P2Z purinoreceptor ligation induces activation of caspases with distinct roles in apoptotic and necrotic alterations of cell death

Myeloic cells express a peculiar surface receptor for extracellular ATP, called the P2Z/P2X7 purinoreceptor, which is involved in cell death signalling. Here, we investigated the role of caspases, a family of proteases implicated in apoptosis and the cytokine secretion. We observed that extracellular ATP induced the activation of multiple caspases including caspase-1, -3 and -8, and subsequent cleavage of the caspase substrates PARP and lamin B. Using caspase inhibitors, it was found that caspases were specifically involved in ATP-induced apoptotic damage such as chromatin condensation and DNA fragmentation. In contrast, inhibition of caspases only marginally affected necrotic alterations and cell death proceeded normally whether or not nuclear damage was blocked. Our results therefore suggest that the activation of caspases by the P2Z receptor is required for apoptotic but not necrotic alterations of ATP-induced cell death.     

Involvement of protein kinase C-delta in DNA damage-induced apoptosis

Apoptosis is a highly orchestrated cell suicidal program required to maintain a balance between cell proliferation and cell death. A defect in apoptotic machinery can cause cancer. Many anticancer drugs are known to kill tumor cells by inducing apoptosis, and a defect in apoptosis can lead to anticancer drug resistance. Apoptosis is regulated by a complex cellular signaling network. Several members of the protein kinase C (PKC) family serve as substrates for caspases and PKCδ isozyme has been intimately associated with DNA damage-induced apoptosis. It can act both upstream and downstream of caspases. In response to apoptotic stimuli, the full-length and the catalytic fragment of PKCδ may translocate to distinct cellular compartments, including mitochondria and the nucleus, to reach their targets. Both activation and intracellular distribution of PKCδ may have significant impact on apoptosis. This review intends to assimilate recent views regarding the involvement of PKCδ in DNA damage-induced apoptosis.     

Paradox of Bcl‐2 (and p53): why may apoptosis‐regulating proteins be irrelevant to cell death?

Although the Bcl-2 family members and p53 are involved in the regulation of apoptosis, the status of apoptotic machinery (eg caspases) plays a major role in determining the mode and timing of cell death. If the apoptotic machinery is lost, inhibited, or intrinsically inactivated, the "death stars", Bcl-2 and p53, may become irrelevant to cell death. In this light, high levels of Bcl-2 may indicate that downstream apoptotic pathways are still functional. This explains why Bcl-2 overexpression can be a marker of chemosensitivity and favorable prognosis in certain cancers and why retention of wild-type p53 may manifest inactivation of caspases in aggressive cancers.     

Spermine oxidation leads to necrosis with plasma membrane phosphatidylserine redistribution in mouse leukemia cells

Oxidation by copper/quinone-containing serum amine oxidases (SAO) is a well-known cause of polyamine cytotoxicity. Spermine oxidation exerts potent immunosuppressive effects in animal cells, but the cell death mechanism involved remains unclear. We compared biochemical and morphological parameters of SAO-mediated cell death in L1210 mouse leukemia cells with normal or amplified ornithine decarboxylase gene expression with those observed during apoptosis induced by deregulated polyamine uptake or by okadaic acid. None of the characteristic features of apoptotic cell death (e.g., chromatin condensation, nuclear fragmentation, internucleosomal DNA cleavage, poly(ADP-ribose) polymerase cleavage) were observed during spermine oxidation-mediated cell death, which was clearly necrotic by morphological criteria. Inhibition of a wide spectrum of caspases did not prevent SAO-dependent cell death, whereas N-acetylcysteine completely abolished the cytotoxic effects of spermine oxidation. Catalase only delayed spermine oxidation-induced cell death without affecting its modality or preventing depletion of intracellular glutathione, suggesting that both H(2)O(2) and aminoaldehydes generated by SAO-mediated spermine oxidation contribute to SAO-induced necrosis. Interestingly, redistribution of phosphatidylserine to the outer leaflet of the plasma membrane, usually a diagnostic feature of apoptosis, preceded necrotic cytolysis triggered by spermine oxidation. Thus, L1210 cell death caused by SAO-mediated spermine oxidation has all the attributes of primary necrosis, but is also accompanied by loss of phospholipid asymmetry, indicating that the latter phenomenon may not be unique to apoptosis. Phosphatidylserine exposure, a potent engulfment signal for phagocytes, might contribute to the immunosuppressive effects of plasma polyamines through a controlled and rapid necrotic process involving SAO.     

Caspases: A Molecular Switch Node in the Crosstalk between Autophagy and Apoptosis

Autophagy and apoptosis are two important catabolic processes contributing to the maintenance of cellular and tissue homeostasis. Autophagy controls the turnover of protein aggregates and damaged organelles within cells, while apoptosis is the principal mechanism by which unwanted cells are dismantled and eliminated from organisms. Despite marked differences between these two pathways, they are highly interconnected in determining the fate of cells. Intriguingly, caspases, the primary drivers of apoptotic cell death, play a critical role in mediating the complex crosstalk between autophagy and apoptosis. Pro-apoptotic signals can converge to activate caspases to execute apoptotic cell death. In addition, activated caspases can degrade autophagy proteins (i.e., Beclin-1, Atg5, and Atg7) to shut down the autophagic response. Moreover, caspases can convert pro-autophagic proteins into pro-apoptotic proteints to trigger apoptotic cell death instead. It is clear that caspases are important in both apoptosis and autophagy, thus a detailed deciphering of the role of caspases in these two processes is still required to clarify the functional relationship between them. In this article, we provide a current overview of caspases in its interplay between autophagy and apoptosis. We emphasized that defining the role of caspases in autophagy-apoptosis crosstalk will provide a framework for more precise manipulation of these two processes during cell death.     

The analysis of biochemical markers of MCF-7 cells apoptosis induced by recombinant analog of lactaptin

Earlier we isolated and characterized human milk pro-apoptotic peptide - lactaptin and generated its engineered analog - RL2. It was shown that both lactaptin and RL2 are capable to induce apoptotic death of MCF-7 cells. In this report we have analyzed biochemical markers of RL2 induced MCF-7 apoptosis. The activation of initiator and effector caspases as well as mitochondrial membrane potential and cytoplasm membrane changes were analyzed using flow cytometry and Western-blot methods. We have found that RL2 induced apoptotic death of MCF-7 cells was accompanied by PS exposure on the plasma membrane surface. It also was shown that RL2 has induced dissipation of mitochondrial membrane potential and resulted in activation of initiator caspases 8, 9 and effector caspase 7.     

Apoptosis-regulating proteins as targets for drug discovery.

Defects in the regulation of apoptosis (programmed cell death) contribute to many diseases, including pathologies associated with cell loss (e.g. stroke, heart failure, neurodegeneration and AIDS), and disorders characterized by a failure to eliminate harmful cells (e.g. cancer, autoimmunity). Apoptosis is caused by activation of intracellular proteases, known as caspases, which are responsible directly or indirectly for the morphological and biochemical events that characterize the apoptotic cell. Numerous caspase regulators have been discovered, which respond to environmental stimuli and influence the decision of cell death and survival. Knowledge of the molecular details of apoptosis regulation, and the three-dimensional structures of proteins constituting the apoptosis core machinery has revealed new strategies for identifying small-molecule drugs that could one day yield more effective treatments for a wide variety of illnesses.     

Differential involvement of initiator caspases in apoptotic volume decrease and potassium efflux during Fas- and UV-induced cell death.

Caspase activation and apoptotic volume decrease are fundamental features of programmed cell death; however, the relationship between these components is not well understood. Here we provide biochemical and genetic evidence for the differential involvement of initiator caspases in the apoptotic volume decrease during both intrinsic and extrinsic activation of apoptosis. Apoptosis induction in Jurkat T lymphocytes by Fas receptor engagement (intrinsic) or ultraviolet (UV)-C radiation (extrinsic) triggered the loss of cell volume, which was restricted to cells with diminished intracellular K(+) ions. These characteristics kinetically coincided with the proteolytic processing and activation of both initiator and effector caspases. Although the polycaspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone completely inhibited the Fas-mediated apoptotic volume decrease and K(+) efflux, it was much less effective in preventing these processes during UV-induced cell death under conditions whereby caspase activities and DNA degradation were blocked. To define the roles of specific initiator caspases, we utilized Jurkat cells genetically deficient in caspase-8 or stably transfected with a dominant-negative mutant of caspase-9. The results show that the activation of caspase-8, but not caspase-9, is necessary for Fas-induced apoptosis. Conversely, caspase-9, but not caspase-8, is important for UV-mediated shrunken morphology and apoptosis progression. Together, these findings indicate that cell shrinkage and K(+) efflux during apoptosis are tightly coupled, but are differentially regulated by either caspase-8 or caspase-9 depending on specific pathways of cell death.     

Multiple species of CPP32 and Mch2 are the major active caspases present in apoptotic cells.

The activity of ICE-like proteases or caspases is essential for apoptosis. Multiple caspases participate in apoptosis in mammalian cells but how many caspases are involved and what is their relative contribution to cell death is poorly understood. To identify caspases activated in apoptotic cells, we developed an approach to simultaneously detect multiple active caspases. Using tumor cells as a model, we have found that CPP32 (caspase 3) and Mch2 (caspase 6) are the major active caspases in apoptotic cells, and are activated in response to distinct apoptosis-inducing stimuli and in all cell lines analyzed. Both CPP32 and Mch2 are present in apoptotic cells as multiple active species. In a given cell line these species remained the same irrespective of the apoptotic stimulus used. However, the species of CPP32 and Mch2 detected varied between cell lines, indicating differences in caspase processing. The strategy described here is widely applicable to identify active caspases involved in apoptosis.     

Caspase independent/dependent regulation of K(+), cell shrinkage, and mitochondrial membrane potential during lymphocyte apoptosis.

The loss of cell volume is a fundamental feature of apoptosis. We have previously shown that DNA degradation and caspase activity occur only in cells which have shrunken as a result of potassium and sodium efflux (Bortner, C. D., Hughes, F. M., Jr., and Cidlowski, J. A. (1997) J. Biol. Chem. 272, 32436-32442). Furthermore, maintaining a normal intracellular potassium concentration represses the cell death process by inhibiting the activity of apoptotic nucleases and suppressing the activation of effector caspases (Hughes, F. M., Jr., Bortner, C. D. Purdy, G. D., and Cidlowski, J. A. (1997) J. Biol. Chem. 272, 30567-30576). We have now investigated the relationship between cell shrinkage, ion efflux, and changes in the mitochondrial membrane potential, in addition to the role of caspases in these apoptotic events. Treatment of Jurkat cells with a series of inducers which act via distinct signal transduction pathways, resulted in all of the cell death characteristics including loss of cell viability, cell shrinkage, K(+) efflux, altered mitochondrial membrane potential, and DNA fragmentation. Interestingly, only cells which shrunk had a loss of mitochondrial membrane potential and the other apoptotic characteristics. Treatment of Jurkat cells with an anti-Fas antibody in the presence of the general caspase inhibitor z-VAD, abrogated these features. In contrast, when Jurkat cells were treated with either the calcium ionophore A23187 or thapsigargin, z-VAD failed to prevent cell shrinkage, K(+) efflux, or changes in the mitochondrial membrane potential, while effectively inhibiting DNA degradation. Treatment of Jurkat cells with various apoptotic agents in the presence of either the caspase-3 inhibitor DEVD, or the caspase-8 inhibitor IETD also blocked DNA degradation, but failed to prevent other characteristics of apoptosis. Together these data suggest that the cell shrinkage, K(+) efflux, and changes in the mitochondrial membrane potential are tightly coupled, but occur independent of DNA degradation, and can be largely caspase independent depending on the particular signal transduction pathway.     

A conserved checkpoint pathway mediates DNA damage--induced apoptosis and cell cycle arrest in C. elegans

To maintain genomic stability following DNA damage, multicellular organisms activate checkpoints that induce cell cycle arrest or apoptosis. Here we show that genotoxic stress blocks cell proliferation and induces apoptosis of germ cells in the nematode C. elegans. Accumulation of recombination intermediates similarly leads to the demise of affected cells. Checkpoint-induced apoptosis is mediated by the core apoptotic machinery (CED-9/CED-4/CED-3) but is genetically distinct from somatic cell death and physiological germ cell death. Mutations in three genes--mrt-2, which encodes the C. elegans homolog of the S. pombe rad1 checkpoint gene, rad-5, and him-7-block both DNA damage-induced apoptosis and cell proliferation arrest. Our results implicate rad1 homologs in DNA damage-induced apoptosis in animals.     

Role of caspases in ionizing radiation-induced apoptosis in the developing cerebellum

Caspase activation and dependence on caspases has been observed in different paradigms of apoptotic cell death in vivo and in vitro. The present study examines the role of caspases in ionizing radiation-induced apoptosis in the developing cerebellum of rats subjected to a single dose (2-Gy gamma rays) of whole-body irradiation at postnatal day 3. Radiation-induced apoptosis in the external granule cell layer, as defined by the presence of cells by extremely condensed, often fragmented nucleus, which were stained with the method of in situ end-labeling of nuclear DNA fragmentation, first appeared at 3 h and peaked at 6 h following irradiation. Increased expression of the precursors of caspase 1 (ICE), 2 (Nedd2), 3 (CPP32), 6 (Mch2), and 8 (Mch5 and FLICE), and increased expression of active caspase 3, as revealed by immunohistochemistry, were observed in the external granule cell layer of the cerebellum. Radiation-induced apoptosis was accompanied by an increase in the expression of the poly(ADP-ribose) polymerase (PARP) fragment of about 89 kD, as revealed by Western blots of cerebellar homogenates. This was not associated with modifications of protein kinase Cdelta and Lamin B. Concomitant injection in the culmen of the cerebellum in irradiated rats of high doses of Y-VAD-cmk, DEV-fmk, or IETD-fmk resulted in decreased expression of the PARP fragment in cerebellar homogenates. This was accompanied by a decrease in the expression of active caspase 3, as shown by immunohistochemistry. These observations suggest caspase activation following ionizing radiation. However, no differences in the number and morphological and biochemical characteristics of apoptotic cells, including strong nuclear and cytoplasmic c-Jun/AP-1 (N) expression, were observed between irradiated and both irradiated and caspase inhibitor-treated rats. Taken together, these observations suggest that the caspases examined are not essential for radiation-induced apoptosis in the developing cerebellum.     

Serine protease inhibitors N-alpha-tosyl-L-lysinyl-chloromethylketone (TLCK) and N-tosyl-L-phenylalaninyl-chloromethylketone (TPCK) are potent inhibitors of activated caspase proteases

Serine protease inhibitors N-alpha-tosyl-L-lysinyl-chloromethylketone (TLCK) and N-tosyl-L-phenylalaninyl-chloromethylketone (TPCK) exhibit multiple effects on cell death pathways in mammalian cells. Thus, they are able to induce apoptosis by itself or promote cell death induced by other cytotoxic stimuli [King et al., 2004; Murn et al., 2004]. On the other hand, TLCK and TPCK were reported to prevent apoptosis by inhibiting the processing of caspases in response to some cell death inducing stimuli [Stefanis et al., 1997; Jones et al., 1998]. We observed that the pretreatment of HL-60 cells with TLCK or TPCK diminished caspases 3 and -7 (DEVDase) and caspase-6 (VEIDase) activity in response to various cell death inducing stimuli such as staurosporine (STS), etoposide (ETP), or N6-(2-isopentenyl)adenosine. In addition, TLCK but not TPCK inhibited collapse of mitochondrial transmembrane potential Delta Psi m (delta psi) in dying HL-60 cells. Such effects used to be considered as protective, however, the protection was only presumable since neither TLCK nor TPCK actually prevented cells from death. Our results further indicated that serine protease inhibitors TLCK and particularly TPCK acted as efficient direct inhibitors of mature caspases. Indeed, experiments with human recombinant caspases provided unequivocal evidence that TLCK and TPCK are very potent but non-specific inhibitors of activated caspases, namely caspases 3, -6, and -7. Interestingly, TPCK exhibited similar efficiency towards human recombinant caspases to that found for panspecific caspase inhibitor Boc-D-CMK. Such properties of TLCK and TPCK, previously considered as specific inhibitors of serine proteases, might offer novel consistent explanation for several protective or protective-like effects on apoptotic cells.     

Oxidative stress induces caspase-independent retinal apoptosis in vitro

Apoptosis is the mode of cell death in retinitis pigmentosa (RP), a heterogeneous group of retinal degenerations. The activation of the caspase proteases forms a pivotal step in the initiation and execution phase of apoptosis in many cells. Inhibition of caspases has been reported to prevent apoptosis in many model systems. However, we demonstrate the absence of caspase activation during retinal cell apoptosis in vitro which involves phosphatidylserine (PS) externalisation, DNA nicking and cell shrinkage. In addition, zVAD-fmk, DEVD-CHO and BD-fmk, inhibitors of the caspases, were unable to alter the characteristics or kinetics of apoptosis, implying that retinal cell death in vitro follows a caspase-independent pathway. We have previously demonstrated the ability of reactive oxygen species (ROS) to act as mediators of retinal cell apoptosis in vitro as well as the ability of antioxidants to prevent retinal cell apoptosis. Here we demonstrate the oxidative inactivation of caspases in this model of retinal apoptosis and provide evidence for an oxidative stress driven cell death pathway that does not involve caspase activity and which retains key features of apoptotic cell death. Furthermore, our data indicates that apoptotic events such as PS exposure, DNA nicking and cell shrinkage may occur independently of caspase activity.     

Comparative analysis of apoptotic pathways in rat, mouse, and hamster spermatozoa

Apoptosis is a type of cell death characterized by the activation of a family of cysteine-proteases called caspases. We made a comparative study to determine the presence of several caspases and other regulators of apoptosis in rat, mouse, and hamster spermatozoa. Our results showed that the three species have both active and inactive caspases-8 and -3, the proapoptotic protein BID, p53, and the endogenous caspase inhibitor cIAP-1. However, we did not find evidence for the presence of active caspase-9. The acrosome reaction (i.e., the exocytic process of sperm acrosome) and sperm viability were not affected by the presence of a general caspase inhibitor. On the other hand, valinomycin, which promotes caspase-dependent cell death in somatic cells, induced caspase-independent cell death in spermatozoa. TRAIL, a ligand whose receptor induces apoptosis in malignant cells, did not have any effect in the viability of mouse spermatozoa, despise the presence of its receptor in rat and mouse, but not in hamster spermatozoa. Therefore, our results strongly suggest that rodent spermatozoa have some components of the apoptotic pathway. However, the role of caspases in mammalian spermatozoa appears to be unrelated to sperm survival or to the acrosome reaction under physiological conditions.     

Time sequence analysis of caspase-3-independent programmed cell death and apoptosis in X-irradiated human leukemic MOLT-4 cells

It has been demonstrated that caspase-3 is responsible for determining the mode of cell death, i.e., apoptosis or necrosis. To characterize the mode of cell death induced by the inhibition of caspase-3, we have studied the effects of Ac-DEVD-CHO, Ac-YVAD-CHO, and Ac-IETD-CHO, inhibitors of caspases, on structural changes in X-irradiated human leukemic MOLT-4 cells. When cells were irradiated with X-rays and incubated in the presence of Ac-DEVD-CHO, the expression of cell death, as measured by the dye exclusion test, was inhibited, whereas no such change was observed in colony-forming ability. The hallmarks of apoptosis, i.e., nuclear condensation and DNA ladder formation, were depressed. However, a new type of nuclear morphology appeared. The sum of the frequencies of apoptosis and this new type of nuclear structure corresponded to the frequency of X-ray-induced apoptosis for cells incubated in the absence of Ac-DEVD-CHO. Removal of Ac-DEVD-CHO during the course of post-irradiation incubation increased apoptotic nuclear condensation accompanied by a slight decrease in the frequency of the new type of nuclear structure. When Ac-IETD-CHO was used in place of Ac-DEVD-CHO, inhibition of cell death (apoptosis) was also observed, but not in the case of Ac-YVAD-CHO. These results suggest that the inhibition of caspase-3 diminishes the expression of apoptotic hallmarks with no effect on cell survival, that the morphology observed in the presence of Ac-DEVD-CHO is an apoptosis-related structure, and that the cell death observed is a programmed cell death independent of caspase-3. The development of this mode of cell death was slower than that of apoptosis by 4 h.     

Analysis of biochemical markers of MCF-7 cell apoptosis induced by a recombinant analogue of lactaptin

Earlier, a pro-apoptotic peptide lactaptin was isolated from human milk and characterized and its recombinant analogue RL2 was generated. Both peptides were demonstrated to be capable of apoptotic cell death induction in human breast adenocarcinoma MCF-7 cells. In the work, biochemical markers of RL2-induced MCF-7 apoptosis are analyzed. Activation of initiator and effector caspases, as well as apoptotic changes in mitochondrial membrane potential and cytoplasm membrane composition in cells treated with RL2, were analyzed using flow cytometry and Western blot techniques. MCF-7 cell death induced by RL2 was found to be associated with phosphatidylserine exposure on the surface of the plasma membrane. Also, RL2 was demonstrated to induce dissipation of the mitochondrial membrane potential and activation of initiator caspases 8 and 9 and an effector caspase 7.     

Inhibition of caspases inhibits the release of apoptotic bodies: Bcl-2 inhibits the initiation of formation of apoptotic bodies in chemotherapeutic agent-induced apoptosis

During apoptosis, the cell actively dismantles itself and reduces cell size by the formation and pinching off of portions of cytoplasm and nucleus as "apoptotic bodies." We have combined our previously established quantitative assay relating the amount of release of [3H]-membrane lipid to the degree of apoptosis with electron microscopy (EM) at a series of timepoints to study apoptosis of lymphoid cells exposed to vincristine or etoposide. We find that the [3H]-membrane lipid release assay correlates well with EM studies showing the formation and release of apoptotic bodies and cell death, and both processes are regulated in parallel by inducers or inhibitors of apoptosis. Overexpression of Bcl-2 or inhibition of caspases by DEVD inhibited equally well the activation of caspases as indicated by PARP cleavage. They also inhibited [3H]-membrane lipid release and release of apoptotic bodies. EM showed that cells overexpressing Bcl-2 displayed near-normal morphology and viability in response to vincristine or etoposide. In contrast, DEVD did not prevent cell death. Although DEVD inhibited the chromatin condensation, PARP cleavage, release of apoptotic bodies, and release of labeled lipid, DEVD-treated cells showed accumulation of heterogeneous vesicles trapped in the condensed cytoplasm. These results suggest that inhibition of caspases arrested the maturation and release of apoptotic bodies. Our results also imply that Bcl-2 regulates processes in addition to caspase activation.