Contribution of autophagic cell death to p53-dependent cell death in human glioblastoma cell line SF126

Apoptosis and autophagic cell death are programmed cell deaths that are involved in cell survival, growth, development and carcinogenesis. p53, the most extensively studied tumor suppressor, regulates apoptosis and autophagy by transactivating its downstream genes. It also stimulates the mitochondrial apoptotic pathway and inhibits autophagy in a transactivation-independent manner. However, the contribution of apoptosis and autophagic cell death to p53-dependent cell death is unclear. Using wild-type (WT) and mutant (MT) p53 inducible cell lines in TP53-null SF126 glioblastoma cells, we examined the apoptosis and autophagic cell death induced by p53. WT p53 expression in SF126 cells induced apoptosis and autophagy, and reduced the cell number. An autophagy inhibitor reduced autophagy, increased the S-phase fraction, and attenuated the inhibition of cell proliferation induced by WT p53. Pan-caspase inhibitor reduced apoptosis but showed weaker inhibition of cell proliferation than the autophagy inhibitor. We concluded that p53-dependent cell death in SF126 cells comprises caspase-dependent and caspase-independent apoptosis and autophagic cell death, and the induction of autophagy as well as apoptosis could be a new strategy to treat some type of WT p53-retaining tumors.     

Down-regulation of Wilms’ tumor 1 expression in glioblastoma cells increases radiosensitivity independently of p53

The Wilms’ tumor 1 (WT1) gene is overexpressed in human glioblastoma and correlates with wild-type p53 status. In other cell types, WT1 inhibits p53-mediated apoptosis in response to DNA damaging agents. However, neither this interaction nor the relationship between WT1 and radiosensitivity has been studied in glioblastoma. To study this interaction, we generated LN-229 glioma cell lines (p53 mutant) stably expressing WT1 isoforms and induced apoptosis by transfecting with different doses of wild-type p53 plasmid expression vector. Constitutive expression of WT1 did not protect against exogenous p53-mediated apoptosis. Likewise, WT1 expression did not protect against endogenous p53-mediated cell death induced by radiotherapy in U87MG cells, which contain functional wild-type p53. We then tested the efficacy of WT1 siRNA in inhibiting WT1 expression and its effect on radiosensitivity. In T98G and LN-18 glioma cells, which possess p53 mutations, WT1 siRNA decreased WT1 protein to almost undetectable levels by 96-h post-transfection. Furthermore, WT1 siRNA transfection caused a significantly larger decrease in viability following irradiation than was seen in untransfected cells in both cell lines after treatment with ED₅₀ of ionizing radiation. In conclusion, WT1 overexpression did not protect against p53-mediated apoptosis or ionizing radiation induced cell death. WT1 siRNA increased the radiosensitivity of two human glioma cell lines independently of p53. Anti-WT1 strategies may, therefore, prove useful in improving the response of glioblastoma to radiotherapy, thus potentially improving patient survival.     

Endogenous p21WAF1/CIP1 status predicts the response of human tumor cells to wild-type p53 and p21WAF1/CIP1 overexpression

Expression of exogenous wild-type (wt) p53 protein can suppress the growth and/or induce apoptosis in different tumor cells. The effect of exogenous p21(WAF1/CIP1) expression is more controversial: while it can induce apoptosis in some cells, it can protect against p53-mediated apoptosis in others. We used adenoviral vectors to introduce p53 and p21(WAF1/CIP1) genes into human tumor cell lines with different p53 and/or p21(WAF1/CIP1) status. The cell growth inhibition and the induction of apoptosis were measured. Overexpression of wt p53 induced more efficient growth inhibition and apoptosis in SW 620 (mutant p53) and HeLa (inactivated p53 protein) than in MCF-7 (wt p53) and CaCo-2 cell line, which was the most resistant to p53 overexpression despite the p53 mutation. Unlike HeLa and SW 620 cells, the basal p21 protein level was readily detected in CaCo-2 and MCF-7 cells. Overexpression of p21(WAF1/CIP1) gene induced somewhat less pronounced growth inhibition of all cell lines tested, but it also induced apoptosis in HeLa and SW 620 cells. These results suggest that the basal, but not the inducible, levels of p21(WAF1/CIP1) protein in tumor cells could protect from p53-mediated apoptosis. On the other hand, overexpression of p21(WAF1/CIP1) gene itself can induce apoptosis in cells with no basal p21(WAF1/CIP1) protein level. Possible mechanisms of the differential response to these genes are discussed.     

Specific TP53 Mutants Overrepresented in Ovarian Cancer Impact CNV,TP53 Activity,Responses to Nutlin-3a,and Cell Survival

Evolutionary Action analyses of The Cancer Gene Atlas data sets show that many specific p53 missense and gain-of-function mutations are selectively overrepresented and functional in high-grade serous ovarian cancer (HGSC). As homozygous alleles, p53 mutants are differentially associated with specific loss of heterozygosity (R273; chromosome 17); copy number variation (R175H; chromosome 9); and up-stream, cancer-related regulatory pathways. The expression of immune-related cytokines was selectively related to p53 status, showing for the first time that specific p53 mutants impact, and are related to, the immune subtype of ovarian cancer. Although the majority (31%) of HGSCs exhibit loss of heterozygosity, a significant number (24%) maintain a wild-type (WT) allele and represent another HGSC subtype that is not well defined. Using human and mouse cell lines, we show that specific p53 mutants differentially alter endogenous WT p53 activity; target gene expression; and responses to nutlin-3a, a small molecular that activates WT p53 leading to apoptosis, providing “proof of principle” that ovarian cancer cells expressing WT and mutant alleles represent a distinct ovarian cancer subtype. We also show that siRNA knock down of endogenous p53 in cells expressing homozygous mutant alleles causes apoptosis, whereas cells expressing WT p53 (or are heterozygous for WT and mutant p53 alleles) are highly resistant. Therefore, despite different gene regulatory pathways associated with specific p53 mutants, silencing mutant p53 might be a suitable, powerful, global strategy for blocking ovarian cancer growth in those tumors that rely on mutant p53 functions for survival. Knowing p53 mutational status in HGSC should permit new strategies tailored to control this disease.Abbreviations: CASP3, caspase 3; CDKN1A, cyclin dependent protein kinase 1, P21; CNV, copy number variation; DNAJC3, DnaJ homolog subfamily C; GFP, green fluorescent protein; GOF, gain-of-function; HGSC, high-grade serous ovarian cancer; HOX, homeobox genes; LOH, loss of heterozygosity; MDM2, mouse double minute 2; TCGA, The Cancer Genome Atlas; TP53, tumor protein 53, p53; UPR, unfolded protein response; WT, wild type     

Activation of p53/p21Waf1/Cip1 pathway by 5-aza-2'-deoxycytidine inhibits cell proliferation, induces pro-apoptotic genes and mitogen-activated protein kinases in human prostate cancer cells

The tumor suppressor gene p53 plays an essential role in cell proliferation and apoptosis. Due to its relevance to cancer therapy, most studies have focused on the cellular consequences of p53 activation in relation to cytotoxic drugs. 5-aza-2'-deoxycytidine (5-aza-CdR) is widely used as an anti-cancer drug for the treatment of leukemia and solid tumors. However, the mechanism by which 5-aza-CdR exerts its anti-neoplastic activity remains unclear. Here, we address the role of p53 in regulating cellular responses to 5-aza-CdR treatment in human prostate cancer cells. We found that 5-aza-CdR induces p53 and p21Waf1/Cip1 expression associated with inhibition of cell proliferation in LNCaP cells (p53 wild-type), but not in DU145 cells (p53 mutant). By using pifithrin-alpha, a chemical inhibitor of p53, we confirmed that the increase in p21Waf1/Cip1 expression and inhibition of cell proliferation in LNCaP cells by 5-aza-CdR is p53-dependent. Also, the activation of p53 and p21Waf1/Cip1 pathway by 5-aza-CdR modified multiple gene expressions including apoptotic target genes and MAP kinases in LNCaP cells. 5-aza-CdR-induced apoptosis in LNCaP cells is assessed by DNA fragmentation analysis. Furthermore, knockdown of p53 by pU6-p53 siRNA vector suggests the involvement of MAP kinases in the process of 5-aza-CdR-mediated activation of p53 pathway to inhibit cell proliferation and induce apoptosis. Finally, the comet or SCGE assay and methylation-sensitive restriction analysis demonstrated that 5-aza-CdR induced p53 and p21Waf1/Cip1 expression as a consequence of DNA damage and independent of DNA demethylation. Our findings suggest that 5-aza-CdR induces anti-neoplastic activity primarily through the activation of p53 pathway in response to DNA damage and subsequently leads to inhibition of cell proliferation as well as induction of apoptosis. Therefore, our data indicate that p53 status in tumor cells may be critical for the clinical efficacy and toxicity of 5-aza-CdR.     

Effects of p53 mutations on cellular sensitivity to ionizing radiation

Mutations in the p53 tumor suppressor gene have been found in more than 50% of human tumors including those in breast, colon, lung, and oral cavity. However, the significance of p53 mutation in radiation sensitivity and its underlying mechanisms still remains unclear. In this study, we have measured the effects of p53 mutation on cell cycle delay, apoptosis, and radiation sensitivity using mouse cells transfected with different forms of p53 mutations. Wild-type p53 and p53-Null mouse embryo fibroblast cells were used as positive and negative controls, respectively. Exponentially growing cells were irradiated with 0- to 9-Gy gamma rays and then assayed for cell survival, p53 expression, cell cycle checkpoint, and apoptosis. Cell survivals determined by clonogenic assay show that p53 mutant cells are generally more sensitive to ionizing radiation than cells with wild-type p53. Western blot analysis indicates that exposure to 6-Gy gamma rays increases the p53 expression levels by two- to threefold in wild-type p53 cells. However, the p53 level remains unchanged in cells with mutant p53 during the same postirradiation period. Irradiation with 6-Gy gamma rays produces G2/M arrest in all cell lines, indicating that p53 is probably not involved in the G2/M checkpoint. However, all mutant cells fail to show any significant G1/S arrest after irradiation, suggesting that G1/S arrest may be implicated in radiation sensitivity. Finally, there is very little apoptosis (<3% by Tat-mediated dUTP nick-end labeling [TUNNEL] and morphologic assays) detected in wild-type and p53 mutant cell lines after 6-Gy gamma rays. Our results suggest that mutant forms of p53 represent a phenotype that affects the radiation sensitivity and is not dependent on the apoptotic pathway.     

Introduction of mutant p53 into a wild-type p53-expressing glioma cell line confers sensitivity to Ad-p53-induced apoptosis

Transient expression of the tumor suppressor gene p53 via adenoviral-mediated gene transfer induces apoptosis in glioma cells expressing mutant p53, while causing cell cycle arrest in cells with wild-type p53. To determine whether a change in p53 status of a wild-type p53-expressing cell line such as U-87 MG would alter its apoptotic resistant phenotype in response to Ad-p53 infection, we generated cell lines U-87-175.4 and U-87-175.13 via retroviral-mediated gene transfer of the p53 (175H) mutant into the U-87 MG parental line. Control cell lines U-87-Lux.6 and U-87-Lux.8 were also generated and express the reporter gene luciferase. Both U-87-175.4 and U-87-175.13, but not control cell lines, exhibited morphology characteristic of apoptosis after Ad-p53 infection. Furthermore, expression of other p53 mutants (248W, 273H) in U-87 MG also sensitized cells to Ad-p53-induced apoptosis. Apoptosis was confirmed by TUNEL and cell cycle analysis. Several p53 response genes were examined in cells infected with Ad-p53, and among these, BCL2, p21WAF1/CIP1, CPP32/caspase 3, and PARP showed differences in expression between U87-175 and U87-Lux cell lines. Taken together, our data demonstrate that the introduction of p53 mutants in U-87 MG promotes an apoptotic response in association with adenoviral-mediated wild-type p53 gene transfer. These results underscore the importance of glioma p53 genotype for predicting tumor response to p53-based gene therapy.     

Anti-cancer efficacy of SREBP inhibitor,alone or in combination with docetaxel,in prostate cancer harboring p53 mutations

Mutant p53 proteins (mutant p53s) have oncogenic gain-of-function properties correlated with tumor grade, castration resistance, and prostate cancer (PCa) tumor recurrence. Docetaxel is a standard first-line treatment for metastatic castration-resistant PCa (mCRPC) after the failure of hormone therapy. However, most mCRPC patients who receive docetaxel experience only transient benefits and rapidly develop incurable drug resistance, which is closely correlated with the p53 mutation status. Mutant p53s were recently reported to regulate the metabolic pathways via sterol regulatory element-binding proteins (SREBPs). Therefore, targeting the SREBP metabolic pathways with docetaxel as a combination therapy may offer a potential strategy to improve anti-tumor efficacy and delay cellular drug resistance in mCRPC harboring mutant p53s. Our previous data showed that fatostatin, a new SREBP inhibitor, inhibited cell proliferation and induced apoptosis in androgen receptor (AR)-positive PCa cell lines and xenograft mouse models. In this study, we demonstrated that mutant p53s activate the SREBP-mediated metabolic pathways in metastatic AR-negative PCa cells carrying mutant p53s. By blocking the SREBP pathways, fatostatin inhibited cell growth and induced apoptosis in metastatic AR-negative PCa cells harboring mutant p53s. Furthermore, the combination of fatostatin and docetaxel resulted in greater proliferation inhibition and apoptosis induction compared with single agent treatment in PCa cells in vitro and in vivo, especially those with mutant p53s. These data suggest for the first time that fatostatin alone or in combination with docetaxel could be exploited as a novel and promising therapy for metastatic PCa harboring p53 mutations.     

Chemosensitivity of human malignant glioma: modulation by p53 gene transfer

Loss of wild-type p53 activity is one of the most common molecular abnormalities in human cancers including malignant gliomas. The p53 status is also thought to modulate sensitivity to irradiation and chemotherapy. Here, we studied the effect of a p53 gene transfer on the chemosensitivity of three human glioma cell lines with different endogenous p53 status (LN-229, wild-type; LN-18, mutant; LN-308, deleted), using the murine temperature-sensitive p53 val¹³⁵ mutant. Expression of mutant p53 enhanced proliferation of LN-308 cells but reduced proliferation in the other cell lines. Expression of wild-type p53 caused reversible growth arrest of all cell lines but failed to induce apoptosis. Growth arrest induced by wild-type p53 was associated with strong induction of p21 expression. Strong induction of BAX expression and loss of BCL-2 expression, which are associated with p53-dependent apoptosis rather than growth arrest, were not observed. Wild-type p53 failed to sensitize glioma cells to cytotoxic drugs including BCNU, cytarabine, doxorubicin, teniposide and vincristine. The combined effects of wild-type p53 gene transfer and drug treatment were less than additive rather than synergistic, suggesting that the intracellular cascades activated by p53 and chemotherapy are rebundant. Unexpectedly, forced expression of mutant p53 modulated drug sensitivity in that it enhanced the toxicity of some drugs but attenuated the effects of others. These effects may represent a dominant negative effect of mutant p53 in LN-229 cells which have wild-type p53 activity but must be considered a gain of function-type effect in the other two cell lines which have no wild-type p53 activity. Importantly, no clear-cut pattern emerged among the three cell lines studied. We conclude that somatic gene therapy based on the reintroduction of p53 will limit the proliferation of human malignant glioma cells but is unlikely to induce clinically relevant sensitization to chemotherapy in these tumors.     

Implication of p53 in growth arrest and apoptosis induced by the synthetic retinoid CD437 in human lung cancer cells.

CD437 is a novel retinoid that can induce apoptosis in a variety of tumor cell types by an unknown mechanism. We found that CD437 up-regulated the expression of p21(WAF1/CIP1), Bax, and Killer/DR5 and induced G1 arrest and rapid apoptosis in three human non-small cell lung carcinoma cell lines with wild-type p53 but not in five cell lines with mutant p53, suggesting a role for p53 in the effects of CD437. Using H460 cells in which wild-type p53 protein was degraded by transfection of the human papillomavirus 16 E6 (HPV-16 E6) gene and H460 cells transfected with a control plasmid only, we found that CD437 increased p53, p21(WAF1/CIP1), Bax, and Killer/DR5 in the control transfectants. In contrast, the constitutive p53 protein level was suppressed, and the ability of CD437 to increase p53 and its downstream genes was compromised in E6 transfectants. In addition, CD437 induced G1 arrest and apoptosis in the control transfectants but not in the E6-transfected cells. These results indicate that p53 plays a role in CD437-induced growth inhibition and apoptosis in human non-small cell lung carcinoma cells.     

Induction of apoptosis in human choriocarcinoma cell lines by treatment with 3,4-dihydro-6-[4-(3,4-dimethoxybenzoyl)-1-piperazinyl]-2(1H)-quinolinone (vesnarinone)

Induction of apoptosis is an attractive strategy in cancer therapy but it clinical practice is not yet sufficient in choriocarcinoma. The quinolinone derivative, vesnarinone, is a novel inotropic agent used for treating congestive heart failure and may also have a potential anticancer activity. It induces apoptosis and differentiation in some tumor cell lines. We examined the antitumor effect of vesnarinone in eight cell lines established from human choriocarcinoma and hydatidiform mole using MTT assay and also analyzed the nuclear fragmentation of tumor cells by DNA electrophoresis assay. Vesnarinone inhibited the proliferation of choriocarcinoma cell lines in a dose-dependent manner and induced DNA fragmentation in cells. However, the BM cell line prepared by subcultivation from hydatidiform mole showed no growth suppression or DNA fragmentation in response to vesnarinone. On the other hand, PCR-SSCP analysis and direct DNA sequencing have shown that a human choriocarcinoma cell line, SCH, has a mutant p53 gene at codon 249. When SCH cells were treated with vesnarinone cellular proliferation was significantly inhibited. Vesnarinone suppressed the proliferation of all choriocarcinoma cell lines and induced apoptosis, regardless of the existence of p53 mutation. In addition, it has been found by RT-PCR that expression of c-Myc mRNA is upregulated by treating choriocarcinoma cells with vesnarinone. The finding suggests that vesnarinone might induce expression of c-Myc gene in choriocarcinoma cells, the product of which may be associated with the inhibition of cell growth and induce apoptosis. These results suggest that vesnarinone is a useful reagent for the treatment of choriocarcinoma.