Decreased proliferation and cell cycle arrest in neoplastic rat pituitary cells is associated with transforming growth factor-beta1-induced expression of p15/INK4B.
Transforming growth factor beta (TGF-beta) is a member of a family of cytokines that regulate differentiation and proliferation in a wide variety of tissues including the pituitary gland. In both the normal pituitary and tumorous cell lines TGF-beta1 has anti-proliferative activity, however the intracellular mechanisms responsible have not been defined. In the pituitary derived cell line GH(3), p27(Kip1), a key regulator of G(1)/S transition is not expressed, suggesting that this protein is not an effector of the anti-proliferative response following TGF-beta1 treatment. Among other TGF-beta responsive cell cycle regulators p15(Ink4b) has been shown to have anti-proliferative effects associated with cell cycle arrest in other cell types. We therefore examined p15(Ink4b) expression in response to TGFbeta-1 to determine if this cyclin dependent kinase inhibitor was responsible for anti-proliferative activity in GH(3) cells. Treatment of GH(3) cells with TGF-beta1 (0.5-30 ng/ml) showed significant dose dependent growth inhibition (P<0.001) as assessed by viable cell counts. Maximum growth inhibition (66%) was observed following treatment with 2 ng/ml TGF-beta1. FACS analysis carried 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 significant 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) arrest over three cycles showed that these changes were not due to either an apoptotic response or cytoxicity, respectively. Semi-quantitative RT-PCR and Western blot analysis showed no change in the expression level of cyclin dependent kinase 4 (CDK4), p16(Ink4a) or p21(Cip1). However, p15(Ink4b) mRNA and protein levels showed a 10 and 8 -fold induction, respectively. Increased levels of p15(Ink4b) were accompanied by a shift in the phosphorylation status of pRb toward its active hypophosphorylated form. Furthermore, studies 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 investigations would suggest that p15(Ink4b) is a functional effector of TGF-beta1 mediated cell cycle arrest in GH(3) cells. However, our present studies cannot determine if it is the sole mediator. Identification of intracellular target(s) that mediate responses to anti-proliferative signals will increase our understanding of these pathways and aberrations responsible for their dysfunction in tumorigenesis.
Frost
S J
SJ
Centre for Cell and Molecular Medicine, School of Postgraduate Medicine, Keele University, North Staffordshire Hospital, Stoke on Trent, ST4 7QB, UK.
Simpson
D J
DJ
Farrell
W E
WE
D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002453:Cell Cycle / Q000187:drug effects*; D018797:Cell Cycle Proteins; D002455:Cell Division / Q000187:drug effects; D051358:Cyclin-Dependent Kinase 4; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics / Q000378:metabolism; D050759:Cyclin-Dependent Kinase Inhibitor p21; D018844:Cyclin-Dependent Kinases / Q000235:genetics / Q000378:metabolism; D016213:Cyclins / Q000235:genetics / Q000378:metabolism; D016193:G1 Phase / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects*; D007700:Kinetics; D010902:Pituitary Gland / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D053773:Transforming Growth Factor beta1; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins
D002352:Carrier Proteins; C493958:Cdkn1a protein, rat; C493969:Cdkn2b protein, rat; D018797:Cell Cycle Proteins; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; C508962:Tgfb1 protein, rat; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D025521:Tumor Suppressor Proteins; C495903:Cdk4 protein, rat; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases