We are studying the effect of melatonin on protein and RNA metabolism in human breast cancer cells. We have found that melatonin rapidly (within 15 minutes) enhances protein synthesis. Enhancement is self limiting and complete within 40 minutes. Analysis of subcellular fractions at 40 minutes indicates that the protein content of mitochondria, microsomes, and cytosol significantly increased. Pulse chase studies at 3 hours demonstrate significant increase in nuclear con;tent of the induced protein. Gel electrophoresis of cytosol from melatonin-treated cells indicates that one or more polypeptides in the 3000 molecular weight range are induced by melatonin. Melatonin also stimulates total cellular RNA synthesis, beginning at 6-12 hours. This stimulation is blocked by cycloheximide and therefore requires the prior action of melatonin on protein synthesis. In vitro studies of nuclear DNA-dependent RNA polymerase indicate that melatonin enhances both polymerase I and II, and therefore stimulates both ribosomal and messenger RNA synthesis. Analysis of 3H-uridine labeled ribosomal RNA by agarose gel electrophoresis indicate that both 18S & 28S RNA are being stimulated. Studies of the in vitro translation products of melatonin stimulated m-RNA suggest that the transcription of message for multiple small polypepitides is being stimulated by melatonin. Studies to further characterize these polypeptides are in progress. We have also used the covalently attaching label 3H-tamoxifen aziridine to study the molecular weight forms of the estrogen receptor in human breast cancer cells. By denaturing gel electrophoresis the estrogen receptor migrates as the subunit protein (67K form.) Under low salt conditions the subunit form aggregates to larger proteins (240K form), which are readily dissociated by the addition of salt. Analysis in the presence of protease inhibitors indicate the presence of two discrete large molecular weight forms which are not dissociated by salt. These forms are felt to be large molecular weight precursors for the estrogen receptor subunit. These methods are now being used to characterize estrogen receptors protein in human breast cancer tumors.
