The cyclin-dependent kinase (cdk) inhibitor flavopiridol induces cell cycle arrest in many solid tumor cell lines. However, cells are sensitized to flavopiridol following recruitment to S phase by synchronization or by treatment with chemotherapy agents that impose S phase delay. The combination of gemcitabine, followed by flavopiridol, produces sequence-dependent cytotoxic synergy. Flavopiridol-mediated cdk inhibition during S phase is expected to prevent the appropriately timed neutralization of E2F-1 activity, resulting in an apoptotic response. In the first specific aim, the effects of flavopiridol in gemcitabine-treated cells will be examined to establish the inappropriate persistence of E2F-1 expression during S phase traversal. Reduced phosphorylation of E2F-1 in the presence of flavopiridol will be confirmed using cells labeled with orthophosphate as well as with phospho-specific antibodies directed at cdk2- and cdk7-phosphorylation sites. The half-life and transcriptional activity of E2F-1 in the presence of flavopiridol will also be determined. Furthermore, siRNAs targeting E2F-1 will be introduced into tumor cells to confirm that flavopiridol-induced apoptosis following recruitment to S phase by gemcitabine is E2F-1-dependent. A dominant negative mutant of E2F-1, retaining DNA binding activity, but lacking transcriptional transactivation activity, will be inducibly expressed to determine if the latter is required for flavopiridol-mediated apoptosis during S phase. In addition, the interaction of E2F-1 with components of the NF-kappaB pathway will be examined. In the second specific aim, xenograft-bearing mice will be treated with gemcitabine and flavopiridol to confirm the sequence dependence of the combination and to determine the optimal interval between the two drugs. The activity of the combination will also be tested in a p27Kip1-deficient animal model of Barrett's-associated esophageal carcinoma. Xenografts and murine tumors will be subjected to immunohistochemistry for appropriate cell cycle proteins and their phosphorylated forms to confirm cdk inhibition by flavopiridol. In the third specific aim, a phase I trial of gemcitabine followed by flavopiridol will be performed in subjects with advanced solid tumors. A novel flavopiridol schedule will be employed, designed to achieve sustained micromolar concentrations that are necessary for cdk inhibition. In addition to pharmacokinetic analyses, the trial will incorporate pharmacodynamic endpoints for flavopiridol activity in tumor tissue, skin and plasma.
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