CONTROL OF CELL PROLIFERATION

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The proliferation of all eukaryotic cells is primarily controlled by a single decision which occurs in the G1 phase of the cell cycle -- either to complete the cell cycle and divide or to withdraw from the cell cycle and enter a quiescent state. In normal cells this decision is regulated by specific intracellular and extracellular signals, such as mitogenic growth factors. In transformed or tumorigenic cells, however, these controls are suppressed and cell proliferation is unconstrained. Important advances have been made recently in understanding the molecular basis for this critical regulatory process. In S. cerevisiae, this decision is called START and completion of START culminates in DNA replication and commits the cell to complete the remainder of the cell division cycle. The biochemical event that underlies this cellular decision is the assembly and activation of complex between a protein kinase, encoded by the CDC28 gene, and a positively acting regulatory subunit of the kinase, a cyclin. Efforts to understand the molecular basis for controlling the proliferation of mammalian cells, therefore, have recently focused upon this family of protein kinases, named the cyclin-dependent kinases, and their regulators, the cyclins. We have isolated a new human cyclin, cyclin E, based upon its ability to functionally substitute for the S. cerevisiae G1 cyclins. We have found, by biochemical and physiological assays, that it probably plays a major role in regulating proliferation during the G1 phase of the mammalian cell cycle --1) Cyclin E binds to and activates a cyclin-dependent kinase specifically during the G1 phase of the human cell cycle; 2) Constitutive expression of cyclin E in primary human fibroblasts accelerates G1, reduces cell size and decreases growth factor requirements for cell proliferation; 3) Two factors that inhibit the cell cycle in G1, rapamycin and TGF-beta, block activation of the cyclin B/kinase complex. Our general goal is to develop a more complete picture of the physiological, biochemical and molecular actions of cyclin E during the mammalian cell cycle, both in fibroblasts and lymphocytes, and to understand how these actions contribute to the control of cell proliferation. Our specific approaches will include analyzing the physiological effects of constitutive cyclin E expression and inhibition of cyclin E expression on regulation of the mammalian cell cycle; studying, by high resolution two dimensional PAGE, cell cycle dependent modifications of cyclin E and cell cycle dependent interactions of cyclin B with other proteins; and correlating the physiological actions of cyclin B with its molecular and biochemical properties by analysis of cyclin E mutations.