Interaction between the product of the breast cancer susceptibility gene BRCA2 and DSS1, a protein functionally conserved from yeast to mammals.

Germ line mutations in the breast cancer susceptibility gene BRCA2 predispose to early-onset breast cancer, but the function of the nuclear protein encoded by the gene is ill defined. Using the yeast two-hybrid system with fragments of human BRCA2, we identified an interaction with the human DSS1 (deleted in split hand/split foot) gene. Yeast and mammalian two-hybrid assays showed that DSS1 can associate with BRCA2 in the region of amino acids 2472 to 2957 in the C terminus of the protein. Using coimmunoprecipitation of epitope-tagged BRCA2 and DSS1 cDNA constructs transiently expressed in COS cells, we were able to demonstrate an association. Furthermore, endogenous BRCA2 could be coimmunoprecipitated with endogenous DSS1 in MCF7 cells, demonstrating an in vivo association. Apparent orthologues of the mammalian DSS1 gene were identified in the genome of the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae. Yeast strains in which these DSS1-like genes were deleted showed a temperature-sensitive growth phenotype, which was analyzed by flow cytometry. This provides evidence for a link between the BRCA2 tumor suppressor gene and a gene required for completion of the cell cycle.

Activation of p53 DNA binding activity by point mutation.

The p53 tumor suppressor protein can adopt both latent, non-DNA binding and active, DNA binding forms, and p53 activity is thought to be regulated in cells, at least in part, through a conformational shift which leads to sequence specific DNA binding. In vitro, this allosteric regulation of DNA binding by p53 has been shown to be mediated through the C-terminus of the protein. We show here that although deletion of the C-terminal 16 amino acids of p53 did not activate DNA binding, deletion of a further eight amino acids resulted in constitutive activation of DNA binding activity. Simultaneous mutation of the three lysine residues within these eight amino acids also resulted in constitutive DNA binding activity, although this was reduced when only two of these lysines were altered. The deletion or point mutants of p53 showing constitutive DNA binding activity did not display clear evidence of DNA binding site specificity, although some binding site preference was seen with the point mutants. Each of the constitutively active p53 mutants retained transcriptional activity and induced both cell cycle arrest and apoptosis in transiently transfected cells at rates comparable with the wild type protein.

Nuclear location and cell cycle regulation of the BRCA2 protein.

Women carrying a germ-line mutation in the BRCA1 or BRCA2 genes have a high risk of developing breast cancer, and loss of the wild-type allele in tumors suggests that these genes function as tumor suppressor genes. The BRCA2 gene encodes a 3418-amino acid protein with no significant sequence similarity to any known protein. To begin to elucidate the cellular role of BRCA2, we have raised antibodies to the BRCA2 protein and used these to study its subcellular localization and expression. We show that BRCA2 is a nuclear protein expressed in response to cell proliferation and that BRCA2 expression is initiated before DNA synthesis.

Sensitivity of p53 lysine mutants to ubiquitin-directed degradation targeted by human papillomavirus E6.

The activity of the p53 tumor suppressor protein is regulated, at least in part, through the stability of the protein. p53 degradation in normal cells is controlled by ubiquitin-dependent proteolysis, and activation of p53 following DNA damage is associated with an increase in the stability of the protein. The human papillomavirus-encoded E6 protein abrogates p53 function by targeting it for rapid degradation, also through the ubiquitin pathway. Although the p53 protein is ubiquitinated following interaction with E6, we show here that none of the lysine residues within p53 are specifically required for E6-targeted degradation. Mutation of lysine residues within the C-terminus of p53 resulted in resistance to E6-mediated degradation in vitro, although the ability of the two proteins to form a complex was not affected. The same mutant was efficiently targeted for degradation in cells, however, illustrating a lack of correlation between the in vitro and the in vivo assays.

Oligomerisation of full length p53 contributes to the interaction with mdm2 but not HPV E6.

The tumour suppressor protein p53 normally functions as a tetramer in a defined conformational state. Mutations within p53 which contribute to cancer development frequently induce a conformational shift in the protein which correlates with loss of wild type growth suppressor functions. Both the cell encoded mdm2 protein and the human papillomavirus oncoprotein E6 can regulate p53 function and we have examined the interaction of these proteins with p53. The E6/p53 association is sensitive to conformational alterations in the p53 protein, although oligomerisation is not necessary for this interaction to occur. Analysis of C-terminal p53 truncations has indicated that the region between residues 327 and 347 may play a role in E6 binding. Since monomeric forms of p53 retain transcriptional and transformation suppressor activities, our results indicate that E6 targets p53 proteins which retain these wild type functions. Conversely, the interaction of p53 with mdm2 is not dependent on the conformation of the p53 protein but is significantly impaired by loss of quaternary structure. It is possible that mdm2 plays a role in mediating activities of p53 which, unlike transcriptional activation, depend on oligomerisation.

Transcriptional activation by p53 correlates with suppression of growth but not transformation.

The tumor suppressor protein p53 shows growth and transformation suppression functions that are frequently lost by mutant proteins detected in cancers. Using a large series of p53 mutants, we have demonstrated an excellent correlation between transcriptional activation and growth suppression in p53-null human cells. Not all transcriptionally active mutants retain the ability to suppress transformation in primary rodent cells, however, and two tumor-derived point mutants displayed some evidence of both transforming and transactivating activity. Transformation by these mutants was not mediated by transdominant repression of endogenous p53 transactivating function, and cell lines expressing these p53 proteins showed elevated p53 transcriptional activity. Our results suggest that activation of transcriptional regulation by p53 will not necessarily result in tumor suppression.

Interaction of p53 with MDM2 is independent of E6 and does not mediate wild type transformation suppressor function.

Using a new series of p53 mutants targeting the conserved regions we have analysed the relationship of various activities of the protein. Mdm-2 and human papillomavirus (HPV) E6, two proteins which interact with and abrogate p53 function, were shown to bind independently. Deletion of the conserved regions of the protein in which most of the naturally occurring mutations are found (boxes II-V) abrogated transcriptional activity and the ability to interact with E6, supporting the importance of this DNA binding domain to these activities. Nevertheless, these mutants retained the ability to interact with mdm2. One mutant, deleted of all the C-terminal sequences, showed loss of mdm2 binding, E6 binding and transcriptional activity. More subtle mutations within the C-terminus of the protein, including alterations of the cdc2 and CKII phosphorylation sites, had no effect on the transcriptional trans-activation, mdm-2 or E6 binding functions, indicating that phosphorylation of these sites is not essential for these activities. Deletion of conserved box I sequences abolished the interaction with mdm-2 without loss of transcriptional activation or transformation suppressor activity, suggesting that mdm-2 is not a downstream effector of p53 function.

The selection of antibodies for targeted therapy of small-cell lung cancer (SCLC) using a human tumour spheroid model to compare the uptake of cluster 1 and cluster w4 antibodies.

Spheroids of a small-cell lung cancer (SCLC) cell line POC were used to evaluate the uptake and penetration of two antibodies recognising different SCLC antigens. Spheroids approximately 300-400 microns in diameter were incubated with 1 microgram ml-1 125I-labelled NY.3D11, an antibody which reacts with the cluster 1 group antigen (neural cell adhesion molecule; NCAM) and [125I]SWA11, which binds to the cluster w4 antigen. The rate of uptake of both antibodies was similar; an initially rapid phase was seen during the first 8 h and maximum uptake occurred by 24 h. The mean uptake per spheroid at 24 h was 0.97 ng for [125I]NY.3D11 and 0.45 ng for [125I]SWA11. An objective measurement of antibody penetration into spheroids was developed using a computerised image analysis of immunostained sections of spheroids. The concentration of antibody and incubation times were varied. Both antibodies penetrated the spheroids to a depth of 50 microns after 30 min. This increased to about 100 microns after 4 h incubation with 1 or 100 micrograms ml-1 SWA11. The results with 1 microgram ml-1 NY.3D11 were similar, but in the presence of 100 micrograms ml-1 NY.3D11 penetration into the spheroid was deep and diffuse. These results demonstrate a major concentration-dependent difference in the uptake and penetration of cluster 1 and cluster w4 antibodies in this spheroid model and they have implications for the selection of antibodies for targeted therapy of SCLC.

Biodistribution and tumour localisation of 131I SWA11 recognising the cluster w4 antigen in patients with small cell lung cancer.

The biodistribution of radiolabelled SWA11, a mouse monoclonal antibody recognising the cluster w4 group antigen associated with small cell lung cancer (SCLC) was studied in patients with SCLC. Five patients were injected intravenously with approximately 5 mCi of 131I conjugated to 1 mg of SWA11. The half-life of the radiolabel in blood was short but there was a prolonged second phase of clearance with a half-life of about 40 h. Tumour was detected by gamma camera imaging two patients. However, most of the whole body radioactivity was located in the bone marrow. At least 35% of the radioactivity in blood 18 h after injection was bound to circulating granulocytes and this probably accounted for the unusual biodistribution of the radiolabel in man. This study shows that the biodistribution of radiolabelled SWA11 in man differs from human tumour xenograft models and that the antibody in unsuitable for targeting therapy to SCLC in man.