SM-induced Cell Death Research Articles

Gentiopicroside and swertiamarin induce non-selective oxidative stress-mediated cytotoxic effects in human peripheral blood mononuclear cells

Gentiopicroside (Gp) and swertiamarin (Sm), secoiridoid glycosides commonly found in plants of the Gentianaceae family, differ in one functional group. They exhibit promising cytotoxic effects in cancer cell lines and overall protective outcomes, marking them as promising molecules for developing novel pharmaceuticals. To investigate potential variations in cellular sensitivity to compounds of similar molecular structures, we analyzed the mode of Gp and Sm induced cell death in human peripheral blood mononuclear cells (PBMCs) after 48 h of treatment. The lowest tested concentration that significantly reduces cell viability, 50 μM, was applied. Oxidative stress parameters were estimated by measuring the levels of prooxidative/antioxidative balance, lipid peroxidation products, and 8-oxo-7,8-dihydro-2-deoxyguanosine, while gene expression of DNA repair enzymes was evaluated by employing quantitative real-time PCR. Cellular morphology was analyzed by fluorescent microscopy, and immunoblot analysis of apoptosis and necroptosis-related proteins was used to assess the type of cell death induced by the treatments. The discriminatory impact of Gp/Sm treatments on apoptosis and necroptosis-induced cell death was evaluated by monitoring the cell survival in co-treatment with specific cell death inhibitors. Obtained results show greater cytotoxicity of Gp than Sm suggesting that variations in the molecular structures of the tested compounds can substantially affect their biological effects. Gp/Sm co-treatment with apoptosis and necroptosis inhibitors revealed a distinct, albeit non-specific mechanism of PBMCs cell death. Although the therapeutic may not directly cause a specific type of cell death, its extent can be pivotal in assessing the safety of therapeutic application and developing phytopharmaceuticals with improved features. Since phytopharmaceuticals affect all exposed cells, identification of cytotoxic mechanisms on PBMCs after Gp and Sm treatment is important for addressing the formulation and dosage of potential phytopharmaceuticals.

Role of RIPK1 in SMAC mimetics-induced apoptosis in primary human HIV-infected macrophages

Macrophages serve as viral reservoirs due to their resistance to apoptosis and HIV-cytopathic effects. We have previously shown that inhibitor of apoptosis proteins (IAPs) confer resistance to HIV-Vpr-induced apoptosis in normal macrophages. Herein, we show that second mitochondrial activator of caspases (SMAC) mimetics (SM) induce apoptosis of monocyte-derived macrophages (MDMs) infected in vitro with a R5-tropic laboratory strain expressing heat stable antigen, chronically infected U1 cells, and ex-vivo derived MDMs from HIV-infected individuals. To understand the mechanism governing SM-induced cell death, we show that SM-induced cell death of primary HIV-infected macrophages was independent of the acquisition of M1 phenotype following HIV infection of macrophages. Instead, SM-induced cell death was found to be mediated by IAPs as downregulation of IAPs by siRNAs induced cell death of HIV-infected macrophages. Moreover, HIV infection caused receptor interacting protein kinase-1 (RIPK1) degradation which in concert with IAP1/2 downregulation following SM treatment may result in apoptosis of macrophages. Altogether, our results show that SM selectively induce apoptosis in primary human macrophages infected in vitro with HIV possibly through RIPK1. Moreover, modulation of the IAP pathways may be a potential strategy for selective killing of HIV-infected macrophages in vivo.

Selective Induction of Cell Death in Human M1 Macrophages by Smac Mimetics Is Mediated by cIAP-2 and RIPK-1/3 through the Activation of mTORC.

Inflammatory macrophages have been implicated in many diseases, including rheumatoid arthritis and inflammatory bowel disease. Therefore, targeting macrophage function and activation may represent a potential strategy to treat macrophage-associated diseases. We have previously shown that IFN-γ-induced differentiation of human M0 macrophages toward proinflammatory M1 state rendered them highly susceptible to the cytocidal effects of second mitochondria-derived activator of caspases mimetics (SMs), antagonist of the inhibitors of apoptosis proteins (IAPs), whereas M0 and anti-inflammatory M2c macrophages were resistant. In this study, we investigated the mechanism governing SM-induced cell death during differentiation into M1 macrophages and in polarized M1 macrophages. IFN-γ stimulation conferred on M0 macrophages the sensitivity to SM-induced cell death through the Jak/STAT, IFN regulatory factor-1, and mammalian target of rapamycin complex-1 (mTORC-1)/ribosomal protein S6 kinase pathways. Interestingly, mTORC-1 regulated SM-induced cell death independent of M1 differentiation. In contrast, SM-induced cell death in polarized M1 macrophages is regulated by the mTORC-2 pathway. Moreover, SM-induced cell death is regulated by cellular IAP (cIAP)-2, receptor-interacting protein kinase (RIPK)-1, and RIPK-3 degradation through mTORC activation during differentiation into M1 macrophages and in polarized M1 macrophages. In contrast to cancer cell lines, SM-induced cell death in M1 macrophages is independent of endogenously produced TNF-α, as well as the NF-κB pathway. Collectively, selective induction of cell death in human M1 macrophages by SMs may be mediated by cIAP-2, RIPK-1, and RIPK-3 degradation through mTORC activation. Moreover, blocking cIAP-1/2, mTORC, or IFN regulatory factor-1 may represent a promising therapeutic strategy to control M1-associated diseases.

Selective killing of human M1 macrophages by Smac mimetics alone and M2 macrophages by Smac mimetics and caspase inhibition.

The inflammatory and anti-inflammatory Mϕs have been implicated in many diseases including rheumatoid arthritis, multiple sclerosis, and leprosy. Recent studies suggest targeting Mϕ function and activation may represent a potential target to treat these diseases. Herein, we investigated the effect of second mitochondria-derived activator of caspases (SMAC) mimetics (SMs), the inhibitors of apoptosis (IAPs) proteins, on the killing of human pro- and anti-inflammatory Mϕ subsets. We have shown previously that human monocytes are highly susceptible whereas differentiated Mϕs (M0) are highly resistant to the cytocidal abilities of SMs. To determine whether human Mϕ subsets are resistant to the cytotoxic effects of SMs, we show that M1 Mϕs are highly susceptible to SM-induced cell death whereas M2a, M2b, and M2c differentiated subsets are resistant, with M2c being the most resistant. SM-induced cell death in M1 Mϕs was mediated by apoptosis as well as necroptosis, activated both extrinsic and intrinsic pathways of apoptosis, and was attributed to the IFN-γ-mediated differentiation. In contrast, M2c and M0 Mϕs experienced cell death through necroptosis following simultaneous blockage of the IAPs and the caspase pathways. Overall, the results suggest that survival of human Mϕs is critically linked to the activation of the IAPs pathways. Moreover, agents blocking the cellular IAP1/2 and/or caspases can be exploited therapeutically to address inflammation-related diseases.

Selective killing of proinflammatory human M1 macrophages by SMAC mimetics and anti-inflammatory M2 macrophages by SMAC mimetics and caspase inhibitors

Abstract The inflammatory and anti-inflammatory macrophages have been implicated in many diseases including rheumatoid arthritis, multiple sclerosis and leprosy. Targeting macrophage function and activation may represent a potential target to treat these diseases. Herein, we investigated the effect of second mitochondria-derived activator of caspases (SMAC) mimetics (SMs), the inhibitors of apoptosis (IAPs) proteins, on the killing of human pro- and anti-inflammatory macrophage subsets. We have shown previously, that human monocytes are susceptible to the cytotoxic effects of SMs, however, differentiated macrophages (M0) are resistant. Whether human macrophage subsets are also resistant to the cytotoxic effects of SM remains unknown. We show that differentiation of M0 macrophages towards M1 state rendered them highly susceptible to SM-induced cell death, whereas M2a, M2b and M2c subsets were resistant, with M2c being the most resistant. SM-induced cell death in M1 macrophages was mediated by apoptosis as well as necroptosis, and activated both extrinsic and intrinsic pathways of apoptosis. The susceptibility of M1 macrophages to SM-induced cell death was attributed to the IFNg-mediated polarization as JAK inhibitor reversed their susceptibility. In contrast, M2c and M0 macrophages experienced cell death through necroptosis pathway following simultaneous blockage of the IAPs pathways by SM and the caspase pathways by the pan-caspase inhibitors. Overall, the results suggest that survival of human macrophages is critically linked to the activation of the IAPs pathways. Moreover, agents blocking the cellular IAP1/2 and/or caspases can be exploited therapeutically to address inflammation-related diseases.

Biomarker profile for prediction of response to SMAC mimetic monotherapy in pediatric precursor B-cell acute lymphoblastic leukemia.

Second mitochondria-derived activator of caspase (SMAC) mimetics (SMs) targeting inhibitor of apoptosis proteins (IAPs) activate cell death pathways, and are currently being evaluated in clinical trials. Their successful therapeutic implementation requires upfront identification of patients who could benefit from a SM-based treatment but biomarkers for SM sensitivity have not yet been described. Here, we analyzed the intrinsic activity of two monovalent (AT406 and LCL161) and two bivalent (Birinapant and BV6) SMs on unselected patient-derived pediatric precursor B-cell acute lymphoblastic leukemia (BCP-ALL) identifying a subset of patient samples to be particularly sensitive to SM-induced cell death. This subset was defined by a characteristic gene expression signature with 127 differentially regulated genes, amongst them TNFRSF1A encoding TNFR1, and a critical role of TNFR1 in SM-induced cell death in sensitive BCP-ALL was confirmed on the functional level. Interestingly, samples with intermediate or low sensitivity to SMs were sensitized to SM-induced cell death by inhibition of caspases using zVAD.fmk or Emricasan, a pan-caspase inhibitor in clinical trials. When we compared our expression data to published data sets, we identified an overlap of four genes to be commonly differentially regulated in SM-sensitive BCP-ALL, that is, TSPAN7, DIPK1C, MTX2 and, again, TNFRSF1A. Functional testing revealed that this set of genes identified samples with high sensitivity to SM treatment. In summary, our data suggest using this gene signature as biomarker predicting response to SM treatment and point to the development of new combinatorial treatments consisting of SMs and pan-caspase inhibitors for a successful clinical implementation of SMs in treatment of BCP-ALL.

Biomarker Profile for Prediction of Patient Response to Smac Mimetic Monotherapy in Pediatric Precursor B-Cell Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is the most common malignancy in childhood. While improved multi-agent chemotherapy regimens with individualized risk stratification have led to increased survival rates of approximately 80 percent, 20 percent of patients respond poorly to therapy or relapse. Therefore, novel therapeutic avenues are urgently needed to improve treatment outcome, overcome resistance and reduce side effects. Failure to undergo cell death represents a key survival mechanism of cancer cells and results in drug resistance and clonal escape. Since inhibitor of apoptosis proteins (IAPs) are often overexpressed in malignant cells and their overexpression correlates with inferior survival rates, they provide an attractive molecular target for therapeutic intervention. Small molecule inhibitors have been developed that act as SMAC mimetics (SMs) to counteract the cell death inhibitory function of IAPs. SMs can activate and/or modulate cell death pathways, and are currently being evaluated in clinical trials. Their successful therapeutic implementation requires identification of patients who could benefit from a SM-based treatment regimen ideally before start of therapy. Here, we analyzed the intrinsic activity of two monovalent (AT406 and LCL161) and two bivalent (Birinapant or BV6) SMs on 29 unselected patient-derived pediatric precursor B-cell (BCP)-ALL samples and identified a subset of BCP-ALL primografts to be sensitive to SM treatment (n=8). When we compared gene expression of SM-sensitive (n=8) and SM-insensitive (n=6) patient-derived BCP-ALL samples, we identified a characteristic gene expression signature with 127 differentially regulated genes, amongst them upregulation of TNFRSF1A (TNFR1) in the SM-sensitive subset. In line with previous reports, we confirmed a critical role of the TNF/TNFR1-axis for SM-induced cell death in BCP-ALL by functional analysis. Expression of TNFRSF1A alone, however, did not correlate with sensitivity to SM-induced cell death indicating that TNFR1 is not the only factor regulating cell fate decisions in response to SM treatment. To identify potential biomarker genes for prediction of patient response to SM monotherapy in BCP-ALL, we compared differentially regulated genes of SM responders and non-responders from our cohort with data from a published cohort. Interestingly, we found 4 genes to overlap between these two cohorts. Of these 4 genes TSPAN7, FAM69C, and TNFRSF1A were upregulated whereas MTX2 was downregulated in SM-sensitive samples. The signature identified may reflect a particular TNF network. Analysis of expression levels of these 4 genes in BCP-ALL cell lines (Nalm6, Reh, UoCB6 and RS4;11) revealed that Reh cells, sensitive to SM-induced cell death, exhibited the biomarker profile of primograft sensitivity, i.e. upregulation of TSPAN7, FAM69C, TNFRSF1A and downregulation of MTX2. Nalm6 cells resembled the expression pattern of SM-insensitive samples with a downregulation of TSPAN7, FAM69C, TNFRSF1A and an upregulation of MTX2 and were resistant to SM-induced cell death. RS4;11 and UoCB6 cells showed no pattern. Based on these findings we hypothesized that the respective expression patterns of TSPAN7, FAM69C, TNFRSF1A and MTX2 could predict sensitivity to SMs. An extended screen of additional primary BCP-ALL samples for their expression levels of TSPAN7, FAM69C, TNFRSF1A and MTX2 and response to SMs substantiated this hypothesis. In summary, the subset of primary BCP-ALL samples with sensitivity to SMs is characterized by a gene signature with MTX2 low and TSPAN7, FAM69C and TNFRSF1A high. By using this expression profile, sensitivity to SMs in BCP-ALL could be identified in cell lines and additional primografts. Based on these results, we suggest the identified gene expression pattern as a biomarker for selecting patients to be treated by SM monotherapy in clinical trials. Disclosures No relevant conflicts of interest to declare.

Differential involvement of TAK1, RIPK1 and NF-κB signaling in Smac mimetic-induced cell death in breast cancer cells.

Smac mimetics (SMs) are considered promising cancer therapeutics. However, the mechanisms responsible for mediating cell death by SMs are still only partly understood. Therefore, in this study, we investigated signaling pathways upon treatment with the bivalent SM BV6 using two SM-sensitive breast cancer cell lines as models. Interestingly, genetic silencing of transforming growth factor (TGF)β activated kinase (TAK)1, an upstream activator of the nuclear factor-kappaB (NF-κB) subunit RelA (p65), increased BV6-induced cell death only in EVSA-T cells, although it reduced BV6-mediated upregulation of tumor necrosis factor (TNF)α in both EVSA-T and MDA-MB-231 cells. By comparison, genetic silencing of p65, a key component of canonical NF-κB signaling, blocked BV6-induced cell death in MDA-MB-231 but not in EVSA-T cells. Similarly, knockdown of NF-κB-inducing kinase (NIK) rescued MDA-MB-231 cells from BV6-induced cell death, while it failed to do so in EVSA-T cells. Consistently, silencing of p65 or NIK reduced BV6-stimulated upregulation of TNFα in MDA-MB-231 cells. In conclusion, TAK1, receptor-interacting kinase 1 (RIPK1) as well as canonical and non-canonical NF-κB signaling are differentially involved in SM-induced cell death in breast cancer cells. These findings contribute to a better understanding of SM-induced signaling pathways.

Anti-apoptotic and moderate anti-inflammatory effects of berberine in sulfur mustard exposed keratinocytes

Skin affections after sulfur mustard (SM) exposure include erythema, blister formation and severe inflammation. An antidote or specific therapy does not exist. Anti-inflammatory compounds as well as substances counteracting SM-induced cell death are under investigation. In this study, we investigated the benzylisoquinoline alkaloide berberine (BER), a metabolite in plants like berberis vulgaris, which is used as herbal pharmaceutical in Asian countries, against SM toxicity using a well-established in vitro approach. Keratinocyte (HaCaT) mono-cultures (MoC) or HaCaT/THP-1 co-cultures (CoC) were challenged with 100, 200 or 300mM SM for 1h. Post-exposure, both MoC and CoC were treated with 10, 30 or 50μM BER for 24h. At that time, supernatants were collected and analyzed both for interleukine (IL) 6 and 8 levels and for content of adenylate-kinase (AK) as surrogate marker for cell necrosis. Cells were lysed and nucleosome formation as marker for late apoptosis was assessed. In parallel, AK in cells was determined for normalization purposes. BER treatment did not influence necrosis, but significantly decreased apoptosis. Anti-inflammatory effects were moderate, but also significant, primarily in CoC. Overall, BER has protective effects against SM toxicity in vitro. Whether this holds true should be evaluated in future in vivo studies.

Abstract 3548: In vivo CRISPR reveals dual activation of apoptosis and necroptosis as means to eradicate drug resistant leukemia

Abstract Dysregulation of apoptosis constitutes a general mechanism for refractory acute lymphoblastic leukemia (ALL) to escape cell death during chemotherapy. We have assessed the potential of SMAC mimetic compounds (SM) to eradicate drug resistant ALL by blocking the pro-survival cellular inhibitor of apoptosis proteins (cIAPs). On the basis of a large xenograft bank of primary ALL samples, obtained by xenotransplantation in immunodeficient NSG mice, we detected exquisite sensitivity to SM in vitro in a third of the cases tested, including drug-resistant and relapsed samples. Potent in vitro activity was highly predictive of in vivo efficacy in the xenograft model. Treatment of transplanted mice with SM not only delayed leukemia progression, but also completely eliminated leukemia burden in established disease for several weeks following treatment. SM activity required the presence of RIP1, since lentiCRISPR mediated gene knockout of RIP1 in patient-derived ALL cells blocked SM-induced cell death in vivo and in vitro. In contrast, RIP1 loss had no significant impact on the response to standard antileukemic therapies, supporting a view that RIP1-dependent death pathways are not likely to be selected against by front line chemotherapy. Furthermore, using a multicolor lentiCRISPR approach for simultaneous disruption of multiple genes, we found that downstream of RIP1, the response to SM was dependent on simultaneous execution of apoptosis and necroptosis, since genetic targeting of both cell death pathways was required to block SM-induced death. Upstream of RIP1, expression levels of TNF receptor II (TNFR2) in primary ALL correlated with sensitivity to SM, and genetic disruption of TNFR2 conferred resistance to SM treatment in vivo. Thus, SM treatment circumvents drug resistance through parallel activation of RIP1-dependent apoptosis and necroptosis, providing a strong rationale for application in salvage treatment of refractory childhood ALL. Citation Format: Júlia Aguadé-Gorgorió, Scott McComb, Gunnar Cario, Martin Stanulla, Cornelia Eckert, Jean-Pierre Bourquin, Beat C. Bornhauser. In vivo CRISPR reveals dual activation of apoptosis and necroptosis as means to eradicate drug resistant leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3548.

Sulfur mustard induced cytokine production and cell death: Investigating the potential roles of the p38, p53, and NF‐κB signaling pathways with RNA interference

Cutaneous and ocular injuries caused by sulfur mustard (SM; bis-(2-chloroethyl) sulfide) are characterized by severe inflammation and death of exposed cells. Given the known roles of p38MAPK and NF-kappaB in inflammatory cytokine production, and the known roles of NF-kappaB and p53 in cell fate, these pathways are of particular interest in the study of SM injury. In this study, we utilized inhibitory RNA (RNAi) targeted against p38 alpha, the p50 subunit of NF-kappaB, or p53 to characterize their role in SM-induced inflammation and cell death in normal human epidermal keratinocytes (NHEK). Analysis of culture supernatant from 200 microM SM-exposed cells showed that inflammatory cytokine production was inhibited by p38 alpha RNAi but not by NF-kappaB p50 RNAi. These findings further support a critical role for p38 in SM-induced inflammatory cytokine production in NHEK and suggest that NF-kappaB may not play a role in the SM-induced inflammatory response of this cell type. Inhibition of NF-kappaB by p50 RNAi did, however, partially inhibit SM-induced cell death, suggesting a role for NF-kappaB in SM-induced apoptosis or necrosis. Interestingly, inhibition of p53 by RNAi potentiated SM-induced cell death, suggesting that the role of p53 in SM injury, may be complex and not simply prodeath.

Mechanisms of Mitochondrial Dysfunction Induced by Sulfur Mustard in Human Epidermal Keratinocytes (HEK)

Exposure of HEK to the chemical warfare agent sulfur mustard (SM) significantly disrupts cell energy metabolism. Our data suggest that one or more enzymes of respiratory complex I may be directly inhibited by SM and that complexes II, III and IV are relatively insensitive. To further localize sites of inhibition, we examined the partial reactions NADH‐ferricyanide reductase and cytochrome c oxidase. 100 μM SM did not inhibit either reaction at 24 hrs post‐exposure. 500 μM inhibited cyt. c oxidase by 25%, possibly secondary to cell death. We conclude that SM inhibits anterior to cytochrome c for succinate and between NADH dehydrogenasecyt. c for NAD‐linked substrates. Our previous data also suggest that SM causes irreversible mitochondrial damage parallel to late stage (18–24 hrs) losses in ATP and cell viability. We examined mitochondrial membrane permeability with JC‐1 and calcein‐AM+CoCl2. At ≤ 8 hrs we saw no changes with ≤ 500 μM SM. At 18–24 hours, 100 μM SM had no effect, but 300 and 500 μM caused losses in membrane potential that, in some cells, coincided with abnormal morphology. The data suggest that collapse of the mitochondrial membrane potential is a key event in SM‐induced cell death, perhaps at the level of the transition pore. Supported by DTRA‐JSTO, Med. S&T Div. The opinions, interpretations, conclusions, and recommendations are those of the author and are not necessarily endorsed by the US Army or the DoD.

Inhibition of poly(ADP-ribose) polymerase (PARP) influences the mode of sulfur mustard (SM)-induced cell death in HaCaT cells

Sulfur mustard (SM) is a bifunctional alkylating agent. Its primary toxic consequence is severe skin damage with blisters, occurring after skin contact. These vesicant properties of SM have been linked to cell death of proliferating keratinocytes in the basal layer of the skin. Catalytic activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP-1) has been demonstrated to be a major event in response to high levels of DNA damage, and PARP-1 activation may be part of apoptotic signaling. In other contexts, overstimulation of PARP-1 triggers necrotic cell death because of rapid consumption of its substrate, beta-nicotinamide adenine dinucleotide (NAD+) and the consequent depletion of ATP. These findings prompted us to evaluate whether SM induces apoptosis in keratinocytes like HaCaT cells and to determine whether blocking of PARP enzyme activity with 3-aminobenzamide (3AB) can influence the mode of cell death. HaCaT cells were exposed to SM (10-1,000 microM; 30 min) and then cultivated in SM-free medium with or without 3AB for up to 48 h. This treatment resulted in a time and SM dose-dependent increase of apoptotic cell death characterized by PARP-1 cleavage and DNA fragmentation during the experimental period. After just 45 min of exposure to 1 mM SM, we observed a significant increase in PARP-1 activity in HaCaT cells. About 6 h after exposure, intracellular ATP levels were diminished by 22%, which seemed to be completely prevented by the addition of 3AB directly after exposure. However, 18 h later, this 3AB effect on the SM concentration-dependent loss of ATP was no longer detectable. Interestingly, the effect of SM on total cell viability was not changed by 3AB. However, the mode of cell death was influenced by 3AB exhibiting an increase of apoptotic cells and a concomitant decrease of necrotic HaCaT cells during the first 24 h after SM exposure. Our results indicate that SM concentrations of 1 mM or higher induce a prominent PARP activation leading to ATP depletion and necrosis. In contrast, lower concentrations of SM cause minor PARP activation and, especially, PARP-1 cleavage by caspase 3 without ATP depletion. Because ATP is required for apoptosis, we suggest that ATP acts as an early molecular switch from apoptotic to necrotic modes of SM-induced cell death, at least at high concentrations (> or =1 mM). Thus, the observed early proapoptotic effect of 3AB at lower SM concentrations may point to the influence of ATP-independent cell-death regulating mechanisms.

Sulfur mustard-induced apoptosis in hairless guinea pig skin.

The present study was aimed to examine whether apoptosis is involved in the pathogenesis of sulfur mustard (SM)-induced basal cell death. Skin sites of the hairless guinea pig exposed to SM vapor for 8 minutes were harvested at 3, 6, 12, 24, and 48 hours postexposure. Immunohistochemical detection of basal cell apoptosis was performed using the ApopTag in situ apoptosis labeling kit. Only occasional apoptotic basal cells (BC)were observed in nonexposed and perilesional control sites. At lesional sites, apoptosis of BC was not detected at 3 hours postexposure. However, at 6 hours and 12 hours postexposure, 18% and 59% of BC were apoptotic, respectively. At 24 and 48 hours postexposure, individual apoptotic basal cells were not clearly recognizable due to necrosis. At the ultrastructural level, degenerating BC exhibited typical apoptotic morphology including nuclear condensation and chromatin margination. The results suggest that apoptotic cell death is a cytotoxic mechanism with the number of BC undergoing apoptosis significantly increasing from 6 to 12 hours postexposure. In addition, because necrosis is preferential at 24 hours postexposure, we believe that SM-induced cell death involves early apoptosis and late necrosis, which temporally overlap to produce a single cell death pathway along an apoptotic-necrotic continuum.

Nuclear Dependence of Sulfur Mustard-Mediated Cell Death

Although sulfur mustard (SM) has been reported to be a DNA alkylating agent, it is not clear how much of the cytotoxicity of this agent is secondary to DNA damage. To test the hypothesis that the presence of a nucleus is required for the toxicity of sulfur mustard, enucleated endothelial cytoplasts were treated with SM. Using a combination of biochemical and microscopic assays, we demonstrate that some aspects of SM-induced cell death may be dependent on the presence of a nucleus, while others may not be. For example, it was found that cytoskeletal changes, such as loss of stress fibers and rounding, proceed in response to sulfur mustard treatment even in the absence of a nucleus. However, significant further increases in caspase activity and the associated phosphatidylserine translocation were not observed in cytoplasts treated with 500 μM SM for 6 h (following a 20-h recovery at the end of cytoplast preparation). In contrast, cytoplasts treated with chelerythrine, an agent previously reported to induce rapid apoptosis, demonstrated increases in caspase activity in cytoplasts comparable to that observed in the nucleated cells. This indicates that sulfur mustard-induced alkylation of nuclear DNA may be an important stimulus for activation of caspases in nucleated cells. Interestingly, the baseline caspase activity in cytoplasts was greater than in nucleated cells. Analysis of the time course of caspase activation in untreated adherent cytoplasts indicated that the activity increases initially and then stabilizes by 8 h to a low level that was comparable to the level observed at 26 h in untreated cytoplasts. This indicates that cytoplasts are able to tolerate stable low levels of caspase activity and not proceed immediately into the execution phase of apoptosis. The cytoplast model may be quite useful in the toxicological assessment of agents that are thought to exert their toxicity through DNA damage.