Flavopiridol is the first potent inhibitor of cyclin-dependent kinases (cdks) to enter clinical trial. Preclinical data suggest promising activity against non-small cell lung cancer (NSCLC), a disease for which there is a pressing need for new agents. In most NSCLC cell lines, cytotoxicity only occurs after prolonged exposures at high concentrations of drug that are difficult to achieve in vivo. However, flavopiridol treatment of cells during early S phase, following release from a hydroxyurea-induced block at the G1/S boundary, results in earlier, markedly enhanced cell death. Similarly, treatment with flavopiridol following an S phase delay imposed by chemotherapeutic agents such as cisplatin or gemcitabine results in enhanced apoptosis, so that there is sequence-dependent synergism. In the first specific aim, the fate of BrdU-labeled cells will be analyzed in apoptosis assays to determine whether cells in S phase are the most sensitive to flavopiridol. In addition, appropriately paired cell lines will be examined to determine whether flavopiridol-mediated apoptosis during S phase is selective for transformed cells and whether it requires p53. In the second specific aim, the mechanism of S phase sensitivity will be investigated. Data will be generated to support a model in which flavopiridol-mediated cyclin A-cdk2 inhibition during S phase prevents the phosphorylation of E2F-l and its partner DP-1, resulting in inappropriately persistent E2F-1 activity and apoptosis. Cyclin A-kinase activity, as well as E2F-1 phosphorylation and its transactivation and DNA binding activities will be assessed in flavopiridol-treated cells following recruitment to S phase. E2F-1 activity will be altered by ectopic expression of wild-type and mutant species to determine whether the apoptotic response to flavopiridol during S phase is affected. Furthermore, flavopiridol-mediated apoptosis will be linked to cdk2 inhibition by the generation of flavopiridol-resistant cell lines and by expression of cdk2 mutants that retain activity but harbor altered ATP binding sites. In the third specific aim, the toxicity and potential anti-tumor activity of the sequential combination of gemcitabine, an S phase-specific agent, followed by flavopiridol, will be assessed in a phase I clinical trial. In vitro studies will be performed to determine whether flavopiridol affects the metabolism of gemcitabine. The pharmacokinetic interactions of the two drugs in patients will also be explored.
Showing the most recent 10 out of 24 publications
