Protein-kinase-C iso-enzymes support DNA synthesis and cell survival in colorectal-tumor cells

Protein-kinase-C: signalling has been blocked in colorectal tumor cells by kinase inhibitors, by TPA down-regulation or by exposure to anti-sense olig onucleotides. This resulted in growth inhibition in all cell lines used. Th e kinase inhibitors H7 and calphostin induced apoptosis, demonstrated by th e appearance of cells with characteristically condensed chromatin and the i nduction of stand-breaks in the DNA. A cell-death-inducing concentration of 15 mu g/ml H7 down-regulated the bcl-2 levels after 9 hr, while bak levels were not affected, Go6976,-an inhibitor of Ca++-dependent PKC iso-enzymes, was not active in growth inhibition or induction of apoptosis, Analysis of DNA synthesis in inhibitor-treated cultures indicated that H7 caused stron g inhibition in all cell lines, while the more specific inhibitor calphosti n was effective only in VACO235 adenoma cells. When down-regulation by TPA or anti-sense oligonucleotides was used to block PKC, effects on cell numbe rs were smaller and delayed. However, induction of apoptosis was significan tly increased in SW480 carcinoma cells 4 days after exposure to anti-epsilo n and anti-zeta oligonucleotides in SW480 and T84 carcinoma cells, Apoptosi s was preceeded by loss of PKC protein and of bcl-2 from day after addition of the oligonucleotides, In VACO235 adenoma cells, no induction of apoptos is could be observed when anti-epsilon and anti-zeta oligonucleotides were used. On the other hand; the adenoma cells were more responsive to anti-alp ha and anti-beta oligonucleotides, which strongly inhibited DNA-synthesis 3 days after addition to the culture medium. Our results indicate that the C a++-dependent PKCs alpha and beta are involved in proliferation signals, wh ile the Ca++-independent PKCs epsilon and zeta are involved in survival pat hways of colorectal tumor cells. (C) 1999 Wiley-Liss, Inc.