L-TRIIODOTHYRONINE EFFECTS ON CELL REGULATION

We will examine thyroid hormone effects on cell growth in cultured GC cells, a thyroid hormone-sensitive cell line that secretes GH. We have synchronized these cells in early Gl and demonstrated that T3 induces growth during the first 4-6h of the Gl period. Growth induction by T3 has characteristics suggesting mediation by the nuclear T3 receptor and demonstrates a requirement for protein synthesis. We now propose to identify the specific proteins and phosphoproteins that are regulated by T3 at the Gl commitment point by pulse-labelling and 2-dimensional gel electrophoresis. The regulation by T3 of specific Gl mRNAs will be determined by differential hybridization of a cDNA library in lambda-gtlO phage vector. The time course and dose-dependence of these Gl responses will be assessed. Since GC cell growth can also be regulated by GC cell conditioned medium, calf serum growth factor, hydrocortisone, EGF and defined medium, we will determine whether these regulators result in biochemical changes that are similar to those induced by T3. We have previously shown that T3 nuclear receptor concentration is augmented in S phase of the cell cycle and is localized to the nuclear matrix, a DNA and salt resistant nuclear fraction that is associated with new DNA synthesis and transcription complexes. We now propose to examine the physico-chemical characteristics of nascent T3 receptor molecules in conjunction with nascent DNA in the S phase of the cell cycle. We propose to clarify the relationship between nuclear T3 receptor and nuclear matrix by determining the role of disulfide bonds and metal ion complexes in this interaction. These studies should elucidate the nature of the physico-chemical association of T3 receptor to DNA and chromatin. We have reported that the rate of GH mRNA synthesis is decreased in S phase of the cell cycle, an interval when nuclear receptor concentration is augmented. To explain the dissociation between T3 receptor concentration and mRNA synthesis, we now propose to measure the transcription rate of GH in GC cell populations that are synchronized in Gl and S phase. Lastly, we will determine whether these synchronized GC cell populations contain cell cycle- specific trans-acting factors which may mediate the cell cycle- specific regulation of GH transcription.