PROTEIN-KINASE-C ISOFORMS IN HUMAN AND RAT COLONIC MUCOSA

The protein kinase C (PKC) family of enzymes plays a key role in the r egulation of cellular events, including cell proliferation and differe ntiation. Work from our laboratory has shown that the effects of dieta ry fat and fiber on colonic cell proliferation were positively correla ted with membrane/cytosol PKC activity ratios (Chapkin et al., 1993, J . Nutr. 123, 649-655). The presence and subcellular distribution of sp ecific PKC isoforms in rat and human colon were therefore determined i n cytosolic and membrane extracts. Tissue extracts were probed with an tibodies to individual PKC isoforms. PKC alpha, beta, delta, epsilon, and zeta were detected in both rat and human colonic mucosa, while PKC eta was detected in human colonic mucosa only. PKC alpha, beta, and z eta were predominantly localized in the cytosolic fraction, whereas th e majority of PKC delta, epsilon, and eta were found in the membrane-a ssociated fraction. Presence of mRNA for individual PKC isoforms was d etermined by reverse transcriptase PCR (RT-PCR). Using rat colonic muc ose, mRNA for PKC alpha, beta, delta, epsilon, eta, and zeta were dete cted by RT-PCR with identity confirmed by sequencing. The relative ste ady-state levels of PKC isoforms in human colon adenocarcinoma as comp ared with normal colonic mucosa were determined, with adenocarcinomas having higher amounts of cytosolic PKC beta, delta, epsilon, eta, and zeta. PKC isoforms were also detected in viable, exfoliated colonic ce lls isolated from human feces, demonstrating that this noninvasive met hod can be utilized to examine PKC expression in colonic cells. These results demonstrate that colonic mucose expresses both calcium-depende nt (classical) and calcium-independent (novel and atypical) PKC isofor ms with distinct subcellular distributions for each. The dynamics of t hese PKC isoforms may have implications in the development of colon ca rcinogenesis. (C) 1994 Academic Press, Inc.