application) All mammalian cells, including prostate epithelial cells, that have been studied require polypeptide growth factors for proliferation. Prostate cell proliferation is additionally regulated by androgen. However, we know very little about how androgen regulates cell proliferation. Progression of cells from G1 into S phase is dependent on events regulated by calcium ion and calmodulin (CaM). Inhibition of CaM at G1/S boundary, prevents cells from entering into S phase. This critical role of CaM appears to be mediated by a specific CaM binding protein of 68 kDa (CaMBP68). CaMBP68 exhibits growth factor dependent translocation from the cytoplasm into the nucleus of cells entering into S phase, where it binds tightly to DNA polymerase-alpha/primase, a component of the replitase complex required for DNA replication. Androgen sensitive (LNCaP) prostate cancer cells also are growth inhibited by antiCaM drugs, suggesting a critical role of CaM also in prostate cancer cell proliferation. Moreover, androgen independent (DU145 and PC3) prostate cancer cells have a markedly higher level of CaMBP68 in their nuclei (compared to that in LNCaP cells) and are 6-8 times less sensitive to the growth inhibitory effect of CaM antagonists than are androgen sensitive cells. Taken together, these observations lead us to postulate that CaM and CaMBPs play an essential role in androgen regulation of prostate cancer cell proliferation. To test this hypothesis we will first determine the point in the cell cycle when androgen is required for progression of synchronized androgen sensitive prostate cancer cells from G1 into S phase and then identify whether androgen-stimulated events at that point in cell cycle are being mediated CaM or CaMBP68. Based on earlier observations with the estrogen receptor, we will determine whether CaM is essential for the binding of androgen receptor (AR) to androgen responsive element (ARE) and for the expression of androgen responsive genes. Since we observed that androgen independent cells, as compared to androgen sensitive cells, contain markedly increased levels of CaMBP68 in their nuclei, and since CaMBP68 is an essential component of the replitase complex, we will examine whether there are any differences in the assembly of replitase complexes between these two cell systems. Elucidating the mechanism by which androgen regulates the cell cycle and identifying the role of CaM in androgen action in these studies may provide new insights and opportunities for controlling prostate cancer cell proliferation and treatment of the disease.
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