Diagnosis of Cell Death by Means of Infrared Spectroscopy

Fourier transform infrared (FTIR) spectroscopy has been established as a fast spectroscopic method for biochemical analysis of cells and tissues. In this research we aimed to investigate FTIR's utility for identifying and characterizing different modes of cell death, using leukemic cell lines as a model system. CCRF-CEM and U937 leukemia cells were treated with arabinoside and doxorubicin apoptosis inducers, as well as with potassium cyanide, saponin, freezing-thawing, and H₂O₂ necrosis inducers. Cell death mode was determined by various gold standard biochemical methods in parallel with FTIR-microscope measurements. Both cell death modes exhibit large spectral changes in lipid absorbance during apoptosis and necrosis; however, these changes are similar and thus cannot be used to distinguish apoptosis from necrosis. In contrast to the above confounding factor, our results reveal that apoptosis and necrosis can still be distinguished by the degree of DNA opaqueness to infrared light. Moreover, these two cell death modes also can be differentiated by their infrared absorbance, which relates to the secondary structure of total cellular protein. In light of these findings, we conclude that, because of its capacity to monitor multiple biomolecular parameters, FTIR spectroscopy enables unambiguous and easy analysis of cell death modes and may be useful for biochemical and medical applications.     

Inhibition of Mitochondrial ATP Generation by Nitric Oxide Switches Apoptosis to Necrosis

Under pathological conditions, the mode of cell death, apoptosis or necrosis, is relevant for the subsequent fate of the tissue. Cell demise may be shaped by endogenous mediators such as nitric oxide (NO) which interfere with subroutines of the death program. Here we show that apoptosis of Jurkat cells elicited by either staurosporine (STS) or anti-CD95 antibodies in glucose-free medium is converted to necrosis by NO donors. In the presence of NO, release of mitochondrial cytochrome c was delayed and activation of execution caspases was prevented. Stimulated cells died nonetheless. The switch in the mode of cell death was due to NO-dependent failure of mitochondrial energy production. Restoration of intracellular ATP by glucose supplementation recovered the cells' ability to activate caspases and undergo apoptosis. In this system, the apoptosis/necrosis conversion promoted by NO was not mediated by cyclic guanosine monophosphate-dependent mechanisms, poly-(ADP-ribose)-polymerase (PARP) activation, or inhibition of caspases due to S-nitrosylation and glutathione depletion. In contrast, depleting intracellular ATP with rotenone, an inhibitor of mitochondrial complex I mimicked the effect of NO. The findings presented here suggest that NO can decide the shape of cell death by lowering intracellular ATP below the level required to allow the coordinated execution of apoptosis.     

Morphological classification of plant cell deaths

Programmed cell death (PCD) is an integral part of plant development and of responses to abiotic stress or pathogens. Although the morphology of plant PCD is, in some cases, well characterised and molecular mechanisms controlling plant PCD are beginning to emerge, there is still confusion about the classification of PCD in plants. Here we suggest a classification based on morphological criteria. According to this classification, the use of the term 'apoptosis' is not justified in plants, but at least two classes of PCD can be distinguished: vacuolar cell death and necrosis. During vacuolar cell death, the cell contents are removed by a combination of autophagy-like process and release of hydrolases from collapsed lytic vacuoles. Necrosis is characterised by early rupture of the plasma membrane, shrinkage of the protoplast and absence of vacuolar cell death features. Vacuolar cell death is common during tissue and organ formation and elimination, whereas necrosis is typically found under abiotic stress. Some examples of plant PCD cannot be ascribed to either major class and are therefore classified as separate modalities. These are PCD associated with the hypersensitive response to biotrophic pathogens, which can express features of both necrosis and vacuolar cell death, PCD in starchy cereal endosperm and during self-incompatibility. The present classification is not static, but will be subject to further revision, especially when specific biochemical pathways are better defined.     

Cell death in head and neck cancer pathogenesis and treatment

Many cancer therapies aim to trigger apoptosis in cancer cells. Nevertheless, the presence of oncogenic alterations in these cells and distorted composition of tumour microenvironment largely limit the clinical efficacy of this type of therapy. Luckily, scientific consensus describes about 10 different cell death subroutines with different regulatory pathways and cancer cells are probably not able to avoid all of cell death types at once. Therefore, a focused and individualised therapy is needed to address the specific advantages and disadvantages of individual tumours. Although much is known about apoptosis, therapeutic opportunities of other cell death pathways are often neglected. Molecular heterogeneity of head and neck squamous cell carcinomas (HNSCC) causing unpredictability of the clinical response represents a grave challenge for oncologists and seems to be a critical component of treatment response. The large proportion of this clinical heterogeneity probably lies in alterations of cell death pathways. How exactly cells die is very important because the predominant type of cell death can have multiple impacts on the therapeutic response as cell death itself acts as a second messenger. In this review, we discuss the different types of programmed cell death (PCD), their connection with HNSCC pathogenesis and possible therapeutic windows that result from specific sensitivity to some form of PCD in some clinically relevant subgroups of HNSCC.Subject terms: Oral cancer, Cell death, Oncogenesis     

Cell death mechanisms: Cross-talk and role in disease

Multiple cell death mechanisms operate in both uni- and multicellular organisms. Hence, research during the past forty years has revealed that apoptosis is not the only cell death program involved in the regulation of tissue homeostasis and the removal of unwanted cells in biological organisms. While the molecular pathways of apoptosis and necrosis are now relatively well established, the precise mechanisms of other cell death modalities, and their cross-talk, require additional study. This is particularly important, since many human disorders can be attributed, directly or indirectly, to defective cell death mechanisms. In this review we shall discuss the characteristics and cross-talk between various modes of cell death and their role in cell death-related disorders, notably, neurodegenerative disease and cancer.     

Macromitophagy,neutral lipids synthesis,and peroxisomal fatty acid oxidation protect yeast from “liponecrosis”, a previously unknown form of programmed cell death

We identified a form of cell death called “liponecrosis.” It can be elicited by an exposure of the yeast Saccharomyces cerevisiae to exogenous palmitoleic acid (POA). Our data imply that liponecrosis is: (1) a programmed, regulated form of cell death rather than an accidental, unregulated cellular process and (2) an age-related form of cell death. Cells committed to liponecrotic death: (1) do not exhibit features characteristic of apoptotic cell death; (2) do not display plasma membrane rupture, a hallmark of programmed necrotic cell death; (3) akin to cells committed to necrotic cell death, exhibit an increased permeability of the plasma membrane for propidium iodide; (4) do not display excessive cytoplasmic vacuolization, a hallmark of autophagic cell death; (5) akin to cells committed to autophagic death, exhibit a non-selective en masse degradation of cellular organelles and require the cytosolic serine/threonine protein kinase Atg1p for executing the death program; and (6) display a hallmark feature that has not been reported for any of the currently known cell death modalities—namely, an excessive accumulation of lipid droplets where non-esterified fatty acids (including POA) are deposited in the form of neutral lipids. We therefore concluded that liponecrotic cell death subroutine differs from the currently known subroutines of programmed cell death. Our data suggest a hypothesis that liponecrosis is a cell death module dynamically integrated into a so-called programmed cell death network, which also includes the apoptotic, necrotic, and autophagic modules of programmed cell death. Based on our findings, we propose a mechanism underlying liponecrosis.     

Types of cell death and methods of their detection in yeast Saccharomyces cerevisiae

The occurrence of programmed cell death in unicellular organisms is a subject that arouses great interest of theoreticians and experimental scientists. Already found evolutionarily conserved genes and metabolic pathways confirmed its existence in yeast, protozoa and even bacteria. In the yeast Saccharomyces cerevisiae, at least three main types of death are distinguished: apoptosis, necrosis and autophagy. Their classification suggested by the Nomenclature Committee on Cell Death initially based on the morphological characteristics has now been extended to include the measurable biochemical characteristics. Several laboratory methods previously used to detect the types of cell death of higher eucaryotes and later developed and successfully used for the analysis of yeast cells are here critically reviewed. Their advantages and limitations are described.     

1,000 Ways to die: natural compounds modulate non-canonical cell death pathways in cancer cells

Over the past decades, apoptosis emerged as a leading strategy to eliminate cancer cells. Nowadays, it becomes progressively clear that apoptosis is not the only programmed cell death mechanism implicated in the elimination of malignant cells. This review will discuss non-apoptotic cell death modalities and will focus on natural compounds acting as inducers of these recently described processes. We will describe the major molecular characteristics of each individual death mechanism and we will detail the effects of natural and naturally-derived compounds on these cellular mechanisms.     

Brain Endothelial Cell Death: Modes,Signaling Pathways,and Relevance to Neural Development,Homeostasis, and Disease

Emerging evidence indicates that brain microvascular endothelial cells play a critical role in brain development, maturation, and homeostasis. Acute or chronic insults, including oxidative stress, oxygen–glucose deprivation, trauma, infections, inflammatory cytokines, DNA damaging agents, β-amyloid deposition, and endoplasmic reticulum stress induce brain endothelial cell dysfunction and damage, which can result in cell death. The homeostatic balance between endothelial cell survival and endothelial cell death is critical for brain development, remodeling, and repair. On the other hand, dysregulation of brain endothelial cell death exacerbates, or even initiates, several inflammatory, ischemic, and degenerative disorders of the central nervous system. In here, the morphological, biochemical, and functional characteristics of the brain endothelium and its contribution to brain homeostasis will be reviewed. Recent insights into modalities and regulatory pathways involved in brain endothelial cell death will be described. The effects of regulated and dysregulated endothelial cell death leading to angiogenesis will be outlined. The relevance of brain endothelial cell dysfunction and death to disease processes will be discussed with special reference to recent findings that could help translate current knowledge on brain endothelial cell apoptosis into new therapeutic strategies for the treatment of certain neurological disorders.     

The morphology of apoptosis

The concept of apoptotic cell death as an essential part of the development and life of complex organisms has been devised in different situations and tested from various angles. This review article discusses the morphological changes during death by apoptosis. In cells undergoing apoptosis, an intracellular signalling pathway operates cell autonomously to implement the death and disposal of the cell. The similarity of the biochemical events during apoptosis in different situations is reflected by a high uniformity of morphological changes in many situations of naturally occurring or experimentally induced cell death. The unifying concept of apoptosis has been derived from the observation of this morphological consistency of dying cells almost 30 years ago. Since then, we have learned much about the intracellular signalling in the apoptotic process and the molecular background has been delineated which guides the initiation of the morphological changes. Here, an attempt is made to present the current knowledge about the molecular events in the development of these morphological alterations and to place these changes in the context of apoptotic cell death.     

Activation and caspase-mediated inhibition of PARP: a molecular switch between fibroblast necrosis and apoptosis in death receptor signaling

Death ligands not only induce apoptosis but can also trigger necrosis with distinct biochemical and morphological features. We recently showed that in L929 cells CD95 ligation induces apoptosis, whereas TNF elicits necrosis. Treatment with anti-CD95 resulted in typical apoptosis characterized by caspase activation and DNA fragmentation. These events were barely induced by TNF, although TNF triggered cell death to a similar extent as CD95. Surprisingly, whereas the caspase inhibitor zVAD prevented CD95-mediated apoptosis, it potentiated TNF-induced necrosis. Cotreatment with TNF and zVAD was characterized by ATP depletion and accelerated necrosis. To investigate the mechanisms underlying TNF-induced cell death and its potentiation by zVAD, we examined the role of poly(ADP-ribose)polymerase-1 (PARP-1). TNF but not CD95 mediated PARP activation, whereas a PARP inhibitor suppressed TNF-induced necrosis and the sensitizing effect of zVAD. In addition, fibroblasts expressing a noncleavable PARP-1 mutant were more sensitive to TNF than wild-type cells. Our results indicate that TNF induces PARP activation leading to ATP depletion and subsequent necrosis. In contrast, in CD95-mediated apoptosis caspases cause PARP-1 cleavage and thereby maintain ATP levels. Because ATP is required for apoptosis, we suggest that PARP-1 cleavage functions as a molecular switch between apoptotic and necrotic modes of death receptor-induced cell death.     

IgH-2 cells: a reptilian model for apoptotic studies

Regulation of proper cell number in tissues depends upon a balance between cell proliferation and cell death. The process of apoptosis has thus far been studied in a variety of multicellular organisms from humans to higher plants. In order to broaden our perspective and identify another metazoan system with which to deepen our understanding of the function and evolution of the apoptotic machinery, we have characterized cell death in a reptilian cell line. We show that the death of IgH-2 Iguana (Iguana iguana) heart cells [Clark, H.F., Cohen, M.M., Karzon, D.T., 1970. Characterization of reptilian cell lines established at incubation temperatures of 23 to 36 degrees. Proc. Soc. Exp. Biol. Med. 133, 1039-1047.] is, in response to DNA damaging agents, accompanied by classic morphological changes of apoptosis including detachment from the substrate, cell shrinkage, nuclear pyknosis and externalization of the plasma membrane phospholipid phosphatidylserine. Our biochemical studies show that the death of IgH-2 cells is accompanied by internucleosomal DNA fragmentation and activation of caspases. Our studies with the pan-caspase inhibitor zVAD.fmk implicate caspases in the apoptotic process we observe. This work represents the first detailed molecular and biochemical analysis of apoptosis in cells of an organism of class Reptilia and establishes IgH-2 cells as a suitable model system with which to investigate the phenomenon of caspase dependent apoptosis and the apoptotic machinery in a reptilian model.     

Which cell death modality wins the contest for photodynamic therapy of cancer?

Photodynamic therapy (PDT) was discovered more than 100 years ago. Since then, many protocols and agents for PDT have been proposed for the treatment of several types of cancer. Traditionally, cell death induced by PDT was categorized into three types: apoptosis, cell death associated with autophagy, and necrosis. However, with the discovery of several other regulated cell death modalities in recent years, it has become clear that this is a rather simple understanding of the mechanisms of action of PDT. New observations revealed that cancer cells exposed to PDT can pass through various non-conventional cell death pathways, such as paraptosis, parthanatos, mitotic catastrophe, pyroptosis, necroptosis, and ferroptosis. Nowadays, immunogenic cell death (ICD) has become one of the most promising ways to eradicate tumor cells by activation of the T-cell adaptive immune response and induction of long-term immunological memory. ICD can be triggered by many anti-cancer treatment methods, including PDT. In this review, we critically discuss recent findings on the non-conventional cell death mechanisms triggered by PDT. Next, we emphasize the role and contribution of ICD in these PDT-induced non-conventional cell death modalities. Finally, we discuss the obstacles and propose several areas of research that will help to overcome these challenges and lead to the development of highly effective anti-cancer therapy based on PDT.Subject terms: Cancer immunotherapy, Cell death and immune response     

The role of Apaf-1 in programmed cell death: from worm to tumor

Apoptosis or programmed cell death is an important process to eliminate unnecessary or hazardous cells. Apaf-1, a mammalian homologue of CED-4 of C. elegans, is the essential adaptor molecule in the mitochondrial pathway of apoptosis. Mice lacking Apaf-1 show accumulation of neurons in the developing central nervous system due to reduced apoptosis. Apaf-1-deficient cells are remarkably resistant to various apoptotic stimuli. Apaf-1-mediated apoptosis plays a role in the prevention of tumorigenesis. However, Apaf-1-independent cell death pathways are also indicated. In this review, we will summarize what has been learned about the role of Apaf-1 by biochemical and genetical approaches.     

Is Saccharomyces cerevisiae apoptotic cell death associated with gene transfer?

Programmed cell death in unicellular organisms is difficult to account for in evolutionary terms. In the budding yeast, Saccharomyces cerevisiae, existence of several morphological and biochemical features of apoptosis has been described, and genes responsible for execution of the death program have been identified. It is here suggested that apoptosis of yeast cells could provide direct benefit to the genes of the dying cells, by facilitating DNA transfer to surrounding cells. The biochemical details of yeast apoptotic death are considered in light of a gene transfer hypothesis.     

Mitochondria-dependent apoptosis and cellular pH regulation

Mitochondria play a critical role in apoptosis induction in response to myriad stimuli. These organelles release proteins into the cytosol which trigger caspase activation or perform other functions relevant to apoptosis, including cytochrome c (cyt-c), caspases, AIF, and SMAC (Diablo). The mechanisms by which these proteins escape from mitochondria remain enigmatic. Moreover, it is unclear whether release of these proteins versus disturbances in core mitochondrial functions represents the cell death commitment mechanism. In this regard, suppression of apoptosis using broad-spectrum caspase inhibitory compounds has been reported in many circumstances to prevent the morphological and biochemical manifestations of apoptosis, and yet not protect cells from death and not preserve clonigenic survival. Thus, while mitochondrial damage can be coupled to caspase activation pathways, cell death commitment often occurs upstream of caspase activation when mitochondria-dependent cell death pathways are invoked. Here, we review evidence implicating dysregulation of cellular pH as a component of the cell death mechanism involving mitochondria. Cell Death and Differentiation (2000) 7, 1155 - 1165     

Annexin V staining due to loss of membrane asymmetry can be reversible and precede commitment to apoptotic death.

Signal-induced apoptosis is a normal phenomenon in which cells respond to changes in their environment through a cascade of intracellular biochemical changes culminating in cell death. However, it is not clear at what point in this process the cell becomes committed to die. An early biochemical change characteristic of cells undergoing apoptosis is the loss of plasma membrane asymmetry, such that high levels of phosphatidylserine become exposed on the outside cell surface. These cells can be recognized by staining with Annexin V, which binds to phosphatidylserine with high affinity. To investigate the mechanisms controlling signal-induced apoptosis we have examined the response of a B cell lymphoma to crosslinking of the membrane immunoglobulin (mIg) receptor. We have found that many of the cells that stain positive for Annexin V are viable and can resume growth and reestablish phospholipid asymmetry once the signal is removed. These results indicate that Annexin V staining, and thus loss of membrane asymmetry, precedes commitment to apoptotic death in this system.     

The role of programmed cell death in Plasmodium-mosquito interactions

Many host-parasite interactions are regulated in part by the programmed cell death of host cells or the parasite. Here we review evidence suggesting that programmed cell death occurs during the early stages of the development of the malaria parasite in its vector. Zygotes and ookinetes of Plasmodium berghei have been shown to die by programmed cell death (apoptosis) in the midgut lumen of the vector Anopheles stephensi, or whilst developing in vitro. Several morphological markers, indicative of apoptosis, are described and evidence for the involvement of a biochemical pathway involving cysteine proteases discussed in relationship to other protozoan parasites. Malaria infection induces apoptosis in the cells of two mosquito tissues, the midgut and the follicular epithelium. Observations on cell death in both these tissues are reviewed including the role of caspases as effector molecules and the rescue of resorbing follicles resulting from inhibition of caspases. Putative signal molecules that might induce parasite and vector apoptosis are suggested including nitric oxide, reactive nitrogen intermediates, oxygen radicals and endocrine balances. Finally, we suggest that programmed cell death may play a critical role in regulation of infection by the parasite and the host, and contribute to the success or not of parasite establishment and host survival.