p53-Dependent accelerated senescence induced by ionizing radiation in breast tumour cells

Ionizing radiation has been reported to promote accelerated or premature senescence in both normal and tumour cell lines. The current studies were designed to characterize the accelerated senescence response to radiation in the breast tumour cell in terms of its dependence on functional p53 and its relationship to telomerase activity, telomere lengths, expression of human telomerase reverse transcriptase (hTERT, the catalytic subunit of telomerase) and human telomerase RNA (hTR, the RNA subunit of telomerase), as well as the induction of cytogenetic aberrations. Studies were performed in p53 wild-type MCF-7 cells, MCF-7/E6 cells with attenuated p53 function, MDA-MB231 cells with mutant p53 and MCF-7/hTERT cells with constitutive expression of hTERT. Telomerase activity was measured by the telomeric repeat amplification protocol (TRAP assay), telomere lengths by the terminal restriction fragment (TRF) assay, hTR and hTERT expression by reverse transcriptase-polymerase chain reaction (RT-PCR), senescence by β-galactosidase staining, and apoptosis by TdT-mediated d-UTP-X nick-end labelling (TUNEL assay). Widespread and extensive expression of β-galactosidase, a marker of cellular senescence, was evident in MCF-7 breast tumour cells following exposure to 10 Gy of ionizing radiation. Radiation did not suppress expression of either hTERT or hTR, alter telomerase activity or induce telomere shortening. Senescence arrest was also observed in irradiated MCF-7/hTERT cells, which have elongated telomeres due to the ectopic expression of the catalytic component of telomerase. In contrast to MCF-7 cells, irradiated MDA-MB231 breast tumour cells and MCF-7/E6 cells failed to senesce and instead demonstrated a delayed apoptotic cell death. Irradiation produced chromosome end associated abnormalities, including end-to-end fusions (an indicator of telomere dysfunction) in MCF-7 cells, MCF-7/hTERT cells, as well as in MCF-7/E6 cells. When cells were maintained in culture following irradiation, proliferative recovery was evident exclusively after senescence while the cell lines which responded to radiation by apoptosis continued to decline in cell number. Accelerated senescence in response to ionizing radiation is p53 dependent and associated with telomer dysfunction but is unrelated to changes in telomerase activity or telomere lengths, expression of hTERT and hTR. In the absence of functional p53, cells are unable to arrest for an extended period, resulting in apoptotic cell death while accelerated senescence in cells expressing p53 is succeeded by proliferative recovery.

[1]  S. Kramer.  From a cellular level. , 2006, Healthcare informatics : the business magazine for information and communication systems.

[2]  L. Elmore,et al.  Evasion of a Single-Step, Chemotherapy-Induced Senescence in Breast Cancer Cells: Implications for Treatment Response , 2005, Clinical Cancer Research.

[3]  Szu-Yu Chen,et al.  Escape from therapy-induced accelerated cellular senescence in p53-null lung cancer cells and in human lung cancers. , 2005, Cancer research.

[4]  M. Manns,et al.  The cellular level of telomere dysfunction determines induction of senescence or apoptosis in vivo , 2005, EMBO reports.

[5]  V. Rotter,et al.  p53-dependent Down-regulation of Telomerase Is Mediated by p21waf1* , 2004, Journal of Biological Chemistry.

[6]  C. Gennings,et al.  Potentiation of cell killing by fractionated radiation and suppression of proliferative recovery in MCF-7 breast tumor cells by the Vitamin D3 analog EB 1089 , 2004, The Journal of Steroid Biochemistry and Molecular Biology.

[7]  R. Kanda,et al.  Sister chromatid exchanges in ring chromosomes following X‐irradiation of human lymphocytes , 2004, International journal of radiation biology.

[8]  J. Murnane,et al.  Human fibroblasts expressing hTERT show remarkable karyotype stability even after exposure to ionizing radiation , 2004, Cytogenetic and Genome Research.

[9]  J. Haveman,et al.  Differential Response to Radiation of TP53-Inactivated Cells by Overexpression of Dominant-Negative Mutant TP53 or HPVE6 , 2004, Radiation research.

[10]  T. Cloughesy,et al.  p53 disruption profoundly alters the response of human glioblastoma cells to DNA topoisomerase I inhibition , 2004, Oncogene.

[11]  H. Schmidberger,et al.  Role of DNA‐dependent protein kinase in the process of radiation‐induced aberration formation , 2004, International journal of radiation biology.

[12]  K. Ang,et al.  C225 antiepidermal growth factor receptor antibody enhances the efficacy of docetaxel chemoradiotherapy. , 2003, International journal of radiation oncology, biology, physics.

[13]  Huichen Wang,et al.  DNA damage checkpoint control in cells exposed to ionizing radiation , 2003, Oncogene.

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

[15]  M. Spitz,et al.  Telomere dysfunction: a potential cancer predisposition factor. , 2003, Journal of the National Cancer Institute.

[16]  Masashi Narita,et al.  Reversal of human cellular senescence: roles of the p53 and p16 pathways , 2003, The EMBO journal.

[17]  A. Rebbaa,et al.  Caspase inhibition switches doxorubicin-induced apoptosis to senescence , 2003, Oncogene.

[18]  Robert L Fine,et al.  Fas-mediated apoptosis is dependent on wild-type p53 status in human cancer cells expressing a temperature-sensitive p53 mutant alanine-143. , 2003, Cancer research.

[19]  J. Remacle,et al.  Down-regulation and decreased activity of cyclin-dependent kinase 2 in H2O2-induced premature senescence. , 2003, The international journal of biochemistry & cell biology.

[20]  I. Germano,et al.  Molecular response of human glioblastoma multiforme cells to ionizing radiation: cell cycle arrest, modulation of the expression of cyclin-dependent kinase inhibitors, and autophagy. , 2003, Journal of neurosurgery.

[21]  W. J. DeCoursey,et al.  Introduction: Probability and Statistics , 2003 .

[22]  T. von Zglinicki.  Replicative senescence and the art of counting. , 2003, Experimental gerontology.

[23]  J. Haveman,et al.  Importance of TP53 and RB in the Repair of Potentially Lethal Damage and Induction of Color Junctions after Exposure to Ionizing Radiation , 2002, Radiation research.

[24]  J. Remacle,et al.  UVB-induced premature senescence of human diploid skin fibroblasts. , 2002, The international journal of biochemistry & cell biology.

[25]  O. Pereira-smith,et al.  Expression of Human Telomerase (hTERT) Does Not Prevent Stress-induced Senescence in Normal Human Fibroblasts but Protects the Cells from Stress-induced Apoptosis and Necrosis* , 2002, The Journal of Biological Chemistry.

[26]  L. Elmore,et al.  Adriamycin-induced Senescence in Breast Tumor Cells Involves Functional p53 and Telomere Dysfunction* , 2002, The Journal of Biological Chemistry.

[27]  J. Campisi,et al.  Reversible Manipulation of Telomerase Expression and Telomere Length , 2002, The Journal of Biological Chemistry.

[28]  P. Harari,et al.  Modulation of radiation response and tumor-induced angiogenesis after epidermal growth factor receptor inhibition by ZD1839 (Iressa). , 2002, Cancer research.

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

[30]  L. Gollahon,et al.  Telomerase Protects Cancer-Prone Human Cells from Chromosomal Instability and Spontaneous Immortalization , 2002, Cancer biology & therapy.

[31]  S. Jackson,et al.  Sensing and repairing DNA double-strand breaks. , 2002, Carcinogenesis.

[32]  A. Smogorzewska,et al.  Senescence Induced by Altered Telomere State, Not Telomere Loss , 2002, Science.

[33]  G. Campiani,et al.  Caspase‐3 is not essential for DNA fragmentation in MCF‐7 cells during apoptosis induced by the pyrrolo‐1,5‐benzoxazepine, PBOX‐6 , 2002, FEBS letters.

[34]  S. Joel,et al.  DNA damage is able to induce senescence in tumor cells in vitro and in vivo. , 2002, Cancer research.

[35]  J. Campisi,et al.  Cellular senescence, cancer and aging: the telomere connection , 2001, Experimental Gerontology.

[36]  Margaret A. Strong,et al.  The Shortest Telomere, Not Average Telomere Length, Is Critical for Cell Viability and Chromosome Stability , 2001, Cell.

[37]  I. Roninson,et al.  If not apoptosis, then what? Treatment-induced senescence and mitotic catastrophe in tumor cells. , 2001, Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy.

[38]  M. Mayberg,et al.  Induction of a Senescence-Like Phenotype in Bovine Aortic Endothelial Cells by Ionizing Radiation , 2001, Radiation research.

[39]  I. H. Engels,et al.  Ionizing radiation but not anticancer drugs causes cell cycle arrest and failure to activate the mitochondrial death pathway in MCF-7 breast carcinoma cells , 2001, Oncogene.

[40]  Y. Hannun,et al.  Role of Ceramide in Mediating the Inhibition of Telomerase Activity in A549 Human Lung Adenocarcinoma Cells* , 2001, The Journal of Biological Chemistry.

[41]  K. Weber,et al.  Induction of Telomerase Activity by Irradiation in Human Lymphoblasts , 2001, Radiation research.

[42]  P. Lambin,et al.  In vivo antitumor effect of vascular targeting combined with either ionizing radiation or anti-angiogenesis treatment. , 2001, International journal of radiation oncology, biology, physics.

[43]  Keiji Suzuki,et al.  Radiation-Induced Senescence-like Growth Arrest Requires TP53 Function but not Telomere Shortening , 2001, Radiation research.

[44]  E. Blackburn,et al.  Telomere states and cell fates , 2000, Nature.

[45]  P. Finnon,et al.  Upregulation of telomerase activity by X-irradiation in mouse leukaemia cells is independent of Tert, Terc, Tnks and Myc transcription. , 2000, Carcinogenesis.

[46]  S. Kyo,et al.  Adenoviral expression of p53 represses telomerase activity through down-regulation of human telomerase reverse transcriptase transcription. , 2000, Clinical cancer research : an official journal of the American Association for Cancer Research.

[47]  S. Grant,et al.  Induction of apoptosis in U937 human leukemia cells by suberoylanilide hydroxamic acid (SAHA) proceeds through pathways that are regulated by Bcl-2/Bcl-XL, c-Jun, and p21CIP1, but independent of p53 , 1999, Oncogene.

[48]  I. Roninson,et al.  Role of p53 and p21waf1/cip1 in senescence-like terminal proliferation arrest induced in human tumor cells by chemotherapeutic drugs , 1999, Oncogene.

[49]  E. Kandel,et al.  A senescence-like phenotype distinguishes tumor cells that undergo terminal proliferation arrest after exposure to anticancer agents. , 1999, Cancer research.

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

[51]  A. W. Boersma,et al.  Distinct p53‐independent apoptotic cell death signalling pathways in testicular germ cell tumour cell lines , 1999, International journal of cancer.

[52]  J. M. Lee,et al.  p53 regulates a G2 checkpoint through cyclin B1. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

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

[54]  M. Hande,et al.  Induction of telomerase activity and chromosome aberrations in human tumour cell lines following X-irradiation. , 1998, Mutation research.

[55]  Alan G. Porter,et al.  Caspase-3 Is Required for DNA Fragmentation and Morphological Changes Associated with Apoptosis* , 1998, The Journal of Biological Chemistry.

[56]  V. Rotter,et al.  p53-dependent cell cycle control: response to genotoxic stress. , 1998, Seminars in cancer biology.

[57]  M. S. Orr,et al.  Influence of ionizing radiation on proliferation, c-myc expression and the induction of apoptotic cell death in two breast tumour cell lines differing in p53 status. , 1997, International journal of radiation biology.

[58]  E. Carosella,et al.  Telomerase activity during the cell cycle and in gamma-irradiated hematopoietic cells , 1997, Leukemia.

[59]  M. S. Orr,et al.  Ionizing radiation and teniposide increase p21(waf1/cip1) and promote Rb dephosphorylation but fail to suppress E2F activity in MCF-7 breast tumor cells. , 1997, Molecular pharmacology.

[60]  C. Bellamy,et al.  p53 and apoptosis. , 1997, British medical bulletin.

[61]  P. Nicotera,et al.  Radiation induced apoptosis. , 1996, Mutation research.

[62]  H. Tanke,et al.  Heterogeneity in telomere length of human chromosomes. , 1996, Human molecular genetics.

[63]  D. Louis,et al.  Mutational analysis of CDKN2 (MTS1/p16ink4) in human breast carcinomas. , 1994, Cancer research.

[64]  A. Haimovitz-Friedman,et al.  Ionizing radiation acts on cellular membranes to generate ceramide and initiate apoptosis , 1994, The Journal of experimental medicine.

[65]  B. Vogelstein,et al.  Participation of p53 protein in the cellular response to DNA damage. , 1991, Cancer research.

[66]  J. Little,et al.  Effects of X-irradiation on cell-cycle progression, induction of chromosomal aberrations and cell killing in ataxia telangiectasia (AT) fibroblasts. , 1985, Mutation research.

[67]  J. Little,et al.  Induction of chromosome aberrations and sister chromatid exchanges by X rays in density-inhibited cultures of mouse 10T1/2 cells. , 1981, Radiation research.

[68]  J. Fried,et al.  Flow cytofluorometric analysis of cell cycle distributions using propidium iodide. Properties of the method and mathematical analysis of the data , 1976, The Journal of cell biology.