RESUMO
METHODS: The activities of cathepsin L and its endogenous inhibitors were analyzed in rat embryo fibroblasts, immortalized and transformed by different genes. RESULTS: Regardless of the transfecting agent used (DNA of adenovirus SA7 or polyomavirus LT gene), the immortal cells showed an increase in the cathepsin L activity (in both lysates and conditioned media) compared to primary fibroblasts. Transformed cells exhibited either an increase (with c-Ha-ras gene) or decrease (with E7 HPV gene) in cathepsin L activity in lysates as opposed to immortal cells. CONCLUSIONS: The data are suggestive of alterations in the trafficking of cathepsin L upon fibroblast transfection with polyomavirus LT gene and E7 HPV gene. An endogenous inhibitor(s) of cysteine proteinase was found in conditioned media, but not in lysates, of all cell cultures studied and its activity in normal fibroblasts was higher than in media of immortal and transformed cells.
Assuntos
Catepsinas/antagonistas & inibidores , Catepsinas/biossíntese , DNA/genética , Fibroblastos/metabolismo , Proteínas E1A de Adenovirus/genética , Adenovirus dos Símios/genética , Animais , Catepsina L , Catepsinas/genética , Linhagem Celular Transformada/citologia , Linhagem Celular Transformada/metabolismo , Cisteína Endopeptidases , Inibidores de Cisteína Proteinase/metabolismo , Embrião de Mamíferos/citologia , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Fibroblastos/citologia , Genes ras/genética , Ratos , TransfecçãoRESUMO
Chromosomal analysis of immortalized rat embryo fibroblasts (IE5) and transformed by HPV18 E6+E7 genes (A4E5) or HPV16 E7 alone (trB4; trF8; trC2) variants have been done. Transformed cell lines represented heterogeneous cell populations containing neardiploid subpopulations with 41-44 chromosomes and also heterogeneous polyploid cells in contrast to immortal cells IE5 that contained normal number of chromosomes-42. In transformed cells the abnormalities of interphase nuclei (giant-, micro-, apoptotic nuclei) were observed which could reflect genomic instability of polyploid cells. Several chromosomal alterations were revealed in immortal IE5 cells, but only reciprocal translocation t(8; 10) (q22q12.3) was stable and kept in cells transformed by HPV18 E6+E7 genes or HPV16 E7 alone. We can conclude that genomic instability and clonal expansion of the cells with specific chromosomal alterations contribute to HPV-mediated transformation.
