Lloyd S. Gray

Distinct Mechanisms for K+ Efflux, Intoxication, and Hemolysis by Bordetella pertussis AC Toxin

Adenylate cyclase (AC) toxin fromBordetella pertussis delivers its catalytic domain to the interior of target cells where it converts host ATP to cAMP in a process referred to as intoxication. This toxin also hemolyzes sheep erythrocytes by a mechanism presumed to include pore formation and osmotic lysis. Intoxication and hemolysis appear at strikingly different toxin concentrations and evolve over different time scales, suggesting that different molecular processes may be involved. The present study was designed to test the hypothesis that intoxication and hemolysis occur by distinct mechanisms. Although the hemolytic activity of AC toxin has a lag of >1 h, intoxication starts immediately. Because of this difference, we sought a surrogate or precursor lesion that leads to hemolysis, and potassium efflux has been observed from erythrocytes treated with other pore-forming toxins. AC toxin elicits an increase in K+efflux from sheep erythrocytes and Jurkat cells, a human T-cell leukemia line, that begins within minutes of toxin addition. The toxin concentration dependence along with the analysis of the time course suggest that toxin monomers are sufficient to elicit release of K+ and to deliver the catalytic domain to the cell interior. Hemolysis, on the other hand, is a highly cooperative event that likely requires a subsequent oligomerization of these individual units. Although induction of K+ efflux shares some structural and environmental requirements with both intoxication and hemolysis, it can occur under conditions in which intoxication is reduced or prevented. The data presented here suggest that the transmembrane pathway by which K+ is released is separate and distinct from the structure required for intoxication but may be related to, or a precursor of, that which is ultimately responsible for hemolysis.

Hemolytic activity of adenylate cyclase toxin from Bordetella pertussis

Adenylate cyclase (AC) toxin fromB. pertussis enters eukaryotic cells where it produces supraphysiologie levels of cAMP. Purification of AC toxin activity [(1989) J. Biol. Chem. 264, 19279] results in increasing potency of hemolytic activity and electroelution of the 216‐kDA holotoxin yields a single protein with AC enzymatic, toxin and hemolytic activities. AC toxin andE. coli hemolysin, which have DNA sequence homology [(1988) EMBO J. 7, 3997] are immunologically cross‐reactive. The time courses of hemolysis elicited by the two molecules are strikingly different, however, with AC toxin eliciting cAMP accumulation with rapid onset, but hemolysis with a lag of ≥ 45 min. Finally, osmotic protection experiments indicate that the size of the putative pore produced by AC toxin is 3 5‐fold smaller than that ofE. coli hemolysin.

The role of K+ in the regulation of the increase in intracellular Ca2+ mediated by the T lymphocyte antigen receptor

The regulation of the increase in intracellular calcium ([Ca2+]i) occurring in cytolytic T lymphocytes (CTLs) upon their interaction with antigen was examined. This [Ca2+]i increase and lytic function were insensitive to verapamil, a Ca channel blocker. An antigen-independent increase in [Ca2+]i was not induced by depolarization of CTLs with excess extracellular K+, suggesting that Ca2+ influx is not mediated by the ubiquitous voltage-gated Ca channel. The antigen-induced [Ca2+]i increase was inhibited by prior membrane hyperpolarization with valinomycin. Hyperpolarization occurred under normal circumstances in CTLs exposed to antigen-receptor-specific antibodies. This potential change was Ca2+-dependent and inhibited by K channel blockade. Conversely, K channel blockade augmented the antigen-specific [Ca2+]i increase while markedly decreasing the K+ efflux associated with CTL lytic function. Therefore, either membrane potential or intracellular K+ regulates the antigen-specific [Ca2+]i increase in CTLs.

T-type calcium channels blockers as new tools in cancer therapies

T-type calcium channels are involved in a multitude of cellular processes, both physiological and pathological, including cancer. T-type channels are also often aberrantly expressed in different human cancers and participate in the regulation of cell cycle progression, proliferation, migration, and survival. Here, we review the recent literature and discuss the controversies, supporting the role of T-type Ca2+ channels in cancer cells and the proposed use of channels blockers as anticancer agents. A growing number of reports show that pharmacological inhibition or RNAi-mediated downregulation of T-type channels leads to inhibition of cancer cell proliferation and increased cancer cell death. In addition to a single agent activity, experimental results demonstrate that T-type channel blockers enhance the anticancer effects of conventional radio- and chemotherapy. At present, the detailed biological mechanism(s) underlying the anticancer activity of these channel blockers is not fully understood. Recent findings and ideas summarized here identify T-type Ca2+ channels as a molecular target for anticancer therapy and offer new directions for the design of novel therapeutic strategies employing channels blockers. Physiological relevance: T-type calcium channels are often aberrantly expressed or deregulated in cancer cells, supporting their proliferation, survival, and resistance to treatment; therefore, T-type Ca2+ channels could be attractive molecular targets for anticancer therapy.

A Role for Protein Kinase CβI in the Regulation of Ca2+ Entry in Jurkat T Cells

T cell activation leading to cytokine production and cellular proliferation involves a regulated increase and subsequent decrease in the intracellular concentration of Ca2+([Ca2+] i ). While much is understood about agonist-induced increases in [Ca2+] i , less is known about down-regulation of this pathway. Understanding the mechanism of this down-regulation is critical to the prevention of cell death that can be the consequence of a sustained elevation in [Ca2+] i . Protein kinase C (PKC), activated by the diacylglycerol produced as a consequence of T cell receptor engagement, has long been presumed to be involved in this down-regulation, although the precise mechanism is not wholly clear. In this report we demonstrate that activation of PKC by phorbol esters slightly decreases the rate of Ca2+ efflux from the cytosol of Jurkat T cells following stimulation through the T cell receptor or stimulation in a receptor-independent manner by thapsigargin. On the other hand, phorbol ester treatment dramatically reduces the rate of Ca2+influx following stimulation. Phorbol ester treatment is without an effect on Ca2+ influx in a different T cell line, HSB. Down-regulation of PKCβI expression by 18-h phorbol ester treatment is associated with a loss of the response to acute phorbol ester treatment in Jurkat cells, suggesting that PKCβI may be the isozyme responsible for the effects on Ca2+ influx. Electroporation of an anti-PKCβI antibody, but not antibodies against PKCα or PKCγ, led to an increase in the rate of Ca2+ influx following stimulation. Taken together, these data suggest that PKCβI may be a component of the down-regulation of increases in [Ca2+] i associated with Jurkat T cell activation.

Translocation-Specific Conformation of Adenylate Cyclase Toxin from Bordetella pertussis Inhibits Toxin-Mediated Hemolysis

Bordetella pertussis adenylate cyclase (AC) toxin belongs to the RTX family of toxins but is the only member with a known catalytic domain. The principal pathophysiologic function of AC toxin appears to be rapid production of intracellular cyclic AMP (cAMP) by insertion of its catalytic domain into target cells (referred to as intoxication). Relative to other RTX toxins, AC toxin is weakly hemolytic via a process thought to involve oligomerization of toxin molecules. Monoclonal antibody (MAb) 3D1, which binds to an epitope (amino acids 373 to 399) at the distal end of the catalytic domain of AC toxin, does not affect the enzymatic activity of the toxin (conversion of ATP into cAMP in a cell-free system) but does prevent delivery of the catalytic domain to the cytosol of target erythrocytes. Under these conditions, however, the ability of AC toxin to cause hemolysis is increased three- to fourfold. To determine the mechanism by which the hemolytic potency of AC toxin is altered, we used a series of deletion mutants. A mutant toxin, DeltaAC, missing amino acids 1 to 373 of the catalytic domain, has hemolytic activity comparable to that of wild-type toxin. However, binding of MAb 3D1 to DeltaAC enhances its hemolytic activity three- to fourfold similar to the enhancement of hemolysis observed with 3D1 addition to wild-type toxin. Two additional mutants, DeltaN489 (missing amino acids 6 to 489) and DeltaN518 (missing amino acids 6 to 518), exhibit more rapid hemolysis with quicker onset than wild-type toxin does, while DeltaN549 (missing amino acids 6 to 549) has reduced hemolytic activity compared to wild-type AC toxin. These data suggest that prevention of delivery of the catalytic domain or deletion of the catalytic domain, along with additional amino acids distal to it, elicits a conformation of the toxin molecule that is more favorable for hemolysis.

A voltage-gated calcium channel is linked to the antigen receptor in Jurkat T lymphocytes

Activation of T lymphocytes results in an increase in intracellular Ca2+ due in large part to influx of extracellular Ca2+. Using the patch clamp technique, an inward current in Jurkat T lymphocytes was observed upon depolarization from a holding potential of −90 mV but not from −60 mV. This whole‐cell current was insensitive to tetrodotoxin, carried by Ba2+, and blocked by Ni2+. Occupancy of the T lymphocyte antigen receptor increased the currents magnitude. These data suggest that antigen receptor‐induced Ca2+ entry in T lymphocytes may be mediated by a voltage‐regulated Ca channel.

A model for the regulation of T-type Ca2+ channels in proliferation: roles in stem cells and cancer

Ca2+ influx at critical points in the cell cycle is required for proliferation. This requirement is so ubiquitous that its occurrence is often treated as background noise. Yet without it, cells stop dividing, suggesting an obvious and potentially effective way to treat cancer. To control proliferation by controlling Ca2+ influx requires that the mechanism be elucidated, but this field of study has been filled with controversy and devoid of therapeutic utility. In this study, the authors present a model for the regulation of Ca2+ influx at the G1/S restriction point in cancer and stem cells that is simple, cohesive and, we believe, reasonably complete. The model illustrates the essential role of T-type Ca2+ channels in mediating influx and points clearly to the therapeutic strategies that have recently entered clinical trials.

Calmodulin regulation of Ca2+ entry in Jurkat T cells

We have previously proposed a role for calmodulin (CaM) in the regulation of initiation of Ca2+ entry in Jurkat T cells, as well as in the regulation of the current that mediates Ca2+ entry, IT. In this report, we provide evidence for the mechanism of CaM action. We have previously shown that activation-induced Ca2+ entry into Jurkat T cells is mediated by a current we have called IT. In the whole cell variation, but not the perforated patch variation, of the patch clamp technique, this current is short-lived (under 6 min) suggesting that the current is under the control of a diffusible component of the cytosol. Addition of CaM to the whole cell recording pipette solution maintained IT for up to 20 min, suggesting that CaM may be this diffusible component. Pharmacological inhibitors of CaM blocked the augmentation of IT normally induced by an activating stimulus. Cells electroporated in the presence of anti-CaM antibodies had reduced influx of extracellular Ca2+, with no change in release of Ca2+ from the internal stores. These observations suggest that T cell receptor engagement initiates Ca2+ influx by a pathway that likely includes CaM, which may in turn regulate IT. Influx of extracellular Ca2+ is required for cellular proliferation, and inhibition of CaM by pharmacological inhibitors reduced cellular proliferation. This same inhibition of proliferation was seen in cells electroporated with anti-CaM antibodies. This suggests that inhibition of CaM and/or IT may be a target for therapeutic inhibition of inappropriate T cell proliferation.

Targetable T-type calcium channels drive glioblastoma

Glioblastoma (GBM) stem-like cells (GSC) promote tumor initiation, progression, and therapeutic resistance. Here, we show how GSCs can be targeted by the FDA-approved drug mibefradil, which inhibits the T-type calcium channel Cav3.2. This calcium channel was highly expressed in human GBM specimens and enriched in GSCs. Analyses of the The Cancer Genome Atlas and REMBRANDT databases confirmed upregulation of Cav3.2 in a subset of tumors and showed that overexpression associated with worse prognosis. Mibefradil treatment or RNAi-mediated attenuation of Cav3.2 was sufficient to inhibit the growth, survival, and stemness of GSCs and also sensitized them to temozolomide chemotherapy. Proteomic and transcriptomic analyses revealed that Cav3.2 inhibition altered cancer signaling pathways and gene transcription. Cav3.2 inhibition suppressed GSC growth in part by inhibiting prosurvival AKT/mTOR pathways and stimulating proapoptotic survivin and BAX pathways. Furthermore, Cav3.2 inhibition decreased expression of oncogenes (PDGFA, PDGFB, and TGFB1) and increased expression of tumor suppressor genes (TNFRSF14 and HSD17B14). Oral administration of mibefradil inhibited growth of GSC-derived GBM murine xenografts, prolonged host survival, and sensitized tumors to temozolomide treatment. Our results offer a comprehensive characterization of Cav3.2 in GBM tumors and GSCs and provide a preclinical proof of concept for repurposing mibefradil as a mechanism-based treatment strategy for GBM. Cancer Res; 77(13); 3479-90. ©2017 AACR.