Induction of senescence in human malignant glioma cells by p16INK4A.

p16INK4A is a G1-specific cell cycle inhibitor which maps to human chromosome 9p21, a region frequently mutated or deleted in cancer cell lines and primary tumors. In glioblastomas the frequency of homozygous deletions is 40-70% making it one of the most common mutations in this tumor type. We have analysed the significance of the loss of this gene in gliomas by introducing the cDNA for p16INK4A into the human glioma cell line U-1242 MG which has a deleted CDKN2 locus. We used the tetracycline repressible vector system and obtained two stably transfected clones that expressed p16INK4A upon induction. p16INK4A expression caused a G1 arrest and enlargement of the cells similar to that of senescent cells. When staining for Senescence-Associated beta-galactosidase activity, described to be specific for senescent cells, we could show that the enlarged cells specifically gave a positive staining reaction. This senescence phenotype was dependent on the continuous expression of p16INK4A since it was reversed upon reintroduction of tetracycline suppression. Thus, the induced expression of p16INK4A in these glioma cells reverted their immortal phenotype and caused an immediate cellular senescence.