Flavopiridol Induces Apoptosis in Chronic Lymphocytic Leukemia Cells Via Activation of Caspase-3 Without Evidence of bcl-2 Modulation or Dependence on Functional p53

Flavopiridol has been reported to induce apoptosis in lymphoid celllines via downregulation of bcl-2. The in vitro activity offlavopiridol against human chronic lymphocytic leukemia (CLL) cells andpotential mechanisms of action for inducing cytotoxicity were studied.The in vitro viability of mononuclear cells from CLL patients (n = 11) was reduced by 50% at 4 hours, 24 hours, and 4 days at aflavopiridol concentration of 1.15 µmol/L (95% confidence intervalCI] ±0.31), 0.18 µmol/L (95% CI±0.04), and 0.16 µmol/L (95% CI ±0.04), respectively. Loss ofviability in human CLL cells correlated with early induction ofapoptosis. Exposure of CLL cells to 0.18 µmol/L of flavopiridolresulted in both decreased expression of p53 protein and cleavage ofthe caspase-3 zymogen 32-kD protein with the appearance of its 20-kD subunit. Contrasting observations of others in tumor cell lines, flavopiridol cytotoxicity in CLL cells did not correlate with changesin bcl-2 protein expression alterations. We evaluated flavopiridol'sdependence on intact p53 by exposing splenocytes from wild-type(p53^+/+) and p53 null (p53^/) micethat demonstrated no preferential cytotoxicity as compared with amarked differential with F-ara-a and radiation. Incubation of CLL cellswith antiapoptotic cytokine interleukin-4 (IL-4) did not alter theLC₅₀ of flavopiridol, as compared with a marked elevationnoted with F-ara-a in the majority of patients tested. These datademonstrate that flavopiridol has significant in vitro activity againsthuman CLL cells through activation of caspase-3, which appears to occurindependently of bcl-2 modulation, the presence of IL-4, or p53 status.Such findings strongly support the early introduction of flavopiridolinto clinical trials for patients with B-CLL.