Attenuation of WAF1/Cip1 expression by an antisense adenovirus expression vector sensitizes glioblastoma cells to apoptosis induced by chemotherapeutic agents 1,3-bis(2-chloroethyl)-1-nitrosourea and cisplatin.

Abstract

Previous studies have shown that the negative cell cycle regulator WAF1/Cip1 is often overexpressed in human gliomas and that WAF1/Cip1 overexpression renders glioma cells resistant to chemotherapy agents. In this study, we investigated whether down-regulation of WAF1/Cip1 would sensitize gliomas to chemotherapy. An adenoviral vector expressing antisense WAF1/Cip1 was constructed and used to infect D54 glioma cells, which express a high level of endogenous WAF1/Cip1. After D54 cells were infected with antisense WAF1/Cip1 adenovirus, Western blotting revealed a significant decrease in the WAF1/Cip1 protein level. Down-regulation of WAF1/Cip1 alone resulted in the cells rounding up and detaching from plates. Electron microscopy revealed some nuclear fragmentation in antisense WAF1/Cip1-infected cells, indicating the initiation of apoptosis. The antisense WAF1/Cip1-infected cells were then treated with the chemotherapeutic agents 1,3-bis(2-chloroethyl)-1-nitrosourea and cisplatin. Other cells were infected with sense WAF1/Cip1 adenovirus or control virus and served as controls. Trypan blue exclusion assay revealed significant cell death in antisense WAF1/Cip1-infected cells. In situ end-labeling assay by flow cytometry revealed that many cells died of apoptosis. Our results show that the attenuation of WAF1/Cip1 expression initiated glioma cell death and sensitized glioma cells to apoptosis induced by 1,3-bis(2-chloroethyl)-1-nitrosourea and cisplatin. Thus, blocking WAF1/Cip1 production may serve as a useful chemosensitization regimen for treating glioma.