Clinical and pathological significance of programmed cell death 1 (PD-1)/programmed cell death ligand 1 (PD-L1) expression in high grade serous ovarian cancer

We investigated programmed cell death 1 (PD-1) / programmed cell death ligand 1 (PD-L1) expression in high grade serous ovarian cancer (HGSOC) and its relationship to tumor infiltrating lymphocytes (TIL) and prognosis. Formalin fixed paraffin embedded (FFPE) samples of 94 HGSOC cases were included in the study. Immunohistochemical analysis (CD3, CD4, CD8, PD-1 and PD-L1) was performed. Samples were analyzed for expression of immune proteins in the peritumoral stromal and intratumoral areas, scored, and expression was correlated with overall survival, stage, and age. PD-L1 staining ratio with a score greater than 0 was found to have lower survival. There were two positive staining patterns, patchy/diffuse and patchy/focal patterns, in 24 (25.5%) cases. Considering the threshold value ≥5%, we demonstrated that the PD-L1 positive cancer cell membrane immunoreactivity rate and patchy/diffuse PD-L1 expression were 9.6% (n = 9). There was statistically significant relationship between high PD-1 scores and PD-L1 cases of ≥ 5%. A statistically significant difference was found between PD-L1 staining and survival in patients with a threshold ≥ 5%. However an appropriate rate for treatment was determined in 9.6% cases. There was a statistically significant correlation between PD-1 positive TIL score and intratumoral CD3, peritumoral stromal CD3, intratumoral CD4 and intratumoral CD8 positive cells. Survival was lower in cases with higher PD-L1 positive stromal TIL score.     

Erythrocyte programmed cell death

Eryptosis, the suicidal death of erythrocytes, is characterised by cell shrinkage, membrane blebbing and cell membrane phospholipid scrambling with phosphatidylserine exposure at the cell surface. Phosphatidylserine-exposing erythrocytes are recognised by macrophages, which engulf and degrade the affected cells. Reported triggers of eryptosis include osmotic shock, oxidative stress, energy depletion, ceramide, prostaglandin E(2), platelet activating factor, hemolysin, listeriolysin, paclitaxel, chlorpromazine, cyclosporine, methylglyoxal, amyloid peptides, anandamide, Bay-5884, curcumin, valinomycin, aluminium, mercury, lead and copper. Diseases associated with accelerated eryptosis include sepsis, malaria, sickle-cell anemia, beta-thalassemia, glucose-6-phosphate dehydrogenase (G6PD)-deficiency, phosphate depletion, iron deficiency, hemolytic uremic syndrome and Wilsons disease. Eryptosis may be inhibited by erythropoietin, adenosine, catecholamines, nitric oxide (NO) and activation of G-kinase. Most triggers of eryptosis except oxidative stress are effective without activation of caspases. Their signalling involves formation of prostaglandin E(2) with subsequent activation of cation channels and Ca2+ entry and/or release of platelet activating factor (PAF) with subsequent activation of sphingomyelinase and formation of ceramide. Ca2+ and ceramide stimulate scrambling of the cell membrane. Ca2+ further activates Ca2+-sensitive K+ channels leading to cellular KCl loss and cell shrinkage and stimulates the protease calpain resulting in degradation of the cytoskeleton. Eryptosis allows defective erythrocytes to escape hemolysis. On the other hand, excessive eryptosis favours the development of anemia. Thus, a delicate balance between proeryptotic and antieryptotic mechanisms is required to maintain an adequate number of circulating erythrocytes and yet avoid noneryptotic death of injured erythrocytes.     

1-Butanol triggers programmed cell death in Populus euphratica cell cultures

1-Butanol, which is a specific inhibitor of phospholipase D, usually inhibits phosphatidic acid (PA) production and blocks the PA-dependent signaling pathway under stress conditions. However, the effects of 1-butanol on plant cells under non-stress condition are still unclear. In this study, we report that 1-butanol induced a dose dependent cell death in poplar (Populus euphratica) cell cultures. In contrast, the control 2-butanol and ethanol had no effects on cell viability. 1-Butanol-treated cells displayed hallmark features of programmed cell death (PCD), such as shrinkage of the cytoplasm, DNA fragmentation, condensed or stretched chromatin and the activation of caspase-3-like protease. Exogenous application of PA markedly inhibited the 1-butanol-induced PCD. 1-Butanol also caused a burst of mitochondrial H₂O₂ ([H₂O₂]_mit) that was usually accompanied by a loss of mitochondrial membrane potential (?Ψm). Supplement of PA, antioxidant enzyme (catalase) and antioxidant (ascorbic acid) reversed this effect. Moreover, a significant increase of nitric oxide (NO) was observed in 1-butanol-treated poplar cells. This NO burst was suppressed by PA or inhibitors of NO synthesis. Further pharmacological experiments indicate that the burst of NO contributed to the 1-butanol-induced inhibition of antioxidant enzymes and subsequent H₂O₂-dependent PCD. In conclusion, we propose that 1-butanol is a potent inducer of PCD in plants and this process is regulated by the PA, NO and H₂O₂.     

Involvement of 5-lipoxygenase in programmed cell death of cancer cells

We investigated the involvement of 5-lipoxygenase activity in the early phases of programmed cell death (PCD) induced by H2O2 or retinoids in different human tumour cells (erythroleukaemia, neuroblastoma, melanoma). Apoptotic cells showed enhanced 5-lipoxygenase activity which was paralleled by decreased superoxide dismutase activity and increased light emission. Ultraweak luminescence, mainly due to membrane lipid peroxidation by lipoxygenase activation, increased in all cell lines tested within 10-15 min after induction of PCD, in a concentration and time-dependent manner. At the same time, we observed a significant increase in the intracellular steady state level of the 5-lipoxygenase metabolite leukotriene B4. Furthermore, 5-lipoxygenase metabolite 5-hydroxyeicosatetraenoic acid was able to induce PCD in all cell lines tested. Conversely, the general lipoxygenase inhibitor nordihydroguaiaretic acid and the selective 5-lipoxygenase inhibitor caffeic acid protected the different tumour cells from H2O2-induced PCD to a similar extent. These results show the activation of the 5-lipoxygenase pathway in PCD of three different cancer cell lines.     

Microgravity-induced programmed cell death in astrocytes

Cultured astrocytes were submitted to simulated microgravity using a Fokker clinostat under continuous rotation (60 rpm) for 15', 30', 1h, 20h and 32h. Samples processing included (i) nuclear stainings using Propidium Iodide and 4,6-diamidino-2-phenilindole, dihydro chloride, (ii) immunohistochemical identification of Caspase-7, (iii) identification of DNA fragmentation using the terminal dUTP nick end labelling and (iv) Scanning Electron Microscope analysis. After 30' at simulated microgravity the glial cells showed morphological evidence of apoptosis: cell shrinkage, chromatin condensation, nuclear blebs and fragmentation. The enzyme caspase-7 was present and DNA fragmentation was evident. After 32h the density of the cell population was much lower than that observed in controls.     

Developmentally programmed cell death in Drosophila

During the development of metazoans, programmed cell death (PCD) is essential for tissue patterning, removal of unwanted cells and maintaining homeostasis. In the past 20 years Drosophila melanogaster has been one of the systems of choice for studies involving developmental cell death, providing an ideal genetically tractable model of intermediary complexity between Caenorhabditis elegans and mammals. The lessons learned from studies using Drosophila indicate both the conserved nature of the many cell death pathways as well as novel and unexpected mechanisms. In this article we review the understanding of PCD during Drosophila development, highlighting the key mechanisms that are evolutionarily conserved as well as apparently unusual pathways, which indicate divergence, but provide evidence of complexity acquired during organismic evolution. This article is part of a Special Section entitled: Cell Death Pathways. Guest Editors: Frank Madeo and Slaven Stekovic.     

Developmental programmed cell death in plants

Mechanisms of plant developmental programmed cell death (PCD) have been intensively studied in recent years. Most plant developmental PCD is triggered by plant hormones, and the 'death signal' may be transduced by hormonal signaling pathways. Although there are some fundamental differences in the regulation of developmental PCD in various eukaryotes of different kingdoms, hormonal control and death signal transduction via pleiotropic signaling pathways constitute a common framework. However, plants possess a unique process of PCD execution that depends on vacuolar lytic function. Comparisons of the developmental PCD mechanisms of plants and other organisms are providing important insights into the detailed characteristics of developmental PCD in plants.     

Apoptotic-like programmed cell death in plants

Programmed cell death (PCD) is now accepted as a fundamental cellular process in plants. It is involved in defence, development and response to stress, and our understanding of these processes would be greatly improved through a greater knowledge of the regulation of plant PCD. However, there may be several types of PCD that operate in plants, and PCD research findings can be confusing if they are not assigned to a specific type of PCD. The various cell-death mechanisms need therefore to be carefully described and defined. This review describes one of these plant cell death processes, namely the apoptotic-like PCD (AL-PCD). We begin by examining the hallmark 'apoptotic-like' features (protoplast condensation, DNA degradation) of the cell's destruction that are characteristic of AL-PCD, and include examples of AL-PCD during the plant life cycle. The review explores the possible cellular 'executioners' (caspase-like molecules; mitochondria; de novo protein synthesis) that are responsible for the hallmark features of the cellular destruction. Finally, senescence is used as a case study to show that a rigorous definition of cell-death processes in plant cells can help to resolve arguments that occur in the scientific literature regarding the timing and control of plant cell death.     

Autophagy functions in programmed cell death

Autophagic cell death is a prominent morphological form of cell death that occurs in diverse animals. Autophagosomes are abundant during autophagic cell death, yet the functional role of autophagy in cell death has been enigmatic. We find that autophagy and the Atg genes are required for autophagic cell death of Drosophila salivary glands. Although caspases are present in dying salivary glands, autophagy is required for complete cell degradation. Further, induction of high levels of autophagy results in caspase-independent autophagic cell death. Our results provide the first in vivo evidence that autophagy and the Atg genes are required for autophagic cell death and confirm that autophagic cell death is a physiological death program that occurs during development.

Addendum to: Berry DL, Baehrecke EH. Growth arrest and autophagy are required for programmed salivary gland cell degradation in Drosophila. Cell 2007; 131:1137-48.     

Caspase function in programmed cell death

The first proapoptotic caspase, CED-3, was cloned from Caenorhabditis elegans in 1993 and shown to be essential for the developmental death of all somatic cells. Following the discovery of CED-3, caspases have been cloned from several vertebrate and invertebrate species. As reviewed in other articles in this issue of Cell Death and Differentiation, many caspases function in nonapoptotic pathways. However, as is clear from the worm studies, the evolutionarily conserved role of caspases is to execute programmed cell death. In this article, I will specifically focus on caspases that function primarily in cell death execution. In particular, the physiological function of caspases in apoptosis is discussed using examples from the worm, fly and mammals.     

Chitosan-induced programmed cell death in plants

Chitosan, CN⁻, or H₂O₂ caused the death of epidermal cells (EC) in the epidermis of pea leaves that was detected by monitoring the destruction of cell nuclei; chitosan induced chromatin condensation and marginalization followed by the destruction of EC nuclei and subsequent internucleosomal DNA fragmentation. Chitosan did not affect stoma guard cells (GC). Anaerobic conditions prevented the chitosan-induced destruction of EC nuclei. The antioxidants nitroblue tetrazolium or mannitol suppressed the effects of chitosan, H₂O₂, or chitosan + H₂O₂ on EC. H₂O₂ formation in EC and GC mitochondria that was determined from 2′,7′-dichlorofluorescein fluorescence was inhibited by CN⁻ and the protonophoric uncoupler carbonyl cyanide m-chlorophenylhydrazone but was stimulated by these agents in GC chloroplasts. The alternative oxidase inhibitors propyl gallate and salicylhydroxamate prevented chitosan- but not CN⁻-induced destruction of EC nuclei; the plasma membrane NADPH oxidase inhibitors diphenylene iodonium and quinacrine abolished chitosan- but not CN⁻-induced destruction of EC nuclei. The mitochondrial protein synthesis inhibitor lincomycin removed the destructive effect of chitosan or H₂O₂ on EC nuclei. The effect of cycloheximide, an inhibitor of protein synthesis in the cytoplasm, was insignificant; however, it was enhanced if cycloheximide was added in combination with lincomycin. The autophagy inhibitor 3-methyladenine removed the chitosan effect but exerted no influence on the effect of H₂O₂ as an inducer of EC death. The internucleosome DNA fragmentation in conjunction with the data on the 3-methyladenine effect provides evidence that chitosan induces programmed cell death that follows a combined scenario including apoptosis and autophagy. Based on the results of an inhibitor assay, chitosan-induced EC death involves reactive oxygen species generated by the NADPH oxidase of the plasma membrane.     

Caspase-mediated programmed cell death pathways as potential therapeutic targets in cancer

The caspase family is well characterized as playing a crucial role in modulation of programmed cell death (PCD), which is a genetically regulated, evolutionarily conserved process with numerous links to many human diseases, most notably cancer. In this review, we focus on summarizing the intricate relationships between some members of the caspase family and their key apoptotic mediators, involving tumour necrosis factor receptors, the Bcl-2 family, cytochrome c, Apaf-1 and IAPs in cancer initiation and progression. We elucidate new emerging types of cross-talk between several caspases and autophagy-related genes (Atgs) in cancer. Moreover, we focus on presenting several PCD-modulating agents that may target caspases-3, -8 and -9, and their substrates PARP-1 and Beclin-1, which may help us harness caspase-modulated PCD pathways for future drug discovery.     

Secretion of human soluble programmed cell death protein 1 by chimeric antigen receptor-modified T cells enhances anti-tumor efficacy

Background aimsChimeric antigen receptor (CAR) T cells have achieved favorable responses in patients with hematologic malignancies, but the outcome has been far from satisfactory in the treatment of tumors with high expression of immunosuppressive molecules. To overcome this limitation, we modified CAR T cells to secrete types of human soluble programmed cell death protein 1 (PD-1) called sPD-1 CAR T cells.MethodsTo compare the effector function between second (conventional second-generation CAR targeting CD19) and sPD-1 CAR T cells, we measured cytotoxicity, cytokine secretion and activation markers incubated with or without tumor cells expressing CD19 and/or programmed cell death ligand 1 (PD-L1). Furthermore, the anti-tumor efficacy of second and sPD-1 CAR T cells was determined using an NSG mouse model bearing NALM-6-PD-L1. Finally, the underlying mechanism was investigated by metabolic parameters and RNA sequencing analysis of different CAR T cells.ResultsCompared with second CAR T cells, sPD-1 CAR T cells enhanced killing efficiency toward CD19⁺PD-L1⁺ tumor cells in vitro. Furthermore, sPD-1 CAR T cells reduced the tumor burden and prolonged overall survival of the NSG (NOD-SCID-IL2rg) mice bearing NALM-6-PD-L1. To explore the effect of soluble PD-1 on CAR T cells, we found that sPD-1 CAR T cells exhibited higher levels of activation and ameliorative profiles of differentiation, exhaustion, glycolysis and apoptosis.ConclusionsWith constitutive soluble PD-1 secretion, sPD-1 CAR T cells have tended to eradicate tumors with a high expression of PD-L1 more effectively than second CAR T cells. This may be due to soluble PD-1 enhancing apoptosis resistance, aerobic metabolism and a more “stem” differentiation of CAR T cells. Overall, our study presents a feasible strategy to increase the efficacy of CAR T cells.     

Autophagy functions in programmed cell death

Autophagic cell death is a prominent morphological form of cell death that occurs in diverse animals. Autophagosomes are abundant during autophagic cell death, yet the functional role of autophagy in cell death has been enigmatic. We find that autophagy and the Atg genes are required for autophagic cell death of Drosophila salivary glands. Although caspases are present in dying salivary glands, autophagy is required for complete cell degradation. Further, induction of high levels of autophagy results in caspase-independent autophagic cell death. Our results provide the first in vivo evidence that autophagy and the Atg genes are required for autophagic cell death and confirm that autophagic cell death is a physiological death program that occurs during development.     

Chlamydia and programmed cell death

Discordant views regarding host cell death induction by Chlamydia are likely owing to the different methods used for evaluation of apoptosis. Apoptotic and non-apoptotic death owing to both caspase-dependent and -independent activation of the Bax protein occur late in the productive growth cycle. Evidence also suggests that Chlamydia inhibits apoptosis during productive growth as part of its intracellular survival strategy. This is in part owing to proteolytic degradation of the BH3-only family of pro-apoptotic proteins in the mitochondrial pathway. Chlamydia also inhibits apoptosis during persistent growth or in phagocytes, but induces apoptosis in T cells, which suggests that apoptosis has an immunomodulatory role in chlamydial infections. The contribution of apoptosis in disease pathogenesis remains a focus for future research.     

Cancer and programmed cell death

A report on the 15th Lorne Cancer Conference, Lorne, Australia, 13-16 February 2003.