Inhibition of the Glioblastoma Cell Cycle by Type I IFNs Occurs at Both the G1 and S phases and Correlates With the Upregulation of P21WAF1/CIP1

Abstract

The antiproliferative effect of IFNα was tested on the human glioblastoma cell lines, U-373MG and T98G. IFNα significantly inhibited the growth of both cell lines, but was more effective in retarding the growth of U-373MG cells. Flow cytometry analysis indicated that synchronized IFNα-treated U-373MG cells showed a strong block in the progression of cells out of the S phase of the cell cycle. T98G cells, on the other hand, showed a moderate delay in the transition of cells from G₁ to S phase and only a slight delay in the S phase, consistent with the decreased antiproliferative effect of IFNα on this cell line. IFNα-treated cells were then tested for the induction of the tumor suppressor gene product, p21^WAF1/CIP1. Higher levels of p21^WAF1/CIP1 were detected in lysates from IFNα-treated U-373MG cells as compared to media controls for as long as 18 h. In IFNα-treated T98G cells, p21^WAF1/CIP1 levels were slightly elevated at 4 and 6 h, but decreased to levels similar to controls thereafter, correlating with the antiproliferative effects of IFNα on each cell line. Immunoprecipitation studies on lysates from IFNα-treated U-373MG and T98G cells indicated that increased amounts of p21^WAF1/CIP1 were complexed to both cyclin D1 and cyclin E. Further, reduced cyclin-dependent kinase 2 (cdk2) activity was found in both IFNα-treated U-373MG and T98G cells, suggesting a mechanism by which p21^WAF1/CIP1 exerted its antiproliferative effects. Lastly, we analyzed the time-dependent production of the cyclins Dl, E, and A. No differences in cyclin D1 levels were found between IFNα-treated and media-treated U-373MG and T98G cells. However, both IFNα-treated U-373MG and T98G cells showed a prolonged elevation in cyclin E, correlating with the G₁ to S phase delays observed in these cell lines. Further, the duration of cyclin E production corresponded with the magnitude of the cell cycle delays seen in IFNα-treated U-373MG and T98G cells. Prolonged elevation of cyclin A was also seen in both IFNα-treated U-373MG and T98G cells, the magnitude of which correlated with the S phase delay observed in these cell lines. Thus, the data indicate that IFNα has significant antiproliferative activity against glioblastoma cells that is mediated, at least in part, by the tumor suppressor gene product, p21^WAF1/CIP1.