G1 EXPRESSION AND MULTISTAGE DYNAMICS OF CYCLIN-A IN HUMAN OSTEOSARCOMA CELLS

Cyclin A was initially characterized as a 'mitotic cyclin', believed t o function exclusively at the G2-to-M phase transition; however, recen t studies have provided compelling evidence that cyclin A additionally functions earlier in the mammalian somatic cell cycle as a putative ' S-phase-promoting factor'. Moreover, numerous inconsistencies have ari sen concerning the temporal induction, subcellular localization, subun it configuration, covalent modification and proteolytic destruction of cyclin A, as well as the physiological function of the cyclin A-assoc iated protein kinase complexes. Utilizing precisely synchronized human MG-63 osteosarcoma cells, the present study demonstrates that cyclin A mRNA and protein are clearly expressed in late G1 prior to S-phase e ntry, as is cyclin A-associated kinase activity and concomitant phosph orylation of the Rb protein. A series of monospecific cyclin A antibod ies were generated and utilized to confirm that multiple covalent modi fications of cyclin A occur during the course of the cell cycle, and t o characterize the subcellular dynamics in additional detail. Pharmaco logical blockade with mimosine was utilized to further delineate cycli n A expression and to distinguish the temporal induction from the mech anisms of enzyme activation. Subcellular fractionation and immunocytoc hemical staining localized nascent cyclin A to the cytoplasm, and reve aled a distinct translocation to the nucleus during the G1-to-S phase transition. The results of these studies support a multistage model of cyclin A metabolism and enzyme activation.