The long range objective of the proposed studies is the development of a new endocrine-based therapy for the treatment of human breast cancer. Our experiments will be performed with the hormone-responsive human breast cancer tissue culture cell line, MCF-7. The major hypothesis to be tested is that estradiol induction of human breast cancer growth is dependent upon calcium; therefore, calcium antagonism constitute a novel ode of human breast cancer endocrine therapy. Sp.
Aim 1 is to determine if calcium channel blockade by verapamil, nifedipine, calmodulin antagonism by W-7, W-13 or protein kinase C antagonism by H-7 are independent and novel mechanisms of antagonism of estrogen-induced breast cancer cell proliferation. Estrogen-induced proliferation will be monitored by induction of c-myc mRNA, 3H-thymidine incorporation, ad cell number. Sp.
Aim 2 is to determine the effects of calmodulin inhibitors, calcium channel blockers, and a protein kinase C inhibitor on MCF-7 estrogen receptor,levels and binding affinities, to determine if these drugs exert antiestrogen actions like tamoxifen by competing with estradiol for estrogen receptor binding sites. Sp.
Aim 3 is to monitor calcium ion fluxes in MCF-7 cells after exposure to estradiol using 45Ca and Fura-2 fluorescence changes to monitor changes in the free cytosolic calcium concentration [and investigate the mechanism of E2 and drug effects on Ca +2 fluxes through voltage-sensitive Ca +2 channels using voltage-patch clamp techniques.] Sp.
Aims 4 and 5 are to assess the effects of estradiol on calmodulin and protein kinase activities in MCF-7 cells and to demonstrate that the specific antiestrogenic activities of the various calmodulin and protein kinase C inhibitors are correlated with inhibition of these biochemical processes. [Particular emphasis is placed on in vivo and in vitro estradiol-stimulated protein phosphorylation in the membrane and cytosol fractions during the first 30 minutes of exposure to estradiol and whether estradiol effects on the calcium channel are mediated by phosphorylation of this channel.]
