THE PROTEIN-KINASE-C INHIBITOR H7 BLOCKS NORMAL HUMAN LYMPHOCYTE STIMULATION AND INDUCES APOPTOSIS OF BOTH NORMAL LYMPHOCYTES AND LEUKEMIA MOLT-4 CELLS

The isoquinoline sulfonamide (H7) is an inhibitor of protein kinase C (PKC) that also inhibits the activity of cyclic nucleotide-dependent protein kinases. The effect of H7 on mitogen stimulation (G0 to G1 transition) of normal human lymphocytes and on their subsequent progression through the cell cycle was investigated and compared with the effect of this inhibitor on proliferation of human lymphocytic leukemic MOLT-4 cells. At H7 concentrations of 10 and 50 muM, the transition of G0 lymphocytes to the cell cycle was suppressed by 45 and 98%, respectively. The cell cycle progression of stimulated lymphocytes was unaffected at 10 muM H7, whereas, at 50 muM, the overall rate of progression was reduced by 50% with no evidence of cell arrest at a specific phase of the cycle. Similar concentrations of H7 (45 muM) suppressed proliferation of MOLT-4 cells by 50%, though, in the latter case, cells underwent transition to higher DNA ploidy, most likely via endoreduplication. Thus, the G0 to G1 transition appears to be the event most sensitive to H7. Exposure of MOLT-4 cells to 100 muM H7 for 24 h induced extensive apoptosis: activation of an endogenous nuclease with preference to internucleosomal linker DNA sections resulted in DNA degradation (revealed by agarose gel electrophoresis and loss of DNA measured by flow cytometry), which was paralleled by intracellular proteolysis, while the integrity of the plasma membrane, mitochondria and lysosomes was preserved. Morphological examination of these apoptotic cells confirmed DNA degradation. However, the perinuclear and fine-granular localization of the remaining DNA and lack of typical chromatin condensation and nuclear fragmentation differed from the classical pattern of apoptosis observed in other cell systems, suggesting that some events of apoptosis (nuclear fragmentation) may be affected by H7. The observed effects are consistent with the possible role of H7 in inhibition of PKC or its direct effect on the ATP-binding domain of DNA topoisomerase II, which shares homology with the H7 binding sites on PKC and the cyclic nucleotide-dependent protein kinases.