Telomere maintenance and cell cycle regulation in spontaneously immortalized T-cell lines from Nijmegen breakage syndrome patients

Nijmegen breakage syndrome (NBS) is a rare genetic instability syndrome associated with a high incidence of lymphoid malignancies. The NBS1 protein has been implicated in telomere biology suggesting that cells from NBS patients might have deficient telomere maintenance capacity. In this study we characterized spontaneously immortalized T-cell lines derived from three NBS patients regarding growth characteristics, telomere biology, expression of cell-cycle regulators, and response to DNA damage to understand the role of NBS1 in the immortalization process. In all the NBS T-cell lines the acquisition of an immortal phenotype was associated with telomere length stabilization, high telomerase activity, and increased mRNA expression of the catalytic subunit of telomerase (hTERT), together with c-myc up-regulation. Our findings provide evidence that telomere length maintenance was intact in the T lymphocytes in the absence of a full-length NBS protein, presumably due to the presence of an alternatively transcribed NBS protein of 70 kDa. Normal protein expression patterns for pRb and p53 in all the immortal lines coincided with altered expression of some cell-cycle proteins as well as with an impaired G1/S arrest after gamma irradiation, despite a seemingly normal p53/p21 pathway. The here described, spontaneously immortalized NBS derived T-cell lines can be useful in future analysis of the biologic effects in the NBS.     

A distinct response to endogenous DNA damage in the development of Nbs1-deficient cortical neurons

Microcephaly is a clinical characteristic for human nijmegen breakage syndrome (NBS, mutated in NBS1 gene), a chromosomal instability syndrome. However, the underlying molecular pathogenesis remains elusive. In the present study, we demonstrate that neuronal disruption of NBS (Nbn in mice) causes microcephaly characterized by the reduction of cerebral cortex and corpus callosum, recapitulating neuronal anomalies in human NBS. Nbs1-deficient neocortex shows accumulative endogenous DNA damage and defective activation of Ataxia telangiectasia and Rad3-related (ATR)-Chk1 pathway upon DNA damage. Notably, in contrast to massive apoptotic cell death in Nbs1-deficient cerebella, activation of p53 leads to a defective neuroprogenitor proliferation in neocortex, likely via specific persistent induction of hematopoietic zinc finger (Hzf) that preferentially promotes p53-mediated cell cycle arrest whilst inhibiting apoptosis. Moreover, Trp53 mutations substantially rescue the microcephaly in Nbs1-deficient mice. Thus, the present results reveal the first clue that developing neurons at different regions of brain selectively respond to endogenous DNA damage, and underscore an important role for Nbs1 in neurogenesis.     

Activation of p53 by nutlin-3a induces apoptosis and cellular senescence in human glioblastoma multiforme

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in adults. Despite concerted efforts to improve current therapies and develop novel clinical approaches, patient survival remains poor. As such, increasing attention has focused on developing new therapeutic strategies that specifically target the apoptotic pathway in order to improve treatment responses. Recently, nutlins, small-molecule antagonists of MDM2, have been developed to inhibit p53-MDM2 interaction and activate p53 signaling in cancer cells. Glioma cell lines and primary cultured glioblastoma cells were treated with nutlin-3a. Nutlin-3a induced p53-dependent G1- and G2-M cell cycle arrest and apoptosis in glioma cell lines with normal TP53 status. In addition, nutlin-arrested glioma cells show morphological features of senescence and persistent induction of p21 protein. Furthermore, senescence induced by nutlin-3a might be depending on mTOR pathway activity. In wild-type TP53 primary cultured cells, exposure to nutlin-3a resulted in variable degrees of apoptosis as well as cellular features of senescence. Nutlin-3a-induced apoptosis and senescence were firmly dependent on the presence of functional p53, as revealed by the fact that glioblastoma cells with knockdown p53 with specific siRNA, or cells with mutated or functionally impaired p53 pathway, were completely insensitive to the drug. Finally, we also found that nutlin-3a increased response of glioma cells to radiation therapy. The results provide a basis for the rational use of MDM2 antagonists as a novel treatment option for glioblastoma patients.     

Influence of Arg72Pro polymorphisms of TP53 on the response of buccal cells to radiotherapy

Polymorphisms in the TP53 gene codon 72 (Arg72Pro) influence apoptosis induction and DNA damage repair. We evaluated how variants of protein p53 (p53Arg and p53Pro) affect cell death and DNA damage repair by analyzing the frequencies of karyorrhexis and micronuclei. There were significant differences in the frequency of karyorrhexis between the three p53 genotypes (Arg/Arg, Arg/Pro, and Pro/Pro), between samples taken before and after radiotherapy, and between patients and controls. The frequency of micronucleated cells increased significantly after radiotherapy. There were no significant differences in the micronucleus frequency in healthy tissues of these patients compared to controls, or in the comparisons between the three genotypes. We conclude that Arg72Pro polymorphism influences cell apoptotic capacity. This is the first study investigating karyorrhexis and micronuclei, as indicators of apoptosis after radiotherapy, and how these indicators are influenced by the TP53 polymorphism Arg72Pro.     

Correlation between unirradiated cell TP53 protein levels and radiosensitivity in MOLT-4 cells.

MOLT-4 cells undergo apoptosis after X irradiation. A radiosensitive variant, MOLT-4N1, and a radioresistant variant, MOLT-4N2, have been studied with respect to their radiosensitivity and its relationship to the levels of TP53 protein (formerly known as p53). X irradiation induces apoptosis in both cell lines with the following difference: The induction of apoptosis in MOLT-4N2 cells occurred later than in MOLT-4N1 cells as determined by the morphological changes and DNA fragmentation. The levels of cell death measured by the dye exclusion test coincided with the levels of apoptosis in both cell lines, suggesting that radiation-induced cell killing is determined by the induction of apoptosis. Unirradiated MOLT-4N1 cells contained a significantly higher intracellular level of TP53 protein and a much higher level of TP53 mRNA compared to MOLT-4N2 cells. X irradiation led to an accumulation of TP53 protein in both cell lines that was greater in MOLT-4N1 cells. This accumulation of TP53 protein preceded changes in DNA degradation and ladder formation and in nuclear morphology. These results strongly suggest that the radiosensitivity of MOLT-4 cells correlates well with the unirradiated control levels of TP53 mRNA and TP53 protein, and that the quantitative levels of TP53 protein must reach a threshold for the cells to undergo apoptosis.     

Distinct functions for WRN and TP53 in a shared pathway of cellular response to 1-beta-D-arabinofuranosylcytosine and bleomycin

Mutations in the WRN or the TP53 genes lead to spontaneous genetic instability, an elevated risk of tumor formation, and sensitivity to compounds that interfere with DNA replication, such as camptothecin and DNA interstrand cross-linking drugs. We investigated the hypothesis that WRN and TP53 are involved in cellular responses to DNA replication-blocking lesions by exposing WRN deficient and TP53 mutant lymphoblastoid cell lines (LCLs) to 1-beta-d-arabinofuranosylcytosine (AraC) and bleomycin. Loss of WRN or TP53 function resulted in induction of apoptosis and lesser proliferative survival in response to AraC and bleomycin. WRN and TP53 operate in a shared DNA damage response pathway, since in cells in which TP53 was inactivated by SV-40 transformation, no difference in AraC and bleomycin sensitivity was found regardless of WRN status. In contrast to TP53 mutant LCLs, WRN-deficient cells showed unaffected cell cycle arrest after AraC and bleomycin exposure, which indicates that WRN is not involved in DNA damage-activated cell cycle arrest. Neither WRN nor TP53 deficiency affected cellular recovery from exposure to AraC and bleomycin, which disagrees with a direct role in repair of these DNA lesions. Our results indicate that WRN and TP53 perform different functions in a shared DNA damage response pathway.     

Gene polymorphisms,apoptotic capacity and cancer risk

Programmed cell death has been implicated in various aspects of cancer development. Apoptotic capacity is a subject of significant interindividual variations, which are largely attributed to hereditary traits. Single nucleotide polymorphisms (SNPs) located within cell death genes may influence cancer risk in various ways. Low activity of apoptosis may favor cancer development because of the failure to eliminate cellular clones carrying DNA damage and propensity to inflammation, but may also protect against malignancy due to preservation of antitumor immune cells. Phenotyping studies assessing cell death rate in cancer patients versus healthy controls are limited in number and produced controversial results. TP53 R72P polymorphism is the only SNP whose functional impact on apoptotic response has been replicated in independent investigations. Intriguingly, meta-analysis of TP53 genotyping studies has provided evidence for the association between apoptosis-deficient TP53 genotype and tumor susceptibility. Systematic analysis of cancer-predisposing relevance of other apoptotic gene SNPs remains to be done. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Absence of p53 enhances growth defects and etoposide sensitivity of human cells lacking the Bloom syndrome helicase BLM

The Bloom syndrome helicase BLM and the tumor-suppressor protein p53 play important roles in preserving genome integrity. Here, we knock out the genes for BLM and p53 in a human pre-B-cell line, Nalm-6. We show that p53 plays an important role in cell proliferation, but not apoptosis, when BLM is absent. Intriguingly, despite the apoptotic function of p53, BLM(/)TP53(/) cells were more sensitive than either single mutant to etoposide, an anticancer agent that poisons DNA topoisomerase II. Our results suggest a direct, BLM-independent role for p53 in etoposide-induced, topoisomerase II-mediated DNA damage in human cells.     

Comparison of endogenous TP53 genomic status with clonogenicity and different modes of cell death after X irradiation

Although extensive data indicate that the tumor suppressor TP53 modifies the radiation responses of human and rodent cells, the exact relationship between TP53 and radiation responsiveness remains controversial. To elucidate the relevance of endogenous TP53 genomic status to radiosensitivity in a cell-type-independent manner, different cells of 10 human tumor cell lines with different tissues of origin were examined for TP53 status. The TP53 status was compared with radiation-related cell survival parameters (D(q), D(0), SF2) and with the mode of cell death. Different modes of cell death were examined by measuring radiation-induced micronucleation, apoptosis and abnormal cells. Alterations of the TP53 gene were detected in eight cell lines. No splicing mutation was found. Five cell lines showed codon 68 polymorphism. Codon 72 alterations were found in four cell lines. "Hot spot" alterations were detected in only two of 10 cell lines. Although the cells differed widely in survival parameters (D(q), D(0), SF2) and modes of cell death (micronucleation/apoptosis/abnormal cells) after irradiation, significant cell-type-independent correlations were obtained between the multiple cell death parameter micronucleation/apoptosis/abnormal cells and SF2 (P < 0.001) and D(q) (P = 0.003). Moreover, cells with a wild-type TP53 gene were more resistant to X rays than cells with a mutated TP53 gene or cells that were TP53-deficient. The alterations within exons 5-10 of the TP53 correlated with a enhanced radiosensitivity. For the first time, we demonstrated a correlation between endogenous genetic alterations within exons 5-10 of TP53 and radiation-related cell survival and cell death. This indicates a new molecular relevance of TP53 status to intrinsic cellular radiosensitivity.     

DNA damage induces two distinct modes of cell death in ovarian carcinomas

Activation of p53 by cellular stress may lead to either cell cycle arrest or apoptotic cell death. Restrictions in a cell's ability to halt the cell cycle might, in turn, cause mitotic catastrophe, a delayed type of cell death with distinct morphological features. Here, we have investigated the contribution of p53 and caspase-2 to apoptotic cell death and mitotic catastrophe in cisplatin-treated ovarian carcinoma cell lines. We report that both functional p53 and caspase-2 were required for the apoptotic response, which was preceded by translocation of nuclear caspase-2 to the cytoplasm. In the absence of functional p53, cisplatin treatment resulted in caspase-2-independent mitotic catastrophe followed by necrosis. In these cells, apoptotic functions could be restored by transient expression of wt p53. Hence, p53 appeared to act as a switch between apoptosis and mitotic catastrophe followed by necrosis-like lysis in this experimental model. Further, we show that inhibition of Chk2, and/or 14-3-3sigma deficiency, sensitized cells to undergo mitotic catastrophe upon treatment with DNA-damaging agents. However, apoptotic cell death seemed to be the final outcome of this process. Thus, we hypothesize that the final mode of cell death triggered by DNA damage in ovarian carcinoma cells is determined by the profile of proteins involved in the regulation of the cell cycle, such as p53- and Chk2-related proteins.     

Activation of calcium-sensing receptor increases TRPC3 expression in rat cardiomyocytes

Tumor protein p53-induced nuclear protein 1 (TP53INP1) is a well known stress-induced protein that plays a role in both cell cycle arrest and p53-mediated apoptosis. Loss of TP53INP1 expression has been reported in human melanoma, breast carcinoma, and gastric cancer. However, TP53INP1 expression and its regulatory mechanism in esophageal squamous cell carcinoma (ESCC) remain unclear. Our findings are in agreement with previous reports in that the expression of TP53INP1 was downregulated in 28% (10/36 cases) of ESCC lesions, and this was accompanied by significant promoter methylation. Overexpression of TP53INP1 induced G1 cell cycle arrest and increased apoptosis in ESCC cell lines (EC-1, EC-109, EC-9706). Furthermore, our study showed that the oncoprotein c-Myc bound to the core promoter of TP53INP1 and recruited DNA methyltransferase 3A to methylate the local promoter region, leading to the inhibition of TP53INP1 expression. Our findings revealed that TP53INP1 is a tumor suppressor in ESCC and that c-Myc-mediated DNA methylation-associated silencing of TP53INP1 contributed to the pathogenesis of human ESCC.     

Apoptosis in erythroid progenitors deprived of erythropoietin occurs during the G1 and S phases of the cell cycle without growth arrest or stabilization of wild-type p53.

Erythropoietin (Epo) inhibits apoptosis in murine proerythroblasts infected with the anemia-inducing strain of Friend virus (FVA cells). We have shown that the apoptotic process in FVA cell populations deprived of Epo is asynchronous as a result of a heterogeneity in Epo dependence among individual cells. Here we investigated whether apoptosis in FVA cells correlated with cell cycle phase or stabilization of p53 tumor suppressor protein. DNA analysis in nonapoptotic FVA cell subpopulations cultured without Epo demonstrated little change in the percentages of cells in G1,S, and G2/M phases over time. Analysis of the apoptotic subpopulation revealed high percentages of cells in G1 and S, with few cells in G2/M at any time. When cells were sorted from G1 and S phases prior to culture without Epo, apoptotic cells appeared at the same rate in both populations, indicating that no prior commitment step had occurred in either G1 or S phase. Steady-state wild-type p53 protein levels were very low in FVA cells compared with control cell lines and did not accumulate in Epo-deprived cultures; however, p53 protein did accumulate when FVA cells were treated with the DNA-damaging agent actinomycin D. These data indicate that erythroblast apoptosis caused by Epo deprivation (i) occurs throughout G1 and S phases and does not require cell cycle arrest, (ii) does not have a commitment event related to cell cycle phase, and (iii) is not associated with conformational changes or stabilization of wild-type p53 protein.     

ATR and Chk1 Suppress a Caspase-3–Dependent Apoptotic Response Following DNA Replication Stress

The related PIK-like kinases Ataxia-Telangiectasia Mutated (ATM) and ATM- and Rad3-related (ATR) play major roles in the regulation of cellular responses to DNA damage or replication stress. The pro-apoptotic role of ATM and p53 in response to ionizing radiation (IR) has been widely investigated. Much less is known about the control of apoptosis following DNA replication stress. Recent work indicates that Chk1, the downstream phosphorylation target of ATR, protects cells from apoptosis induced by DNA replication inhibitors as well as IR. The aim of the work reported here was to determine the roles of ATM- and ATR-protein kinase cascades in the control of apoptosis following replication stress and the relationship between Chk1-suppressed apoptotic pathways responding to replication stress or IR. ATM and ATR/Chk1 signalling pathways were manipulated using siRNA-mediated depletions or specific inhibitors in two tumour cell lines or fibroblasts derived from patients with inherited mutations. We show that depletion of ATM or its downstream phosphorylation targets, NBS1 and BID, has relatively little effect on apoptosis induced by DNA replication inhibitors, while ATR or Chk1 depletion strongly enhances cell death induced by such agents in all cells tested. Furthermore, early events occurring after the disruption of DNA replication (accumulation of RPA foci and RPA34 hyperphosphorylation) in ATR- or Chk1-depleted cells committed to apoptosis are not detected in ATM-depleted cells. Unlike the Chk1-suppressed pathway responding to IR, the replication stress-triggered apoptotic pathway did not require ATM and is characterized by activation of caspase 3 in both p53-proficient and -deficient cells. Taken together, our results show that the ATR-Chk1 signalling pathway plays a major role in the regulation of death in response to DNA replication stress and that the Chk1-suppressed pathway protecting cells from replication stress is clearly distinguishable from that protecting cells from IR.