| Summary: | 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<sup>CIP1/WAF1</sup> is critical to Cdt-induced apoptosis. Cdt induces increases in the levels of p21<sup>CIP1/WAF1</sup> 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<sup>CIP1/WAF1</sup> were accompanied by a significant decline in the levels of phosphorylated p21<sup>CIP1/WAF1</sup>. The significance of Cdt-induced p21<sup>CIP1/WAF1</sup> increase was assessed by preventing these changes with a two-pronged approach; pre-incubation with the novel p21<sup>CIP1/WAF1</sup> inhibitor, UC2288, and development of a p21<sup>CIP1/WAF1</sup>-deficient cell line (Jurkat<sup>p21−</sup>) using clustered regularly interspaced short palindromic repeats (CRISPR)/cas9 gene editing. UC2288 blocked toxin-induced increases in p21<sup>CIP1/WAF1</sup>, and Jurkat<sup>WT</sup> cells treated with this inhibitor exhibited reduced susceptibility to Cdt-induced apoptosis. Likewise, Jurkat<sup>p21−</sup> cells failed to undergo toxin-induced apoptosis. The linkage between Cdt, p21<sup>CIP1/WAF1</sup>, 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<sup>CIP1/WAF1</sup> as these changes were not observed in Jurkat<sup>p21−</sup> cells. Finally, we determined that the p21<sup>CIP1/WAF1</sup> increases were dependent upon toxin-induced increases in the level and activity of the chaperone heat shock protein (HSP) 90. We propose that p21<sup>CIP1/WAF1</sup> plays a key pro-apoptotic role in mediating Cdt-induced toxicity.
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