Mutations in TP53 and PIK3CA genes in hepatocellular carcinoma patients are associated with chronic Schistosomiasis

The purpose of this study was to evaluate the genetic variation of the PIK3CA gene and the histopathological changes in liver tissue of patients with chronic Schistosomiasis to predict hepatocellular carcinoma. In this retrospective, the study samples were taken from 20 patients, divided into chronic schistosomiasis infected group of people (S) and chronic schistosomiasis uninfected group of people (C). The liver tissue biopsy samples for histological examinations were obtained only from chronic Schistosomiasis patients (n = 9). The blood samples were obtained from groups S and C for the mutational analysis of the PIK3CA and TP53 genes. The results suggest that the patients diagnosed with chronic Schistosomiasis were 9 (55%), and healthy patients without Schistosomiasis were 11 (45%). Histological results found that proliferation of fibrosis was observed in the hepatocytes of schistosomiasis patients. A total of 8 mutations (5 male, 3 female) were detected in PIK3CA and TP53 genes. Including 1634 A > G substitution mutations in PIK3CA, which was the only mutation found in males and females among the 8 mutations, accounting 22.22%. PIK3CA gene mutations were found more predominant in male groups as compared to other TP53 gene mutations. In conclusion, this study found that patients with chronic Schistosomiasis are at risk of PIK3CA gene mutations, eventually leading to hepatocytes fibrosis and liver cancer.     

Suppression of apoptosis and clonogenic survival in irradiated human lymphoblasts with different TP53 status

The influence of radiation-induced apoptosis on radiosensitivity was studied in a set of closely related human lymphoblastoid cell lines differing in TP53 status. The clonogenic survival of irradiated TK6 cells (expressing wild-type TP53), WTK1 cells (overexpressing mutant TP53), and TK6E6 cells (negative for TP53 owing to transfection with HPV16 E6) was assessed in relation to the induction of apoptosis and its suppression by caspase inhibition or treatment with PMA as well as after treatment with caffeine. Measurements using the alkaline comet assay and pulsed-field electrophoresis of the induction and repair of DNA strand breaks showed similar kinetics of the processing of early DNA damage in these cell lines. The cytochalasin B micronucleus assay revealed identical levels of residual damage in the first postirradiation mitosis of these cells. Abrogation of TP53-dependent apoptosis in TK6E6 cells resulted in a distinct increase in radioresistance. Further suppression of apoptosis as observed in WTK1 cells overexpressing mutant TP53 apparently was not responsible for the high radioresistance of WTK1 cells, since other means of highly efficient suppression of apoptosis (caspase inhibition or PMA treatment) increased the clonogenic survival of irradiated TK6 cells only to levels similar to those of TK6E6 cells with abrogated TP53-dependent apoptosis. Considering the similar levels of residual chromosomal damage in TK6E6 cells and WTK1 cells, a hitherto unknown mechanism of tolerance needs to be inferred for these TP53 mutant cells. This residual damage tolerance, however, appears to require an intact G2/M-phase checkpoint function since the relative radioresistance of the WTK1 cells was completely lost upon caffeine treatment, which also resulted in a failure of the TK6 and TK6E6 cells to execute apoptosis. In this situation, the cellular response seems to be dominated entirely by TP53-independent mitotic failure.     

Low-dose-rate radiation attenuates the response of the tumor suppressor TP53

Acute low-dose irradiation (0.1-1 Gy, 1.33 Gy/min) of cells of a human glioblastoma cell line, A-172, induced a dose-dependent monophasic accumulation of TP53 (formerly known as p53) and CDKN1A (formerly known as WAF1). In contrast, chronic gamma irradiation (0.001 Gy/min) produced a clear biphasic response of accumulation TP53 with the first peak at 1.5 h (0.09 Gy) and the second peak at 10 h (0.54 Gy). Significantly, when the cells were preirradiated with a chronic dose of gamma irradiation for 24 h (1.44 Gy) or 50 h (3 Gy), they no longer responded to an acute challenging dose to produce a dose-dependent response of the TP53 pathway. These findings suggest that chronic irradiation at low dose rate alters the TP53-dependent signal transduction pathway. Wearing away of the TP53 pathway by chronic exposure to radiation may have important implications for radiation protection.     

CDK4-mediated MnSOD activation and mitochondrial homeostasis in radioadaptive protection

Mammalian cells are able to sense environmental oxidative and genotoxic conditions such as the environmental low-dose ionizing radiation (LDIR) present naturally on the earth’s surface. The stressed cells then can induce a so-called radioadaptive response with an enhanced cellular homeostasis and repair capacity against subsequent similar genotoxic conditions such as a high dose radiation. Manganese superoxide dismutase (MnSOD), a primary mitochondrial antioxidant in mammals, has long been known to play a crucial role in radioadaptive protection by detoxifying O₂^•− generated by mitochondrial oxidative phosphorylation. In contrast to the well-studied mechanisms of SOD2 gene regulation, the mechanisms underlying posttranslational regulation of MnSOD for radioprotection remain to be defined. Herein, we demonstrate that cyclin D1/cyclin-dependent kinase 4 (CDK4) serves as the messenger to deliver the stress signal to mitochondria to boost mitochondrial homeostasis in human skin keratinocytes under LDIR-adaptive radioprotection. Cyclin D1/CDK4 relocates to mitochondria at the same time as MnSOD enzymatic activation peaks without significant changes in total MnSOD protein level. The mitochondrial-localized CDK4 directly phosphorylates MnSOD at serine-106 (S106), causing enhanced MnSOD enzymatic activity and mitochondrial respiration. Expression of mitochondria-targeted dominant negative CDK4 or the MnSOD-S106 mutant reverses LDIR-induced mitochondrial enhancement and adaptive protection. The CDK4-mediated MnSOD activation and mitochondrial metabolism boost are also detected in skin tissues of mice receiving in vivo whole-body LDIR. These results demonstrate a unique CDK4-mediated mitochondrial communication that allows cells to sense environmental genotoxic stress and boost mitochondrial homeostasis by enhancing phosphorylation and activation of MnSOD.     

Prognostic value of mutations in TP53 and RAS genes in breast cancer

The identification of molecular indicators of higher risk for specific subgroups of cancer patients may allow to develop more aggressive therapeutic strategies aimed at cases with the highest likelihood of response. This would avoid unnecessary toxicity to patients and alleviate the burden of cancer care for healthcare systems. Activated oncogenes and mutated tumor suppressor genes are causal determinants of the appearance and progression of tumors in man. They therefore represent potential indicators of prognosis and/or response to therapy. However, even in cases of well-studied oncogenes and tumor suppressor genes such as TP53 and RAS, their attributed prognostic and predictive value is often based on studies of insufficient statistical power that often lead to conflicting conclusions. Findings in favor or against the use of TP53 and RAS as prognostic and predictive indicators in breast cancer are reviewed and discussed here.     

Association of TP53 mutation, p53 overexpression, and p53 codon 72 polymorphism with susceptibility to apoptosis in adult patients with diffuse astrocytomas

Clarification of TP53 alterations is important to understand the mechanisms underlying the development of diffuse astrocytomas. It has been suggested that the alleles of TP53 at codon 72 differ in their ability to induce apoptosis in human cancers. The aim of this study was to analyze the possible association of TP53 mutation, p53 overexpression, and p53 codon 72 polymorphism with susceptibility to apoptosis in adult Brazilian patients with diffuse astrocytomas. We analyzed 56 surgical specimens of diffuse astrocytomas for alterations of TP53, using polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) direct sequencing. p53 and cleaved caspase 3 protein expression were assessed by immunohistochemistry. We found TP53 mutations in 19.6% (11 out of 56) of tumors tested, with the lowest mutation rate found in the cases of glioblastomas (8.8%) (p = 0.03). Only 16.1% of tumors tested showed cleaved caspase 3-positive staining, demonstrating that apoptosis is very inhibited in these tumors. All tumors having TP53 mutation and p53 accumulation had no expression of cleaved caspase 3. Additionally, no association was observed in tumors having proline and arginine alleles and expression of cleaved caspase 3. We concluded that clarification of the TP53 alterations allows a better understanding of the mechanisms involved in the progression of diffuse astrocytomas, and the allele status at codon 72 was not associated with apoptosis in these tumors.     

Frequent and simultaneous epigenetic inactivation of TP53 pathway genes in acute lymphoblastic leukemia

Aberrant DNA methylation is one of the most frequent alterations in patients with Acute Lymphoblastic Leukemia (ALL). Using methylation bead arrays we analyzed the methylation status of 807 genes implicated in cancer in a group of ALL samples at diagnosis (n = 48). We found that 154 genes were methylated in more than 10% of ALL samples. Interestingly, the expression of 13 genes implicated in the TP53 pathway was downregulated by hypermethylation. Direct or indirect activation of TP53 pathway with 5-aza-2′-deoxycitidine, Curcumin or Nutlin-3 induced an increase in apoptosis of ALL cells. The results obtained with the initial group of 48 patients was validated retrospectively in a second cohort of 200 newly diagnosed ALL patients. Methylation of at least 1 of the 13 genes implicated in the TP53 pathway was observed in 78% of the patients, which significantly correlated with a higher relapse (p = 0.001) and mortality (p<0.001) rate being an independent prognostic factor for disease-free survival (DFS) (p = 0.006) and overall survival (OS) (p = 0.005) in the multivariate analysis. All these findings indicate that TP53 pathway is altered by epigenetic mechanisms in the majority of ALL patients and correlates with prognosis. Treatments with compounds that may reverse the epigenetic abnormalities or activate directly the p53 pathway represent a new therapeutic alternative for patients with ALL.     

Inducible heat-shock protein 70 is involved in the radioadaptive response

Park, S-H., Lee, S-J., Chung, H-Y., Kim, T-H., Cho, C-K., Yoo, S-Y. and Lee, Y-S. Inducible Heat-Shock Protein 70 Is Involved in the Radioadaptive Response. The thermoresistant (TR) clone of radiation-induced fibrosarcoma (RIF) cells showed an adaptive response, i.e. a reduced effect, after exposure to a higher challenging dose (4 Gy) when the priming dose (1 cGy) was given 4 or 7 h earlier, but RIF cells did not. Since inducible Hsp70 expression was different in cells of these two cell lines, the role of inducible Hsp70 in the adaptive response was examined. When inducible Hsp70 was transfected into RIF cells, the adaptive response was acquired. Transfection of inducible Hsp70 to NIH 3T3 mouse embryo cells also conferred radioresistance to the cells as assayed by clonogenic survival, [(3)H]thymidine incorporation, and an ELISA cell death detection kit. An increased tendency for the induction of an adaptive response was also observed. Interestingly, basal levels of Ca(2+)-dependent and independent Pkc activities were increased by transfection with inducible Hsp70 compared to those of control vector cells. Irradiation with gamma rays induced activation of Pkc within minutes in control vector cells, while transfection with inducible Hsp70 did not. Cellular redistribution to particulate fractions of Pkca, d and z after exposure gamma rays also was not detected. Furthermore, radioresistance by transfection with inducible Hsp70, as tested by clonogenic survival, disappeared after pretreatment with Pkc inhibitors, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), prolonged treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), and GF109203X. Taken together, these data suggest that radioresistance inducible by Hsp70 is associated with an elevated level of Pkc activity.     

Involvement of TP53 in apoptosis induced in human lymphoblastoid cells by fast neutrons

We investigated the involvement of TP53 in apoptosis induced by fast neutrons in cells of three human B-lymphoblast cell lines derived from the same donor and differing in TP53 status: TK6 (wild-type TP53), WTK1 (mutant TP53) and NH32 (knockout TP53). Cells were exposed to X rays or to fast neutrons at doses ranging from 0.5 to 8 Gy. Apoptosis was determined by measurements of the sub-G0 /G1-phase DNA content and by the externalization of phosphatidylserine. Fast neutrons induced extensive apoptosis in TK6 cells, as shown by the formation of hypodiploid particles, the externalization of phosphatidylserine, and the activation of caspases. In contrast, cell death was triggered at a significantly lower rate in cells lacking functional TP53. However, TP53-independent cell death also expressed the morphological and biochemical hallmarks of apoptosis. Proliferation tests and clonogenic assays showed that fast neutrons can nevertheless kill WTK1 and NH32 cells efficiently. The absence of functional TP53 only delays radiation-induced cell death, which is also mediated by caspases. These results indicate that fast-neutron irradiation activates two pathways to apoptosis and that the greater relative biological effectiveness of fast neutrons reflects mainly an increase in clonogenic cell death.     

Mammalian DNA damage-inducible genes associated with growth arrest and apoptosis

Mammalian cells are exposed to a wide variety of genotoxic stresses from both endogenous and exogenous sources. Cells typically exhibit cell cycle delays, or checkpoints, in response to acute genotoxic stress. Other types of cellular responses to DNA damage include apoptosis and probably increases in DNA repair levels. These response pathways are altered in cancer cells, by genetic alterations such as overexpression or mutation of oncogenes, or loss of tumor suppressor gene functions. As cancer chemotherapy relies primarily on the selective killing of cancer cells by DNA-damaging agents, genetic alterations affecting cellular stress response pathways may affect the outcome of cancer treatment.     

Low-dose radiation hypersensitivity is associated with p53-dependent apoptosis

Exposure to environmental radiation and the application of new clinical modalities, such as radioimmunotherapy, have heightened the need to understand cellular responses to low dose and low-dose rate ionizing radiation. Many tumor cell lines have been observed to exhibit a hypersensitivity to radiation doses <50 cGy, which manifests as a significant deviation from the clonogenic survival response predicted by a linear-quadratic fit to higher doses. However, the underlying processes for this phenomenon remain unclear. Using a gel microdrop/flow cytometry assay to monitor single cell proliferation at early times postirradiation, we examined the response of human A549 lung carcinoma, T98G glioma, and MCF7 breast carcinoma cell lines exposed to gamma radiation doses from 0 to 200 cGy delivered at 0.18 and 22 cGy/min. The A549 and T98G cells, but not MCF7 cells, showed the marked hypersensitivity at doses <50 cGy. To further characterize the low-dose hypersensitivity, we examined the influence of low-dose radiation on cell cycle status and apoptosis by assays for active caspase-3 and phosphatidylserine translocation (Annexin V binding). We observed that caspase-3 activation and Annexin V binding mirrored the proliferation curves for the cell lines. Furthermore, the low-dose hypersensitivity and Annexin V binding to irradiated A549 and T98G cells were eliminated by treating the cells with pifithrin, an inhibitor of p53. When p53-inactive cell lines (2800T skin fibroblasts and HCT116 colorectal carcinoma cells) were examined for similar patterns, we found that there was no hyperradiosensitivity and apoptosis was not detectable by Annexin V or caspase-3 assays. Our data therefore suggest that low-dose hypersensitivity is associated with p53-dependent apoptosis.     

TP53 and mutations in human cancer

TP53 is the most frequently mutated gene in human cancer, with a predominance of missense mutations scattered over 200 codons. In many cancers, specific mutation patterns can be identified, which are shaped by site-specific mutagenesis and by biological selection. In tobacco-related cancers (lung, head and neck), organ-specific patterns are observed, with many mutations compatible with the ones experimentally induced by tobacco carcinogens. In several other cancers, such as squamous cell carcinoma of the oesophagus or hepatocellular carcinoma (HCC), mutation patterns show geographic variations between regions of high and low incidence, suggesting a role for region-specific risk factors. HCC from high-incidence regions showing also a high prevalence of a specific Ser-249 TP53 mutation is one of the most striking examples of a mutagen fingerprint. All such assessments are useful to generate clues on the mutagenic mechanisms involved in human cancer. Moreover, it has been shown that DNA retrieved from plasma can be successfully used for detection of TP53 mutations, which gives hope for earlier more accurate detection of human cancers.     

Crosstalk between the FGFR2 and TP53 genes in breast cancer: data from an association study and epistatic interaction analysis

To evaluate the potential for gene-gene interaction effects in sporadic breast cancer (BC) risk, we studied combinations of the fibroblast growth factor receptor 2 (FGFR2) rs1219648 and tumor protein 53 (TP53) rs1042522, rs1625895, and rs17878362 polymorphisms in BC patients (n=388) and healthy persons (n=275). In addition to a single-locus effect manifested by the association of FGFR2 rs1219648 and TP53 rs1042522 polymorphisms with high BC risk, depending on menopause status (0.001相似文献   

Polymorphisms in insulin-related genes predispose to specific KRAS2 and TP53 mutations in colon cancer

Risk factors for colon cancer may not only influence the overall risk of cancer but also the risk for specific types of mutations. We evaluated the effect of polymorphisms in four insulin-related genes (G972R in IRS1, G1057D in IRS2, a CA repeat in IGFI and an A/C polymorphism at -202 of IGFBP3) on the risk of microsatellite instability and KRAS2 and TP53 mutations in a population-based set of 1788 cases of colon cancer and 1981 controls. The GR/RR IRS1 genotypes were associated with an increased risk of colon cancers with the KRAS2 G12D mutation (OR 2.3, 95% CI 1.5, 3.5 versus controls, OR 1.7, 95% CI 1.1, 2.6 versus KRAS2 wild type), the "no 192" IGFI genotype increased the risk of the KRAS2 G13D mutation (OR 2.3, 95% CI 1.2, 4.2 versus controls, OR 2.1, 95% CI 1.1, 4.0 versus wild type), and the DD IRS2 genotype increased the risk of the G12V KRAS2 mutation (OR 1.8, 95% CI 0.9, 3.5 versus controls, OR 2.0, 95% CI 1.0, 4.0 versus wild type). Polymorphisms in IRS1 and IGF1 were also associated with an approximately two-fold increased risk of specific TP53 mutations relative to controls without cancer. We conclude that polymorphisms in some insulin-related genes are associated with an increased risk of colon cancer with specific KRAS2 and TP53 mutations, implying a link between these genetic changes and specific mutational pathways in carcinogenesis.     

Accelerated decline in lung function in cigarette smokers is associated with TP53/MDM2 polymorphisms

In vitro studies have shown that p53 mediates a protective response against DNA damage by causing either cell-cycle arrest and DNA repair, or apoptosis. These responses have not yet been demonstrated in humans. A common source of DNA damage in humans is cigarette smoke, which should activate p53 repair mechanisms. As the level of p53 is regulated by MDM2, which targets p53 for degradation, the G-allele of a polymorphism in intron 1 of MDM2 (rs2279744:G/T), that results in higher MDM2 levels, should be associated with a reduced p53 response and hence more DNA damage and corresponding tissue destruction. Similarly, the alleles of rs1042522 in TP53 that encode arginine (G-allele) or proline (C-allele) at codon 72, which cause increased pro-apoptotic (G-allele) or cell-cycle arrest activities (C-allele), respectively, may moderate p53’s ability to prevent DNA damage. To test these hypotheses, we examined lung function in relation to cumulative history of smoking in a population-based cohort. The G-alleles in MDM2 and TP53 were found to be associated with accelerated smoking-related decline in lung function. These data support the hypothesis that p53 protects from DNA damage in humans and provides a potential explanation for the variation in lung function impairment amongst smokers.     

Primordial germ cell deficiency in the connexin 43 knockout mouse arises from apoptosis associated with abnormal p53 activation

Connexin 43 knockout (Cx43alpha1KO) mice exhibit germ cell deficiency, but the underlying cause for the germ cell defect was unknown. Using an Oct4-GFP reporter transgene, we tracked the distribution and migration of primordial germ cells (PGCs) in the Cx43alpha1KO mouse embryo. Analysis with dye injections showed PGCs are gap-junction-communication competent, with dye coupling being markedly reduced in Cx43alpha1-deficient PGCs. Time-lapse videomicroscopy and motion analysis showed that the directionality and speed of cell motility were reduced in the Cx43alpha1KO PGCs. This was observed both in E8.5 and E11.5 embryos. By contrast, PGC abundance did not differ between wild-type and heterozygous/homozygous Cx43alpha1KO embryos until E11.5, when a marked reduction in PGC abundance was detected in the homozygous Cx43alpha1KO embryos. This was accompanied by increased PGC apoptosis and increased expression of activated p53. Injection of alpha-pifithrin, a p53 antagonist, inhibited PGC apoptosis and prevented the loss of PGC. Analysis using a cell adhesion assay indicated a reduction in beta1-integrin function in the Cx43alpha1KO PGCs. Together with the abnormal activation of p53, these findings suggest the possibility of anoikis-mediated apoptosis. Overall, these findings show Cx43alpha1 is essential for PGC survival, with abnormal p53 activation playing a crucial role in the apoptotic loss of PGCs in the Cx43alpha1KO mouse embryos.