The Cell-Cycle Regulatory Protein p21CIP1/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 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.

Cdt B internalization leads to irreversible cell-cycle arrest and eventually apoptotic cell death. These toxic effects were originally attributable to CdtB's ability to function as a DNase, thereby causing DNA damage which in turn leads to G2/M arrest and death [9,[15][16][17][18][19][20][21][22][23]. Over the past several years, our studies suggested an alternative paradigm to account for Aggregatibacter actinomycetemcomitans Cdt-mediated toxicity which is based upon a novel molecular mode of action for CdtB. In this regard, we demonstrated that, in addition to exhibiting DNase activity, CdtB is a potent lipid phosphatase capable of converting the signaling lipid phosphatidylinositol (PI)-3,4,5triphosphate (PIP3) to PI-3,4-diphosphate [24][25][26][27][28]. Moreover, our investigations demonstrated that the ability of CdtB to function as a PIP3 phosphatase enables this toxin subunit to intoxicate cells via blockade of the PI-3K signaling pathway. Indeed, we demonstrated that the toxic effects of Cdt on lymphocytes, macrophages, and mast cells results in PI-3K signaling blockade characterized by decreases in PIP3, leading to concomitant reductions in the phosphorylation status of downstream targets: Akt and GSK3β. Additionally, we demonstrated that the induction of both G2/M arrest and apoptosis is dependent upon CdtB-mediated PI-3K blockade.
In order to more accurately define the molecular mechanisms that link CdtB-mediated PI-3K blockade with G2/M arrest and apoptosis, we investigated the role of the cyclin-dependent kinase inhibitor known as CDK-interacting protein 1 (Cip1) and wild-type p53-activated fragment 1 (WAF1) (p21 CIP1/WAF1 ). P21 CIP1/WAF1 was originally identified as a negative regulator of the cell cycle, as well as a tumor suppressor. However, recent studies demonstrated additional functions for p21 CIP1/WAF1 that are associated with regulation of a number of cellular processes including cell differentiation, migration, senescence, and apoptosis [29][30][31][32][33]. Thus, it is not surprising that several investigators demonstrated an association between p21 CIP1/WAF1 expression and exposure to Cdt [16,[34][35][36][37]. It should be noted, however, that these studies did not provide any information as to whether the p21 CIP1/WAF1 levels were mechanistically linked to and/or required for Cdt toxicity. In this study, we investigated the relationship between lymphocyte exposure to A. actinomycetemcomitans Cdt, altered p21 CIP1/WAF1 levels, and induction of toxicity. We now report that Cdt-treated human lymphocytes exhibit dose-dependent increases in levels of p21 CIP1/WAF1 and the chaperone HSP90 within 4-16 h of exposure to the toxin. To study the biologic consequence of these increases, we employed a twopronged approach to modify the ability of Cdt to alter expression of p21 CIP1/WAF1 : gene editing and pharmacologic intervention. Additionally, these interventions were assessed for their ability to alter cell susceptibility to Cdt toxicity. Our results indicate a requisite role for p21 CIP1/WAF1 in Cdt-induced apoptosis.

Cdt Induces Elevations in Lymphocyte Levels of p21 CIP1/WAF1
Cdt derived from A. actinomycetemcomitans, Haemophilus ducreyi, and Helicobacter hepaticus were shown to induce increases in p21 CIP1/WAF1 within 24-48 h in several cell lines including fibroblasts, lymphocytes, enterocytes, and hepatocytes [16,[34][35][36][37][38]. Likewise, we now demonstrate that A. actinomyetemcomitans Cdt induces increases in p21 CIP1/WAF1 levels in Jurkat cells in a time-and dosedependent manner. Jurkat cells were treated with varying amounts of Cdt (0-400 pg/mL) for 4, 8, and 16 h and then analyzed by Western blot to assess total p21 CIP1/WAF1 levels ( Figure 1A,B). Analysis indicates that a small, but consistent, increase in p21 CIP1/WAF1 was detected within 4 h in cells exposed to the highest concentration of Cdt (400 pg/mL). Following an 8-h exposure, significant increases of nine-and 18-fold were observed in cells exposed to 100 and 400 pg/mL Cdt, respectively. After a 16-h exposure, the relative levels of p21 CIP1/WAF1 remained elevated; cells treated with 25, 100, and 400 pg/mL Cdt exhibited three-, six-, and seven-fold increases, respectively. Figure 1. Effect of cytolethal distending toxin (Cdt) on p21 CIP1/WAF1 levels in Jurkat cells. Jurkat cells were exposed to 0-400 pg/mL Cdt for 4, 8, and 16 h; cells were then harvested, and extracts were fractionated by SDS-PAGE and analyzed by Western blot for the presence of p21 CIP1/WAF1 . Panel (A) shows representative Western blots of p21 CIP1/WAF1 for cells treated with each dose of toxin at 4, 8, and 16 h; glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as a gel loading control. Panel (B) shows the results from multiple blots which were analyzed by digital densitometry; the value of relative intensity obtained from digital densitometry is presented as the mean ± standard error of the mean (SEM) of four experiments. Panel (C) shows the relative levels of pp21 CIP1/WAF1 in cells treated with 50 pg/mL Cdt or 0.5 μM GSK690693 for 16 h expressed as a percentage observed in control (medium only) cells. A representative blot is shown along with compiled results from three experiments: results are expressed as the percentage (mean ± SEM) of pp21 CIP1/WAF1 observed in control cells; * indicates statistical significance (p < 0.05) when compared to untreated cells.
To verify that the effects of Cdt on p21 CIP1/WAF1 levels were not unique to the Jurkat cell line, the cutaneous T-cell lymphoma line, MyLa, was also assessed for altered p21 CIP1/WAF1 levels when exposed to the same doses of Cdt. As shown in Figure 2A,B, MyLa cells treated with 0-400 pg/mL Cdt for 16 h exhibited significant increases in p21 CIP1/WAF1 levels: 4.5-(100 pg/mL Cdt) and 5.7-fold (400 pg/mL Cdt) over control levels. In addition to lymphoid cell lines, p21 CIP1/WAF1 levels were assessed in primary human lymphocytes (HPBMCs). As shown in Figure 2A,B, exposure to 25 pg/mL Cdt resulted in a detectable but not statistically significant increase in p21 CIP1/WAF1 . Significant increases in p21 CIP1/WAF1 levels were observed in the presence of 100 and 400 pg/mL Cdt leading to 361% ± 82% and 673% ± 185% over that observed in untreated control cells. Figure 2. Effect of Cdt on p21 CIP1/WAF1 levels in primary human lymphocytes (HPBMCs) and MyLa cells. HPBMC and MyLa cells were treated with 0-400 pg/mL Cdt for 16 h. The cells were harvested, and extracts were fractionated by SDS-PAGE and analyzed by Western blot for the presence of p21 CIP1/WAF1 . Panel (A) shows representative Western blots, and panel (B) shows results from four experiments assessed by digital densitometry; results (intensity) are expressed as the mean ± SEM. Panel (C) shows the effect of 50 pg/mL of Cdt containing wild-type CdtB (CdtB WT ) or CdtB containing mutations (CdtB R117A , CdtB R144A , and CdtB A163R ) on p21 CIP1/WAF1 levels in MyLa cells. A representative Western blot is shown, as well as results from four experiments that were analyzed by digital densitometry; results are expressed as a percentage of p21 CIP1/WAF1 levels observed in control (untreated) cells; * indicates statistical significance (p < 0.05) when compared to untreated cells.
As noted earlier, we previously demonstrated that Cdt toxicity in lymphocytes (both primary and cell lines), macrophages, and mast cells is dependent upon PIP3 phosphatase activity exhibited by the active Cdt subunit, CdtB [24][25][26][27][28]. We generated and previously reported on the characterization of the enzymatic and toxic activities of Cdts containing targeted mutations within the CdtB subunit [24]. In each instance, we observed that the retention of lipid phosphatase activity, and not DNase activity, was a requisite for both Cdt-induced cell-cycle arrest and apoptosis in lymphocytes, as well as the induction of pro-inflammatory responses in macrophages. Therefore, we next determined the requirement for PIP3 phosphatase activity in Cdt-induced increases in the levels of p21 CIP1/WAF1 . Cdt containing CdtB mutant proteins that were previously described were employed [24]: CdtB A163R retains lipid phosphatase activity, lacks DNase activity, and is toxic; CdtB R144A exhibits low lipid phosphatase activity, exhibits increased DNase activity and is not toxic; CdtB R117A exhibits low lipid phosphatase activity, retains DNase activity, and is not toxic. As shown in Figure 2C, elevations in p21 CIP1/WAF1 levels were only observed when MyLa cells were treated with toxin containing the active wild-type subunit, CdtB WT (2.5-fold increase), or the CdtB mutant, CdtB A163R (3.2-fold increase) [24]. Cells exposed to CdtB mutant proteins that we previously demonstrated to be deficient in phosphatase activity and lack toxicity (CdtB R144A and CdtB R117A ) did not exhibit significant changes in levels of p21 CIP1/WAF1 : 1.2-fold and 1.7 fold, respectively. It is noteworthy that we previously reported that A. actinomycetemcomitans Cdt-induced cell-cycle arrest and apoptosis in human lymphocytes does not involve activation of the DNA damage response (DDR) [24,39]. These observations were confirmed as we now demonstrate that Cdt containing CdtB WT , as well as the other CdtB mutant proteins employed in this study, does not induce phosphorylation of the histone H2AX, a commonly employed indicator of DDR activation due to DNA damage ( Figure S1, Supplementary Materials).
The biologic activity of p21 CIP1/WAF1 is governed by post-translational modifications such as phosphorylation; therefore, we also assessed Cdt-treated Jurkat cells for changes in phosphorylation status (T145; (pp21 CIP1/WAF1 )) ( Figure 1C). Baseline levels of p21 CIP1/WAF1 were very low, but detectable; as described above, these levels increased at 8-16 h in the presence of Cdt (see above). In contrast, the relative amount present as phosphorylated p21 CIP1/WAF1 (pp21 CIP1/WAF1 ) significantly declined in the presence of Cdt. Cells treated with 50 pg/mL Cdt for 16 h exhibited a reduction in the amount of pp21 CIP1/WAF1 to 20.6% ± 3.4% of the amount observed in untreated control cells. Interestingly, the PI-3K signaling pathway exhibits cross-talk with other regulatory pathways; specifically, p21 CIP1/WAF1 was shown to be a downstream target of activated Akt (pAkt) [40]. Furthermore, we showed that Cdt-treated cells exhibit reduced levels of pAkt (reduced kinase activity) [24]. These observations, along with our current findings that toxin-treated cells also exhibit reduced levels of pp21 CIP1/WAF1 , are consistent with the proposed molecular mode of action for CdtB which involves PIP3 phosphatase activity leading to PI-3K signaling blockade. To further support our findings and provide additional "proof-of-principle" evidence for a relationship between pAkt and p21 CIP1/WAF1 , we employed GSK690693, an Akt inhibitor [41]. Jurkat cells treated for 16 h with 0.5 μM GSK690693, exhibited a reduction in pp21 CIP1/WAF1 to 0.7% ± 0.3% of that observed with untreated cells ( Figure 1C).

Blockade of Cdt-Induced Increases in p21 CIP1/WAF1 Results in Reduced Jurkat Cell Susceptibility to Cdt Toxicity
We next extended our investigation to address the biological significance of Cdt-induced increases in p21 CIP1/WAF1 by utilizing a two-pronged approach: pharmacologic modulation and altered expression using gene editing. The novel inhibitor of p21 CIP1/WAF1 , UC2288, was demonstrated to reduce p21 CIP1/WAF1 levels [42]; therefore, we firstly employed UC2288 as a potential inhibitor of Cdt toxicity by virtue of its ability to block increases in p21 CIP1/WAF1 . As shown in Figure 3A, pre-treatment of Jurkat WT cells with UC2288 (2.5-10 μM) resulted in reduced Cdt-induced apoptosis in the presence of the highest concentration of the inhibitor (10 μM); interestingly, this is the same concentration reported to be effective in other studies [42]. Untreated control cells exhibited 7.7% ± 1.1% terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL)-positive cells and, in the presence of 50 pg/mL Cdt, the percentage of TUNEL-positive cells increased to 35.0% ± 1.9%. Pretreatment of cells with UC2288 reduced the percentage of TUNEL-positive cells to 19.7% ± 1.4%. Two inactive analogues of UC2288 were employed: UC1770 and UC1472; neither of these altered Cdtinduced apoptosis, as the percentage of TUNEL-positive cell was not reduced below 31% when cells were treated with equivalent concentrations of these analogues. The ability of UC2288 to block Cdtinduced increases in p21 CIP1/WAF1 levels was also confirmed ( Figure 3B). Results are expressed as a percentage of p21 CIP1/WAF1 levels observed in cells exposed to toxin alone (100%); the latter represents almost a five-fold increase over control cells (medium only). Pre-treatment with 10 μM UC2288 resulted in a significant reduction of p21 CIP1/WAF1 levels to 24.7% of that observed with cells treated with toxin alone. Effect of UC2288 on Cdt-induced apoptosis and on p21 CIP1/WAF1 levels. Jurkat WT cells were pre-incubated with 0-10 μM UC2288 or its inactive analogues UC1770 and UC1472 for 30 min. Cdt (50 pg/mL) was added to the cell cultures, and the cells were harvested 24 h later and analyzed for apoptosis using the TUNEL assay as described (panel (A)). Results are expressed as a percentage of apoptotic cells versus drug concentration; the mean ± SEM for four experiments is plotted. Panel (B) shows the effect of UC2288 on Cdt-induced p21 CIP1/WAF1 levels at 16 h. A representative blot is shown along with results from four experiments. Western blots were analyzed by digital densitometry; the data are expressed as a percentage of the p21 CIP1/WAF1 levels observed in cells treated with Cdt alone; * indicates statistical significance (p < 0.05) when compared to untreated cells.
To confirm our observations with UC2288 and to better understand the biological significance of Cdt-induced elevations in p21 CIP1/WAF1 levels, we utilized CRISPR/Cas9 gene editing to establish a stable Jurkat cell line deficient in p21 CIP1/WAF1 expression (Jurkat p21− ). As shown in Figure 4A, Jurkat p21− cells were unable to express p21 CIP1/WAF1 when challenged with either Cdt or etoposide for 16 h; in contrast, Jurkat WT cells exhibited clear elevations in this protein when challenged with either agent under identical conditions. As noted above, we established that Cdt-induced toxicity is dependent on the CdtB subunit's ability to function as a PIP3 phosphatase, thereby mediating blockade of the PI-3K signaling pathway [26,27]. Therefore, we next verified that Jurkat p21− cells remained susceptible to toxin-induced signaling blockade. Specifically, cells were assessed after 2 and 4 h of exposure to Cdt for changes in the phosphorylation status of two downstream PI-3K signaling targets: Akt and GSK3β. Jurkat WT and Jurkat p21− cells were treated with 50 pg/mL Cdt and then analyzed by Western blot for the presence of Akt, pAkt, GSK3β, and pGSK3β at 0, 2, and 4 h. Figure 4B shows representative immunoblots indicating that the levels of both pAkt and pGSK3β were reduced at 2 and 4 h in both Jurkat WT and Jurkat p21− ; in contrast, the total amount of these proteins (Akt and GSK3β) remained unchanged. In previous studies, we reported that decreases in pAkt and pGSK3β within Jurkat WT cells were statistically significant with levels of pAkt reduced to 48.7% ± 9.3% (2 h) and 45.5% ± 11.6% (4 h) relative to untreated control cells; likewise, pGSK3β levels were reduced to 55.7% ± 6.0% and 47.6% ± 6.1% of control values at 2 and 4 h, respectively [24]. Figure 4C shows similar compiled results from multiple experiments for Jurkat p21− cells. Akt and GSK3β levels remained relatively constant for the 4-h period. In contrast, pAkt levels were reduced to 56.4% ± 19% (2 h) and to 34.8% ± 16.7% (4 h); pGSK3β levels were reduced to 59.7% ± 22.7% (2 h) and 49.5% ± 21.7% (4 h). Jurkat WT and Jurkat p21− cells were next assessed and compared for their susceptibility to Cdtinduced apoptosis using the TUNEL assay ( Figure 4D) following a 24-h treatment with toxin. Consistent with our previous findings, Jurkat WT cells exhibit dose-dependent apoptosis; the percentages of TUNEL-positive were 4.2% ± 0.9% (0 Cdt), 28.3% ± 1.6% (25 pg/mL Cdt), 47.7% ± 1.7% (100 pg/mL Cdt), and 63.5% ± 0.9% (400 pg/mL Cdt). In contrast, Jurkat p21− cells were resistant to Cdtinduced apoptosis; cells incubated with 0-400 pg/mL toxin exhibited 5.7% ± 1.7%, 8.9% ± 2.6%, 10.5% ± 3.0%, and 12.6% ± 3.0% apoptotic cells. It is noteworthy that Jurkat p21− cells retained the capacity to undergo apoptotic cell death as they remained sensitive to paclitaxel ( Figure S2, Supplementary  Materials).
Previously, we demonstrated that Cdt-induced apoptosis involves the intrinsic apoptotic pathway and, in particular, development of the mitochondrial permeability transition (MPT) [39,43]. Therefore, we assessed and compared Jurkat WT and Jurkat p21− for expression of pro-apoptotic members of the Bcl-2 protein family in response to Cdt. Treatment of Jurkat WT cells with 50 pg/mL Cdt resulted in significant increases in both Bid and Bax at 8 hrs ( Figure 5A); Bid levels increased by 498.3% ± 290.5% relative to control cells and Bax levels increased by 637.0% ± 274.7%. A consistent, but not statistically significant, increase was observed for Bak to 160% ± 38.0%. In comparison, Cdt failed to induce increases in the levels of any of the three pro-apoptotic proteins in Jurkat p21− cells; relative to untreated cells, Bid, Bax, and Bak expression levels were 73.2% ± 7.5%, 94.2% ± 10.5%, and 80.4% ± 20.4%, respectively. Representative blots are shown on top for Bid, Bax, Bak, and GAPDH for Jurkat WT cells exposed to medium and Cdt-treated, as well as for Jurkat p21− cells exposed to medium and Cdt-treated. Western blots were analyzed by digital densitometry; results represent the levels of protein expressed as a percentage of that observed in respective control cells. The mean ± SEM for five experiments is shown; * indicates statistical significance (p < 0.05) when compared to untreated cells. Panels (B-I) show the effect of Cdt (0-400 pg/mL) on the ΔΨm in both Jurkat WT (panels (B-E)) and Jurkat p21− cells (panels (F-I)). ΔΨm was determined using DIOC6 (3), and the percentage of cells exhibiting a reduction the membrane potential was determined using the analytical gate indicated; the numbers represent the mean ± SEM of three experiments, each performed in duplicate.
Cdt was also assessed for its ability to induce the MPT in both Jurkat WT and Jurkat p21− cells by measuring a decline in the mitochondrial transmembrane potential (ΔΨm) using the fluorochrone DIOC6(3) [39,43]. As shown in Figure 5B-E, Jurkat WT cells treated with 0-400 pg/mL Cdt exhibited a dose-dependent increase in the percentage of cells exhibiting a decline in the ΔΨm, with 9% in control cells versus 34.4%, 41.5%, and 45.5% in cells exposed to 100, 200, and 400 pg/mL Cdt. In contrast, Jurkat p21− did not exhibit a significant change in the ΔΨm; 15.3% of control cells exhibited reduced ΔΨm, and Cdt treatment resulted in a slight, but not statistically significant increase to 21.2%, 23.1%, and 23% in the presence of 100, 200, and 400 pg/mL Cdt, respectively ( Figure 5 F-I).

Cdt-Induced Increases in p21 CIP1/WAF1 Levels and Induction of Apoptosis Are Blocked by the Chaperone HSP90.
The intracellular levels of p21 CIP1/WAF1 are controlled transcriptionally, as well as posttranslationally, by the proteasome. Experiments were carried out to account for the mode of Cdtinduced increases in p21 CIP1/WAF1 . We firstly addressed the effect of Cdt on p21 CIP1/WAF1 messenger RNA (mRNA) levels in Jurkat cells. As shown in Figure 6A, p21 CIP1/WAF1 mRNA levels were marginally elevated at high toxin doses within 4 h and declined in the presence of the same doses of Cdt at 16 h; however, these changes were not statistically significant. It should also be noted that A. actinomycetemcomitans Cdt-induced increases in lymphocyte p21 CIP1/WAF1 levels were previously shown to be p53-independent [35]. These findings were confirmed as we demonstrate that Cdtinduced p21 CIP1/WAF1 increases were observed in the presence of the p53 inhibitor, pifithrin-α ( Figure  S3, Supplementary Materials); this inhibitor also failed to block Cdt-induced apoptosis. Similar results were observed with Molt-4 cells (data not shown). On the post-translational side, the chaperone HSP90 was shown to stabilize p21 CIP1/WAF1 and prevent its degradation by the proteasome [44][45][46]. Jurkat cells were assessed for changes in the levels of HSP90 by Western blot following 4 and 8 h of exposure to Cdt ( Figure 6B,C). Untreated cells exhibited detectable levels of HSP90 at both time points. Exposure of Jurkat cells to Cdt for 4 h resulted in 1.5-(25 pg/mL), 2.0-(100 pg/mL), and 1.9fold (400 pg/mL) increases in HSP90 levels. Treatment with the same doses of Cdt for 8 h resulted in increases of 1.6-, 1.5-, and 3.6-fold. The dependence of Cdt-induced increases in HSP90 on CdtBassociated PIP3 phosphatase activity was also assessed ( Figure 6D). Jurkat cells treated with Cdt containing the active CdtB subunits CdtB WT and CdtB A163R exhibited increased HSP90 levels of 5.1fold and 4.6-fold over levels observed in control cells, respectively. The HSP90 levels in cells treated with the PIP3 phosphatase-inactive CdtB units, CdtB R117A and CdtB R144A , did not change.
The relationship between HSP90 and Cdt-induced increases in p21 CIP1/WAF1 levels, as well as the induction of apoptosis, was demonstrated by employing geldanamycin (GM), an inhibitor of HSP90 ATPase activity [47]. As shown in Figure 6E, GM was assessed for its ability to inhibit HSP90 and in turn Cdt-induced apoptosis. Jurkat cells were pre-treated with medium or GM (0-5 μM) for 1 h, followed by the addition of medium or Cdt (50 pg/mL). Cells were assessed for apoptosis (TUNELpositive) 24 h later ( Figure 6E). Cells treated with medium exhibited 6.2% ± 1.8% TUNEL-positive cells; in comparison, cells treated with 50 pg/mL Cdt exhibited 63.7% ± 8.8% TUNEL-positive cells. Pre-treatment of cells with GM followed by the addition of toxin resulted in a dose-dependent reduction in apoptosis. In the presence of 1.25, 2.5, and 5 μM GM, the percentage of TUNEL-positive cells was reduced to 21.6% ± 0.7%, 16.4% ± 1.7%, and 8.7% ± 2.3%. It should be noted that GM alone exhibited low levels of toxicity (net 5-10% over untreated cells). In addition to evaluating the effects of GM on Cdt-induced apoptosis, Jurkat cells were also assessed for p21 CIP1/WAF1 levels by Western blot ( Figure 6F). Untreated Jurkat cells contained marginally detectable levels of p21 CIP1/WAF1 ; cells treated with diluent (DMSO) or GM (2.5 μM) exhibited slight increases in p21 CIP1/WAF1 to 1.2 ± 1.0 and 1.1 ± 0.98, respectively. Cells exposed to Cdt alone exhibited a significant increase in p21 CIP1/WAF1 levels to 2.4 ± 1.0; pretreatment of cells with 2.5 μM GM reduced p21 CIP1/WAF1 to near baseline levels (0.56 ± 0.19).

Functional Significance of Cdt-induced Increases in the Levels of p21 CIP1/WAF1
We now report that A. actinomycetemcomitans Cdt induces intracellular increases of p21 CIP1/WAF1 in human lymphocyte cell lines, as well as primary HPBMCs. These observations are consistent with those of other investigators who also demonstrated similar increases in response to Cdt derived from either A. actinomycetemcomitans or Haemophilus ducreyi [16,18,35,36]. P21 CIP1/WAF1 increases were observed in a murine B-cell hybridoma cell line, fibroblasts, the Hep-2 carcinoma cell line, and a gingival squamous cell carcinoma cell line, Ca9-22. These investigators did not demonstrate the biological significance of the observed Cdt-dependent changes in cellular levels of p21 CIP1/WAF1 . As noted earlier, increases in p21 CIP1/WAF1 levels are typically observed in response to cell stress or following DNA damage. The binding of p21 CIP1/WAF1 to cyclins and their CDK binding partner (e.g., CDK1 or CDK2) results in inhibition of the kinase complex, resulting in cell-cycle arrest (reviewed in References [29,[31][32][33]). It is generally accepted that p21 CIP1/WAF1 regulates both cell-cycle arrest, i.e., checkpoint activation, and apoptosis. Collectively, the actions of p21 CIP1/WAF1 promote cell survival and provide time for cells to undergo DNA repair before completing the cell cycle. However, it is becoming increasingly clear that the role of p21 CIP1/WAF1 in regulating apoptosis is more complex than originally thought [30,48]. For example, several investigators demonstrated that pro-apoptotic agents induce increases in p21 CIP1/WAF1 and, furthermore, over-expression of p21 CIP1/WAF1 enhances apoptosis [49][50][51][52]. Likewise, cells and animals deficient in p21 CIP1/WAF1 expression exhibit reduced susceptibility to apoptotic cell death [53,54].
In the current study, we employed a combination of gene editing (CRISPR/cas9) and pharmacologic intervention to assess the functional consequence of Cdt-induced increases in p21 CIP1/WAF1 . Collectively, our observations indicate that Cdt-induced apoptosis is dependent upon increased levels of p21 CIP1/WAF1 . Jurkat p21− cells failed to exhibit apoptotic death following treatment with Cdt. Nonetheless, Jurkat p21− cells retained their susceptibility to other pro-apoptotic agents such as paclitaxel. Additionally, we employed the novel p21 CIP1/WAF1 inhibitor UC2288 whose structure is based upon sorafenib, another inhibitor of p21 CIP1/WAF1 . UC2288 is a new-generation inhibitor that exhibits greater selectivity with higher potency to attenuate p21 CIP1/WAF1 levels [41,55]. Similar to our findings with Jurkat p21− cells, Jurkat WT cells treated with Cdt in the presence of 10 μM UC2288 also failed to exhibit increased levels of p21 CIP1/WAF1 ; moreover, the drug suppressed Cdt-induced apoptosis. It should also be noted that the effective dose of UC2288 used in this study was identical to that employed in other studies [41,55].
Our results demonstrate a pivotal role for p21 CIP1/WAF1 in Cdt-induced apoptosis ( Figure 7); these observations encompass a non-conventional role for p21 CIP1/WAF1 as it proposes a pro-apoptotic function when increases in this regulatory protein are commonly thought to, at least initially, serve a pro-survival role. It is well established that the pleiotropic effects of p21 CIP1/WAF1 are regulated by posttranscriptional modification. For example, phosphorylation of p21 CIP1/WAF1 is known to alter its function which includes a shift in the balance between its pro-and anti-apoptotic effects [29,31,56,57]. In this regard, p21 CIP1/WAF1 was shown to serve as a downstream target for phosphorylation (pp21 CIP1/WAF1 (T-145)) by pAkt, the active form of Akt [58,59]. Consistent with these findings is our observation that Cdt-induced increases in p21 CIP1/WAF1 were not only associated with the induction of apoptosis but also accompanied by a substantial shift in the distribution of pp21 CIP1/WAF1 levels relative to untreated cells. It is noteworthy that, in addition to inducing p21 CIP1/WAF1 increases, we previously established that treatment of lymphocytes with A. actinomycetemcomitans Cdt results in blockade of the PI-3K signaling cascade and, furthermore, Cdt toxicity is dependent upon perturbation of these early signaling events. Cdt-mediated PI-3K blockade is the result of potent lipid phosphatase activity, specifically PIP3 phosphatase activity, associated with the active Cdt subunit, CdtB [26]. Cells exposed to Cdt exhibit PI-3K signaling blockade within 2 h; this is characterized by PIP3 depletion and reduced Akt activity [24,27,28]. It is important to note that Jurkat p21− cells retained their susceptibility to Cdt-induced PI-3K signaling blockade but were unable to undergo apoptosis due to their inability to produce p21 CIP1/WAF1 . We propose that depletion of pAkt in Cdt-treated cells limits phosphorylation of p21 CIP1/WAF1 (Figure 7); this relationship is further supported by our finding that the Akt inhibitor, GSK690693, also depletes Jurkat WT cells of pp21 CIP1/WAF1 . Collectively, these findings support a critical pro-apoptotic role for non-phosphorylated p21 CIP1/WAF1 in mediating Cdt-induced cell death. Figure 7. Overview of the proposed mechanism via which Cdt induces p21 CIP1/WAF1 -dependent apoptosis. Based upon the data presented in this paper, the proposed pathway of Cdt-induced p21 CIP1/WAF1 -dependent apoptosis is shown with red arrows. The dotted line shows the proposed relationship between Akt and the putative downstream target, p21 CIP1/WAF1 . Finally, the link between increased p21 CIP1/WAF1 levels and the onset of apoptosis was established in experiments in which we assessed the ability of Cdt to alter the expression of pro-apoptotic members of the Bcl-2 family. In previous studies, we, and others, established that Cdt-induced apoptosis involves development of the mitochondrial permeability transition state characterized by a decrease in the transmembrane potential and an increase in production of reactive oxygen species [43,60,61]; these events were followed by activation of the caspase cascade. Of particular relevance to our current study were our previous findings that over-expression of Bcl-2 blocked Cdt-induced apoptosis [39]. In this context, the pro-apoptotic requirement for p21 CIP1/WAF1 in Cdt-induced apoptosis is further supported by our current observation that the toxin induces p21 CIP1/WAF1 -dependent upregulation of the pro-apoptotic proteins Bid, Bax, and Bak. P21 CIP1/WAF1 -dependency was demonstrated by the observation that the levels of these proteins were not altered in Cdt-treated Jurkat p21− cells. Moreover, Jurkat p21− cells also failed to exhibit changes in the ΔΨm as we observed with Cdt-susceptible cells. It is noteworthy that Gogada et al. [62] demonstrated that p21 CIP1/WAF1 played a critical role in circumin-induced apoptosis by altering mitochondrial permeability, thereby facilitating the release of cytochrome c.

Cdt Toxicity Requires HSP90-Dependent Increases in the Levels of p21 CIP1/WAF1
Eukaryotic cell-cycle progression and cell survival are regulated at multiple checkpoints involving a number of critical regulatory proteins. One such regulatory protein is p21 CIP1/WAF1 which functions as a cyclin-dependent kinase inhibitor [29]. Elevated levels of p21 CIP1/WAF1 are critical to its function; typically, p21 CIP1/WAF1 is expressed at low levels under normal growth conditions as an unstable protein with a short half-life. It is well established that p21 CIP1/WAF1 can be upregulated via a number of p53-dependent and -independent mechanisms (reviewed in Reference [31]). Likewise, Cdt-induced p21 CIP1/WAF1 increases were reported to be both p53-dependent and -independent (reviewed in Reference [1]); these findings led others to propose that the role of p53 with respect to Cdt is dependent upon both the specific target cell and the source of toxin. Of particular relevance to this study, Sato et al. [35] demonstrated that A. actinomycetemcomitans Cdt-treated lymphocytes exhibit p53-independent increases in p21 CIP1/WAF1 . We confirmed these observations by demonstrating that A. actinomycetemcomitans Cdt-treated lymphocytes exhibited increases in p21 CIP1/WAF1 and apoptosis when pre-exposed to the p53 inhibitor pifithrin-α.
Stress and/or DNA damage are known to induce increases in p21 CIP1/WAF1 levels as a result of elevated transcription, RNA stability and/or decreased proteasomal degradation (reviewed in References [29,33]). Cdt-induced increases in p21 CIP1/WAF1 were not accompanied by significant changes in mRNA levels. These findings led us to explore the possibility that the observed increases were instead due to protein stabilization and reduced proteasomal degradation. It is in this context that we considered the role of the heat shock protein HSP90 in both Cdt-induced increases in p21 CIP1/WAF1 and toxicity. As noted above, p21 CIP1/WAF1 is typically expressed at low levels as an unstable protein with a short half-life in normally growing cells. HSP90 is known to function as a chaperone that is critical to stabilizing proteins involved in several cellular processes [46]. In particular, HSP90 was shown to be recruited to p21 CIP1/WAF1 where it binds via the WAF1/CIP1 stabilizing protein 39 (WISp39). The complex of HSP90, WISp39, and p21 CIP1/WAF1 was reported to stabilize and protect the cell-cycle inhibitor from proteasomal degradation [44,45].
We extended these observations to Cdt-induced stress in lymphocytes by firstly assessing toxintreated Jurkat cells for changes in both WISp39 and HSP90. Jurkat cells constitutively express WISp39, and we observed that exposure to Cdt did not result in further increases (data not shown). In contrast, Cdt-treated cells exhibited significant increases in HSP90 levels within 4 h, prior to the peak elevation in p21 CIP1/WAF1 . It should also be noted that the ability of CdtB to alter HSP90 levels, as demonstrated for p21 CIP1/WAF1 , was found to be dependent upon PIP3 phosphatase activity, since toxin-containing CdtB subunit mutants that lacked phosphatase activity were unable to induce increases in HSP90 levels. To further investigate the relationship between HSP90, p21 CIP1/WAF1 , and apoptosis, we employed the HSP90 inhibitor, GM. Specifically, our observations demonstrate that cells pre-treated with GM prior to exposure to Cdt prevented toxin induces increases in p21 CIP1/WAF1 ; moreover, these cells exhibited decreased susceptibility to toxin-induced apoptosis, demonstrating a linkage between HSP90, changes in p21 CIP1/WAF1 levels, and toxicity.

Conclusions
In summary, this study advances our understanding of the molecular events leading to A. actinomycetemcomitans Cdt-mediated toxicity in lymphocytes. We previously demonstrated that Cdt toxicity is dependent upon PI-3K signaling blockade in lymphocytes, mast cells, and macrophages [2,[24][25][26]28,63,64]. A key to impairment of this signaling pathway is the ability of the active Cdt subunit, CdtB, to function as a PIP3 phosphatase. Our current observations utilized both pharmacologic and gene editing approaches to demonstrate that toxin-induced apoptosis is also dependent upon increased levels of p21 CIP1/WAF1 . Furthermore, toxin-induced increases in this critical regulatory protein are dependent upon HSP90. Moreover, the timeline for these and previous observations suggest a sequence in which the earliest events involve: Cdt binding to membrane cholesterol via CdtC and CdtB, internalization of CdtB, and depletion of PIP3, leading to a concomitant decrease in pAkt (loss of activity), increased expression/activation of HSP90, increased levels of p21 CIP1/WAF1 , and increased levels of pro-apoptotic Bcl-2 family proteins [28,65]. We propose that the ability of Cdt to impair lymphocyte proliferation and promote cell death therefore compromises the host response to Cdt-producing organisms. Our observations are of particular significance, as Cdts are produced by not only A. actinomycetemcomtans, but also by over 30 γ-and ε-Proteobacteria [1,2]. Therefore, we further propose that the action of this putative virulence factor contributes to the pathogenesis of a range of diseases leading to persistent infection by Cdt-producing pathogens. Our current findings not only contribute to a greater understanding of the molecular events critical to Cdt toxicity, but also provide avenues for developing novel approaches to attenuating the immunoinhibitory effects of the toxin.

Reagents and Antibodies
We previously reported on the construction and expression of the plasmids which contain the cdt genes for the holotoxin (pUCAacdtABC his ), as well as those constructs containing CdtB mutations [10]. The histidine-tagged holotoxin was isolated by nickel affinity chromatography as previously described [66]. All antibodies were obtained from commercial sources as indicated. UC2288 was provided by RH Weiss), GM was purchased from Thermofisher Scientific (Waltham, MA, USA), and GSK690962 was purchased from Cayman Chemical (Ann Arbor, MI, USA).
To generate p21 CIP1/WAF1 -deficient Jurkat cells (Jurkat p21− ), we utilized CRISPR/cas9 technology (Santa Cruz Biotechnology; Santa Cruz, CA, USA) as previously described [68]. Cells were transfected (Amaxa Nucleofector system; Lonza, Basel) with a pool of three plasmids, each encoding the Cas9 nuclease and a p21 CIP1/WAF1 -specific 20-nt guide RNA (gRNA). Cells were co-transfected with a pool of three plasmids each containing a homology-directed DNA repair (HDR) template; this corresponded to sites generated by the p21 CIP1/WAF1 CRISPR/cas9 knockout plasmids. HDR plasmids insert the puromycin resistance gene that facilitates selection of stable knockout cells. Cells were firstly incubated for a five-day period following transfection and then for an additional seven days in incubation in puromycin (5 μg/mL). Limiting dilution of surviving cells was utilized to clone cells; clones were expanded and assessed by Western blot analysis for both the presence of p21 CIP1/WAF1 their ability to increase p21 CIP1/WAF1 levels in response to Cdt or etoposide. Clones determined to be deficient in p21 CIP1/WAF1 were then cloned a second time using limiting dilution ( Figure 4A). Jurkat p21− cell lines were maintained in medium containing puromycin (1 μg/mL); experiments were conducted in medium without puromycin.

Assessment of Apoptosis
Jurkat WT and Jurkat p21− cells were challenged with Cdt or medium (control) for 24 h, and apoptosis was assessed by measuring DNA fragmentation (In Situ Cell Death Detection Kit; Sigma Aldrich, St. Louis, MI, USA) [43]. Briefly, after 24 h of incubation, the cells were re-suspended in freshly prepared 4% formaldehyde and permeabilized with 0.1% Triton X-100 for 2 min at 4 °C; then, they were washed and stained with a solution containing FITC-labeled nucleotide and terminal deoxynucleotidyl transferase (TdT). Flow cytometry was employed to measure FITC fluorescence with a laser at 488 nm to excite the fluorochrome; fluorescence emission was measured through a 530/30-nm bandpass filter.
To measure development of the permeability phase transition, Jurkat cells were incubated for 18 h in medium alone or containing Cdt under conditions described above. Changes in the transmembrane potential (ΔΨm) were determined using 4 nM 3,3′-dihexyloxacarbocyanine (DIOC6(3); Thermofisher) [39,43]. Cells were stained for 15 min (37 °C) with the fluorochrome and fluorescence measured following excitation with a laser at 488 nm (250 mW), and emission was monitored through a 530/30-nm bandpass filter; at least 10,000 cells were analyzed per sample.

Statistical Analysis
The mean ± standard error of the mean was calculated for replicate experiments. Significance was determined using a Student's t-test using SigmaPlot Software (Systat; San Jose, CA); a p-value of less than 0.05 was considered to be statistically significant.
Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1: Figure S1: Effect of Cdt containing CdtB WT or one of the CdtB mutants on phosphorylation of H2AX; Figure S2: Susceptibility of Jurkat p21− cells paclitaxel; Figure S3: Effect of pifithrin-α (PFT) on Cdt-induced increases in p21 CIP1/WAF1 and apoptosis.