Adenovirus-mediated p16 gene transfer prevents drug-induced cell death through G₁ arrest in human glioma cells

This study examined the effects of full-length p16 gene transfer by recombinant adenovirus on cell growth and on sensitivity to CDDP or ACNU chemotherapies. We developed a recombinant adenovirus expressing the full- length human p16 gene (AxCA-hp16) by the COS-TPC method. AxCA-hp16 was infected into the p16-null human glioma cell line, U251MG. AxCA-hp16 infection inhibited proliferation of U251MG cells. A proliferation assay employing MTT showed that AxCA-hp16 infection induced chemoresistance, preventing CDDP-induced cell death (11- to 15-fold) and ACNU-induced cell death (80- to 92-fold). In the absence of AxCA-hp16, cell death was induced with CDDP or ACNU at 3 to 5 days after treatment, as demonstrated by Trypan- blue exclusion. Flow-cytometric analysis showed that CDDP or ACNU arrested cells in the G₂ phase on day 1 and that cells re-entered the cycle on day 3. However, the cells infected with AxCA-hp 16 after CDDP or ACNU treatment showed G ₁ arrest on day 5 after re-entering the cycle from G₂ arrest on day 3. The cells infected with ÅxCA-hp16 before CDDP or ACNU treatment showed G₁ arrest over the 5 days after the infection. This study demonstrated that G₁ arrest induced with p16-gene expression prevents ACNU- or CDDP-induced cell death. The cell death induced by ACNU and CDDP therefore appears to occur in the phase after the G₁/S check point.