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.     

Teratogen-induced activation of caspase-6 and caspase-7 in early postimplantation mouse embryos

BACKGROUND: Previous work has shown that teratogens such as hyperthermia (HS), 4-hydroperoxycyclophosphamide (4CP), and staurosporine (ST) induce cell death in day 9 mouse embryos by activating the mitochondrial apoptotic pathway. Key to the activation of this pathway is the activation of a caspase cascade involving the cleavage-induced activation of an initiator procaspase, caspase-9, and the downstream effector procaspase, caspase-3. For example, procaspase-3, an inactive proenzyme of 32 kDa is cleaved by activated caspase-9 to generate a large subunit of approximately 17 kDa and a small subunit of approximately 10 kDa. In turn, caspase-3 is known to target a variety of cellular proteins for proteolytic cleavage as part of the process by which dying cells are eliminated. Previous work has also shown that neuroepithelial cells are sensitive to teratogen-induced activation of this pathway and subsequent cell death whereas cells of the heart are resistant. Although caspase-3 is a key effector caspase activated by teratogens, two other effector caspases, caspase-6 and caspase-7, are known; however, their role in teratogen-induced cell death is unknown. METHODS: Because cleavage-induced generation of specific subunits is the most specific assay for activation of caspases, we have used antibodies that recognize the procaspase and one of its active subunits and a Western blot approach to assess the activation of caspase-6 and caspase-7 in day 9 mouse embryos (or heads, hearts and trunks isolated from whole embryos) exposed to HS, 4CP, and ST. To probe the relationship between teratogen-induced activation of caspase-9/caspase-3 and the activation of caspase-6/caspase-7, we used a mitochondrial-free embryo lysate with or without the addition of cytochrome c, recombinant active caspase-3, or recombinant active caspase-9. RESULTS: Western blot analyses show that these three teratogens, HS, 4CP, and ST, induce the activation of procaspase-6 (appearance of the 13 kDa subunit, p13) and caspase-7 (appearance of the 19 kDa subunit, p19) in day 9 mouse embryos. In vitro studies showed that both caspase-6 and caspase-7 could be activated by the addition of cytochrome c to a lysate prepared from untreated embryos. In addition, caspase-6 could be activated by the addition of either recombinant caspase-3 or caspase-9 to a lysate prepared from untreated embryos. In contrast, caspase-7 could be activated by addition of recombinant caspase-3 but only minimally by recombinant caspase-9. Like caspase-9/caspase-3, caspase-6 and caspase-7 were not activated in hearts isolated from embryos exposed to these three teratogens. CONCLUSIONS: HS, 4CP and ST induce the cleavage-dependent activation of caspase-6 and caspase-7 in day 9 mouse embryos. Results using DEVD-CHO, a caspase-3 inhibitor, suggest that teratogen-induced activation of caspase-6 is mediated by caspase-3. In addition, our data suggest that caspase-7 is activated primarily by caspase-3; however, we cannot rule out the possibility that this caspase is also activated by caspase-9. Finally, we also show that teratogen-induced activation of caspase-6 and caspase-7 are blocked in the heart, a tissue resistant to teratogen-induced cell death.     

Roles of DNA fragmentation factor and poly(ADP-ribose) polymerase in an amplification phase of tumor necrosis factor-induced apoptosis.

During apoptosis, endonucleases cleave DNA into 50-300-kb fragments and subsequently into internucleosomal fragments. DNA fragmentation factor (DFF) is implicated in apoptotic DNA cleavage; this factor comprises DFF45 and DFF40 subunits, the former of which acts as a chaperone and inhibitor of the catalytic subunit and whose cleavage by caspase-3 results in DFF activation. Disruption of the DFF45 gene blocks internucleosomal DNA fragmentation and confers resistance to apoptosis in primary thymocytes. The role of DFF-mediated DNA fragmentation in apoptosis was investigated in primary fibroblasts from DFF45(-/-) and control (DFF45(+/+)) mice. DFF45 deficiency rendered fibroblasts resistant to apoptosis induced by tumor necrosis factor (TNF). TNF induced rapid cleavage of DNA into approximately 50-kb fragments in DFF45(+/+) fibroblasts but not in DFF45(-/-) cells, indicating that DFF mediates this initial step in DNA processing. The TNF-induced activation of poly(ADP-ribose) polymerase (PARP), which requires PARP binding to DNA strand breaks, and the consequent depletion of the PARP substrate NAD were markedly delayed in DFF45(-/-) cells, suggesting a role for DFF in PARP activation. The activation of caspase-3 and mitochondrial events important in apoptotic signaling, including the loss of mitochondrial membrane potential and the release of cytochrome c, induced by TNF were similarly delayed in DFF45(-/-) fibroblasts. DFF45(-/-) and DFF45(+/+) cells were equally sensitive to the DNA-damaging agent and PARP activator N-methyl-N'-nitro-N-nitrosoguanidine. Inhibition of PARP by 3-aminobenzamide partially protected DFF45(+/+) cells against TNF-induced death and inhibited the associated release of cytochrome c and activation of caspase-3. These results suggest that the generation of 50-kb DNA fragments by DFF, together with the activation of PARP, mitochondrial dysfunction, and caspase-3 activation, contributes to an amplification loop in the death process.     

Co-localization of active caspase-3 and DNA fragmentation (TUNEL) in normal and hyperthermia-induced abnormal mouse development

BACKGROUND: Previous work has shown that caspase-3 activation and DNA fragmentation, two hallmarks of apoptosis, are induced in day 9 mouse embryos exposed to hyperthermia (43 degrees C); however, the methods used to assess caspase-3 activation (Western blot) and DNA fragmentation (gel electrophoresis) did not allow these apoptotic events to be localized to specific cells within the embryo. METHODS: To co-localize active caspase-3 and DNA fragmentation to specific cells, we used paraffin sections of day 13 mouse limb buds, sections of control and hyperthermia-treated day 9 mouse embryos, and sequential immunohistochemical staining for caspase-3 and TUNEL staining for DNA fragmentation. We used a primary rabbit antibody specific for the active, p17 subunit of caspase-3 and a goat anti-rabbit secondary antibody conjugated to Alexa 594 fluorochrome (red fluorescence) to localize active caspase-3. To co-localize DNA fragmentation, we subsequently processed the same sections by the TUNEL method using fluorescein-labeled dUTP (green fluorescence). RESULTS: Using this dual labeling approach, we show that active caspase-3 (caspase-3 positive) and DNA fragmentation (TUNEL positive) occur in a sub-population of interdigital mesenchyme cells of day 13 mouse limb buds. Using the same approach, we detected a small number of caspase-3 positive and TUNEL-positive cells in the central nervous system and in the mesenchyme of the first branchial arch of untreated day 9 mouse embryos. The number of caspase-3 and TUNEL-positive cells are greatly increased 5 hr after a brief exposure to hyperthermia (43 degrees C, 13 min). Caspase-3 and TUNEL-positive cells were most abundant in the neuroepithelium of the developing central nervous system, mesenchyme of the first pharyngeal arch, and somitic mesoderm. In contrast, the heart, mesencephalic mesenchyme, and the visceral yolk sac contained few, if any, caspase-3 and TUNEL-positive cells. CONCLUSIONS: This is the first demonstration that activation of caspase-3 and DNA fragmentation co-localize in cells programmed to die in the interdigital mesenchyme of day 13 limb buds and in the neuroepithelium and branchial arch mesenchyme of day 9 mouse embryos. Similarly, our results represent the first co-localization of teratogen-induced activation of caspase-3 and DNA fragmentation in specific cells of early postimplantation mouse embryos, and confirm that cells of the developing central nervous system are acutely sensitive to the cell death-inducing potential of hyperthermia, whereas cells of the heart are resistant. Finally, we show for the first time that, like cells of the heart, cells of the mesencephalic mesenchyme and the visceral yolk sac are also resistant to hyperthermia-induced apoptosis.     

Cleavage of Poly(ADP-ribose) polymerase measured in situ in individual cells: relationship to DNA fragmentation and cell cycle position during apoptosis

Poly(ADP-ribose) polymerase (PARP), a nuclear enzyme involved in DNA repair, is a target of caspases during apoptosis: its cleavage onto 89- and 24-kDa fragments is considered to be a hallmark of the apoptotic mode of cell death. Another hallmark is the activation of endonuclease which targets internucleosomal DNA. The aim of the present study was to reveal cell cycle phase specificity as well as the temporal and sequence relationships of PARP cleavage vis-à-vis DNA fragmentation in two model systems of apoptosis, one induced by DNA damage via cell treatment with camptothecin (CPT) (mitochondria-induced pathway) and another by the cytotoxic ligand tumor necrosis factor alpha (TNF-alpha) (cell surface death receptor pathway). PARP cleavage was detected immunocytochemically using antibody which recognizes its 89-kDa fragment (PARP p89) while DNA fragmentation was assayed by in situ labeling of DNA strand breaks. The frequency and extent of PARP cleavage as well as DNA fragmentation were measured by mutiparameter flow and laser scanning cytometry. PARP cleavage, selective to S phase cells, was detected 90 min after administration of CPT. PARP cleavage in the cells treated with TNF-alpha was not selective to any cell cycle phase and was seen already after 30 min. DNA fragmentation trailed PARP cleavage by about 30 min and showed a similar pattern of cell cycle specificity. PARP p89 was present in nuclear chromatin but at least in the early phase of apoptosis it did not colocalize with DNA strand breaks. The rate of cleavage of PARP molecules in individual cells whether induced by CPT or TNF-alpha was rapid as reflected by the paucity of cells with a mixture of cleaved and noncleaved PARP molecules. In contrast, DNA fragmentation proceeded stepwise before reaching the maximal number of DNA strand breaks. Although time windows for PARP cleavage vs DNA fragmentation were different at early stages of apoptosis, a good overall correlation between the cytometric assays of apoptotic cells identification based on these events was observed in both CPT- and TNF-alpha-treated cultures.     

Myrtucommulone from <Emphasis Type="Italic">Myrtus communis</Emphasis> induces apoptosis in cancer cells via the mitochondrial pathway involving caspase-9

Myrtucommulone (MC) is a unique, nonprenylated acylphloroglucinol contained in the leaves of myrtle (Myrtus communis). Here, we addressed the potential of MC to induce apoptosis of cancer cells. MC potently induced cell death of different cancer cell lines (EC₅₀ 3–8 μM) with characteristics of apoptosis, visualized by the activation of caspase-3, -8 and -9, cleavage of poly(ADP-ribose)polymerase (PARP), release of nucleosomes into the cytosol, and DNA fragmentation. MC was much less cytotoxic for non-transformed human peripheral blood mononuclear cells (PBMC) or foreskin fibroblasts (EC₅₀ cell death = 20–50 μM), and MC up to 30 μM hardly caused processing of PARP, caspase-3, -8 and -9 in human PBMC. MC-induced apoptosis was mediated by the intrinsic rather than the extrinsic death pathway. Thus, MC caused loss of the mitochondrial membrane potential in MM6 cells and evoked release of cytochrome c from mitochondria. Interestingly, Jurkat cells deficient in caspase-9 were resistant to MC-induced cell death and no processing of PARP or caspase-8 was evident. In cell lines deficient in either CD95 (Fas, APO-1) signalling, FADD or caspase-8, MC was still able to potently induce cell death and PARP cleavage. Conclusively, MC induces apoptosis in cancer cell lines, with marginal cytotoxicity for non-transformed cells, via the mitochondrial cytochrome c/Apaf-1/caspase-9 pathway. I. Tretiakova and D. Blaesius contributed equally to this work.     

Involvement of caspase-3 activation in squamocin-induced apoptosis in leukemia cell line HL-60

Annonaceous acetogenins have potent antitumor effect in vitro and in vivo. Squamocin is one of the annonaceous acetogenins and has been reported to have antiproliferative effect on cancer cells. Our results from this study showed that squamocin inhibited proliferation of HL-60 cells with IC50 value of 0.17 microg/ml and induced apoptosis of HL-60 cells. Investigation of the mechanism of squamocin-induced apoptosis revealed that treatment of HL-60 cells with squamocin resulted in extensive nuclear condensation. DNA fragmentation, cleavage of the death substrate poly (ADP-ribose) polymerase (PARP) and induction of caspase-3 activity. Pretreatment of HL-60 cells with caspase-3 specific inhibitor DEVD-CHO prevented squamocin-induced DNA fragmentation, PARP cleavage and cell death. The expression levels of protein bcl-2, bax have no change in response to squamocin treatment in HL-60 cells, whereas stress-activated protein kinase (SAPK/JNK) was activated after treatment with squamocin in HL-60 cells. These results suggest that apoptosis of HL-60 cells induced by squamocin requires caspase-3 activation and is related to SAPK activation.     

Caspase-3 activation during the process of apoptosis induced by a vacuolar type H(+)-ATPase inhibitor

Concanamycin A, a specific inhibitor of vacuolar type H(+)-ATPases, induced DNA fragmentation in B cell hybridoma HS-72 cells. Immunoblot analysis revealed that the exposure of concanamycin A to HS-72 cells induced the cleavage of retinoblastoma protein (Rb) and poly(ADP-ribose) polymerase (PARP). The cytosol from concanamycin A-treated HS-72 cells induced DNA fragmentation in nuclei from untreated cells in a cell-free system. This fragmentation was suppressed by a specific inhibitor of caspase-3. The cytosol induced Rb proteolysis in vitro, which was inhibited by a caspase-3 inhibitor. These findings indicate that caspase-3 induces DNA fragmentation and cleavage of Rb and PARP during the process of apoptosis induced by concanamycin A.     

Failure of Poly(ADP-Ribose) Polymerase Cleavage by Caspases Leads to Induction of Necrosis and Enhanced Apoptosis

Activation of poly(ADP-ribose) polymerase (PARP) by DNA breaks catalyzes poly(ADP-ribosyl)ation and results in depletion of NAD+ and ATP, which is thought to induce necrosis. Proteolytic cleavage of PARP by caspases is a hallmark of apoptosis. To investigate whether PARP cleavage plays a role in apoptosis and in the decision of cells to undergo apoptosis or necrosis, we introduced a point mutation into the cleavage site (DEVD) of PARP that renders the protein resistant to caspase cleavage in vitro and in vivo. Here, we show that after treatment with tumor necrosis factor alpha, fibroblasts expressing this caspase-resistant PARP exhibited an accelerated cell death. This enhanced cell death is attributable to the induction of necrosis and an increased apoptosis and was coupled with depletion of NAD+ and ATP that occurred only in cells expressing caspase-resistant PARP. The PARP inhibitor 3-aminobenzamide prevented the NAD+ drop and concomitantly inhibited necrosis and the elevated apoptosis. These data indicate that this accelerated cell death is due to NAD+ depletion, a mechanism known to kill various cell types, caused by activation of uncleaved PARP after DNA fragmentation. The present study demonstrates that PARP cleavage prevents induction of necrosis during apoptosis and ensures appropriate execution of caspase-mediated programmed cell death.     

Caspase-dependent apoptosis and -independent poly(ADP-ribose) polymerase cleavage induced by transforming growth factor beta1

Apoptosis is an important cell suicide program which involves the caspases activation and is implicated in physiological and pathological processes. Poly(ADP-ribose) polymerase (PARP) cleavage is often associated with apoptosis and has been served as one hallmark of apoptosis and caspase activation. In this study, we aimed to determine TGF-beta1-induced apoptosis and to examine the involvement of caspases and its relationship with PARP cleavage. TGF-beta1 induces strong apoptosis of AML-12 cells which can be detected by DNA fragmentation, FACS, and morphological assays. Z-VAD-fmk, a selective caspase inhibitor, partially inhibits the TGF-beta1-induced apoptosis; but has no effect on TGF-beta1-induced DNA fragmentation and PARP cleavage. However, BD-fmk, a broad-spectrum caspase inhibitor, completely suppresses TGF-beta1-induced apoptosis, but unexpectedly does not inhibit TGF-beta1-induced PARP cleavage. Furthermore, Z-VAD-fmk treatment is able to completely inhibit the daunorubicin-induced apoptosis in A-431 cells, but only slightly blocks the daunorubicin-induced PARP cleavage, whereas BD-fmk can inhibit both daunorubicin-induced apoptosis and PARP cleavage completely. In addition, we observed that both TGF-beta1-induced apoptosis and PARP degradation in AML-12 cells can be completely blocked by inhibiting the protein synthesis with cycloheximide. These results demonstrate for the first time that TGF-beta1-induced caspase-dependent apoptosis is associated with caspase-independent PARP cleavage that requires the TGF-beta1-induced synthesis of new proteins. The results indicate that caspase-3 is not a major caspase involved in TGF-beta1-induced apoptosis in AML-12 cells, and is not required for apoptosis-associated DNA fragmentation. The results also suggest that PARP cleavage may occur as an independent event that can be disassociated with cell apoptosis.     

Reactive oxygen species and caspase activation mediate silica-induced apoptosis in alveolar macrophages

Alveolar macrophages (AMs) are the principal target cells of silica and occupy a key position in the pathogenesis of silica-related diseases. Silica has been found to induce apoptosis in AMs, whereas its underlying mechanisms involving the initiation and execution of apoptosis are largely unknown. The main objective of the present study was to examine the form of cell death caused by silica and the mechanisms involved. Silica-induced apoptosis in AMs was evaluated by terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling assay and cell cycle/DNA content analysis. The elevated level of reactive oxygen species (ROS), caspase-9 and caspase-3 activation, and poly(ADP-ribose) polymerase (PARP) cleavage in silica-treated AMs were also determined. The results showed that there was a temporal pattern of apoptotic events in silica-treated AMs, starting with ROS formation and followed by caspase-9 and caspase-3 activation, PARP cleavage, and DNA fragmentation. Silica-induced apoptosis was significantly attenuated by a caspase-3 inhibitor, N-acetyl-Asp-Glu-Val-Asp aldehyde, and ebselen, a potent antioxidant. These findings suggest that apoptosis is an important form of cell death caused by silica exposure in which the elevated ROS level that results from silica exposure may act as an initiator, leading to caspase activation and PARP cleavage to execute the apoptotic process.     

Survival and proliferation of cells expressing caspase-uncleavable Poly(ADP-ribose) polymerase in response to death-inducing DNA damage by an alkylating agent

To determine whether caspase-3-induced cleavage of poly(ADP-ribose) polymerase (PARP), a DNA damage-sensitive enzyme, alters the balance between survival and death of the cells following DNA damage, we created stable cell lines that express either caspase-uncleavable mutant or wild type PARP in the background of PARP (-/-) fibroblasts. The survival and apoptotic responses of these cells were compared after exposure to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a DNA-damaging agent that activates PARP, or to tumor necrosis factor-alpha, which causes apoptosis without initial DNA damage. In response to MNNG, the cells with caspase-uncleavable PARP were very resistant to loss of viability or induction of apoptosis. Most significantly, approximately 25% of these cells survived and retained clonogenicity at a level of DNA damage that eliminated the cells with wild type PARP or PARP (-/-) cells. Expression of caspase-uncleavable PARP could not protect the cells from death induced by tumor necrosis factor, although there was a slower progression of apoptotic events in these cells. Therefore, one of the functions for cleavage of PARP during apoptosis induced by alkylating agents is to prevent survival of the extensively damaged cells.     

Poly(ADP-ribose) Polymerase Activation and Cell Injury in the Course of Rat Heart Heterotopic Transplantation

Free radicals and other reactive species generated during reperfusion of ischemic tissues may cause DNA damage and, consequently, the activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP). An excessive PARP activation may result in a depletion of intracellular NAD + and ATP, hence cell suffering and, ultimately, cell death. The present study is aimed at clarifying the role of PARP in a heart transplantation procedure and the contribution of myocyte necrosis and/or apoptosis to this process. In our experimental model, rat heart subjected to heterotopic transplantation, low temperature global ischemia (2 h) was followed by an in vivo reperfusion (30 or 60 &#117 min). Under these conditions clear signs of oxidative stress, such as lipoperoxidation and DNA strand breaks, were evident. In addition to a marked activation, accompanied by a significant NAD + and ATP depletion, PARP protein levels significantly increased after 60 &#117 min of reperfusion. Ultrastructural analysis showed nuclear clearings, intracellular oedema and plasma membrane discontinuity. Other relevant observations were the absence of typical signs of apoptosis like caspase-3 activation and PARP cleavage, random DNA fragmentation, rise in serum levels of heart damage markers. Our results suggest that during heart transplantation, the activation of PARP, causing energy depletion, results in myocardial cell injury whose dominant feature, at least in our experimental model, is necrosis rather than apoptosis.     

Poly(ADP-ribose) polymerase activation and cell injury in the course of rat heart heterotopic transplantation

Free radicals and other reactive species generated during reperfusion of ischemic tissues may cause DNA damage and, consequently, the activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP). An excessive PARP activation may result in a depletion of intracellular NAD + and ATP, hence cell suffering and, ultimately, cell death. The present study is aimed at clarifying the role of PARP in a heart transplantation procedure and the contribution of myocyte necrosis and/or apoptosis to this process. In our experimental model, rat heart subjected to heterotopic transplantation, low temperature global ischemia (2 h) was followed by an in vivo reperfusion (30 or 60 λmin). Under these conditions clear signs of oxidative stress, such as lipoperoxidation and DNA strand breaks, were evident. In addition to a marked activation, accompanied by a significant NAD + and ATP depletion, PARP protein levels significantly increased after 60 λmin of reperfusion. Ultrastructural analysis showed nuclear clearings, intracellular oedema and plasma membrane discontinuity. Other relevant observations were the absence of typical signs of apoptosis like caspase-3 activation and PARP cleavage, random DNA fragmentation, rise in serum levels of heart damage markers. Our results suggest that during heart transplantation, the activation of PARP, causing energy depletion, results in myocardial cell injury whose dominant feature, at least in our experimental model, is necrosis rather than apoptosis.     

Caspase-dependent activation of calpain during drug-induced apoptosis

We have previously demonstrated that calpain is responsible for the cleavage of Bax, a proapoptotic protein, during drug-induced apoptosis of HL-60 cells (Wood, D. E., Thomas, A., Devi, L. A., Berman, Y., Beavis, R. C., Reed, J. C., and Newcomb, E. W. (1998) Oncogene 17, 1069-1078). Here we show the sequential activation of caspases and calpain during drug-induced apoptosis of HL-60 cells. Time course experiments using the topoisomerase I inhibitor 9-amino-20(S)-camptothecin revealed that cleavage of caspase-3 substrates poly(ADP-ribose) polymerase (PARP) and the retinoblastoma protein as well as DNA fragmentation occurred several hours before calpain activation and Bax cleavage. Pretreatment with the calpain inhibitor calpeptin blocked calpain activation and Bax cleavage but did not inhibit PARP cleavage, DNA fragmentation, or 9-amino-20(S)-camptothecin-induced morphological changes and cell death. Pretreatment with the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk) inhibited PARP cleavage, DNA fragmentation, calpain activation, and Bax cleavage and increased cell survival by 40%. Interestingly, Z-VAD-fmk-treated cells died in a caspase- and calpain-independent manner that appeared morphologically distinct from apoptosis. Our results suggest that excessive or uncontrolled calpain activity may play a role downstream of and distinct from caspases in the degradation phase of apoptosis.     

α-Santalol, a derivative of sandalwood oil, induces apoptosis in human prostate cancer cells by causing caspase-3 activation

The anticancer effects of α-santalol, a major component of sandalwood oil, have been reported against the development of certain cancers such as skin cancer both in vitro and in vivo. The primary objectives of the current study were to investigate the cancer preventive properties of α-santalol on human prostate cancer cells PC-3 (androgen independent and P-53 null) and LNCaP (androgen dependent and P-53 wild-type), and determine the possible mechanisms of its action. The effect of α-santalol on cell viability was determined by trypan blue dye exclusion assay. Apoptosis induction was confirmed by analysis of cytoplasmic histone-associated DNA fragmentation using both an apoptotic ELISA kit and a DAPI fluorescence assay. Caspase-3 activity was determined using caspase-3 (active) ELISA kit. PARP cleavage was analyzed using immunoblotting. α-Santalol at 25-75 μM decreased cell viability in both cell lines in a concentration and time dependent manner. Treatment of prostate cancer cells with α-santalol resulted in induction of apoptosis as evidenced by DNA fragmentation and nuclear staining of apoptotic cells by DAPI. α-Santalol treatment also resulted in activation of caspase-3 activity and PARP cleavage. The α-santalol-induced apoptotic cell death and activation of caspase-3 was significantly attenuated in the presence of pharmacological inhibitors of caspase-8 and caspase-9. In conclusion, the present study reveals the apoptotic effects of α-santalol in inhibiting the growth of human prostate cancer cells.     

Photodynamic therapy induces caspase-3 activation in HL-60 cells

Caspases have been shown to play a crucial role in apoptosis induced by various deleterious and physiologic stimuli. In this study, we show for the first time that photodynamic therapy (PDT), using benzoporphyrin derivative monoacid ring A (BPD-MA, verteporfin) as the photosensitizer, induces the complete cleavage and subsequent activation of caspase-3 (CPP32/Yama/Apopain) but not caspase-1 (ICE) in human promyelocytic leukemia HL-60 cells. Poly(ADP-ribose) polymerase (PARP) and the catalytic subunit of DNA dependent protein kinase (DNA PK(CS)) were cleaved within 60 min of light activation of BPD-MA. The general caspase inhibitor Z-Asp-2,6 dichlorobenzoyloxymethylketone (Z-Asp-DCB) blocked PARP cleavage while the serine protease inhibitors 3,4-dichloroisocoumarin (DCI) and N-tosyl-lysyl chloromethyl ketone (TLCK) blocked the cleavage of caspase-3 suggesting that they act upstream of caspase-3 activation. All three inhibitors were able to block DNA fragmentation that was induced by treatment with BPD-MA followed by light application. These studies demonstrate that protease activity, particularly that of caspase-3, is triggered in HL-60 cells treated with lethal levels of BPD-MA and visible light.     

Poly (ADP-ribose) polymerase cleavage monitored in situ in apoptotic cells

During apoptosis, the activation of a family of cysteine proteases, or caspases, results in proteolytic cleavage of numerous substrates. Antibody probes specific for neoepitopes on protein fragments generated by caspase cleavage provide a means to monitor caspase activity at the level of the individual cell. Poly (ADP-ribose) polymerase (PARP), a nuclear enzyme involved in DNA repair, is a well-known substrate for caspase-3 cleavage during apoptosis. Its cleavage is considered to be a hallmark of apoptosis. Here, we demonstrate that an affinity-purified polyclonal antibody to the p85 fragment of PARP is specific for apoptotic cells. Western blots show that the antibody recognizes the 85-kDa (p85) fragment of PARP but not full-length PARP. We demonstrate a time course of PARP cleavage and DNA fragmentation in situ using the PARP p85 fragment antibody and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) in Jurkat cells treated with anti-Fas. Furthermore, our results indicate that the p85 fragment of PARP resulting from caspase cleavage during apoptosis is rapidly localized outside the condensed chromatin but not in the cytoplasm.     

Role of protein kinase Cdelta in UV-B-induced apoptosis of macrophages in vitro

We have previously reported that murine peritoneal macrophages exposed to ultraviolet B (UV-B; 100 mJ/cm2) undergo apoptosis, as indicated by alterations in cell morphology, caspase-3 activation, poly (ADP-ribose) polymerase (PARP) cleavage, DNA fragmentation, sustained activation of p38/c-Jun N-terminal kinase (JNK) mitogen-activated protein kinases (MAPKs) and inactivation of p42/44 MAPKs. It is now reported that macrophages undergoing UV-B-induced apoptosis show enhanced expression of protein kinase Cdelta (PKCdelta) in a time-dependent manner. Pretreatment of macrophages with PKCdelta-specific inhibitor rottlerin prior to the UV-B irradiation inhibits activation of caspase-3, PARP cleavage, DNA fragmentation and release of intracellular Ca2+. Inhibition of PKCdelta also blocks the sustained activation of p38 and JNK MAPKs as well as inactivation of p42/44 MAPKs. PKCalpha and PKCbeta1 expression also increases during UV-B-induced apoptosis in macrophages. Inhibition of these two isoforms with Go6976 slightly suppresses caspase-3 activation, PARP cleavage, DNA fragmentation and release of intracellular Ca2+, but has no effect on the sustained activation of p38/JNK MAPKs or inactivation of p42/44 MAPKs. It is, therefore, suggested that activation of PKCdelta might play an important role in the UV-B-induced apoptosis and that specific activated isoforms of PKC may have distinct functions in cell death.