Characterisation of the promoter region of the human DNA-repair gene Rad51.

PURPOSE OF INVESTIGATION: Regulatory elements of the 5'-flanking region of the DNA-repair gene Rad51 were analysed to characterise pathological alterations of Rad51 mRNA expression during tumour development. METHODS: Various fragments of the Rad51 promoter were cloned into the pGL3 reporter vector and the respective promoter activity was determined by luciferase assays in transfected U2-OS cells. Transcription factor binding was identified using Protein/DNA arrays. RESULTS: The region encompassing base pairs -204 to -58 was identified as crucial for Rad51 gene transcription. Down regulator sequences are present upstream (-305 to -204) and downstream (-48 and +204) of this core promoter element. Promoter activity is significantly enhanced by substituting G at the polymorphic positions +135 and +172 for C and T, respectively. Transcription factors Ets1/PEA3, E2F1, p53, EGR1, and Stat5 were identified as relevant for regulating expression of Rad51. CONCLUSION: We identified three separate cis-sequence elements within the Rad51 transcriptional promoter, one ensuring basal levels of expression and two elements limiting expression to relatively low levels. The characterisation of transcription factor binding might help to explain high-level expression of Rad51 in a variety of solid tumours. The polymorphic sites appear important for the increased risk of breast and/or ovarian cancer for BRCA2 mutation carriers.

Autoantibodies in sera of pancreatic cancer patients identify recombination factor Rad51 as a tumour-associated antigen.

PURPOSE: The recombination factor Rad51 is highly expressed in human pancreatic adenocarcinoma. In this study we asked whether high-level expression of Rad51 antigen stimulates a B-cell response leading to Rad51-specific autoantibodies in human pancreatic cancer patients. METHODS: Sera of patients suffering from pancreatic cancer (57) as well as sera of healthy donors (86) were screened for Rad51 autoantibodies by Western-blot analysis. Rad51 over-expressing cell lines were used as antigen source. RESULTS: Four out of 57 (7%) sera tested were found positive for Rad51 autoantibodies of IgG subclass, while all 86 control sera were negative. CONCLUSION: This observation identifies Rad51 as a tumour-associated antigen in pancreatic adenocarcinoma. Since high-level expression of Rad51 is restricted to tumour cells, Rad51 is also a tumour-specific antigen. Further analyses should reveal whether Rad51 might also function as a tumour-specific transplantation antigen (TSTA) and whether it might serve as a target for new immunotherapeutical approaches.

Over-expression of wild-type Rad51 correlates with histological grading of invasive ductal breast cancer.

Breast cancer is a major cause of cancer-related death in women. BRCA1 tumour-suppressor function is abolished in sporadic breast cancer by down-regulation of the protein level. This down-regulation inversely correlates with tumour grading. BRCA1 is part of a multiprotein complex, which also contains the recombination factor Rad51. Here we describe that in contrast to BRCA1, histological grading of sporadic invasive ductal breast cancer significantly correlates with over-expression of wild-type Rad51. These data suggest that in addition to the absence of the tumour-suppressor protein BRCA1, over-expression of wild-type Rad51 also contributes to the pathogenesis of a significant percentage of sporadic breast cancers and that other mechanisms than mutations must be responsible for this altered expression.

DNA repair and recombination factor Rad51 is over-expressed in human pancreatic adenocarcinoma.

Molecular processes that could contribute to differences in chemo- and radioresistance include variations in DNA repair mechanisms. In mammalian cells, the product of the rad51 gene mediates DNA repair via homologous recombination. We describe that in contrast to conventional monolayer cell systems Rad51 protein accumulates to high-levels in three-dimensional cell culture models as well as in orthotopic xeno-transplants of human pancreatic cancer cells. Strikingly, over-expression of wild-type Rad51 was also found in 66% of human pancreatic adenocarcinoma tissue specimens. Functional analysis revealed that Rad51 over-expression enhances survival of cells after induction of DNA double strand breaks. These data suggest that perturbations of Rad51 expression contribute to the malignant phenotype of pancreatic cancer. Oncogene (2000).

Interaction of p53 with the human Rad51 protein.

p53 is thought to function in the maintenance of genomic stability by modulating transcription and interacting with cellular proteins to influence the cell cycle, DNA repair and apoptosis. p53 mutations occur in >50% of human cancers, and cells which lack wild type p53 accumulate karyotypic abnormalities such as amplifications, deletions, inversions and translocations. We propose that p53 hinders these promiscuous recombinational events by interacting with cellular recombination and repair machinery. We recently reported that p53 can directly bind in vivo to human Rad51 (hRad51) protein and in vitro to its bacterial homologue RecA. We used GST-fusion and his-tagged protein systems to further investigate the physical interaction between p53 and hRad51, homologue of the yeast Rad51 protein that is involved in recombination and DNA double strand repair. The hRad51 binds to wild-type p53 and to a lesser extent, point mutants 135Y, 249S and 273H. This binding is not mediated by a DNA or RNA intermediate. Mapping studies using a panel of p53 deletion mutants indicate that hRad51 could bind to two regions of p53; one between amino acids 94 and 160 and a second between 264 and 315. Addition of anti-p53 antibody PAb421 (epitope 372-381 amino acids) inhibited the interaction with hRad51. In contrast, p53 interacts with the region between aa 125 and 220 of hRad51, which is highly conserved among Rad51 related proteins from bacteria to human. In Escherichia coli ecA protein, this region is required for homo-oligomerization, suggesting that p53 might disrupt the interaction between RecA and Rad51 subunits, thus inhibiting biochemical functions of Rad51 like proteins. These data are consistent with the hypothesis that p53 interaction with hRAD51 may influence DNA recombination and repair and that additional modifications of p53 by mutation and protein binding may affect this interaction.

Isolation and characterization of six monoclonal antibodies raised against human RAD51 recombinant protein.

Monoclonal antibodies were produced against recombinant human RAD51 recombination protein. The antibodies of IgG subclasses were isolated from serum-free cell culture medium and purified by affinity chromatography on protein A-Sepharose. The antibodies can be used to detect specifically RAD51 protein on immunoblots of total cell lysates. Native RAD51 protein is specifically precipitated from lysates of human cells. In addition, these antibodies readily detect RAD51 in the cell nucleus by immunofluorescence staining. Epitope mapping on overlapping peptides spanning the complete primary amino acid sequence of human RAD51 revealed that three monoclonals recognize an epitope on RAD51 very close to the N-terminus of the protein (amino acids 16 to 20); the other three monoclonals interact with amino acids 85 to 95 of human RAD51.

p53 is linked directly to homologous recombination processes via RAD51/RecA protein interaction.

The tumour suppressor p53 prevents tumour formation after DNA damage by halting cell cycle progression to allow DNA repair or by inducing apoptotic cell death. Loss of wild-type p53 function renders cells resistant to DNA damage-induced cell cycle arrest and ultimately leads to genomic instabilities including gene amplifications, translocations and aneuploidy. Some of these chromosomal lesions are based on mechanisms that involve recombinatorial events. Here we report that p53 physically interacts with key factors of homologous recombination: the human RAD51 protein and its prokaryotic homologue RecA. In vitro, wild-type p53 inhibits defined biochemical activities of RecA protein, such as three-way DNA strand exchange and single strand DNA-dependent ATPase activity. In vivo, temperature-sensitive p53 forms complexes with RAD51 only in wild-type but not in mutant conformation. These observations suggest that functional wild-type p53 may select directly the appropriate pathway for DNA repair and control the extent and timing of the production of genetic variation via homologous recombination. Gene amplification an other types of chromosome rearrangements involved in tumour progression might occur not only as result of inappropriate cell proliferation but as a direct consequence of a defect in p53-mediated control of homologous recombination processes due to mutations in the p53 gene.

Abrogation of p53-induced apoptosis by the hepatitis B virus X gene.

The p53 tumor suppressor gene product is a transcriptional transactivator and a potent apoptotic inducer. The fact that many of the DNA tumor virus oncoproteins bind to p53 and affect these p53 functions indicates that this interaction is an important step in oncogenic transformation. We and others have recently demonstrated that the hepatitis B virus oncoprotein, HBx, can form a complex with p53 and inhibit its DNA consensus sequence binding and transcriptional transactivator activity. Using a microinjection technique, we report here that HBx efficiently blocks p53-mediated apoptosis and describe the results of studies exploring two possible mechanisms of HBx action. First, inhibition of apoptosis may be a consequence of the failure of p53, in the presence of HBx, to upregulate genes, such as p21WAF1, Bax, or Fas, that are involved in the apoptotic pathway. Data consistent with this hypothesis include HBx reduction of p53-mediated p21WAF1 expression. Alternatively, HBx could affect p53 binding to the TFIIH transcription-nucleotide excision repair complex as HBx binds to the COOH terminus of p53 and inhibits its binding to XPB or XPD. Binding of p53 to these constituents of the core TFIIH is a process that may be involved in apoptosis. Because the HBx gene is frequently integrated into the genome of hepatocellular carcinoma cells, inhibition of p53-mediated apoptosis by HBx may provide a clonal selective advantage for hepatocytes expressing this integrated viral gene during the early stages of human liver carcinogenesis.

A C-terminal alpha-helix plus basic region motif is the major structural determinant of p53 tetramerization.

The p53 gene product has been implicated in both human and animal tumorigenesis. p53 forms heterologous complexes with the transforming proteins encoded by several different DNA tumor viruses. p53 also assembles into stable homo-oligomers. We demonstrate that the major structural determinant for the tetramerization of p53 is an alpha-helical plus basic region motif near the C-terminus of the protein. A monomeric p53 mutant adopts a conformation distinct from both 'wild-type' and 'mutant' form as defined by PAb1620 and PAb240 monoclonal antibody recognition. Nevertheless, monomeric and dimeric mutant p53 proteins retain the ability to suppress SV40 origin-directed DNA replication in vivo. Thus, p53-p53 interaction and expression of the PAb1620 epitope is not a prerequisite for such activity. We present data suggesting that suppression of replication by p53 may occur by a mechanism that is independent of detectable p53-T antigen association.

A human tumour-derived mutant p53 protein induces a p34cdc2 reversible growth arrest in fission yeast.

We have expressed wild-type and human tumour-derived mutant p53 cDNA genes in the fission yeast Schizosaccharomyces pombe. In the case of one mutant this resulted in a growth arrest of recipient yeast cells. In contrast, wild-type p53 and three other mutant proteins tested did not block outgrowth of colonies. Human and yeast cdc2 acted as functionally equivalent extragenic suppressors of the mutant-induced growth arrest allowing the establishment of viable p53 expressor strains. In cotransformation assays the mutant allele was found to be dominant over wt p53. Our results provide the first evidence of a functional relationship between p53 and p34cdc2 in an in-vivo system and suggest that the wide variety of mutant proteins present in human tumours may fall into functionally distinct subclasses.

p53 interacts with p34cdc2 in mammalian cells: implications for cell cycle control and oncogenesis.

The p53 gene product has been implicated in both human and animal tumorigenesis. p53 complexes with the transforming proteins encoded by several different DNA tumour viruses. We demonstrate that human p53 is phosphorylated by the mammalian p34cdc2 kinase in vitro and coprecipitates with p34cdc2 in vivo. Our observations suggest that phosphorylation of p53 by p34cdc2 kinase may regulate the known activities of p53 in the initiation steps of DNA replication in mammalian cells.

The p53 nuclear localisation signal is structurally linked to a p34cdc2 kinase motif.

We have identified a region of human p53 protein with striking homology to a sequence motif on Simian Virus 40 T antigen which includes the nuclear localisation signal. Mutation of basic amino acid residues in this region of p53 (residues 312 to 323; SSSPQPKKKP) compromises transport of p53 protein to the nucleus. The sequence functions efficiently as a nuclear localisation signal when fused to E. coli beta galactosidase. Serine 315 within this p53 structural motif is phosphorylated in vitro by the cell cycle kinase p34cdc2. Thus in both T antigen and p53, nuclear localisation signal and p34cdc2 kinase acceptor residue map to a contiguous region of primary amino acid sequence.

Evolutionary conservation of the biochemical properties of p53: specific interaction of Xenopus laevis p53 with simian virus 40 large T antigen and mammalian heat shock proteins 70.

We have investigated the biochemical properties of Xenopus laevis p53. With an in vitro binding assay, we can detect a specific association between X. laevis p53 and simian virus 40 large T antigen. Furthermore, X. laevis p53 expressed in monkey COS cells is stably associated with this viral antigen. Like mammalian p53, X. laevis p53 in complex with simian virus 40 large T antigen exhibits a 20-fold increase of its half-life. On the other hand, X. laevis p53 is unable to associate either in vivo or in vitro with adenovirus type 5 E1B 55-kilodalton protein. We show by an immunological technique that X. laevis p53 forms specific complexes with mammalian hsp72 and hsp73 heat shock proteins only at a temperature well above the optimal growth temperature for X. laevis. Our results suggest that the protein-binding properties of p53 are closely related to the functional activity of the protein.

Two distinct regions of the murine p53 primary amino acid sequence are implicated in stable complex formation with simian virus 40 T antigen.

We mapped regions of the mouse p53 primary amino acid sequence implicated in stable complex formation with simian virus 40 T antigen. A number of mutant p53 proteins failed to complex stably with T antigen in vivo but formed stable complexes with T antigen in in vitro association assays. In contrast to an earlier report (T.-H. Tan, H. Wallis, and A. J. Levine, J. Virol. 59:574-583, 1986), our study showed that two distinct regions of p53 primary amino acid sequence, highly conserved between mouse and Xenopus laevis, were implicated in stable complex formation. Our data support the proposal that, when in complex, T antigen may occupy a site on p53 that is implicated in the normal function of the protein.

Mouse p53 blocks SV40 DNA replication in vitro and downregulates T antigen DNA helicase activity.

Immunopurified mouse p53 proteins were used to gain experimental access to the mechanisms underlying nonprimate p53 directed suppression of SV40 origin directed DNA replication in vivo. In replication competent HeLa cell extracts containing exogenous T antigen, mouse p53 blocks T antigen dependent DNA synthesis as in vivo. However, in transcription competent HeLa extracts, mouse p53 has no effect either on overall transcription or on the ability of immunopurified T antigen to downregulate SV40 early transcription. We show that although mouse p53 has no significant effect on T antigen encoded activities such as ATPase and DNA binding, helicase activity is somewhat reduced suggesting that the in vivo suppression by mouse p53 of SV40 replication may be due, at least in part, to direct modulation of T antigen function.

Characterization of mutant p53-hsp72/73 protein-protein complexes by transient expression in monkey COS cells.

Several mutant, but not wild-type, p53 proteins form complexes with hsp72/73 heat shock-related proteins in simian virus 40-transformed monkey COS cells. We carried out a detailed biochemical and structural mapping analysis of p53 and report here that p53-hsp72/73 complex formation showed considerable structural specificity. Such complexes were remarkably stable, but unlike analogous complexes formed between p53 and simian virus 40 T antigen, they did not form in in vitro association assays. p53-hsp72/73 complex formation in vivo appears to be dependent on aspects of mutant p53 protein conformation. However, absence of the conformation-sensitive epitope recognized by monoclonal antibody PAb 246 was not reliably diagnostic of such complexes, nor was p53-hsp72173 binding reliably diagnostic of oncogenic activation.

Mouse p53 inhibits SV40 origin-dependent DNA replication.

p53 is a cellular phosphoprotein that is present at elevated concentrations in cells transformed by different agents. p53 complementary DNA expression-constructs immortalize primary cells in vitro and co-operate with an activated ras oncogene in malignant transformation. Several reports have implicated p53 in mammalian cell cycle control and specifically with events occurring at the G0-G1 boundary. p53 forms specific complexes with simian virus 40 (SV40) large-T antigen, and such complexes are found associated with both replicating and mature SV40 DNA in lytically infected cells. In an accompanying paper Gannon and Lane report that in in vitro plate-binding assays, mouse p53 can displace polymerase alpha from complex with T-antigen. We have examined the in vivo consequences of expressing wild-type and mutant p53 proteins from other species in SV40-transformed monkey cells. We report here that expression of mouse p53 results in a substantial and selective inhibition of SV40 origin-dependent DNA replication. In addition to any function in the G0-G1 transition, the data presented suggest that p53 may affect directly the initiation or maintenance of replicative DNA synthesis.

Enhanced protein phosphorylation in SV40-transformed and -infected cells.

We have studied the phosphorylation of cellular phosphoproteins and, in more detail, of SV40 T antigen and the cellular protein p53 in SV40 tsA-transformed cells. As detected by radiolabeling cold-sensitive tsA1499- or heat-sensitive tsA58-transformed rat fibroblasts with [32P]orthophosphate or by in vitro labeling extracts with [gamma-32P]ATP the hyperphosphorylation of certain cellular phosphoproteins including p53 and also of free SV40 large T antigen and T antigen complexed with p53 is strictly correlated with the expression of the transformed phenotype. This hyperphosphorylation can be observed as early as 30 min after shifting to the temperature where the cells expressed the transformed phenotype and, furthermore, it is dependent on protein synthesis. To evaluate the influence of a functional T antigen and to exclude properties of individual transformants we 32P labeled in vitro cellular proteins from rat F111, mouse NIH 3T3, and monkey TC-7 cells infected with tsA58 or tsA1499. In tsA58-infected cells we found a heat-sensitive enhancement of protein phosphorylation just as in tsA58 transformants. In tsA1499-infected monkey cells we observed a heat-sensitive and in abortively infected rat or mouse cells a cold-sensitive hyperphosphorylation of proteins. Thus in tsA-transformants and in various tsA-infected cells we found a strong correlation among the transformed phenotype, functions of T antigen, and the phosphorylation of various cellular proteins and in particular T antigen and p53.

Mutant p53 proteins bind hsp 72/73 cellular heat shock-related proteins in SV40-transformed monkey cells.

We have examined the expression of a series of mouse mutant, as well as wild-type, p53 proteins in SV40-transformed monkey COS cells. Wild-type mouse p53 binds predominantly to SV40 large T antigen in these cells. However, several of the mutants co-precipitate exclusively with proteins of approximately 68 Kd relative molecular mass. We show by immunological and proteolytic mapping techniques that these proteins are identical to the hsp 72/73 heat shock proteins. p53 mutants in complex with hsp 72/73 have an altered subcellular location compared to the wild-type protein and the hsp 72/73 binding p53 mutants fail to exhibit an epitope recognized by monoclonal antibody PAb 246. The existence of at least two antigenically distinct subclasses of hsp 72/73 complexed to mutant p53 is shown.