EFFECT OF CAMPTOTHECIN ON MITOGENIC STIMULATION OF HUMAN-LYMPHOCYTES - INVOLVEMENT OF DNA TOPOISOMERASE-I IN CELL TRANSITION FROM G0 TO G1 PHASE OF THE CELL-CYCLE AND IN DNA-REPLICATION

The possible involvement of DNA topoisomerase I in cell transition from G0 to G1 and in progression through the cell cycle was studied by estimating the ability of human peripheral blood lymphocytes to undergo mitogenic stimulation in the presence of the topoisomerase I inhibitor camptothecin (CAM). Exposure of quiescent G0 lymphocytes to up to 3-mu-M CAM for 24 h had no significant effect on their ability to subsequently undergo mitogenic stimulation in the presence of phytohemagglutinin (PHA); higher doses of CAM, although not immediately cytotoxic, impaired the mitogenic response. Stimulation of lymphocytes with PHA in the presence of less-than-or-equal-to 1.5-mu-M CAM resulted in unperturbed transition of these cells from G0 to G1 characterized as an increase in cellular rRNA content, appearance of interleukin-2 receptor, and, after removal of CAM, response to interleukin-2 by entering S phase of the cell cycle. However, lymphocytes were prevented from entering S phase in the presence of CAM at a concentration of greater-than-or-equal-to 30 nM, and their rate of progression through S was minimal even at CAM concentration as low as 3 nM. When cycling lymphocytes (48 h after stimulation by PHA) were treated with CAM, the cell progression through S and G2 was also very sensitive to the inhibitor: the cells were "frozen" in S and G2 at greater-than-ot-equal-to 6 nM CAM. These cells died within 24 h; their selective loss from the cultures (with only G0/G1 cells remaining) coincided with the appearance of cells with fractional DNA content, typical of apoptotic cells. Human lymphocytic leukemic MOLT-4 cells were arrested in S and G2 at greater-than-or-equal-to 7.5 nM CAM. Thus, progressions through S and G2 of both normal and leukemic lymphocytes were perturbed at approximately two orders of magnitude lower CAM concentration than the G0 to G1 transition. These data suggest that DNA replication and chromosomal events during G2 are more sensitive to inhibition of DNA topoisomerase I, compared with the early events of lymphocyte stimulation, which involve activation and transcription of numerous genes associated with the G0 to G1 transition. The antitumor properties of CAM may be related to its high cytostatic/cytotoxic activity toward cycling cells and relative resistance of cells in G0 or undergoing transition from G0 to G1.