Relatively little is known about the molecular mechanisms of tumor promotion/progression in mammary carcinogenesis. Increased protein kinase C (PKC) activity is known to promote tumor formation in several tissues; however, its role in mammary carcinogenesis is not yet known. To determine if individual PKCs may selectively regulate properties of mammary tumor cells, we compared PKC isozyme levels in mammary tumor cell lines with low, moderate and high metastatic potential. All three cell lines expressed a, d, e and z PKCs; however, PKCd levels were relatively increased in the highly metastatic cells. To determine if increased PKCd could contribute to promotion/progression, we overexpressed PKCd in the low and moderately metastatic cell lines. PKCd overexpression had no signi®cant e ect on growth of adherent cells, but signi®cantly increased anchorage-independent growth. Conversely, expressing the regulatory domain of PKCd (RDd), a putative PKCd inhibitory fragment, inhibited anchorage-independent growth. The e cacy of RDd as a PKCd inhibitor was demonstrated by showing that RDd selectively interfered with PKCd subcellular location and signi®cantly interfered with phosphorylation of the PKC cytoskeletal substrate, adducin. PKC-dependent phosphorylation of cytoskeletal substrate proteins, such as adducin, provides a mechanistic link between increased PKCd activity and phenotypic changes in cytoskeletaldependent processes such as migration and attachment, two processes that are relevant to metastatic potential. The reciprocal growth e ects of expressing PKCd and RDd as gain and loss of function constructs, respectively, provide strong evidence that PKCd regulates processes important for anchorage-independent growth in these mammary tumor cells.

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