Somatic cell hybridization of growth hormone-producing rat pituitary cells and mouse fibroblasts results in extinction of growth hormone expression via a defect in growth hormone RNA production.
In the GH3 line of pituitary cells, growth hormone (GH) production is stimulated by thyroid and glucocorticoid hormones, yet non-pituitary cells, for example mouse fibroblast cells, which also contain receptors for these hormones do not produce GH. We have chosen to study both types of control over GH production in GH3 rat pituitary x GH nonexpressing LB82 mouse fibroblast hybrid cells. Most hybrid cells fail to produce GH in either the absence or presence of the inducers triiodothyronine and dexamethasone although these hormone receptors are present. Rat and mouse GH genes were detected in the hybrid cells' DNA by Southern hybridization following restriction enzyme digestion and gel electrophoresis. The probe was a cloned, full length rat GH cDNA which cross-hybridizes with the mouse GH gene. Analyses with multiple restriction enzymes that cut inside and outside the rat and mouse GH coding sequences were performed. We conclude that 1) both rat and mouse GH genes are present in the hybrid cells in copy number equivalent to those in the parental cell lines, and 2) the rat GH structural gene and flanking sequences are identical in the GH3 and hybrid cell DNA at the level of sensitivity afforded by restriction enzyme analyses. GH mRNA was not detected in total cell or poly(A+) RNA isolated from control or hormone-treated hybrid cells assayed by translation in 2 in vitro systems or by Northern hybridization analyzes. The latter experiments showed that no hybridizable GH mRNA sequences were present in the hybrids as either incomplete transcripts, mature mRNA, or unprocessed high molecular weight precursors. We conclude that extinction of GH in these hybrid cells results from a transcriptional block and/or rapid transcript degradation.
D000818:Animals; D002455:Cell Division; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D004262:DNA Restriction Enzymes; D005910:Glioma; D013006:Growth Hormone / Q000235:genetics*; D006822:Hybrid Cells / Q000502:physiology*; D007739:L Cells / Q000502:physiology; D051379:Mice; D009693:Nucleic Acid Hybridization; D011061:Poly A / Q000235:genetics; D012313:RNA / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics*; D051381:Rats; D014158:Transcription, Genetic
D012333:RNA, Messenger; D011061:Poly A; D012313:RNA; D013006:Growth Hormone; D004247:DNA; D004262:DNA Restriction Enzymes