Showing papers in "Apoptosis in 1996"

Induction of apoptosis by the mistletoe lectins: A review on the mechanisms of cytotoxicity mediated by Viscum album L.

Abstract: This review focuses on the cytotoxic properties of Viscum album L. (VAL). Apart from well-established results of protein synthesis inhibition by the mistletoe lectins (MLs), namely their catalytic A chain, there is now convincing evidence that the VAL-mediated cytotoxicity is mainly due to an induction of apoptosis. Among the more than 1,000 proteins detected in VAL, the MLs and the viscotoxins (VTs) are the predominant toxic proteins. Using purified components, such as the D-galactose-specific ML I, the N-acetyl-D-galactosamine-specific ML II and ML III, crude VTs and oligosaccharides, only the MLs induced apoptosis. The in vitro studies suggest that interaction of lectin B chains with appropriate receptors on the cell surface activates distinct signalling pathways that ultimately leads to apoptosis in a large fraction of cells, while others survive, however, with a conservation of their DNA. Inhibition of protein synthesis by the A chain of the hololectin probably accelerates the B chain-induced course of events.

Cell acidification in apoptosis

Abstract: Programmed cell death is a stereotyped and highly conserved process leading to deletion of unwanted cells. The process may be initiated in response to physiologic signals (Fas ligation, removal of extra-cellular matrix or growth factors), or pathologic events (DNA damage, hypoxia/reperfusion injury, viral infection). Some of the signal transduction pathways between a specific stimulus and the commitment to apoptosis are being worked out, although these may not represent general pathways for all triggers of apoptosis. In addition, the cell is able to integrate a variety of signals, some favoring apoptosis and some favoring survival, and to make a life-or-death decision. This has been termed the ‘judgment phase’, whereas once the cell is irreversibly committed to apoptosis, the ‘execution phase’ is initiated. The biochemical features of the ‘execution phase’ are still unclear; DNA cleavage probably represents one of the final events of the execution phase, but what about the multitude of proteases that participate in the process, phosphatidylserine externalization, transglutaminase activation, and, of course, the subject of this discussion, cytoplasmic acidification? Where do these events fit into the process, and what are the relationships of one to the other? This review will address the following points: (1) Is cytoplasmic acidification a universal feature of apoptosis, and is it essential? The reported cases examined to date will be evaluated. (2) How is cytoplasmic acidification accomplished? Is acidification sufficient for apoptosis to occur? (3) What are the consequences of acidification, particularly with respect to other biochemical features of apoptosis? (4) Finally, I would like to advance the hypothesis that acidification may represent the cellular mechanism for integration of multiple signals; cytoplasmic acidification could represent the point of no return on the road to apoptosis.

The cell biology of apoptosis: Evidence for the implication of mitochondria

Abstract: The apoptotic process can be subdivided into three phases: a death-stimulus-dependent heterogeneous induction phase, a common effector phase during which the central apoptotic executioner is activated, and a common degradation phase during which cells acquire the biochemical and morphological features of end-stage apoptosis. Recently, it has become clear that the central apoptosis executioner is dictated by cytoplasmic (non-nuclear) events and that nuclear changes that define apoptosis (chromatin condensation and oligonucleosomal DNA fragmentation) only become manifest beyond the point-of-no-return of apoptosis, during the late degradation phase. It appears that one obligatory event of the apoptotic cascade involves a characteristic change in mitochondrial function, namely the so-called mitochondrial permeability transition. Permeability transition leading to disruption of the mitochondrial transmembrane potential precedes nuclear and plasma membrane features of apoptosis. Induction of permeability transition in cells suffices to cause the full-blown picture of apoptosis. In vitro induction of permeability transition in isolated mitochondria provokes the release of a factor capable of inducing apoptotic changes in isolated nuclei. Permeability transition is subject to regulation by multiple endogenous effectors, including members of the bcl-2 gene family. Its inhibition by pharmacological agents or hyperexpression of Bcl-2 prevents apoptosis, indicating that PT is a central coordinating event of the apoptotic effector stage.

The neutral comet assay is sufficient to identify an apoptotic ‘window’ by visual inspection

Abstract: As a sensitive technique for measuring DNA damage, the alkaline comet assay (capable of detecting and distinguishing apoptotic and necrotic damage) shows significantly greater ability to detect DNA breaks than a neutral counterpart. Using a heat shock model, we show that the fraction of visually detectable comets decreases using the neutral assay as cell death shifts from apoptosis to necrosis. We also show a virtual absence of neutral comets in cells dying by necrosis in another model. We conclude that the non-denaturing assay allows identification of putative apoptotic ‘windows’ by showing sensitivity to apoptotic, but not necrotic, DNA damage.

Lack of gp120-induced anergy and apoptosis in chimpanzees is correlated with resistance to AIDS

Abstract: Human immunodeficiency virus-1 (HIV-1) infects both humans and chimpanzees, but in the chimpanzee, HIV-1 infection leads only very rarely to loss of CD4 T cells or to AIDS-like disease. The pathogenetic basis for this difference in host range is not understood. In previous studies, using CD4 T cells from HIV-1 seronegative human donors, we demonstrated that crosslinking of CD4-bound gp120, followed by signaling through the T cell receptor for antigen (TCR), resulted in cell death by apoptosis. To determine whether activation-induced apoptosis correlates with progression to AIDS, we studied the chimpanzee. Our data suggest that, although human CD4 T cells respond to CD4 ligation with anergy and apoptosis upon activation, chimpanzee CD4 T cells do not undergo apoptosis after cross-linking of CD4-bound gp120, followed by signaling through the TCR. In addition, proliferation assays show that chimpanzee CD4 T cells do not become anergic after CD4 ligation. Thus, it is possible that, in the chimpanzee, the absence of cellular anergy and apoptotic cell death after CD4 ligation by HIV-1 gp120 protect this primate species from progression to AIDS-like disease.

Apoptosis of cells lacking mitochondrial DNA

Abstract: The mitochondrial genome of animals encodes a few subcomponents of the respiratory chain complexes I, III and IV, whereas nuclear DNA encodes the overwhelming majority, both in quantitative and qualitative terms, of mitochondrial proteins. Complete depletion of mitochondrial DNA (mtDNA) can be achieved by culturing cells in the presence of inhibitors of mtDNA replication or mitochondrial protein synthesis, giving rise to mutant cells (ϱ∘ cells) which carry morphological near-to-intact mitochondria with respiratory defects. Such cells can be used to study the impact of mitochondrial respiration on apoptosis. ϱ∘ cells do not undergo cell death in response to determined stimuli, yet they conserve their potential to undergo full-blown apoptosis in many experimental systems. This indicates that mtDNA and associated functions (in particular mitochondrial respiration) are irrelevant to apoptosis execution. However, the finding that mtDNA-deficient mitochondria can undergo apoptosis does not argue against the involvement of mitochondria in the apoptotic process, since mitochondria from ϱ∘ cells conserve most of their functions including those involved in the execution of the death programme: permeability transition and release of one or several intermembrane proteins causing nuclear apoptosis.

Neuronal apoptosis versus necrosis induced by glutamate or free radicals

Abstract: The type of cell death encountered in neuronal cell cultures exposed to excitatory amino acids — such as glutamate, the major excitatory neurotransmitter in the central nervous system, or free radicals, such as nitric oxide (NO.) and superoxide anoin (O2. −), which react to form peroxynitrite (ONOO−) — appears to depend on the intensity of the exposure and may involve two temporarily distinct phases. Following relatively fulminant insults, an initial phase of necrosis — associated with extreme energy depletion — may simply reflect the failure of neurons to carry out the ‘default’ apoptotic death program used to efficiently dispose of aged or otherwise unwanted cells. Neurons recovering mitochondrial energy potential after an initial fulminant insult or following a more subtle inciting injury may subsequently undergo apoptosis, possibly associated with a factor released from mitochondria that triggers this death program. The maintenance of balanced energy production may be a decisive factor in detemining the degree, type, and progression of neuronal injury caused by ‘excitotoxins’ and free radicals. Similar events could possibly occur in vivo after ischemia or other insults.

Immunohistochemical localization of BAX and BAD in the normal and BCL-2 null gastrointestinal tract

Abstract: BAX and BAD are members of the BCL-2 family of proteins. The over-expression of BAX protein has been shown to accelerate apoptosis and increasedbax mRNA levels have also been shown to be associated with the initiation of apoptosis. BAD has also been shown to accelerate apoptosis. In this paper we describe the localization of BAD and BAX expression throughout the gastrointestinal tract of the mouse and the effect that BCL-2 has on the expression of these two proteins. We have discussed the distribution of BAX and BAD in relation to the differences between the small and large bowel in (i) the susceptibility of stem cells to apoptosis and (ii) tumour incidence.

The CD95 (Fas/APO-1) receptor is phosphorylatedin vitro andin vivo and constitutively associates with several cellular proteins

Abstract: Different CD95 (Fas/APO-1) isoforms and phosphory lated CD95 species were identified in human T and B cell lines. We had shown previously that the CD95 intracellular domain (IC), expressed as a glutathione S-transferase (GST) fusion protein in murine L929 fibroblasts, was phosphorylatedin vivo. GST-CD95IC was phosphorylatedin vitro by a kinase present in extracts from the human lymphocytic cell lines Jurkat and MP-1 and from murine L929 cells. Phosphoamino acid analysis indicated that phosphorylation occurred at multiple threonine residues and also at tyrosine (Tyr232 and Tyr291) and serine. Amino acids 191 to 275 of CD95 were sufficient for phosphorylation at threonine, tyrosine and serine and also mediated interaction with a 35 kDa cellular protein. Immuno-precipitation of CD95 and chemical cross-linking revealed CD95-associated proteins of approximately 35, 45 and 75 kDa. GST-CD95IC affinity chromatography detected binding of the 35 and 75 kDa protein species. The 75 kDa species may correspond to the CD95-associated proteins RIP or FAF1 and the 35 kDa protein may represent a TRADD analogue. These data indicate that several cellular proteins interact with CD95, possibly in a multi-protein complex, and that a kinase activity is associated with CD95 not onlyin vitro but alsoin vivo. Therefore, receptor phosphorylation may play a role in CD95 signal transduction.

Apoptosis in non-small cell lung cancer as related to drug resistance and prognosis

Abstract: The percentage of apoptotic cells among the tumor cells (apoptotic index) was determined in a series of 178 non-small cell lung carcinomas with a long term clinical follow-up by a terminal desoxynucleotidyl transferase mediated dUTP nick end labelling technique. In survival analysis, we found no statistically significant correlation between the apoptotic index and survival times. We estimated also the sensitivity of specimens to doxorubicin by a short term test. Tumors with a high apoptotic activity were more sensitive to doxorubicin than tumors with a less apoptotic index. Thus, our data indicate that apoptosis may be involved in drug response of lung tumors and it could be useful for the chemotherapeutic strategy to design drugs which trigger apoptosis.

Increased cyclin B1/CDC 2 kinase activity and phosphorylation of Bcl-2 associated with paclitaxel-induced apoptosis in human nasopharyngeal carcinoma cells

Abstract: Paclitaxel is a potential cancer chemotherapeutic agent for ovary, breast, and head and neck cancers; its effects on nasopharyngeal carcinoma (NPC) have not been reported previously. This study investigated the cytotoxic mechanism of paclitaxel in two NPC cell lines, NPC-TW01 and NPC-TW04. NPC cells treated with pacli-taxel showed convoluted nuclei, condensed chromatin and decreased cellular and nuclear volume, and also exhibited genomic DNA degradation into multiple oligonucleosomal fragments, suggesting that pacli-taxel induced apoptosis in these cells. The effects of paclitaxel on apoptosis-related proteins including Bcl-2, Bax and CDC 2 were also detected. Although the levels of Bcl-2 and Bax were not changed in NPC cells following treatment with 5 nM-1 μM of paclitaxel, phosphorylation of Bcl-2 was significantly observed in the cells treated with 1 μM of paclitaxel for 12 hours. In addition, cyclin B1-associated CDC 2 kinase was highly activated in the NPC cells exposed to paclitaxel even at low (5 nM) concentration, and this result is associated with the finding that low concentration of paclitaxel is able to induce apoptosis in NPC cells.

Infrequent but significant p21/WAF1/CIP1 gene alteration in synchronous or metachronous transitional cell carcinoma

Abstract: p53 inducible cyclin dependent kinase inhibitor, p21/WAF1/CIP1(p21), played a pivotal role for G1 arrest when cells received genotoxic stimuli. p21 could be a putative tumor suppressor gene, since its dysfunction may lead to accumulation of genomic alteration. We investigated the p21 and p53 status using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and immunohistochemical analyses, in eight patients who had synchronous or metachronous urothelial tumors. Loss of heterozygosity (LOH) of p21 gene was detected in one coincidental tumor in one case. p21 positive cells were detected by immuno-histochemical staining in all tumors in one case, and in one coincidental tumor in two cases. Among p21 positive cells in these three cases, no p53 mutations were detected, whereas no p21 positive cells were detected in other cases with a p53 mutation. These findings suggested that in transitional cell carcinoma (TCC) p21 gene mutation is infrequent like the p53 gene mutation, but that LOH might be important in the inactivation of p21.

GL331-induced disruption of cyclin B1/CDC 2 complex and inhibition of CDC 2 kinase activity

Abstract: GL331, a new homologue of etoposide (VP-16), was developed to cope with the multiple drug resistance occurring in certain malignant tumours. We previously indicated that GL331, like VP-16 and other major cancer chemotherapeutic agents, induced apoptosis in a variety of human cancer cell lines including nasopharyngeal carcinoma (NPC) NPC-TW01 and NPC-TW04 cells. In this study, we further explored the effect of GL331 on the cell cycle progression of NPC cells. Flow cytometric analysis of DNA content was first used to demonstrate the ability of GL331 to induce cell growth arrest at S-G2 phase in most NPC cells. Besides acting as a topoisomerase II inhibitor, GL331 inhibited cellular cyclin B1-associated CDC 2 kinase activity 6 h after treatment, accounting partly at least for its induction of the cell cycle arrest. As with cyclin A, D1, E, CDK 2 and PCNA, the levels of cyclin B1 and CDC 2 proteins were not changed after GL331 treatment; however, the ability to form complex between cyclin B1 and CDC 2 was obviously affected in GL331-treated NPC cells, which associates with the inhibition of cyclin B1/CDC 2 kinase activity elicited by GL331. These data could provide more principal bases for future therapeutic application of this potential anti-cancer agent.

Epidermal growth factor induced apoptosis

Abstract: Apoptosis, or programmed cell death, plays an important role in the pathogenesis of a number of human diseases, including cancers, autoimmune diseases, and neurodegenerative disorders. Recent evidence suggests that EGF induced signal transduction pathways which govern cell proliferation and cell cycle progression also mediate antiproliferative effects leading to increased apoptosis in cells that express high levels of epidermal growth factor receptors. Treatments designed to increase apoptosis have potential to change the natural progression of cancer and eventually lead to its successful control.

Apoptosis: identification by a critical electrolyte concentration method

Abstract: A critical electrolyte concentration method previously proposed to distinguish RNA from DNA was considered for a rapid screening of apoptosis at the cellular level. For the monitoring of the apoptotic phenomenon, preparations subjected to anin situ 3′ end DNA labelling assay were also analyzed. The results indicated that the critical electrolyte concentration assay as used here is a simple and useful tool for the rapid identification of apoptotic cells and could contribute to the determination of their frequency, localization and other characteristics.

Induction of apoptosis by a calpain stimulator, ONO-3403

Abstract: The effects of a synthetic serine protease inhibitor, FOY-305, and its derivatives, ONO-3403 and FO-349, on the proliferation of mouse NIH3T3 cells were investigated. At concentrations between 10 and 100 μg/ml, three protease inhibitors induced a moderate suppression of cell growth. However, only ONO-3403 showed severe cytotoxicity at concentrations higher than 200 μg/ml. Results of TUNEL staining and DNA fragmentation analysis indicated that ONO-3403 induced apoptosis at the high concentrations. Biochemical analysis has shown that ONO-3403 directly enhanced the amidolytic activity of purified μ-calpain at a concentration higher than 100 μg/ml while FOY-305 and FO-349 showed less effects. When the cell extract was incubated in the presence of ONO-3403, specific degradation of a few proteins including protein kinase C was observed. Similar degradation was also observed by addition of μ-calpain to the extract. These results imply that ONO-3403 is a specific stimulator of calpain. It seems reasonable to conclude that increase in calpain activity results in apoptosis.

Ras-mediated cell proliferation and cell death: some clues from the interleukin-2 receptor system

Abstract: Oncoproteins of the Ras family have been extensively studied because of their implication in human cancer. Their roles have been primarily assigned to the commandment of cell proliferation and suppression of apoptosis, which has also been demonstrated by the involvement of Ras activation in the signal transduction pathways triggered by most cytokine receptors. Nevertheless, the functions of Ras proteins have been extended in the last years by the findings showing that they can also act as promoters or enhancers of apoptosis in various systems and conditions. These considerations have raised the issue as to how the signals delivered by Ras are regulated and translated in terms of cellular responses, suggesting that signal complementation may direct the final fate of cells. As an example, the interleukin-2 receptor system may represent a useful model in which the meaning of Ras signals may be evaluated in terms of interactions with other simultaneous signalling events, since knowledge of the biochemical events triggered by the interaction of interleukin-2 with its cell surface receptor in lymphocytes has allowed the proposal of a complete signalling model arranged in three independent channels, one of which is mediated by Ras.

Involvement of the TNF-α system and the Fas system in the induction of apoptosis of mouse mammary glands after weaning

Abstract: Mammary gland involution after cessation of milk production is associated with extensive loss of secretory epithelial cells. In order to study the mechanism of mammary gland involution, litters were removed on day 20 of lactation and morphological and biochemical changes were examined in GR/A mice andlprcg mice lacking functional Fas. DNA fragmentation occurred 24 h after weaning both in GR/A mice andlprcg mice indicating that apoptotic cell death occurs during involution of mammary glands.In situ 3′-end labelling method revealed apoptotic cells in epithelial cells lining alveolar lumens and in cells shed into the alveolar lumen. The number of apoptotic cells plateaued on day 2 of weaning in mammary glands of GR/A mice. Reverse transcriptase polymerase chain reaction (RT-PCR) showed that expression of bcl-2, Fas ligand, keratinocyte growth factor (KGF) and transforming growth factor-β1 (TGF-β1) increased on day 1 of weaning in the process of involution of mammary glands in GR/A mice. In mammary glands oflprcg mice, RT-PCR showed that expression of bcl-2, Fas ligand, KGF, tumour necrosis factor-α(TNF-α) and TGF-βs increased in the process of involution. These results suggest that the Fas ligand, TGF-β1 and TNF-α are involved in the involution of mammary glands after weaning. TNF-α and anti-Fas antibody directly killed more than 80% of mammary cells from p53 knockout micein vitro within 24 h in the presence of actinomycin D, supporting the hypothesis that Fas and/or TNF-α are involved in the induction of apoptosis of mouse mammary glands.

Retinoids and apoptosis in cancer therapy

Abstract: Retinoids serve as physiologic and pharmacologic mediators of proliferation, differentiation and apoptosis in normal and malignant cell types. All-trans-retinoic acid (tRA), a natural metabolite of vitamin A, induces differentiation and subsequent apoptosis in several types of malignant cells with immature phenotypes. Clinically, tRA has been approved for the treatment of patients with acute promyelocytic leukemia. Several synthetic retinoids induce apoptosis without differentiation in a variety of malignant epithelial cells in vitro. The synthetic derivative, N-(4-hydroxyphenyl)retinamide (HPR), shows significant promise as a chemo-preventive and therapeutic anti-cancer agent in light of its minimal toxicity and broad activity in experimental cancer models representing common human malignancies. This paper reviews the role of retinoids as mediators of differentiation and apoptosis in malignant cells, and the impact this activity could have on clinical oncology.

Photoreversion of ultraviolet induced apoptosis in Rat Kangaroo cells

Abstract: Rat kangaroo(Potorous tridactylus) cells efficiently repair 254 nm ultraviolet light (UV) induced cyclobutane pyrlmidine dimers (CPDs) through photoreactivation, leading to an enhancement of survival when cells are exposed to photoreactivation light (PRL) immediately after UV-irradiation. This work presents evidence that at least part of the UV-irradiated cells die through apoptosis, as demonstrated by DNA fragmentation and chromatin condensation. The induction of this kind of cell death can be reversed through photoreactivation immediately after irradiation, indicating that CPDs are essential signals for the initiation of apoptosis by UV-irradiation. Exposure to PRL 24 h after UV-irradiation does not reverse the induction of apoptosis, implying that the cells are committed to die at this time after irradiation. Inhibition of DNA synthesis during this period of time following UV-irradiation, and before exposure to PRL, does not avoid apoptosis. Since similar results were obtained in Go confluent and G1/S synchronized cells, the signals for the UV-induced apoptosis do not seem to be related to a specific phase of cell cycle. Nevertheless, by adding 3-aminobenzamide (3AB—an inhibitor of poly(ADP-ribose) polymerase) in the cell medium after UV-irradiation, apoptosis endpoints were partially reversed if cells are exposed to PRL 24 h later. This result strongly indicates that poly(ADP-ribose) is an intermediary signal for UV-induced apoptosis in mammalian cells.

Human eosinophils: Apoptosis versus survival in the mediation of inflammation

Abstract: Eosinophil-derived mediators are thought to make a major contribution to the inflammation underlying a number of allergic diseases, most notably asthma. The toxic potential of eosinophils at tissue sites of inflammation might be limited if they were cleared by the process of programmed cell death or apoptosis. In this review we have examined the relationship between the signal transduction pathways important in controlling cytokine-induced prolonged survival and the mechanisms responsible for the induction and control of apoptosis in the eosinophil. A greater understanding of these processes might result in the development of novel therapeutic agents which would promote the safe and rapid removal by apoptosis of this important pro-inflammatory cell.

Failure of zinc to inhibit hyperthermia-induced apoptosis predicts differential nuclease activity detection by comet assay

Abstract: Apoptotic DNA cleavage generally proceeds in two stages, first producing large 50–300kb fragments, and later oligonucleosomal pieces which create the characteristic DNA ladder. We show that zinc treatment of hyperthermia-induced apoptotic cultures is sufficient to prevent ladder formation, but not apoptosis (all features of which were inhibited by actinomycin D and cycloheximide). DNA damage measured in single cells using the comet (single cell gel) assay is detectable in zinc treated cultures using both alkaline and non-denaturing conditions. Both assays predict the same fraction of cells undergoing apoptosis, and damage is detectable earlier than shown by DNA ladder appearance. We conclude that the comet assay is detecting damage consistent with the initial 50–300kb fragments. Additionally, various cell lines when heattreated follow different temporal pathways or display differential apoptotic phenotypes. Also, we were unable to demonstrate an ‘apoptotic window’ for cells refractory to hyperthermia by increasing the heat load.

DNA fragmentation during thymic apoptosis is catalyzed by DNase γ

Abstract: The physiological and pathological importance of cell death by apoptosis has recently been recognized. One of the hallmarks of apoptosis is the enzymatic cleavage of genomic DNA into nucleosomal oligomers. The identification of an endonuclease responsible for apoptosis might help to explain how this cell suicide is regulated and why DNA is cleaved. Here, we found that γ type of DNase was retained in apoptotic rat thymocyte nuclei. Homogeneously purified DNase γ (Mr = 33 kDa) from the apoptotic nuclei was revealed to be a Ca2+/Mg2+-dependent endonuclease and inhibited by Zn2+. This enzyme cleaved chromosomal DNA with 3′-hydroxyl (OH) and 5′-phosphoryl (P) ends. The cleavage ends and its divalent cation dependencies match those observed in apoptotic thymocytes induced by X-irradiation or glucocorticoid treatment, indicating that this endonuclease is a central component of the thymic apoptosis machinery.

Block of bcr-abl expression and induction of apoptosis by cisplatinum in a human chronic myeloid leukaemia cell line

Abstract: Chronic myeloid leukaemia (CML) cell lines expressing the bcr-abl fusion gene are resistant to drug-induced apoptosis. Using a human CML cell line (EM2), we investigated the effects of cisplatinum (DDP), Doxorubicin and Tallimustine on the level of p210, the product of the hybrid bcr-abl gene, and on the induction of apoptosis. DDP exposure of this cell line resulted in a decrease of p210 levels with a concomitant activation of apoptosis. At all the concentrations tested, neither Doxorubicin nor Tallimustine were able to induce DNA fragmentation nor to reduce the levels of the fusion protein p210. The reduction in the p210 levels induced by DDP were also observed at mRNA level as observed with RT-PCR, suggesting that, at least in part, the decrease in p210 levels was the result of a reduction in the transcription of the bcr-abl fusion gene. The DDP-induced DNA fragmentation and decrease in p210 levels, were observed in EM2 cells but not in another human CML cell line (K562) which overexpresses the fusion gene. In K562 cells the levels of bcr-abl, although decreased, remained well detectable after DDP treatment. Data indicate that it may be possible to investigate compounds able to contrast the resistance to DNA-damage induced apoptosis of CML cell lines.

Tissue transglutaminase in mesenchymal tumour cells

Abstract: During our search for novel transformation-sensitive proteins whose synthesis is abolished in tumour cells we found a cDNA clone coding for tissue transglutaminase. This enzyme was identified, at the protein as well as the mRNA level, in normal human fibroblasts, but was completely missing in their matched SV40 transformed counterparts. Since tissue transglutaminase has been implicated in cell cycle regulation and apoptosis, we investigated the possibility of whether this enzyme might represent a negative marker for tumour cells. We found that its synthesis varied largely among 10 cell lines derived from spontaneous mesenchymal tumours. While cells from a rhabdomyosarcoma and a chondrosarcoma did not produce it at all, an extremely high expression was observed in cells from an osteosarcoma and a liposarcoma. Thus, tissue transglutaminase is not a tumour-related marker.

Molecular mechanisms of programmed cell death

Abstract: Programmed cell death is currently under active investigation. A recent meeting focused on the molecular machinery of programmed cell death and on its role in the pathogenesis of human diseases.

FasL and TNF interactions: their roles in immunoregulation and disease

Abstract: The TNF receptor superfamily comprises type I membrane glycoproteins homologous to the receptors for TNF (TNFR) and nerve growth factor (NGF). 1 Included in this family of receptors are CD27, CD30, CD40, 4-1BB, Ox40, TNFR (p55), TNFR (p75), TNFR-related protein (TNFR-rp), and Fas/APO-1 (CD95). The respective ligands for these receptors have also been identified and form a similar family of related type II membrane glycoproteins with homology to TNF.2A common feature of TNF superfamily members is that they are expressed on activated T cells, and ligation of their respective receptors on T cells suboptimally activated by CD3 mAb leads to T cell proliferation. Two members of this r e c e p t o r / l i g a n d s u p e r f a m i l y FasL/Fas and TNF/TNFR stand out in particular in that they also mediate apoptotic death of appropriately activated target cells. The fact that both proliferative and apoptotic signals can be mediated by ligation of these two receptors is both interesting and potentially of significant biological importance. Early studies indicated that the dichotomy of outcomes (i.e., co-stimulation vs. apoptosis) after ligation of TNFR was due to differential signalling events mediated by the two distinct receptors for TNE Thus, ligation of murine TNFR (p55) mediates cytotoxicity, whereas ligation of murine TNFR (p75) results in proliferative signals. 3 In contrast, ligation of Fas on

Immunolocalization of EGF receptor (EGFr) in intestinal epithelium: recognition of apoptotic cells

Abstract: The EGF-like family of growth factors are known to be involved in the control of the intestinal epithelium. The intracellular events are mediated by the EGF receptor (EGFr), a transmembrane glycoprotein which is overexpressed in many malignancies and also in many radiosensitive cell types. The precise mode of action of the receptor in controlling proliferation and whether the factor is also involved in controlling apoptosis in this tissue is not clear. Using polyclonal antibodies raised against a cytoplasmic region of the receptor distant to the phosphorylation site and one raised against the peptide sequence DVVDADEYLIPQ, which is present in the cytoplasmic tail phosphorylation site of the EGFr, we have examined the immunostaining in normal and irradiated murine intestine. The former antibody labelled the basolateral membranes of the epithelial cells in the proliferative zones of both the small intestine and colon, in both control and irradiated tissue. The latter antibody however, strongly labelled the Goblet cells and the microvilli of the enterocyte apical membrane in control tissue. Following irradiation\ the apical labelling redistributed and was localized in the apical cytoplasm and in a paranuclear region. Furthermore, strong labelling was now seen in many of the apoptotic cells of the small intestinal epithelium. The greatly differing results with the two antibodies indicates that interpretation of such immunostaining must be viewed with caution and may relate to the availability of each particular epitope. These results also suggest that antibodies to DVVDADEYLIPQ may be a useful marker of apoptotic calls and could imply a correlation between high levels of epitope availability, the radiosensitive (frequently p53 expressing) cells of the crypt epithelium and the induction of apoptosis.

Upregulation of Fas-induced apoptosis by interferon-γ accompanied by increased ICE expression in renal cell cancer cells

Abstract: The integration of Fas/Apo-1 (CD95) by Fas ligand or anti-Fas antibody induces apoptosis, and this system plays a pivotal role for the lysis of target cells by cytotoxic T lymphocytes. Fas-mediated apoptosis is also increased by a prior incubation of Fas-bearing cells with interferon(IFN)-γ. Interleukin-1-β converting enzyme (ICE) and/or CPP32, or other members of ICE family act as direct cell death executors downstream of this mechanism, and a tetrapeptide inhibitor of these cysteine proteases blocks Fas-mediated apoptosis. In this study, we examined the effect of IFN-γ on Fas-mediated apoptosis in ACHN cells. IFN-γ augmented apoptosis in a dose dependent manner and reached a plateau at 400 U/ml when exposed for 48 h before the end of culture. The kinetics revealed a significant increase in apoptosis after 24 h. Exposing ACHN cells to IFN-γ increased pro-ICE expression accompanied with a decrease of pro-CPP32. These results suggest that direct enhancement of ICE expression and/or upregulation of conversion of pro-CPP32 to active form increases Fas-mediated apoptosis by IFN-γ in ACHN cells.

AACR meeting on programmed cell death, Lake George, 19–23 October 1996

Abstract: Martin Raft (University College London) opened the meeting, which was organized by Stan Korsmeyer (Washington University), Shigekazu Nagata (Osaka Bioscience Institute) and Andrew Wyllie (Edinburgh), by reviewing the history ofapoptosis as a field of study and then raising a number of pertinent questions that still need to be answered; for example, how do Bcl-2 protein family members regulate cell death and what is the role of individual death inducing caspase proteases and their target substrates? Raft presented evidence that caspases are involved in normal developmental cell deaths in addition to death by insult as the caspase inhibitor zVADfmk blocks neural tube closure in the chick embryo. He also provided further evidence for his theorems that all cells (except blastomeres) require extracellular signals for survival, and that all cells possess the required components to undergo apoptosis without de novo transcription. Raft summarized data demonstrating that single cell organisms such as Dictyostelium, trypanosomes and bacteria undergo programmed cell death. The role for proteases in programmed cell death in mammals was likened to the E. coli Lit protease which is activated by the head protein of bacteriophage T4 (through a proteolysis step) which in turn cleaves EF-Tu leading to cell death.