Protein kinase C isoenzyme expression in normal mouse mammary epithelial cells grown in primary culture.

Abstract

Mammary epithelial cells isolated from midpregnant BALB/c mice were grown within collagen gels and maintained in serum-free media containing 10 ng/ml epidermal growth factor (EGF) for an 8-day culture period. Western blot and scanning densitometric image analysis showed the presence of protein kinase C(PKC)alpha (82 kDa), delta(75 kDa), eta(90 and 78 kDa), and zeta(82, 74, and 65 kDa), whereas PKCbeta, gamma, and theta were not detected in either the cytosolic or membrane fractions in these cells. Cytosolic and membrane levels of PKCalpha and 82 kDa PKCzeta band progressively increased throughout the 8-day culture period. During this same time, cytosolic PKCdelta levels decreased, while membrane levels of PKCdelta showed no change. Cytosolic and membrane levels of PKCeta and the 74- and 65-kDa PKCzeta bands displayed some fluctuations but remained relatively constant during the 8-day culture period. Other studies showed that 24-hr treatment with 100 nM of phorbol 12-myristate 13-acetate (PMA), resulted in the downregulation of PKCalpha, delta, and eta, and the 82-kDa PKCzeta band. However, PMA treatment had no effect on cytosolic and membrane levels of the 74- and 65-kDa PKCzeta bands. Since PKC activation is associated with hormone- and growth factor-dependent mammary epithelial cell proliferation, these findings suggest that increases and/or decreases in the relative levels of the different PKC isoenzymes in proliferating cells may indicate their possible role in mediating or regulating EGF-dependent mitogenesis.