Cadmium induces p53-dependent apoptosis in human prostate epithelial cells

Cadmium, a widespread toxic pollutant of occupational and environmental concern, is a known human carcinogen. The prostate is a potential target for cadmium carcinogenesis, although the underlying mechanisms are still unclear. Furthermore, cadmium may induce cell death by apoptosis in various cell types, and it has been hypothesized that a key factor in cadmium-induced malignant transformation is acquisition of apoptotic resistance. We investigated the in vitro effects produced by cadmium exposure in normal or tumor cells derived from human prostate epithelium, including RWPE-1 and its cadmium-transformed derivative CTPE, the primary adenocarcinoma 22Rv1 and CWR-R1 cells and LNCaP, PC-3 and DU145 metastatic cancer cell lines. Cells were treated for 24 hours with different concentrations of CdCl(2) and apoptosis, cell cycle distribution and expression of tumor suppressor proteins were analyzed. Subsequently, cellular response to cadmium was evaluated after siRNA-mediated p53 silencing in wild type p53-expressing RWPE-1 and LNCaP cells, and after adenoviral p53 overexpression in p53-deficient DU145 and PC-3 cell lines. The cell lines exhibited different sensitivity to cadmium, and 24-hour exposure to different CdCl(2) concentrations induced dose- and cell type-dependent apoptotic response and inhibition of cell proliferation that correlated with accumulation of functional p53 and overexpression of p21 in wild type p53-expressing cell lines. On the other hand, p53 silencing was able to suppress cadmium-induced apoptosis. Our results demonstrate that cadmium can induce p53-dependent apoptosis in human prostate epithelial cells and suggest p53 mutation as a possible contributing factor for the acquisition of apoptotic resistance in cadmium prostatic carcinogenesis.     

Selective COX-2 inhibitor (celecoxib) decreases cellular growth in prostate cancer cell lines independent of p53

Abstract

Celecoxib is a clinically available COX-2 inhibitor that has been reported to have antineoplastic activity. It has been proposed as a preventative agent for several types of early neoplastic lesions. Earlier studies have shown that sensitivity of prostatic carcinoma (PCa) to celecoxib is associated with apoptosis; however, these studies have not demonstrated adequately whether this effect is dependent on p53 status. We studied the relation between sensitivity to celecoxib and the phenotypic p53 status of PCa cells lines, LNCaP (wild type p53), PC3 (null p53) and DU145 (mutated p53). Cellular growth was assessed at 24, 48, 72 and 96 h after celecoxib treatment at concentrations of 0, 10, 30, 50, 70 and 100 μM using an MTT assay. Cellular proliferation (Ki-67 expression) was determined by immunocytochemistry. Phenotypic expression of p53 was analyzed by western blotting. The effects of celecoxib on cellular growth and its association with p53 were assessed after down-regulation of p53 using synthetic interfering RNAs (siRNA) in LNCaP cells. Expression of p53 and COX-2 at mRNA levels was assessed by quantitative real time polymerase reaction (qRT-PCR). We found that celecoxib inhibited cellular growth and proliferation in a dose-dependent manner in all three cell lines; LNCaP cells with a native p53 were the most sensitive to celecoxib. We observed a down- regulation effect on p53 in LNCaP cells exposed to ≥ 30 μM celecoxib for 72 h, but found no significant changes in the p53 levels of DU145 cells, which have a mutated p53. Reduced COX-2 expression was found with decreased p53 in LNCaP and PC-3 cells that were exposed to ≥ 20 μM of celecoxib for 72 h, but COX-2 expression was increased in DU145 cells. All three cell lines demonstrated pan-cytotoxicity when exposed to 100 μM celecoxib. When p53 expression was inhibited using siRNA in LNCaP cells, the inhibitory effects on cellular growth usually exerted by celecoxib were not changed significantly. Celecoxib reduces the growth of prostate cancer cell lines in part by decreasing proliferation, which suggests that the inhibition of growth of LNCaP cells by celecoxib is independent of normal levels of native p53.     

淋巴细胞凋亡与p53蛋白表达关系的研究

培养B95-8细胞,分离EB病毒,转染外周血和扁桃体淋巴细胞,建立永生化的LCLs和TLCL细胞株; 带有wt P₅₃基因的LCLs在DNA损伤剂——顺铂处理前未检出p53蛋白,经顺铂处理后,LCLs随作用时间延长细胞存活率明显下降、p53蛋白水平升高、DNA电泳显出梯状带;含mt P₅₃基因的淋巴瘤细胞在顺铂处理前可检出高浓度的p53蛋白,经顺铂处理后,细胞存活率与p53蛋白并无明显改变.这些结果表明：顺铂引起细胞DNA损伤、激活wt p53蛋白的表达、继而wt p53蛋白又促进了DNA损伤细胞凋亡.     

Accumulation of mutant p53(V143A) modulates the growth, clonogenicity, and radiochemosensitivity of malignant glioma cells independent of endogenous p53 status

Alterations of the p53 gene have been attributed a major role in the development and resistance to therapy of several human cancers. Accumulation of p53 in tumor cells may result from mutations associated with prolonged half-life or from stabilization of wild-type p53 by different mechanisms. To address the role of p53 accumulation in the response of malignant glioma cells to radiochemotherapy, we expressed the p53 mutant p53(V143A) in five human malignant glioma cell lines with different genetic and functional p53 status. Accumulation of p53(V143A) modulated proliferation in three and clonogenicity in four of five cell lines without a clear pattern with regard to their endogenous p53 status. p53(V143A) inhibited the camptothecin-induced accumulation of p21(WAF1/CIP1) in cell lines with p53 functional wild-type activity, but not in cell lines lacking p53 activity, consistent with a transdominant-negative effect of p53(V143A). Irradiation induced a moderate G2/M arrest in all cell lines, irrespective of the p53 status, that was unaffected by p53(V143A). Radiosensitivity as well as sensitivity to BCNU, teniposide (VM26), topotecan, vincristine, Taxol, and cisplatin both in cytotoxic cell death and in clonogenic cell death was unchanged in p53(V143A)-transfected cells with few exceptions. These data do not support the hypothesis that accumulation of mutant p53 is a major determinant of the response to adjuvant radiochemotherapy in human malignant glioma cells.     

Phenylbutyrate-induced apoptosis and differential expression of Bcl-2, Bax, p53 and Fas in human prostate cancer cell lines

OBJECTIVE: To assess the mechanisms of action of phenylbutyrate (PB), an investigational chemotherapeutic agent for prostate cancer (PCa), in apoptosis induction in PCa cell lines in vitro. STUDY DESIGN: We analyzed the differential expression of different apoptosis modulators, Bcl-2, Bax, p53 and Fas, for their potential role in PB-induced apoptosis using relative quantitative flow cytometry (FCM). Both androgen-dependent (LNCaP) and androgen-independent (C-4-2, PC-3-PF and DU145) human PCa cell lines were examined. RESULTS: PB induced apoptosis in PCa cell lines in a dose-dependent manner. Fifty percent apoptosis could be induced by 5-10 mM PB. Bcl-2 was down-regulated 30-75% and the Bax:Bcl-2 ratio elevated in apoptotic PCa cell lines regardless of their androgen dependency or p53 status. FCM revealed a heterogeneous stimulatory effect on the expression of Bax and Bcl-2 in PC3-PF cells at 0.5-2.5 mM PB. In a p53-positive cell line (DU145), p53 was repressed by 70% and Fas elevated sixfold with 10 mM PB. CONCLUSION: Our data show that PB-induced PCa apoptosis is associated with the relative repression of Bcl-2 and with up-regulation of Bax and Fas proteins and that this PB-induced apoptosis is independent of p53 and androgen-dependency status of PCa cell lines.     

p53-dependent and p53-independent activation of apoptosis in mammary epithelial cells reveals a survival function of EGF and insulin

The p53 tumor suppressor protein has been implicated as a mediator of programmed cell death (PCD). A series of nontransformed mammary epithelial cell (MEC) lines were used to correlate p53 function with activation of PCD. Treatment of MECs expressing mutant, inactive, or no p53 with DNA-damaging agents did not induce apoptosis. Upon introduction of temperature-sensitive p53 into HC11 cells, which lack wild-type (wt) p53, PCD was observed after mitomycin treatment at 32 degrees, when the ts p53 protein is in wt conformation. Thus, wt p53 mediates activation of PCD in response to mitomycin in HC11 cells. Treatment of the MCF10-A cells, which express wt p53, with various DNA- damaging agents led to nuclear accumulation of p53. Only mitomycin treatment led to an increase in the number of apoptotic nuclei. ErbB-2- transformed MCF10-A cells responded to mitomycin, cisplatin, and 5-Fl- uracil, suggesting that signaling from activated ErbB-2 enhances the cells ability to respond to DNA damage. A combination of high cell density and serum-free medium induces apoptosis in all MECs tested, irrespective of their p53 status. Under these conditions, EGF or insulin act as survival factors in preventing PCD. These data might elucidate some aspects of breast involution and tumorigenesis.     

Trifluorothymidine Induces Cell Death Independently of p53

Trifluorothymidine (TFT), a potent anticancer agent, inhibits thymidylate synthase (TS) and is incorporated into the DNA, both events resulting in cell death. Cell death induction related to DNA damage often involves activation of p53. We determined the role of p53 in TFT cytotoxicity and cell death induction, using, respectively, the sulforhodamine B-assay and FACS analysis, in a panel of cell lines with either wild type, inactive, or mutated p53. Neither TFT cytotoxicity nor cell death induction changed with TFT exposure in cell lines with wt, inactive or mutated p53. Conclusion: sensitivity to TFT is not dependent on the expression of wt p53.     

Trifluorothymidine induces cell death independently of p53

Trifluorothymidine (TFT), a potent anticancer agent, inhibits thymidylate synthase (TS) and is incorporated into the DNA, both events resulting in cell death. Cell death induction related to DNA damage often involves activation of p53. We determined the role of p53 in TFT cytotoxicity and cell death induction, using, respectively, the sulforhodamine B-assay and FACS analysis, in a panel of cell lines with either wild type, inactive, or mutated p53. Neither TFT cytotoxicity nor cell death induction changed with TFT exposure in cell lines with wt, inactive or mutated p53. Conclusion: sensitivity to TFT is not dependent on the expression of wt p53.     

Potentiation of a tumor cell susceptibility to autologous CTL killing by restoration of wild-type p53 function

Inactivation of p53 has been implicated in many types of tumors particularly in non-small cell lung carcinoma, one of the most common cancers in which p53 mutation has been frequently identified. The aim of this study was to investigate the influence of p53 status on the regulation of tumor susceptibility to specific CTL-mediated cell death. For this purpose, we used a cytotoxic T lymphocyte clone, Heu127, able to lyse the human autologous lung carcinoma cell line, IGR-Heu, in a HLA-A2-restricted manner. Direct genomic DNA sequencing revealed that IGR-Heu expresses a mutated p53 at codon 132 of the exon 5 which results in the loss of p53 capacity to induce the expression of the p53-regulated gene product p21(waf/CIP1). Initial experiments demonstrated that IGR-Heu was resistant to Fas, TNF, and TRAIL apoptotic pathways. This correlated with the lack of p55 TNFRI, Fas, DR4, and DR5 expression. The effect of wild-type (wt) p53 restoration on the sensitization of IGR-Heu to autologous CTL clone lysis was investigated following infection of the tumor cell line with a recombinant adenovirus encoding the wt p53 (Adwtp53). We demonstrate that the restoration of wt p53 expression and function resulted in a significant potentiation of target cell susceptibility to CTL-mediated lysis. The wt p53-induced optimization of tumor cell killing by specific CTL involves at least in part Fas-mediated pathway via induction of CD95 expression by tumor cells but does not appear to interfere with granzyme B cytotoxic pathway.     

miR-155 通过p53/p21 调控前列腺癌细胞周期

目的:探讨miR-155对前列腺癌细胞周期的影响及其分子机制。方法:通过转染anti-miR-155抑制前列腺癌DU145和PC-3细胞中miR-155水平后,采用流式细胞术观察细胞周期的变化,western blot和RT-PCR观察p53和p21蛋白及CDK2和cyclin蛋白和m RNA表达的变化。结果:与对照组相比,DU145和PC-3细胞转染anti-miR-155后,G2/M期细胞阻滞,S期细胞数比例显著增加(P0.05),p53和p21蛋白和m RNA表达水平显著增加(P0.01),CDK2和cyclin E蛋白和m RNA表达均显著降低(P0.01)。结论:miR-155可影响人前列腺癌细胞的周期,可能与其调节p53、p21及其下游的CDK2和cyclin E的表达相关。     

Accumulation of Mutant p53 Modulates the Growth, Clonogenicity, and Radiochemosensitivity of Malignant Glioma Cells Independent of Endogenous p53 Status

Alterations of the p53 gene have been attributed a major role in the development and resistance to therapy of several human cancers. Accumulation of p53 in tumor cells may result from mutations associated with prolonged half-life or from stabilization of wild-type p53 by different mechanisms. To address the role of p53 accumulation in the response of malignant glioma cells to radiochemotherapy, we expressed the p53 mutant p53^V143A in five human malignant glioma cell lines with different genetic and functional p53 status. Accumulation of p53^V143A modulated proliferation in three and clonogenicity in four of five cell lines without a clear pattern with regard to their endogenous p53 status. p53^V143A inhibited the camptothecin-induced accumulation of p21^WAF1/CIP1 in cell lines with p53 functional wild-type activity, but not in cell lines lacking p53 activity, consistent with a transdominant-negative effect of p53^V143A. Irradiation induced a moderate G2/M arrest in all cell lines, irrespective of the p53 status, that was unaffected by p53^V143A. Radiosensitivity as well as sensitivity to BCNU, teniposide (VM26), topotecan, vincristine, Taxol, and cisplatin both in cytotoxic cell death and in clonogenic cell death was unchanged in p53^V143A-transfected cells with few exceptions. These data do not support the hypothesis that accumulation of mutant p53 is a major determinant of the response to adjuvant radiochemotherapy in human malignant glioma cells.     

Bivariate analysis of the p53 pathway to evaluate Ad-p53 gene therapy efficacy.

BACKGROUND: Gene therapy of human tumors with adenovirus vectors presents a clinical research challenge and a potential opportunity in cancer therapy. One of the research challenges is that endpoints like tumor reduction, time to recurrence, and survival do not provide information about whether a potential therapeutic infects the targeted cells or whether the transferred gene functions or induces a cellular response. Therefore, a flow cytometric approach was developed for a wildtype, p53 encoding adenoviral vector (Ad-p53) that provides (1) the relative level of p53 transferred by p53 immunoreactivity, (2) mdm2 immunoreactivity as an assay of p53 activity, and (3) estimates of the percentage of infected cells by dual parameter analysis (p53 versus mdm2). METHODS: Three prostate cancer cell lines (PC-3, LNCaP, DU 145) that are null, wild-type, and mutant for p53, respectively, and two ovarian cancer cell lines (PA1, MDAH 2774) that are wild-type and mutant for p53, respectively, were tested for immunoreactivity and lack of cross-reactivity with the monoclonal antibodies, DO-7 (anti-p53) and IF2 (anti-mdm2). Optimal dual staining conditions for a flow cytometric assay employing saturating levels of antibody were developed and tested by infection of PC-3, PA1, and MDAH 2774 with Ad-p53 or a control virus, Ad-luc. Dual staining with DO-7 and propidium iodide was used to determine any biological effect of the transferred gene. RESULTS: Neither DO-7 nor IF2 showed appreciable cross-reactions by Western blot analysis of representative prostate or ovarian cell lines. By flow cytometric titration, DO-7 appears to be a high avidity antibody (saturation staining of 10(6) DU 145 cells with 0.5ug) whereas IF2 appears less so (optimum signal to noise ratio at 1ug/10(6) cells). Infection with Ad-p53 was detected at 6 to 48 hours post infection as a uniform relative increase in p53 levels over background p53 levels. Coincident increases in mdm2 immunoreactivity were also detected. DNA content measurements of PA1 and MDAH 2774 cells indicated that G1 arrest and/or apoptosis occurred subsequent to Ad-p53 infection. p53 and mdm2 levels and DNA content distributions for Ad-luc infected cells were equivalent to uninfected cells. CONCLUSIONS: A flow cytometric approach to measure the efficacy of an Ad-p53 gene therapy vector was developed that detects not only the gene transferred but also the activity of the transferred gene product.     

Anti-Fas antibody-induced apoptosis in human colorectal carcinoma cell lines: role of the p53 gene

The cell surface Fas antigen transducts an apoptotic signal by its crosslinking with Fas ligand or anti-Fas antibody in a variety of human cultured cells. In this study, we examined the expression of Fas antigen and its mediation of apoptosis in six human colorectal carcinoma cell lines. A flow cytometric analysis revealed that LoVo, DLD-1, WiDr and SW837 cell lines showed higher expression levels of Fas antigen, in contrast to lower expression in COLO201 and COLO320DM. Interferon- enhanced the expression of Fas antigen in all of the cell lines examined. Both Fas ligand and Fas-associated phosphatase-1 (FAP-1) were expressed only in COLO320DM. Anti-Fas antibody induced apoptosis in LoVo carrying wild-type p53 gene, but not in the other five cell lines carrying mutated p53 gene. The transfection of wild-type p53 gene using an adenovirous vector upregulated P53 protein in WiDr and SW837 cells, both of which showed, however, no increase in apoptotic cells by anti-Fas antibody treatment. These results indicated that (1) Fas antigen was variably expressed, regardless of the p53 gene status and (2) the susceptibility to anti-Fas antibody-mediated apoptosis did not correlate to Fas, Fas ligand or FAP-1 expression levels. Therefore, we conclude that wild-type P53 expression might not necessarily be essential for Fas-mediated apoptosis in human colorectal carcinoma cell lines.     

Retained platinum uptake and indifference to p53 status make novel transplatinum agents active in platinum-resistant cells compared to cisplatin and oxaliplatin

Despite the clinical success of platinum-containing drugs in the treatment of solid tumors, acquired resistance remains a major obstacle. We previously identified a group of novel transplanaramine or transplatinum compounds based on distinct activity profiles in the NCI-60 panel. In the present study, parental KB-3.1 cells with wild-type p53 and its cisplatin- and oxaliplatin-resistant sublines harboring mutant p53 proteins were used to contrast several transplatinum compounds with cisplatin and oxaliplatin. The transplatinum compounds retained cytotoxic activity in the resistant cell lines. While intracellular accumulation and DNA platination of cisplatin and oxaliplatin was decreased in the resistant cells, the transplatinum compounds both accumulated intracellularly and platinated DNA at comparable levels in all cell lines. Cytoflow analysis confirmed that cisplatin and oxaliplatin alter the cell cycle distribution and result in apoptosis; however, at comparably toxic concentrations, the transplatinum compounds did not alter the cell cycle distribution. Analysis of the cytoplasmic fraction treated with acetone showed that cisplatin and oxaliplatin readily bound to macromolecules in the pellet, whereas a larger percentage of the transplatinum compounds remained in the supernatant. We concluded that, distinct from platinum compounds currently in use, transplatinum compounds accumulate intracellularly in resistant cells at levels comparable to those in drug-sensitive cells, do not affect the cell cycle and thus retain cytotoxicity independent of p53 status and likely have cytoplasmic targets that are important in their activity.     

Electrogene therapy with p53 of murine sarcomas alone or combined with electrochemotherapy using cisplatin

The aim of our study was to evaluate feasibility and therapeutic potential of electrogene therapy with p53 alone or combined with electrochemotherapy using cisplatin on two murine sarcomas with different p53 status. Antitumor effectiveness of three consecutive electrogene treatments with p53 was more effective in wild-type LPB tumors than mutated SA-1 tumors, resulting in 21.4% of tumor cures in LPB tumors and 12.5% in SA-1 tumors. Pretreatment of tumors with electrogene therapy with p53 enhanced chemosensitivity of both tumor models treated by electrochemotherapy with cisplatin. After only one application of this treatment combination in the LPB tumor model, specific tumor growth delay was prolonged in the combined treatment group compared to electrogene therapy with p53 or electrochemotherapy with cisplatin alone, whereas in SA-1 tumors this treatment combination resulted in 31.6% of cured animals. Results of our study show that electrogene therapy with p53 alone or combined with electrochemotherapy is feasible and effective treatment of tumors. The combination of electrogene therapy and electrochemotherapy after only one application resulted in complete regression of tumors.     

E2F-1 has properties of a radiosensitizer and its regulation by cyclin A kinase is required for cell survival of fibrosarcoma cells lacking p53.

Negative regulation of E2F-1 DNA binding function by cyclin A kinase represents part of an S-phase checkpoint control system that, when activated, leads to apoptosis. In this study, we examined the cellular sensitivity and resistance of isogenic mouse fibrosarcoma cell lines, differing primarily in their p53 status, to ectopic expression of wild-type (wt) E2F-1 and cyclin A kinase binding-defective mutants of it. We found that E2F-1 (wt) potently affected the survival of p53+/+ tumor cells but not that of p53-/- cells. In contrast, expression of cyclin A kinase binding-defective E2F-1 species interfered with cell survival of fibrosarcoma cells irrespective of their p53 status. Finally, expression of E2F-1 (wt) in p53-/- fibrosarcoma cells enhanced the cytotoxic effect of ionizing radiation in vitro and in vivo in a mouse tumor model. These results suggest that E2F-1-dependent activation of an S-phase checkpoint is p53 independent and that E2F-1 possesses radiosensitizing properties in the absence of p53.     

Prolonged wild-type p53 protein accumulation and cisplatin resistance

The major limitation for the chemotherapeutic use of DNA-damaging agent cisplatin is the development of resistance in initially responsive tumors. One of the main pathways regulating cell survival following DNA damage is the p53 pathway. In this study we compared the cisplatin-induced response of p53 protein and its downstream targets p21WAF-1 and Mdm2 in the cisplatin-sensitive ovarian carcinoma cell line A2780 and its cisplatin-resistant derivative CP70. A higher dose of cisplatin and a longer exposure time was required to achieve the same level of p53, p21WAF-1, and Mdm2 protein accumulation in the cisplatin-resistant CP70 cells versus cisplatin-sensitive A2780 cells. A significant difference between the two cell lines was observed in cisplatin-induced stabilization of p53 protein. The p53 half-life increased 31-fold in CP70 cells compared to only 6-fold in A2780 cells. In contrast, there was no difference in p21WAF-1 half-life between the two cell lines. These results demonstrate that in A2780 and CP70 cells resistance to cisplatin correlates with prolonged p53 protein stabilization and accumulation.     

Increased BNip3 and decreased mutant p53 in cisplatin-sensitive PDT-resistant HT29 cells

We have reported previously the isolation of three photodynamic therapy (PDT)-resistant human colon carcinoma HT29 cell lines that show increased expression of the Hsp27 and BNip3 protein and a decreased expression of the mutant p53 protein compared to parental HT29 cells. Since mutant p53 and increased expression of Hsp27 have been associated with resistance to various chemotherapeutic agents, whereas BNip3 is a potent inducer of apoptosis, we were interested in determining whether these PDT-resistant cells were cross-resistant to other cytotoxic agents. We report here that the PDT-resistant HT29 cell lines showed a significant increase in cisplatin sensitivity and an increase in both spontaneous and cisplatin-induced apoptosis compared to parental HT29 cells. In addition, the cisplatin sensitivity of the PDT-resistant HT29 variants and several other clonal variants of HT29 cells correlated with increased BNip3 and decreased mutant p53 protein levels, but not Hsp27 protein levels.