Basal Cell Death Research Articles

JAK-STAT activation contributes to cytotoxic T cell–mediated basal cell death in human chronic lung allograft dysfunction

Chronic lung allograft dysfunction (CLAD) is the leading cause of death in lung transplant recipients. CLAD is characterized clinically by a persistent decline in pulmonary function and histologically by the development of airway-centered fibrosis known as bronchiolitis obliterans. There are no approved therapies to treat CLAD, and the mechanisms underlying its development remain poorly understood. We performed single-cell RNA-Seq and spatial transcriptomic analysis of explanted tissues from human lung recipients with CLAD, and we performed independent validation studies to identify an important role of Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling in airway epithelial cells that contributes to airway-specific alloimmune injury. Specifically, we established that activation of JAK-STAT signaling leads to upregulation of major histocompatibility complex 1 (MHC-I) in airway basal cells, an important airway epithelial progenitor population, which leads to cytotoxic T cell-mediated basal cell death. This study provides mechanistic insight into the cell-to-cell interactions driving airway-centric alloimmune injury in CLAD, suggesting a potentially novel therapeutic strategy for CLAD prevention or treatment.

Generation of gene edited hiPSC from familial Alzheimer's disease patient carrying N141I missense mutation in presenilin 2

Alzheimer's disease (AD) is the major cause of dementia worldwide. Early-onset familial AD accounts for about 0.5% of all AD and is caused by single major gene mutations and autosomal dominant inheritance. An N141I missense mutation is associated with a significant increase in basal cell death and apoptosis. In this work we generated hiPSC from skin fibroblasts obtained from an AD patient carrying a N141I missense mutation in PSEN2. The generated iPSC colonies grew and were characterized by pluripotency marker staining; the N141I missense mutation was corrected using genome editing technology.

Microglia are both a source and target of extracellular cyclophilin A

Glioblastoma (GBM) are lethal primary brain tumours whose pathogenesis is aided, at least partly, via a pro-tumorigenic microenvironment. This study investigated whether microglia, a cell component of the GBM microenvironment, mediates pro-tumorigenic properties via the action of cyclophilin A (CypA), a potent secretable chemokine and cytoprotectant that signals via the cell surface receptor, CD147. To this end, intracellular and secreted CypA expression was assessed in human primary microglia and BV2 microglial cells treated with the endotoxin, lipopolysaccharide (LPS) and the oxidative stress inducer, LY83583. We report that human primary microglia and BV2 microglia both express CypA and CD147, and that BV2 microglial cells secrete CypA in response to pro-inflammatory and oxidative stimuli. We also demonstrate for the first time that recombinant CypA (rCypA; 1nM–1000nM) dose-dependently increased wound healing and reduced basal cell death in BV2 microglial cells. To determine the cell–signalling pathways involved, we probed microglial cell lysates for changes in ERK1/2 and AKT phosphorylation, IκB degradation, and IL-6 secretion using Western blot and ELISA analysis. In summary, BV2 microglial cells secrete CypA in response to inflammatory and oxidative stress, and that rCypA increases cell viability and chemotaxis. Our findings suggest that rCypA is a pro-survival chemokine for microglia that may influence the GBM tumour microenvironment.

CD209 activation promotes survival of lymphoblastic human B cells

Abstract The role of B cells in host protection and antibody production depends in part on their ability to recognize antigens and pathogen associated molecular patterns (e.g. carbohydrates and nucleic acids) mostly through membrane bound receptors. CD209 (i.e. DC-SIGN) is a C-type lectin receptor expressed on dendritic cells, macrophages and B cells which binds to different oligosaccharides and plays a role in antigen capture and cell adhesion. CD209 also functions as a receptor for many bacterial, fungal and viral pathogens. In B cells, CD209 expression enhances infections by HHV8 and H5N1 viruses and in HIV positive patients, promotes trans-infection of CD4+ T cells. Besides its role in cell adhesion and pathogen recognition, the role of CD209 in B cell physiology remains elusive. Our goal is to identify and characterize CD209-induced signaling pathways in human B cells and determine how these pathways relate to normal and pathologic B cell responses. Our data shows that CD209+ lymphoblastic B cells have a higher level of basal cell death suggesting that expression sensitizes B cells to apoptosis. Stimulating CD209+ B cells using an anti-CD209 antibody or Mannan, promotes cell survival. Moreover, CD209 stimulation inhibits BCR-mediated programmed cell death and modifies the expression of several genes involved in cell survival and apoptosis (e.g. Bim, BclXL, Xiap). Elucidating the role of CD209 in B cell physiology will have important implications towards understanding the development of the humoral immune response to pathogens such as HHV8, Zika, Dengue and HIV. Moreover, our results will set the groundwork for future studies on vaccine development and biomarker discovery. Supported by NIGMS-INBRE P20 GM103475-14

Media formulation influences chemical effects on neuronal growth and morphology.

Screening for developmental neurotoxicity using in vitro, cell-based systems has been proposed as an efficient alternative to performing in vivo studies. One tool currently used for developmental neurotoxicity screening is automated high-content imaging of neuronal morphology. While high-content imaging (HCI) has been demonstrated to be useful in detection of potential developmental neurotoxicants, comparison of results between laboratories or assays can be complicated due to methodological differences. In order to determine whether high-content imaging-based developmental neurotoxicity assays can be affected by differences in media formulation, a systematic comparison of serum-supplemented (Dulbecco's modified Eagle's media (DMEM) + 10% serum) and serum-free (Neurobasal A + B27) culture media on neuronal morphology was performed using primary rat cortical neurons. Concentration-response assays for neuritogenesis, axon and dendrite outgrowth, and synaptogenesis were performed in each media type using chemicals with previously demonstrated effects. Marked qualitative and quantitative differences in the characteristics of neurons cultured in the two media types were observed, with increased neuronal growth and less basal cell death in Neurobasal A + B27. Media formulation also affected assay sensitivity and selectivity. Increases in assay sensitivity were observed in Neurobasal A + B27 media as compared to serum-supplemented DMEM. In some instances, a greater difference between effective concentrations for cell death and neurodevelopmental-specific endpoints was also observed in Neurobasal A + B27 media as compared to serum-supplemented DMEM. These data show that media formulation must be considered when comparing data for similar endpoints between studies. Neuronal culture maintained in Neurobasal A + B27 media had several features advantageous for HCI applications including less basal cell death, less cell clustering and neurite fasciculation, and a tendency towards increased sensitivity and selectivity in chemical concentration-response studies.

Priming Mesenchymal Stem Cells with Endothelial Growth Medium Boosts Stem Cell Therapy for Systemic Arterial Hypertension

Systemic arterial hypertension (SAH), a clinical syndrome characterized by persistent elevation of arterial pressure, is often associated with abnormalities such as microvascular rarefaction, defective angiogenesis, and endothelial dysfunction. Mesenchymal stem cells (MSCs), which normally induce angiogenesis and improve endothelial function, are defective in SAH. The central aim of this study was to evaluate whether priming of MSCs with endothelial growth medium (EGM-2) increases their therapeutic effects in spontaneously hypertensive rats (SHRs). Adult female SHRs were administered an intraperitoneal injection of vehicle solution (n = 10), MSCs cultured in conventional medium (DMEM plus 10% FBS, n = 11), or MSCs cultured in conventional medium followed by 72 hours in EGM-2 (pMSC, n = 10). Priming of the MSCs reduced the basal cell death rate in vitro. The administration of pMSCs significantly induced a prolonged reduction (10 days) in arterial pressure, a decrease in cardiac hypertrophy, an improvement in endothelium-dependent vasodilation response to acetylcholine, and an increase in skeletal muscle microvascular density compared to the vehicle and MSC groups. The transplanted cells were rarely found in the hearts and kidneys. Taken together, our findings indicate that priming of MSCs boosts stem cell therapy for the treatment of SAH.

Reactive oxygen species initiate luminal but not basal cell death in cultured human mammary alveolar structures: a potential regulator of involution

Post-lactational involution of the mammary gland is initiated within days of weaning. Clearing of cells occurs by apoptosis of the milk-secreting luminal cells in the alveoli and through stromal tissue remodeling to return the gland almost completely to its pre-pregnant state. The pathways that specifically target involution of the luminal cells in the alveoli but not the basal and ductal cells are poorly understood. In this study we show in cultured human mammary alveolar structures that the involution process is initiated by fresh media withdrawal, and is characterized by cellular oxidative stress, expression of activated macrophage marker CD68 and finally complete clearing of the luminal but not basal epithelial layer. This process can be simulated by ectopic addition of reactive oxygen species (ROS) in cultures without media withdrawal. Cells isolated from post-involution alveoli were enriched for the CD49f+ mammary stem cell (MaSC) phenotype and were able to reproduce a complete alveolar structure in subcultures without any significant loss in viability. We propose that the ROS produced by accumulated milk breakdown post-weaning may be the mechanism underlying the selective involution of secretory alveolar luminal cells, and that our culture model represents an useful means to investigate this and other mechanisms further.

Switching of N-Methyl-d-aspartate (NMDA) Receptor-favorite Intracellular Signal Pathways from ERK1/2 Protein to p38 Mitogen-activated Protein Kinase Leads to Developmental Changes in NMDA Neurotoxicity

Excitotoxicity mediated by overactivation of N-methyl-D-aspartate receptors (NMDARs) has been implicated in a variety of neuropathological conditions in the central nervous system (CNS). It has been suggested that N-methyl-D-aspartate (NMDA) neurotoxicity is developmentally regulated, but the definite pattern of the regulation has been controversial, and the underlying mechanism remains largely unknown. Here, we show that NMDA treatment leads to significant cell death in mature (9 and 12 days in vitro) hippocampal neurons or hippocampi of young postnatal day 12 and adult rats but not in immature (3 and 6 days in vitro) neurons or embryonic day 18 and neonatal rat hippocampi. In contrast, NMDA promotes survival of immature neurons against tropic deprivation. Interestingly, it is found that NMDA preferentially activates p38 MAPK in mature neuron and adult rat hippocampus, but it favors ERK1/2 activation in immature neuron and postnatal day 0 rat hippocampus. Moreover, it is shown that NMDA neurotoxicity in mature neuron is mediated via p38 MAPK activation, and neuroprotection in immature neuron is mediated via ERK1/2 activation, whereas all these effects are NR2B-containing NMDAR-dependent, as well as Ca(2+)-dependent. We also revealed that mature and immature neurons showed no difference in the amplitude of NMDA-induced intracellular calcium ([Ca(2+)](i)) increase. However, the basal level of [Ca(2+)](i) is shown to elevate with the maturation of neuron, and this elevation is attributable to the changes in NMDA neurotoxicity but not to the switch of the NMDAR signaling pathway. Taken together, our results suggest that a switch of NMDA receptor-favorite intracellular signal pathways from ERK1/2 to p38 MAPK and the elevated basal level of [Ca(2+)](i) with age might be critical for the developmental changes in NMDA neurotoxicity in the hippocampal neuron.

Peroxiredoxin II Restrains DNA Damage-induced Death in Cancer Cells by Positively Regulating JNK-dependent DNA Repair

The 2-Cys peroxiredoxins (Prx) belong to a family of antioxidant enzymes that detoxify reactive oxygen and nitrogen species and are distributed throughout the intracellular and extracellular compartments. However, the presence and role of 2-Cys Prxs in the nucleus have not been studied. This study demonstrates that the PrxII located in the nucleus protects cancer cells from DNA damage-induced cell death. Although the two cytosolic 2-Cys Prxs, PrxI and PrxII, were found in the nucleus, only PrxII knockdown selectively and markedly increased cell death in the cancer cells treated with DNA-damaging agents. The increased death was completely reverted by the nuclearly targeted expression of PrxII in an activity-independent manner. Furthermore, the antioxidant butylated hydroxyanisole did not influence the etoposide-induced cell death. Mechanistically, the knockdown of Prx II expression impaired the DNA repair process by reducing the activation of the JNK/c-Jun pathway. These results suggest that PrxII is likely to be attributed to a tumor survival factor positively regulating JNK-dependent DNA repair with its inhibition possibly sensitizing cancer cells to chemotherapeutic agents.

Effects of Vanadium Pentoxide on the Histological and Sperm Parameters of Male Guinea Pigs

The pharmacological effects of intraperitoneal administration of different doses of vanadium pentoxide (V2O5) on the histological and sperm parameters of male guinea pigs were investigated. Also investigatedwere the effects of oral pretreatment with different doses of vitamin E (a known protein kinase C inhibitor) on the V2O5 -induced responses of the testis and liver of male guinea pigs. In n = 5 experiments, vanadium pentoxide in the dose range of 4.5-12.5mg/kg caused destruction of the testicular and liver architecture. This was characterized by areduction in spermatogonia, destruction of seminiferous tubules, necrosis of the testicular tissues, necrosis of liver cells, fatty cells infiltration and vacoulation. Oral administration/ pretreatment with vitamin E in the dose range of 500-2000I.U caused a reversal of the vanadium pentoxide –induced histological damages of the testis and the liver cells. Furthermore, in n = 5 animals experiments, Vanadium pentoxide (4.5-12.5 mg/kg/) caused a statistically significant increase in the percentage basal cell death, from 5.0 to 75.0 ± 1.0%, reduction in sperm motility from 90.0 to 31.0 ± 3.9%, reduction in sperm count from 80.0 x106cells/ml to 25.0± 4.0 x 106cells/ml and alteration in the spermatic cell morphology ( i.e. causing a change in the cellular structure of sperm cells and an increase in abnormal cells count) of the male guinea pigs. These inhibitory effects were significant at P < 0.05 (ANOVA). These effects were all dose- and time-dependent and may have a role in oxidative pathology of vanadium pentoxide.

14-3-3 Mediated regulation of the tumor suppressor protein, RASSF1A

Death receptor-dependent apoptosis is an important mechanism of growth control. It has been demonstrated that Ras association domain family protein 1A (RASSF1A) is a tumor suppressor protein involved in death receptor-dependent apoptosis. However, it is unclear how RASSF1A-mediated cell death is initiated. We have now detailed 14-3-3 dependent regulation of RASSF1A-mediated cell death. We demonstrate that basal association of RASSF1A with 14-3-3 was lost following stimulation with tumor necrosis factor alpha (TNFalpha) or TNFalpha related apoptosis inducing ligand (TRAIL). Subsequent to the loss of 14-3-3 association, RASSF1A associated with modulator of apoptosis (MOAP-1) followed by death receptor association with either TNFalpha receptor 1 (TNF-R1) or TRAIL receptor 1 (TRAIL-R1). 14-3-3 association required basal phosphorylation by the serine/threonine kinase, glycogen synthase kinase 3beta (GSK-3beta), on serine 175, 178, and 179. Mutation of these critical serines resulted in the loss of 14-3-3 association and earlier recruitment of RASSF1A to MOAP-1, TNF-R1, and TRAIL-R1. Furthermore, stable cells containing a triple serine mutant of RASSF1A [serine (S) 175 to alanine (A) [S175A], S178A, and S179A] resulted in increased basal cell death, enhanced Annexin V staining and enhanced cleavage of poly (ADP-ribose) polymerase (PARP) following TNFalpha stimulation when compared to stable cells containing wild type RASSF1A. RASSF1A-mediated cell death is, therefore, tightly controlled by 14-3-3 association.

Regional differences in the vulnerability of substantia nigra dopaminergic neurons in weaver mice.

Vulnerability of midbrain dopaminergic (DA) neurons in the weaver mouse was studied at postnatal (P) days 8 and 90, in chosen coronal levels throughout the anteroposterior (AP) extent of the substantia nigra pars compacta (SNc). Wild-type (+/+) and homozygous weaver (wv/wv) mice used were the offspring of pregnant dams injected in several cases with tritiated thymidine on embryonic days 11-15. DA neurons were identified for their tyrosine hydroxylase immunoreactivity. Data reveal that at P8, the frequency of both +/+ and wv/wv late-generated DA cells increases from rostral to caudal SNc. No apparent DA-cell loss was observed at P8 in the mutant genotype, irrespective of the AP level considered. However, throughout the AP, there was a significant reduction in the number of these neurons at any level in 90-day-old weavers. Comparison of P8 and P90 +/+ SNc suggests that cell death is not a major aspect in the developmental regulation of normal DA neurons, although numerical cell depletion in the postnatal development of weaver SNc probably results from the amplification of a basal cell-death process, which affected all the coronal levels studied.

TRAILs and tribulation

TRAILs and tribulation

Molecular determination of laminin‐5 degradation: a biomarker for mustard gas exposure diagnosis and its mechanism of action

Laminin-5, a heterotrimer of laminin alpha3, beta3 and gamma2 subunits, is a component of epithelial cell basement membranes. Laminin-5 functions as a ligand of the alpha3beta1 and alpha6beta4 integrins to regulate cell adhesion, migration and morphogenesis. In the skin, laminin-5 facilitates the assembly of basement membranes; thus it is essential for a stable attachment of the epidermis to the dermis and recovery of damaged skin. Sulphur mustard (SM), also known as mustard gas, is a vesicant that has been employed as a chemical weapon in various conflicts during the twentieth century. Skin exposure to SM results in fluid-filled blisters; proposed mechanisms are inflammation, protease stimulation, basal cell death and separation of the epidermis from the dermis apparently because of the degradation of attachment proteins like laminin-5. Therefore, we investigated the effects of SM exposure on the degradation of laminin-5 and its three subunits, alpha3, beta3 and gamma2 by exposing normal human epidermal keratinocytes (NHEK) to SM (0-300 microM, 1-24 h). We found that SM degraded laminin-5 and its two subunits beta3 and gamma2, but not alpha3. Preincubation of cells with a serine protease inhibitor (PMSF), or a metalloprotease inhibitor (1,10-phenanthroline) prior to SM exposure partially prevented SM-induced degradation of laminin-5 subunits, beta3 and gamma2. Specificity studies showed that the degradation of laminin-5 gamma2 was due to a bifunctional mustard compound such as SM, but not due to the other alkylating agents tested. Our results support that laminin-5 degradation is an important mechanism of SM injury as well as a useful biomarker of SM exposure. The knowledge of the mechanisms of laminin-5 degradation in SM-exposed NHEK has potential application in developing cutaneous therapeutics against SM.

Neurosteroids in the fetus and neonate: Potential protective role in compromised pregnancies

Complications during pregnancy and birth asphyxia lead to brain injury, with devastating consequences for the neonate. In this paper we present evidence that the steroid environment during pregnancy and at birth aids in protecting the fetus and neonate from asphyxia-induced injury. Earlier studies show that the placental progesterone production has a role in the synthesis and release of neuroactive steroids or their precursors into the fetal circulation. Placental precursor support leads to remarkably high concentrations of allopregnanolone in the fetal brain and to a dramatic decline with the loss of the placenta at birth. These elevated concentrations influence the distinct behavioral states displayed by the late gestation fetus and exert a suppressive effect that maintains sleep-like behavioral states that are present for much of fetal life. This suppression reduces CNS excitability and suppresses excitotoxicity. With the availability of adequate precursors, mechanisms within the fetal brain ultimately control neurosteroid levels. These mechanisms respond to episodes of acute hypoxia by increasing expression of 5α-reductase and P450scc enzymes and allopregnanolone synthesis in the brain. This allopregnanolone response, and potentially that of other neurosteroids including 5α-tetrahydrodeoxycorticosterone (TH-DOC), reduces hippocampal cell death following acute asphyxia and suggests that stimulation of neurosteroid production may protect the fetal brain. Importantly, inhibition of neurosteroid synthesis in the fetal brain increases the basal cell death suggesting a role in controlling developmental processes late in gestation. Synthesis of neurosteroid precursors in the fetal adrenal such as deoxycorticosterone (DOC), and their conversion to active neurosteroids in the fetal brain may also have a role in neuroprotection. This suggests that the adrenal glands provide precursor DOC for neurosteroid synthesis after birth and this may lead to a switch from allopregnanolone alone to neuroprotection mediated by allopregnanolone and TH-DOC.

Loss of Caspase-9 Provides Genetic Evidence for the Type I/II Concept of CD95-mediated Apoptosis

The death receptor CD95 triggers apoptosis upon formation of a death-inducing signaling complex and the activation of caspase-8. Two types of CD95-mediated apoptosis have been distinguished that differ in their efficiency of death-inducing signaling complex formation and the requirement of mitochondria for caspase activation. The validity of the type I/II model, however, has been challenged, as Bcl-2 expression or the use of various CD95 agonists resulted in different apoptosis effects. By identifying a caspase-9-deficient T cell line, we now provide genetic evidence for the two-pathway model of CD95-mediated apoptosis and demonstrate that type II cells strongly depend on caspase-9. Caspase-9-deficient cells revealed strongly impaired apoptosis, caspase activation, and mitochondrial membrane depolarization upon CD95 triggering, whereas, surprisingly, activation of Bak and cytochrome c release were not inhibited. Furthermore, caspase-9-deficient cells did not switch to necrosis, and reconstitution of caspase-9 expression restored CD95 sensitivity. Finally, we also show that different death receptors have a distinct requirement for caspase-9.

Growth hormone releasing peptide‐6 acts as a survival factor in glutamate‐induced excitotoxicity

Chronic systemic treatment given to adult male rats with growth hormone releasing peptide-6, an agonist of the ghrelin receptor, increases insulin-like growth factor I levels in various brain regions, including the hypothalamus and cerebellum. Furthermore, intracellular signalling cascades normally associated with anti-apoptotic actions are activated in the same areas and are coincident with decreased basal cell death. Because abnormally high concentrations of glutamate can lead to overexcitation of neurones leading to cell damage and/or death, we investigated whether administration of growth hormone releasing peptide-6 attenuates monosodium glutamate-induced apoptosis in the rat hypothalamus and cerebellum. Glutamate increased activation of caspase 9 followed by cleavage of caspase 7, which in turn fragmented poly(ADP-ribose) polymerase, terminating in cell death in both the hypothalamus and cerebellum. Growth hormone releasing peptide-6 reversed glutamate-induced cell death by decreasing activation of caspases 9 and 7 and poly(ADP-ribose) polymerase fragmentation. These results provide a better understanding of the neuroprotective role of growth hormone secretagogues and the mechanisms involved.

Neuroprotective actions of endogenous interleukin‐1 receptor antagonist (IL‐1ra) are mediated by glia

The pro-inflammatory cytokine interleukin-1 (IL-1), contributes to neuronal inflammation and cell death induced by ischemia, excitotoxicity, or trauma, while administration of IL-1 receptor antagonist (IL-1ra) reduces neuronal injury. The aim of the present study was to test the hypothesis that endogenous IL-1ra is neuroprotective in vivo and in vitro, and to identify its mechanism of actions. Mice lacking IL-1ra (IL-1ra knock-out (KO]) exhibited a dramatic increase in neuronal injury (3.6-fold increase in infarct size) induced by transient cerebral ischemia compared to wild-type (WT) animals. Basal cell death of cultured cortical neurons from WT and IL-1ra KO was identical, and treatment with NMDA or AMPA (20 microM) increased cell death to the same extent in WT and IL-1ra KO neurons. However, basal and NMDA- or AMPA-induced cells death was significantly higher in glial-neuronal co-cultures from IL-1ra KO than from WT mice. We further showed that pure microglial cultures, but not pure astrocytes cultures, released IL-1ra in response to treatment with conditioned medium from NMDA- or AMPA-treated primary neurons. These results demonstrate that endogenous IL-1ra produced by microglia is neuroprotective in cerebral ischemia or excitotoxicity.

A Convenient Fluorometric Method to Study Sulfur Mustard–Induced Apoptosis in Human Epidermal Keratinocytes Monolayer Microplate Culture

Sulfur mustard [SM; bis-(2-chloroethyl) sulfide], which causes skin blistering or vesication [(1991). Histo- and cytophatology of acute epithelial lesions. In: Papirmeister, B., Feister, A. J., Robinson, S. I., Ford, R. D., eds. Medical Defense Against Mustard Gas: Toxic Mechanisms and Pharmacological Implications. Boca Raton: CRC Press, pp. 43–78.], is a chemical warfare agent as well as a potential terrorism agent. SM-induced skin blistering is believed to be due to epidermal–dermal detachment as a result of epidermal basal cell death via apoptosis and/or necrosis. Regarding the role of apoptosis in SM pathology in animal skin, the results obtained in several laboratories, including ours, suggest the following: 1) cell death due to SM begins via apoptosis that proceeds to necrosis via an apoptotic–necrotic continuum and 2) inhibiting apoptosis decreases SM-induced microvesication in vivo. To study the mechanisms of SM-induced apoptosis and its prevention in vitro, we have established a convenient fluorometric apoptosis assay using monolayer human epidermal keratinocytes (HEK) adaptable for multiwell plates (24-, 96-, or 384-well) and high-throughput applications. This assay allows replication and multiple types of experimental manipulation insister cultures so that the apoptotic mechanisms and the effects of test compounds can be compared statistically. SM affects diverse cellular mechanisms, including endoplasmic reticulum (ER) Ca2 + homeostasis, mitochondrial functions, energy metabolism, and death receptors, each of which can independently trigger apoptosis. However, the biochemical pathway in any of these apoptotic mechanisms is characterized by a pathway-specific sequence of caspases, among which caspase-3 is a key member. Therefore, we exposed 80–90% confluent HEK cultures to SM and monitored apoptosis by measuring the fluorescence generated due to hydrolysis of a fluorogenic caspase-3 substrate (acetyl- or benzyl oxycarbonyl-Asp-Glu-Val-Asp-fluorochrome, also designated as AC-or Z-DEVD- fluorochrome) added to the assay medium. Fluorescence was measured using a plate reader. We used two types of substrates, one (Sigma-Aldrich, CASP-3-F) required cell disruption and the other (Beckman-Coulter CellProbe HT Caspase-3/7 Whole Cell Assay Kit) was cell permeable. The latter substrate was useful in experiments such as determining the time-course of apoptosis immediately following SM exposure without disruption (e.g., due to cell processing). In SM-exposed HEK, fluorescence generated from the fluorogenic caspase-3 substrate hydrolysis increased in a time (0–24 h) and concentration (0.05, 0.1, 0.15, 0.2, 0.3, 0.5 mM) dependent manner. SM caused maximum fluorescence at about 0.5 mM. However, at 2 mM SM, fluorescence decreased compared with 0.5 mM, which remains to be explained. Following 0.3 mM SM exposure, which is considered to be the in vitro equivalent of a vesicating dose in vivo (Smith W. J., Sanders, K. M., Ruddle, S.E., Gross, C. L., (1993). Cytometric analysis of DNA changes induced by sulfur mustard. J. Toxicol.-Cut. Ocular Toxicol. 12(4): 337–347.), a small fluorescence increase was observed at 6 to 8 h, which was markedly higher at 12 h. At 24 h, all SM concentrations increased fluorescence. Fluorescence increase due to SM was prevented 100% by a caspase-3–specific peptide inhibitor AC-DEVD-CHO (acetyl-Asp-Glu-Val-Asp-aldehyde, 0.1 mM), but less effectively by a general caspase inhibitor Z-VAD-FMK (benzyl oxycarbonyl-Val-Ala-Asp-fluoromethylketone, 0.01 mM), indicating that the fluorescence increase was due to caspase-3–mediated apoptosis. These results suggest potential applications of this method to study apoptosis mechanisms involving caspase-3 substrates and possibly those involving other caspase substrates.

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.