CONSTITUTIVE EXPRESSION OF HUMAN BCL-2 MODULATES NITROGEN-MUSTARD ANDCAMPTOTHECIN INDUCED APOPTOSIS

Bcl-2 is a novel protooncogene which prolongs cell survival and suppre sses apoptosis. We examined whether constitutive expression of transfe cted human bcl-2 conferred resistance to two different DNA damaging dr ugs, nitrogen mustard (HN2) and camptothecin (CPT) in a murine, IL-3 d ependent cell line (FL5.12). HN2 treatment produced 2-fold less cell d eath and DNA degradation in cells overexpressing bcl-2 relative to con trol cells transfected with a construct bearing only the neo(R) gene. DNA degradation was characterized by oligonucleosomal length fragments indicating that programmed cell death or apoptosis had occurred. Equi molar HN2 produced similar extents of interstrand cross-link formation and repair in each cell line. Cell cycle characteristics were similar for both cell lines following equimolar HN2 treatment, exhibiting a b rief S phase delay followed by a longer G2 arrest. Time course studies indicated that DNA fragmentation occurred following peak G2 arrest in control cells and 12 h later in bcl-2 transfected cells. Equimolar CP T exposure also induced 2-fold less death and apoptotic DNA fragmentat ion in bcl-2 transfected compared to control cells. DNA single strand break formation and resealing kinetics were comparable in both cell li nes following equimolar CPT treatment. CPT caused similar cell cycle p erturbations in both cell lines, with a brief S phase block detectable 12 h after an equimolar drug dose. Kinetic studies showed apoptosis o ccurred following maximal S phase arrest in control and 12 h later in bcl-2 transfected cells. By contrast, IL-3 withdrawal produced rapid a nd extensive DNA degradation and apoptosis in controls 24 h postwithdr awal, and this process was inhibited 3-4-fold in bcl-2 transfectants. Cell cycle analysis showed both cell lines arrested in G0/G1 following IL-3 removal. In summary, bcl-2 transfection affords a 2-fold protect ion from HN2 and CPT cytotoxicity and decreases drug induced apoptosis in FL5.12 cells, despite the different mechanisms of action and cell cycle effects of each agent. Bcl-2 overexpression appears to represent a novel drug resistance mechanism of potential clinical significance.