We constructed replication-defective adenoviruses containing CDKIs to investigate mechanistically the anti-proliferative effects of ectopic overexpression of these gene products on human brain tumors and to determine if they could be used therapeutically. Replication-defective (E1-deleted) adenovirus 5 was further modified by the addition of a prokaryotic Tn 10 (tet) promoter system (for robust expression that could be repressed by anhydrous tetracycline) together with multimers of a glial fibrillary acidic protein(GFAP) enhancer element (to enable glial-specific expression). Genes inserted into the adenovirus system included the CDKIs p16, p19, p21, p27 andβGalactosidase (control). Colorimetric assay of adenovirus-βGalatosidase-infected cells demonstrated very high-level expression restricted to malignant astrocytoma lines that was inhibited by tetracycline; no expression was detected in other human tumor lines or in normal cells including neurons. Anti-proliferative effects of over expressed CDKIs were demonstrated by decreased [3H]thymidine incorporation, increased cell division time and G1 cell cycle arrest. There was no detectable effect on apoptosis or differentiation. Overexpressed p21and p27 were associated with decreased CDC2, CDK2, CDK4 and CDK 6 activity. For CDC2, but not the other CDKs, decreased activity was associated with a corresponding decrease in the amount of protein. Overexpression of p21and p27 was associated with a marked decrease in the accumulation of aneuploid cells. Aneuploidy in the astrocytoma cell line tested (U373) occured as the result of a defective spindle checkpoint mechanism: mitotically arrested cells cycle (manifested by phase-specific CDK activity) exhibited ongoing DNA synthesis in the absence of chromosome segregation. p27 inhibited aneuploid accumulation by blocking DNA replication via induction of G1 arrest. Finally, decreased malignant potential of glioma cells was manifested by the loss of anchorage-independent growth in soft agar and the failure to induce tumorigenesis in both subcutaneous and intracerebral xenografts using a stereotactic frame and detection of tumor formation by MRI. This pre-clinical investigation supports the further development of these constructs for human gene therapy studies to treat post-operative microscopic residual disease resistant to conventional chemoradiotherapy.
