Rb localization and phosphorylation kinetics correlate with the cellular phenotype of cultured breast adenocarcinoma...

Retinoblastoma protein (Rb) expression has been correlated with state of differentiation, proliferation rate, and metastatic potential in breast adenocarcinomas and established cell lines. These observations, based on immunoreactivity of total Rb rather than hypophosphorylated protein, do not address the relationship between functional Rb and indicators of an aggressive transformed cellular phenotype. We hypothesized that the distribution of functional Rb and the kinetics of Rb phosphorylation would differ between cell lines representing immortalized mammary epithelium (MCF10A), differentiated nonmetastatic mammary adenocarcinoma (MCF-7), and poorly differentiated, highly metastatic mammary adenocarcinoma (MDA-MB-231) and that these differences would be informative of the cellular phenotype. Direct immunofluorescence microscopy was used to compare qualitatively the subcellular localization of total and hypophosphorylated Rb protein in synchronized and asynchronous cells. This technique was also used to quantitatively assess the amounts of hypophosphorylated Rb throughout the cell cycle in these representative cell lines. Total Rb stained more prominently than hypophosphorylated Rb in the nucleus of all asynchronous cells. Rb phosphorylation was more rapid in MCF-7 cells than in MCF10A cells, whereas Rb dephosphorylation appeared deregulated in MDA-MB-231 cells. We conclude that assessment of hypophosphorylated Rb may be more useful than assessment of total Rb for the evaluation of transformed breast adenocarcinoma phenotypes.