Abstract
Previous studies have demonstrated elevated levels of protein kinase C (PKC) activity in multidrug-resistant human breast carcinoma MCF-7/ADR cells compared to control drug-sensitive MCF-7/WT cells (R. L. Fine, J. Patel, and B. A. Chabner, Proc. Natl. Acad. Sci. USA, 85: 582–586, 1988). In our present studies, immunohistochemical localization analysis using a polyclonal PKC antibody recognizing the α, β, and γ subtypes of PKC demonstrates that immunoreactivity is enhanced in MCF-7/ADR cells, with pronounced staining noted in the nuclear region. Other studies with purified nuclei isolated from MCF-7/ADR cells also show a marked increase in the intensity of immunostaining for PKC when compared to nuclei prepared from control MCF-7/WT cells. Western blot analysis of proteins extracted from purified nuclear preparations further establishes an increase in PKC enzyme protein associated with the nuclear fraction of MCF-7/ADR cells. Subcellular fractionation studies also indicate that MCF-7/ADR cells have 4–8 times higher nuclear PKC activity compared to that of control MCF-7/WT cells. MCF-7/ADR cells also possess 3-5-fold elevated cytosolic PKC activity, while a <2-fold increase is found in PKC activity associated with the plasma membrane fraction of MCF-7/ADR cells. Examination of these extracts with PKC isotype-specific antisera, as well as by DEAE-cellulose chromatography, reveals that nuclei prepared from MCF-7/ADR cells contain markedly elevated amounts of a slightly altered form of PKCα. These results suggest that elevated levels of a modified form of PKCα at the nucleus may play a role in modulating nuclear events to promote the development of multidrug resistance in MCF-7 cells.
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