Is over-expression of protein kinase C alpha required to induce a hormone-independent phenotype in T47D breast cancer cells?

Stable transfection of protein kinase C alpha (PKCα) in T47D:A18 cells results in hormone-independence as characterized by growth in the absence or presence of 17α-estradiol (E2) and 4-hydroxytamoxifen (4-OHT) in cell culture (Tonetti et al., Br. J. Cancer, 2000, 83:782-791). Tumors derived from this cell line also grow without hormone treatment and in the presence of tamoxifen. We have shown that stable transfection of PKCα in T47D:A18 cells and tumors results in upregulation of PKCs β and δ. Other laboratories have also documented cross-regulation of other PKC isozymes by PKCα overexpression. Methods: To elucidate the importance of PKCβ and δ in the manifestation of the unique hormone-independent phenotype, we have established T47D:A18/PKCβ and T47D:A18/PKCδ stable clones and examined PKC isozyme expression by Western blot analysis. Proliferation assays were done to determine growth response to E2 and 4-OHT. ERE-luciferase reporter assays were employed to examine ER function. Results: Two T47D: A18/PKCβII clones similarly show cross-upregulation of PKCα but not PKCδ. Stable transfection of PKCδ resulted in a clone that overexpressed only PKCβ11, but not PKCα. None of the T47D:A18/PKCβ nor T47D:A18/PKCδ clones examined exhibited hormone-independent growth in cell culture. ER function is not diminished as assessed by activation of an ERE-luciferase reporter. Conclusion: These results suggest that overexpression of either PKCβ and PKCδ or PKCβ11 and PKCα is not sufficient to confer hormone-independence. These results suggest that coordinate overexpression of PKCα, β and δ is required to elicit hormone-independence in T47D human breast cancer cells. These studies were supported by an NIH/NCI Grant R01CA7984.