[Show abstract][Hide abstract] ABSTRACT: Zinc has been known for many years to inhibit apoptosis but the mechanism remains unclear. Originally thought to inhibit an apoptotic endonuclease, zinc has subsequently been shown to inhibit steps earlier in the pathway. Since many additional steps in apoptosis have now been defined, we have re-evaluated the steps inhibited by zinc. In response to activation of the chemical-mediated death pathway by anisomycin, 0.3 mM zinc inhibited Bax and Bak activation, cytochrome c release, and all of the subsequent steps in apoptosis. In the receptor-mediated death pathway initiated by Fas or tumor necrosis factor, 3 mM zinc was required to inhibit apoptosis as judged by inhibition of caspase 3 activity and DNA digestion, but it failed to inhibit cytochrome c release, activation of Bax and Bak, or upstream signaling events in this pathway. These results are consistent with zinc selectively inhibiting activation of BH3-only proteins required in the chemical pathway but inhibiting downstream caspase activation in the death-receptor pathway.
Cell Death and Differentiation 07/2003; 10(6):652-61. · 8.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Deoxyribonuclease IIalpha (DNase IIalpha) is one of many endonucleases implicated in DNA digestion during apoptosis. We produced mice with targeted disruption of DNase IIalpha and defined its role in apoptosis. Mice deleted for DNase IIalpha die at birth with many tissues exhibiting large DNA-containing bodies that result from engulfed but undigested cell corpses. These DNA-containing bodies are pronounced in the liver where fetal definitive erythropoiesis occurs and extruded nuclei are degraded. They are found between the digits, where apoptosis occurs, and in many other regions of the embryo. Defects in the diaphragm appear to cause death of the mice due to asphyxiation. The DNA in these bodies contains 3'-hydroxyl ends and therefore stain positive in the TUNEL assay. In addition, numerous unengulfed TUNEL-positive cells are observed throughout the embryo. Apoptotic cells are normally cleared rapidly from a tissue; hence the persistence of the DNA-containing bodies and TUNEL-positive cells identifies sites where apoptosis occurs during development. These results demonstrate that DNase IIalpha is not required for the generation of the characteristic DNA fragmentation that occurs during apoptosis but is required for degrading DNA of dying cells and this function is necessary for proper fetal development.
Cell Death and Differentiation 10/2002; 9(9):956-62. · 8.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Acidic endonuclease activity is present in all cells in the body and much of this can be attributed to the previously cloned and ubiquitously expressed deoxyribonuclease II (DNase II). Database analysis revealed the existence of expressed sequence tags and genomic segments coding for a protein with considerable homology to DNase II. This report describes the cloning of this cDNA, which we term deoxyribonuclease IIbeta (DNase IIbeta) and comparison of its expression to that of the originally cloned DNase II (now termed DNase IIalpha). The cDNA encodes a 357 amino acid protein. This protein exhibits extensive homology to DNase IIalpha including an amino-terminal signal peptide and a conserved active site, and has many of the regions of identity that are conserved in homologs in other mammals as well as C. elegans and Drosophila. The gene encoding DNase IIbeta has identical splice sites to DNase IIalpha. Human DNase IIbeta is highly expressed in the salivary gland, and at low levels in trachea, lung, prostate, lymph node, and testis, whereas DNase IIalpha is ubiquitously expressed in all tissues. The expression pattern of human DNase IIbeta suggests that it may function primarily as a secreted enzyme. Human saliva was found to contain DNase IIalpha, but after immunodepletion, considerable acid-active endonuclease remained which we presume is DNase IIbeta. We have localized the gene for human DNase IIbeta to chromosome 1p22.3 adjacent (and in opposing orientation) to the human uricase pseudogene. Interestingly, murine DNase IIbeta is highly expressed in the liver. Uricase is also highly expressed in mouse but not human liver and this may explain the difference in expression patterns between human and mouse DNase IIbeta.
[Show abstract][Hide abstract] ABSTRACT: Transforming growth factor beta (TGF-beta) is a multifunctional cytokine capable of regulating diverse cellular processes. In this study we investigated the effect of autocrine TGF-beta signaling on tumor necrosis factor (TNF) alpha-induced cell death. We abrogated the TGF-beta autocrine loop by overexpression of a truncated TGF-beta type II receptor in MCF-7 breast carcinoma cells and found that this generated resistance to TNF-alpha-induced cytotoxicity. To elucidate the molecular basis of the influence of TGF-beta on TNF-alpha-induced cytotoxicity, we evaluated the expression levels or activities of proteins involved in TNF-alpha signal transduction or the regulation of apoptosis in general in TGF-beta-responsive and TGF-beta-nonresponsive MCF-7 cells. We observed no significant difference in the expression of TNF-alpha receptors or the TNF receptor-associated death domain protein. In addition, downstream activation of nuclear factor kappaB by TNF-alpha was not altered in cells that had lost TGF-beta responsiveness. Analysis of members of the Bcl-2 family of apoptosis-regulatory proteins revealed that Bcl-X(L) and Bax expression levels were not changed by disruption of TGF-beta signaling. In contrast, the TGF-beta-nonresponsive cells expressed much higher levels of Bcl-2 protein and mRNA than did cells with an intact TGF-beta autocrine loop. Furthermore, restoration of a TGF-beta signal to MCF-7 cells that had spontaneously acquired resistance to TGF-beta caused a reduction in Bcl-2 protein expression. Taken together, our data indicate that loss of autocrine TGF-beta signaling results in enhanced resistance to TNF-alpha-mediated cell death and that this is likely to be mediated by derepression of Bcl-2 expression.
Cell growth & differentiation: the molecular biology journal of the American Association for Cancer Research 03/2001; 12(2):109-17.
[Show abstract][Hide abstract] ABSTRACT: Chemotherapeutic agents induce alterations in intracellular signal transduction cascades that culminate in the initiation of the apoptotic program. Here, the relationship between the mitogen-activated protein kinase (MAPK) response and apoptosis in ML-1 cells treated with vinblastine and paclitaxel was investigated. We show that these compounds elicit different effects on MAPKs with vinblastine, but not paclitaxel, increasing both c-Jun-NH2-terminal kinase (JNK) and p38 activity. However, vinblastine and paclitaxel both induced apoptosis with similar kinetics, suggesting that increased JNK and p38 activity is not required for apoptosis that is induced by microtubule interfering agents. Strikingly, the abrogation of extracellular signal-regulated kinase (ERK)-signaling by the MAPK/ERK kinase (MEK)1/2 inhibitor PD098059 in combination with vinblastine robustly induced apoptosis in ML-1 cells at a rate much faster than treatment with vinblastine alone and occurred at all phases of the cell cycle. This apoptotic induction was attributed to JNK activation because: (a) non-JNK-activating concentrations of vinblastine failed to increase apoptosis in the presence of PD098059; (b) apoptosis induced by paclitaxel, which did not activate JNK, was not potentiated by PD098059; and (c) transduction of an inhibitor of JNK activity partially suppressed both JNK activity and apoptosis induced by vinblastine plus PD098059. Additionally, we found that the activation of JNK by vinblastine occurred upstream of effector caspase activation because treatment with a pan-specific caspase inhibitor (valine-alanine-aspartate-fluoromethylketone) resulted in complete abrogation of apoptosis with no effect on MAPK signaling. Taken together, these data suggest that inhibition of the MEK-->ERK signal transduction cascade alleviates cell cycle dependence for vinblastine-induced apoptosis by a mechanism that requires JNK activation.
Cancer Research 03/2001; 61(4):1533-40. · 8.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Deoxyribonuclease II (DNase II) has been implicated in diverse functions including degradation of foreign DNA, genomic instability, and in mediating the DNA digestion associated with apoptosis. The production of a mouse deleted for DNase II would clearly help to discriminate these functions. We have cloned and sequenced the mouse gene encoding DNase II. It was found to have a similar intron/exon structure to the human gene, although introns 3 and 5 are considerably shorter. The gene is located on mouse chromosome 8. The order of genes at this locus is mGCDH, mEKLF, mDNase II, mSAST, which is the same order that these genes are found on human chromosome 19. The GenBank database contains incorrect expressed sequence tags (ESTs) for the 3' end of the mouse mRNA. Furthermore, the gene structure of two of the three homologs in C. elegans is also incorrectly predicted in the database. We have established the correct intron/exon structure for these genes and show the conserved sequence and structure of the C. elegans, murine and human genes.
[Show abstract][Hide abstract] ABSTRACT: We have previously reported that UCN-01 (7-hydroxystaurosporine), a protein kinase inhibitor that is under clinical trials as an anti-cancer agent in the USA and Japan, enhanced the anti-tumor activity of mitomycin C (MMC) in vitro and in vivo. Subsequent studies from other laboratories revealed that UCN-01 could selectively enhance cytotoxicity of DNA damaging agents in p53 defective cells and that this was mediated by abrogation of S and /or G(2) arrest by UCN-01. In this study, we report that UCN-01 selectively enhances the cytotoxicity of MMC in human p53 mutant cell lines. In contrast, UCN-01 showed little, if any, effect on MMC cytotoxicity in human p53 wild-type cell lines. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-nick end-labeling (TUNEL) assay revealed that the combination of MMC with UCN-01 increased DNA breaks consistent with apoptosis in p53 defective A431 epidermoid carcinoma cells. In p53 wild-type MCF-7 breast carcinoma cells, the cyclin-dependent kinase inhibitor protein p21/WAF1 was markedly induced after the treatment with MMC alone, although this response was significantly delayed from the time of MMC treatment. Detailed cell-cycle studies revealed that UCN-01 abrogated S and G(2) phase accumulation induced by MMC in p53 defective cells and to a lesser extent in p53 wild-type cell lines. The abrogation of arrest in p53 wild-type cells was observed prior to significant induction of the p53 response. Since MMC was less effective against p53 defective cell lines than against p53 wild-type cell lines and UCN-01 selectively enhanced MMC cytotoxicity in p53 defective cell lines, UCN-01 may provide a new modality of MMC-based cancer chemotherapy, particularly in p53 defective cancer patients.
International Journal of Cancer 04/2000; 85(5):703-9. · 6.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: DNA-damaging agents such as cisplatin arrest cell cycle progression at either the G1, S, or G2 phase, although the G1 arrest is seen only in cells expressing the wild-type p53 tumor suppressor protein. Caffeine has been shown to abrogate the S and G2 arrest in p53-defective cells and to enhance cytotoxicity, but at concentrations too toxic to administer to humans. We have reported that 7-hydroxystaurosporine (UCN-01) also overcomes S and G2 phase arrest and enhances the cytotoxicity of cisplatin. We show here that UCN-01 at non-cytotoxic concentrations abrogated S and G2 arrest induced by cisplatin in two p53-defective human breast cancer cell lines. UCN-01 pushed the cells through S phase and mitosis, with subsequent apoptosis. Inhibition of mitosis with nocodazole reduced the apoptosis induced by UCN-01 plus cisplatin. Seven staurosporine analogs were compared for their ability to abrogate cell cycle arrest. Staurosporine was as effective as UCN-01 at abrogating S and G2 arrest, but the concentrations required were cytotoxic. K252a abrogated S phase arrest but failed to abrogate G2 arrest because alone it induced G2 arrest. Hence, K252a did not enhance cisplatin-induced cytotoxicity because it failed to push the cells through a lethal mitosis. None of the other analogs influenced cell cycle progression at the concentrations tested. Accordingly, UCN-01 was the only analog that overcame cell cycle arrest and enhanced the cytotoxicity of cisplatin while exhibiting no cytotoxicity of its own. Hence, UCN-01 remains the most promising candidate for testing clinically in combination with cisplatin.
[Show abstract][Hide abstract] ABSTRACT: While investigating endonucleases potentially involved in apoptosis, an antisera was raised to bovine deoxyribonuclease II, but it recognized a smaller protein of 26 kDa protein in a variety of cell lines. The 26 kDa protein underwent proteolytic cleavage to 22 kDa concomitantly with DNA digestion in cells induced to undergo apoptosis. Sequencing of the 26 kDa protein identified it as the Rho GDP-dissociation inhibitor D4-GDI. Zinc, okadaic acid, calyculin A, cantharidin, and the caspase inhibitor z-VAD-fmk, all prevented the cleavage of D4-GDI, DNA digestion, and apoptosis. The 26 kDa protein resided in the cytoplasm of undamaged cells, whereas following cleavage, the 22 kDa form translocated to the nucleus. Human D4-GDI, and D4-GDI mutated at the caspase 1 or caspase 3 sites, were expressed in Chinese hamster ovary cells which show no detectable endogenous D4-GDI. Mutation at the caspase 3 site prevented D4-GDI cleavage but did not inhibit apoptosis induced by staurosporine. The cleavage of D4-GDI could lead to activation of Jun N-terminal kinase which has been implicated as an upstream regulator of apoptosis in some systems. However, the results show that the cleavage of D4-GDI and translocation to the nucleus do not impact on the demise of the cell.
Cell Death and Differentiation 06/1999; 6(5):412-9. · 8.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Apoptosis is mediated by members of the caspase family of proteases which can be activated by release of mitochondrial cytochrome c. Additional members of the caspase family are activated at the cell surface in response to direct stimulus from the external environment such as by activation of the Fas receptor. It has been suggested that these upstream caspases directly activate the downstream caspases which would obviate a role for cytochrome c in apoptosis induced by the Fas receptor. We demonstrate that cytochrome c is released from mitochondria of Jurkat cells in response to both staurosporine and an agonistic anti-Fas antibody and that only the latter is inhibited by the caspase inhibitor z-VAD-FMK. This suggests that an upstream caspase such as caspase-8 is required for the Fas-mediated release of mitochondrial cytochrome c. The protein phosphatase inhibitor calyculin A prevented cytochrome c release and apoptosis induced by both agents, suggesting that release of cytochrome c is required in both models. Zinc, once thought of as an endonuclease inhibitor, has previously been shown to prevent the activation of caspase-3. We show that zinc prevents the activation of downstream caspases and apoptosis induced by both insults, yet does not prevent release of mitochondrial cytochrome c. The ability of calyculin A and zinc to prevent DNA digestion implies that the mitochondrial pathway is important for induction of apoptosis by both agents. These results do not support an alternative pathway in which caspase-8 directly activates caspase-3. These results also demonstrate that a critical protein phosphatase regulates the release of cytochrome c and apoptosis induced by both insults.
Experimental Cell Research 04/1999; 247(2):505-13. · 3.56 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: During apoptosis, DNA fragmentation and intracellular acidification occur concurrently. Previous results have shown that intracellular acidification is not required for DNA fragmentation, while the alternative, that acidification is a consequence of DNA fragmentation was analyzed here. To obviate the requirement of any nuclear function in acidification, apoptosis was induced by staurosporine in cytoplasts made from the breast tumor cell line MDA-MB-468. Both cells and cytoplasts demonstrated externalization of phosphatidylserine that was prevented by the pan-caspase inhibitor zVAD-fluoromethylketone or by expression of Bcl-2. Intracellular acidification was observed in both cells and cytoplasts and this was also inhibited by both zVAD-fluoromethylketone and Bcl-2. These results show that intracellular acidification and DNA fragmentation are independent consequences of caspase action during apoptosis.
Biochemical and Biophysical Research Communications 02/1999; 254(3):821-7. · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have previously implicated deoxyribonuclease II (DNase II) as an endonuclease responsible for DNA digestion during apoptosis. The full-length human cDNA has now been cloned. The cDNA contains an open reading frame of 1078 bases coding for a 40-kDa protein. This protein is 10 kDa larger than commercially supplied enzyme, which has been proteolytically cleaved at an internal aspartate residue. The gene is located at chromosome 19p13.2, and has no significant homology to other human proteins, but has >30% identity to three predicted genes in Caenorhabditis elegans. To determine whether overexpression of DNase II induces apoptosis in Chinese hamster ovary cells, the cDNA was cotransfected with a plasmid encoding green fluorescent protein. Within 24 h, a significant proportion of green fluorescent protein-positive cells contained condensed chromatin, whereas vector-only controls remained viable. Considering that DNase II is normally active only at low pH, it was surprising that transfection induced chromatin condensation. To confirm that transfection was not activating another endonuclease, cells were incubated with the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-(O-methyl)-fluoromethylketone; this failed to inhibit chromatin condensation induced by DNase II. These results demonstrate that DNase II acts downstream of caspase activation and that it may be activated by an as yet unknown mechanism to induce DNA digestion during apoptosis.
Journal of Biological Chemistry 12/1998; 273(47):30909-14. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Members of both the mitogen activated protein (MAP) kinase and BCL2 gene families, acting in concert with other gene products, are involved in the regulation of cell viability. However, the relationship between these families, and the signal transduction networks that control viability-regulating genes, are only beginning to be elucidated. MCL1 is a viability-promoting member of the BCL2 family that exhibits a rapid increase in expression in response to specific differentiation- and apoptosis-inducing stimuli. The signal transduction pathway involved in eliciting this increase has now been investigated. In the ML-1 human myeloblastic leukemia cell line, a rapid and sustained increase in phosphorylation of the extracellular signal-regulated kinase (ERK) members of the MAP kinase family was found to precede the increase in MCL1 expression produced by 12-O-tetradecanoylphorbol 13-acetate (TPA) or the microtubule-disrupting agents colchicine and vinblastine. ERK activation was necessary for the increase in MCL1, as inhibition of the increase in ERK phosphorylation (with the inhibitor PD 98059) prevented the increase in MCL1 expression and caused rapid cell death by apoptosis. In addition, other agents that markedly increased ERK phosphorylation (lipopolysaccharide, okadaic acid) also increased MCL1 expression. In contrast, agents that did not have this marked effect did not increase MCL1. Upstream components in this ERK-mediated pathway were also identified, where the pathway was found to be stimulated by microtubule disruption acting through protein kinase C (PKC). These results indicate that expression of the MCL1 viability-enhancing gene is regulated through a cytoskeletal disruption-induced ERK-mediated signal transduction pathway. They therefore suggest a mechanism through which the cytoskeleton and MAP kinases can exert effects on cell viability.
[Show abstract][Hide abstract] ABSTRACT: A variety of agents, such as caffeine, have been shown to abrogate the DNA damage-dependent G2 checkpoint and enhance cytotoxicity. However, these agents are too toxic for clinical use. We have reported that the potent protein kinase inhibitor 7-hydroxystaurosporine (UCN-01) at nontoxic doses abrogates the G2 arrest caused by the DNA-damaging agent cisplatin. Here, using Chinese hamster ovary cells, we show that cisplatin causes predominantly an S-phase arrest; UCN-01 abrogates this S-phase arrest, causing progression of cells to G2 and, subsequently, apoptotic cell death. In searching for an explanation for this accelerate DNA synthesis, we discovered that UCN-01 caused translocation of proliferating cell nuclear antigen (PCNA) to the detergent-insoluble, DNA-bound fraction. PCNA acts as a sliding clamp for DNA polymerase delta. Sequestering of PCNA by p21waf1/cip1 is required for p53-dependent G1 arrest in damaged cells. However, the S-phase arrest occurs independently of p53 and p21waf1/cip1. Our results suggest that PCNA is also a component of this S-phase checkpoint, despite the fact that CHO cells are defective for p53, and no increase in p21waf1/cip1 was observed. The mechanism by which PCNA is sequestered in the absence of p21waf1/cip1 and the mechanism by which UCN-01 disrupts this sequestration remain to be elucidated.
Cell growth & differentiation: the molecular biology journal of the American Association for Cancer Research 08/1997; 8(7):779-88.
[Show abstract][Hide abstract] ABSTRACT: n-Butyrate inhibits the growth of colon cancer cell lines. In the HCT 116 cell line, butyrate-induced growth inhibition is almost fully reversible, whereas in the VACO 5 cell line, a subpopulation undergoes apoptosis within 30 hr of treatment with butyrate. Concurrent treatment of VACO 5 cells with butyrate and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) accelerates and increases the incidence of cell death to nearly 100% of the population, whereas HCT 116 cells largely remain alive during treatment with this combination. The action of butyrate as an inhibitor of histone deacetylase was assessed in these cell lines by examining extracted core histones for their electrophoretic mobility in Triton/acid/urea gels. The concentrations of butyrate that were effective for inducing apoptosis were similar to the concentrations that caused hyperacetylation of core histones in the VACO 5 cell line. Furthermore, an examination of other carboxylic acids for induction of apoptosis revealed a rank order that corresponded to the order of potency in causing hyperacetylation of core histones. Specifically, the active acids were 3-5 carbons in length and lacked substitution at the 2-position. Isovaleric and propionic acids, in particular, proved to be effective inducers of both hyperacetylation and apoptosis at 5 mM concentrations, a finding of potential relevance to the unusual pancytopenia occurring after acidotic episodes in isovaleric and propionic acidemias. The duration of butyrate treatment required for chromatin fragmentation (10-20 hr) corresponded to the time required for histone H4 to become predominantly tetraacetylated. Furthermore, trichostatin A, a structurally dissimilar inhibitor of histone deacetylase, mimicked butyrate-induced apoptosis of VACO 5 cells and growth inhibition of HCT 116 cells. The dramatic enhancement of VACO 5 cell death by TPA, and the high level resistance of HCT 116 cells to butyrate were not evident from histone acetylation determinations. Thus, applications of butyrate for cytoreduction therapy will benefit from pharmacodynamic assessment of histone acetylation, but will require additional work to predict susceptibility to butyrate-induced death.
[Show abstract][Hide abstract] ABSTRACT: Apoptosis is commonly associated with DNA digestion, but it remains controversial as to which endonuclease is involved. The ability of zinc to inhibit DNA digestion in intact cells, and inhibit a Ca2+/Mg2+-dependent endonuclease in cell lysates, has been used frequently to suggest this is the endonuclease involved. However, zinc has many other effects on cells, and here it is shown that zinc also prevents many upstream events in apoptosis. These studies were performed in human ML-1 cells following incubation with etoposide. During apoptosis, these cells undergo intracellular acidification, increased accumulation of Hoechst 33342, DNA digestion and chromatin condensation. Zinc inhibited all of these events. An upstream event in apoptosis is activation of ICE/CED-3 proteases which is commonly observed as proteolysis of a substrate protein, poly(ADP-ribose) polymerase (PARP). The ICE/CED-3 proteases are themselves activated by proteolysis, and this was detected here by cleavage of one family member CPP32. Zinc prevented cleavage of both CPP32 and PARP. We recently demonstrated that dephosphorylation of the retinoblastoma susceptibility protein Rb was a marker of an event even further upstream in apoptosis; zinc was also found to inhibit Rb dephosphorylation. Therefore, zinc must protect cells at a very early step in the apoptotic pathway, and not as a direct inhibitor of an endonuclease.
Cell Death and Differentiation 03/1997; 4(2):125-9. · 8.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Apoptosis occurs during development and tissue homeostasis, and under conditions of physical and chemical stress. During apoptosis, cells digest their DNA, decrease intracellular pH, shrink, exhibit protein phosphatase activity, and activate members of the ICE/CED-3 family of proteases. This protease activity is identified by cleavage of poly(ADP-ribose) polymerase (PARP). Phosphatase activity during apoptosis is observed as dephosphorylation of the retinoblastoma susceptibility protein (Rb). Serine/threonine phosphatase inhibitors can prevent dephosphorylation of Rb and apoptosis, suggesting that Rb dephosphorylation is an indication of a critical regulator of apoptosis. The experiments described here were designed to establish the temporal relationship between these events. Apoptosis was induced in human ML-1 cells by the topoisomerase inhibitor etoposide. An inhibitor of the ICE/CED-3 protease family, z-VAD-fluoromethylketone (FMK), showed concentration-dependent protection from PARP cleavage, intracellular acidification, DNA digestion, early changes in membrane permeability, and cell shrinkage, thereby placing all of these events downstream of the ICE/CED-3 protease action. However, z-VAD-FMK did not prevent the dephosphorylation of Rb, placing this change upstream of the protease. These results suggest that the imbalance between protein phosphatase and kinase that is responsible for the dephosphorylation of Rb is also responsible for the activation of ICE/CED-3 proteases, which in turn is responsible for all the other events associated with apoptosis.
Experimental Cell Research 02/1997; 230(1):22-7. · 3.56 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Intracellular acidification appears to be a frequent and possibly universal occurrence during apoptosis. We have used carboxy SNARF-1 to measure intracellular pH by flow cytometry. We report here that the addition of Hoechst 33342 concurrently with carboxy SNARF-1 provides a clearer discrimination of the apoptotic population. Apoptotic cells accumulate Hoechst 33342 more rapidly than do viable nonapoptotic cells. The cells with greater Hoechst 33342 fluorescence are the same cells as those with decreased intracellular pH; these cells also have decreased volume. The different parameters in this analysis are presented for three models of apoptosis: human myeloblastoid ML-1 cells incubated with etoposide, murine cytotoxic T cells following withdrawal of interleukin-2, and Chinese hamster ovary cells incubated with staurosporine. In most circumstances, carboxy SNARF-1 provides excellent resolution of viable and apoptotic cells. However, the addition of Hoechst 33342 is particularly valuable when carboxy SNARF-1 alone can not fully discriminate the two cell populations. This situation occurs in Chinese hamster ovary cells, which undergo a smaller degree of acidification than do the other cell models. This situation also occurs when the extracellular pH is experimentally reduced to investigate the mechanism of pH dysregulation; the apoptotic cells appear to retain the ability to regulate intracellular pH at low pH, although they are defective in proton export at neutral pH.
[Show abstract][Hide abstract] ABSTRACT: The ability of Bcl-2 to inhibit cell death is well documented but its mechanism of action remains elusive. Recent reports have suggested that Bcl-2 prevents apoptosis by inhibiting the release of Ca2+ from the thapsigargin-sensitive Ca2+ store. The mobilization of Ca2+ from this store has been implicated as a signal regulating apoptotic cell death induced by glucocorticoids and by interleukin-3 withdrawal. The present study was designed to determine if Bcl-2 would still inhibit apoptosis after depletion of intracellular Ca2+ stores. We compared the response of two Chinese hamster ovary cell lines (5AHSmyc and 5A300bcl-2.2) following incubation with the calcium ionophore ionomycin to deplete intracellular Ca2+ stores. Continued incubation of 5AHSmyc cells in calcium-free media induced substantial apoptotic DNA fragmentation within 4 h and >95% loss of viability within 48 h. However, 5A300bcl-2.2 cells showed no evidence of DNA fragmentation or loss of viability over the same time period. Intracellular Ca2+ was analyzed with the Ca2+-sensitive fluorescent dye INDO-1 and confirmed that ionomycin was capable of releasing Ca2+ from intracellular stores in both cell lines. These results show that depletion of intracellular Ca2+ stores induces apoptosis and that these Ca2+ stores are not required for the protection afforded by Bcl-2.
Journal of Biological Chemistry 12/1996; 271(44):27739-43. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Butyrate is a potentially selective therapeutic agent for many adenocarcinomas. Butyrate causes reversible growth arrest as well as some death of VACO 5 colon cancer cells. Combined treatment with butyrate and the phorbol ester TPA leads instead only to cell death, while TPA causes little death on its own. Cells dying during treatment with TPA and butyrate, as well as those dying in the presence of butyrate alone, exhibit features typical of apoptosis, including detachment, shrinkage and internucleosomal DNA cleavage. Pre-treating VACO 5 cell cultures with TPA for as little as 6 hr prior to butyrate addition led to a markedly diminished enhancement of butyrate-induced apoptosis. Treatment with a distinct PKC activator, bryostatin 1, was ineffective in enhancing butyrate-induced death and, furthermore, counteracted the death-enhancing actions of TPA. Such antagonism was apparent when bryostatin was added after 12 hr of TPA/butyrate treatment but was much less effective thereafter. The duration of TPA/butyrate treatment required for depressing cell survival by >95% was thereby estimated to be 24 hr. Other colon cancer cell lines were examined for the extent of cell death following treatment with TPA/butyrate. In each of these lines, butyrate inhibited cell replication in a reversible manner, similar to that seen in VACO 5. However, the combination of butyrate and TPA led to high levels of cell death in only a subset of these lines. TPA/butyrate-treated cultures of COLO 201 exhibited extensive apoptosis, similar in timing and magnitude to the response by VACO 5, whereas HCT 116 was reversibly growth-arrested. Our findings indicate that the PKC system plays a critical role in maintaining cell survival during butyrate-induced growth arrest.
International Journal of Cancer 09/1996; 67(5):715-23. · 6.20 Impact Factor