Susan C. Kiley

Increased protein kinase Cδ in mammary tumor cells: relationship to transformation and metastatic progression

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 α, δ, ε and ζ PKCs; however, PKCδ levels were relatively increased in the highly metastatic cells. To determine if increased PKCδ could contribute to promotion/progression, we overexpressed PKCδ in the low and moderately metastatic cell lines. PKCδ overexpression had no significant effect on growth of adherent cells, but significantly increased anchorage-independent growth. Conversely, expressing the regulatory domain of PKCδ (RDδ), a putative PKCδ inhibitory fragment, inhibited anchorage-independent growth. The efficacy of RDδ as a PKCδ inhibitor was demonstrated by showing that RDδ selectively interfered with PKCδ subcellular location and significantly 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 PKCδ activity and phenotypic changes in cytoskeletal-dependent processes such as migration and attachment, two processes that are relevant to metastatic potential. The reciprocal growth effects of expressing PKCδ and RDδ as gain and loss of function constructs, respectively, provide strong evidence that PKCδ regulates processes important for anchorage-independent growth in these mammary tumor cells.

Protein kinase C isozymes and substrates in mammary carcinogenesis.

Protein kinase C (PKC) comprises a family of ubiquitously expressed phospholipid-dependent enzymes that regulate cell growth and differentiation. Several effectors that modify mammary cell biology work at least partially through PKC-dependent pathways. Studies with mammary epithelial cells and tissues have demonstrated probable roles for the PKCs in processes associated with carcinogenesis including proliferation, estrogen sensitivity, and apoptosis. The involvement of PKCs in this wide variety of responses may in part be explained by the expression of multiple PKCs in breast tissue and the possibility that individual PKCs selectively phosphorylate different proteins and preferentially mediate different biological responses. Further understanding of the role of individual PKCs in mammary cell growth and tumor promotion/progression is likely to lead to new insights for breast cancer diagnosis and treatment.

Immunocytochemical Localization of Protein Kinase C

Protein kinase C (PKC) is a key enzyme involved in regulation of growth and differentiated function (Nishizuka, 1986). In vitro, the enzymatic activity requires both calcium and phospholipids. The neutral lipid, diacylglycerol, also interacts with PKC and decreases the Kact for calcium to concentrations compatible with those found in the cytoplasm. DAG is considered to be the key regulator of PKC activation state in vivo. This is due to the fact that cellular levels of DAG are tightly coupled to hormonal activation of phospholipase C. Tumor promoting phorbol esters mimic diacylglycerol with respect to binding to and activating PKC. Infact, PKC is the major cellular receptor for phorbol esters (Castagna, et al., 1982; Niedel, et al., 1983). It is for this reason that phorbol esters are so often used as specific tools for exploring the role of PKC in biological processes.