Cancer‐specific mutations in p53 induce the translation of Δ160p53 promoting tumorigenesis

Wild‐type p53 functions as a tumour suppressor while mutant p53 possesses oncogenic potential. Until now it remains unclear how a single mutation can transform p53 into a functionally distinct gene harbouring a new set of original cellular roles. Here we show that the most common p53 cancer mutants express a larger number and higher levels of shorter p53 protein isoforms that are translated from the mutated full‐length p53 mRNA. Cells expressing mutant p53 exhibit “gain‐of‐function” cancer phenotypes, such as enhanced cell survival, proliferation, invasion and adhesion, altered mammary tissue architecture and invasive cell structures. Interestingly, Δ160p53‐overexpressing cells behave in a similar manner. In contrast, an exogenous or endogenous mutant p53 that fails to express Δ160p53 due to specific mutations or antisense knock‐down loses pro‐oncogenic potential. Our data support a model in which “gain‐of‐function” phenotypes induced by p53 mutations depend on the shorter p53 isoforms. As a conserved wild‐type isoform, Δ160p53 has evolved during millions of years. We thus provide a rational explanation for the origin of the tumour‐promoting functions of p53 mutations.     

Nanosecond pulsed electric fields induce apoptosis in p53-wildtype and p53-null HCT116 colon carcinoma cells

Non-ionizing radiation produced by nanosecond pulsed electric fields (nsPEFs) is an alternative to ionizing radiation for cancer treatment. NsPEFs are high power, low energy (non-thermal) pulses that, unlike plasma membrane electroporation, modulate intracellular structures and functions. To determine functions for p53 in nsPEF-induced apoptosis, HCT116p53^+/+ and HCT116p53^−/− colon carcinoma cells were exposed to multiple pulses of 60 kV/cm with either 60 ns or 300 ns durations and analyzed for apoptotic markers. Several apoptosis markers were observed including cell shrinkage and increased percentages of cells positive for cytochrome c, active caspases, fragmented DNA, and Bax, but not Bcl-2. Unlike nsPEF-induced apoptosis in Jurkat cells (Beebe et al. 2003a) active caspases were observed before increases in cytochrome c, which occurred in the presence and absence of Bax. Cell shrinkage occurred only in cells with increased levels of Bax or cytochrome c. NsPEFs induced apoptosis equally in HCT116p53^+/+ and HCT116p53^−/− cells. These results demonstrate that non-ionizing radiation produced by nsPEFs can act as a non-ligand agonist with therapeutic potential to induce apoptosis utilizing mitochondrial-independent mechanisms in HCT116 cells that lead to caspase activation and cell death in the presence or absence of p-53 and Bax. This work was supported by the U.S. Air Force Office of Scientific Research/DOD MURI grant on Subcellular Responses to Narrow Band and Wide Band Radio Frequency Radiation, administered by Old Dominion University, and the American Cancer Society.     

Apoptosis and cell recovery in response to oxidative stress in p53-deficient prostate carcinoma cells

We have studied the effects of different concentrations of H(2)O(2) on the proliferation of PC-3 prostate carcinoma cells. Since this cell line lacks functional p53, we sought to characterize whether apoptotic response to the oxidative insult was altered such that, unlike in cells containing functional p53 apoptosis may be reduced and replaced by other mechanisms of cellular arrest and death. We did not observe necrosis in PC-3 cells treated with H(2)O(2) concentrations of up to 500 microM. In the presence of 50 microM H(2)O(2), arrest was observed in the G2-phase of the cell cycle, along with p53-independent apoptosis. In the presence of 500 microM H(2)O(2), addition of l-buthionine sulfoximine increased the percentage of apoptotic cell death. Senescence-associated cell arrest was never observed. Moreover, some of the treated cells seemed to be resistant to oxidative damage. These cells re-entered the cell cycle and proliferated normally. Analysis of the expression of p21(waf1) and of p21 protein levels, as well as the activity of caspase-3 and caspase-8, allowed us to characterize some aspects of the arrest of PC-3 cells in G2 and the apoptotic response to oxidative stress in the absence of functional p53.     

Gain-of-function p53 protein transferred via small extracellular vesicles promotes conversion of fibroblasts to a cancer-associated phenotype

1. Download : Download high-res image (201KB)
2. Download : Download full-size image

     

Transfection of mutant p53 gene depresses X-ray- or CDDP-induced apoptosis in a human squamous cell carcinoma of the head and neck

The present study examined whether X-ray- and CDDP-sensitivities depend on p53 gene status in human squamous cell carcinoma of the head and neck (SAS cells) showing dominant negative nature of mutant p53 protein. SAS cells were transfected with a vector carrying a mutant p53 gene (SAS/Trp248 cells) or neomycin resistant gene control vector (SAS/neo cells). Sensitivities of the transfected cells to X-ray or CDDP were measured with colony formation assay. The incidence of apoptosis by X-ray or CDDP was analyzed with Hoechst staining or DNA ladder formation assay. The activation of caspase-3 was estimated as an indicator of apoptosis by the detection of fragmentation of caspase-3 or poly (ADP ribose) polymerase (PARP) with Western blot. SAS/Trp248 cells showed X-ray- and CDDP-resistance due to the dominant negative nature of mutant p53, compared with SAS/neo cells. The incidence of DNA ladders and apoptotic bodies increased markedly in SAS/neo cells after X-ray irradiation or CDDP treatment, but increased only slightly in SAS/Trp248 cells. Fragmentation of caspase-3 and PARP was observed in SAS/neo cells, but almost no such fragmentation was observed in SAS/Trp248 cells after X-ray irradiation or CDDP treatment. The present results strongly suggest that the X-ray- and CDDP-sensitivities of human squamous cell carcinomas are p53-dependent, and that the sensitivities are tightly correlated with the induction of apoptosis through caspase-3 activation. The p53-dependent X-ray- or CDDP-sensitivity was supported by results from p53-null human lung cancer H1299 cells which were transfected with wild-type or mutant p53 gene.     

Analysis of the p53 gene alterations in mouse embryo fibroblast cell line Balb 3T12 and its derivative 312

We have analysed the status of the p53 gene in the mouse embryo fibroblast cell line Balb 3T12 (TD₅₀=10⁶) and its transformed clonal derivative 312 (TD₅₀=10⁴) with an aim to determine whether there exists a correlation between increased tumorigenicity and clonal expansion of cells bearing a mutation in the p53 gene. While Southern hybridizations did not show any obvious changes in the p53 gene organization in 3T12 and 312 cells, sequencing the p53 cDNA revealed that 3T12 is mutated at the amino acid residue 233 (Tyr→ Asp) whereas 312 is mutated at the residue 132 (Cys→Trp). Exploiting the altered RFLP pattern due to mutations, we identified that 3T12 contains p53 alleles that are different from the already identified mutant p53. On the basis of these observations, we conclude that 3T12 and 312 have evolved independently.     

Linc-ROR regulates apoptosis in esophageal squamous cell carcinoma via modulation of p53 ubiquitination by targeting miR-204-5p/MDM2

The long intergenic noncoding RNA, regulator of reprogramming (linc-ROR) has been reported to participate in tumorigenesis, while its functions and fundamental mechanisms in esophageal squamous cell carcinoma (ESCC) remain unclear. In this study, gain-of-function assays showed that linc-ROR upregulation enhanced cell viability, promoted cell proliferation, and inhibited apoptosis. Mechanistically, the regulatory network of linc-ROR/miR-204-5p/MDM2 was established with bioinformatics analysis and online databases, then validated via dual-luciferase reporter assays, RNA immunoprecipitation assays in ESCC cells. Linc-ROR positively regulates the expression of MDM2 as a molecular sponge of miR-204-5p. Moreover, results of western blot and coimmunoprecipitation indicated that linc-ROR overexpression enhanced the ubiquitination level of p53, and its downstream apoptosis-related genes have showed higher bcl-2 expression, lower bax, and cleaved caspase-3 expressions, while miR-204-5p could counteract with this effect. Finally, small interfering RNAs tailored to linc-ROR were established to further evaluate its effects on ESCC comprehensively. In summary, this study revealed that linc-ROR modulated cell apoptosis and regulated p53 ubiquitination via targeting miR-204-5p/MDM2 axis, which provides a novel therapeutic insight into treatments for ESCC.     

Determination of sensitive positions to mutations in human p53 protein

Over last several years, we demonstrated that the mutations are more likely to occur at randomly unpredictable amino acid pairs in a protein. We therefore can in principle predict the amino acid pairs sensitive to the future mutations in a protein. However, we still need to predict the positions at which the sensitive amino acid pairs are located in a protein. In this study, we use a probabilistic approach to analyze the effect of 191 mutations in human p53 protein and can approximately estimate the sensitive positions to mutations in human p53 protein.     

CDK11p110 plays a critical role in the tumorigenicity of esophageal squamous cell carcinoma cells and is a potential drug target

Esophageal squamous cell carcinoma (ESCC) is a serious malignancy with limited options for targeted therapy. The exploration of novel targeted therapies for combating ESCC is urgently needed. Cyclin-dependent kinases (CDKs) play important roles in the progression of cancers; however, the function of CDK11^p110 (cyclin-dependent kinase 11^p110) in ESCC is still unknown. Here, we investigated the effects and molecular mechanisms of CDK11^p110 in the proliferation and growth of ESCC by examining the expression of CDK11^p110 in ESCC tissues and by detecting phenotypic changes in ESCC cells after CDK11^p110 knockdown or overexpression in vitro and in vivo. According to the tissue microarray analysis, compared with its expression level in normal tissues, the expression level of CDK11^p110 was significantly elevated in ESCC tissues; this result was in concordance with the data in TCGA (The Cancer Genome Atlas) datasets. In addition, RNAi-mediated CDK11^p110 silencing exerted a substantial inhibitory effect on the proliferation, clonogenicity and migration ability of ESCC cells. Further study indicated that CDK11^p110 knockdown arrested ESCC cells in the G2/M phase of the cell cycle and induced cell apoptosis. Moreover, stable shRNA-mediated CDK11^p110 knockdown inhibited tumor growth in an ESCC xenograft model, and overexpression of CDK11^p110 enhanced tumor growth. In addition, the Ki67 proliferation index was closely associated with the elevation or depletion of CDK11^p110 in vivo. In summary, this study provides evidence that CDK11^p110 play a critical role in the tumorigenicity of ESCC cells, which suggests that CDK11^p110 may be a promising therapeutic target in ESCC.

Abbreviations: CDKs: cyclin-dependent kinases; CDK11: Cyclin-dependent kinase 11; CDK11^p110: Cyclin-dependent kinase 11^p110, the larger isomer of cyclin-dependent kinase 11; ESCC: esophageal squamous cell carcinoma; FACS: fluorescence-activated cell sorting; FDA: the Food and Drug Administration; TCGA: The Cancer Genome Atlas; TMA: tissue microarray.     

Establishment and characterization of a human cell line derived from a uterine papillary serous carcinoma with wild-type p53 function

Uterine papillary serous carcinoma is an uncommon histologic subtype of endometrial cancer that behaves aggressively and has a poor prognosis. We successfully established a uterine papillary serous carcinoma cell line. The population-doubling time was approximately 16 h. Although loss of p53 function is considered critical for the molecular pathogenesis of uterine papillary serous carcinoma, p53 was not only mutated but functionally active in this cell line. This newly established cell line should be useful for investigating the characteristics of uterine papillary serous carcinoma.     

p53-independent pRB degradation contributes to a drug-induced apoptosis in AGS cells

The retinoblastoma （RB） tumor suppressor protein, pRB, plays an important role in the regulation of mammalian cell cycle. Furthermore, several lines of evidence suggest that pRB also involves in the regulation of apoptosis. In the present study, the degradation of pRB was observed in apoptotic gastric tumor cells treated with a new potent anti-tumor component, tripchlorolide （TC）. The inhibition of pRB degradation by a general cysteine protease inhibitor IDAM resulted in the reduction of the apoptotic cells. Furthermore, the survival of the gastric tumor cells under the TC treatment was enhanced by an over-expression of exogenous pRB. These results suggest that the pRB degradation of the gastric tumor cells under the TC treatment involves in the apoptotic progression. In addition, the same extent of TCinduced pRB-degradation was detected in the gastric tumor cells containing a p53 dominant-negative construct, indicat- ing that this kind of pRB degradation is p53-independent.     

The p33ING1b tumor suppressor cooperates with p53 to induce apoptosis in response to etoposide in human osteosarcoma cells

p33ING1b induces cell cycle arrest and stimulates DNA repair, apoptosis and chemosensitivity. The magnitude of some p33ING1b effects may be due to activation of the tumor suppressor p53. To investigate if the p33ING1b protein affected chemosensitivity of osteosarcoma cells, we overexpressed p33ING1b in p53+/+ U2OS cells or in p53-mutant MG63 cells, and then assessed for growth arrest and apoptosis after treatment with etoposide. p33ING1b increased etoposide-induced growth inhibition and apoptosis to a much greater degree in p53+/+ U2OS cells than in p53-mutant MG63 cells. Moreover, ectopic expression of p33ING1b markedly upregulated p53, p21WAF1 and bax protein levels and activated caspase-3 protein kinase in etoposide-treated U2OS cells. Together, our data indicate that p33ING1b prominently enhances etoposide-induced apoptosis through p53-dependent pathways in human osteosarcoma cells. p33ING1b may be an important marker and/or therapeutic target in the prevention and treatment of metastatic osteosarcoma.     

Determination of amino acid pairs in human p53 protein sensitive to mutations/variants by means of a random approach

This is the continuation of our studies using random approaches to analyse the p53 protein family. In this data-based theoretical analysis, we use the random approach to analyse the amino acid pairs in human p53 protein in order to determine which amino acid pairs are more sensitive to 190 human p53 mutations/variants. The rationale of this study is based on our hypothesis and findings that a harmful mutation is more likely to occur at randomly unpredictable amino acid pairs, and a harmless mutation is more likely to occur at randomly predictable amino acid pairs. This is because we argue that the randomly predictable amino acid pairs should not be deliberately evolved, whereas the randomly unpredictable amino acid pairs should be deliberately evolved with a connection to protein function. The results show, for example, that 93.16% of 190 mutations/variants occur at randomly unpredictable amino acid pairs. Thus, the randomly unpredictable amino acid pairs are more sensitive to mutations/variants in human p53 protein. The results also suggest that the human p53 protein has a tendency for the occurrence of mutation/variants.     

p53在诱导性多潜能干细胞向肿瘤细胞转化中的作用

p53基因是抑制肿瘤形成的抑癌基因,p53通过阻断细胞周期、诱导衰老细胞凋亡和修复受损的DNA从而抑制肿瘤的形成。而突变的p53则具有相反的功能,被认为是促进细胞重编程的关键。这些发现表明p53在去分化过程中具有重要的作用,而肿瘤的形成与细胞重编程存在高度的相似性,所以探讨p53与诱导性多能干细胞(induced pluripotent stem cell,iPSC)和肿瘤形成之间的联系具有一定的意义。     

PKCdelta requires p53 for suppression of the transformed phenotype in human colon cancer cells

We have previously demonstrated that the delta isoform of Protein Kinase C (PKCdelta) acts as a tumor suppressor in HCT116 human colon cancer cells, and that p21(waf1/cip1) is an essential downstream effector of PKCdelta. Our data suggested that p53 might also be involved in the suppression of the neoplastic phenotype induced by PKCdelta. Here we show that homozygous knockout of p53 renders the HCT116 cell line unresponsive to PKCdelta overexpression. Whereas reconstitution of p53 alone did not modify the morphology and growth properties of HCT116/p53null cells, overexpression of both p53 and PKCdelta induced a number of alterations indicating suppression of the transformed phenotype. Interestingly, PKCdelta was ineffective when overexpressed in HT29 cells, a human colon cancer line characterized by the Arg273His dominant-negative mutation of p53. Thus, our data indicate that wild-type p53 is an essential effector of PKCdelta in human colon cancer cells.     

p53 is important for the anti-proliferative effect of ibuprofen in colon carcinoma cells

S-ibuprofen which inhibits the cyclooxygenase-1/-2 and R-ibuprofen which shows no COX-inhibition at therapeutic concentrations have anti-carcinogenic effects in human colon cancer cells; however, the molecular mechanisms for these effects are still unknown. Using HCT-116 colon carcinoma cell lines, expressing either the wild-type form of p53 (HCT-116 p53^wt) or being p(HCT-116 p53^−/−), we demonstrated that both induction of a cell cycle block and apoptosis after S- and R-ibuprofen treatment is in part dependent on p53. Also in the in vivo nude mice model HCT-116 p53^−/− xenografts were less sensitive for S- and R-ibuprofen treatment than HCT-116 p53^wt cells. Furthermore, results indicate that induction of apoptosis in HCT-116 p53^wt cells after ibuprofen treatment is in part dependent on a signalling pathway including the neutrophin receptor p75^NTR, p53 and Bax.