Molecular and Cellular Endocrinology 127 (1997) 41 – 47 GH3 cells expressing constitutively active Gs (Q227L) show enhanced hormone secretion and proliferation J. Ham a, *, M. Ivan b , D. Wynford-Thomas b , M.F. Scanlon a a Department of Medicine, Uniersity of Wales College of Medicine, Cardiff CF44XN, UK b Department of Pathology, Uniersity of Wales College of Medicine, Cardiff CF44XN, UK Received 23 October 1996; accepted 20 November 1996 Abstract cAMP levels in GH3gsp cells (Q227L mutation of Gs ), in comparison with uninfected GH3 and GH3vt (with vector alone) cells, were two to three fold (P 0.01) higher (basal), and 10–20 fold (P 0.001) higher (in the presence of isobutyl methylxanthine, (IBMX)). Proliferation of GH3gsp cells after 7 days in culture, as determined by cell number and [ 3 H]thymidine incorporation, were up to 25% (respectively P 0.001 and P 0.02) higher. After chronic (4 days) but not acute (15 min) exposure to forskolin (10  M) or dibutyryl cAMP (50  M) all cell types showed a greater than 200% (P 0.001) increase in [ 3 H]thymidine incorporation. Secretion of prolactin and growth hormone by GH3gsp cells were two to four fold (P 0.001) higher than GH3 and GH3vt cells after 4 h and 10–12 fold (P 0.001) higher after 8 h. In conclusion GH3 cells possessing Q227L have a higher proliferation rate and secrete higher levels of prolactin and growth hormone which are associated with higher levels of cAMP. © 1997 Elsevier Science Ireland Ltd. Keywords: GH3 cells; Gs mutation; cAMP; Proliferation; Prolactin; Growth hormone 1. Introduction Mutations of Gs can cause constitutive activation of adenylyl cyclase and increased intracellular cAMP lev- els. The most common mutation, found in about 30% of growth-hormone (GH) secreting pituitary adenomas [1,2] and recently in 40% of autonomously functioning thyroid adenomas [3] is the substitution of leucine for glutamine at amino acid position 227, a guanine nucle- otide binding site. Gs is thereby stabilised in its active form because GTPase activity is inhibited [4]. It has been hypothesised that such constitutive activation of Gs is a cause of increased cell proliferation and secre- tory activity [5,6]. However, cAMP can be either growth inhibitory or growth promoting dependent on cell type. It is thus conceivable that in cells in which cAMP has a negative effect on proliferation then the presence of a mutation in Gs would lead to further growth inhibition. The mutated gene, Q227L has been previously incor- porated into Swiss 3T3 cells [7], but such infected cells did not show increased proliferation possibly due to a concomitant increase in phosphodiesterase activity al- lowing only a small increase in cAMP levels. These cells however were particularly sensitive to cAMP-stimulat- ing agents such as forskolin and Ro 20-1724 and showed an enhanced mitogenic response in comparison with uninfected cells and cells infected with wild-type Gs. On the other hand in thyroid FRTL-5 cells which were similarly infected, growth occurred in the absence of TSH whereas cell division in uninfected cells re- quired TSH [8]. Thus in this endocrine cell system, mutated Gs can induce both proliferation and differ- entiation. In this study we have infected GH3 cells (a rat pituitary tumour cell line) with the retroviral expres- * Corresponding author. Tel.: +44 1222 744571; fax: +44 1222 744671; e-mail: Jack Ham@CF.AC.UK 0303-7207/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S0303-7207 ( 96 ) 03987-1