Impairment of the DNA Repair and Growth Arrest Pathways by p53R2 Silencing Enhances DNA Damage–Induced Apoptosis in a p53-Dependent Manner in Prostate Cancer Cells

p53R2 is a p53-inducible ribonucleotide reductase that contributes to DNA repair by supplying deoxynucleotide triphosphate pools in response to DNA damage. In this study, we found that p53R2 was overexpressed in prostate tumor cell lines compared with immortalized prostatic epithelial cells and that the protein was induced upon DNA damage. We investigated the effects of p53R2 silencing on DNA damage in LNCaP cells (wild-type p53). Silencing p53R2 potentiated the apoptotic effects of ionizing radiation and doxorubicin treatment as shown by increased sub-G1 content and decreased colony formation. This sensitizing effect was specific to DNA-damaging agents. Comet assay and γ-H2AX phosphorylation status showed that the decreased p53R2 levels inhibited DNA repair. Silencing p53R2 also reduced the levels of p21WAF1/CIP1 at the posttranscriptional level, suggesting links between the p53-dependent DNA repair and cell cycle arrest pathways. Using LNCaP sublines stably expressing dominant-negative mutant p53, we found that the sensitizing effect of p53R2 silencing is mediated by p53-dependent apoptosis pathways. In the LNCaP sublines (R273H, R248W, and G245S) that have defects in inducing p53-dependent apoptosis, p53R2 silencing did not potentiate DNA damage–induced apoptosis, whereas p53R2 silencing was effective in a LNCaP subline (P151S) which retains the ability to induce p53-dependent apoptosis. This study shows that p53R2 is a potential therapeutic target that could be used to enhance the effectiveness of ionizing radiation or DNA-damaging chemotherapy in a subset of patients with prostate cancer. (Mol Cancer Res 2008;6(5):808–18)

[1]  S. Natsugoe,et al.  Expression of p53R2 Is Related to Prognosis in Patients with Esophageal Squamous Cell Carcinoma , 2006, Clinical Cancer Research.

[2]  T. Oyama,et al.  P53R2, p53 inducible ribonucleotide reductase gene, correlated with tumor progression of non-small cell lung cancer. , 2006, Anticancer research.

[3]  Y. Lee,et al.  GTI-2501, an antisense agent targeting R1, the large subunit of human ribonucleotide reductase, shows potent anti-tumor activity against a variety of tumors. , 2006, International journal of oncology.

[4]  J. Gregg,et al.  Profiling of gene expression changes caused by p53 gain‐of‐function mutant alleles in prostate cancer cells , 2005, The Prostate.

[5]  R. Schilsky,et al.  A phase I study of antisense oligonucleotide GTI-2040 given by continuous intravenous infusion in patients with advanced solid tumors. , 2005, Annals of oncology : official journal of the European Society for Medical Oncology.

[6]  A. Mizuno,et al.  Silencing of the p53R2 gene by RNA interference inhibits growth and enhances 5-fluorouracil sensitivity of oral cancer cells. , 2005, Cancer letters.

[7]  Wafik S El-Deiry,et al.  P53 and radiation responses , 2003, Oncogene.

[8]  Yusuke Nakamura,et al.  Impaired function of p53R2 in Rrm2b-null mice causes severe renal failure through attenuation of dNTP pools , 2003, Nature Genetics.

[9]  V. Lam,et al.  GTI-2040, an antisense agent targeting the small subunit component (R2) of human ribonucleotide reductase, shows potent antitumor activity against a variety of tumors. , 2003, Cancer research.

[10]  R. deVere White,et al.  Androgen-independent growth of LNCaP prostate cancer cells is mediated by gain-of-function mutant p53. , 2003, Cancer research.

[11]  A. Chabes,et al.  Sequences downstream of the transcription initiation site are important for proper initiation and regulation of mouse ribonucleotide reductase R2 gene transcription. , 2003, European journal of biochemistry.

[12]  Lijun Xue,et al.  Wild-type p53 regulates human ribonucleotide reductase by protein-protein interaction with p53R2 as well as hRRM2 subunits. , 2003, Cancer research.

[13]  V. Rotter,et al.  The onset of p53-dependent DNA repair or apoptosis is determined by the level of accumulated damaged DNA. , 2002, Carcinogenesis.

[14]  S. Chi,et al.  Expression and mutation analyses of P53R2, a newly identified p53 target for DNA repair in human gastric carcinoma , 2002, International journal of cancer.

[15]  R. deVere White,et al.  Complex functions of mutant p53 alleles from human prostate cancer , 2002, The Prostate.

[16]  Y. Nakamura,et al.  p53R2-dependent pathway for DNA synthesis in a p53-regulated cell cycle checkpoint. , 2001, Cancer research.

[17]  Yusuke Nakamura,et al.  Mammalian p53R2 Protein Forms an Active Ribonucleotide Reductasein Vitro with the R1 Protein, Which Is Expressed Both in Resting Cells in Response to DNA Damage and in Proliferating Cells* , 2001, The Journal of Biological Chemistry.

[18]  X. Tu,et al.  GTI-2501. Lorus Therapeutics. , 2001, Current opinion in investigational drugs.

[19]  Z. Fan,et al.  Protection against chemotherapy-induced cytotoxicity by cyclin-dependent kinase inhibitors (CKI) in CKI-responsive cells compared with CKI-unresponsive cells , 2001, Oncogene.

[20]  J. Stubbe,et al.  Ribonucleotide reductases: the link between an RNA and a DNA world? , 2000, Current opinion in structural biology.

[21]  Yusuke Nakamura,et al.  p53AIP1, a Potential Mediator of p53-Dependent Apoptosis, and Its Regulation by Ser-46-Phosphorylated p53 , 2000, Cell.

[22]  F. Kashanchi,et al.  Overexpression of p21waf1 in Human T-Cell Lymphotropic Virus Type 1-Infected Cells and Its Association with Cyclin A/cdk2 , 2000, Journal of Virology.

[23]  T. Taniguchi,et al.  Reprimo, a New Candidate Mediator of the p53-mediated Cell Cycle Arrest at the G2 Phase* , 2000, The Journal of Biological Chemistry.

[24]  K. Kinzler,et al.  Cooperative effects of genes controlling the G(2)/M checkpoint. , 2000, Genes & development.

[25]  A. Sartorelli,et al.  Triapine (3-aminopyridine-2-carboxaldehyde- thiosemicarbazone): A potent inhibitor of ribonucleotide reductase activity with broad spectrum antitumor activity. , 2000, Biochemical pharmacology.

[26]  S. Lowe,et al.  PERP, an apoptosis-associated target of p53, is a novel member of the PMP-22/gas3 family. , 2000, Genes & development.

[27]  Yusuke Nakamura,et al.  A ribonucleotide reductase gene involved in a p53-dependent cell-cycle checkpoint for DNA damage , 2000, Nature.

[28]  K. Krejcy,et al.  Gemcitabine in Non-Small Cell Lung Cancer (NSCLC) , 2000, Investigational New Drugs.

[29]  D. Bruner,et al.  Prostate cancer elder alert. Living with treatment choices and outcomes. , 2000, Journal of gerontological nursing.

[30]  H. Saya,et al.  A point mutation of human p53, which was not detected as a mutation by a yeast functional assay, led to apoptosis but not p21Waf1/Cip1/Sdi1 expression in response to ionizing radiation in a human osteosarcoma cell line, Saos-2. , 1999, International journal of radiation oncology, biology, physics.

[31]  K. Kinzler,et al.  14-3-3σ is required to prevent mitotic catastrophe after DNA damage , 1999, Nature.

[32]  M. Kaneuchi,et al.  Induction of apoptosis by the p53‐273L (Arg→Leu) mutant in HSC3 cells without transactivation of p21Waf1/Cip1/Sdi1 and bax , 1999, Molecular carcinogenesis.

[33]  K. Kinzler,et al.  Disruption of p53 in human cancer cells alters the responses to therapeutic agents. , 1999, The Journal of clinical investigation.

[34]  G. Ross.  Induction of cell death by radiotherapy. , 1999, Endocrine-related cancer.

[35]  A. Levine,et al.  Mutant p53 gain of function: differential effects of different p53 mutants on resistance of cultured cells to chemotherapy , 1999, Oncogene.

[36]  K. Kinzler,et al.  Requirement for p53 and p21 to sustain G2 arrest after DNA damage. , 1998, Science.

[37]  D. Hunting,et al.  p21-induced cycle arrest in G1 protects cells from apoptosis induced by UV-irradiation or RNA polymerase II blockage , 1998, Oncogene.

[38]  V. Pedraza,et al.  Relationship between DNA damage, rejoining and cell killing by radiation in mammalian cells. , 1996, Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology.

[39]  C C Ling,et al.  Radiation-induced apoptosis: relevance to radiotherapy. , 1995, International journal of radiation oncology, biology, physics.

[40]  K. Kohn,et al.  p53 gene mutations are associated with decreased sensitivity of human lymphoma cells to DNA damaging agents. , 1994, Cancer research.

[41]  Y. Oshika,et al.  P-glycoprotein-mediated acquired multidrug resistance of human lung cancer cells in vivo. , 1996, British Journal of Cancer.

[42]  Lijun Xue,et al.  Ribonucleotide reductase small subunit p53R2 facilitates p21 induction of G1 arrest under UV irradiation. , 2007, Cancer research.

[43]  A. Jemal,et al.  Cancer Statistics, 2007 , 2007, CA: a cancer journal for clinicians.

[44]  N. Dubrawsky.  Cancer statistics , 1989, CA: a cancer journal for clinicians.