Nijmegen breakage syndrome: The clearance pathway for mutant nibrin protein is allele specific

The autosomal recessive disorder Nijmegen breakage syndrome (NBS) is caused by mutations in the NBN gene which codes for the protein nibrin (NBS1; p95). In the majority of cases, a 5 bp deletion, a founder mutation, leads to a hypomorphic 70 kD protein, p70-nibrin, after alternative initiation of translation. Protein levels are of relevance for the clinical course of the disease, particularly with regard to malignancy. Here, mechanisms and efficiency of mutant protein clearance were examined in order to establish whether these have an impact on nibrin abundance. Cell lines from NBS patients and retroviral transductants were treated with proteasome and lysosome inhibitors and examined by semi-quantitative immunoblotting for p70-nibrin and p95-nibrin levels. The results show that p70-nibrin is degraded by the proteasome with varying efficiency in cell lines from different NBS patients leading to lower or higher steady state levels of this partially active protein fragment. In contrast, a previously described NBN missense mutation, which disturbs protein folding due to the substitution of a critical arginine by tryptophan, was found to be cleared by lysosomal microautophagy leading also to lower cellular levels. The data show that truncated nibrin and misfolded nibrin have different clearance pathways.     

Roles of nibrin and AtM/ATR kinases on the G2 checkpoint under endogenous or radio-induced DNA damage

Checkpoint response to DNA damage involves the activation of DNA repair and G2 lengthening subpathways. The roles of nibrin (NBS1) and the ATM/ATR kinases in the G2 DNA damage checkpoint, evoked by endogenous and radio-induced DNA damage, were analyzed in control, A-T and NBS lymphoblast cell lines. Short-term responses to G2 treatments were evaluated by recording changes in the yield of chromosomal aberrations in the ensuing mitosis, due to G2 checkpoint adaptation, and also in the duration of G2 itself. The role of ATM/ATR in the G2 checkpoint pathway repairing chromosomal aberrations was unveiled by caffeine inhibition of both kinases in G2. In the control cell lines, nibrin and ATM cooperated to provide optimum G2 repair for endogenous DNA damage. In the A-T cells, ATR kinase substituted successfully for ATM, even though no G2 lengthening occurred. X-ray irradiation (0.4 Gy) in G2 increased chromosomal aberrations and lengthened G2, in both mutant and control cells. However, the repair of radio-induced DNA damage took place only in the controls. It was associated with nibrin-ATM interaction, and ATR did not substitute for ATM. The absence of nibrin prevented the repair of both endogenous and radio-induced DNA damage in the NBS cells and partially affected the induction of G2 lengthening.     

Modulation of DNA damage response and induction of apoptosis mediates synergism between doxorubicin and a new imidazopyridine derivative in breast and lung cancer cells

DNA damage response machinery (DDR) is an attractive target of cancer therapy. Modulation of DDR network may alter the response of cancer cells to DNA damaging anticancer drugs such as doxorubicin. The aim of the present study is to investigate the effects of a newly developed imidazopyridine (IAZP) derivative on the DDR after induction of DNA damage in cancer cells by doxorubicin. Cytotoxicity sulphrhodamine-B assay showed a weak anti-proliferative effect of IAZP alone on six cancer cell lines (MCF7, A549, A549DOX11, HepG2, HeLa and M8) and a normal fibroblast strain. Combination of IAZP with doxorubicin resulted in synergism in lung (A549) and breast (MCF7) cancer cells but neither in the other cancer cell lines nor in normal fibroblasts. Molecular studies revealed that synergism is mediated by modulation of DNA damage response and induction of apoptosis. Using constant-field gel electrophoresis and immunofluorescence detection of γ-H2AX foci, IAZP was shown to inhibit the repair of doxorubicin-induced DNA damage in A549 and MCF7 cells. Immunoblot analysis showed that IAZP suppresses the phosphorylation of the ataxia lelangiectasia and Rad3 related (ATR) protein, which is an important player in the response of cancer cells to chemotherapy-induced DNA damage. Moreover, IAZP augmented the doxorubicin-induced degradation of p21, activation of p53, CDK2, caspase 3/7 and phosphorylation of Rb protein. These effects enhanced doxorubicin-induced apoptosis in both cell lines. Our results indicate that IAZP is a promising agent that may enhance the cytotoxic effects of doxorubicin on some cancer cells through targeting the DDR. It is a preliminary step toward the clinical application of IAZP in combination with anticancer drugs and opens the avenue for the development of compounds targeting the DDR pathway that might improve the therapeutic index of anticancer drugs and enhance their cure rate.     

NBS1 and TRF1 colocalize at promyelocytic leukemia bodies during late S/G2 phases in immortalized telomerase-negative cells. Implication of NBS1 in alternative lengthening of telomeres

Nijmegen breakage syndrome, a chromosomal instability disorder, is characterized in part by cellular hypersensitivity to ionizing radiation. The NBS1 gene product, p95 (NBS1 or nibrin) forms a complex with Rad50 and Mre11. Cells deficient in the formation of this complex are defective in DNA double-strand break repair, cell cycle checkpoint control, and telomere length maintenance. How the NBS1 complex is involved in telomere length maintenance remains unclear. Here we show that the C-terminal region of NBS1 interacts directly with a telomere repeat binding factor, TRF1, by both yeast two-hybrid and in vivo DNA-coimmunoprecipitation assays. NBS1 and Mre11 colocalize with TRF1 at promyelocytic leukemia (PML) nuclear bodies in immortalized telomerase-negative cell lines, but rarely in telomerase-positive cell lines. The translocation of NBS1 to PML bodies occurs specifically during late S to G(2) phases of the cell cycle and coincides with active DNA synthesis in these NBS1-containing PML bodies. These results suggest that NBS1 may be involved in alternative lengthening of telomeres in telomerase-negative immortalized cells.     

Distinct functional domains of nibrin mediate Mre11 binding, focus formation, and nuclear localization

The inherited chromosomal instability disorder Nijmegen breakage syndrome (NBS) results from truncating mutations in the NBS1 gene, which encodes the protein nibrin. Nibrin is part of a nuclear multiprotein complex that also contains the DNA repair proteins Mre11 and Rad50. Upon irradiation, this complex redistributes within the nucleus, forming distinct foci that have been implicated as sites of DNA repair. In NBS cells, nibrin is absent and Mre11 and Rad50 are cytoplasmic. In this study, the interacting domains on nibrin and Mre11 were mapped using the yeast two-hybrid system and expression of epitope-tagged constructs in NBS fibroblasts. Deletion of the carboxy-terminal 101 amino acids of nibrin eliminated its ability to interact with Mre11 and to complement the radiation sensitivity of NBS cells. However, this truncated form of nibrin could localize to the nucleus and form radiation-inducible foci. Expression of a carboxy-terminal 354-amino-acid fragment of nibrin was sufficient to direct the nuclear localization of nibrin, as well as that of Mre11 and Rad50. Despite providing some partial complementation of the radiation-sensitive phenotype, the nibrin-Mre11-Rad50 complexes in these cells were unable to form foci. These results indicate that nibrin directs not only the nuclear localization of the nibrin-Mre11-Rad50 complexes but also radiation-induced focus formation. However, direct interaction between nibrin and Mre11 is required for normal cellular survival postirradiation. Distinct domains of nibrin are required for each of these functions, focus formation, nuclear localization, and Mre11 interaction.     

The MRE11 complex: starting from the ends

The maintenance of genome stability depends on the DNA damage response (DDR), which is a functional network comprising signal transduction, cell cycle regulation and DNA repair. The metabolism of DNA double-strand breaks governed by the DDR is important for preventing genomic alterations and sporadic cancers, and hereditary defects in this response cause debilitating human pathologies, including developmental defects and cancer. The MRE11 complex, composed of the meiotic recombination 11 (MRE11), RAD50 and Nijmegen breakage syndrome 1 (NBS1; also known as nibrin) proteins is central to the DDR, and recent insights into its structure and function have been gained from in vitro structural analysis and studies of animal models in which the DDR response is deficient.     

The Nijmegen breakage syndrome protein is essential for Mre11 phosphorylation upon DNA damage.

The Nijmegen breakage syndrome (NBS), a chromosomal instability disorder, is characterized in part by cellular hypersensitivity to ionizing radiation. Repair of DNA double-strand breaks by radiation is dependent on a multifunctional complex containing Rad50, Mre11, and the NBS1 gene product, p95 (NBS protein, nibrin). The role of p95 in these repair processes is unknown. Here it is demonstrated that Mre11 is hyperphosphorylated in a cell cycle-independent manner in response to treatment of cells with genotoxic agents including gamma irradiation. This response is abrogated in two independently established NBS cell lines that have undetectable levels of the p95 protein. NBS cells are also deficient for radiation-induced nuclear foci containing Mre11, while those with Rad51 are unaffected. An analysis of the kinetic relationship between Mre11 phosphorylation and the appearance of its radiation-induced foci indicates that the former precedes the latter. Together, these data suggest that specific phosphorylation of Mre11 is induced by DNA damage, and p95 is essential in this process, perhaps by recruiting specific kinases.     

DNA damage in Nijmegen Breakage Syndrome cells leads to PARP hyperactivation and increased oxidative stress

Nijmegen Breakage Syndrome (NBS), an autosomal recessive genetic instability syndrome, is caused by hypomorphic mutation of the NBN gene, which codes for the protein nibrin. Nibrin is an integral member of the MRE11/RAD50/NBN (MRN) complex essential for processing DNA double-strand breaks. Cardinal features of NBS are immunodeficiency and an extremely high incidence of hematological malignancies. Recent studies in conditional null mutant mice have indicated disturbances in redox homeostasis due to impaired DSB processing. Clearly this could contribute to DNA damage, chromosomal instability, and cancer occurrence. Here we show, in the complete absence of nibrin in null mutant mouse cells, high levels of reactive oxygen species several hours after exposure to a mutagen. We show further that NBS patient cells, which unlike mouse null mutant cells have a truncated nibrin protein, also have high levels of reactive oxygen after DNA damage and that this increased oxidative stress is caused by depletion of NAD+ due to hyperactivation of the strand-break sensor, Poly(ADP-ribose) polymerase. Both hyperactivation of Poly(ADP-ribose) polymerase and increased ROS levels were reversed by use of a specific Poly(ADP-ribose) polymerase inhibitor. The extremely high incidence of malignancy among NBS patients is the result of the combination of a primary DSB repair deficiency with secondary oxidative DNA damage.     

The roles of p27Kip1 and DNA damage signalling in the chemotherapy‐induced delayed cell cycle checkpoint

DNA lesions trigger the DNA damage response (DDR) machinery, which protects genomic integrity and sustains cellular survival. Increasing data underline the significance of the integrity of the DDR pathway in chemotherapy response. According to a recent work, persistent exposure of A549 lung carcinoma cells to doxorubicin induces an initial DDR‐dependent checkpoint response, followed by a later DDR‐independent, but p27^Kip1‐dependent one. Prompted by the above report and to better understand the involvement of the DDR signaling after chemotherapeutic stress, we examined the potential role of the canonical DDR pathway in A549 cells treated with doxorubicin. Exposure of A549 cells, prior to doxorubicin treatment, to ATM, ATR and DNA‐PKcs inhibitors either alone or in various combinations, revealed that the earlier documented two‐step response was DDR‐dependent in both steps. Notably, inhibition of both ATM and ATR or selective inhibition of ATM or DNA‐PKcs resulted in cell‐cycle re‐entry despite the increased levels of p27^Kip1 at all time points analyzed. We further investigated the regulation of p27^Kip1 protein levels in the particular setting. Our results showed that the protein status of p27^Kip1 is mainly determined by p38‐MAPK, whereas the role of SKP2 is less significant in the doxoroubicin‐treated A549 cells. Cumulatively, we provide evidence that the DNA damage signaling is responsible for the prolonged cell cycle arrest observed after persistent chemotherapy‐induced genotoxic stress. In conclusion, precise identification of the molecular mechanisms that are activated during the chemotherapeutic cycles could potentially increase the sensitization to the therapy applied.     

UV-induced histone H2AX phosphorylation and DNA damage related proteins accumulate and persist in nucleotide excision repair-deficient XP-B cells

DNA double strand breaks (DSB) may be caused by ionizing radiation. In contrast, UV exposure forms dipyrimidine photoproducts and is not considered an inducer of DSB. We found that uniform or localized UV treatment induced phosphorylation of the DNA damage related (DDR) proteins H2AX, ATM and NBS1 and co-localization of γ-H2AX with the DDR proteins p-ATM, p-NBS1, Rad51 and FANCD2 that persisted for about 6h in normal human fibroblasts. This post-UV phosphorylation was observed in the absence of nucleotide excision repair (NER), since NER deficient XP-B cells (lacking functional XPB DNA repair helicase) and global genome repair-deficient rodent cells also showed phosphorylation and localization of these DDR proteins. Resolution of the DDR proteins was dependent on NER, since they persisted for 24h in the XP-B cells. In the normal and XP-B cells p53 and p21 was detected at 6h and 24h but Mdm2 was not induced in the XP-B cells. Post-UV induction of Wip1 phosphatase was detected in the normal cells but not in the XP-B cells. DNA DSB were detected with a neutral comet assay at 6h and 24h post-UV in the normal and XP-B cells. These results indicate that UV damage can activate the DDR pathway in the absence of NER. However, a later step in DNA damage processing involving induction of Wip1 and resolution of DDR proteins was not observed in the absence of NER.     

Interplay between oncogene-induced DNA damage response and heterochromatin in senescence and cancer

Two major mechanisms have been causally implicated in the establishment of cellular senescence: the activation of the DNA damage response (DDR) pathway and the formation of senescence-associated heterochromatic foci (SAHF). Here we show that in human fibroblasts resistant to premature p16(INK4a) induction, SAHF are preferentially formed following oncogene activation but are not detected during replicative cellular senescence or on exposure to a variety of senescence-inducing stimuli. Oncogene-induced SAHF formation depends on DNA replication and ATR (ataxia telangiectasia and Rad3-related). Inactivation of ATM (ataxia telangiectasia mutated) or p53 allows the proliferation of oncogene-expressing cells that retain increased heterochromatin induction. In human cancers, levels of heterochromatin markers are higher than in normal tissues, and are independent of the proliferative index or stage of the tumours. Pharmacological and genetic perturbation of heterochromatin in oncogene-expressing cells increase DDR signalling and lead to apoptosis. In vivo, a histone deacetylase inhibitor (HDACi) causes heterochromatin relaxation, increased DDR, apoptosis and tumour regression. These results indicate that heterochromatin induced by oncogenic stress restrains DDR and suggest that the use of chromatin-modifying drugs in cancer therapies may benefit from the study of chromatin and DDR status of tumours.     

Five dysfunctional telomeres predict onset of senescence in human cells

Replicative senescence is accompanied by a telomere-specific DNA damage response (DDR). We found that DDR+ telomeres occur spontaneously in early-passage normal human cells and increase in number with increasing cumulative cell divisions. DDR+ telomeres at replicative senescence retain TRF2 and RAP1 proteins, are not associated with end-to-end fusions and mostly result from strand-independent, postreplicative dysfunction. On the basis of the calculated number of DDR+ telomeres in G1-phase cells just before senescence and after bypassing senescence by inactivation of wild-type p53 function, we conclude that the accrual of five telomeres in G1 that are DDR+ but nonfusogenic is associated with p53-dependent senescence.     

The canonical NF-κB pathway differentially protects normal and human tumor cells from ROS-induced DNA damage

DNA damage responses (DDR) invoke senescence or apoptosis depending on stimulus intensity and the degree of activation of the p53-p21(Cip1/Waf1) axis; but the functional impact of NF-κB signaling on these different outcomes in normal vs. human cancer cells remains poorly understood. We investigated the NF-κB-dependent effects and mechanism underlying reactive oxygen species (ROS)-mediated DDR outcomes of normal human lung fibroblasts (HDFs) and A549 human lung cancer epithelial cells. To activate DDR, ROS accumulation was induced by different doses of H(2)O(2). The effect of ROS induction caused a G2 or G2-M phase cell cycle arrest of both human cell types. However, ROS-mediated DDR eventually culminated in different end points with HDFs undergoing premature senescence and A549 cancer cells succumbing to apoptosis. NF-κB p65/RelA nuclear translocation and Ser536 phosphorylation were induced in response to H(2)O(2)-mediated ROS accumulation. Importantly, blocking the activities of canonical NF-κB subunits with an IκBα super-repressor or suppressing canonical NF-κB signaling by IKKβ knock-down accelerated HDF premature senescence by up-regulating the p53-p21(Cip1/Waf1) axis; but inhibiting the canonical NF-κB pathway exacerbated H(2)O(2)-induced A549 cell apoptosis. HDF premature aging occurred in conjunction with γ-H2AX chromatin deposition, senescence-associated heterochromatic foci and beta-galactosidase staining. p53 knock-down abrogated H(2)O(2)-induced premature senescence of vector control- and IκBαSR-expressing HDFs functionally linking canonical NF-κB-dependent control of p53 levels to ROS-induced HDF senescence. We conclude that IKKβ-driven canonical NF-κB signaling has different functional roles for the outcome of ROS responses in the contexts of normal vs. human tumor cells by respectively protecting them against DDR-dependent premature senescence and apoptosis.     

Overexpression of NBS1 contributes to transformation through the activation of phosphatidylinositol 3-kinase/Akt

Nijmegen breakage syndrome (NBS) is a chromosomal instability syndrome associated with cancer predisposition, radiosensitivity, microcephaly, and growth retardation. The NBS gene product, NBS1 (p95) or nibrin, is a part of the hMre11 complex, a central player associated with double strand break repair. We previously demonstrated that c-Myc directly activates NBS1 expression. Here we have shown that constitutive expression of NBS1 in Rat1a and HeLa cells induces/enhances their transformation. Repression of endogenous NBS1 levels using short interference RNA reduces the transformation activity of two tumor cell lines. Increased NBS1 expression is observed in 40-52% of non-small cell lung carcinoma, hepatoma, and esophageal cancer samples. NBS1 overexpression stimulates phosphatidylinositol (PI) 3-kinase activity, leading to increased phosphorylation levels of Akt and its downstream targets such as glycogen synthase kinase 3beta and mammalian target of rapamycin in different cell lines and tumor samples. Transformation induced by NBS1 overexpression can be inhibited by a PI3-kinase inhibitor (LY294002). Repression of endogenous Akt expression by short interference RNA decreases the transformation activity of Rat1a cells overexpressing NBS1. These results indicate that overexpression of NBS1 is an oncogenic event that contributes to transformation through the activation of PI3-kinase/Akt.     

Poly(ADP-ribose) polymerase inhibition enhances p53-dependent and -independent DNA damage responses induced by DNA damaging agent

Targeting DNA repair with poly(ADP-ribose) polymerase (PARP) inhibitors has shown a broad range of anti-tumor activity in patients with advanced malignancies with and without BRCA deficiency. It remains unclear what role p53 plays in response to PARP inhibition in BRCA-proficient cancer cells treated with DNA damaging agents. Using gene expression microarray analysis, we find that DNA damage response (DDR) pathways elicited by veliparib (ABT-888), a PARP inhibitor, plus topotecan comprise the G1/S checkpoint, ATM, and p53 signaling pathways in p53-wildtype cancer cell lines and BRCA1, BRCA2 and ATR pathway in p53-mutant lines. In contrast, topotecan alone induces the G1/S checkpoint pathway in p53-wildtype lines and not in p53-mutant cells. These responses are coupled with G2/G1 checkpoint effectors p21CDKN1A upregulation, and Chk1 and Chk2 activation. The drug combination enhances G2 cell cycle arrest, apoptosis and a marked increase in cell death relative to topotecan alone in p53-wildtype and p53-mutant or -null cells. We also show that the checkpoint kinase inhibitor UCN-01 abolishes the G2 arrest induced by the veliparib and topotecan combination and further increases cell death in both p53-wildtype and -mutant cells. Collectively, PARP inhibition by veliparib enhances DDR and cell death in BRCA-proficient cancer cells in a p53-dependent and -independent fashion. Abrogating the cell-cycle arrest induced by PARP inhibition plus chemotherapeutics may be a strategy in the treatment of BRCA-proficient cancer.     

Rac1 Protein Signaling Is Required for DNA Damage Response Stimulated by Topoisomerase II Poisons

To investigate the potency of the topoisomerase II (topo II) poisons doxorubicin and etoposide to stimulate the DNA damage response (DDR), S139 phosphorylation of histone H2AX (γH2AX) was analyzed using rat cardiomyoblast cells (H9c2). Etoposide caused a dose-dependent increase in the γH2AX level as shown by Western blotting. By contrast, the doxorubicin response was bell-shaped with high doses failing to increase H2AX phosphorylation. Identical results were obtained by immunohistochemical analysis of γH2AX focus formation, comet assay-based DNA strand break analysis, and measuring the formation of the topo II-DNA cleavable complex. At low dose, doxorubicin activated ataxia telangiectasia mutated (ATM) but not ATM and Rad3-related (ATR). Both the lipid-lowering drug lovastatin and the Rac1-specific inhibitor NSC23766 attenuated doxorubicin- and etoposide-stimulated H2AX phosphorylation, induction of DNA strand breaks, and topo II-DNA complex formation. Lovastatin and NSC23766 acted in an additive manner. They did not attenuate doxorubicin-induced increase in p-ATM and p-Chk2 levels. DDR stimulated by topo II poisons was partially blocked by inhibition of type I p21-associated kinases. DDR evoked by the topoisomerase I poison topotecan remained unaffected by lovastatin. The data show that the mechanisms involved in DDR stimulated by topo II poisons are agent-specific with anthracyclines lacking DDR-stimulating activity at high doses. Pharmacological inhibition of Rac1 signaling counteracts doxorubicin- and etoposide-stimulated DDR by disabling the formation of the topo II-DNA cleavable complex. Based on the data we suggest that Rac1-regulated mechanisms are required for DNA damage induction and subsequent activation of the DDR following treatment with topo II but not topo I poisons.     

Independent roles for nibrin and Mre11-Rad50 in the activation and function of Atm

The Atm protein kinase and Mre11-Rad50-nibrin (MRN) complex play an integral role in the cellular response to DNA double-strand breaks. Mutations in Mre11 and nibrin result in the radiosensitivity disorders ataxia-telangiectasia-like disorder (ATLD) and Nijmegen breakage syndrome (NBS), respectively. Cells from ATLD and NBS patients are deficient in activation of the Atm protein kinase and phosphorylation of downstream Atm targets following irradiation. However, the roles of individual MRN complex proteins in Atm function are not clear, because the mutations in NBS and ATLD cells result in global effects on the MRN complex. Previously we showed that the C-terminal 100 amino acids of nibrin were necessary and sufficient to translocate the MRN complex to the nucleus. Here we have taken advantage of this feature of nibrin to create isogenic cell lines lacking either nibrin or Mre11-Rad50 in the nucleus. We found that nuclear expression of Mre11-Rad50, but not nibrin, stimulated Atm activation at early times after low doses of radiation. At later times or higher doses of irradiation, Atm activation was independent of Mre11-Rad50 or nibrin. The requirement of MRN complex proteins for downstream Atm phosphorylation events following irradiation was more complex. Phosphorylation of nibrin and Chk2 by Atm required Mre11-Rad50 expression in the nucleus at early times after irradiation, reflecting the stimulation of Atm activation by Mre11-Rad50. By contrast, autophosphorylation of Chk2 and phosphorylation of Smc1 at Ser-957 was dependent on the MRN complex 60 min after irradiation, even though Atm was activated at that time point. These results indicate an independent role for Mre11-Rad50 in the activation of Atm and suggest nibrin and/or Mre11-Rad50 also act as adaptors for some downstream Atm phosphorylation events.     

The Role of Hypoxia Inducible Factor-1 Alpha in Bypassing Oncogene-Induced Senescence

Oncogene induced senescence (OIS) is a sustained anti-proliferative response acutely induced in primary cells via activation of mitogenic oncogenes such as Ras/BRAF. This mechanism acts as an initial barrier preventing normal cells transformation into malignant cell. Besides oncogenic activation and DNA damage response (DDR), senescence is modulated by a plethora of other factors, and one of the most important one is oxygen tension of the tissue. The aim of this study was to determine the impact of hypoxia on Ras^V12-induced senescence in human diploid fibroblasts (HDFs). We showed here that hypoxia prevents execution of oncogene induced senescence (OIS), through a strong down-regulation of senescence hallmarks, such as SA- β-galactosidase, H3K9me3, HP1γ, p53, p21^CIP1 and p16^INK4a in association with induction of hypoxia inducible factor-1α (HIF-1α). In addition, hypoxia also decreased marks of H-Ras^V12-induced DDR in both cell lines through down-regulation of ATM/ATR, Chk1 and Chk2 phosphorylation as well as decreased γ-H2AX positivity. Utilizing shRNA system targeting HIF-1α we show that HIF-1α is directly involved in down regulation of p53 and its target p21^CIP1 but not p16^INK4a. In line with this finding we found that knock down of HIF-1α leads to a strong induction of apoptotic response, but not restoration of senescence in Ras expressing HDFs in hypoxia. This indicates that HIF-1α is an important player in early steps of tumorigenesis, leading to suppression of senescence through its negative regulation of p53 and p21^CIP1. In our work we describe a mechanism through which hypoxia and specifically HIF-1α preclude cells from maintaining senescence-driven anti proliferative response. These findings indicate the possible mechanism through which hypoxic environment helps premalignant cells to evade impingement of cellular failsafe pathways.