Protein-kinase-C signalling has been blocked in
colorectal tumor cells by
kinase inhibitors, by TPA down-regulation or by exposure to
anti-sense oligonucleotides. This resulted in growth inhibition in all cell lines used. The
kinase inhibitors H7 and
calphostin induced apoptosis, demonstrated by the appearance of cells with characteristically condensed
chromatin and the induction of stand-breaks in the
DNA. A cell-death-inducing concentration of 15 microgram/ml H7 down-regulated the bcl-2 levels after 9 hr, while bak levels were not affected. Gö6976,-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 strong inhibition in all cell lines, while the more specific inhibitor
calphostin was effective only in VACO235
adenoma cells. When down-regulation by TPA or
anti-sense oligonucleotides was used to block PKC, effects on cell numbers were smaller and delayed. However, induction of apoptosis was significantly increased in SW480
carcinoma cells 4 days after exposure to anti-epsilon and anti-zeta
oligonucleotides in SW480 and T84
carcinoma cells. Apoptosis was preceeded by loss of PKC
protein and of bcl-2 from day 1 after addition of the
oligonucleotides. In VACO235
adenoma cells, no induction of apoptosis could be observed when anti-epsilon and anti-zeta
oligonucleotides were used. On the other hand, the
adenoma cells were more responsive to anti-alpha and anti-beta
oligonucleotides, which strongly inhibited
DNA-synthesis 3 days after addition to the culture medium. Our results indicate that the Ca(++)-dependent
PKCs alpha and beta are involved in proliferation signals, while the Ca(++)-independent
PKCs epsilon and zeta are involved in survival pathways of
colorectal tumor cells.