p53-dependent regulation of Mcl-1 contributes to synergistic cell death by ionizing radiation and the Bcl-2/Bcl-XL inhibitor ABT-737

Treatment with the Bcl-2/Bcl-XL inhibitor ABT-737 is a promising novel strategy to therapeutically induce apoptotic cell death in malignant tumors such as glioblastomas. Although many studies have demonstrated that ABT-737 acts synergistically with chemotherapeutic drugs, the possibility of a combined treatment with ionizing radiation (IR) and ABT-737 has not yet been thoroughly investigated. Similarly, the relationship between p53 function and the pro-apoptotic effects of ABT-737 are still obscure. Here, we demonstrate that IR and ABT-737 synergistically induce apoptosis in glioblastoma cells. The sensitivity to ABT-737-mediated cell death is significantly increased by the IR-dependent accumulation of cells in the G2/M cell cycle phase. Wild type p53 function inhibits the efficacy of a combined IR and ABT-737 treatment via a p21-dependent G1 cell cycle arrest. Moreover, mutant as well as wild type p53 counteract the pro-apoptotic activity of ABT-737 by maintaining the expression levels of the Mcl-1 protein. Thus, p53 regulates the sensitivity to ABT-737 of glioblastoma cells. Our results warrant a further evaluation of a novel combination therapy using IR and ABT-737. The efficacy of such a therapy could be substantially enhanced by Mcl-1-lowering strategies.     

APC/C(Cdc20) controls the ubiquitin-mediated degradation of p21 in prometaphase

During the G1/S transition, p21 proteolysis is mediated by Skp2; however, p21 reaccumulates in G2 and is degraded again in prometaphase. How p21 degradation is controlled in mitosis remains unexplored. We found that Cdc20 (an activator of the ubiquitin ligase APC/C) binds p21 in cultured cells and identified a D box motif in p21 necessary for APC/C(Cdc20)-mediated ubiquitylation of p21. Overexpression of Cdc20 or Skp2 destabilized wild-type p21; however, only Skp2, but not Cdc20, was able to destabilize a p21(D box) mutant. Silencing of Cdc20 induced an accumulation of p21, increased the fraction of p21 bound to Cdk1, and inhibited Cdk1 activity in p21(+/+) prometaphase cells, but not in p21(-/-) cells. Thus, in prometaphase Cdc20 positively regulates Cdk1 by mediating the degradation of p21. We propose that the APC/C(Cdc20)-mediated degradation of p21 contributes to the full activation of Cdk1 necessary for mitotic events and prevents mitotic slippage during spindle checkpoint activation.     

Alterations in DNA repair efficiency are involved in the radioresistance of esophageal adenocarcinoma

To study radioresistance in esophageal adenocarcinoma, we generated an isogenic cell line model by exposing OE33 esophageal adenocarcinoma cells to clinically relevant fractionated doses of radiation (cumulative dose 50 Gy). A clonogenic assay confirmed enhanced survival of the radioresistant OE33 subline (OE33 R). To our knowledge, we are the first to generate an isogenic model of radioresistance in esophageal adenocarcinoma. This model system was characterized in terms of growth, cell cycle distribution and checkpoint operation, apoptosis, reactive oxygen species generation and scavenging, and DNA damage. While similar properties were found for both the parental OE33 (OE33 P) cells and radioresistant OE33 R cells, OE33 R cells demonstrated greater repair of radiation-induced DNA damage. Our results suggest that the radioresistance of OE33 R cells is due at least in part to increased DNA repair.     

Poly(ADP-ribose) polymerase-1 is a positive regulator of the p53-mediated G1 arrest response following ionizing radiation

Poly(ADP-ribose) polymerase-1 (PARP-1) and the p53 tumor suppressor protein are both involved in the cellular response to genotoxic stress. Upon binding to the site of DNA strand breakage, PARP-1 is activated, leading to rapid and transient poly(ADP-ribosyl)ation of nuclear proteins using NAD+ as substrate. To investigate the role of PARP-1 in the p53 response to ionizing radiation in human cells, PARP-1 function was disrupted in wild-type p53 expressing MCF-7 and BJ/TERT cells using two strategies: chemical inhibition with 1,5-dihydroxyisoquinoline, and trans-dominant inhibition by overexpression of the PARP-1 DNA-binding domain. Although a number of proteins can catalyze poly(ADP-ribosyl)ation in addition to PARP-1, we show that PARP-1 is the only detectable active species in BJ/TERT and MCF-7 cells. 1,5-Dihydroxyisoquinoline treatment prior to ionizing radiation delayed and attenuated the induction of two p53-responsive genes, p21 and mdm-2, and led to suppression of the p53-mediated G1-arrest response in MCF-7 and BJ/TERT cells. Trans-dominant inhibition of PARP-1 by overexpression of the PARP-1 DNA-binding domain in MCF-7 cells also led to a delay and attenuation in p21 induction and suppression of the p53-mediated G1 arrest response to ionizing radiation. Hence, inhibition of endogenous PARP-1 function suppresses the transactivation function of p53 in response to ionizing radiation. This study establishes PARP-1 as a critical regulator of the p53 response to DNA damage.     

Downregulation of PCNA potentiates p21-mediated growth inhibition in response to hyperoxia

Prolonged exposure to hyperoxia inhibits cell proliferation in G1 via increased expression of p21. While p21 inhibits proliferating cell nuclear antigen (PCNA)-dependent DNA synthesis, it can also directly lower PCNA abundance; however, it is unclear whether loss of PCNA contributes to growth arrest. Here, we investigate how PCNA loss affects ability of p21 to exert G1 growth arrest of lung epithelial cells exposed to hyperoxia. In A549 cells that express p21 and growth arrest in G1 during hyperoxia, small interfering RNA (siRNA) knockdown of p21 led to G1 checkpoint bypass, increased cell death, and restoration of PCNA expression. Conditional overexpression of the PCNA binding domain of p21 in H1299 cells that do not normally express p21, or exposure to hyperoxia, caused a time-dependent loss of PCNA. Titrating PCNA levels using siRNA to approximate the low amount observed in cells expressing p21 resulted in S phase arrest. While lowering PCNA by itself caused S phase arrest, the combination of hyperoxia and siRNA against PCNA dramatically reduced PCNA abundance resulting in G1 arrest. G1 growth arrest was markedly enhanced upon the addition of p21 to these cells. Our findings suggest a model in which reducing expression of the abundant protein PCNA allows the less abundant protein p21 to be more effective at suppressing the processivity functions of remaining PCNA, thereby fully exerting the G1 checkpoint. Given that high p21 expression is often associated with lower PCNA abundance, our findings are suggestive of a global growth inhibitory mechanism involving p21-mediated PCNA suppression.     

Phenotypic alterations of small cell lung carcinoma induced by different levels of wild-type p53 expression

p53 induces both growth arrest and apoptosis in cancer cells. To clarify whether the level of p53 expression determines the response of small cell lung carcinoma (SCLC) cells, we assessed the effect of various p53 levels on a p53-null SCLC cell line, N417, using a tetracycline (Tc)-regulated inducible p53 expression system. Apoptosis was induced in SCLC cells with high p53 expression. Although low levels of p53 induced G1 arrest accompanied by p21 expression, cells with G1 arrest seemed to undergo apoptosis after further cultivation. Expression of exogenous p21 induced G1 arrest but not apoptosis in SCLC cells, suggesting that p53-mediated G1 arrest was induced through p21 expression. Moreover, high level of p53 expression down-regulated Bcl-2 expression in SCLC cells, while Bax was consistently expressed irrespective to the level of p53 expression. These results suggest that p53-mediated apoptosis and G1 arrest depend on level of p53 expression in SCLC cells and that the relative dominancy of Bax to Bcl-2 is involved in the induction of apoptosis by high level of p53 expression.     

黄酮和黄酮醇通过诱导PIG3表达引发人食管癌细胞凋亡

采用两株人食管癌细胞(鳞癌KYSE-510和腺癌OE33)为肿瘤模型,在体外探讨黄酮、黄酮醇化合物诱导细胞凋亡的分子机制．DNA片段化、吖啶橙染色以及流式细胞术的分析结果表明,3种黄酮(木犀草素、白杨素、芹菜素)和3种黄酮醇(槲皮素、山奈酚、杨梅素)均能诱导两株食管癌细胞发生凋亡．荧光定量RT-PCR和Western-blot分析结果表明,黄酮和黄酮醇是通过诱导PIG3 mRNA和蛋白质的表达、经线粒体途径以非p53依赖的方式,引发两株食管癌细胞凋亡．同时,这一过程可能受到p63和p73的调节．     

ErbB2 potentiates breast tumor proliferation through modulation of p27(Kip1)-Cdk2 complex formation: receptor overexpression does not determine growth dependency

Overexpression of the ErbB2 receptor, a major component of the ErbB receptor signaling network, contributes to the development of a number of human cancers. ErbB2 presents itself, therefore, as a target for antibody-mediated therapies. In this respect, anti-ErbB2 monoclonal antibody 4D5 specifically inhibits the growth of tumor cells overexpressing ErbB2. We have analyzed the effect of 4D5-mediated ErbB2 inhibition on the cell cycle of the breast tumor cell line BT474. 4D5 treatment of BT474 cells resulted in a G(1) arrest, preceded by rapid dephosphorylation of ErbB2, inhibition of cytoplasmic signal transduction pathways, accumulation of the cyclin-dependent kinase inhibitor p27(Kip1), and inactivation of cyclin-Cdk2 complexes. Time courses demonstrated that 4D5 treatment redirects p27(Kip1) onto Cdk2 complexes, an event preceding increased p27(Kip1) expression; this correlates with the downregulation of c-Myc and D-type cyclins (proteins involved in p27(Kip1) sequestration) and the loss of p27(Kip1) from Cdk4 complexes. Similar events were observed in ErbB2-overexpressing SKBR3 cells, which exhibited reduced proliferation in response to 4D5 treatment. Here, p27(Kip1) redistribution resulted in partial Cdk2 inactivation, consistent with a G1 accumulation. Moreover, p27(Kip1) protein levels remained constant. Antisense-mediated inhibition of p27(Kip1) expression in 4D5-treated BT474 cells further demonstrated that in the absence of p27(Kip1) accumulation, p27(Kip1) redirection onto Cdk2 complexes is sufficient to inactivate Cdk2 and establish the G(1) block. These data suggest that ErbB2 overexpression leads to potentiation of cyclin E-Cdk2 activity through regulation of p27(Kip1) sequestration proteins, thus deregulating the G(1)/S transition. Moreover, through comparison with an ErbB2-overexpressing cell line insensitive to 4D5 treatment, we demonstrate the specificity of these cell cycle events and show that ErbB2 overexpression alone is insufficient to determine the cellular response to receptor inhibition.     

Phosphorylation and nuclear accumulation are distinct events contributing to the activation of p53

It has been recently shown that ionizing radiation (IR) and the mRNA synthesis inhibitor 5,6-dichloro-1-b-D-ribofuranosylbenzimidazole (DRB) act in synergy to induce p53-mediated transactivation of reporter plasmids in human cells [Oncogene 19 (2000) 3829]. We have extended these studies and show that ionizing radiation and DRB also act in synergy to induce ATM-mediated phosphorylation of the ser15 site of p53 and enhance the expression of endogenous p21 protein. Examination of the localization of p53 revealed that while DRB did not induce phosphorylation of the ser15 site of p53 but efficiently accumulated p53 in the nucleus, ionizing radiation induced phosphorylation of the ser15 site of p53 without prolonged nuclear accumulation. Importantly, the combination of DRB and IR resulted in a strong accumulation of phosphorylated p53 in the nucleus that was more persistent then p53 accumulation after IR alone. Furthermore, the nuclear export inhibitor leptomycin B showed a similar synergy with IR as did DRB regarding ser15 phosphorylation of p53 and p21 induction. These results suggest that the synergistic activation of the p53 response by the combination treatment is due to the activation of two distinct pathways where DRB causes the prolonged nuclear accumulation of p53 while ionizing radiation activates p53 by ATM-mediated phosphorylation.     

Mechanism-based screen establishes signalling framework for DNA damage-associated G1 checkpoint response

DNA damage activates checkpoint controls which block progression of cells through the division cycle. Several different checkpoints exist that control transit at different positions in the cell cycle. A role for checkpoint activation in providing resistance of cells to genotoxic anticancer therapy, including chemotherapy and ionizing radiation, is widely recognized. Although the core molecular functions that execute different damage activated checkpoints are known, the signals that control checkpoint activation are far from understood. We used a kinome-spanning RNA interference screen to delineate signalling required for radiation-mediated retinoblastoma protein activation, the recognized executor of G(1) checkpoint control. Our results corroborate the involvement of the p53 tumour suppressor (TP53) and its downstream targets p21(CIP1/WAF1) but infer lack of involvement of canonical double strand break (DSB) recognition known for its role in activating TP53 in damaged cells. Instead our results predict signalling involving the known TP53 phosphorylating kinase PRPK/TP53RK and the JNK/p38MAPK activating kinase STK4/MST1, both hitherto unrecognised for their contribution to DNA damage G1 checkpoint signalling. Our results further predict a network topology whereby induction of p21(CIP1/WAF1) is required but not sufficient to elicit checkpoint activation. Our experiments document a role of the kinases identified in radiation protection proposing their pharmacological inhibition as a potential strategy to increase radiation sensitivity in proliferating cancer cells.     

Rapamycin-mediated mTOR inhibition attenuates survivin and sensitizes glioblastoma cells to radiation therapy

Survivin, an antiapoptotic protein, is elevated in most malignancies and attributes to radiation resistance in tumors including glioblastoma multiforme. The downregulation of survivin could sensitize glioblastoma cells to radiation therapy. In this study, we investigated the effect of rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), in attenuating survivin and enhancing the therapeutic efficacy for glioblastoma cells, and elucidated the underlying mechanisms. Here we tested various concentrations of rapamycin (1-8 nM) in combination with radiation dose 4 Gy. Rapamycin effectively modulated the protein kinase B (Akt)/mTOR pathway by inhibiting the phosphorylation of Akt and mTOR proteins, and this inhibition was further enhanced by radiation. The expression level of survivin was decreased in rapamycin pre-treatment glioblastoma cells followed by radiation; meanwhile, the phosphorylation of H2A histone family member X (H2AX) at serine-139 (γ-H2AX) was increased. p21 protein was also induced on radiation with rapamycin pre-treatment, which enhanced G1 arrest and the accumulation of cells at G0/subG1 phase. Furthermore, the clonogenic cell survival assay revealed a significant dose-dependent decrease in the surviving fraction for all three cell lines pre-treated with rapamycin. Our studies demonstrated that targeting survivin may be an effective approach for radiosensitization of malignant glioblastoma.     

Hzf, a p53-responsive gene, regulates maintenance of the G2 phase checkpoint induced by DNA damage

The hematopoietic zinc finger protein, Hzf, is induced in response to genotoxic and oncogenic stress. The Hzf protein is encoded by a p53-responsive gene, and its overexpression, either in cells retaining or lacking functional 53, halts their proliferation. Enforced expression of Hzf led to the appearance of tetraploid cells with supernumerary centrosomes and, ultimately, to cell death. Eliminating Hzf mRNA expression by use of short hairpin (sh) RNAs had no overt effect on unstressed cells but inhibited the maintenance of G2 phase arrest following ionizing radiation (IR), thereby sensitizing cells to DNA damage. Canonical p53-responsive gene products such as p21Cip1 and Mdm2 were induced by IR in cells treated with Hzf shRNA. However, the reduction in the level of Hzf protein was accompanied by increased polyubiquitination and turnover of p21Cip1, an inhibitor of cyclin-dependent kinases whose expression contributes to maintaining the duration of the G2 checkpoint in cells that have sustained DNA damage. Thus, two p53-inducible gene products, Hzf and p21Cip1, act concomitantly to enforce the G(2) checkpoint.     

Initiation of DNA synthesis by human papillomavirus E7 oncoproteins is resistant to p21-mediated inhibition of cyclin E-cdk2 activity.

The E6 and E7 proteins from the high-risk human papillomaviruses (HPVs) bind and inactivate the tumor suppressor proteins p53 and Rb, respectively. In HPV-positive cells, expression of E6 proteins from high-risk types results in increased turnover of p53, which leads to an abrogation of p21-mediated G1/S arrest in response to DNA-damaging agents. In contrast, keratinocytes which express E7 alone have increased levels of p53 but, interestingly, also fail to undergo a G1/S arrest. We investigated the mechanism by which E7 bypasses this p21 arrest by using both keratinocytes which stably express E7 as well as U20S cells which stably or transiently express E7. We observed that E7 does not affect the induction of p21 synthesis by p53. While glutathione S-transferase (GST)-E7 bound a low level of in vitro-translated p21, we were unable to detect E7 and p21 in the same complex by GST-E7 binding assays or immunoprecipitations from cell extracts. Furthermore, E7 did not prevent p21-mediated inhibition of cyclin E kinase activity. In keratinocytes expressing E7, increased levels of p53, p21, and cyclin E, as well as increased cyclin E kinase activity, were observed. To determine if this increase in cyclin E activity was necessary for E7's ability to overcome p21-mediated G1/S arrest, we examined U20S cells in which cyclin E levels are not increased in response to E7 expression. U20S cells which stably express E7 were found to initiate DNA synthesis in the presence of DNA-damaging agents despite the inhibition of cyclin E activity by p21. In transient assays, cotransfection of E7 or E2F-1 along with p21 into U20S cells rescued G1 arrest and resulted in S-phase entry, as measured by the ability to incorporate bromodeoxyuridine. These data indicate that E7 is able to overcome G1/S arrest without directly affecting p21 function and likely acts through deregulation of E2F activity.