The Cell-Cycle Regulatory Protein p21(CIP1/WAF1) Is Required for Cytolethal Distending Toxin (Cdt)-Induced Apoptosis

The Aggregatibacter actinomycetemcomitans cytolethal distending toxin (Cdt) induces lymphocytes to undergo cell-cycle arrest and apoptosis; toxicity is dependent upon the active Cdt subunit, CdtB. We now demonstrate that p21(CIP1/WAF1) is critical to Cdt-induced apoptosis. Cdt induces increases in the levels of p21(CIP1/WAF1) in lymphoid cell lines, Jurkat and MyLa, and in primary human lymphocytes. These increases were dependent upon CdtB’s ability to function as a phosphatidylinositol (PI) 3,4,5-triphosphate (PIP3) phosphatase. It is noteworthy that Cdt-induced increases in the levels of p21(CIP1/WAF1) were accompanied by a significant decline in the levels of phosphorylated p21(CIP1/WAF1). The significance of Cdt-induced p21(CIP1/WAF1) increase was assessed by preventing these changes with a two-pronged approach; pre-incubation with the novel p21(CIP1/WAF1) inhibitor, UC2288, and development of a p21(CIP1/WAF1)-deficient cell line (Jurkat(p21−)) using clustered regularly interspaced short palindromic repeats (CRISPR)/cas9 gene editing. UC2288 blocked toxin-induced increases in p21(CIP1/WAF1), and Jurkat(WT) cells treated with this inhibitor exhibited reduced susceptibility to Cdt-induced apoptosis. Likewise, Jurkat(p21−) cells failed to undergo toxin-induced apoptosis. The linkage between Cdt, p21(CIP1/WAF1), and apoptosis was further established by demonstrating that Cdt-induced increases in levels of the pro-apoptotic proteins Bid, Bax, and Bak were dependent upon p21(CIP1/WAF1) as these changes were not observed in Jurkat(p21−) cells. Finally, we determined that the p21(CIP1/WAF1) increases were dependent upon toxin-induced increases in the level and activity of the chaperone heat shock protein (HSP) 90. We propose that p21(CIP1/WAF1) plays a key pro-apoptotic role in mediating Cdt-induced toxicity.