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

We sought to characterize the pathway by which the multifunctional cytokine transforming growth factor-beta (TGF-beta) inhibits the proliferation of n ormal astrocytes, and we analyzed the alterations in the TGF-beta pathway i n 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 astroc ytes revealed that the expression of the cyclin-dependent kinase inhibitor (CdkI) p15(INK4B) was significantly up-regulated upon TGF-beta treatment wi thout a change in other CdkI levels. The retinoblastoma protein (Rb) became hypophosphorylated, and Cdk2 activity decreased. Analysis of Smad3 null mo use astrocytes showed a significant loss of both TGF-beta-mediated growth i nhibition and p15(INK4B) induction compared with wild-type mouse astrocytes . Infection of rat astrocytes by SMAD3 and SMAD4 adenoviruses failed to ind uce increased expression of p15(INK4B), implying indirect transcriptional r egulation of p15(INK4B) by SMAD3, High-grade human gliomas secrete TGF-beta , yet are resistant to its growth inhibitory effects. Analysis of the effec ts of TGF-beta on 12 human glioma cell lines showed that TGF-beta mildly in hibited the growth of six lines, had no effect on four lines, and stimulate d the growth of two lines. The majority of glioma lines had homozygous dele tions of the p15(INK4B) gene, except for two lines that expressed p15(INK4B ) protein, which was induced further upon TGF-beta treatment. Three lines m ildly 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 inc reased malignant behavior. This suggests that the loss of p15(INK4B) may ex plain, in part, the selective loss of growth inhibition by TGF-beta in glio mas to form a more aggressive tumor phenotype.