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Protein-kinase-C iso-enzymes support DNA synthesis and cell survival in colorectal-tumor cells
K Hochegger1, G Partik, M Schörkhuber
1Institute of Tumor Biology/Cancer Research, University of Vienna, Vienna, Austria.
Abstract:
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.
Insights
Blocking Protein Kinase C (PKC) signaling in colorectal tumor cells inhibits growth and induces apoptosis. Different PKC isoforms have distinct roles, with Ca(++)-dependent PKCs involved in proliferation and Ca(++)-independent PKCs in survival pathways.
Area of Science:
- Molecular Biology
- Cancer Research
- Cell Signaling
Background:
- Protein Kinase C (PKC) signaling plays a critical role in cellular processes.
- Dysregulation of PKC signaling is implicated in the development of colorectal tumors.
- Targeting PKC pathways offers a potential therapeutic strategy for colorectal cancer.
Purpose of the Study:
- To investigate the role of different Protein Kinase C (PKC) isoforms in colorectal tumor cell proliferation and survival.
- To determine the effects of inhibiting specific PKC isoforms on tumor cell growth and apoptosis.
- To elucidate the distinct functions of Ca(++)-dependent and Ca(++)-independent PKC pathways in colorectal cancer.
Main Methods:
- Inhibition of PKC signaling using kinase inhibitors (H7, calphostin, Gö6976).
- Down-regulation of PKC using TPA and anti-sense oligonucleotides targeting specific PKC isoforms (alpha, beta, epsilon, zeta).
- Assessment of cell growth, DNA synthesis, and apoptosis (chromatin condensation, DNA strand breaks, bcl-2/bak levels) in various colorectal tumor cell lines (SW480, T84, VACO235).
Main Results:
- Kinase inhibitors H7 and calphostin inhibited growth and induced apoptosis in colorectal tumor cells.
- H7 down-regulated bcl-2 levels, a key apoptosis regulator, without affecting bak.
- Ca(++)-dependent PKC inhibitors showed varied effects, with Gö6976 being inactive.
- Anti-sense oligonucleotides targeting Ca(++)-independent PKCs (epsilon, zeta) induced apoptosis.
- Anti-sense oligonucleotides targeting Ca(++)-dependent PKCs (alpha, beta) inhibited DNA synthesis, particularly in adenoma cells.
- PKC epsilon and zeta down-regulation led to apoptosis preceded by loss of PKC protein and bcl-2.
Conclusions:
- Ca(++)-dependent PKC isoforms (alpha, beta) are crucial for proliferation signals in colorectal tumor cells.
- Ca(++)-independent PKC isoforms (epsilon, zeta) are involved in survival pathways, protecting cells from apoptosis.
- Targeting specific PKC isoforms may offer differential therapeutic strategies for colorectal cancer, depending on the tumor's molecular profile.