RESUMO
Disruption of the retinoblastoma (RB) tumor suppressor pathway is a common and important event in breast carcinogenesis. To examine the role of the retinoblastoma protein (pRB) in this process, we created human mammary epithelial cells (HMEC) deficient for pRB by infecting primary outgrowth from breast organoids with the human papillomavirus type 16 (HPV16) E7 gene. HPV16 E7 binds to and inactivates pRB and also causes a significant down-regulation of the protein. Culturing normal HMEC in a reconstituted basement membrane (rBM) provides a correct environment and signaling cues for the formation of differentiated, acini-like structures. When cultured in this rBM, HMEC+E7 were found to respond morphologically as normal HMEC and form acinar structures. In contrast to normal HMEC, many of the cells within the HMEC+E7 structures were not growth arrested, as determined by a 5-bromo-2'-deoxyuridine incorporation assay. pRB deficiency did not affect polarization of these structures, as indicated by the normal localization of the cell-cell adhesion marker E-cadherin and the basal deposition of a collagen IV membrane. However, in HMEC+E7 acini, we were unable to detect by immunofluorescence microscopy the milk protein lactoferrin or cytokeratin 19, both markers of differentiation expressed in the normal HMEC structures. These data suggest that loss of RB in vivo would compromise differentiation, predisposing these cells to future tumor-promoting actions.
Assuntos
Mama/patologia , Transformação Celular Neoplásica , Proteínas Oncogênicas Virais/genética , Proteína do Retinoblastoma/fisiologia , Mama/metabolismo , Caderinas/metabolismo , Diferenciação Celular , Células Cultivadas , Colágeno/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Matriz Extracelular/fisiologia , Humanos , Queratinas/biossíntese , Lactoferrina/biossíntese , Proteínas Oncogênicas Virais/metabolismo , Proteínas E7 de Papillomavirus , Proteína do Retinoblastoma/antagonistas & inibidores , Proteína do Retinoblastoma/biossíntese , Transdução GenéticaRESUMO
Previous studies identified as putative preneoplastic lesions 1) enzyme-altered foci in sections of methacrylate-embedded colon and 2) aberrant crypts in methylene blue-stained unembedded (whole-mount) colon and established that aberrant crypts embedded in methacrylate had enzyme alterations. We have now studied histochemically demonstrable hexosaminidase activity in unembedded or whole-mount preparations of colons from carcinogen-treated rats. These preparations have revealed two populations of crypts that are enzyme-altered: those that are morphologically altered or aberrant and those that are morphologically normal. Both populations can be quantified rigorously in less than an hour with whole-mount preparations reacted for hexosaminidase. The demonstration of phenotypic characteristics with histochemical techniques in whole-mount preparations should have wide applicability to functional studies in many normal and diseased tissues.