Human cyclin A1 is expressed at its highest levels in spermatocytes but also in leukemic cell lines and leukemic cells obtained from patients with acute myeloid leukemia (AML), particularly acute promyelocytic leukemia (APL). Interestingly, in mouse leukemia models and in preliminary investigations on the human APL cell line NB4, the localization of cyclin A1 is predominantly cytoplasmic, unlike its nuclear localization in germ cells. The hypothesis that will be tested is that the cytoplasmic accumulation of cyclin A1 contributes to its transforming potential. NB4 cells will be examined for cell cycle-specific cyclin A1 subcellular localization patterns. The cytoplasmic distribution of cyclin A1 will be determined by sub-cellular fractionation followed by immunoblotting. Coimmunoprecipitation assays will be used to determine the binding partners of cyclin A1, including its partner kinase(s) and substrates. Kinase assays will be performed to evaluate the activity of cyclin A1-CDK complexes. Finally, the contribution of cytoplasmic cyclin A1 localization to the transforming phenotype will be examined by fusing cytoplasmic organelle-specific targeting sequences to cyclin A1 constructs.
