Decreased proliferation and cell cycle arrest in neoplastic rat pituitary cells is associated with transforming growth factor-beta 1-induced expression of p15/INK4B

Transforming growth factor beta (TGF-beta) is a member of a family of cytok ines that regulate differentiation and proliferation in a wide variety of t issues including the pituitary gland. In both the normal pituitary and tumo rous cell lines TGF-beta1 has anti-proliferative activity, however the intr acellular mechanisms responsible have not been defined. In the pituitary de rived cell line GH(3), p27(Kip1), a key regulator of G(1)/S transition is n ot expressed, suggesting that this protein is not an effector of the anti-p roliferative response following TGF-beta1 treatment. Among other TGF-beta r esponsive cell cycle regulators p15(Ink4b) has been shown to have anti-prol iferative effects associated with cell cycle arrest in other cell types. We therefore examined p15(Ink4b) expression in response to TGF beta -1 to det ermine if this cyclin dependent kinase inhibitor was responsible For anti-p roliferative activity in GH(3) cells. Treatment of GH(3) cells with TGF-bet a1 (0.5-30 ng/ml) showed significant dose dependent growth inhibition (P < 0.001) as assessed by viable cell counts. Maximum growth inhibition (66%) w as observed following treatment with 2 ng/ml TGF-beta1. FAGS analysis carri ed out in parallel with the growth studies showed treatment was associated with a decrease in the proportion of cells in S-phase (22-9%) and a signifi cant increase in the G(1) fraction from 58 to 75% relative to controls (P < 0.001), The absence of a sub G(1) fraction and reversibility of the G(1) a rrest over three cycles showed that these changes were not due to either an apoptotic response or cytoxicity, respectively. Semi-quantitative RT-PCR a nd Western blot analysis showed no change in the expression level of cyclin dependent kinase 4 (CDK4), p16(Ink4a) or p21(Cip1). However, p15(Ink4b) mR NA and protein levels showed a 10 acid 8 -fold induction, respectively. Inc reased levels of p15(Ink4b) were accompanied by a shift in the phosphorylat ion status of pRb toward its active hypophosphorylated form. Furthermore, s tudies of the kinetics of p15(Ink4b) induction showed that arrest of cells in G(1) is preceded by induction of p15(Ink4b) mRNA and protein. These inve stigations would suggest that p15(Ink4b) is a functional effector of TGF-be ta1 mediated cell cycle arrest in GH(3) cells. However, our present studies cannot determine if it is the sole mediator. Identification of intracellul ar target(s) that mediate responses to anti-proliferative signals will incr ease our understanding of these pathways and aberrations responsible for th eir dysfunction in tumorigenesis. (C) 2001 Elsevier Science Ireland Ltd. Al l rights reserved.