Transfection of an inducible p16/CDKN2A construct mediates reversible growth inhibition and G(1) arrest in the AtT20 pituitary tumor cell line

Recent studies have shown that methylation of the CPG island within the p16 /CDKN2A gene is associated with an absence of p16 protein in human pituitar y tumors. However, the effect of restoration of p16 protein expression in t his tumor type has not been investigated. In the absence of an available human pituitary cell line we first assessed the suitability of the mouse corticotroph cell line AtT20 as a model system . Initial experiments showed that the p16/CDKN2A gene was not expressed, wh ereas a transcript for RSI was detected as assessed by RT-PCR. Further stud ies showed the p16/CDKN2A gene to be homozygously deleted, the absence of p 16/CDKN2A and presence of RB1, the downstream effector of p16-mediated cell cycle arrest confirmed the suitability of the AtT20 cell line as a model s ystem. Stable transfectants were generated in which p16/CDKN2A is regulated by an inducible promoter. The regulatory effects of p16/CDKN2A expression on cell proliferation were assessed and complemented by fluorescence-activa ted cell sorting (FACS) analysis of cell cycle profile. Induced expression of p16/CDKN2A resulted in a profound inhibition of cell growth and G(1) arr est (80-82%). Western blot analysis showed concomitant expression of p16 pr otein in arrested cells and a shift in the phosphorylation status of pRB to ward its hypophosphorylated form. To further confirm that expression of plG /CDKN2A mimicked its in vivo role, reversibility was assessed using alterna te cycles in the presence and absence of inducer (isopropyl-1-thio-beta-D-g alactopyranoside). Over three cycles the absence of induced expression of p 16/CDKN2A resulted in release from G(1) arrest. These results show that, in a pituitary cell line model, restoration of pie expression is indeed sufficient to arrest cells in G(1) and inhibit cell p roliferation and is reversible. Thus restoration of p16 expression through novel strategies, including gene therapy or demethylating agents, may offer successful therapeutic intervention in human forms of this disease.