Induction Of Apoptosis And Micronuclei By Bleomycin Sulfate In Different Cell Cycle Stages Of Human Lymphocytes

Document Type : Original Article




Introduction: Bleomycin sulfate is a DNA damaging agent used in cancer chemotherapy. The effect of this drug on various cell cycle stages might be different, thus inducing different modes of death (apoptotic or mitotic death). The aim of this investigation was to study the effects of bleomycin on human peripheral blood lymphocytes at various cell cycle stages by two different end points (induction of apoptosis or micronuclei).
Material and Methods: Human peripheral blood lymphocytes were treated with various doses of bleomycin at G0, G1, and G2 phases of the cell cycle and the percentages of apoptosis (AP) and micronuclei (MN) were determined. The peripheral lymphocytes were isolated by ficoll hypaque and suspended in RPMI-1640 containing 15 % fetal calf serum. The isolated lymphocytes were stimulated by phytohemagglutinin (PHA), cultured again inRPMI-1640, harvested after 64 hrs and 96 hrs, and stained with acridine orange and ethidiumbromide to determine the percentage of apoptotic cells. MN assay was done according to the standard in vitro micronucleus assay.
Results: The results showed that the percentages of apoptotic cells and MN at G2 stage were significantly higher than those of G0 and G1 stages. At higher doses, MN formation and apoptotic cells were increased; however with increasing time, the percentage of MN decreased while the percentage of apoptotic cells generally increased in all the cell cycle stages.
Conclusion: The results indicate that bleomycin is a potent inducer of both micronuclei and apoptosis. The incidence of apoptotic cells following bleomycin treatment in G0 and G1 was much higher than the incidence of micro nucleated cells at the two sampling times. The percentage of AP cells following bleomycin treatment remained constant across cell cycle stages.