A convergence of diverse experimentation has led to the identification of a new class of potentially oncogenic serine/threonine protein kinases and an appreciation of the regulatory role of """"""""cyclin"""""""" proteins in normal and neoplastic cell growth. Previous studies by the Principal Investigator characterized the substrate specificity, subunit configuration, growth factor sensitivity, and viral subversion of mammalian cyclin-dependent protein kinases (Cdks), providing biochemical evidence linking specific Cdk complexes to the regulation of tumor suppressor proteins and cell cycle transitions. In the course of investigating the temporal expression and enzymology of cyclin D1 (a.k.a. the PRAD1 oncogene) in human osteosarcoma cells (NSF grant# DCB 9104769), a novel and unique human G1 cyclin was discovered in this laboratory. This novel G1 cyclin, designated Cyclin X, is first expressed in Mid G1, well after the induction of Cyclin D (Early G1) yet prior to Cyclins E and A (late G1). This proposal seeks to further investigate the cellular and molecular biology of Cyclin X, and to characterize its functional role in the mammalian somatic cell cycle. The working hypothesis is that G1 cyclins function in a concerted manner as S Phase Promoting Factors, and that Cyclin X, and its Cdk2 catalytic subunit, participate in a progressive series of biochemical events that ultimately lead to S phase commitment. Conversely, it is postulated that disruption of upstream contingencies and/or Cyclin X expression will impair cell cycle progression. The proposal has four Specific Aims: (1) To characterize Cyclin X and related family members, by molecular cloning. (2) To characterize and modulate the expression of Cyclin X in precisely synchronized cells. (3) To investigate the biochemical mechanism of Cyclin X/Cdk2 activation in Late G1. (4) To assess the role of Cyclin X in regulating Late G1 and/or S phase events. The experimental strategy will employ molecular, biochemical, and immunological approaches to characterize the expression and dynamics of Cyclin X in a variety of human cell lines and murine Cdc2(ts) mutants. A combination of pharmacological blocks, immunoperturbation studies, and antisense strategies will be used to assess the functional significance of the Cyclin X/Cdk2 complex. It is anticipated that these studies will further elucidate the regulatory mechanisms of cell cycle progression, and will identify a novel target for potential therapeutic intervention.
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