Wild-type P53 Protein Levels Research Articles

P53 proteins accumulated by heat stress associate with heat shock proteins HSP72/HSC73 in human glioblastoma cell lines.

We investigated the accumulation of p53 proteins after heat stress and their association with HSP72/HSC73 using four human glioblastoma cell lines. Human glioblastoma cell lines U-87MG and A-172 exhibited no mutation in the region between the 2nd and 11th exons of the p53 gene, whereas A-7 and T98G had mutations in exon 5 and exon 7 of the p53 gene, respectively. In U-87MG and A-172, the levels of wild-type p53 protein were slightly increased by heat stress. Levels of mutant p53 protein were apparently increased by heat stress in A-7, but not in T98G. Furthermore, wild-type p53 proteins in both U-87MG and A-172 co-immunoprecipitated with anti-HSP72/HSC73 antibody and HSP72 and HSC73 in them co-immunoprecipitated with anti-p53 antibody as did the mutant p53 proteins. These findings suggest that p53 proteins accumulated by heat stress are associated with HSP72 and HSC73.

EFFECT OF ACTINOMYCIN-D ON THE CELL-CYCLE PROGRESSION AND THE EXPRESSION OF P53, WAF1/CIP1, GADD45, AND MDM-2 GENES IN HUMAN ORAL KERATINOCYTES - IMPLICATION OF HUMAN PAPILLOMAVIRUS INFECTION

Exposure of human oral keratinocytes immortalized by transfection with 'high risk' HPV DNA to chemical carcinogens converts the cells to malignant phenotype, but it does not transform normal cells. To investigate the underlying mechanism for different chemical carcinogen susceptibility of normal and the HPV-immortalized oral keratinocytes to genotoxic agent, we studied the progression of cell cycle and the expression of p53, WAF1/CIP1, gadd45 and mdm-2 genes in normal and in the HPV-immortalized oral keratinocytes after exposing cells to actinomycin D. Normal oral keratinocytes demonstrated transient G(1) arrest after the exposure, but the HPV-immortalized cells did not. Actinomycin D significantly increased the levels of intranuclear wild-type p53 and mdm-2 proteins and the transcripts of WAF1/CIP1, gadd45 and mdm-2 in normal cells, but it did not increase them in the HPV-immortalized cells. These data indicate that actinomycin D-induced transient cell cycle arrest may be associated with enhanced level of wild-type p53 protein and the transcripts of WAF1/CIP1 and gadd45 in normal human oral keratinocytes. This study also suggests that overexpressed mdm-2 may not be enough to abrogate the cell cycle arrest mediated by wild-type p53. Our data support the idea that conversion of the HPV-immortalized oral keratinocytes to tumorigenic cells may, in part, be due to the incapability of the cells to arrest the cell cycle when exposed to genotoxic agents, which would result in improper repair of damaged DNA and subsequent inheritance of genetic error.

Transient Stabilization of p53 in Non-Small Cell Lung Carcinoma Cultures Arrested for Growth by Retinoic Acid

Proliferation of five non-small cell lung carcinoma (NSCLC) cultures was inhibited after 16 h exposure to retinoic acid. We investigated whether expression of the p53 protein correlated with the growth pattern of NSCLC lines observed in the presence of retinoic acid. Levels of wild-type p53 protein underwent fivefold increases in lines H460a and H226b after 16 to 48 h treatment with 5 μM retinoic acid but then decreased to undetectable amounts in these cell lines after 72 h retinoic acid treatment. Levels of p53 transcripts remained unchanged during the time of increases in protein expression in retinoic acid-treated H460a cells, suggesting that a post-translational mechanism was involved in the increased expression of the protein. Pulse—chase analysis demonstrated that wild-type p53 was significantly more stabile in H460a cells treated with retinoic acid, exhibiting a half-life greater than 6 h, in contrast to 3 h for the protein in untreated control cells. The retinoic acid-mediated effect was specific for wild-type p53, since expression of mutant p53 in the H596b and H322j cell lines remained relatively unchanged even after 72 h exposure to retinoic acid. We conclude that retinoic acid induces stabilization of wild-type p53 in NSCLC cells by a post-translational mechanism. Furthermore, increases in expression of p53 were not responsible for the retinoic acid-induced transient inhibition of growth of NSCLC cells, since the growth of H358 p53-null cells also was inhibited by retinoic acid.

Apoptosis in erythroid progenitors deprived of erythropoietin occurs during the G1 and S phases of the cell cycle without growth arrest or stabilization of wild-type p53.

Erythropoietin (Epo) inhibits apoptosis in murine proerythroblasts infected with the anemia-inducing strain of Friend virus (FVA cells). We have shown that the apoptotic process in FVA cell populations deprived of Epo is asynchronous as a result of a heterogeneity in Epo dependence among individual cells. Here we investigated whether apoptosis in FVA cells correlated with cell cycle phase or stabilization of p53 tumor suppressor protein. DNA analysis in nonapoptotic FVA cell subpopulations cultured without Epo demonstrated little change in the percentages of cells in G1,S, and G2/M phases over time. Analysis of the apoptotic subpopulation revealed high percentages of cells in G1 and S, with few cells in G2/M at any time. When cells were sorted from G1 and S phases prior to culture without Epo, apoptotic cells appeared at the same rate in both populations, indicating that no prior commitment step had occurred in either G1 or S phase. Steady-state wild-type p53 protein levels were very low in FVA cells compared with control cell lines and did not accumulate in Epo-deprived cultures; however, p53 protein did accumulate when FVA cells were treated with the DNA-damaging agent actinomycin D. These data indicate that erythroblast apoptosis caused by Epo deprivation (i) occurs throughout G1 and S phases and does not require cell cycle arrest, (ii) does not have a commitment event related to cell cycle phase, and (iii) is not associated with conformational changes or stabilization of wild-type p53 protein.

Apoptosis in erythroid progenitors deprived of erythropoietin occurs during the G1 and S phases of the cell cycle without growth arrest or stabilization of wild-type p53.

Erythropoietin (Epo) inhibits apoptosis in murine proerythroblasts infected with the anemia-inducing strain of Friend virus (FVA cells). We have shown that the apoptotic process in FVA cell populations deprived of Epo is asynchronous as a result of a heterogeneity in Epo dependence among individual cells. Here we investigated whether apoptosis in FVA cells correlated with cell cycle phase or stabilization of p53 tumor suppressor protein. DNA analysis in nonapoptotic FVA cell subpopulations cultured without Epo demonstrated little change in the percentages of cells in G1,S, and G2/M phases over time. Analysis of the apoptotic subpopulation revealed high percentages of cells in G1 and S, with few cells in G2/M at any time. When cells were sorted from G1 and S phases prior to culture without Epo, apoptotic cells appeared at the same rate in both populations, indicating that no prior commitment step had occurred in either G1 or S phase. Steady-state wild-type p53 protein levels were very low in FVA cells compared with control cell lines and did not accumulate in Epo-deprived cultures; however, p53 protein did accumulate when FVA cells were treated with the DNA-damaging agent actinomycin D. These data indicate that erythroblast apoptosis caused by Epo deprivation (i) occurs throughout G1 and S phases and does not require cell cycle arrest, (ii) does not have a commitment event related to cell cycle phase, and (iii) is not associated with conformational changes or stabilization of wild-type p53 protein.

Elevated Wild-Type p53 Protein Levels in Human Epithelial Cell Lines Immortalized by the Human Papillomavirus Type 16 E7 Gene

The role tumor suppressors p53 and retinoblastoma (RB) play in the transformation process has become central to understanding the pathogenesis of DNA tumor viruses. The two oncoproteins of human papillomavirus (HPV)-16, E6 and E7, bind to p53 and RB, respectively, thus inactivating the function of these tumor suppressor genes. Immortalization of primary human foreskin epithelial cells by HPV requires expression of the E7 protein, and the E6 protein greatly enhances the immortalization frequency. Two of three cell lines immortalized by the HPV-16 E7 oncoprotein expressed wild-type p53 and only one of the three cell lines had acquired a p53 mutation and loss of heterozygosity at 17p during the immortalization process. All three E7-immortalized lines contained higher steady-state levels of p53 protein. Mutation of the p53 gene is not required for immortalization in the absence of the HPV-16 E6 inactivation of the p53 protein, and 16E7 expression leads to the stabilization of wild-type p53.

The p53-mdm-2 autoregulatory feedback loop.

The p53 protein can bind to a set of specific DNA sequences, and this may activate the transcription of genes adjacent to these DNA elements. The mdm-2 gene is shown here to contain a p53 DNA-binding site and a genetically responsive element such that expression of the mdm-2 gene can be regulated by the level of wild-type p53 protein. The mdm-2 protein, in turn, can complex with p53 and decrease its ability to act as a positive transcription factor at the mdm-2 gene-responsive element. In this way, the mdm-2 gene is autoregulated. The p53 protein regulates the mdm-2 gene at the level of transcription, and the mdm-2 protein regulates the p53 protein at the level of its activity. This creates a feedback loop that regulates both the activity of the p53 protein and the expression of the mdm-2 gene.

Comparative Study of p53 Gene and Protein Alterations in Human Astrocytic Tumors

The p53 gene is a tumor suppressor gene involved in many common malignancies, including astrocytomas. Genetic analysis of the p53 gene and immunohistochemistry of the p53 protein have each been used to screen astrocytomas. To compare these methods, we performed immunohistochemistry with the monoclonal antibody PAb 1801 and single-strand conformational polymorphism (SSCP) with sequence analysis on 34 astrocytic tumors (WHO grades II, III and IV). Seven cases had detectable p53 protein and gene mutations, while twelve cases had neither detectable protein nor gene mutations. Four tumors had frameshift mutations in the p53 gene that were not revealed by immunohistochemistry. One tumor had a genetic polymorphism and no detectable p53 protein. Ten tumors had p53 protein accumulation but no mutations by SSCP; these cases may represent p53 mutations outside of the conserved exons or elevated levels of wild-type p53 protein. Thus, some p53 mutations are missed with PAb 1801 immunohistochemistry alone. p53 immunohistochemistry, however, may reveal p53 accumulation independent of mutations in the conserved portions of the gene. Finally, we suggest that glioblastomas with p53 mutations in the conserved region of the gene may be a subset that are more common in women and in younger patients.

Low p53 level in immortal, non-tumorigenic oral keratinocytes harboring HPV-16 DNA.

The p53 protein level was determined in normal oral keratinocytes and two non-tumorigenic, immortal oral keratinocyte lines harboring human papillomavirus-16 (HPV-16)DNA. The p53 mRNA level in the immortal cells was higher than the normal counterpart, but the p53 protein level was notably lower in the immortalised cells. The half-life of p53 protein in the normal and immortal cells was < 1 h, and the p53 cDNA sequence of these cells showed no mutation. The immortal cells transcribed a high amount of E6/E7 mRNA encoded by HPV-16, but normal cells did not. These observations suggest that the immortal keratinocytes may translate normal level of wild-type p53 protein, and the low p53 level in these cells may be due to the enhanced degradation of the protein by HPV-16 E6 protein.

The community health center as a training site for a multidisciplinary approach to hypertension control: Robert M. Palmer, University of Southern California School of Medicine, 2025 Zonal Avenue, Los Angeles, California; Jean Mathison

We investigated the accumulation of p53 proteins after heat stress and their association with HSP72/HSC73 using four human glioblastoma cell lines. Human glioblastoma cell lines U-87MG and A-172 exhibited no mutation in the region between the 2nd and 11th exons of the p53 gene, whereas A-7 and T98G had mutations in exon 5 and exon 7 of the p53 gene, respectively. In U-87MG and A-172, the levels of wild-type p53 protein were slightly increased by heat stress. Levels of mutant p53 protein were apparently increased by heat stress in A-7, but not in T98G. Furthermore, wild-type p53 proteins in both U-87MG and A-172 co-immunoprecipitated with anti-HSP72HSC73 antibody and HSP72 and HSC73 in them co-immunoprecipitated with anti-p53 antibody as did the mutant p53 proteins. These findings suggest that p53 proteins accumulated by heat stress are associated with HSP72 and HSC73.