| Summary: | Thesis (Ph.D.)--Memorial University of Newfoundland, 1994. Medicine Bibliography: leaves 181-210. Epidermal growth factor (EGF), in pharmacological concentrations, inhibits the cellular proliferation of MDA-468 human breast cancer cells. In this study, we characterized this unusual phenomenon by means of cell cycle and Northern blot analysis. Following EGF treatment, cell number in Gl phase increased, with a concomitant depletion of cells in S and G2/M phases of the cell cycle, as revealed by flow cytometric analysis of DNA content. DNA synthesis, as measured by incorporation of [3H] thymidine, was reduced to about 35% of that measured in control cells after 48 hours of EGF treatment, confirming the earlier observation of G1 arrest. Moreover, DNA synthesis returned to normal following the removal of EGF from the growth arrested cells. Northern blot analysis revealed that EGF treatment did not alter the induction of early Gl marker, c-myc, nor expression of the late G1 markers, proliferating cell nuclear antigen and thymidine kinase. However, EGF treatment resulted in the downregulation of p53 and histone 3.2 steady-state mRNA levels. Increased levels of these gene transcripts are observed at the Gl/S boundary and in S phase, respectively. These results indicated that EGF reversibly blocks the cell cycle of MDA-468 cells at the G1/S boundary. -- The observation of lowered mRNA levels of p53 (a point mutant, p53273.His) led us to hypothesize its possible involvement in EGF-mediated G1 arrest. The wild- type p53 is generally regarded as a tumour suppressor and mutations in p53 are commonly seen in a wide variety of cancers. Since it has been suggested that this particular mutant p53273.His, might have gained an alternative function and act positively to enhance cell proliferation, we hypothesized that EGF-induced G1 arrest might be mediated by changes induced in p53273.His. In order to test this hypothesis, an in-depth analysis of EGF effect on p53273.Hiswas undertaken. -- In our studies, no immediate effects of EGF-treatment were observed with regard to mRNA and protein levels, protein stability, and protein synthesis of p53273.His in MDA-468 cells. Interestingly, an EGF-dependent altered conformation of p53 was indicated by immunofluorescence studies. These experiments demonstrated a decreased PAb 240 (mutant-specific anti-p53) reactivity of nuclear p53273.His in EGF-treated cells, while PAb 1801 and PAb 1620 (pan-specific and wild- type-specific anti-p53 antibodies respectively) continued to detect the nuclear presence of p53273.His. Further studies indicated a decreased phosphorylation of p53273.His in EGF-treated cells. The EGF-dependent conformation shift and lowered phosphorylation levels of nuclear p53273.His were detected early enough to be attributed as causative of EGF-mediated cell cycle arrest. -- In order to obtain further confirmation for the observed EGF-dependent altered conformation, and to test its functional significance in terms of transcriptional regulation by p53273.His, DNA-binding and transactivation assays were performed. We detected specific complexes of p53273.His with both CON and FRA oligonucleotides, two of the known p53-DNA binding sites. Furthermore, in transient transfection assays, these sequences mediated p53-specific transcriptional modulation, namely transactivation through CON, and repression through FRA. These experiments, indicated a distinct function for p53273.His in MDA-468 cells. Interestingly enough, EGF-treatment of MDA-468 cells, resulted in increased DNA- binding ability of p53273.His to both CON and FRA. In addition, EGF potentiated p53-mediated transcription from a minimal promoter. Taken together, this study, has provided significant insights into EGF-mediated growth inhibition in MDA-468 breast cancer cells and furnished enough evidence to implicate the involvement of an endogenous mutant p53 in EGF signal transduction. Furthermore, the data presented, suggest a novel and unique function for p53273.His which may be cell-type specific.
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