p53 status in radiation-induced soft-tissue sarcomas

BACKGROUND: Following therapeutic irradiation after a latency period of many years radiation-induced tumors, often sarcomas, can arise. Results of radiation-induced DNA damage can be 1. p53 over-expression, inducing growth arrest or apoptosis, and 2. occurrence of mutations, frequently including the p53 gene, as one molecular promotor for carcinogenesis. We were interested whether radiation-induced sarcomas are associated with alterations of the p53 status. MATERIAL AND METHODS: Samples from 11 radiation-induced soft-tissue sarcomas (STS) were studied by a non-radioactive PCR-SSCP sequencing analysis and by immunohistochemistry with five antibodies for their p53 status. RESULTS: A tumor of one patient possessed a G->A transition in codon 280 (exon 8). Of 11 tumors, 9 showed nuclear p53 positivity, detected by monoclonal antibody DO-1. Of these 9 patients, 7 died during the observation period, whereas the 2 patients with DO-1 negative tumor samples are still alive. CONCLUSIONS: p53 over-expression and p53 mutation occur in radiation-induced STS. p53 status is expected to have prognostic impact for radiation-induced STS.     

Cloning and sequence of a processed p53 pseudogene from rat: a potential source of false 'mutations' in PCR fragments of tumor DNA

We describe here the nucleotide (nt) sequence of a p53 processed pseudogene (psi-gene) from the normal F344 rat genome. Exon-derived primers were utilized to amplify and clone a 1447-bp polymerase chain reaction (PCR) product corresponding to the coding regions of exons 2-11 of the functional gene. This psi-gene is a cDNA-like sequence possessing 87% homology with the functional rat p53. We have also partially characterized two additional and distinctly different putative rat p53 psi-genes, focussing on the sequences surrounding the reported rat p53 mutational hot spots of codons 202R and 211R within exon 6/7. Each of these three psi-gene sequences contained various single- and/or double-nt substitutions, small deletions and insertions that distinguish them from p53. One substitution, 211R CGG-->CAG, found both in the cloned psi-gene and in one of the partially characterized, putative psi-genes, corresponded precisely with the sequence that has been reported as a mutation at one of the hot spots. Co-amplification of one or more of the p53 psi-genes with portions of the functional p53 is likely, if exon-based primers are utilized for PCR amplification of rat p53. Consequently, psi-gene sequences are potential sources of sequence variations that can be misidentified as somatic cell mutations by direct sequencing of inappropriately generated PCR products.     

p53 induces angiogenesis-restricted dormancy in a mouse fibrosarcoma

The p53 tumor-suppressor gene is inactivated in over 50% of all human cancers. In normal cells, p53 induces growth arrest and apoptosis in response to DNA damage. We show that p53 acts as potent tumor-suppressor gene independent of its well-documented effects on tumor-cell proliferation and apoptosis. p53 activates target genes in a murine fibrosarcoma cell-line but does not affect tumor cell-cycle progression or survival. Exogenous expression of wt-p53 does, however, block the angiogenic potential of the tumor cells resulting in formation of dormant tumors in vivo. These data provide evidence that: (1) p53 acts as a tumor suppressor gene independent of its anti-proliferative effects; (2) By inhibiting angiogenesis p53 can indirectly induce apoptosis in vivo but not in vitro; (3) p53-gene therapy which alters a tumors angiogenic potential, can revert tumors to a dormant phenotype.     

Wild-type p53 triggers a rapid senescence program in human tumor cells lacking functional p53

The p53 tumor suppressor gene has been shown to play an important role in determining cell fate. Overexpression of wild-type p53 in tumor cells has been shown to lead to growth arrest or apoptosis. Previous studies in fibroblasts have provided indirect evidence for a link between p53 and senescence. Here we show, using an inducible p53 expression system, that wild-type p53 overexpression in EJ bladder carcinoma cells, which have lost functional p53, triggers the rapid onset of G1 and G2/M growth arrest associated with p21 up-regulation and repression of mitotic cyclins (cyclin A and B) and cdc2. Growth arrest in response to p53 induction became irreversible within 48-72 h, with cells exhibiting morphological features as well as specific biochemical and ultrastructural markers of the senescent phenotype. These findings provide direct evidence that p53 overexpression can activate the rapid onset of senescence in tumor cells.     

Structures and functions of the tumor suppressor p53

The tumour suppressor p53 plays a crucial role in the cellular response to DNA damage. The p53 protein is able both to detect sites of DNA damage and to interact with DNA in a sequence-specific manner and function in the regulation of target gene expression. These two properties map to discrete functional domains of the protein, the C-terminus and the central core domain respectively. They are essential for integration of a normal cellular response to DNA damage, with initiation of either G1 cell cycle arrest or apoptosis. This review considers the domain structure of p53 in relation to the protein's various functions, together with the importance of tertiary structure and conformational flexibility. The precise regulation of p53 function remains to be established, although the protein is known to be phosphorylated/de-phosphorylated by a number of specific protein kinases/phosphatases. A recent discovery indicates that p53 may be activated by autoproteolysis and that proteolytic cleavage is induced by direct interaction with sites of DNA damage. This process is reminiscent of the bacterial Lex A system and would provide one mechanism for activation of p53 in response to cellular DNA damage.     

Relationship between p21 expression and mutation of the p53 tumor suppressor gene in normal and malignant ovarian epithelial cells

In many cell types, p53-mediated growth inhibition is dependent on induction of p21, which is an inhibitor of cyclin-dependent kinases that are required for cell cycle progression. Failure of mutant p53 proteins to transactivate p21 may lead to uncontrolled proliferation. Because many ovarian cancers have mutations in the p53 gene, we examined p21 levels in normal and malignant ovarian epithelial cells to determine whether p21 expression is dependent on wild-type p53. Normal ovarian epithelial cells and two ovarian cancer cell lines with wild-type p53 expressed readily detectable levels of p21, whereas in p53 null and mutant cell lines, expression of p21 was diminished strikingly. A correlation between the status of the p53 gene and p21 expression also was noted in 23 primary epithelial ovarian cancers. Normal levels of p21 RNA were seen in 4/7 (57%) cancers with wild-type p53, whereas 14/16 (88%) cancers with mutant p53 had reduced p21 expression (P < 0.05). In addition, we found that lambda-irradiation of normal and malignant ovarian epithelial cells with wild-type, but not mutant, p53 resulted in induction of p21. These data are suggestive that induction of p21 is a feature of p53-mediated growth inhibition in normal ovarian epithelial cells. Conversely, mutation of the p53 gene in ovarian cancers usually is associated with decreased p21 expression. The lack of an absolute correlation between p21 expression and the status of the p53 gene in ovarian cancers is consistent with other studies that have suggested that p21 may also be regulated by p53-independent pathways.     

Human papillomavirus type 16 E6 and E7 oncogenes abrogate radiation-induced DNA damage responses in vivo through p53-dependent and p53-independent pathways

E6 and E7 oncoproteins from high risk human papillomaviruses (HPVs) transform cells in tissue culture and induce tumors in vivo. Both E6, which inhibits p53 functions, and E7, which inhibits pRb, can also abrogate growth arrest induced by DNA-damaging agents in cultured cells. In this study, we have used transgenic mice that express HPV-16 E6 or E7 in the epidermis to determine how these two proteins modulate DNA damage responses in vivo. Our results demonstrate that both E6 and E7 abrogate the inhibition of DNA synthesis in the epidermis after treatment with ionizing radiation. Increases in the levels of p53 and p21 proteins after irradiation were suppressed by E6 but not by E7. Through the study of p53-null mice, we found that radiation-induced growth arrest in the epidermis is mediated through both p53-dependent and p53-independent pathways. The abrogation of radiation responses in both E6 and E7 transgenic mice was more complete than was seen in the p53-null epidermis. We conclude that E6 and E7 each have the capacity to modulate p53-dependent as well as p53-independent cellular responses to radiation. Additionally, we found that the conserved region (CR) 1 and CR2 domains in E7 protein, which are involved in the inactivation of pRb function and required for E7's transforming function, were also required for E7 to modulate DNA damage responses in vivo. Thus pRb and/or pRb-like proteins likely mediate both p53-dependent and p53-independent responses to radiation.     

Homologous nonallelic recombinations between the iduronate-sulfatase gene and pseudogene cause various intragenic deletions and inversions in patients with mucopolysaccharidosis type II

About 20% of patients with mucopolysaccharidosis type II (MPS II) have gross structural rearrangements involving the iduronate-sulfatase (IDS) gene in Xq27.3-q28. A nearby IDS pseudogene (IDS-2) promotes nonallelic recombination between highly homologous sequences. Here we describe major rearrangements due to gene/pseudogene recombination. In two unrelated patients, partial IDS gene deletions were found joining introns 3 and 7 of the IDS gene together with gene to pseudogene conversion in the area of breakpoints. In a third patient, a junction between intron 3 of IDS-2 and intron 7 of IDS was seen that was due to a deletion and inversion of the 5' part of the gene. Characterisation of breakpoints in six patients with large inversions revealed that all recombinations of this type occurred in the same area of homology between IDS and IDS-2; they were molecularly balanced, and accompanied by gene conversions in most cases. Apart from diagnostic implications, such naturally occurring recombination 'hot spots' may allow some insight into general features of crossover events in mammals.     

Mutations of the p53 tumor suppressor gene occur infrequently in Wilms' tumor

Mutations of the p53 tumor suppressor gene occur frequently in a variety of adult-onset tumors, including colon, breast, lung, and brain, yet are infrequently identified in pediatric malignancies. Wilms' tumor, a common solid tumor of childhood, can be associated with mutations of the WT1 gene. Alterations of the p53 gene have been shown to modulate the ability of WT1 to transactivate its targets. Although positive p53 immunostaining has been demonstrated in Wilms' tumors, the correlation to p53 gene mutations is not clear. We examined Wilms' tumor samples for p53 mutations utilizing polymerase chain reaction-single-strand conformation polymorphism analysis and single-strand DNA sequencing. Mutations in the coding region of the p53 gene were demonstrated in 2 of 21 (9.5%) Wilms' tumors. Each mutation yielded a substitution of amino acid residues. One mutation was located in exon 6 and the other in exon 7. Both mutations were found in tumors from patients with advanced stage disease. Focal anaplasia was demonstrated in one of these tumors. Our data suggest that although p53 mutations occur infrequently in Wilms' tumor, they may be associated with advanced disease.     

Tumor-suppressor p53: implications for tumor development and prognosis

The p53 protein plays a central role in modulating cellular responses to cytotoxic stresses by contributing to both cell-cycle arrest and programmed cell death. Loss of p53 function during tumorigenesis can lead to inappropriate cell growth, increased cell survival, and genetic instability. p53 gene mutations occur in approximately half of all malignancies from a wide range of human tumors. In some tumor types, these p53 mutations are associated with poor prognosis and treatment failure. Based on these insights, new approaches are being developed to prevent, diagnose, and treat cancer.     

The p53-deficient mouse: a model for basic and applied cancer studies

Inactivation of the p53 gene in the germline of mice by gene targeting has provided researchers with a model similar in many respects to the analogous human inherited cancer predisposition Li-Fraumeni syndrome. The viability of p53 null mice has allowed unexpected opportunities to study the role of p53 in many different in-vivo and in-vitro contexts. Null (p53-/-) mice have an average time to tumor development of 4.5 months, while half of the heterozygous (p53+/-) mice develop tumors by 18 months. The p53-deficient mice have been particularly valuable in examining the effects of p53 loss on tumor progression. In addition, the mice hold significant promise as tools to assess carcinogens, teratogens, chemopreventative agents, and cancer therapeutic regimens.     

Bcl-2 protects murine erythroleukemia cells from p53-dependent and -independent radiation-induced cell death

To better understand the molecular basis of radiation-induced cell death, we studied the role of the bcl-2 oncogene and the p53 tumor suppressor gene in this process. A temperature-sensitive mutant of murine p53 (p53Val-135) and/or bcl-2 was transfected into murine erythroleukemia cells (MEL, DP16-1, which are null in p53). We demonstrate that radiation-induced cell death occurs by both p53-dependent and -independent pathways and overexpression of bcl-2 modulates both pathways. When viability was measured 24 h post-radiation, cells that had been briefly exposed to wtp53 immediately after X-ray irradiation had decreased survival as compared to unirradiated cells expressing wtp53 or X-ray irradiated DP16-1 cells. However, at later times X-ray irradiated parental DP16-1 cells also had decreased survival compared to the unirradiated control. This decrease in survival began 48 h following radiation. Bcl-2 prevented radiation-induced cell death in DP16-1 cells expressing wtp53 and delayed radiation-induced cell death in DP16-1 cells without wtp53. X-ray irradiated cells expressing wtp53 displayed microscopic and biochemical characteristics consistent with cell death due to apoptosis. DP16-1 cells which were untransfected or co-transfected with wtp53 and bcl-2 displayed characteristics of cells undergoing necrosis. These results suggest that radiation-induced cell death occurs by both p53-dependent and p53-independent pathways. The p53-dependent pathway results in cell death via apoptosis and occurs approximately 24 h following radiation. The p53-independent pathway does not appear to involve apoptosis and occurs at a later time, starting 48 h after X-ray exposure. Thus, bcl-2 protects cells from p53-dependent radiation-induced apoptotic cell death and attenuates p53-independent radiation-induced cell death.     

Specific mismatch recognition in heteroduplex intermediates by p53 suggests a role in fidelity control of homologous recombination

We demonstrate that wild-type p53 inhibits homologous recombination. To analyze DNA substrate specificities in this process, we designed recombination experiments such that coinfection of simian virus 40 mutant pairs generated heteroduplexes with distinctly unpaired regions. DNA exchanges producing single C-T and A-G mismatches were inhibited four- to sixfold more effectively than DNA exchanges producing G-T and A-C single-base mispairings or unpaired regions of three base pairs comprising G-T/A-C mismatches. p53 bound specifically to three-stranded DNA substrates, mimicking early recombination intermediates. The KD values for the interactions of p53 with three-stranded substrates displaying differently paired and unpaired regions reflected the mismatch base specificities observed in recombination assays in a qualitative and quantitative manner. On the basis of these results, we would like to advance the hypothesis that p53, like classical mismatch repair factors, checks the fidelity of homologous recombination processes by specific mismatch recognition.