Signaling of DNA damage is not sufficient to induce p53 response: (re)activation of wt p53 protein strongly depends on cellular context

It is generally accepted that exposure of cells to a variety of DNA-damaging agents leads to up-regulation and activation of wild-type (wt) p53 protein. We investigated the (re)-activation of p53 protein in two human cancer cell lines in which the gene for this tumor suppressor is not mutated: HeLaS(3) cervix carcinoma and MCF-7 breast cancer cells, by induction via different genotoxic and cytotoxic stimuli. Treatment of human cells with the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or different anti-cancer drugs resulted in a strong DNA damage as evidenced by Comet assay and a marked increase in site-specific phosphorylation of H2AX. Unlike in MCF-7 cells, in HeLaS(3) cells the expression of p53 protein did not increase after MNNG treatment despite a strong DNA damage. However, other agents for example doxorubicin markedly induced p53 response in HeLaS(3) cells. After exposure of these cells to MNNG, the ATM-dependent effector proteins Chk2 and NBS1 were phosphorylated, thereby evidencing that MNNG-induced DNA breakage was recognized and properly signaled. In HeLaS(3) cells wt p53 protein is not functional due to E6-mediated targeting for accelerated ubiquitylation and degradation. Therefore, the activation of a p53 response to genotoxic stress in HeLaS(3) cells seems to depend on the status of E6 oncoprotein. Indeed, the induction of p53 protein in HeLaS(3) cells in response to distinct agents inversely correlates with the cellular level of E6 oncoprotein. This implicates that the capability of different agents to activate p53 in HeLaS(3) cells primarily depends on their inhibitory effect on expression of E6 oncoprotein.     

The Imprinted Gene PEG3 Inhibits Wnt Signaling and Regulates Glioma Growth

The imprinted gene PEG3 confers parenting and sexual behaviors, alters growth and development, and regulates apoptosis. However, a molecular mechanism that can account for the diverse functions of Peg3/Pw1 is not known. To elucidate Peg3-regulated pathways, we performed a functional screen in zebrafish. Enforced overexpression of PEG3 mRNA during zebrafish embryogenesis decreased β-catenin protein expression and inhibited Wnt-dependent tail development. Peg3/Pw1 also inhibited Wnt signaling in human cells by binding to β-catenin and promoting its degradation via a p53/Siah1-dependent, GSK3β-independent proteasomal pathway. The inhibition of the Wnt pathway by Peg3/Pw1 suggested a role in tumor suppression. Hypermethylation of the PEG3 promoter in primary human gliomas led to a loss of imprinting and decreased PEG3 mRNA expression that correlated with tumor grade. The decrease in Peg3/Pw1 protein expression increased β-catenin, promoted proliferation, and inhibited p53-dependent apoptosis in human CD133⁺ glioma stem cells. Thus, mammalian imprinting utilizes Peg3/Pw1 to co-opt the Wnt pathway, thereby regulating development and glioma growth.     

p53 regulates the expression of the tumor suppressor gene maspin

Maspin has been shown to inhibit tumor cell invasion and metastasis in breast tumor cells. Maspin expression was detected in normal breast and prostate epithelial cells, whereas tumor cells exhibited reduced or no expression. However, the regulatory mechanism of maspin expression remains unknown. We report here a rapid and robust induction of maspin expression in prostate cancer cells (LNCaP, DU145, and PC3) and breast tumor cells (MCF7) following wild type p53 expression from an adenovirus p53 expression vector (AdWTp53). p53 activates the maspin promoter by binding directly to the p53 consensus-binding site present in the maspin promoter. DNA-damaging agents and cytotoxic drugs induced endogenous maspin expression in cells containing the wild type p53. Maspin expression was refractory to the DNA-damaging agents in cells containing mutant p53. These results, combined with recent studies of the tumor metastasis suppressor gene KAI1 and plasminogen activator inhibitor 1 (PAI1), define a new category of molecular targets of p53 that have the potential to negatively regulate tumor invasion and/or metastasis.     

Cyclic AMP-induced p53 destabilization is independent of EPAC in pre-B acute lymphoblastic leukemia cells in vitro

Context: Activation of the tumor suppressor protein p53 facilitates the cellular response to genotoxic stress. Thus, releasing the wild-type p53 from indirect suppression would be crucial to successful killing of cancer cells by DNA-damaging therapeutic agents.

Objective: The aim of this study was to investigate the inhibitory role of cyclic adenosine monophosphate (cAMP) levels on p53 protein in acute lymphoblastic leukemia (ALL) cells. More importantly, we were interested to show through which receptor cAMP acts to promote p53 degradation.

Materials and methods: In cell cultures, we investigated the effects of forskolin/3-isobutyl-1-methylxanthine (IBMX) on stimulated p53 of ALL cell lines. Western blotting analysis was performed to detect the expression of p53, phospho-p53, acetylated-p53, phospho-cAMP response element-binding protein (CREB), and Mdm2 proteins. Flow cytometry was applied to analyze apoptosis. The gene expression of p53 and its target genes was examined by real-time polymerase chain reaction.

Results: We show that elevation of cAMP levels in ALL cells exposed to DNA damage attenuates p53 accumulation. Inhibition of proteosome function with MG-132 reversed the inhibitory effect of cAMP on p53. However, targeting the p53–Mdm2 interaction did not rescue accumulated p53 from the destabilizing signal of cAMP. The specific agonist of the cAMP receptor exchange protein activated by cAMP had no effect on p53 expression in doxorubicin-treated NALM-6 cells, whereas PKA activators decreased p53 accumulation.

Discussion and conclusion: Our studies demonstrate that cAMP-PKA pathway regulates the sensitivity toward DNA-damaging agents via inhibition of a p53-dependent pathway in B-cell precursor ALL (BCP-ALL) cells.     

Different cell cycle mechanisms between UV-induced and X-ray-induced apoptosis in WiDr colorectal carcinoma cells

Although DNA-damaging agents such as ultraviolet (UV) and X-ray can induce apoptosis, the difference in the apoptotic mechanism is not clearly understood. In the present study, we investigated the effects of these two genotoxic agents on the induction of DNA damage and subsequent apoptotic cell death from the viewpoint of cell cycle regulation by using WiDr cells. Transient G1 arrest was observed after UV exposure, whereas G2 but not G1 arrest was induced after X-ray irradiation. UV-exposure could induce G1 arrest in both mutant-type (mt-p53) and wild-type p53 (wt-p53) cells, but obvious G1 arrest was not observed in the cells lacking in p53 expression. An increase in the DNA fragmentation was observed at S phase in UV-irradiated cells and at G2 phase in X-irradiated cells, respectively. UV-irradiated cells showed an increase production of p53 protein and accumulation of p21 protein. On the contrary, both p53 and p21 proteins remained at a low level in X-irradiated cells. Treatment with aphidicolin, an S phase blocking agent, prolonged cell cycle arrest and reduced the rate of apoptotic cell death in both UV-irradiated and X-irradiated cells. From these results, it is suggested that UV-induced apoptosis occurs mainly at S phase and is regulated by increased production of p53 and p21 proteins, while X-ray-induced apoptosis occurs after G2 blockade and may be independent of p53.     

Bioluminescence detection of cells having stabilized p53 in response to a genotoxic event

Inactivation of p53 is one of the most frequent molecular events in neoplastic transformation. Approximately 60% of all human tumors have mutations in both p53 alleles. Wild-type p53 activity is regulated in large part by the proteosome-dependent degradation of p53, resulting in a short p53 half-life in unstressed and untransformed cells. Activation of p53 by a variety of stimuli, including DNA damage induced by genotoxic drugs or radiation, is accomplished by stabilization of wild-type p53. The stabilized and active p53 can result in either cell-cycle arrest or apoptosis. Surprisingly, the majority of tumor-associated, inactivating p53 mutations also result in p53 accumulation. Thus, constitutive elevation of p53 levels in cells is a reliable measure of p53 inactivation, whereas transiently increased p53 levels reflect a recent genotoxic stress. In order to facilitate noninvasive imaging of p53 accumulation, we here describe the construction of a p53-luciferase fusion protein. Induction of DNA damage in cells expressing the fusion protein resulted in a time-dependent accumulation of the fusion that was noninvasively detected using bioluminescence imaging and validated by Western blot analysis. The p53-Luc protein retains p53 function because its expression in HCT116 cells lacking functional p53 resulted in activation of p21 expression as well as induction of apoptosis in response to a DNA damaging event. Employed in a transgenic animal model, the proposed p53-reporter fusion protein will be useful for studying p53 activation in response to exposure to DNA-damaging carcinogenic agents. It could also be used to study p53 stabilization as a result of inactivating p53 mutations. Such studies will further our understanding of p53's role as the "guardian of the genome" and its function in tumorigenesis.     

Tumor-specific induction of apoptosis by a p53-reactivating compound

The tumor suppressor function of p53 is disabled in the majority of tumors, either by a point mutation of the p53 gene, or via MDM2-dependent proteasomal degradation. We have screened a chemical library using a cell-based assay and identified a low molecular weight compound named MITA which induced wild-type p53-dependent cell death in a variety of different types of human tumor cells, such as lung, colon and breast carcinoma cells, as well as in osteosarcoma and fibrosarcoma-derived cells. MITA inhibited p53-MDM2 interaction in vitro and in cells, which in turn prevented MDM2-mediated ubiquitination of p53 and resulted in a prolonged half-life and accumulation of p53 in tumor cells. Notably, p53 induction by MITA resulted in upregulated expression of p53 target genes MDM2, Bax, Gadd45 and PUMA, on protein and mRNA level. Importantly, neither p53 nor these target genes were induced in normal human fibroblasts (HDFs), which correlated with the absence of growth suppression in fibroblasts after treatment with MITA. However, upon activation of oncogenes in fibroblasts an induction and activation of p53 was observed, suggesting that activation of p53 by MITA occurs predominantly in tumor cells.     

Tumor suppressor protein Pdcd4 inhibits translation of p53 mRNA

The tumor suppressor protein Pdcd4 is thought to suppress translation of mRNAs containing structured 5'-UTRs by interacting with translation initiation factor eIF4A and inhibiting its helicase activity. However, natural target mRNAs regulated by Pdcd4 so far are mostly unknown. Here, we identified p53 mRNA as a translational target of Pdcd4. We found that Pdcd4 is associated with p53 mRNA and suppresses its translation. The inhibitory effect of Pdcd4 on the translation of p53 mRNA depends on the ability of Pdcd4 to interact with eIF4A and is mediated by the 5'-UTR of p53 mRNA, which is able to form a stable stem-loop structure. We show that treatment of cells with DNA-damaging agents decreases the expression of Pdcd4. This suggests that translational suppression by Pdcd4 plays a role in maintaining a low level of p53 in unstressed cells and that this suppression is abrogated due to low levels of Pdcd4 after DNA damage. Overall, our work demonstrates for the first time that Pdcd4 is directly involved in translational suppression of a natural mRNA with a 5'-structured UTR and provides novel insight into the translational control of p53 expression.     

p53-mediated up-regulation of CD95 is not involved in genotoxic drug-induced apoptosis of human breast tumor cells

Induction of CD95 (Fas/APO-1) and CD95 ligand during chemotherapeutic treatment may contribute to the death by apoptosis of some tumor cells. In this study, we have analyzed the role of the CD95 system in genotoxic drug-induced death of human breast tumor cells. Incubation of the breast tumor cell lines MCF-7 and EVSA-T with doxorubicin or methotrexate caused apoptosis after 48 h of treatment. These drugs induced a marked increase in the level of CD95 mRNA and protein in wild-type p53-expressing MCF-7 cells. On the contrary, the breast cancer cell line EVSA-T that expresses high levels of an inactive form of p53, did not up-regulate CD95 upon drug treatment. Elevation of CD95 expression by DNA-damaging drugs was notably blocked in MCF-7 cells expressing the human papillomavirus type 16 E6 protein (E6 cells) which prevented p53 accumulation upon DNA damage. However, E6 cells were still killed by the drugs. Furthermore, the genotoxic drugs did not induce the expression of CD95 ligand in MCF-7 cells at doses that caused apoptosis in these breast tumor cells. Moreover, drug-induced apoptosis of breast tumor cells was not prevented in the presence of either a CD95 antagonistic antibody or a CD95 ligand blocking antibody. We also observed a strong synergism between lower doses of DNA-damaging drugs and CD95 agonistic antibody in the induction of apoptosis in MCF-7 cells. In summary, our data indicate that drug-induced apoptosis of breast tumor cells occurs by a CD95/CD95L-independent mechanism although by elevating the tumor suppressor proteins p53 and CD95, genotoxic drugs may sensitize breast tumor cells to CD95-mediated apoptosis.     

The Inhibition of Autophagy Sensitises Colon Cancer Cells with Wild-Type p53 but Not Mutant p53 to Topotecan Treatment

Background

Topotecan produces DNA damage that induces autophagy in cancer cells. In this study, sensitising topotecan to colon cancer cells with different P53 status via modulation of autophagy was examined.

Methodology/Principal Findings

The DNA damage induced by topotecan treatment resulted in cytoprotective autophagy in colon cancer cells with wild-type p53. However, in cells with mutant p53 or p53 knockout, treatment with topotecan induced autophagy-associated cell death. In wild-type p53 colon cancer cells, topotecan treatment activated p53, upregulated the expression of sestrin 2, induced the phosphorylation of the AMPKα subunit at Thr172, and inhibited the mTORC1 pathway. Furthermore, the inhibition of autophagy enhanced the anti-tumour effect of topotecan treatment in wild-type p53 colon cancer cells but alleviated the anti-tumour effect of topotecan treatment in p53 knockout cells in vivo.

Conclusions/Significance

These results imply that the wild-type p53-dependent induction of cytoprotective autophagy is one of the cellular responses that determines the cellular sensitivity to the DNA-damaging drug topotecan. Therefore, our study provides a potential therapeutic strategy that utilises a combination of DNA-damaging agents and autophagy inhibitors for the treatment of colon cancer with wild-type p53.     

p53 mediates repression of the BRCA2 promoter and down-regulation of BRCA2 mRNA and protein levels in response to DNA damage

Adriamycin and other DNA-damaging agents have been shown to reduce BRCA2 mRNA levels in breast cancer cell lines, but the mechanism by which this occurs is unknown. In this study, we show that adriamycin and mitomycin C, but not other DNA-damaging agents, repress BRCA2 promoter activity in a dose- and time-dependent manner. We demonstrate that the effect is dependent on wild type p53 and that adriamycin and p53 mediate repression of the BRCA2 promoter by inhibiting binding of an upstream stimulatory factor protein complex to the promoter. In addition, we present evidence indicating that adriamycin and other DNA-damaging agents reduce BRCA2 mRNA and protein levels by altering both BRCA2 mRNA stability and protein stability. Thus, BRCA2 levels in the cell are regulated by three independent mechanisms in a p53-dependent manner.