Neoplasia can result from the disruption of carefully regulated signal transduction pathways. These pathways which control critical decisions such as the balance between proliferation and differentiation are, in part, controlled by changes in the transcription rates of growth-responsive genes. Therefore, understanding the transcriptional control of cell growth is an essential aspect of understanding oncogenesis. A signal transduction pathway that has led to insight in the control of cell growth is initiated by the addition of serum growth factors to quiescent mammalian fibroblasts. This results in the transduction of extracellular signals to the cell nucleus, with a concomitant change in expression of growth-responsive genes. At the transition from Gl to S phase, a set of late serum-response genes involved in nucleotide biosynthesis are transcriptionally activated. One such gene is the DHFR gene which is required for the synthesis of glycine, purines, and thymidylate. Although the DHFR promoter has been shown to be growth-regulated at the transcriptional level, the cis elements that are responsible for the regulation have not yet been defined. Using serum starvation and restimulation of cells transfected with constructs bearing the DHFR promoter driving the luciferase cDNA, we will determine if luciferase activity can be detected with late-response kinetics. If so, we will then determine which cis element is responsible for the growth regulation by mutating previously identified transcription factor binding sites in the DHFR promoter. Purified protein, antibodies, and cDNA or genomic clones of the growth-responsive factors will be prepared to investigate how the cell achieves growth regulation via these particular factors. Changes in the amounts and modifications of the regulatory proteins will be analyzed. To fully understand the growth regulation of the DHFR promoter, it is essential to understand the biochemical mechanisms through which the transcriptional regulation is achieved. We will determine the mechanism of action of the growth-regulatory transcription factors using an in vitro system which can reproduce the transcriptional activation of the late-response DHFR promoter. Our studies will help to understand the growth-responsive events that occur prior to the activation of DHFR and will advance our long-term objectives which are to trace a signal transduction pathway from the addition of serum to the growth medium to the activation of late-response genes required for DNA replication.
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