Major Apoptosis Pathways Research Articles

Chicken blastoderms and primordial germ cells possess a higher expression of DNA repair genes and lower expression of apoptosis genes to preserve their genome stability

DNA is susceptible to damage by various sources. When the DNA is damaged, the cell repairs the damage through an appropriate DNA repair pathway. When the cell fails to repair DNA damage, apoptosis is initiated. Although several genes are involved in five major DNA repair pathways and two major apoptosis pathways, a comprehensive understanding of those gene expression is not well-understood in chicken tissues. We performed whole-transcriptome sequencing (WTS) analysis in the chicken embryonic fibroblasts (CEFs), stage X blastoderms, and primordial germ cells (PGCs) to uncover this deficiency. Stage X blastoderms mostly consist of undifferentiated progenitor (pluripotent) cells that have the potency to differentiate into all cell types. PGCs are also undifferentiated progenitor cells that later differentiate into male and female germ cells. CEFs are differentiated and abundant somatic cells. Through WTS analysis, we identified that the DNA repair pathway genes were expressed more highly in blastoderms and high in PGCs than CEFs. Besides, the apoptosis pathway genes were expressed low in blastoderms and PGCs than CEFs. We have also examined the WTS-based expression profiling of candidate pluripotency regulating genes due to the conserved properties of blastoderms and PGCs. In the results, a limited number of pluripotency genes, especially the core transcriptional network, were detected higher in both blastoderms and PGCs than CEFs. Next, we treated the CEFs, blastoderm cells, and PGCs with hydrogen peroxide (H2O2) for 1 h to induce DNA damage. Then, the H2O2 treated cells were incubated in fresh media for 3–12 h to observe DNA repair. Subsequent analyses in treated cells found that blastoderm cells and PGCs were more likely to undergo apoptosis along with the loss of pluripotency and less likely to undergo DNA repair, contrasting with CEFs. These properties of blastoderms and PGCs should be necessary to preserve genome stability during the development of early embryos and germ cells, respectively.

Reciprocal amplification of caspase-3 activity by nuclear export of a putative human RNA-modifying protein, PUS10 during TRAIL-induced apoptosis

Pus10 is a pseudouridine synthase present in Archaea and Eukarya, but not in Bacteria and yeast. It has been suggested that the human PUS10 (DOBI) gene is needed during TRAIL-induced apoptosis. We analyzed the role of PUS10 in TRAIL-induced apoptosis by immunofluorescence, immunoblotting and several indicators of apoptosis. We examined several TRAIL-sensitive cell lines and we also examined some resistant cell lines after treatment with cycloheximide. PUS10 is mainly present in the nucleus. Early during apoptosis, PUS10 translocates to mitochondria via CRM1-mediated export with the concurrent release of cytochrome c and SMAC. Caspase-3 is required for PUS10 translocation, which reciprocally amplifies the activity of caspase-3 through the intrinsic/mitochondrial pathway. This suggests that in addition to cytoplasmic factors, nuclear factors also have a direct role in the major apoptosis pathways. However, p53 is not involved in TRAIL-induced PUS10 movement. The caspase-3-mediated movement of PUS10 and the release of mitochondrial contents enhancing caspase-3 activity creates a feedback amplification loop for caspase-3 action. Therefore, any defect in the movement or interactions of PUS10 would reduce the TRAIL sensitivity of tumor cells.

Flavored little cigar smoke induces cytotoxicity and apoptosis in airway epithelia

Addition of flavors reduces the harsh taste of tobacco, facilitating the initiation and maintenance of addiction among youths. Flavored cigarettes (except menthol) are now banned. However, the legislation on little cigars remains unclear and flavored little cigars are currently available for purchase. Since inhaled tobacco smoke directly exerts toxic effects on the lungs, we tested whether non-flavored and flavored little cigar smoke exposure had the potential for harm in cultured pulmonary epithelia. We cultured Calu-3 lung epithelia on both 96-well plates and at the air–liquid interface and exposed them to smoke from non-flavored Swisher Sweets and flavored (sweet cherry, grape, menthol, peach and strawberry) Swisher Sweets little cigars. Irrespective of flavor, acute little cigar smoke exposure (10×35 ml puffs) significantly increased cell death and decreased the percentage of live cells. Chronic exposure (10×35 ml puffs per day for 4 days) of smoke to Calu-3 cultures significantly increased lactate dehydrogenase release, further indicating toxicity. To determine whether this exposure was associated with increased cell death/apoptosis, a protein array was used. Chronic exposure to smoke from all types of little cigars induced the activation of the two major apoptosis pathways, namely the intrinsic (mitochondrial-mediated) and the extrinsic (death receptor-mediated) pathways. Both flavored and non-flavored little cigar smoke caused similar levels of toxicity and activation of apoptosis, suggesting that flavored and non-flavored little cigars are equally harmful. Hence, the manufacture, advertisement, sale and use of both non-flavored and flavored little cigars should be strictly controlled.

Colistin-induced nephrotoxicity in mice involves the mitochondrial, death receptor, and endoplasmic reticulum pathways.

Nephrotoxicity is the dose-limiting factor for colistin, but the exact mechanism is unknown. This study aimed to investigate the roles of the mitochondrial, death receptor, and endoplasmic reticulum pathways in colistin-induced nephrotoxicity. Mice were intravenously administered 7.5 or 15 mg of colistin/kg of body weight/day (via a 3-min infusion and divided into two doses) for 7 days. Renal function, oxidative stress, and apoptosis were measured. Representative biomarkers involved in the mitochondrial, death receptor, and endoplasmic reticulum pathways were investigated, and the key markers involved in apoptosis and autophagy were examined. After 7-day colistin treatment, significant increase was observed with blood urea nitrogen, serum creatinine, and malondialdehyde, while activities of superoxide dismutase (SOD) and catalase decreased in the kidneys. Acute tubular necrosis and mitochondrial dysfunction were detected, and colistin-induced apoptosis was characterized by DNA fragmentation, cleavage of poly(ADP-ribose) polymerase (PARP-1), increase of 8-hydroxydeoxyguanosine (8-OHdG), and activation of caspases (caspase-8, -9, and -3). It was evident that colistin-induced apoptosis involved the mitochondrial pathway (downregulation of Bcl-2 and upregulation of cytochrome C [cytC] and Bax), death receptor pathway (upregulation of Fas, FasL, and Fas-associated death domain [FADD]), and endoplasmic reticulum pathway (upregulation of Grp78/Bip, ATF6, GADD153/CHOP, and caspase-12). In the 15-mg/kg/day colistin group, expression of the cyclin-dependent kinase 2 (CDK2) and phosphorylated JNK (p-JNK) significantly increased (P < 0.05), while in the 7.5-mg/kg/day colistin group, a large number of autophagolysosomes and classic autophagy were observed. Western blot results of Beclin-1 and LC3B indicated that autophagy may play a protective role in colistin-induced nephrotoxicity. In conclusion, this is the first study to demonstrate that all three major apoptosis pathways and autophagy are involved in colistin-induced nephrotoxicity.

Apoptoza limfocytów pęcherzykowych. Część 1—Szlaki apoptozy limfocytów

Apoptosis is a form of programmed cell death essential for maintaining homeostasis, including onset, progress and resolution of immune reactions. Two major apoptosis pathways: extrinsic (mediated by death receptors) and intrinsic (mitochondrial), were distinguished. Lymphocytes with cytotoxic activity may also initiate apoptosis of target cells by granzyme/perforin (pseudoreceptor) pathway. The specific apoptotic processes, i.e. activation induced cell death (AICD) and neglect induced death (NID), are types of extrinsic and intrinsic pathways, respectively. They both seem to be crucial in apoptosis of antigen-primed T cells during the contraction phase of inflammation. Alveolar lymphocytes (AL) are almost exclusively T effector cells, engaged in interstitial lung disease (ILD) pathophysiologies. The AL numbers in lower airways depends on recruitment to the lung, proliferation and local apoptosis. According to the references, it should be noted that AL usually do not proliferate in alveoli; their apoptosis rate accounts, on average, for 1% of cells in healthy subjects, and this is significantly decreased in disorders with lymphocytic alveolitis such as sarcoidosis and extrinsic allergic alveolitis (EAA). The mechanisms of AL apoptosis have not been completely explained. However, it is the NID process that is probably critical for the culling of T-cell response, as in EAA or sarcoidosis remission, with AICD as an auxiliary and/or modulating mechanism only. It should be emphasised that many ILDs are chronic disorders with no remission or improvement, and it is difficult to describe the AL response in terms of immune expansion/contraction.

Apaf-1- and Caspase-8-independent apoptosis

Two major apoptosis pathways, the mitochondrial and death receptor pathways, are well recognized. Here we established cell lines from the fetal thymus of Apaf-1-, Caspase-9-, or Bax/Bak-deficient mice. These cell lines were resistant to apoptosis induced by DNA-damaging agents, RNA or protein synthesis inhibitors, or stress in the endoplasmic reticulum. However, they underwent efficient apoptosis when treated with kinase inhibitors such as staurosporine and H-89, indicating that these inhibitors induce a caspase-dependent apoptosis that is different from the mitochondrial pathway. CrmA, a Caspase-8 inhibitor, did not prevent staurosporine-induced apoptosis of fetal thymic cell lines, suggesting that the death receptor pathway was also not involved in this process. The staurosporine-induced cell death was inhibited by okadaic acid, a serine/threonine phosphatase inhibitor, suggesting that dephosphorylation of a proapoptotic molecule triggered the death process, or that phosphorylation of an antiapoptotic molecule could block the process. Cells of various types (fetal thymocytes, bone marrows, thymocytes, and splenocytes), but not embryonic fibroblasts, were sensitive to the noncanonical staurosporine-induced apoptosis, suggesting that the noncanonical apoptosis pathway is tissue specific.

MiR-133b Targets Antiapoptotic Genes and Enhances Death Receptor-Induced Apoptosis

Despite the importance of microRNAs (miRs) for regulation of the delicate balance between cell proliferation and death, evidence for their specific involvement during death receptor (DR)-mediated apoptosis is scarce. Transfection with miR-133b rendered resistant HeLa cells sensitive to tumor necrosis factor-alpha (TNFα)-induced cell death. Similarly, miR-133b caused exacerbated proapoptotic responses to TNF-related apoptosis-inducing ligand (TRAIL) or an activating antibody to Fas/CD95. Comprehensive analysis, encompassing global RNA or protein expression profiling performed by microarray experiments and pulsed stable isotope labeling with amino acids in cell culture (pSILAC), led to the discovery of the antiapoptotic protein Fas apoptosis inhibitory molecule (FAIM) as immediate miR-133b target. Moreover, miR-133b impaired the expression of the detoxifying protein glutathione-S-transferase pi (GSTP1). Expression of miR-133b in tumor specimens of prostate cancer patients was significantly downregulated in 75% of the cases, when compared with matched healthy tissue. Furthermore, introduction of synthetic miR-133b into an ex-vivo model of prostate cancer resulted in impaired proliferation and cellular metabolic activity. PC3 cells were also sensitized to apoptotic stimuli after transfection with miR-133b similar to HeLa cells. These data reveal the ability of a single miR to influence major apoptosis pathways, suggesting an essential role for this molecule during cellular transformation, tumorigenesis and tissue homeostasis.

Proteomic and transcriptomic study on the action of a cytotoxic saponin (Polyphyllin D): Induction of endoplasmic reticulum stress and mitochondria‐mediated apoptotic pathways

Polyphyllin D (PD) is a potent cytotoxic saponin found in Paris polyphylla. In the present study, bioinformatic, proteomic and transcriptomic analyses were performed to study the mechanisms of action of PD on human nonsmall cell lung cancer (NSCLC) cell line (NCI-H460). Using a gene expression-based bioinformatic tool (connectivity map), PD was identified as a potential ER stress inducer. Our proteomic and transcriptomic analyses revealed that PD treatment led to upregulation of typical ER stress-related proteins/genes including glucose-regulated protein 78 (BiP/GRP78) and protein disulfide isomerase (PDI). In particular, elevated expression of C/EBP homologous transcription factor (chop) and activation of caspase-4 occurred at early time point (8 h) of PD treatment, signifying an initial ER stress-mediated apoptosis. Induction of tumor suppressor p53, disruption of mitochondrial membrane, activation of caspase-9 and caspase-3 were detected upon prolonged PD treatment. Collectively, these data revealed that PD induced the cytotoxic effect through a mechanism initiated by ER stress followed by mitochondrial apoptotic pathway. The ability of activating two major pathways of apoptosis makes PD an attractive drug lead for anticancer therapeutics.

Molecular Mechanism of Apoptosis Induction by Resveratrol, a Natural Cancer Chemopreventive Agent

In efforts to identify naturally occurring compounds that act as protective agents, resveratrol, a phytoalexin existing in wine, has attracted much interest because of its diverse pharmacological characteristics. Considering that apoptosis induction is the most potent defense approach for cancer treatment, we have tried to summarize our present understanding of apoptosis induction by resveratrol based on the two major apoptosis pathways.

Cellular FLIP can substitute for the herpes simplex virus type 1 latency-associated transcript gene to support a wild-type virus reactivation phenotype in mice

Latency-associated transcript (LAT) deletion mutants of herpes simplex virus type 1 (HSV-1) have reduced reactivation phenotypes. Thus, LAT plays an essential role in the latency-reactivation cycle of HSV-1. We have shown that LAT has antiapoptosis activity and demonstrated that the chimeric virus, dLAT-cpIAP, resulting from replacing LAT with the baculovirus antiapoptosis gene cpIAP, has a wild-type HSV-1 reactivation phenotype in mice and rabbits. Thus, LAT can be replaced by an alternative antiapoptosis gene, confirming that LAT's antiapoptosis activity plays an important role in the mechanism by which LAT enhances the virus' reactivation phenotype. However, because cpIAP interferes with both of the major apoptosis pathways, these studies did not address whether LAT's proreactivation phenotype function was due to blocking the extrinsic (Fas-ligand-, caspase-8-, or caspase-10-dependent pathway) or the intrinsic (mitochondria-, caspase-9-dependent pathway) pathway, or whether both pathways must be blocked. Here we constructed an HSV-1 LAT(-) mutant that expresses cellular FLIP (cellular FLICE-like inhibitory protein) under control of the LAT promoter and in place of LAT nucleotides 76 to 1667. Mice were ocularly infected with this mutant, designated dLAT-FLIP, and the reactivation phenotype was determined using the trigeminal ganglia explant model. dLAT-FLIP had a reactivation phenotype similar to wild-type virus and significantly higher than the LAT(-) mutant dLAT2903. Thus, the LAT function responsible for enhancing the reactivation phenotype could be replaced with an antiapoptosis gene that primarily blocks the extrinsic signaling apoptosis pathway.

Apoptosis Induced by Extracts of Helleborus Niger in Different Lymphoma and Leukemia Cell Lines and Primary Lymphoblasts of Children with ALL Is Independent of Smac-Overexpression and Executed Via the Mitochondrial Pathway.

Helleborus niger, also known as Christmas Rose, belongs to the family of Ranunculaceae, a family of flowering plants with about 2500 different species. In complementary medicine Helleborus niger is used as adjuvant drug in the treatment of non-metastasised and metastasised forms of bronchial cancer, abdominal tumours and prostate cancer. It is also applied in myeloproliferative diseases like Hodgkin and Non-Hodgkin lymphoma, leukaemic disorders and AIDS- related diseases like the Kaposi sarcoma. Until now, there is no clinical or preclinical data regarding the effects of Helleborus niger in vivo, ex vivo or in vitro. For this purpose, we investigated the cytotoxic effects of four different standardized aqueous Helleborus niger extracts from the companies Hiscia and Helixor on various cancer cell lines. We used one whole plant extract, one root extract, one leave extract and one containing only the blossom of Helleborus niger. After 4h of treatment with the extracts no significant LDH release was measured, thus excluding an unspecific, necrotic damage of the cell membrane. After 24h a dose dependent inhibition of proliferation up to 69% could be found and after 48h a distinction into early (45,2%) and late apoptotic (45,5%) cells was detected via Annexin/PI staining. The cell cycle analysis revealed characteristic hypodiploid DNA fragments after 72h, once more identifying apoptosis as cause of the cell death. In the Western Blot analysis a processing of Caspase-3 could be found after 36 h incubation with the extract. Apoptotic cell death was detected in the Burkitt-like lymphoma cell line BJAB, the three human acute lymphoblastic leukemia cell lines NALM-6, Sup-B-15 and REH and the melanoma cell line MEL-HO. The apoptosis induction caused by the root extract was higher than the apoptotic cell death in the other extracts. There are two major pathways of apoptosis, the extrinsic pathway via death receptors like FADD and the intrinsic pathway via the mitochondria. In BJAB cells a breakdown of the mitochondrial membrane potential and dose-dependent mitochondrial permeability transition was detected after 48h, revealing that apoptosis is executed via the mitochondrial pathway. Furthermore, we found a decreased apoptosis induction in BCL-2 overexpressing melanoma cells. The dependency of Bcl-2 expression is another sign of apoptosis via the mitochondrial pathway. In contrast, apoptosis induction by Helleborus niger seems to be independent of Smac overexpression, which could be shown in Jurkat cells. In combination with the vinca alkaloid vincristine, which is used in the treatment of ALL, a synergistic effect could be detected. The apoptosis induction was up to 16% higher in combination than in the single treatment. Finally, we evaluated the effect on primary leukemia cells ex vivo. Interestingly, we could show a significant apoptosis induction in primary leukemia cells from 2 patients with ALL or AML in childhood, which were resistant to the treatment with the anthracycline doxorubicin. For the first time, we were able to show that extracts of Helleborus niger induce apoptosis in different cancer cell lines and primary leukemia cells. Apoptosis is executed via the intrinsic pathway and is independent of Smac overexpression. Thus, we present an interesting baseline for the design of upcoming in vivo experiments or clinical trials.

A focus on cell proliferation and death

As you have noticed, the Editorial Office has made major efforts to redesign and improve the journal appearance and contents. This has included the simplification of the submission formats and the introduction of Commentaries to highlight important manuscripts. I believe that this will bring an increased visibility of interesting manuscripts to a broader scientific audience. The September issue starts with another novelty: focusing on a specific topic in a given issue. As these papers are not invited but are regular submissions, focusing on certain topics will depend on the manuscripts we have “in-house.” The September issue presents a collection of manuscripts that are loosely related to DNA analysis, cell proliferation, and death. It starts with an exciting review by the group of Darzynkiewicz (page 648) on ATM activation and histone H2AX phosphorylation to determine DNA damage by cytometric tools. The review elegantly shows the value of flow- and image-cytometry in studying factors that affect DNA repair and explore the linkage between DNA damage, cell cycle checkpoints, and initiation of apoptosis. King et al. (page 668) investigated apoptosis induction in Jurkat cells using three different flow cytometric methods: cytochrome c release, loss of mitochondrial cardiolipin, and decrease of mitochondrial membrane potential. They demonstrated that in their model system, cytochrome c release is a good marker but underestimates the fraction of apoptotic cells. Hollier et al. (page 675) evaluated two assays for apoptosis detection in clinical samples. They proposed two independent novel assays that enable determination of not only the major apoptosis pathways but also the cell types affected. Shi et al. (page 686) investigated the influence of over-expression of glutamate-cysteine ligase (GCL) on the resistance of single-strand breaks to peroxide treatment in two cell lines. Their study yielded the important finding that genetic factors affecting GCL expression have an impact on DNA single-strand breaks and thus affect susceptibility towards cancer. In the study of Slaninova et al. (page 700), quaternary benzo[c]phenanthridine binding to DNA of viable cells was demonstrated. Because of their fluorescent characteristics, the nuclear architecture, chromosomes, and apoptotic fragments could be visualized in fluorescence microscopy and flow cytometry. The authors also stated that a cell cycle analysis would be possible by using the alkaloid macarpine. Intracellular Glutathione (GSH) is attracting attention in the investigation of many diseases due to its role in cell protection. Hamilton et al. (page 709) focused on rapid assessment of GSH levels in clinical settings. Using three different drug treatments they demonstrated that the changes in GSH concentration could be detected semi-quantitatively by CM-SNARF. Quantitation of DNA content and cell cycle are prerequisites for the analysis of changes in the cell cycle. Wang and Huang (page 716) proposed a mixture-model classification for DNA content analysis. They demonstrated in a model system the sensitivity of their approach for detecting cell cycle differences in mixed populations. Lin et al. (page 724) showed a multi-model approach for quantitative studies of 3D confocal microscope images. With their method, nuclear segmentation and classification of heterogenous populations of cell nuclei—like in tissues with multiple cell types and different nuclear features—could be analyzed simultaneously. Since its early years, the journal Cytometry stood out by publishing groundbreaking technologies like DNA flow cytometry of fresh-frozen (1) or paraffin embedded tissues (2), both being standard methods today. Over the last few years new methods, in particular analysis of apoptosis (3), have been proposed. Such novel techniques are released regularly in our journal and obtain the status of standard protocols. These papers belong to the most cited ones in Cytometry. As an example, the paper by Vindelov et al. (1) has been cited over 1,500 times. Tissue Cytometry was introduced in Cytometry Part A as an alternative method to perform quantitative analysis in sectioned material (4). Supporting this concept Mosch et al. (5) proposed quantitative DNA cell cycle analysis of brain sections by Slide Based Cytometry (SBC). Recently, the group clearly demonstrated that SBC analyses of the cell cycle distribution are equal to other independent molecular biological methods (6). This highly emphasizes the great relevance of quantitative imaging and automated analysis of the cell cycle in tissues. The journal Cytometry was in the past and will be in the future an important source for biomedical research and novel technologies for determining cell cycle and death in a quantitative fashion as demonstrated by the manuscripts of this issue. I hope to see your exciting research sent to our journal in the future.

Two optimized combination assays to examine apoptosis pathways in clinical samples

A consequence of a number of diseases is an alteration in apoptosis. Currently, there is no single assay that measures the main stages of apoptosis, requiring that multiple assays be performed. This hinders studies on clinical samples that have limited cell numbers. Our objective was to combine and optimize assays that target specific stages of apoptosis for use in a typical clinical blood sample. Two flow cytometric assays were developed for use on peripheral blood mononuclear cells (PBMC) collected in two 8-ml tubes from a single draw. One measures caspase-12 activity, the level of active caspase-3 and DNA fragmentation. The second assesses depolarization of the mitochondria and phosphatidylserine externalization. Cell populations present within the samples were determined by flow cytometry. Apoptosis was validated by ELISA. Each assay was optimized for use with cell numbers and sample volumes typical of clinical blood samples. Each combination assay effectively distinguished apoptotic from nonapoptotic blood cells. This combined optimized method comprised of two independent assays makes it possible to assay the major pathways of apoptosis in addition to determining the blood cell subsets that are affected.

The Secreted Protease Factor CPAF Is Responsible for Degrading Pro-apoptotic BH3-only Proteins in Chlamydia trachomatis-infected Cells

The Secreted Protease Factor CPAF Is Responsible for Degrading Pro-apoptotic BH3-only Proteins in Chlamydia trachomatis-infected Cells

Life and death of lymphocytes: a role in immunesenescence.

Human aging is associated with progressive decline in immune functions, increased frequency of infections. Among immune functions, a decline in T cell functions during aging predominates. In this review, we will discuss the molecular signaling in two major pathways of apoptosis, namely death receptor pathway and mitochondrial pathway, and their alterations in both T and B lymphocytes in human aging with a special emphasis on naïve and different memory subsets of CD8+ T cells. We will also discuss a possible role of lymphocyte apoptosis in immune senescence.

Apoptosis: Programmed cell death at a molecular level

Objectives: To characterize cell surface receptors, their ligands, and their proteins in the 2 major pathways of apoptosis; the components that promote/suppress these interactions; the noninflammatory removal of apoptotic bodies by dendritic cells; and methods of assay in studies of cell death. To describe: how deregulation of apoptosis may contribute to autoimmunity, cancer, and neurodegenerative disorders and strategies some viruses have evolved that interfere with the host's apoptotic pathways. Methods: The authors reviewed and compiled literature on the extrinsic (tumor necrosis factor [TNF] receptor superfamily and ligands) and intrinsic (mitochondria-associated) apoptotic pathways, the pro- and antiapoptotic proteins of the B-cell follicular lymphoma (Bcl)-2 family, the nuclear factor (NF)-κB family of proteins, commonly used laboratory methods to distinguish apoptosis from necrosis, the recognition and removal by phagocytosis of apoptotic cells by dendritic cells, and viral strategies to avoid a host's apoptotic response. Results: The 2 major pathways of apoptosis are (1) FasL and other TNF superfamily ligands induce trimerization of cell-surface death receptors and (2) perturbated mitochondria release cytochrome c, the flavoprotein apoptosis-inducing factor, and second mitochondria-derived activator of caspases/DIABLO (a protein that directly neutralizes inhibitors of apoptotic proteins and activates proteases). Catalytically inactive cysteine proteases, called caspases, and other proteases are activated, ultimately leading to cell death with characteristic cellular chromatin condensation and DNA cleavage to fragments of approximately 180 bp. The inhibitory/promoting action of Bcl-2 family members is involved in the release of cytochrome c, an essential factor for the mitochondrial-associated pathway. A balance between inhibition/promotion determines a cell's fate. The NF-κB family in the cytoplasm of cells activates various genes carrying the NF-κB response element, such as members of the inhibitor of apoptotic proteins family. A few of the more common methods to detect apoptotic cell death are described, which use immunochemical, morphologic and flow cytometric methods, and genetic markers. Exposed phosphatidylserine at the outer leaflet of the plasma membrane of the apoptotic cell serves as a possible receptor for phagocytosis by immature dendritic cells. These cells phagocytize both apoptotic and necrotic cells, but only the latter induce maturation to become fully functional antigen-presenting cells. Viral inhibitors of apoptosis allow increased virus replication in cells, possibly resulting in their oncogenicity. Conclusions: Balanced apoptosis is crucial in development and homeostasis, and all multicellular organisms have a physiologically programmed continuum of pathways to apoptotic cell death. Further studies of the control at the molecular level of key components and promoters/suppressors of apoptosis may provide better approaches to treatment of autoimmune diseases, malignancies, and neurodegenerative disorders. Many important questions remain regarding the advantages of modifying apoptotic programs in clinical situations. Semin Arthritis Rheum 32:345-369. © 2003 Elsevier Inc. All rights reserved.

Tumor regression mechanisms by IL-13 receptor-targeted cancer therapy involve apoptotic pathways.

IL-13 cytotoxin, composed of IL-13 and a truncated form of Pseudomonas exotoxin, targets IL-13R-overexpressing tumor cell lines in vitro and in vivo. To reveal the molecular mechanism of IL-13 cytotoxin-induced cell death in vivo, we demonstrate activation of apoptotic pathways in 2 s.c. growing human SCCHN tumor models in immunodeficient mice after i.t. administration of IL-13 cytotoxin. Treatment of HN12 tumor bearing mice with i.p. or i.t. administration of IL-13 cytotoxin mediated marked regression of established tumors with complete remission. Interestingly, after a single i.t. administration, IL-13 cytotoxin disappeared within 6 hr but accumulation of caspase-3, -8 and -9 and cleavage of procaspase-3 and PARP continued within the tumors for a prolonged period. We further demonstrate that IL-13 cytotoxin also utilizes an alternate pathway of cell death via the release of cytochrome c from mitochondria to the cytosol. Our results indicate that IL-13 cytotoxin induces 2 major pathways of apoptosis, which may play a role in tumor regression. In addition, apoptotic molecules may serve as surrogate molecular markers of tumor response to IL-13R-directed cytotoxin therapy.

2001 Warkany lecture: to die or not to die, the role of apoptosis in normal and abnormal mammalian development.

Cell death is a common and reproducible feature of the development of many mammalian tissues/organs. Two well-known examples of programmed cell death (PCD) are the cell deaths associated with fusion of the neural folds and removal of interdigital mesenchymal cells during digit formation. Like normal development, abnormal development is also associated with increased cell death in tissues/organs that develop abnormally after exposure to a wide variety of teratogens. At least in some instances, teratogens induce cell death in areas of normal PCD, suggesting that there is a link between programmed and teratogen-induced cell death. Although researchers recognized early on that cell death is an integral part of both normal and abnormal development, little was known about the mechanisms of cell death. In 1972, Kerr et al. ('72) showed conclusively that cell deaths, induced in a variety of contexts, followed a reproducible pattern, which they termed apoptosis. The next breakthrough came in the 1980s when Horvitz and his colleagues identified specific cell death genes (ced) that controlled PCD in the roundworm, Caenorhabditis elegans (C. elegans). Identification of ced genes in the roundworm quickly led to the isolation of their mammalian homologues. Subsequent research in the 1990s led to the identification of a cadre of proteins controlling cell death in mammals, i.e., receptors/ligands, caspases, cytochrome c, Apaf-1, Bcl-2 family proteins, and IAPs. Two major pathways of apoptosis have now been elucidated, the receptor-mediated and the mitochondrial apoptotic pathways. The latter pathway, induced by a wide variety of toxic agents, is activated by the release of cytochrome c from mitochondria. Cytochrome c then facilitates the activation of a caspase cascade involving caspase-9 and -3. Activation of these caspases results in the cleavage of a variety of cellular proteins leading to the orderly demise of the cell. Work from my laboratory in the last 5 years has shown that teratogens, such as hyperthermia, 4-hydroperoxycyclophosphamide, and staurosporine, induce cell death in day 9 mouse embryos by activating the mitochondrial apoptotic pathway, i.e., mitochondrial release of cytochrome c, activation of caspase-9 and -3, inactivation of poly (ADP-ribose) polymerase (PARP), and systematic degradation of DNA. Our work, as well as the work of others, has also shown that different tissues within the early post implantation mammalian embryo are differentially sensitive to the cell death inducing potential of teratogens, from exquisite sensitivity of cells in the developing central nervous system to complete resistance of cells in the developing heart. More importantly, we have shown that the resistance of heart cells is directly related to the failure to activate the mitochondrial apoptotic pathway in these cells. Thus, whether a cell dies in response to a teratogen and therefore contributes to the pathogenesis culminating in birth defects, depends, at least in part, by the cell's ability to regulate the mitochondrial apoptotic pathway. Future research aimed at understanding this regulation should provide insight not only into the mechanism of teratogen-induced cell death but also the role of cell death in the genesis of birth defects.

Regulation of caspase 8- and caspase 9-induced apoptosis by the herpes simplex virus type 1 latency-associated transcript.

The latency-associated transcript (LAT) is the only herpes simplex virus type 1 (HSV-1) gene that is abundantly transcribed during latency. Plasmids expressing LAT inhibit apoptosis induced by etoposide and ceramide in transiently transfected cells. LAT also inhibits apoptosis in trigeminal ganglia of rabbits and promotes spontaneous reactivation, suggesting these events are coupled. In this study, we compared caspase cleavage (activation) in cells infected with dLAT2903 (LAT-null mutant) versus wild-type McKrae or the rescued LAT-null mutant (dLAT2903R). Neuro-2A cells (mouse neuroblastoma), but not NIH3T3 cells infected with dLAT2903, contained higher levels of cleaved caspase 9 compared to cells infected with McKrae. Cleaved caspase 9 was also readily detected in neuro-2A cells, but not NIH3T3 cells, after ultraviolet (UV) light treatment, suggesting that the ability of cells to process caspases and undergo apoptosis influences the antiapoptotic properties of LAT. HSV-1 expresses numerous genes in addition to LAT that can block apoptosis during productive infection of cultured cells. Because these genes may mask the effects of LAT on apoptosis, transient transfection assays were performed to test whether LAT can inhibit caspase 8- and caspase 9-induced apoptosis. A plasmid expressing nucleotides 1 to 4658 of LAT efficiently inhibited caspase 8- and caspase 9-induced apoptosis in transiently transfected neuro-2A cells. These studies indicate that LAT has the potential to inhibit the two major pathways of apoptosis in the absence of other viral genes. Furthermore, these studies support a role for the antiapoptotic properties of LAT in the latency-reactivation cycle.

Signal transduction mediated by Bid, a pro-death Bcl-2 family proteins, connects the death receptor and mitochondria apoptosis pathways.

Two major apoptosis pathways have been defined in mammalian cells, the Fas/TNF-R1 death receptor pathway and the mitochondria pathway. The Bcl-2 family proteins consist of both anti-apoptosis and pro-apoptosis members that regulate apoptosis, mainly by controlling the release of cytochrome c and other mitochondrial apoptotic events. However, death signals mediated by Fas/TNF-R1 receptors can usually activate caspases directly, bypassing the need for mitochondria and escaping the regulation by Bcl-2 family proteins. Bid is a novel pro-apoptosis Bcl-2 family protein that is activated by caspase 8 in response to Fas/TNF-R1 death receptor signals. Activated Bid is translocated to mitochondria and induces cytochrome c release, which in turn activates downstream caspases. Such a connection between the two apoptosis pathways could be important for induction of apoptosis in certain types of cells and responsible for the pathogenesis of a number of human diseases.