): The objective of this proposal is to identify mechanisms linking RA-induced tumor differentiation to changes in the cell cycle leading to G1-S arrest. Elucidating how initiation of tumor differentiation is coupled to the cell cycle machinery should lead to a greater fulfillment of the potential of maturation-based cancer therapy. The human germ cell cancer (teratocarcinoma) cell line, NT2/D1, arrests in G1 during RA-induced neuronal differentiation. Preliminary data indicate in response to RA, NT2/D1 cells show a progressive d e c l ine in cyclin D1 expression that parallels a decline in cell proliferation. Phenotypic maturation and accumulation of cells in G1 occur thereafter. An RA-resistant clone, NT2/D1-R1, shown not to express the retinoid receptor-gamma (RARgamma), did not accumulate in G1 and persistently overexpressed cyclin D1 and p53. Exogenous RARgamma expression reversed RA resistance and restored RA-mediated decline of cyclin D1. This proposal is designed to learn how RA signals those cell cycle changes causing growth suppression. The first specific aim identifies the mechanisms by which RA achieves G1 arrest by comparing the biochemical activity and expression of known mediators of G1-S progression in RA-sensitive and resistant NT2/D1 cells. The second specific aim determines whether RA-induced cell cycle changes are causal in mediating induced growth suppression and maturation by studying the RA sensitivity of cells engineered to overexpress cyclin D1, p53 and other highlighted cell cycle components. The third specific aim explores the role of RARs in signaling RA-induced changes in the cell cycle by individually over-expressing specific retinoid receptors in RA-sensitive and resistant NT2/D1 cells. The direct role of RARgamma in tumor differentiation is addressed in Specific Aim 4 by engineering lines to express a dominant-negative RARgamma construct. This research addresses the mechanisms of induced growth suppression and differentiation of human tumor cells. The successful completion of these specific aims should aid in the rational use of retinoids in cancer therapy and prevention.
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