens the repair of specific lesions distinct from its ubiquitous role in checkpoint abrogation.

Introduction:

Cells have evolved complex mechanisms of genome surveillance and

DNA repair to maintain genetic stability in the face of bombardment by

exogenous insult (1-3). Cell cycle checkpoint pathways are examples of

evolutionarily conserved responses to DNA damage (4). Following recognition of

DNA lesions, such as those induced by ultraviolet radiation (UV) and ionizing radiation (IR), cell cycle checkpoints are elicited to limit the propagation of deleterious mutations to daughter cells. Several checkpoint proteins play essential roles in the maintenance of appropriate DNA damage response. A critical mediator of cell cycle control involved in the DNA damage checkpoint is the retinoblastoma tumor suppressor protein (RB). During early G1 phase of the

cell cycle, hypophosphorylated RB is active and binds to members of the E2F

transcription factor family to antagonize their function. The RB-E2F complex

forms on the promoters of a multitude of E2F target genes to repress

transcription. E2F is known to regulate many downstream targets that are involved in cell cycle progression (e.g. cyclin A, cyclin E, cdc2, and cdk2) and

DNA replication (e.g. proliferating cell nuclear antigen (PCNA), mini-chromosome maintenance-7 (MCM-7), topoisomerase IIα, thymidine kinase) (5,6). Due to the requisite nature of these target genes, RB-mediated transcriptional repression inhibits progression into S-phase. Control of RB binding to E2Fs is exerted in mid

G1 by the activation of cdk4/cyclin D1 and cdk2/cyclin E which phosphorylate and

inactivate RB thereby allowing S-phase entry (7-9). DNA damage has the

58 general influence of activating RB by promoting dephosphorylation. Following

DNA damage, the presence of RB is required for cell cycle inhibition (10-13).

This response has typically been assessed using mouse embryonic fibroblasts, wherein RB is believed to facilitate arrest by transcriptional repression of key targets. However, prior studies have been limited to analysis of the effect of chronic RB loss, rather than the acute inactivation evident in cancer.

It has been reported that RB function is impaired in the majority of cancers as the activities of several disparate mechanisms result in its functional inactivation (14-18). Presumably, RB loss contributes to genetic instability by allowing cells to evade cell cycle regulation and facilitating DNA damage checkpoint bypass. Consistent with this idea, it has been shown that RB suppresses the development of aneuploidy following damage (19). While RB is implicated in gross chromosome instability, its effect on DNA repair remains unexplored. However, a role for RB in repair has recently been suggested by the finding that several RB/E2F regulated genes are involved in the repair of UV and

IR damage (20-24). Therefore, it can be envisioned that RB loss and downstream target deregulation could have distinct effects upon the cellular response to genotoxic insult, including both checkpoint deregulation and aberrant repair.

To probe these responses, we investigated the role of RB in UV and IR damage signaling, checkpoint activation, and lesion repair in adult primary cells containing acute RB loss. Here, we report that RB function is critical for induction of a rapid cell cycle checkpoint in response to these agents. Additionally, we find

59 that the DNA damage checkpoint bypass is concomitant with RB deletion and

downstream target deregulation. Abrogation of the DNA damage checkpoint was

associated with accelerated pyrimidine pyrimidone photoproduct (6-4 PP) repair and rapid engagement of DNA damage repair factors. Taken together, our data demonstrate that RB loss facilitates abrogation of transient cell cycle arrest following environmentally and therapeutically relevant doses of UV and IR, while contributing specifically to the acceleration of UV lesion repair.

60 Materials and Methods:

Isolation of primary RbloxP/loxP murine adult fibroblasts:

Floxed Rb mice (RbloxP/loxP) of mixed 129/FVBN background (25), at least

5 weeks of age, were sacrificed by CO2 anaesthetization followed by cervical

dislocation. Fibroblasts were isolated from the peritoneal fascia by excision,

mincing of the peritoneum, and constant agitation for 40 min at 37°C in 0.2

mg/mL collagenase (Type I, Sigma) supplemented with 100 U Dnase I (Roche).

The dissociated tissue was washed with PBS and subsequently incubated for 20

min at 37°C in 0.25% trypsin (Gibco) with constant agitation. After two PBS

washes, the isolated cells were plated in tissue culture dishes.

Cell culture, recombinant adenoviral infections:

RbloxP/loxP MAFs were subcultured in DMEM containing 10% fetal bovine

serum supplemented with 100 U/mL penicillin/streptomycin and 2 mM L-

glutamine at 37°C in air containing 5% CO2. In this study, all primary cells were

between passages 2 and 4. Replication defective recombinant adenovirus

expressing green fluorescent protein (Ad-GFP) or GFP in addition to Cre

recombinase (Ad-GFP-Cre) were obtained from G. Leone (Department of

Molecular Genetics, Ohio State University). The conditional RB knockout in

primary RbloxP/loxP MAFs was attained by infecting cells with adenovirus at

approximately 2x107 virus particles per dish to achieve an infection efficiency of

90-95% as determined by GFP immunofluorescence. Cells were cultured for at

least 4 days post-adenoviral infection prior to use while the passage number and

61 length of time post-infection remained consistent throughout all experiments

unless otherwise stated.

Immunoblotting:

Cells infected with Ad-GFP or Ad-GFP-Cre were harvested by

trypsinization and lysed in RIPA buffer. Equal amounts of protein, as determined

by Bio-RAD DC assay, were resolved by SDS-PAGE. Specific proteins were

detected by standard immunoblotting procedures using the following primary

antibodies: (Santa Cruz, 1:500 dilution) PCNA (pc10), Cyclin E (HE12), Cyclin A

(C-19), MCM-7 (141.2), Cyclin B1 (GNS1), Lamin B (sc-6217), anti-RB (G3-245,

Becton Dickson, 1:100 dilution), total p53 Ab-3 (Oncogene OP29, 1:250 dilution) and phospho-p53 ser18 (Cell Signaling 9284S, 1:500 dilution).

RT-PCR analysis of recombination:

RT-PCR analysis was performed to verify adenoviral-Cre-mediated

recombination in primary MAFs. Total RNA was extracted using Trizol (Gibco)

and cDNA was synthesized from 1µg of RNA with the SuperScript RT-PCR

system (Gibco) according to the manufacturers protocol. cDNAs were amplified

using PCR and the following primers: (sense) 5’-CCTTGAACCTGCTTGTCCTC -

3’ and (antisense) 5’-GAAGGCGTGCACAGAGTGTA -3’. PCR conditions

consisted of initial denaturation for 2 min at 94°C, followed by 30 cycles of 30 s at

94°C, 30 s at 52°C and 1 min at 72°C, followed by a final extension for 5 min at

62 72°C. 10 µl of PCR product was run on a 2% agarose gel and visualized by

ethidium bromide staining.

DNA damage, PCNA extraction, and immunofluorescence:

Primary MAFs infected with Ad-GFP or Ad-GFP-Cre were seeded on coverslips in 6-well dishes and allowed to attach. Cells were treated at room temperature either with ionizing radiation through exposure to 137Cs (dose rate:

0.67 Gy/min) in tissue culture media or with ultraviolet irradiation (UVC) (low

pressure mercury lamp; Mineralight lamp model UVG-11;UVP, Inc. San Gabriel,

CA) following removal of DMEM and washing the cells twice with PBS. Treated

cells were labeled with BrdU (Amersham Pharmacia Biotech) to detect DNA

synthesis, then washed, fixed in 3.7% formaldehyde, and processed to detect

and quantitate BrdU incorporation by immunofluorescence and cell scoring as

previously described (26). All BrdU results are expressed as a percentage of

untreated control cells set to 100%. PCNA extraction and immunofluorescence

was performed as previously described (27) using the monoclonal pc10 PCNA

antibody (Santa Cruz). γH2AX immunofluorescence was performed as previously

described (28) using an anti-phospho-H2AX ser 139 mouse primary antibody

(Upstate Biotechnology). CPD and 6-4 photoproduct (6-4 PP) staining was

performed as described in Q.-e. Wang et al (29) using antibodies generously

contributed by Dr. Tsukasa Matsunaga (Kanazawa University, Japan). Relative

staining intensities of γH2AX, CPD lesions, and 6-4 PP were quantified by

capturing images of equal exposure using microscopy and performing

63 densitomery using Metamorph. All data are from 10 nuclei captured on random

fields.

Immunoassay for repair of CPD and 6-4 PP:

CPD and 6-4 PP present in cellular DNA were detected and quantified by

slot blot. Total genomic DNA was extracted using the DNEasy tissue kit (Qiagen)

according to manufacturers instructions. DNA was quantified through

spectrophotometry and gel electrophoresis prior to denaturation through boiling

and sonication. Increasing concentrations of DNA were loaded for slot blot transfer using a vacuum blotter and hybridization onto a Nytran membrane. The membrane was blocked in 10% milk / 1x saline-tween and probed with either

1:1000 primary monoclonal antibody specific for CPD or 6-4 PP DNA lesions (T.

Matsunaga). Filters were then processed according to standard Western blot protocol and lesion abundance was quantified using densitometry following the subtraction of background. To control for equal loading, experiments were done in triplicate and percent reduction was calculated between equal DNA concentrations from different time points within the same cell type.

64 Results:

Acute downregulation of RB protein abrogates the DNA damage

checkpoint response to UV and IR

Studies of RB function have historically utilized mouse embryo fibroblasts

(MEFs) harboring loss of RB throughout development or extensively cultured tumor lines. There is a caveat in these models, in that RB loss is compensated

by RB related pocket proteins (i.e. p107 and p130) during development (30,31).

By contrast, RB is acutely lost in the majority of cancer cases (14-18). Thus, we

utilized a conditional knockout system involving mice harboring a conditional Rb

allele in which loxP sites flank Rb exon 19 (RbloxP/loxP mice)(25). Through

adenoviral expression of Cre recombinase, acute RB loss can be achieved in

genetically stable murine adult fibroblasts (MAFs). To examine the action of RB

in cells, MAFs were initially infected with recombinant adenoviruses expressing

both GFP and Cre recombinase (Ad-GFP-Cre) or GFP alone (Ad-GFP) as a

control. Efficient infection of cells was evident following 16-24 hours, as greater

than 90% of Ad-GFP-Cre infected cells demonstrated high levels of GFP

fluorescence (data not shown). Confirmation of Cre-mediated recombination was

performed by RT-PCR analysis using primers in exons 18 and 20 of the Rb gene.

RNA was prepared from uninfected MAFs or those infected with Ad-GFP-Cre at

72 hours post-infection. RT-PCR analysis revealed loss of Rb RNA and

accumulation of the ∆exon19 transcript in the infected cells relative to the control

(Fig. 1A). Immunoblotting with anti-RB monoclonal antibody revealed that the

65 Cre-mediated recombination resulted in acute downregulation of RB protein in

Ad-GFP-Cre infected MAFs (Fig. 1B).

To delineate the consequence of conditional RB ablation on the RB/E2F signaling axis, MAFs infected with either Ad-GFP or Ad-GFP-Cre were harvested five days post-infection and levels of specific RB target proteins were analyzed by immunoblot. Relative to control (Fig. 1C, lane 1), the Ad-GFP-Cre infected

MAFs exhibited increased levels of proteins downstream of RB signaling including, PCNA, cyclin E, and cyclin A (lane 2). No changes were detected in lamin B protein levels, which served as a loading control. Therefore, RB deletion in primary adult cells results in target gene deregulation.

To evaluate the role of RB in the DNA damage response of adult fibroblasts, asynchronously proliferating Ad-GFP or Ad-GFP-Cre infected MAFs were exposed to 0, 10, or 20 J/m2 UV and subsequently cultured for 10 hours to

elicit the checkpoint response. Cells were pulse labeled with bromodeoxyuridine

(BrdU) for 2 hours and the replicative fraction of treated cells was determined by

immunofluorescent detection of BrdU incorporation. MAFs containing functional

RB exhibited a dose-dependent cell cycle inhibition (relative to untreated control), whereas cells lacking RB exhibited minimal responses at each dose (Fig. 1D, top panel). Similar results were evident when the response to therapeutic doses of

IR was investigated in the same manner. Following exposure to 0, 2.5, or 5 Gy

IR, Ad-GFP infected MAFs exhibited a robust dose dependent inhibition of cell cycle in which BrdU incorporation was reduced greater than 75%, while Ad-GFP-

Cre infected cells were largely unaffected (Fig. 1D, bottom panel). Taken

66 together, these data demonstrate that acute deletion of RB in primary adult cells results in abrogation of the DNA damage checkpoint response to both IR and UV irradiation.

RB loss compromises the rapid checkpoint response to IR/ UV

Traditionally, RB has been characterized as participating in checkpoint responses with delayed kinetics. In part, this is due to the use of chemotherapeutic agents wherein the induction of DNA-damage is delayed due

to drug action (32-34). Thus, one of the advantages of studying the cellular

response to UV or IR is the immediate induction of DNA damage. To understand

the kinetics of cell cycle attenuation following exposure to UV and IR, Ad-GFP

and Ad-GFP-Cre infected MAFs were exposed to either 0, 10, or 20 J/m2 UV or

0, 2.5, or 5 Gy IR and cultured. Following damage, cells were pulsed with BrdU

for the final 2 hours in culture prior to harvesting at 2, 4, and 6 hours. The DNA

damage checkpoint was evident as early as 2 hours following exposure to either

UV or IR as determined by immunofluorescent detection of BrdU incorporation

(Fig. 2A). Additionally, this response was maintained for at least 6 hours following damage. Surprisingly, this rapid response to UV and IR damage was compromised in RB deficient cells, as Ad-GFP-Cre infected MAFs largely bypassed cell cycle inhibition at each time point. These data indicate that RB loss is sufficient to bypass the rapid checkpoint response to DNA damage.

Since it is postulated that RB signals via repression of target genes, we investigated the rapid action of DNA damage on RB target genes. In response to

67 IR or UV damage, ser18 of p53 (homologous to human ser15) is known to be

rapidly phosphorylated by ATM family kinases (35). Therefore, as a control for

upstream signaling, immunoblotting for total p53 and phospho-p53 ser18 was

performed. To analyze rapid DNA damage signaling, MAFs infected with either

Ad-GFP or Ad-GFP-Cre were treated with 10 J/m2 UV or 2.5 Gy IR and

harvested 2 hours following treatment and analyzed for total p53 expression by

immunoblot (Fig. 2B, top panel). Lamin B serves as a control for equal loading.

As expected, both cultures exhibited similar inductions of p53 following either UV

or IR damage. To further probe the induction kinetics of p53, phospho-p53

expression was analyzed at 0, 1, 3, and 5 hours post treatment (Fig. 2B, bottom

panel). Not surprisingly, both cultures exhibited relatively equal kinetics of phospho-p53 induction (10,13). However, phospho-p53 induction was slightly

faster in response to IR than UV and its response to UV persisted longer than

that from IR exposure. To subsequently characterize expression levels of

downstream RB targets, Ad-GFP and Ad-GFP-Cre infected MAFs were analyzed

at 0, 3, and 5 hours following 10 J/m2 UV (Fig. 2C, top panel) or 2.5 Gy IR (Fig.

2C, bottom panel) exposure. Interestingly, expression levels of PCNA, MCM-7,

and cyclin A remained relatively constant during the rapid response to UV and IR indicating that RB action did not affect levels of these downstream targets during checkpoint induction. Equal loading was verified by lamin B immunoblot. These data argue that the rapid function of RB in cell cycle arrest following DNA damage does not apparently involve attenuation of target genes.

68 RB loss is directly coupled with target gene deregulation to promote

abrogation of the DNA damage checkpoint response

Although it is understood that RB function is necessary for proper

regulation of downstream targets and the DNA damage checkpoint response, the

kinetic ordering of these events has not been established. Specifically, the data

shown above suggests that while RB may not actively cause the checkpoint, RB

loss could enable checkpoint bypass through the accumulation of RB target gene

products. To determine whether loss of RB protein or target gene deregulation is

more closely coupled to loss of checkpoint function, we examined the discrete kinetics of this pathway. Asynchronous MAFs were infected (T=0h) and harvested for immunoblot every 12 hours for 48 hours. Immunoblot analysis revealed complete loss of RB protein by 24 hours post Ad-GFP-Cre infection

(Fig. 3A). Analysis of RB target genes showed that the expression of several downstream targets including, MCM-7, PCNA, cyclin B1, and cyclin E, all became deregulated concurrent with RB loss.

Since disruption of checkpoint function could simply be a manifestation of chronic RB loss, we examined the nature of checkpoint function throughout the

RB knockout time course. In parallel with the previously outlined experiments,

Ad-GFP-Cre infected MAFs were treated with UV or IR every 12 hours post- infection for 96 hours. Following damage, the cells were pulsed with BrdU for 4 hours and harvested for checkpoint analysis (Fig. 3B). Scoring of the populations of DNA damaged cells throughout the time course of RB knockout revealed that

DNA damage checkpoint function remained intact in response to 0, 10, or 20

69 J/m2 UV irradiation through 28 hours post-infection. However, by 40 hours post-

infection the checkpoint response became impaired (Fig. 3C, top panel),

concurrent with maximal deregulation of target genes following RB protein loss

(Fig 3A). Similarly, the equivalent experimental setup was employed to

investigate checkpoint function in response to IR damage signaling. Abrogation of proper checkpoint function occurred slightly more rapidly in response to 0, 2.5, or 5 Gy IR, such that cells were able incorporate BrdU in the presence of DNA damage by 28 hours post-infection (Fig. 3C, bottom panel). Together, these data reveal that the kinetics of RB loss are concomitant with target gene deregulation and impaired DNA damage checkpoint response. Therefore, the closely coupled dynamics of these events suggest that RB functional inactivation coincident with downstream target deregulation is required for the loss of proper cell cycle arrest in response to DNA damage.

Acute RB loss differentially influences UV- and IR-induced DNA lesion removal

Checkpoint responses are by definition reversible, presumably due to

DNA damage repair (36). Consistent with this notion, Ad-GFP infected cells were able to recover from the UV and IR induced checkpoints and re-enter the cell cycle. As before, following 0, 2.5, or 5 Gy IR, Ad-GFP infected cells were labeled with BrdU for the final 4 hours prior to harvest at 4 or 24 hours post-damage. The results demonstrated a significant increase in BrdU incorporation compared to untreated controls (set to 100%) in RB-proficient MAFs 24 hours following damage, suggesting that the doses of IR used in these experiments are

70 repairable (Fig. 4A). Since IR directly elicits DNA double-strand breaks, we next

investigated the influence of RB loss upon the accumulation and relative repair of

these lesions. Here, we employed immunofluorescence with antibodies

recognizing γH2AX, an efficient measure of double-strand break accumulation, to

demonstrate the extent to which these lesions are repaired in the 24 hours

following damage (Fig. 4B, top panel). Interestingly, RB loss had no significant

effects upon γH2AX foci intensity among images of the cell population taken at

equal exposures following IR damage. Both Ad-GFP and Ad-GFP-Cre infected

MAFs displayed a similar increase in staining intensity during the first 5 hours

post-damage and a similar kinetic decrease in intensity from hours 5 to 24 (Fig.

4B, bottom panel).

In order to determine the corresponding impact of UV repair on the cell

cycle, the ability of the RB-proficient cells to recover from the checkpoint and re-

enter cell cycle was investigated. Ad-GFP infected MAFs were treated with 0, 10,

20 J/m2 UV and propagated in culture while being pulse labeled with BrdU prior

to harvest at 4 and 24 hours. Detection of BrdU incorporation revealed the ability

of RB-proficient cells to significantly recover from the UV-induced DNA damage

checkpoint and resume cell cycle progression. The percentage of BrdU positive cells significantly increased from 4 to 24 hours following each dose of UV damage as compared to untreated controls (set to 100%) (Fig. 4C). To monitor the induction of UV lesions in the single-cell, the abundance of 6-4 PPs was

examined by immunofluorescence immediately following damage (T=0h) and

after 24 hours of recovery (T=24h) (Fig. 4D). These lesions are clearly induced in

71 MAFs by 10 J/m2 UV at T=0h and are largely resolved by 24 hours. To more

closely examine the kinetics of 6-4 PP repair, Ad-GFP and Ad-GFP-Cre infected

cells were treated with 0, 10, or 20 J/m2 UV and harvested for 6-4 PP analysis at

0, 2, 5, and 24 hours following damage. The average pixel intensities of images taken at equal exposures were compared using Metamorph software and displayed graphically to reveal that RB-deficient cells exhibit a kinetic difference in the loss of 6-4 PP staining intensities, as compared to RB-proficient cells (Fig.

4E, top panel). To ensure that acceleration of 6-4 PP resolution was not due to infection with Ad-GFP-Cre, wild type MAF control cells retaining Rb were cultured

and infected with Ad-GFP or Ad-GFP-Cre as before. Wild type cells were treated

with UV and monitored for 6-4PP repair as in the top panel of Fig. 4E, to reveal

that it is indeed RB loss which enhances 6-4 PP repair rather than Ad-GFP-Cre

infection (Fig. 4E, bottom panel). Together, these data indicate that while RB loss

plays no apparent role in the repair of IR lesions (e.g. DNA double-strand breaks), the loss of RB accelerates the repair of UV-induced 6-4 PPs.

Acute RB loss accelerates UV-induced DNA damage repair

Since the role of RB in UV-induced damage repair has been largely unexplored, we dissected the consequence of RB loss on cyclobutane pyrimidine dimmers (CPD) and 6-4 PP repair kinetics in greater detail using a more quantitative analysis. Ad-GFP and Ad-GFP-Cre infected MAFs exposed to 0 or

10 J/m2 UV were harvested at 0, 5, and 10 hours post-UV treatment and lysates

were used to purify genomic DNA. Increasing amounts of DNA were spotted onto

Nytran membranes and immunoblotted for the abundance of both CPD and 6-4

72 PP lesions. In confirmation with other studies, we reveal that MAFs are

compromised for CPD repair (37-40) and RB had no apparent effect upon lesion

repair during the time course examined (Fig 5A, top panel). However, the murine

system has competent 6-4 PP repair pathways (41). Clearly, 6-4 PP repair is

functional in both RB-proficient and RB-deficient MAFs. However, Ad-GFP-Cre

infected MAFs exhibited a greater reduction in 6-4 PP lesions (~68%) as

compared to cells infected with Ad-GFP control (~25%) at 5 and 10 hours post- treatment (Fig 5A, bottom panel), indicating that RB loss contributes to the

increased kinetics of UV damage-induced lesion repair. Despite the early

differential in repair kinetics, both RB-proficient and -deficient MAFs

demonstrated largely complete repair of 6-4 PPs induced by 10 J/m2 UV by 24

hours (Fig. 5B). Thus, RB loss accelerates 6-4 PP repair kinetics.

RB modifies repair factor dynamics

There are two possible explanations for the differential 6-4 PP repair

kinetics among RB-proficient and deficient cells. First, RB has recently been implicated in the negative regulation of a wide array of repair factors (e.g. PCNA,

RAD50, RAD51, MLH1, MSH2, and FEN1) (21-24), thus it is possible that the

elevated levels of these factors in RB-deficient cells may allow for accelerated

repair of DNA damage. Second, loss of RB-dependent DNA damage checkpoint

function and resulting ongoing replication in RB- deficient cells may more quickly

initiate repair factor engagement with the DNA lesion, thereby enhancing repair.

73 In order to examine these possibilities, we investigated the role of RB loss on the kinetics of PCNA engagement with chromatin following UV damage.

PCNA is an interesting repair factor, not only because it is regulated by RB, but also because it performs dual activities upon recruitment to chromatin in DNA replication and DNA repair (42-44). Therefore, to dissect the significance of

PCNA involvement in UV damage repair, chromatin extractions were performed on Ad-GFP and Ad-GFP-Cre infected MAFs at 0, 3, and 5 hours following UV treatment. Next, PCNA immunofluorescence was used to determine the percent of cells in the population that exhibited chromatin tethered PCNA with respect to unextracted controls. RB-proficient cell populations exhibited a slight decrease in

PCNA chromatin tethering from 0 to 3 hours followed by a marked increase by 5 hours following damage (Fig. 6A). In contrast, PCNA engagement with chromatin was accelerated in the RB-deficient populations, peaking at 3 hours and becoming similar to Ad-GFP infected controls by 5 hours post-damage. In confirmation with previous studies, these results reveal an increase in PCNA tethering following UV exposure, indicating the involvement of PCNA in nucleotide excision repair. In addition, this involvement in repair is accelerated in

RB-deficient cells. These experiments also revealed differential PCNA chromatin tethering patterns within the nuclei of cells following UV damage which indicated that a shift in PCNA function was occurring during UV damage repair (Fig. 6B).

Approximately 90 percent of tethered PCNA staining appeared in large foci in undamaged cells, indicative of replication foci (Fig. 6C). However, 3 hours after exposure to 10 J/m2 UV, nearly 100 percent of both RB-proficient and -deficient

74 populations of cells exhibited diffuse small punctate patterns of PCNA tethering within the nucleus. The global nature of this PCNA staining pattern, suggests that

PCNA function has shifted to repair foci at this time point. Strikingly, 5 hours post-UV damage, nearly 100 percent of Ad-GFP infected cells continue to exhibit diffuse punctate PCNA chromatin tethering while more than 75 percent of Ad-

GFP-Cre infected cells demonstrate localized focal patterning, as observed in the undamaged state. These data indicate that the RB-deficient cells are able to initiate UV-induced damage repair with enhanced kinetics and that in these cells the PCNA distribution more rapidly shifted its association with repair foci to replication foci following damage.

75 Discussion:

Consistent with previous studies in MEFs, here we observed impairment of the DNA damage checkpoint and deregulated cell cycle progression following

IR and UV damage in adult primary cells harboring acute RB loss (Fig 1)(10,26).

Several models have been proposed which aim to describe how RB could be

functioning to inhibit cell cycle progression following DNA damage. One model

places RB in direct contact with replication machinery to inhibit replication (45-

52), while another suggests that RB inhibits replication indirectly, via repression

of downstream targets (31). Kinetic analysis of the DNA damage response in

MAFs allowed us to probe the nature of RB function in checkpoint activation. Our

data indicates that downstream targets such as PCNA, cyclin A, and MCM-7 are

not repressed by RB during the induction of the rapid cell cycle checkpoint (Fig

2). This data a priori supports the first model, wherein RB acts directly to inhibit

replication and arrest cell cycle following recognition of DNA damage. However, it

is equally possible that the vast target gene deregulation which occurs

concomitant with RB loss facilitates checkpoint bypass. In an attempt to

differentiate these possibilities, we closely examined the kinetics of RB deletion

and DNA damage response in adult cells. Results presented indicate that RB

loss is intimately coupled with target deregulation, together facilitating checkpoint

abrogation (Fig 3). Thus, either model of RB function in checkpoint induction

could be appropriate, in that RB loss prevents its direct action in replication

inhibition while concurrently disrupting its function in control of downstream

transcriptional targets.

76 The ability of cells to recognize damaged DNA and elicit cell cycle checkpoints following genotoxic insult depends upon complex signaling pathways. Although many of the downstream pathway components in the G1/S

checkpoint are involved in signaling from both UV and IR induced lesions, many

of the initial upstream components vary. In the case of IR-induced DNA double-

strand break signaling, ATM kinase activity is immediately stimulated to

phosphorylate a number of downstream effectors including histone H2AX, p53,

and chk2 (35,53-57). Similarly, ATM and the rad 3 related (ATR) protein senses

UV damage and participates in signal transduction via phosphorylation of many

of the same effectors as ATM, such as chk2 and p53 (58). Our studies reveal

that rapid phosphorylation of H2AX and p53 following IR and UV (p53 only) are

not compromised by acute RB loss, despite the observed impairment of DNA damage checkpoint function (Fig. 2). As such, these results are consistent with literature that places RB downstream of H2AX and p53 phosphorylation in the

DNA damage signaling and repair pathway.

As the function of DNA damage signaling effectors were unaffected by RB loss, we sought to understand the consequence of the differential DNA damage checkpoint function on the cellular response to DNA damage. Because control cells exhibiting a functional checkpoint were able to resume cell cycle progression 24 hours following damage, we assumed that these cells must have been able to repair a significant portion of the DNA lesions. Thus, we sought to monitor the actual induction and removal of these lesions imparted by IR and UV damage. Although monitoring the reduction in γH2AX to analyze DNA double-

77 strand break repair following IR damage is rather qualitative, our studies revealed

that RB did not play a dramatic role in IR induced damage repair (Fig. 4). This may suggest that participants in the DNA double-strand break repair pathway are upstream of the RB/E2F axis or that those repair factors deregulated via RB loss are not rate-limiting for repair. Next, we explored the role of RB in repair of UV- induced 6-4 PPs. Our investigations revealed that RB loss significantly accelerated removal of 6-4 PPs during the first 10 hours following exposure to

UV. However, by 24 hours post-damage, the RB-proficient cells exhibited equal levels of lesion removal with the RB-deficient cells (Fig. 4&5). This is the first evidence indicating that RB inactivation modifies DNA damage repair. The underlying mechanisms for this change could be multiple. First, recent evidence

suggests that RB regulates the expression of several DNA damage repair factors

involved in UV damage repair processes including: FEN1, XPC, RPA2-3, RFC4,

and PCNA (20-24). As we found that the rate of repair of UV-induced damage

was modified by RB loss, we investigated its effect upon the function of an RB-

regulated UV damage repair factor, PCNA. We presume that high basal levels of

proteins such as PCNA enable the RB-deficient cells to complete repair

processes more quickly, as we found that PCNA protein expression rapidly

shifted from diffuse punctate patterning (indicative of its function in repair) to focal

expression (evident in normal replication) (Fig 6). Therefore, the observed UV

repair factor dynamics are consistent with the kinetics of 6-4 PP lesion removal

for each cell type. Second, the loss of cell cycle arrest following damage may

initiate faster repair, modifying the conventional view of checkpoint function being

78 necessary for efficient lesion resolution. Traditionally, DNA damage checkpoints

are viewed as providing necessary time for recruitment of repair or apoptotic

factors. However, due to the fact that RB-deficient cells demonstrate elevated

basal levels of a variety of repair factors, recruitment time may inevitably be

shortened, thereby eliminating the necessity for cell cycle arrest.

In summary, the data presented indicate that RB participates in the

response to UV and IR damage signaling in a differential manner. Although RB

loss abrogates the checkpoint in response to both forms of DNA damage, the

consequence of this loss in each instance differs. Following IR, the loss of

checkpoint function did not affect lesion repair. This suggests that RB-deficient

cells were replicating in the presence of DNA damage for a prolonged period of time, possibly inducing secondary lesions which contribute to genomic instability and activation of apoptotic pathways. However, following UV, RB-deficient cells rapidly repaired 6-4 PPs, potentially limiting the accumulation of detrimental replication-mediated lesions. Although the consequences of checkpoint bypass have not been fully elucidated, there exist several possible influences of the observed rapid repair of UV lesions in RB-deficient cells. First, accelerated 6-4

PP repair kinetics may facilitate secondary lesion development, which would explain the clinical observations that RB-deficient tumor cells are more sensitive to death upon challenge (59,60) and that hereditary retinoblastoma survivors are at an increased risk for melanoma (61,62). Second, loss of cell cycle arrest coupled with rapid 6-4 PP lesion repair may prevent proper activation of apoptotic cascades in cells harboring other UV-induced lesions (e.g. CPD),

79 facilitating the propagation of mutations. Because all cells are assaulted by

damaging environmental signals and a high proportion of RB-deficient cancers

are treated with DNA damaging therapy, understanding the effect of RB

inactivation on the response to DNA damage will enhance our perceptions of

tumorigenesis and cancer therapeutics. Our results provide the framework for

understanding the critical role of RB in the DNA damage response. These data

indicate that RB is required for the rapid induction of cell cycle arrest following

recognition of both UV and IR damage in adult primary cells. Additionally, RB

loss in these cells is closely coupled with target gene deregulation and

contributes to abrogation of checkpoint function. Lastly, we demonstrate that

although damage signaling remains unaffected, RB loss accelerates UV lesion repair and modifies repair factor dynamics.

Acknowledgements: We are grateful to Drs. Karen Knudsen and Christopher

Mayhew for their helpful comments on the manuscript and all members of the

Knudsen laboratories for insightful discussions. This work was supported by

NIEHS Core grant E30-ES-06096 and ACS grant RSG-01-254-01-CCG to ESK.

EEB is supported by DOD BCRP Grant BC030315 and the Albert J. Ryan

Foundation.

80 References:

1. Hartwell, L. (1992) Defects in a cell cycle checkpoint may be responsible

for the genomic instability of cancer cells. Cell, 71, 543-546.

2. Friedberg, E.C. (1995) Out of the shadows and into the light: the

emergence of DNA repair. Trends Biochem Sci, 20, 381.

3. Hoeijmakers, J.H. (2001) Genome maintenance mechanisms for

preventing cancer. Nature, 411, 366-374.

4. Hartwell, L.H. and Weinert, T.A. (1989) Checkpoints: controls that ensure

the order of cell cycle events. Science, 246, 629-634.

5. Chellappan, S.P., Hiebert, S., Mudryj, M., Horowitz, J.M. and Nevins, J.R.

(1991) The E2F transcription factor is a cellular target for the RB protein.

Cell, 65, 1053-1061.

6. Stevaux, O. and Dyson, N.J. (2002) A revised picture of the E2F

transcriptional network and RB function. Curr Opin Cell Biol, 14, 684-691.

7. Sherr, C.J. (1996) Cancer cell cycles. Science, 274, 1672-1677.

8. Harbour, J.W., Luo, R.X., Dei Santi, A., Postigo, A.A. and Dean, D.C.

(1999) Cdk phosphorylation triggers sequential intramolecular interactions

that progressively block Rb functions as cells move through G1. Cell, 98,

859-869.

9. Bartek, J., Bartkova, J. and Lukas, J. (1997) The retinoblastoma protein

pathway in cell cycle control and cancer. Exp Cell Res, 237, 1-6.

81 10. Harrington, E.A., Bruce, J.L., Harlow, E. and Dyson, N. (1998) pRB plays

an essential role in cell cycle arrest induced by DNA damage. Proc Natl

Acad Sci U S A, 95, 11945-11950.

11. Knudsen, E.S., Buckmaster, C., Chen, T.T., Feramisco, J.R. and Wang,

J.Y. (1998) Inhibition of DNA synthesis by RB: effects on G1/S transition

and S-phase progression. Genes Dev, 12, 2278-2292.

12. Herrera, R.E., Sah, V.P., Williams, B.O., Makela, T.P., Weinberg, R.A. and

Jacks, T. (1996) Altered cell cycle kinetics, gene expression, and G1

restriction point regulation in Rb-deficient fibroblasts. Mol Cell Biol, 16,

2402-2407.

13. Mayhew, C.N., Perkin, L.M., Zhang, X., Sage, J., Jacks, T. and Knudsen,

E.S. (2004) Discrete signaling pathways participate in RB-dependent

responses to chemotherapeutic agents. Oncogene, 23, 4107-4120.

14. Yamasaki, L. (2003) Role of the RB tumor suppressor in cancer. Cancer

Treat Res, 115, 209-239.

15. Harbour, J.W., Lai, S.L., Whang-Peng, J., Gazdar, A.F., Minna, J.D. and

Kaye, F.J. (1988) Abnormalities in structure and expression of the human

retinoblastoma gene in SCLC. Science, 241, 353-357.

16. Lee, W.H., Bookstein, R. and Lee, E.Y. (1988) Studies on the human

retinoblastoma susceptibility gene. J Cell Biochem, 38, 213-227.

17. Knudson, A., Jr. (1993) Genetics of tumors of the head and neck. Arch

Otolaryngol Head Neck Surg, 119, 735-737.

82 18. Bookstein, R. (1994) Tumor suppressor genes in prostatic oncogenesis. J

Cell Biochem Suppl, 19, 217-223.

19. Lan, Z., Sever-Chroneos, Z., Strobeck, M.W., Park, C.H., Baskaran, R.,

Edelmann, W., Leone, G. and Knudsen, E.S. (2002) DNA damage invokes

mismatch repair-dependent cyclin D1 attenuation and retinoblastoma

signaling pathways to inhibit CDK2. J Biol Chem, 277, 8372-8381.

20. Cam, H., Balciunaite, E., Blais, A., Spektor, A., Scarpulla, R.C., Young, R.,

Kluger, Y. and Dynlacht, B.D. (2004) A common set of gene regulatory

networks links metabolism and growth inhibition. Mol Cell, 16, 399-411.

21. Ishida, S., Huang, E., Zuzan, H., Spang, R., Leone, G., West, M. and

Nevins, J.R. (2001) Role for E2F in control of both DNA replication and

mitotic functions as revealed from DNA microarray analysis. Mol Cell Biol,

21, 4684-4699.

22. Polager, S., Kalma, Y., Berkovich, E. and Ginsberg, D. (2002) E2Fs up-

regulate expression of genes involved in DNA replication, DNA repair and

mitosis. Oncogene, 21, 437-446.

23. Markey, M.P., Angus, S.P., Strobeck, M.W., Williams, S.L., Gunawardena,

R.W., Aronow, B.J. and Knudsen, E.S. (2002) Unbiased analysis of RB-

mediated transcriptional repression identifies novel targets and distinctions

from E2F action. Cancer Res, 62, 6587-6597.

24. Classon, M. and Harlow, E. (2002) The retinoblastoma tumour suppressor

in development and cancer. Nat Rev Cancer, 2, 910-917.

83 25. Marino, S., Vooijs, M., van Der Gulden, H., Jonkers, J. and Berns, A.

(2000) Induction of medulloblastomas in p53-null mutant mice by somatic

inactivation of Rb in the external granular layer cells of the cerebellum.

Genes Dev, 14, 994-1004.

26. Knudsen, K.E., Booth, D., Naderi, S., Sever-Chroneos, Z., Fribourg, A.F.,

Hunton, I.C., Feramisco, J.R., Wang, J.Y. and Knudsen, E.S. (2000) RB-

dependent S-phase response to DNA damage. Mol Cell Biol, 20, 7751-

7763.

27. Sever-Chroneos, Z., Angus, S.P., Fribourg, A.F., Wan, H., Todorov, I.,

Knudsen, K.E. and Knudsen, E.S. (2001) Retinoblastoma tumor

suppressor protein signals through inhibition of cyclin-dependent kinase 2

activity to disrupt PCNA function in S phase. Mol Cell Biol, 21, 4032-4045.

28. Paull, T.T., Rogakou, E.P., Yamazaki, V., Kirchgessner, C.U., Gellert, M.

and Bonner, W.M. (2000) A critical role for histone H2AX in recruitment of

repair factors to nuclear foci after DNA damage. Curr Biol, 10, 886-895.

29. Wang, Q.E., Zhu, Q., Wani, M.A., Wani, G., Chen, J. and Wani, A.A.

(2003) Tumor suppressor p53 dependent recruitment of nucleotide

excision repair factors XPC and TFIIH to DNA damage. DNA Repair

(Amst), 2, 483-499.

30. Sage, J., Miller, A.L., Perez-Mancera, P.A., Wysocki, J.M. and Jacks, T.

(2003) Acute mutation of retinoblastoma gene function is sufficient for cell

cycle re-entry. Nature, 424, 223-228.

84 31. Dyson, N. (1998) The regulation of E2F by pRB-family proteins. Genes

Dev, 12, 2245-2262.

32. Siddik, Z.H. (2003) Cisplatin: mode of cytotoxic action and molecular basis

of resistance. Oncogene, 22, 7265-7279.

33. Van Triest, B., Pinedo, H.M., Giaccone, G. and Peters, G.J. (2000)

Downstream molecular determinants of response to 5-fluorouracil and

antifolate thymidylate synthase inhibitors. Ann Oncol, 11, 385-391.

34. Lawrence, T.S., Blackstock, A.W. and McGinn, C. (2003) The mechanism

of action of radiosensitization of conventional chemotherapeutic agents.

Semin Radiat Oncol, 13, 13-21.

35. Chao, C., Saito, S., Kang, J., Anderson, C.W., Appella, E. and Xu, Y.

(2000) p53 transcriptional activity is essential for p53-dependent apoptosis

following DNA damage. Embo J, 19, 4967-4975.

36. Bartek, J. and Lukas, J. (2001) Pathways governing G1/S transition and

their response to DNA damage. FEBS Lett, 490, 117-122.

37. Hwang, B.J., Toering, S., Francke, U. and Chu, G. (1998) p48 Activates a

UV-damaged-DNA binding factor and is defective in xeroderma

pigmentosum group E cells that lack binding activity. Mol Cell Biol, 18,

4391-4399.

38. Bohr, V.A., Smith, C.A., Okumoto, D.S. and Hanawalt, P.C. (1985) DNA

repair in an active gene: removal of pyrimidine dimers from the DHFR

gene of CHO cells is much more efficient than in the genome overall. Cell,

40, 359-369.

85 39. van der Horst, G.T., van Steeg, H., Berg, R.J., van Gool, A.J., de Wit, J.,

Weeda, G., Morreau, H., Beems, R.B., van Kreijl, C.F., de Gruijl, F.R. et

al. (1997) Defective transcription-coupled repair in Cockayne syndrome B

mice is associated with skin cancer predisposition. Cell, 89, 425-435.

40. Schul, W., Jans, J., Rijksen, Y.M., Klemann, K.H., Eker, A.P., de Wit, J.,

Nikaido, O., Nakajima, S., Yasui, A., Hoeijmakers, J.H. et al. (2002)

Enhanced repair of cyclobutane pyrimidine dimers and improved UV

resistance in photolyase transgenic mice. Embo J, 21, 4719-4729.

41. Mitchell, D.L. (1988) The relative cytotoxicity of (6-4) photoproducts and

cyclobutane dimers in mammalian cells. Photochem Photobiol, 48, 51-57.

42. Rieger, K.E. and Chu, G. (2004) Portrait of transcriptional responses to

ultraviolet and ionizing radiation in human cells. Nucleic Acids Res, 32,

4786-4803.

43. Paunesku, T., Mittal, S., Protic, M., Oryhon, J., Korolev, S.V., Joachimiak,

A. and Woloschak, G.E. (2001) Proliferating cell nuclear antigen (PCNA):

ringmaster of the genome. Int J Radiat Biol, 77, 1007-1021.

44. Cleaver, J.E., Karplus, K., Kashani-Sabet, M. and Limoli, C.L. (2001)

Nucleotide excision repair "a legacy of creativity". Mutat Res, 485, 23-36.

45. Takemura, M., Kitagawa, T., Izuta, S., Wasa, J., Takai, A., Akiyama, T.

and Yoshida, S. (1997) Phosphorylated retinoblastoma protein stimulates

DNA polymerase alpha. Oncogene, 15, 2483-2492.

46. Sterner, J.M., Dew-Knight, S., Musahl, C., Kornbluth, S. and Horowitz,

J.M. (1998) Negative regulation of DNA replication by the retinoblastoma

86 protein is mediated by its association with MCM7. Mol Cell Biol, 18, 2748-

2757.

47. Pennaneach, V., Salles-Passador, I., Munshi, A., Brickner, H., Regazzoni,

K., Dick, F., Dyson, N., Chen, T.T., Wang, J.Y., Fotedar, R. et al. (2001)

The large subunit of replication factor C promotes cell survival after DNA

damage in an LxCxE motif- and Rb-dependent manner. Mol Cell, 7, 715-

727.

48. Gladden, A.B. and Diehl, J.A. (2003) The cyclin D1-dependent kinase

associates with the pre-replication complex and modulates RB.MCM7

binding. J Biol Chem, 278, 9754-9760.

49. Kennedy, B.K., Barbie, D.A., Classon, M., Dyson, N. and Harlow, E.

(2000) Nuclear organization of DNA replication in primary mammalian

cells. Genes Dev, 14, 2855-2868.

50. Dimitrova, D.S. and Berezney, R. (2002) The spatio-temporal organization

of DNA replication sites is identical in primary, immortalized and

transformed mammalian cells. J Cell Sci, 115, 4037-4051.

51. Bosco, G., Du, W. and Orr-Weaver, T.L. (2001) DNA replication control

through interaction of E2F-RB and the origin recognition complex. Nat Cell

Biol, 3, 289-295.

52. Harbour, J.W. and Dean, D.C. (2000) The Rb/E2F pathway: expanding

roles and emerging paradigms. Genes Dev, 14, 2393-2409.

87 53. Bakkenist, C.J. and Kastan, M.B. (2003) DNA damage activates ATM

through intermolecular autophosphorylation and dimer dissociation.

Nature, 421, 499-506.

54. Khanna, K.K., Keating, K.E., Kozlov, S., Scott, S., Gatei, M., Hobson, K.,

Taya, Y., Gabrielli, B., Chan, D., Lees-Miller, S.P. et al. (1998) ATM

associates with and phosphorylates p53: mapping the region of

interaction. Nat Genet, 20, 398-400.

55. Siliciano, J.D., Canman, C.E., Taya, Y., Sakaguchi, K., Appella, E. and

Kastan, M.B. (1997) DNA damage induces phosphorylation of the amino

terminus of p53. Genes Dev, 11, 3471-3481.

56. Hirao, A., Kong, Y.Y., Matsuoka, S., Wakeham, A., Ruland, J., Yoshida,

H., Liu, D., Elledge, S.J. and Mak, T.W. (2000) DNA damage-induced

activation of p53 by the checkpoint kinase Chk2. Science, 287, 1824-

1827.

57. Stiff, T., O'Driscoll, M., Rief, N., Iwabuchi, K., Lobrich, M. and Jeggo, P.A.

(2004) ATM and DNA-PK function redundantly to phosphorylate H2AX

after exposure to ionizing radiation. Cancer Res, 64, 2390-2396.

58. Motoyama, N. and Naka, K. (2004) DNA damage tumor suppressor genes

and genomic instability. Curr Opin Genet Dev, 14, 11-16.

59. Nahle, Z., Polakoff, J., Davuluri, R.V., McCurrach, M.E., Jacobson, M.D.,

Narita, M., Zhang, M.Q., Lazebnik, Y., Bar-Sagi, D. and Lowe, S.W.

(2002) Direct coupling of the cell cycle and cell death machinery by E2F.

Nat Cell Biol, 4, 859-864.

88 60. Samuelson, A.V. and Lowe, S.W. (1997) Selective induction of p53 and

chemosensitivity in RB-deficient cells by E1A mutants unable to bind the

RB-related proteins. Proc Natl Acad Sci U S A, 94, 12094-12099.

61. Moll, A.C., Imhof, S.M., Bouter, L.M. and Tan, K.E. (1997) Second primary

tumors in patients with retinoblastoma. A review of the literature.

Ophthalmic Genet, 18, 27-34.

62. Fletcher, O., Easton, D., Anderson, K., Gilham, C., Jay, M. and Peto, J.

(2004) Lifetime risks of common cancers among retinoblastoma survivors.

J Natl Cancer Inst, 96, 357-363.

89 Figure 1. D. Post UV120 A. T= 12h 100 Ad-GFP-Cre infected MAFs 0 72 (h) 80 LoxP/LoxP Ad-GFP Rb Ad-GFP-Cre Rb ∆ exon 19 60 1 2 B. 40 Cre FP FP- d-G d-G 20 A A BrdU control untreated percent RB 0 Lamin B 01020 Post IR UV (J/m2) 1 2 120 C. T= 12h 100 -Cre GFP FP Ad- d-G A 80 PCNA

Ad-GFP Cyclin E 60 Ad-GFP Ad-GFP-CreAd-GFP-Cre Cyclin A Lamin B 40 1 2 20 BrdUcontrol untreated percent

0 02.55 IR (Gy)

Figure 1. Acute downregulation of RB protein abrogates the DNA damage

checkpoint response to UV and IR. (A) Asynchronously proliferating primary

RbloxP/loxP MAFs were infected with Ad-GFP or Ad-GFP-Cre adenovirus. RNA

was isolated at 0 and 72 h post-infection and RT-PCR was performed employing

primers specific for regions flanking the loxP sites in the murine RB gene. The appearance of the smaller transcript post-infection indicates recombination at the

floxed RB locus. (B) MAFs infected with adenoviruses encoding either GFP (lane

1) or GFP-Cre (lane 2) were harvested 5 days post infection in RIPA buffer.

Equal amounts of protein were separated by SDS-PAGE and immunoblotted with

90 an anti-RB monoclonal antibody. Lysates were immunoblotted with the polyclonal

Lamin B antibody to control for equal loading. (C) MAFs infected with Ad-GFP

(lane 1) or Ad-GFP-Cre (lane 2) were harvested 5 days post-infection and equal

concentrations of each protein were separated by electrophoresis. The effect of

acute RB loss on downstream target expression was analyzed by immunoblotting

for PCNA, cyclin E, and cyclin A. Lamin B serves as a loading control. (D) Top

Panel: Asynchronously proliferating primary RbloxP/loxP MAFs infected with either

Ad-GFP control or Ad-GFP-Cre adenoviruses were irradiated with 0, 10, or 20

J/m2 UV. Treated cells were cultured for 12 hours and labeled with BrdU for the

final 2 hours prior to harvest. The proliferative fraction of treated cells was determined with respect to untreated control through immunofluorescence using an anti-BrdU antibody. Bottom Panel: Ad-GFP and Ad-GFP-Cre infected MAFs were exposed to 0, 2.5, or 5 Gy gamma irradiation and were cultured for 12 hours while in the presence of BrdU for the final 2 hours prior to harvest.

Immunofluorescence for BrdU was performed to determine the percent of cells that progressed through S-phase during the labeling period.

91 Figure 2.

Ad-GFP Ad-GFP-Cre

A. B. V V U U 2 2 IR IR Post UV: T= 2h T=4h T=6h m /m y / y J J G G 2 hours post-treatment: 0 0 5 100 0 1 5 0 1 Total p53 Lamin B 80 Ad-GFP 1 2 3 4 5 6 Ad-GFP-Cre 60 Ad-GFP Ad-GFP-Cre Hours post-treatment: 0 1 3 5 0 1 3 5 Phospho- 2.5 Gy IR 40 p53 10 J/m2 UV (ser18)

BrdU percent untreated control 1 2 3 4 5 6 7 8 20

0 C. Ad-GFP Ad-GFP-Cre 0 10 20 0 10 20 0 10 20 2 UV (J/m2) Hours post-10 J/m UV: 0 3 5 0 3 5 PCNA Post IR: T= 2h T=4h T=6h 100 Cyclin A MCM-7 80 Lamin B 1 2 3 4 5 6 Ad-GFP 60 Ad-GFP-Cre Ad-GFP Ad-GFP-Cre

40 Hours post-2.5 Gy IR: 0 3 5 0 3 5 PCNA Cyclin A 20 BrdU percent untreatedcontrol MCM-7

0 Lamin B 0 2.5 5 0 2.5 5 0 2.5 5 123456

Figure 2. Acute RB loss compromises the rapid checkpoint response to UV and

IR. (A) Top Panel: Ad-GFP and Ad-GFP-Cre infected MAFs were exposed to 0,

10, or 20 J/m2 UV and cultured for 2, 4, or 6 hours in the presence of BrdU for the final 2 hours. Immunofluorescence was utilized to detect BrdU labeled nuclei which were then scored and represented as percent untreated control. Bottom panel: The aforementioned experiment was repeated following treatment with 0,

2.5, or 5 Gy IR. (B) To assess the role of RB in rapid DNA damage signaling, asynchronously growing MAFs infected with either Ad-GFP or Ad-GFP-Cre were exposed to 2.5 Gy IR or 10 J/m2 UV and harvested at various time points in RIPA

92 buffer. Equal amounts of protein were separated by electrophoresis and immunoblotting for total p53 (top panel) and phosphorylated p53 (ser18) (bottom panel) (C) To analyze the role of RB in DNA damage signaling to downstream targets, cells were treated and harvested as in B and immunoblotting for PCNA, cyclin A, and MCM-7 (top and bottom panels) was performed. Lamin B serves as a control for equal loading.

93 Figure 3. 10 J/m2 2 A. C. 100 20J/m

Hours post-infection: 0 12 24 36 48 80

RB 60 Cyclin E Cyclin B1 40

MCM-7 20

PCNA BrdU positive percent untreated control 0 Lamin B UV post infection: 4h 16h 28h 40h 52h 76h 100h 1 2 3 4 5

2.5 Gy 5 Gy B. 100 0, 12, 24, 36, 4, 16, 28, 40, 80 Ad-GFP-Cre 48, 72, 96h Treat with 52, 76, 100h UV or IR infection Harvest 60 of MAFs and BrdU label for 4h 40

20 BrdU positivepercent untreated control 0 IR post infection: 4h 16h 28h 40h 52h 76h 100h

Figure 3. Kinetics of RB loss is concomitant with target gene deregulation to

promote abrogation of the DNA damage checkpoint response. (A)

Asynchronously proliferating primary MAFs were infected with Ad-GFP-Cre and

harvested every 12 hours for a period of 48 hours. Equal protein concentrations from each lysate were separated by electrophoresis and immunoblotted for the expression of RB, cyclin E, cyclin B1, MCM-7, and PCNA. Immunoblot for lamin

B was used to control for equal loading. (B) This schematic illustrates the experimental approach used to determine whether acute RB loss or target gene deregulation more directly influence the kinetics of DNA damage checkpoint bypass. Asynchronously proliferating MAFs were infected with Ad-GFP-Cre and irradiated at various timepoints. Following irradiation, cells were cultured with

94 BrdU for 4 hours prior to harvesting at their indicated times post-infection.

Immunofluorescence was performed to determine the proliferative fraction of

treated cells as compared to the untreated controls. (C) As illustrated in (B),

MAFs were infected with Ad-GFP-Cre and the temporal cell cycle response to 0,

10, or 20 J/m2 UV during the RB knockout was examined and represented as a

percent of BrdU positive untreated controls. (D) Cells were treated with 0, 2.5, or

5 Gy IR and analyzed for BrdU incorporation as described in (B).

95 Figure 4. A. Ad-GFP B. Post IR Ad-GFP MAFs γH2AX 0 Gy 2.5 Gy T=2h 2.5 Gy T=24h 100

60 2.5 Gy 5 Gy

20 BrdU percent untreated control BrdU untreated percent Post IR: 0 4 h 24 h 0h 2h 5h 12h 24h 2.5

Ad-GFP Ad-GFP-Cre 2

1.5

1 H2AX intensity increase intensity H2AX γ fold fold 0.5

0 0 2.5 5 0 2.5 5 0 2.5 5 0 2.5 5 0 2.5 5 IR (Gy) D. C. Ad-GFP Ad-GFP MAFs 6-4PP Post UV 0 J/m2 10 J/m2 T=0h 10 J/m2 T=24h 70

60 10 J/m2 20 J/m2 50

BrdU percent untreated control 20

0 4 h 24 h

96 E. Post UV: 0h 2h 5h 10h 6

4 Ad-GFP Ad-GFP-Cre

2 Fold 6-4 PP intensity increase intensityPP Fold 6-4 1

0 0 10 20 0 10 20 0 10 20 0 10 20 UV (J/m2) WT MAF Post UV: 0h 2h 5h 10h 6

Ad-GFP 3 Ad-GFP-Cre

Fold 6-4 PP intensity increase 1

0 0 10 20 0 10 20 0 10 20 0 10 20 UV (J/m2)

Figure 4. Differential effects of acute RB loss on UV- and IR-induced DNA lesion removal. (A) Ad-GFP or Ad-GFP-Cre infected MAFs were exposed to 0, 2.5, or

5 Gy IR and cultured for 4 or 24 hours in the presence of BrdU for the final 4 hours prior to harvest. Immunofluorescent detection was employed and BrdU positive cells were scored and represented as percent untreated control. (B)

Asynchronously growing MAFs infected with either Ad-GFP or Ad-GFP-Cre were exposed to 0 or 2.5 Gy IR. At 0, 2, 5, 12, and 24 hours after cell irradiation, samples were fixed and analyzed for γH2AX foci formation by immunofluorescence using an anti-γH2AX monoclonal antibody (top panel). The

97 relative abundance of γH2AX foci was determined through quantification of

staining intensity in images taken at equal exposures using Metamorph software

(bottom panel). These data are represented graphically as the relative increase

in γH2AX intensity. (C) Adenovirally infected MAFs were treated with 0, 10, 20

J/m2 UV and cultured for 4 or 24 hours in the presence of BrdU for the final 4 hours. Scoring of BrdU immunofluorescence revealed the percent of treated cells that were BrdU positive with respect to untreated controls. (D) Ad-GFP and Ad-

GFP-Cre infected MAFs were treated with 0 or 10 J/m2 UV and harvested at 0

and 24 hours post treatment for 6-4 PP immunofluorescence. Images of equal

exposure were taken. (E) MAFs from Fig. 4D were treated with 0, 10, or 20 J/m2

UV and cultured for 0, 2, 5, or 10 hours post-UV. Cells were then harvested and immunofluorescence for 6-4 PP was performed. Micropscopic images of equal exposures were obtained and the relative abundance of 6-4 PPs were quantified using Metamorph software. The data are represented graphically as fold 6-4 PP intensity increase (top panel). Wild type MAF control cells lacking loxP sites were cultured and infected with Ad-GFP or Ad-GFP-Cre. Five days post infection, wild type cells were treated with UV and analyzed for 6-4 PP repair as in top panel

(bottom panel).

98 Figure 5. A. B. Ad-GFP Ad-GFP-Cre CPD 6-4 PP DNA (µg) : 0 .25 .5 .75 1 0 .25 .5 .75 1 DNA (µg) : 0 .25 .5 .75 1 0 .25 .5 .75 1 UV (J/m2): 0 J/m2 CPD 0 T=0h Ad-GFP 10 J/m2 T=0h 2 10 T=0h 10 J/m T=24h 0 J/m2 10 T=5h Ad-GFP-Cre 10 J/m2 T=0h 10 J/m2 T=24h 0 T=0h 6-4 PP 10 T=0h 10 T=5h 10 T=10h

80 CPD 6-4 PP

50 Ad-GFP Ad-GFP-Cre 40

20 Percent intensity reduction 10

0 5h 5h 10h

Figure 5. Acute RB loss accelerates UV-induced DNA damage repair. (A) To examine the role of RB in the kinetics of UV-induced CPD and 6-4 PP lesion repair, Ad-GFP and Ad-GFP-Cre infected MAFs were treated with 0 or 10 J/m2

UV and harvested for genomic DNA purification at 0, 5, and 10 hours. Increasing concentrations of denatured genomic DNA were vacuum blotted onto a membrane and probed with anti-CPD and anti-6-4 PP antibodies (top panel). The lesion intensities in the top panel were quantified using Metmorph and plotted as percent reduction in lesion intensity from cells treated with 10 J/m2 UV and

harvested at T=0 to those harvested at the indicated timepoint (bottom panel).

(B) As performed in (A), cells were treated with UV and cultured prior to

harvesting at 0 and 24 hours. Dot blot analysis was performed as previously

99 described to confirm previous reports of impaired repair of CPD lesions in murine cells and to reveal the effect of acute RB loss on acceleration of 6-4 PP repair.

100 Figure 6. A. B. DAPI Tethered PCNA Merge UV dose: 200 Post UV: T= 3h T= 5h 2 180 0 J/m Ad-GFP 160 Ad-GFP-Cre 140 2 120 10 J/m Ad-GFP T=3h 100 80

60 2 percent untreated control untreated percent 10 J/m Chromatin tethered PCNA 40 T=5h 20

0 01010 UV (J/m2) 0 J/m2 C. Ad-GFP Ad-GFP-Cre Post UV: T= 3h T=5h T= 3h T=5h 100 Ad-GFP- 10 J/m2 Cre T=3h 80 focal punctate 60 10 J/m2 T=5h 40 tethered PCNA

20 Percent chromatin total Percent of

0 0101001010 UV (J/m2)

Figure 6. RB modifies repair factor dynamics following DNA damage. (A) Ad-

GFP and Ad-GFP-Cre infected MAFs treated with 0 or 10 J/m2 UV were harvested for chromatin extraction at 3 and 5 hours post-treatment.

Immunofluorescence for PCNA abundance was performed on extracted cells in parallel with unextracted controls. PCNA positive cells in each sample were counted and set as a percent of their respective unextracted control. These data were graphed as percent chromatin tethered PCNA of the unirradiated control.

(B) Images of cells from (A) were taken at equal exposures to display the differential PCNA chromatin tethering patterns throughout the UV damage repair time course. (C) The PCNA tethering patterns of the irradiated chromatin extracted populations of MAFs from (A) were analyzed as displaying discrete

“foci” colocalized with heterchromatin, indicating PCNA is involved in replication,

101 or diffuse “punctate” staining throughout the nucleus, suggesting PCNA is engaged in global repair. The chromatin tethered PCNA positive cells were scored with respect to their staining pattern and represented as a percent of the total chromatin tethered PCNA positive cells.

102 Chapter IV: RB Modifies the Therapeutic Response of Breast

Cancer

Emily E. Bosco1, Jack T. Zilfou2, Scott W. Lowe2, 3, Huan Xu4, Bruce J. Aronow4,

and Erik S. Knudsen1*

1Department of Cell Biology, Vontz Center for Molecular Studies, University of

Cincinnati, College of Medicine, Cincinnati, OH, 2Cold Spring Harbor Laboratory,

Cold Spring Harbor, NY, 3Howard Hughes Medical Institute, Cold Spring Harbor,

NY, 4Cincinnati Children’s Hospital Medical Center, Cincinnati OH

*Corresponding author, Tel: (513) 558-8885; Fax: (513) 558-4454

Email: [email protected]

Keywords: RB, E2F, tamoxifen, DNA damage, breast cancer

Nonstandard abbreviations: CDK, cyclin dependent kinase; CDT, charcoal

dextran treated; CDDP, cis-diamminedichloroplatinum II; E2, 17β-estradiol pellet;

ER, estrogen receptor; IR, ionizing radiation; MCM7, minichromosome

maintenance 7; PCNA, proliferating cell nuclear antigen; RB, retinoblastoma

tumor suppressor

103 Abstract:

The retinoblastoma tumor suppressor protein (RB) is functionally inactivated in the majority of human cancers, and lost in one third of all breast cancers. RB regulates G1/S phase cell cycle progression and is a critical mediator of anti-proliferative signaling. Here the specific impact of RB loss on

E2F-regulated gene expression, tumorigenic proliferation, and the response to two distinct lines of therapy was interrogated in breast cancer cells. RB loss resulted in RB/E2F target gene deregulation and accelerated tumorigenic proliferation, thereby demonstrating that even in the context of a complex tumor cell genome, RB status exerts significant control over proliferation. Furthermore, the loss of RB compromised the short-term cell cycle inhibition following cisplatin, ionizing radiation, and anti-estrogen therapy. In the context of DNA damaging agents this bypass resulted in increased sensitivity to these agents in cell culture and xenograft models. In contrast, the bypass of anti-estrogen signaling resulted in continued proliferation and xenograft tumor growth in the presence of tamoxifen. These effects of RB loss were recapitulated by ectopic E2F expression, indicating that control of downstream target genes was an important determinant of the observed responses. Specific analyses of the RB/E2F gene expression signature in 60 human patients indicated that deregulation of this pathway was associated with early recurrence following tamoxifen monotherapy.

Thus, because the RB-pathway is a critical determinant of tumorigenic proliferation and differential therapeutic response, it may represent a critical basis for directing therapy in the treatment of breast cancer.

104

Introduction:

Breast cancer is the leading non-cutaneous cancer diagnosis in American

women, impacting over 240,000 new patients per year. Treatment options for

breast cancer are governed by the estrogen dependence of the tumor cells. Two-

thirds of all breast cancers are estrogen receptor (ER) positive, and in these

tumors ER serves as a molecular target for hormone ablation therapy (1). Anti-

estrogens, such as the widely used tamoxifen, are the first line therapy for ER-

positive tumors and efficiently elicit a G0/G1 phase arrest in hormone-dependent

cancer cells (2, 3). This class of drugs is initially effective in curbing the growth of

ER-positive tumors, however many patients whose tumors initially respond to

anti-estrogen treatment develop cellular resistance to tamoxifen while

maintaining ER-positive disease (4-6). This suggests that genetic lesions down

stream of ER bypass the effectiveness of therapy. Second line therapies for tumors that exhibit resistance to anti-estrogens have traditionally included radiation and chemotherapies that function by damaging DNA (e.g. cisplatin).

Importantly, the critical determinants for therapeutic response to either anti- estrogens or DNA damaging agents are largely unknown.

The retinoblastoma tumor suppressor (RB) plays a central role in cell cycle control and regulates the cellular response to diverse therapeutic agents. In quiescent cells, RB is hypophosphorylated and assembles transcriptional repressor complexes on the promoters of E2F-regulated genes to block cell cycle progression. In response to mitogenic factors, including estrogen in breast

105 cancer cells, RB is inactivated through hyperphosphorylation catalyzed by the cyclin D-cyclin dependent kinase (cdk) 4 and cyclin E-cdk2 complexes (7-9).

These modifications are sufficient to disrupt the interaction of RB with E2F proteins, thereby relieving transcriptional repression and permitting cell cycle progression. In contrast, anti-mitogenic factors activate RB, inhibiting cell cycle progression. For example, RB activity is instrumental in the DNA-damage induced cell cycle checkpoint and is necessary for G1 and S-phase arrest following DNA damaging events. Correspondingly, tamoxifen and other anti- estrogens function to block RB phosphorylation and engage RB-mediated transcriptional repression of E2F (2, 3, 10, 11).

RB is lost in approximately 30% of breast cancers (12) and has been associated with poor disease outcome. Specifically, analyses of LOH at the Rb locus or loss of the RB protein is routinely observed in primary breast cancer specimens (12, 13). Additionally, the over-production of cyclin D1 and cyclin E which mediate the inactivation of RB are relatively common events in breast cancer (14). Lastly, microarray analyses have indicated that deregulation of E2F- target genes can be associated with poor prognosis (15, 16). The basis for these effects on breast cancer remains largely unknown and have not been explicitly interrogated in the context of breast cancer therapies. In this regard, there is clear evidence for cohort dependent effects of RB which could have implications for the therapeutic strategies employed.

Resistance to conventional therapy is one of the main causes of patient death associated with breast cancer. Given the frequent disruption of RB function

106 in breast cancer the effect of this event upon the response to therapeutic agents

is imperative for the optimal design of treatment strategies. Here, we show that

RB loss in breast cancer cells resulted in deregulation of E2F-regulated genes and a growth advantage in vitro which was recapitulated by accelerated tumor development in xenograft models. The effect of RB loss was determined in the

context of radiation, cisplatin, or hormone ablation therapy. RB-deficiency

enabled cells to inappropriately progress through the cell cycle following

challenge with all therapeutic modalities tested. In the context of DNA damaging

therapeutics, the loss of RB increased therapeutic sensitivity in both cell culture

and xenograft models. In contrast, following tamoxifen therapy the bypass of cell

cycle inhibition enabled proliferation in the presence of therapy and

corresponding therapeutic failure in xenograft models. Similar to RB loss,

ectopic expression of E2F3 bypassed the cell cycle arrest mediated by therapeutic agents, suggesting that deregulated E2F activity underlies the changes in therapeutic response in RB-deficient breast cancer cells. Using a signature of 59 RB/E2F regulated genes to probe microarray data from breast cancer patients treated with tamoxifen, we revealed that highly-elevated gene

expression levels correlated with failure of tamoxifen therapy. Together, these results demonstrate that RB loss facilitates accelerated tumorigenic proliferation of breast cancer cells and differential resistance to two major breast cancer

treatment modalities.

107 Materials and Methods:

Cell lines and culture

The MCF7 cell line was obtained from American Type Culture Collection

(Manassas VA, USA) and propagated in DMEM containing 10% FBS

supplemented with 100 U/mL penicillin/streptomycin and 2 mM L-glutamine at

37°C in air containing 5% CO2. Cells were infected with adenovirus encoding either, E2F3 (Ad-E2F3 was a generous gift from Dr. James DeGregori) or the lacZ gene (Ad-lacZ was kindly supplied by Dr. Nancy Ratner) as a control. Cells were infected with Ad-lacZ at a multiplicity of infection of 50, at which 95% of cells were infected (as determined by plaque assay in 293 cells) or with 2.7x1011

virus particles/mL of Ad-E2F3 and cultured for 3 days prior to use. RB

knockdown or control MCF7 cells were created through transfection with either

an shRNA plasmid directed against Rb (MSCV-Rb3C; targeted sequence: 5’ cgc

ata ctc cgg tta gga ctg tta tga a 3’ or a control plasmid (MSCV-Donor) using

FuGENE transfection reagent (Roche). Following selection with 2.5 µg/mL

puromycin for 3-4 days, stable clones were isolated and characterized.

To study the effect of estrogen depletion, cells were cultured for 72 hours in phenol red free DMEM supplemented with 10% charcoal dextran treated

(CDT) serum with addition of 10-9M tamoxifen (Sigma) or 10-6M ICI 182780

(Tocris Bioscience) where indicated. Cell growth assays were performed by trypsinizing cells and counting by trypan blue exclusion every three days.

Immunoblot analysis, immunofluorescence, and DNA damage

108 Cells were harvested by trypsinization and lysed in RIPA buffer. Equal

amounts of protein, as determined by Bio-RAD DC assay, were resolved by

SDS-PAGE. Specific proteins were detected by standard immunoblotting

procedures using the following primary antibodies: (Santa Cruz, 1:500 dilution)

PCNA (pc10), Cyclin E (HE12), Cyclin A (C-19), MCM7 (141.2), E2F3 (C-18),

cdk4 (H-22), and anti-RB (G3-245, Becton Dickson, 1:100 dilution).

Immunofluorescence staining for RB was performed on cells growing on

coverslips by fixing them in 3.7% formaldehyde in PBS for 10 minutes. Cells were permeabilized in 0.3% Triton X100 in PBS for 20 minutes and blocked in

5% fetal goat serum in PBS for 1 hour. Cells were incubated in blocking solution

with anti-RB antibody (G3-245, Becton Dickson 1:25 dilution) for 1 hour at 37°C

followed by PBS washing and incubation with Alexa 488 secondary antibody

(Molecular Probes 1:100) and counterstained with DAPI (Sigma).

Cells treated with ionizing radiation (IR) were exposed to 137Cs (dose rate:

0.67 Gy/min) at room temperature in tissue culture media. Culture with clinical

grade cis-diamminedichloroplatinum II (CDDP) for 18 hours was performed for all

CDDP treatments. For all proliferation studies, cells were labeled with BrdU for

10 hours and BrdU immunofluorescence was performed as previously described

(17). All BrdU results are expressed as a percentage of untreated control cells

set to 100% unless otherwise noted.

Xenograft studies and immunohistochemistry

5-8 week old female ovariectomized athymic nude mice (Harlan Sprague)

were anesthetized and a 17β-estradiol pellet (E2, 1.7 mg/pellet 90 day release,

109 Innovative Research of America) or placebo was surgically implanted in the back.

Following implant, 200µL of a PBS and phenol red free Matrigel matrix basement

membrane (Becton Dickinson) solution (3:1) containing 2x106 cells was injected

subcutaneously into the flank, or contralaterally when noted. Tumor volume was measured with calipers every 4 days using the equation; V= 0.52

(width)2x(length). In the therapeutic studies, when tumor volume reached 100-

120 mm3 animals were placed into one of three therapeutic groups (each group containing at least 6 animals); control animals retaining the estrogen pellet, anti- estrogen treated animals, or CDDP treated animals. The anti-estrogen treatment group was anesthetized and the estrogen pellet was surgically removed and a tamoxifen pellet (5mg/pellet 60 day release, Innovative Research of America) implanted. The CDDP treated animals received 5mg/kg CDDP injected i.p. on a q4dx5 schedule (every 4 days for 5 courses). All animals were injected i.p. with

150mg/kg BrdU (Sigma) 1 hour prior to euthanization. Xenograft tumors were weighed and fixed in 10% neutral buffered formalin, paraffin embedded, and cut

into 5µm sections. For immunohistochemical staining, sections were

deparaffinized in xylenes and rehydrated through a graded series of ethanol/water solutions. A BrdU detection kit (Zymed, San Francisco, CA) was

utilized as recommended by the manufacturer. BrdU incorporation was scored

blind and at least 500 cells per section were counted from several random fields.

Microarray Analysis

Microarray data (Series GSE1378 and GSE1379) from Ma XJ, et al (15)

was obtained from the National Center for Biotechnology Information Gene

110 Expression Omnibus(GEO) website

(http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GPL1223) and manipulated

using GeneSpring GX software (version 7.2) (Agilent Technologies). For each

series, the raw data was obtained from GEO as Log base 2 of normalized

Cy5/Cy3 ratio, where tumor sample RNA and human universal reference RNA

were labeled with Cy5 and Cy3 respectively. The raw data was transformed from

Log base 2 to linear values followed by PerGene median normalization in

GeneSpring. A 120 sample experiment was then created by combining the normalized data of the two series (60 tumors, macro and microdissected). The expression levels of 59 RB target genes known from various sources to be upregulated upon RB loss in primary cells (18-20)(Markey MP, et al in preparation) were analyzed in the combined experiment. The 59 genes were first

clustered according to their expression patterns using standard correlation as similarity measurement. A condition tree based on distance correlation was then

created to examine the similarity among samples.

111 Results:

Efficient RB knockdown confers increased growth kinetics

To investigate the influence of RB function in breast cancer we targeted its expression in the ER-positive and estrogen dependent breast cancer cell line

MCF7. Specific and stable knockdown of RB was achieved using a vector

encoding shRNA directed against Rb. Multiple independent clones transfected

with either the shRNA against Rb or vector control were isolated (supplemental

Fig. 1). RB protein levels were determined using immunofluorescence and

immunoblot for RB (Fig. 1A, B top panel). These results demonstrated that RB

protein levels had been reduced to virtually undetectable levels. Since it has

been postulated that nearly all cancer cells harbor compromised RB function, we

initially determined the consequence of RB loss on E2F target gene expression.

Thus, levels of the well-documented RB/E2F targets cyclin A, cyclin E, and

minichromosome maintenance 7 (MCM7) were evaluated by immunoblotting.

The expression levels of all of these proteins were increased in the absence of

RB (Fig. 1B). Analyses of cell cycle progression by BrdU incorporation (Fig. 1C, data from multiple clones in supplemental Fig. 2) or cellular proliferation (Fig. 1D) demonstrated that loss of RB resulted in enhanced proliferation. Thus, loss of RB alters the proliferative kinetics in established breast cancer cells.

To interrogate the biological consequence of RB inactivation in tumorigenesis, we utilized nude mouse xenografts. RB-proficient and deficient

MCF7 cells (2x106) were injected contralaterally into the flanks of nude mice

supplemented with estrogen pellets to support tumor growth. RB knockdown

112 cells produced measureable tumors earlier than the controls and continued to

grow significantly faster (Fig. 2A), such that by 30 days the RB knockdown

tumors had grown to more than double the size of the control tumors. At this

time, mice were injected with BrdU and euthanized. Tumors were excised and

weighed (Fig. 2B), confirming their larger size. Additionally, BrdU

immunohistochemstry demonstrated that the proliferative index was significantly

higher in the RB-deficient tumors (Fig. 2C). Thus, even in the context of an

established breast cancer cell line, RB plays a pivotal role in modulating

tumorigenic proliferation.

Abrogation of the DNA damage checkpoint in RB knockdown cells causes

increased sensitivity

RB is known to be critical for induction of DNA damage checkpoints and

therapies inducing DNA damage are used as an additional line of therapy for ER-

positive breast cancers resistant to hormonal therapy. Therefore, we interrogated

the role of RB in the response to IR and CDDP. The breast cancer clones were

either treated for 18 hours with 0, 8, or 16 µM CDDP or irradiated with 0, 2.5, or 5

Gy IR. Cells were subsequently labeled with BrdU for 10 hours to determine the

corresponding effect of the respective agent on cell cycle progression. These

studies demonstrated that RB-proficient MCF7 cells initiate a dose-dependent checkpoint to both forms of DNA damage. In contrast, RB-deficient cells continued to incorporate BrdU efficiently following DNA damage (Fig. 3A). To

determine the long term effect of DNA damage therapy upon proliferation, cell

113 growth assays were performed wherein cells were plated at equal density and

treated with 2.5 or 5 Gy IR. Cell counting revealed that the RB knockdown cells

were more sensitive to IR in both the 2.5 and 5 Gy conditions as displayed by the greater number of RB-proficient cells in culture following treatment (Fig. 3B).

These data demonstrate that the ability of the RB knockdown cells to progress through the cell cycle in the presence of DNA damage was associated with increased sensitivity to these agents.

The xenograft model system was then utilized to test the response of RB- deficient tumors to CDDP therapy. Tumors were developed in the flanks of mice by injecting MCF7 donor or siRb cells into the flanks of mice and implanting an

E2 pellet into the back. When tumors reached 100-110 mm3, mice received 5

mg/kg CDDP every 4 days for 5 courses. These experiments revealed that both

tumor types regressed during CDDP treatment, however the RB-deficient tumors

regressed more rapidly throughout the 5 courses of therapy (Fig. 3C) and failed

to demonstrate any recovery following the completion of therapy. Upon excision,

all tumors were weighed and the results demonstrate that tumors lacking RB

function weighed less than half of the weight of control tumors following CDDP

therapy (Fig. 3D), indicating that RB-deficient tumors respond more favorably to

DNA damage therapy.

RB-deficient cells are able to bypass estrogen ablation therapy

To determine the impact of RB loss on the response to first line breast cancer anti-estrogen therapies, cells were exposed to several therapeutically

114 relevant conditions. Specifically, the MCF7 clones were cultured in the absence of estrogen (CDT), in the presence of tamoxifen (CDT/Tam), or the pure anti- estrogen, ICI 182780 (CDT/ICI). Following treatment with these modalities, cells were labeled with BrdU to determine the influence of each agent on cell cycle progression and the corresponding influence of RB loss on this response (Fig.

4A, data from multiple clones in supplemental Fig. 3). All conditions limiting estrogen function elicited cell cycle inhibition in cells harboring functional RB.

However, this action of each agent was significantly abrogated by the depletion of RB. These results indicate that RB-deficient cells are able to partially bypass the cell cycle blockade elicited by anti-estrogen therapy. To elucidate the long term growth effects of these therapies, cell proliferation assays were performed over 9 days. Cells were seeded at equal densities and cultured in

CDT/Tamoxifen (Fig. 4B). As previously described, control MCF7 cells did not exhibit cell proliferation (2). However, cells lacking RB were able to continue to proliferate in this hormone deprived environment.

To assess the role of RB in the therapeutic response of MCF7 cells in vivo, the xenograft model was again employed. Upon the attainment of tumors of approximately 100-120 mm3, mice were deprived of estrogen and treated with tamoxifen. Tumor measurement at four day intervals demonstrated that RB- proficient tumors respond to tamoxifen by regressing to nearly immeasurable sizes (Fig. 4C). However, the RB-deficient counterparts did not regress and indeed, increased in size from approximately 110mm3 to 150mm3 in the presence of tamoxifen. All tumors were weighed upon excision and RB-deficient tumors

115 were greater than three times heavier than control tumors following tamoxifen

therapy (Fig. 4D). Together, these data demonstrate that hormone deprivation

therapy is compromised in breast cancers harboring functional inactivation of the

RB pathway.

RB target gene upregulation is a prognostic indicator in human breast cancers

RB performs a myriad of functions, the most well understood being

repression of the E2F family of transcription factors. To determine the specific

influence of the E2F axis on bypassing therapy, an activator E2F was

overexpressed in wild type MCF7 cells. Specifically, cells were infected either with an adenovirus encoding E2F3 (Ad-E2F3) or a control virus (Ad-LacZ) and were harvested 3 days post-infection for immunoblot analysis of levels of known

RB-E2F targets (Fig. 5A). Relative to control (Lane 1), the Ad-E2F3 infected

MCF7 cells (Lane 2) exhibited significantly increased protein levels of E2F target genes, including proliferating cell nuclear antigen (PCNA) and MCM7. As expected, no changes were detected in RB levels and CDK4 served as a loading control. In order to assess the response to therapeutic intervention, 3 days post- infection Ad-E2F3 or Ad-LacZ infected MCF7 cells were separated into 2 major treatment groups; hormone therapy or DNA damage therapy. The estrogen ablation group was then cultured in the absence of estrogen and in the presence of tamoxifen or ICI 182780 as before. Alternatively, the cells in the DNA damage therapy group were treated with 16µM CDDP for 18 hours prior to washing or 5

Gy IR. Cells from both therapy groups were BrdU labeled and the replicative

116 fraction of treated cells was determined with respect to untreated control cells

(Fig 5B). Cells overexpressing E2F3 exhibited significantly reduced levels of cell

cycle arrest in each therapeutic condition as compared to the control infected

cells. This result indicates that the ability of RB-deficient breast cancer cells to

bypass therapeutic cell cycle arrest is due to unrestrained E2F activity,

suggesting RB/E2F-regulated target gene expression is an important marker of

therapeutic response.

To determine the significance of RB/E2F target gene expression in human

breast cancer we analyzed a tumor microarray data set of 60 breast cancer

patients with ER-positive disease who were treated with tamoxifen monotherapy.

The tumor specimens in all cases had been both micro and macrodissected (15).

The expression levels of 59 known RB target genes within this 120 point data set

(60 tumors, macro and microdissected) were analyzed and are displayed as a

condition tree (Fig. 6A). This map shows three major regions of gene

coregulation, low, medium, and high RB target gene expression (blue, yellow,

and red regions respectively). This clustering placed the macro and

microdissected samples from 59 of 60 patients in the same gene expression

groups with only one tumor signature split between the high and medium gene

expression groups on the condition tree. Patient recurrence data revealed that the patients in the “high” target gene group have an increased incidence of

cancer recurrence (65%) relative to the patients in the other two groups (38%).

The high levels of RB target genes in this group would suggest that these tumors

are functionally disrupted for the RB-pathway, and their poor response to

117 tamoxifen therapy would correspond with data described herein. The average expression levels of these 59 RB target genes in each of the three groups are quantitatively displayed as a box plot to reveal an approximate 3 fold increase in gene expression from the low to the high group (Fig. 6B). For each patient the time to disease recurrence is known and a recurrence-free survival curve was generated (Fig. 6C). Patients in the high expression group respond poorly to tamoxifen wherein the median recurrence-free survival is 62.5 months and only

35% remained recurrence-free. In contrast, patients in the low/medium RB target gene expression groups have an improved prognosis on tamoxifen therapy, with greater than 62% remaining disease-free. Thus, compromised control of the

RB/E2F axis is associated with poor response to tamoxifen in human breast cancer.

118 Discussion:

The RB tumor suppressor is functionally inactivated in a large fraction of

human cancers (21). Traditionally, this event is associated with the genesis of

cancer as opposed to the effect on the therapeutic response of a given tumor

type. Here we evaluated the influence of RB loss in the context of established

breast cancer cells and found that while RB loss did accelerate cellular and

tumorigenic proliferation, it also had a profound influence on the response to key

therapeutic modalities utilized in the treatment of breast cancer.

It is widely held that RB functions as a negative regulator of cell cycle

progression that is targeted at high-frequency in human cancers by a myriad of

mechanisms. The frequency of this event has led to the hypothesis that most

cancers functionally inactivate RB during tumor progression (21-23). Such a model would suggest that although the RB protein is expressed in many tumor types it is functionally inert due to upstream deregulation of RB phosphorylation.

In this work we investigated the influence of RB loss on the MCF7 breast cancer cell line. We found that loss of RB led to the enhanced expression of E2F-target genes, suggesting that the RB protein present in MCF7 cells is, in fact, functional. Subsequent analyses of cell cycle progression and cellular proliferation indicated that, similar to what is observed in primary cells, loss of RB induces a modest proliferative advantage (24). Strikingly, loss of RB also facilitated tumorigenic proliferation. Thus, while all tumors may impinge on the

RB-pathway via distinct mechanisms, the RB status still influences the biological activity of these tumor cells.

119 Breast cancer, like all cancers, is a heterogeneous disease. In the case of

ER-positive disease, tamoxifen or similar anti-estrogenic compounds are utilized

to treat the cancer. However, many ER-positive cases develop resistance to anti-

estrogen therapy and an alternative line of therapy such as either radiation or

chemotherapy is required. It has been found in primary cells that the RB/E2F

pathway plays an important role in mediating cell cycle inhibition following

exposure to such agents (25, 26). In MCF7 cells, we observed a similar

dependence on RB/E2F function as either the loss of RB or overexpression of

E2F3 enabled bypass of the DNA damage checkpoint. Strikingly, this loss of

checkpoint function increased the sensitivity of RB-deficient cells or tumors to IR

and CDDP respectively. These findings are in agreement with prior studies in

primary murine cells where RB-deficiency enhances susceptibility to death

following DNA damage (27, 28) which is most likely due to deregulation of cell

cycle and proapoptotic genes (29, 30). Thus, loss of RB, while uncoupling cell

cycle responses, leads to enhanced sensitivity to cytotoxic therapeutics which

function by damaging DNA. This is clinically important for patients harboring

either ER-negative or ER-positive tamoxifen resistant tumors.

First-line therapy for ER-positive breast cancer exploits the estrogen

dependence of these cells. Treatment of estrogen sensitive ER-positive tumors

with estrogen antagonists results in inhibition of tumor growth (31) and

corresponding tumor regression. However, up to 50% of ER-positive tumors fail

to respond to such therapeutics (32). Here we determined the influence of

compromising the RB/E2F pathway on response to estrogen antagonists. Cells

120 with a disruption in this pathway failed to undergo cell cycle inhibition following hormone therapy. However, unlike the situation with DNA-damaging agents, the

RB-deficient breast cancer cells continued to proliferate in the presence of tamoxifen. As a result, RB-deficient tumors continued to progress in the presence of tamoxifen and thus failed therapy. Such a finding has significant clinical impact since nearly all of the patients whose tumors initially respond to tamoxifen eventually develop cellular resistance (33).

The involvement of RB function in breast cancer therapy has not previously been dissected. In human disease, disruption of the RB pathway occurs with relatively high-frequency (>80%) (14, 34) and is often associated with poor prognosis.

Since RB function can be disrupted via mechanisms that do not directly target the protein (e.g. point mutations) and deregulated E2F-activity could similarly bypass tamoxifen, we reasoned that analyses of RB/E2F-target genes could result in an important determinant of tamoxifen response. Consistent with this idea, it has been reported that high levels of the RB/E2F targets, cyclin A and cyclin E can influence tamoxifen resistance (35, 36). Furthermore, we found that high expression of RB/ E2F-target genes was associated with poor response to tamoxifen in the context of monotherapy. This clinical data would suggest that disruption of the RB/E2F pathway plays a role in the progression of breast tumors to anti-estrogen resistance. In fact, RB loss has been well documented to correspond with ER-negativity in breast cancers (13, 37, 38), indicating that RB inactivation could be a crucial step in the progression to advanced disease.

However, the extent to which these findings apply to a larger cohort of breast

121 cancer patients remains under analyses. Together, these studies strongly

suggest that disruption of the RB/E2F-axis has a deleterious influence on

hormone therapy and could be utilized as a metric for informing therapeutic

choice.

Acknowledgements:

The authors would like to thank Drs. Karen Knudsen and Chris Mayhew for thoughtful comments and critical reading of the manuscript. This work was supported by a small grant to ESK from the U.C. Cancer Center and NCI CA-

10647-02 and a grant from the NIH to SWL (AG16379). EEB is supported by the

Albert J. Ryan Foundation and DOD BCRP Predoctoral Grant

W81XWH0410329.

122 References:

1. Jensen, E.V., and Jordan, V.C. 2003. The estrogen receptor: a model for molecular medicine. Clin Cancer Res 9:1980-1989.

2. Sutherland, R.L., Green, M.D., Hall, R.E., Reddel, R.R., and Taylor, I.W.

1983. Tamoxifen induces accumulation of MCF 7 human mammary carcinoma cells in the G0/G1 phase of the cell cycle. Eur J Cancer Clin Oncol 19:615-621.

3. Osborne, C.K., Boldt, D.H., Clark, G.M., and Trent, J.M. 1983. Effects of tamoxifen on human breast cancer cell cycle kinetics: accumulation of cells in early G1 phase. Cancer Res 43:3583-3585.

4. Encarnacion, C.A., Ciocca, D.R., McGuire, W.L., Clark, G.M., Fuqua, S.A., and Osborne, C.K. 1993. Measurement of steroid hormone receptors in breast cancer patients on tamoxifen. Breast Cancer Res Treat 26:237-246.

5. Howell, A., and Robertson, J. 1995. Response to a specific antioestrogen

(ICI 182780) in tamoxifen-resistant breast cancer. Lancet 345:989-990.

6. Robertson, J.F. 1996. Oestrogen receptor: a stable phenotype in breast cancer. Br J Cancer 73:5-12.

7. Broceno, C., Wilkie, S., and Mittnacht, S. 2002. RB activation defect in tumor cell lines. Proc Natl Acad Sci U S A 99:14200-14205.

8. Foster, J.S., Henley, D.C., Bukovsky, A., Seth, P., and Wimalasena, J.

2001. Multifaceted regulation of cell cycle progression by estrogen: regulation of

Cdk inhibitors and Cdc25A independent of cyclin D1-Cdk4 function. Mol Cell Biol

21:794-810.

123 9. Sutherland, R.L., and Musgrove, E.A. 2004. Cyclins and breast cancer. J

Mammary Gland Biol Neoplasia 9:95-104.

10. Carroll, J.S., Prall, O.W., Musgrove, E.A., and Sutherland, R.L. 2000. A pure estrogen antagonist inhibits cyclin E-Cdk2 activity in MCF-7 breast cancer cells and induces accumulation of p130-E2F4 complexes characteristic of quiescence. J Biol Chem 275:38221-38229.

11. Watts, C.K., Brady, A., Sarcevic, B., deFazio, A., Musgrove, E.A., and

Sutherland, R.L. 1995. Antiestrogen inhibition of cell cycle progression in breast cancer cells in associated with inhibition of cyclin-dependent kinase activity and decreased retinoblastoma protein phosphorylation. Mol Endocrinol 9:1804-1813.

12. Borg, A., Zhang, Q.X., Alm, P., Olsson, H., and Sellberg, G. 1992. The retinoblastoma gene in breast cancer: allele loss is not correlated with loss of gene protein expression. Cancer Res 52:2991-2994.

13. Ceccarelli, C., Santini, D., Chieco, P., Taffurelli, M., Gamberini, M., Pileri,

S.A., and Marrano, D. 1998. Retinoblastoma (RB1) gene product expression in breast carcinoma. Correlation with Ki-67 growth fraction and biopathological profile. J Clin Pathol 51:818-824.

14. Malumbres, M., and Barbacid, M. 2001. To cycle or not to cycle: a critical decision in cancer. Nat Rev Cancer 1:222-231.

15. Ma, X.J., Wang, Z., Ryan, P.D., Isakoff, S.J., Barmettler, A., Fuller, A.,

Muir, B., Mohapatra, G., Salunga, R., Tuggle, J.T., et al. 2004. A two-gene expression ratio predicts clinical outcome in breast cancer patients treated with tamoxifen. Cancer Cell 5:607-616.

124 16. van de Vijver, M.J., He, Y.D., van't Veer, L.J., Dai, H., Hart, A.A., Voskuil,

D.W., Schreiber, G.J., Peterse, J.L., Roberts, C., Marton, M.J., et al. 2002. A

gene-expression signature as a predictor of survival in breast cancer. N Engl J

Med 347:1999-2009.

17. Bosco, E.E., Mayhew, C.N., Hennigan, R.F., Sage, J., Jacks, T., and

Knudsen, E.S. 2004. RB signaling prevents replication-dependent DNA double-

strand breaks following genotoxic insult. Nucleic Acids Res 32:25-34.

18. Markey, M.P., Angus, S.P., Strobeck, M.W., Williams, S.L., Gunawardena,

R.W., Aronow, B.J., and Knudsen, E.S. 2002. Unbiased analysis of RB-mediated

transcriptional repression identifies novel targets and distinctions from E2F

action. Cancer Res 62:6587-6597.

19. Ishida, S., Huang, E., Zuzan, H., Spang, R., Leone, G., West, M., and

Nevins, J.R. 2001. Role for E2F in control of both DNA replication and mitotic

functions as revealed from DNA microarray analysis. Mol Cell Biol 21:4684-4699.

20. Polager, S., Kalma, Y., Berkovich, E., and Ginsberg, D. 2002. E2Fs upregulate expression of genes involved in DNA replication, DNA repair and mitosis. Oncogene 21:437-446.

21. Sherr, C.J., and McCormick, F. 2002. The RB and p53 pathways in cancer. Cancer Cell 2:103-112.

22. Hanahan, D., and Weinberg, R.A. 2000. The hallmarks of cancer. Cell

100:57-70.

23. Dimova, D.K., and Dyson, N.J. 2005. The E2F transcriptional network: old acquaintances with new faces. Oncogene 24:2810-2826.

125 24. Sage, J., Mulligan, G.J., Attardi, L.D., Miller, A., Chen, S., Williams, B.,

Theodorou, E., and Jacks, T. 2000. Targeted disruption of the three Rb-related genes leads to loss of G(1) control and immortalization. Genes Dev 14:3037-

3050.

25. Mayhew, C.N., Perkin, L.M., Zhang, X., Sage, J., Jacks, T., and Knudsen,

E.S. 2004. Discrete signaling pathways participate in RB-dependent responses to chemotherapeutic agents. Oncogene 23:4107-4120.

26. Harrington, E.A., Bruce, J.L., Harlow, E., and Dyson, N. 1998. pRB plays an essential role in cell cycle arrest induced by DNA damage. Proc Natl Acad Sci

U S A 95:11945-11950.

27. Almasan, A., Linke, S.P., Paulson, T.G., Huang, L.C., and Wahl, G.M.

1995. Genetic instability as a consequence of inappropriate entry into and

progression through S-phase. Cancer Metastasis Rev 14:59-73.

28. Harbour, J.W., and Dean, D.C. 2000. Rb function in cell-cycle regulation

and apoptosis. Nat Cell Biol 2:E65-67.

29. Nahle, Z., Polakoff, J., Davuluri, R.V., McCurrach, M.E., Jacobson, M.D.,

Narita, M., Zhang, M.Q., Lazebnik, Y., Bar-Sagi, D., and Lowe, S.W. 2002. Direct

coupling of the cell cycle and cell death machinery by E2F. Nat Cell Biol 4:859-

864.

30. Bracken, A.P., Ciro, M., Cocito, A., and Helin, K. 2004. E2F target genes:

unraveling the biology. Trends Biochem Sci 29:409-417.

126 31. Leung, B.S., and Potter, A.H. 1987. Mode of estrogen action on cell

proliferative kinetics in CAMA-1 cells. I. Effect of serum and estrogen. Cancer

Invest 5:187-194.

32. Jordan, V.C., and Morrow, M. 2003. Re: Trends in use of adjuvant multi-

agent chemotherapy and tamoxifen for breast cancer in the United States: 1975-

1999. J Natl Cancer Inst 95:683-684; author reply 684-685.

33. Jordan, V.C., and Murphy, C.S. 1990. Endocrine pharmacology of

antiestrogens as antitumor agents. Endocr Rev 11:578-610.

34. Oesterreich, S., and Fuqua, S.A. 1999. Tumor suppressor genes in breast

cancer. Endocr Relat Cancer 6:405-419.

35. Michalides, R., van Tinteren, H., Balkenende, A., Vermorken, J.B.,

Benraadt, J., Huldij, J., and van Diest, P. 2002. Cyclin A is a prognostic indicator

in early stage breast cancer with and without tamoxifen treatment. Br J Cancer

86:402-408.

36. Dhillon, N.K., and Mudryj, M. 2002. Ectopic expression of cyclin E in

estrogen responsive cells abrogates antiestrogen mediated growth arrest.

Oncogene 21:4626-4634.

37. Lee, E.Y., To, H., Shew, J.Y., Bookstein, R., Scully, P., and Lee, W.H.

1988. Inactivation of the retinoblastoma susceptibility gene in human breast cancers. Science 241:218-221.

38. Loden, M., Stighall, M., Nielsen, N.H., Roos, G., Emdin, S.O., Ostlund, H., and Landberg, G. 2002. The cyclin D1 high and cyclin E high subgroups of

127 breast cancer: separate pathways in tumorogenesis based on pattern of genetic aberrations and inactivation of the pRb node. Oncogene 21:4680-4690.

128 Figure 1. r1 8 o 2 n b o iR d s A. B. 7 7 F F MCF7 donor1 MCF7 siRb28 C C M M RB

DAPI CYCLIN A

MCM7

CYCLIN E RB CDK4 1 2 D. C. 60 10

50 8 ) 6 40 6 MCF7 donor1 30 MCF7 siRb28 4 20 Cell Count (x 10 % BrdU positive 2 10

0 0 MCF7 donor1 MCF7 siRb28 0369 Time (days)

Figure 1. Efficient RB knockdown in MCF7 cells causes deregulation of RB/E2F target genes and increased proliferation kinetics. (A.) MCF7 cells transfected with

MSCV donor or MSCV siRB plasmids were selected with puromycin to isolate stable clones. Clones were screened by RB immunofluorescence as shown for

MCF7 donor1 and siRb28. Images were captured at equal exposures. (B.)

Lysates from MCF7 donor1 and siRb28 clones were immunoblotted for expression levels of RB, cyclin A, MCM7, and cyclin E. Cdk4 served as a loading control. (C.) Cells from A were BrdU labeled for 10 hours and BrdU immunofluorescence was performed and scored. (D.) Cells from A were seeded at 3x105, and cell growth assays were carried out for 9 days while counting every

3 days.

129

Figure 2.

A. B. 30 days post implantation 350 140 p=.030 MCF7 300 120 donor1 3 n=7 n=7 MCF7 250 100 siRb28

200 80 MCF7 donor1 MCF7 siRb28 n=11 150 60

Tumor volume mm volume Tumor 100

n=11 Tumor weight (mg) 40

50 20

0 1418 22 26 30 0 Days post implantation MCF7 donor1 MCF7 siRb28 C. p<.003 18 16 14 12 10 8 6 4 % BrdU positive 2 0 MCF7 donor1 MCF7 siRb28

Figure 2. Tumor growth in nude mouse xenografts is accelerated in RB knockdown cells. (A.) MCF7 donor1 or siRb28 cells were harvested and resuspended in 3:1 PBS/Matrigel mixture. 2x106 cells in 150 µl of mixture were injected subcutaneously in a contralateral manner in flanks of ovariectomized nude mice. Mice were supplemented with E2 pellets and tumors were measured every 4 days. (B.) Excised tumors were weighed 30 days post implantation.

Tumor weights are plotted and a two tailed t-test assuming unequal variances was used to determine significance. Relative tumor size post excision 30 days following implantation (small inset). Scale bar equals 1cm. (C.) Nude mice from A

130 were injected with BrdU 1 hour prior to sacrifice. Sectioned tumors were immunohistochemically stained and scored for BrdU incorporation and statistical analyses were carried out as in B.

131 Figure 3. A. 120 120

100 100 MCF7 donor1 MCF7 donor1 MCF7 siRb28 MCF7 siRb28 80 80

60 60

40 40

20 20 BrdU positive % untreated control BrdU positive % untreated control 0 0 0816 0 2.5 5 CDDP dose (µM) IR dose (Gy)

B. 2.5 Gy 5 Gy MCF7 donor1 1.5 MCF7 donor1 1.5 ) ) MCF7 siRb28

6 MCF7 siRb28 6

1.0 1.0

0.5 0.5 Cell Count (xCell 10 Cell Count (xCell 10 0 0 03 6912 03 6912 Days post-treatment Days post-treatment C. D. 140 160 MCF7donor1 120 MCF7 donor1 140 MCF7 siRb28

3 MCF7 siRb28 100 120 n=6 100 80 80 60 n=6 60 40 n=6

Tumor volume mm volume Tumor p=.05 Tumor weight (mg) 40 20 20 n=6 0 0 481216 20 24 CDDP Days post initial CDDP injection

132 Figure 3. RB-deficiency enables bypass of the DNA damage checkpoint resulting in increased sensitivity. (A.) MCF7 donor1 and siRb28 clones were treated either with 0, 8, or 16 µM CDDP for 18 hours prior to washing (left panel) or with 0, 2.5, or 5 Gy IR (right panel) and BrdU labeled for 10h. Cells were then fixed and BrdU immunofluorescence was performed and scored. (B.) MCF7 donor1 or siRb28 cells were seeded at 3x105, treated with 2.5 (left panel) or 5 Gy IR (right panel), and cell growth assays were performed for 12 days while counting every 3 days.

(C.) Harvested MCF7 donor1 and siRb28 cells were resuspended 3:1 in

PBS/Matrigel and injected subcutaneously into the flanks of mice supplemented with E2 pellets. When xenograft tumors reached approximately 110mm3 during tumor development, mice were treated with CDDP (retain E2 pellet and inject

5mg/kg CDDP IP every 4 days x 5) and tumor size was monitored by caliper measurement. Tumor measurements are plotted and a two tailed t-test assuming unequal variances was used to determine significance of curves. (D.) Tumors from 3C were weighed upon excision.

133 Figure 4. A. B. 3.0 100 CDT/Tam MCF7 donor1 2.5 ) 6 80 MCF7 siRb28 2.0 60 1.5

40 MCF7 donor1 1.0 MCF7 siRb28 Cell Count (x 10 Count Cell

20 0.5

0 0 BrdU positive % untreated control 03 69 1234 FBS CDT CDT/Tam CDT/ICI Days post-treatment

C. D. 160 180

160 140 n=8 3 140 120 n=8

120 100 MCF7 donor1 100 MCF7 donor1 80 MCF7 siRb28 80 MCF7 siRb28 60 60 n=8 Tumor volume mm volume Tumor

Tumor weight (mg) weight Tumor 40 40

20 n=8 20

0 0 4 8 1216202428323640 Tamoxifen Days post Tam implantation -E2

Figure 4. RB is necessary for sensitivity to anti-estrogen therapy and long term growth arrest. (A.) MCF7 donor1 and siRb28 clones were cultured in media containing FBS, CDT, CDT/Tam, or CDT/ICI for 3 days while BrdU labeling for the final 10h. Cells were then fixed and BrdU immunofluorescence was performed and scored. (B.) MCF7 donor1 or siRb28 cells were seeded at 3x105

and cell growth assays were performed for 9 days while cells were cultured in

CDT/Tam and counted every 3 days. (C.) When xenograft tumors (as in 3C)

reached 100-120mm3, mice were treated with tamoxifen (remove E2 pellet, add

tamoxifen pellet). Tumor size of the tamoxifen treated animals was monitored by

calipers. (D.) Final tumor weights of all tumors from 4C upon excision.

134 Figure 5. A. B. 120 Z 3 c F la 2 - -E d d 100 A A 7 7 F F C C MCF7 Ad-lacZ M M 80 MCF7 Ad-E2F3 E2F3 60 RB 40 MCM7 PCNA 20

CDK4 BrdU positive % untreated control

1 2 0 FBS CDT CDT/Tam CDT/ICI 16µM CDDP 5Gy IR

Figure 5. E2F3 overexpression in MCF7 cells allows bypass of anti-mitogenic

checkpoints. (A.) MCF7 cells infected with adenoviral vectors encoding either lacZ or E2F3 were harvested 3 days post-infection, lysed, separated by SDS-

PAGE, and immunoblotted for E2F3, RB, MCM7, and PCNA expression levels.

Cdk4 served as a loading control. (B.) The adenovirus infected cells from A were cultured in media containing FBS, CDT, CDT/Tam, or CDT/ ICI for 3 days or were treated as previously described with 16µM CDDP or 5Gy IR prior to BrdU labeling for 10h. Cells were then fixed and BrdU immunofluorescence and scoring was performed.

135 Figure 6. A. 60 breast tumors micro and macrodissected (120 total) 5...

59 genes: 10864 (RAD21) 8868 (BRCA1) 21049 (ECT2) 15941 (KIF11) 8480 (SMC4L1) 5151 (TOPBP1) 20893 (STK6) 10383 (STK6) 16983 (KIF20A) 8143 (CDC25C) 11438 (CCNB1) 16894 (CDC20) 8577 (CDCA8) 2770 (KIF2C) 15350 (BIRC5) 12771 (CDC45L) 13532 (CDCA3) 10608 (PRC1) 2667 (CCNB2) 3150 (MK167) 13258 (RAD51) 12176 (CDCA5) 11994 (BRRN1) 20771 (TTK) 19457 (KIF23) 17783 (BUB1) 6798 (CENPA) 5608 (CCNA2) 11104 (RRM2) 10591 (TRIP13) 18977 (EZH2) 16888 (MAD2L1) 12415 (TOP2A) 9670 (RAD51AP1) 6259 (TYMS) 20095 (PCNA) 14538 (HMGB2) 6643 (FEN1) 7196 (NEK2) 14056 (CKS2) 15278 (CHEK1) 13907 (CDC6) 3836 (GMNN) 3534 (FIGNL1) 5487 (TMPO) 18243 (TCF19) 4855 (LIG1) 8241 (MCM2) 2830 (MCM3) 6292 (TCF19) 9272 (BUB1) 7151 (BRCA2) 15292 (SMC2L1) 14372 (PRiM1) 2029 (RFC5) 15589 (CDK2) 8536 (CDCA7) 21597 (PLTP) 19972 (TYRO3) Low Medium High

B. 2.5

2.0

1.5 Normalized intensity Normalized 1.0

Low Medium High

C. Percent Survival Percent p=.0088

Disease Free Survival (months)

136 Figure 6. RB/E2F downstream target deregulation correlates with poor prognosis

in human breast cancers treated with tamoxifen monotherapy. (A.) Gene

expression data from 60 ER-positive human breast tumors that were both micro

and macrodissected was analyzed for RB target gene expression using

GeneSpring. The expression patterns of 59 known RB target genes are

displayed in a condition tree for each of the 2 tissue samples from each patient

(120 samples). The average RB target gene expression levels of all 59 genes

were categorized into three groups, low, medium, and high. (B.) The RB target

gene expression levels in each group from C were averaged and displayed as a

box and whiskers plot. A two tailed t-test assuming unequal variances was

utilized to determine significance (p=7.2x10-12 low-medium and p=1.3x10-14 medium- high). (C.) The survival data for each of the 60 patients from the low/medium and high gene expression groups from C was compiled into a disease-free survival curve. Statistical tests were performed as in D.

137 Supplemental Figures:

1. 1 7 1 8 2 2 44 or b1 b b b on R R R iR si si d si s 7 7 7 7 7 F F F F F C C C C C M M M M M RB

cdk4

1 2 3 4 5 2. 3. 60 120 MCF7 donor1 MCF7 siRb17 100 50 MCF7 siRb21 MCF7 siRb28 40 80 MCF7 siRb44

30 60

20 40 % BrdU positive 10 20

0 0 MCF7 MCF7 MCF7 MCF7 MCF7 BrdU positive % untreatedcontrol FBS CDT/TAM CDT/ICI donor1 siRb17 siRb21 siRb28 siRb44

Supplemental Figure 1. Stable RB knockdown in several MCF7 clones. Several stable RB-deficient MCF7 clones (siRb17, siRb21, siRb28, siRb44) were generated through transfection of an shRNA plasmid and tested for RB expression by immunoblot and compared to the RB-proficient control clone

(MCF7 donor1, Lane 1). Cdk4 served as a loading control.

Supplemental Figure 2. RB-deficiency enables accelerated cell cycle progression. RB-deficient clones (siRb17, siRb21, siRb28, siRb44) and donor control cells were labeled with BrdU in culture and BrdU immunofluoresence was performed and scored.

138

Supplemental Figure 3. Bypass of anti-estrogen checkpoint is evident in several

RB-deficient MCF7 clones. Clones from supplemental figure 1 were cultured in

media containing FBS, CDT/Tam, or CDT/ICI for 3 days while BrdU labeling for

the final 10h. Cells were then fixed and BrdU immunofluorescence was performed and scored.

139 Chapter V: Summary and Conclusions

RB-mediated transcriptional repression and maintenance of cell cycle control

RB is a critical regulator of cell cycle control that is targeted for inactivation at high frequency in human cancers (1). Disruption of RB function contributes to tumorigenesis via a wide variety of mechanisms because RB regulates hundreds of target genes involved in cellular processes critical for tumor suppression including: cell cycle transitions, cell cycle checkpoints, apoptosis, DNA replication and repair, and maintenance of chromosome structure, among others (2, 3).

Therefore, the work described herein interrogates the role of RB in tumor suppression with respect to several of these contexts, specifically the anti- mitogenic signaling checkpoints (Chapter II-IV) and DNA damage repair (Chapter

III).

Prior to the work described here, it was understood that in embryonic models of RB loss the DNA damage checkpoint was bypassed allowing cells to proliferate despite unfavorable conditions (4, 5). Correspondingly, RB-deficient embryonic cells were shown to be more sensitive to such damage (6). However, because cancer is most commonly a sporadic disease occurring in adult tissues with minimal functional compensation from the other RB pocket proteins, we utilized the Cre-LoxP system to delete RB in mouse adult fibroblasts. In this model, we demonstrated that RB loss in adult cells enabled cell cycle progression in the presence of DNA lesions from DNA damaging therapeutics such as cisplatin and camptothecin. Consequently, attempts to replicate through

140 existing DNA lesions propagated the development of secondary lesions, such as

DNA double-strand breaks (Chapter II). Subsequent work from our lab has

shown that this ability of RB-deficient cells allows for the accumulation of

aberrant ploidy (7). Thus, we now understand that mutation burden caused by

DNA damage in RB-deficient cells can either lead to tumorigenesis or activation

of apoptotic pathways, both of which have important implications for cancer

development and treatment. This and other work suggests that an important

function of RB is to limit genome instability following genotoxic insult.

Microarray analysis has been an invaluable tool for uncovering hundreds of new RB/E2F target genes. However, much work lies ahead in order to understand the specific mechanisms of their regulation and the downstream consequences of their deregulation following RB inactivation. Because several

DNA repair proteins have been recently identified by microarray analysis to be

RB target genes (3, 8), we aimed to understand the previously unstudied role of

RB in DNA repair as it pertains to tumorigenesis (Chapter III). The significance of this goal was twofold; first, childhood retinoblastoma patients are at increased risk for development of other cancers throughout life (particularly skin cancer due to sun exposure) which could be due to a DNA repair defect, and second, RB- deficient tumors treated with DNA damaging therapies may respond differently to therapy based on their varied DNA repair capacities. Our studies again involved the use of primary mouse adult fibroblasts and their response to therapeutically and environmentally relevant doses of ionizing radiation (IR) and ultraviolet radiation (UV). We found that RB deficient cells were able to bypass the DNA

141 damage checkpoint induced by both forms of damage as seen before with other agents. RB inactivation did not affect the repair of IR-induced DNA lesions and

DNA damage signaling upstream of RB remained intact, suggesting that other proteins were able to compensate for RB-loss to carry out repair of DNA double- strand breaks, the most detrimental lesion caused by IR. In contrast, following

UV damage we found that RB deficient cells were able to repair the major lesion induced by UV more rapidly than control cells due to the deregulation of proteins necessary for this specific type of repair. Presumably, the higher abundance of

RB target genes involved in repair upon RB loss, such as PCNA, enables accelerated repair. Following our work, Berton et.al. addressed a similar question from a new angle, looking at the effect of overexpression of E2F (rather than RB loss) on UV damage repair. In confirmation of our studies, this work found that overexpression of E2F1 lead to enhanced repair of UV-induced lesions and suppression of apoptosis (9). This latter effect was surprising since E2F1 is understood to promote apoptosis following DNA damage. Although our studies did not address the consequence of the accelerated lesion removal in RB- deficient cells to determine its effect upon apoptosis, it is possible that the hyperproliferative effect of RB-loss is sufficient to overcome any protective effect of enhanced DNA repair. Thus, explaining the increased incidence of skin cancers in retinoblastoma survivors due to UV exposure. The studies performed by Berton et. al. confirms that deregulation of E2F underlies the effects of RB loss on DNA repair demonstrated by our work.

142 The role of RB in the development and progression of specific tumor types

is an important area of study since RB is rendered inactive by several different

mechanisms in virtually all cancers. Because it is known that RB-deficient breast

cancers are typically more aggressive tumors associated with poor prognosis

(10, 11), we chose to study the effect of RB on cell cycle control and tumorigenic

proliferation in the MCF7 breast cancer model (Chapter IV). Traditionally, RB has

been viewed as a brake on the cell cycle which becomes critically important for tumor suppression only when cells are challenged following anti-mitogenic signaling. However, long term growth studies with MCF7 cells in vitro and in nude mouse xenografts revealed that RB-deficiency enables faster growth even in the context of an established cancer cell line and that RB regulates cell cycle progression even in the absence of challenge. These findings support clinical data that RB loss is associated with increased aggressiveness in breast cancer

(10).

In summary, this initial work on the role of RB in cell cycle control

(Chapters II-IV) reveals that RB-deficiency and subsequent target gene deregulation allows for increased cell proliferation, bypass of DNA damage checkpoints, and altered DNA repair kinetics. Combined, all of these factors can contribute to tumorigenesis by compromising genomic stability.

Role of RB in therapeutic response

These studies put forth the question: how does the role of RB in cell cycle control impinge on cancer therapy? To address this question we again utilized the breast cancer model (Chapter IV) because treatment of this disease is two-

143 pronged, as first line treatments for ER+ tumors are anti-estrogen therapies and

second line therapies function by damaging DNA.

Previous evidence linking functional RB inactivation and deregulation of

RB/E2F target genes with cellular resistance to anti-estrogen therapy suggested

that RB modifies breast cancer therapeutic response (12-15). We tested this possibility by overexpressing E2F3 or knocking down RB expression in MCF7 cells and xenograft tumors and revealed the ability of these cells to proliferate in

the absence of hormone. This effect has not proven to be cell line specific, as

ongoing experiments with T47D ER+ breast cancer cells have yielded similar

results. This suggests that disruption of the RB/E2F axis enables bypass of the

normal estrogen signaling pathways; however, this topic has remained largely

unstudied. It has been previously shown that RB is dephosphorylated/activated

when estrogen-dependent cells are treated with tamoxifen (16) and the molecular signaling from tamoxifen that arrests cell cycle progression is known to involve the down-regulation of several RB regulated genes (17, 18). Thus, together these data further support a role for RB in the molecular response to anti-estrogenic therapies for breast cancer.

Resistance to first line anti-estrogenic therapy for ER+ breast cancer is a significant clinical problem, and these patients are treated in the same manner as

ER- patients, with DNA damaging therapies. Our work in primary cell lines has

revealed that the DNA damage checkpoint is abrogated in cells with a

compromised RB/E2F pathway (Chapters II-III). Therefore, we tested the ability

of RB-deficient breast cancer cells to proliferate following CDDP and IR therapy.

144 Both overexpression of E2F3 and stable knockdown of RB in MCF7 cells enabled bypass of the DNA damage checkpoint, suggesting that deregulation of downstream RB/E2F targets are largely responsible. Our earlier studies in primary fibroblasts informed us that DNA damage checkpoint bypass could have various consequences for RB-deficient cells, namely, tumor progression or cell death. To address these two outcomes we performed long term studies of breast cancer cell and xenograft tumor growth which demonstrated increased sensitivity of the RB-deficient cells to DNA damage. This is consistent with our work in primary cells and the current understanding in the field as bypass of the DNA damage checkpoint causes loss of genomic integrity and initiation of apoptosis.

Furthermore, it is likely that RB-deficient cells may be more susceptible to death following DNA damage due to deregulation of cell cycle and proapoptotic genes.

Can RB status inform therapy?

Increasing numbers of studies in various tumor types have analyzed the effect of RB loss on clinical prognosis. Many of these investigations have indicated that RB loss correlates with advanced disease, however, RB functional inactivation is a difficult phenotype to directly assess as RB is rarely deleted in cancers. As such, many studies now interrogate RB function through analysis of levels of RB/E2F target genes (e.g. cyclin A). In order to test the applicability of our cell culture observations that RB inactivation promotes anti-estrogen resistance, we probed the expression of RB/E2F target genes in human tumor microarrays (19, 20). These data sets revealed that many patients have ER+ tumors expressing high levels of RB/E2F targets such as, cdc25a-c, cyclin A,

145 Rad51, MCMs, PCNA, and MAD2, indicative of functional RB inactivation.

Correspondingly, patients with this type of genetic tumor signature have a poor prognosis following tamoxifen monotherapy. This clinical data suggests that functional inactivation of RB plays a role in the progression of breast tumors to anti-estrogen resistance. In fact, RB loss has been well documented to correspond with ER-negativity in breast cancers (10, 11), indicating that RB inactivation could be a crucial step in the progression to advanced therapy resistant disease. In sum, this clinical outcome data coupled with our results in the primary and breast cancer cell model systems demonstrating that RB- deficiency synergizes with the DNA damaging agents to make them more effective imply that DNA damage (rather than anti-estrogen) therapy would be

the most beneficial for patients with ER+, RB-deficient breast tumors. However,

additional studies must be performed to directly demonstrate that RB function is

required for sensitivity to anti-estrogen therapy on the clinical level, and thus

could be used as an effective marker at diagnosis to inform clinical options.

Interesting questions and future investigations

This and others work support the idea that RB is an important target of

estrogens and that its functional inactivation can contribute to anti-estrogen

resistance. However, the molecular mechanisms underlying this therapeutic

resistance are not understood, possibly because the relationship between ER

and RB remains unclear. Microarray data with RB-proficient and -deficient MCF7

cells from our lab revealed that RB regulates the expression of many classical

ER transcriptional target genes (data not shown), suggesting that RB could

146 function as a coactivator of ER. Such an idea is not entirely novel in the hormone receptor field as recent reports have demonstrated that, contrary to the traditional functions of RB, it is involved in activation of the transcriptional activity of several nuclear receptors. Specifically, RB enhances the activity of the glucocorticoid receptor through interaction with the Brm/BRG1 components of the SWI/SNF chromatin remodeling complex (21) and the androgen receptor independently of

BRG1 (22, 23). In the context of ER, RB has been shown to interact with the retinoblastoma-interactingzinc finger protein (RIZ) which interacts with ER in an estrogen dependent manner (24) and also potentiates the action of SRC coactivators on ER-dependent transcription (25). Consistent with our microarray results, these studies indicate that RB may act as a direct or indirect transcriptional activator of ER. Therefore, RB functional inactivation in breast cancer could negatively impact ER signaling pathways and may explain the correlation between RB loss and antiestrogen resistance as demonstrated herein. However, more work is necessary to substantiate a functional link between these two proteins.

Cancer therapy is extremely heterogeneous, such that patients receive varying doses of different drugs at different intervals. Such realities make studying the link between molecular tumor composition and therapeutic response in the clinic quite difficult. Thus, in order to build upon the work outlined in this thesis it would be important for us to first test our hypothesis in a true mouse model of breast cancer. To accomplish this, mice with a conditional mammary gland knockout of RB (26) could be mated to mice that are prone to breast

147 cancer development (MMTV-v-Ras or MMTV-c-myc) such that we could interrogate the role of RB in both mammary gland tumorigenesis and therapeutic response. Additionally, it would be of interest to begin performing tumor tissue arrays on patient samples where we could analyze the coincidence of other known prognostic markers in breast cancer (e.g. her2/neu, PR, ER) with RB/E2F pathway deregulation. We believe that such studies are necessary for the future development of more individualized therapies for the treatment of breast cancer.

148 References:

1. Malumbres, M., and Barbacid, M. 2001. To cycle or not to cycle: a critical

decision in cancer. Nat Rev Cancer 1:222-231.

2. Polager, S., Kalma, Y., Berkovich, E., and Ginsberg, D. 2002. E2Fs up-

regulate expression of genes involved in DNA replication, DNA repair and

mitosis. Oncogene 21:437-446.

3. Markey, M.P., Angus, S.P., Strobeck, M.W., Williams, S.L., Gunawardena,

R.W., Aronow, B.J., and Knudsen, E.S. 2002. Unbiased analysis of RB-

mediated transcriptional repression identifies novel targets and distinctions

from E2F action. Cancer Res 62:6587-6597.

4. Harrington, E.A., Bruce, J.L., Harlow, E., and Dyson, N. 1998. pRB plays

an essential role in cell cycle arrest induced by DNA damage. Proc Natl

Acad Sci U S A 95:11945-11950.

5. Knudsen, K.E., Booth, D., Naderi, S., Sever-Chroneos, Z., Fribourg, A.F.,

Hunton, I.C., Feramisco, J.R., Wang, J.Y., and Knudsen, E.S. 2000. RB-

dependent S-phase response to DNA damage. Mol Cell Biol 20:7751-

7763.

6. Wang, J.Y., Naderi, S., and Chen, T.T. 2001. Role of retinoblastoma

tumor suppressor protein in DNA damage response. Acta Oncol 40:689-

695.

7. Mayhew, C.N., Bosco, E.E., Fox, S.R., Okaya, T., Tarapore, P.,

Schwemberger, S.J., Babcock, G.F., Lentsch, A.B., Fukasawa, K., and

149 Knudsen, E.S. 2005. Liver-specific pRB loss results in ectopic cell cycle

entry and aberrant ploidy. Cancer Res 65:4568-4577.

8. Ishida, S., Huang, E., Zuzan, H., Spang, R., Leone, G., West, M., and

Nevins, J.R. 2001. Role for E2F in control of both DNA replication and

mitotic functions as revealed from DNA microarray analysis. Mol Cell Biol

21:4684-4699.

9. Berton, T.R., Mitchell, D.L., Guo, R., and Johnson, D.G. 2005. Regulation

of epidermal apoptosis and DNA repair by E2F1 in response to ultraviolet

B radiation. Oncogene 24:2449-2460.

10. Ceccarelli, C., Santini, D., Chieco, P., Taffurelli, M., Gamberini, M., Pileri,

S.A., and Marrano, D. 1998. Retinoblastoma (RB1) gene product

expression in breast carcinoma. Correlation with Ki-67 growth fraction and

biopathological profile. J Clin Pathol 51:818-824.

11. Loden, M., Stighall, M., Nielsen, N.H., Roos, G., Emdin, S.O., Ostlund, H.,

and Landberg, G. 2002. The cyclin D1 high and cyclin E high subgroups of

breast cancer: separate pathways in tumorogenesis based on pattern of

genetic aberrations and inactivation of the pRb node. Oncogene 21:4680-

4690.

12. Michalides, R., van Tinteren, H., Balkenende, A., Vermorken, J.B.,

Benraadt, J., Huldij, J., and van Diest, P. 2002. Cyclin A is a prognostic

indicator in early stage breast cancer with and without tamoxifen

treatment. Br J Cancer 86:402-408.

150 13. Nielsen, N.H., Arnerlov, C., Emdin, S.O., and Landberg, G. 1996. Cyclin E

overexpression, a negative prognostic factor in breast cancer with strong

correlation to oestrogen receptor status. Br J Cancer 74:874-880.

14. Keyomarsi, K., Tucker, S.L., Buchholz, T.A., Callister, M., Ding, Y.,

Hortobagyi, G.N., Bedrosian, I., Knickerbocker, C., Toyofuku, W., Lowe,

M., et al. 2002. Cyclin E and survival in patients with breast cancer. N Engl

J Med 347:1566-1575.

15. Dhillon, N.K., and Mudryj, M. 2002. Ectopic expression of cyclin E in

estrogen responsive cells abrogates antiestrogen mediated growth arrest.

Oncogene 21:4626-4634.

16. Watts, C.K., Brady, A., Sarcevic, B., deFazio, A., Musgrove, E.A., and

Sutherland, R.L. 1995. Antiestrogen inhibition of cell cycle progression in

breast cancer cells in associated with inhibition of cyclin-dependent kinase

activity and decreased retinoblastoma protein phosphorylation. Mol

Endocrinol 9:1804-1813.

17. Leung, B.S. 1987. Mode of estrogen action on cell proliferation in CAMA-1

cells: III. Effect of antiestrogen. Anticancer Res 7:219-225.

18. Nielsen, N.H., Emdin, S.O., Cajander, J., and Landberg, G. 1997.

Deregulation of cyclin E and D1 in breast cancer is associated with

inactivation of the retinoblastoma protein. Oncogene 14:295-304.

19. Ma, X.J., Wang, Z., Ryan, P.D., Isakoff, S.J., Barmettler, A., Fuller, A.,

Muir, B., Mohapatra, G., Salunga, R., Tuggle, J.T., et al. 2004. A two-gene

151 expression ratio predicts clinical outcome in breast cancer patients treated

with tamoxifen. Cancer Cell 5:607-616.

20. van de Vijver, M.J., He, Y.D., van't Veer, L.J., Dai, H., Hart, A.A., Voskuil,

D.W., Schreiber, G.J., Peterse, J.L., Roberts, C., Marton, M.J., et al. 2002.

A gene-expression signature as a predictor of survival in breast cancer. N

Engl J Med 347:1999-2009.

21. Singh, P., Coe, J., and Hong, W. 1995. A role for retinoblastoma protein in

potentiating transcriptional activation by the glucocorticoid receptor.

Nature 374:562-565.

22. Lu, J., and Danielsen, M. 1998. Differential regulation of androgen and

glucocorticoid receptors by retinoblastoma protein. J Biol Chem

273:31528-31533.

23. Yeh, S., Miyamoto, H., Nishimura, K., Kang, H., Ludlow, J., Hsiao, P.,

Wang, C., Su, C., and Chang, C. 1998. Retinoblastoma, a tumor

suppressor, is a coactivator for the androgen receptor in human prostate

cancer DU145 cells. Biochem Biophys Res Commun 248:361-367.

24. Abbondanza, C., Medici, N., Nigro, V., Rossi, V., Gallo, L., Piluso, G.,

Belsito, A., Roscigno, A., Bontempo, P., Puca, A.A., et al. 2000. The

retinoblastoma-interacting zinc-finger protein RIZ is a downstream effector

of estrogen action. Proc Natl Acad Sci U S A 97:3130-3135.

25. Batsche, E., Desroches, J., Bilodeau, S., Gauthier, Y., and Drouin, J.

2005. Rb enhances p160/SRC coactivator-dependent activity of nuclear

receptors and hormone responsiveness. J Biol Chem 280:19746-19756.

152 26. Simin, K., Wu, H., Lu, L., Pinkel, D., Albertson, D., Cardiff, R.D., and Van

Dyke, T. 2004. pRb inactivation in mammary cells reveals common

mechanisms for tumor initiation and progression in divergent epithelia.

PLoS Biol 2:E22.

153