RESUMO
Tractional force exerted by tissue cells in 3D collagen matrices can be utilized for matrix remodeling or cell migration. The interrelationship between these motile processes is not well understood. The current studies were carried out to test the consequences of oncogenic Ras (H-Ras(V12)) transformation on human fibroblast contraction and migration in 3D collagen matrices. Beginning with hTERT-immortalized cells, we prepared fibroblasts stably transformed with E6/E7 and with the combination HPV16 E6/E7 and H-Ras(V12). Oncogenic Ras-transformed cells lost contact inhibition of cell growth, formed colonies in soft agar and were unable to make adherens junctions. We observed no changes in the extent or growth factor dependence of collagen matrix contraction (floating or stress-relaxation) by oncogenic Ras-transformed cells. On the other hand, transformed cells in nested collagen matrices lost not only growth factor selectivity, but also cell-matrix density-dependent inhibition of migration. These findings demonstrate differential regulation of collagen matrix contraction and cell migration in 3D collagen matrices.
Assuntos
Técnicas de Cultura de Células , Movimento Celular/fisiologia , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Proteínas ras/metabolismo , Animais , Transformação Celular Neoplásica , Células Cultivadas , Matriz Extracelular/química , Fibroblastos/citologia , Genes ras , Humanos , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Proteínas ras/genéticaRESUMO
Leishmania (Viannia) braziliensis is the major causative agent of American tegumentary leishmaniasis, a disease that encompasses a broad spectrum of clinical manifestations. In a previous study, we showed that Brazilian and Colombian L. braziliensis strains, isolated from patients with distinct clinical manifestations, display different pattern of metalloprotease activities. Following these results, we investigated the cellular localization of these molecules and their relation to the major surface protease (gp63) of Leishmania. Comparative analyses of metalloprotease expression among different clinical isolates as well as an evaluation of the effect of long-term in vitro passage on the expression pattern of these metalloproteases were also performed. Western blot analysis, using an anti-gp63 antibody, revealed polypeptide patterns with a similar profile to that observed in zymographic analysis. Flow cytometry and fluorescence microscopy analyses corroborated the presence of metalloproteases with homologous domains to gp63 in the parasites and revealed differences in the expression level of such molecules among the isolates. The cellular distribution of metalloproteases, assessed by confocal analysis, showed the existence of intracellular metalloproteases with homologous domains to gp63, predominantly located near the flagellar pocket. Finally, it was observed that differential zymographic profiles of metalloproteases exhibited by L. (V.) braziliensis isolates remain unaltered during prolonged in vitro culture, suggesting that the proteolytic activity pattern is a stable phenotypic characteristic of these parasites.