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

• Main Authors: Bruce J. Shenker, Lisa M. Walker, Ali Zekavat, Robert H. Weiss, Kathleen Boesze-Battaglia
• Format: Article
• Language: English
• Published: MDPI AG 2020-01-01
• Series: Pathogens
• Subjects: <i>aggregatibacter actinomycetemcomitans</i>; cytolethal distending toxin; lymphocytes; apoptosis; virulence
• Online Access: https://www.mdpi.com/2076-0817/9/1/38

The <i>Aggregatibacter actinomycetemcomitans</i> 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&#8217;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&#8722;) 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&#8722; 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&#8722; 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.