Biochimica et Biophysica Acta 910 (1987) 213-223 213 Elsevier BBA 91760 Defective distal regulatory element at the 5' upstream of rat prolaetin gene of steroid-nonresponsive GH-subclone Vipin Kumar, David T.W. Wong, Sally G. Pasion and Debajit K. Biswas Laboratory of Pharmacology, Harvard School of Dental Medicine and Department of Pharmacology, Harvard Medical School, Boston, MA (U.S.A.) (Received 18 May 1987) (Revised manuscript received 15 July 1987) Key words: Hormone regulation; Prolactin gene; Regulatory element; (Rat cell) The prolactin-nonproducing (PRL-) GH cell strains (rat pituitary tumor cells in culture). GHt2C ! and F I BGH 12C 1, do not respond to steroid hormones estradiol or hydrocortisone (HC). However, the stimula- tory effect of estradiol and the inhibitory effect of hydrocortisone on prolactin synthesis can be demon- strated in the prolactin-producing GH cell strain, GH 4Ca • In this investigation we have examined the 5' end flanking region of rat prolactin (rat PRL) gene of steroid-responsive, GH4C t cells to identify the positive and negative regulatory elements and to verify the status of these elements in steroid-nonresponsive FIBGH!2C ! cells. Results presented in this report demonstrate that the basel level expression of the co-transferred Neo gene (neomycin phosphoribosyi transferase) is modulated by the distal upstream regulatory elements of rat PRL gene in response to steroid hormones. The expression of adjacent Neo gene is inhibited by dexamethasone and is stimulated by estradiol in transfectants carrying distal regulatory elements (SRE) of steroid-responsive cells. These responses are not observed in transfectants with the rat PRL upstream sequences derived from steroid-nonresponsive cells. The basal level expression of the host cell a-2 tubulin gene is not affected by dexamethasone. We report here the identification of the distal steroid regulatory element (SRE) located between 3.8 and 7.8 kb upstream of the transcription initiation site of rat PRL gene. Both the positive and the negative effects of steroid hormones can be identified within this upstream sequence. This distal SRE appears to be nonfunctional in steroid-nonresponsive cells. Though the proximal SRE is functional, the defect in the distal SRE makes the GH substrain nonresponsive to steroid hormones. These results suggest that both the proximal and the distal SREs are essential for the mediation of action of steroid hormones in GH cells. Introduction GH cells are prolactin (PRL) and growth hormone- (GH) producing rat pituitary tumor cells in culture [1,2]. Although GH cells are trans- formed, these cells respond to other steroid or Correspondence: D.K. Biswas, Laboratory of Pharmacology, Harvard School of Dental Medicine, 188 Longwood Avenue, Boston, MA 02115, U.S.A. peptide hormones in a fashion similar to the nor- mal rat pituitary #and. Different subclones of GH cells synthesize and secrete different amounts of one or both of the hormones. Hydrocortisone or its structural analogue, dexamethasone, stimu- lates GH synthesis and simultaneously inhibits the synthesis of PRL in the GH cell strain GHaC t (steroid-responsive). Another steroid hormone, estradiol, on the other hand, inhibits GH synthesis and stimulates PRL synthesis in the same GH cell 0167-4781/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)