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Open AccessArticle

The Cell-Cycle Regulatory Protein p21CIP1/WAF1 Is Required for Cytolethal Distending Toxin (Cdt)-Induced Apoptosis

1
Department of Pathology, University of Pennsylvania School of Dental Medicine, Philadelphia, PA 19104, USA
2
Department of Medicine, University of California at Davis School of Medicine, Sacramento, CA 95616, USA
3
Department of Biochemistry, University of Pennsylvania School of Dental Medicine, Philadelphia, PA 19104, USA
*
Author to whom correspondence should be addressed.
Pathogens 2020, 9(1), 38; https://doi.org/10.3390/pathogens9010038
Received: 14 November 2019 / Revised: 22 December 2019 / Accepted: 28 December 2019 / Published: 2 January 2020
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 p21CIP1/WAF1 is critical to Cdt-induced apoptosis. Cdt induces increases in the levels of p21CIP1/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 p21CIP1/WAF1 were accompanied by a significant decline in the levels of phosphorylated p21CIP1/WAF1. The significance of Cdt-induced p21CIP1/WAF1 increase was assessed by preventing these changes with a two-pronged approach; pre-incubation with the novel p21CIP1/WAF1 inhibitor, UC2288, and development of a p21CIP1/WAF1-deficient cell line (Jurkatp21−) using clustered regularly interspaced short palindromic repeats (CRISPR)/cas9 gene editing. UC2288 blocked toxin-induced increases in p21CIP1/WAF1, and JurkatWT cells treated with this inhibitor exhibited reduced susceptibility to Cdt-induced apoptosis. Likewise, Jurkatp21− cells failed to undergo toxin-induced apoptosis. The linkage between Cdt, p21CIP1/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 p21CIP1/WAF1 as these changes were not observed in Jurkatp21− cells. Finally, we determined that the p21CIP1/WAF1 increases were dependent upon toxin-induced increases in the level and activity of the chaperone heat shock protein (HSP) 90. We propose that p21CIP1/WAF1 plays a key pro-apoptotic role in mediating Cdt-induced toxicity. View Full-Text
Keywords: Aggregatibacter actinomycetemcomitans; cytolethal distending toxin; lymphocytes; apoptosis; virulence Aggregatibacter actinomycetemcomitans; cytolethal distending toxin; lymphocytes; apoptosis; virulence
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Shenker, B.J.; Walker, L.M.; Zekavat, A.; Weiss, R.H.; Boesze-Battaglia, K. The Cell-Cycle Regulatory Protein p21CIP1/WAF1 Is Required for Cytolethal Distending Toxin (Cdt)-Induced Apoptosis. Pathogens 2020, 9, 38.

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