G1 arrest by p16INK4A uncouples growth from cell cycle progression in leukemia cells with deregulated cyclin E and c-Myc expression

The cell cycle inhibitor p16INK4A is frequently inactivated in acute lymphoblastic T-cell leukemia (T-ALL). We analyzed mechanisms and consequences of p16INK4A reconstitution in T-ALL cells lacking this tumor suppressor. CCRF-CEM cells with tetracycline-regulated p16INK4A expression underwent stable G1-phase cell cycle arrest for 72 h followed by massive apoptosis. p16INK4A expression caused pRB hypophosphorylation and repression of certain E2F target genes. Interestingly, cyclin E and c-Myc were not affected, suggesting pRB/E2F-independent expression of these E2F targets. Cyclin E/CDK2, however, was inactive due to stabilization and redistribution of p27Kip1 from CDK4/CDK6 to CDK2. Analyses of c-Myc target genes suggested that c-Myc was transcriptionally inactive, which correlated with hypophosphorylation of the c-Myc inhibitor p107. Thus, p16INK4A, although unable to repress the expression of deregulated cyclin E and c-Myc, functionally inactivated these potential oncogenes. p16INK4A-arrested cells showed morphologic changes, induction of T-cell-specific surface markers and repression of telomerase activity, suggesting differentiation. Moreover, p16INK4A reconstitution was associated with increased cellular volume, normal protein synthesis rates and elevated ATP levels. Taken together, p16INK4A reconstitution in p16INK4A-deficient T-ALL cells induced cell cycle arrest in the presence of cyclin E and c-Myc expression, uncoupled growth from cell cycle progression and caused a sequential process of growth, differentiation and apoptosis.