title: Transforming growth factor-beta-mediated p15(INK4B) induction and growth inhibition in astrocytes is SMAD3-dependent and a pathway prominently altered in human glioma cell lines.
abstract: We sought to characterize the pathway by which the multifunctional cytokine transforming growth factor-beta (TGF-beta) inhibits the proliferation of normal astrocytes, and we analyzed the alterations in the TGF-beta pathway in human glioma cell lines. Upon TGF-beta treatment, primary rat astrocytes showed a significant decrease in DNA synthesis upon thymidine incorporation with a cell cycle arrest in the G(1) phase. Western analysis of the astrocytes revealed that the expression of the cyclin-dependent kinase inhibitor (CdkI) p15(INK4B) was significantly up-regulated upon TGF-beta treatment without a change in other CdkI levels. The retinoblastoma protein (Rb) became hypophosphorylated, and Cdk2 activity decreased. Analysis of Smad3 null mouse astrocytes showed a significant loss of both TGF-beta-mediated growth inhibition and p15(INK4B) induction compared with wild-type mouse astrocytes. Infection of rat astrocytes by SMAD3 and SMAD4 adenoviruses failed to induce increased expression of p15(INK4B), implying indirect transcriptional regulation of p15(INK4B) by SMAD3. High-grade human gliomas secrete TGF-beta, yet are resistant to its growth inhibitory effects. Analysis of the effects of TGF-beta on 12 human glioma cell lines showed that TGF-beta mildly inhibited the growth of six lines, had no effect on four lines, and stimulated the growth of two lines. The majority of glioma lines had homozygous deletions of the p15(INK4B) gene, except for two lines that expressed p15(INK4B) protein, which was induced further upon TGF-beta treatment. Three lines mildly induced CdkI p21(WAF1) expression in response to TGF-beta. Most tumor lines retained other TGF-beta-mediated responses, including extracellular matrix protein and angiogenic factor secretion, which may contribute to increased malignant behavior. This suggests that the loss of p15(INK4B) may explain, in part, the selective loss of growth inhibition by TGF-beta in gliomas to form a more aggressive tumor phenotype.
authors.affiliation: Division of Neurology, Duke University Medical Center, Durham, North Carolina 27710, USA. rich0001@mc.duke.edu
authors.forename: J N
authors.initials: JN
authors.lastname: Rich
authors.forename: M
authors.initials: M
authors.lastname: Zhang
authors.forename: M B
authors.initials: MB
authors.lastname: Datto
authors.forename: D D
authors.initials: DD
authors.lastname: Bigner
authors.forename: X F
authors.initials: XF
authors.lastname: Wang
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D015139:Blotting, Southern; D015153:Blotting, Western; D042846:CDC2-CDC28 Kinases; D002352:Carrier Proteins / Q000378:metabolism*; D018797:Cell Cycle Proteins; D002455:Cell Division / Q000187:drug effects; D051357:Cyclin-Dependent Kinase 2; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050759:Cyclin-Dependent Kinase Inhibitor p21; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D016213:Cyclins / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism* / Q000502:physiology; D005434:Flow Cytometry; D016193:G1 Phase / Q000187:drug effects; D005910:Glioma / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D051379:Mice; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction; D051900:Smad3 Protein; D015534:Trans-Activators / Q000378:metabolism* / Q000502:physiology; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins
chemical_list: C493956:CDKN1A protein, human; C493966:CDKN2B protein, human; D002352:Carrier Proteins; C493957:Cdkn1a protein, mouse; C493958:Cdkn1a protein, rat; C493968:Cdkn2b protein, mouse; 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; D004268:DNA-Binding Proteins; C497790:SMAD3 protein, human; D051900:Smad3 Protein; C497791:Smad3 protein, mouse; C497787:Smad3 protein, rat; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D025521:Tumor Suppressor Proteins; D011494:Protein Kinases; C102333:histone H1 kinase; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; C495893:Cdk2 protein, mouse; C495892:Cdk2 protein, rat; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases