Pyruvate secreted by human lymphoid cell lines protects cells from hydrogen peroxide mediated cell death

Reactive oxygen species (ROS) released from polymorphonuclear leukocytes and macrophages could cause DNA damage, but also induce cell death. Therefore inhibition of cell death must be an important issue for accumulation of genetic changes in lymphoid cells in inflammatory foci. Scavengers in the post culture medium of four lymphoid cell lines, lymphoblastoid cell lines (LCL), Raji, BJAB and Jurkat cells, were examined. Over 80% of cultured cells showed cell death 24 h after xanthine (X)/xanthine oxidase (XOD) treatment, which was suppressed by addition of post culture medium from four cell lines in a dose-dependent manner. H₂O₂ but not O^·-₂ produced by the X/XOD reaction was responsible for the cytotoxity, thus we used H₂O₂ as ROS stress thereafter. The H₂O₂-scavenging activity of post culture media from four cell lines increased rapidly at the first day and continued to increase in the following 2–3 days for LCL, Raji and BJAB cells. The scavenging substance was shown to be pyruvate, with various concentrations in the cultured medium among cell lines. Over 99% of total pyruvate was present in the extracellular media and less than 1% in cells. α-Cyano-4-hydroxycinnamate, a specific inhibitor of the H⁺-monocarbohydrate transporter, increased the H₂O₂-scavenging activity in the media from all four cell lines via inhibition of pyruvate re-uptake by cultured cells from the media. These findings suggest that lymphoid cells in inflammatory foci could survive even under ROS by producing pyruvate, so that accumulation of lymphoid cells with DNA damage is possible.     

Activation of pannexin-1 mediates triglyceride-induced macrophage cell death

The accumulation of triglycerides (TGs) in macrophages induces cell death, a risk factor in the pathogenesis of atherosclerosis. We had previously reported that TG-induced macrophage death is triggered by caspase-1 and -2, therefore we investigated the mechanism underlying this phenomenon. We found that potassium efflux is increased in TG-treated THP-1 macrophages and that the inhibition of potassium efflux blocks TG-induced cell death as well as caspase-1 and -2 activation. Furthermore, reducing ATP concentration (known to induce potassium efflux), restored cell viability and caspase-1 and -2 activity. The activation of pannexin-1 (a channel that releases ATP), was increased after TG treatment in THP-1 macrophages. Inhibition of pannexin-1 activity using its inhibitor, probenecid, recovered cell viability and blocked the activation of caspase-1 and -2 in TG-treated macrophages. These results suggest that TG-induced THP-1 macrophage cell death is induced via pannexin-1 activation, which increases extracellular ATP, leading to an increase in potassium efflux.     

Inhibition of DNA synthesis and cell death

The association between DNA synthesis inhibition and cell death in mouse L-cells was investigated using the drug hydroxyurea. This drug produces a preferential labelling of low molecular weight DNA and dose-response studies revealed a correlation between this effect and cytotoxicity. Investigation of the reassociation kinetics of DNA labelled during hydroxyurea inhibition showed an over-replication of middle repetitive sequences, but the concentration dependence of this effect was quite different to that of cytotoxicity.     

Inhibition of p38 MAPK enhances ABT-737-induced cell death in melanoma cell lines: novel regulation of PUMA

The mitogen-activated protein kinase (MAPK) pathway is constitutively activated in the majority of melanomas, promoting cell survival, proliferation and migration. In addition, anti-apoptotic Bcl-2 family proteins Mcl-1, Bcl-xL and Bcl-2 are frequently overexpressed, contributing to melanoma’s well-documented chemoresistance. Recently, it was reported that the combination of MAPK pathway inhibition by specific MEK inhibitors and Bcl-2 family inhibition by BH3-mimetic ABT-737 synergistically induces apoptotic cell death in melanoma cell lines. Here we provide the first evidence that inhibition of another key MAPK, p38, synergistically induces apoptosis in melanoma cells in combination with ABT-737. We also provide novel mechanistic data demonstrating that inhibition of p38 increases expression of pro-apoptotic Bcl-2 protein PUMA. Furthermore, we demonstrate that PUMA can be cleaved by a caspase-dependent mechanism during apoptosis and identify what appears to be the PUMA cleavage product. Thus, our findings suggest that the combination of ABT-737 and inhibition of p38 is a promising, new treatment strategy that acts through a novel PUMA-dependent mechanism.     

Programmed cell death 4 (PDCD4) mediates the sensitivity of gastric cancer cells to TRAIL-induced apoptosis by down-regulation of FLIP expression

Tumor necrosis factor-related apoptosis induced ligand (TRAIL) is an important apoptosis inducer in a variety of tumor cells. In the present study, we determined the underlying molecular mechanisms by which certain gastric cancer cells are resistant to TRAIL. We first detected expression of programmed cell death 4 (PDCD4) in three gastric cancer cell lines and identified its association with the sensitivity of gastric cancer cells to TRAIL. We then stably transfected PDCD4 cDNA or shRNA into these gastric cell lines. Our data showed that restoration of PDCD4 expression induced TRAIL sensitivity, whereas knockdown of PDCD4 expression reduced the sensitivity of these tumor cells to TRAIL treatment. PDCD4 was able to suppress expression of FLICE-inhibiting protein (FLIP), a negative regulator of apoptosis. Knockdown of FLIP expression using FLIP shRNA had similar effects as those of restored PDCD4 expression. Furthermore, the proteasome inhibitor MG132 was able to inhibit expression of FLIP mRNA and protein and upregulate the sensitivity of these cells to TRAIL treatment. Taken together, the results from the current study demonstrated that PDCD4 plays an important role in mediating the sensitivity of gastric cancer cells to TRAIL-induced apoptosis through FLIP suppression. Therefore, the proteasome inhibitor MG132 should be further evaluated for combination therapy with TRAIL.     

Uncoupling protein 2 mediates resistance to gemcitabine-induced apoptosis in hepatocellular carcinoma cell lines

Oxidative stress induction is a common effector pathway for commonly used chemotherapeutic agents like gemcitabine (GEM) in hepatocellular carcinoma (HCC) patients. However, GEM alone or in combination with oxiplatin hardly renders any survival benefits to HCC patients. Mitochondrial uncoupling protein 2 (UCP2) is known to suppress mitochondrial reactive oxygen species (ROS) generation, thus mitigating oxidative stress-induced apoptosis. We demonstrate in the present study, using a panel of HCC cell lines that sensitivity to GEM in HCC well correlate with the endogenous level of UCP2 protein expression. Moreover, ectopic overexpression of UCP2 in a HCC cell line with low endogenous UCP2 expression, HLE, significantly decreased mitochondrial superoxide induction by the anti-cancer drug GEM. Conversely, UCP2 mRNA silencing by RNA interference in HCC cell lines with high endogenous UCP2 expression significantly enhanced GEM-induced mitochondrial superoxide generation and apoptosis. Cumulatively, our results suggest a critical role for mitochondrial uncoupling in GEM resistance in HCC cell lines. Hence, synergistic targeting of UCP2 in combination with other chemotherapeutic agents might be more potent in HCC patients.     

Caspase-independent killing of Burkitt lymphoma cell lines by rituximab

Caspase-independent cell death may have a critical role to play in the therapeutic destruction of tumours. Recently it has been suggested that one of the mechanisms by which rituximab, a therapeutic anti-CD20 antibody, kills B cells is caspase-independent. In this study we show that rituximab can induce death in a variety of Burkitt lymphoma derived cell lines. Rituximab-treated cells show leakage of adenylate kinase, surface expression of phosphatidylserine, upregulation of the cellular stress protein HSP70, phosphorylation of the survival protein Akt, and depolarisation of the mitochondrial membrane but no loss of cytochrome c or apoptosis inducing factor. Caspase inhibitors do not block these events. In support of these data there is no cleavage of caspases 3, 8 and 9, poly(ADP-ribose) polymerase, BH3 interacting domain death agonist or genomic DNA. Morphologically, cells show nuclear enlargement and cytoplasmic vacuolisation. Triggering of receptor mediated death in CD95 responsive lines results in “classical” apoptosis indicating that the internal machinery necessary for apoptosis is intact in these lines. The results suggest that rituximab can kill human B cells via a caspase-independent form of programmed cell death that shares features of apoptosis and necrosis. This pathway may be relevant to the clinical efficacy of rituximab.     

Modelling cell death in human tumour cell lines exposed to the anticancer drug paclitaxel

Most anti-cancer drugs in use today exert their effects by inducing a programmed cell death mechanism. This process, termed apoptosis, is accompanied by degradation of the DNA and produces cells with a range of DNA contents. We have previously developed a phase transition mathematical model to describe the mammalian cell division cycle in terms of cell cycle phases and the transition rates between these phases. We now extend this model here to incorporate a transition to a programmed cell death phase whereby cellular DNA is progressively degraded with time. We have utilised the technique of flow cytometry to analyse the behaviour of a melanoma cell line (NZM13) that was exposed to paclitaxel, a drug used frequently in the treatment of cancer. The flow cytometry profiles included a complex mixture of living cells whose DNA content was increasing with time and dying cells whose DNA content was decreasing with time. Application of the mathematical model enabled estimation of the rate constant for entry of mitotic cells into apoptosis (0.035 per hour) and the duration of the period of DNA degradation (51 hours). These results provide a dynamic model of the action of an anticancer drug that can be extended to improve the clinical outcome in individual cancer patients.Revised version: 9 October 2003     

Inhibition of polyamine oxidase in rats improves the sensitivity of urinary polyamines as markers for cell death.

In this study we investigated polyamine metabolism during inhibition of two polyamine-catabolizing enzymes. This was performed by treating rats with aminoguanidine [an inhibitor of Cu-dependent amine oxidase (CuAO)], NN'-bis(buta-2,3-dienyl)butane-1,4-diamine [MDL 72527, an inhibitor of FAD-dependent polyamine oxidase (PAO)], tetrachloromethane (hepatotoxic agent) and combinations of these compounds. Emphasis was laid on the origin and possible clinical usefulness of two polyamine metabolites: acetylisoputreanine-gamma-lactam and N1N12-diacetylspermine. Acetylisoputreanine-gamma-lactam is a normal constituent of human and rat urine. Treatment of rats with aminoguanidine led to undetectable urinary levels of acetylisoputreanine-gamma-lactam, whereas MDL 72527 treatment resulted in a 12-fold increase. Under normal conditions this compound represents a minor CuAO catabolite of N1-acetylspermidine, but may become of more importance under CuAO-induced conditions. N1N12-diacetylspermine was undetectable in urine samples from non-pregnant adults and rats, but became detectable after treating rats with MDL 72527. Additional tetrachloromethane poisoning resulted in a 35-fold increase of N1N12-diacetylspermine in urine and its appearance in liver. Hence urinary excretion of N1N12-diacetylspermine during PAO inhibition may serve as a sensitive marker for cell death. This was confirmed by myeloid-leukaemia-bearing rats treated with MDL 72527, which also excreted N1N12-diacetylspermine in urine in relatively high amounts from at least day 14 until spontaneous death.     

Gastrin mediates resistance to hypoxia-induced cell death in xenografts of the human colorectal cancer cell line LoVo

Aim: Gastrins act as growth factors for the normal and neoplastic colorectal mucosa. The aim of this study was to determine the role of gastrins in the response of human colorectal cancer (CRC) cells to hypoxia in vitro and in vivo. Methods: Expression of the gastrin gene in the human CRC cell line LoVo was examined under normoxia and hypoxia by quantitative PCR and by radioimmunoassay. Gastrin expression was knocked down with shRNA, and the effect on cell proliferation was measured by cell counting, on cell apoptosis by annexin V staining, and on cell migration by Boyden chamber assay. The effect of gastrin knockdown on tumourigenesis in mouse xenografts was analysed by measurement of tumour volumes and weights, and by immunohistochemistry. Results: Gastrin gene expression in LoVo cells was stimulated by hypoxia via binding of hypoxia-inducible factor-1α to the gastrin promoter. The viability of gastrin knockdown cells exposed to hypoxia (1% O₂) in vitro was diminished because of loss of resistance against hypoxia-induced apoptosis, and the effect was partly reversed by treatment with non-amidated, but not amidated, gastrin. Conditioned medium from control LoVo cells under hypoxia simulated proliferation but not migration, and the effect was blocked by an inhibitor of non-amidated gastrins, but not by an inhibitor of amidated gastrins. In xenografts in mice exposed to hypoxia (10% O₂) for 21 days, tumour necrosis was significantly increased by knocking down gastrin expression. Conclusion: These results provide evidence that non-amidated gastrins are involved in the adaptation of CRCs to hypoxic microenvironments through increasing resistance to apoptosis.     

Differential expression of Rab3 isoforms in high- and low-secreting mast cell lines

The expression of several isoforms of the small-molecular-weight Rab3 GTP-binding proteins is a characteristic feature of all cell types undergoing regulated exocytosis, in which Rab3 proteins are considered to regulate the assembly/disassembly of a fusion complex between granule and plasma membrane in a positive and negative manner through interaction with effector proteins. The pattern of Rab3 protein expression may, therefore, provide a subtle means of regulating exocytosis. To investigate the relationship between Rab3 expression and secretory activity, we assessed the differential expression of individual Rab3 proteins in high- and low-secreting clones of the rat basophilic (RBL) cell line. mRNAs for Rab3 isoforms (a-d) were analyzed by constructing cDNA libraries of high- and low-secreting RBL clones. The relative abundance of mRNAs for Rab3 isoforms was initially determined from the clonal frequency of corresponding cDNA clones. RT-PCR using isoform-specific primers was successfully applied to the quantitation of Rab3a mRNA. The presence of individual Rab3 proteins was revealed by SDS-PAGE and immunoblotting, and also by in situ immunofluorescence confocal microscopy. We present evidence that Rab3a and Rab3c are expressed at high levels in the low-secreting variant, while Rab3d is predominant in the high secretor. Levels of the Rab3 effector proteins, Rabphilin and Noc2, are similar in both RBL cell lines. Subcellular fractionation of unstimulated high and low secretor RBL clones revealed that in both cell types Rab3a has a cytoplasmic location while Rab3d is present in a membrane/organelle fraction containing secretory vesicles. Differences in the pattern of expression of Rab3 isoforms in the two RBL cell lines and their localisation may influence the secretory potential. Furthermore, the presence of Rab3 and effector proteins indicates that the mechanism for regulated exocytosis in cells of mast cells/basophil lineage appears similar to that in pre-synaptic vesicles and pancreatic beta-cells.     

Progestin inhibition of cell death in human breast cancer cell lines

Previously, we have shown that progestins both stimulate proliferation of the progesterone receptor (PR)-rich human breast cancer cell line T47D and protect from cell death, in charcoal-stripped serum-containing medium. To lessen the variability inherent in different preparations of serum, we decided to further characterize progestin inhibition of cell death using serum starvation to kill the cells, and find that progestins protect from serum-starvation-induced apoptosis in T47D cells. This effect exhibits specificity for progestins and is inhibited by the antiprogestin RU486. While progestin inhibits cell death in a dose–responsive manner at physiological concentrations, estradiol-17β surprisingly does not inhibit cell death at any concentration from 0.001 nM to 1 μM. Progestin inhibition of cell death also occurs in at least two other human breast cancer cell lines, one with an intermediate level of PR, MCF-7 cells, and, surprisingly, one with no detectable level of PR, MDA-MB-231 cells. Further, we have found progestin inhibition of cell death caused by the breast cancer chemotherapeutic agents doxorubicin and 5-fluorouracil. These data are consistent with the building body of evidence that progestins are not the benign hormones for breast cancer they have been so long thought to be, but may be harmful both for undiagnosed cases and those undergoing treatment.     

Inhibition of deoxyribonuclease I by actin is to protect cells from premature cell death

Deoxyribonuclease I (Dnase1) is the major extracellular endonuclease. It is secreted by digestive glands into the alimentary tract and into the plasma, lacrimal fluid and urine by hepatocytes, lacrimal glands and renal proximal tubular cells, respectively. In many species the activity of Dnase1 is inhibited by monomeric actin. However, the biological significance of this high affinity interaction is unknown. We generated a Dnase1 mutant with extremely reduced actin binding capacity. EGFP-constructs of wild-type and mutant Dnase1 were transfected into MCF-7 breast cancer cells and apoptosis or necrosis was induced by staurosporine or oxidative stress. During apoptosis faster chromatin fragmentation occurred in cells transfected with mutant Dnase1. When wt (wild-type)- or mutated Dnase1 were added to cells after induction of necrosis, faster chromatin degradation occurred in the presence of mutant Dnase1. Inclusion of actin under these conditions inhibited chromatin degradation by wt- but not by mutated Dnase1. Thus, inhibition of Dnase1 by actin may serve as a self-protection mechanism against premature DNA degradation during cell damage.     

Resistance to cisplatin does not affect sensitivity of human ovarian cancer cell lines to mifepristone cytotoxicity

Background  

The prototypical antiprogestin mifepristone exhibits potent growth inhibition activity towards ovarian cancer cells in vitro and in vivo. The aim of this research was to establish whether mifepristone is capable of inhibiting cell proliferation and inducing apoptotic cell death regardless of the degree of sensitivity ovarian cancer cells exhibit to cisplatin.     

Activation or suppression of NFkappaB by HPK1 determines sensitivity to activation-induced cell death

Restimulation of the T-cell receptor (TCR) in activated T cells induces CD95 (Fas/Apo-1)-mediated activation-induced cell death (AICD). The TCR-proximal mechanisms leading to AICD are elusive. Here we characterize hematopoietic progenitor kinase 1 (HPK1) as a differentially regulated TCR-proximal signaling protein involved in AICD of primary T cells. We show that HPK1 is a functional component of the endogenous IkappaB kinase (IKK) complex and is crucial for TCR-mediated NFkappaB activation. While full-length HPK1 enhances IKKbeta phosphorylation, siRNA-mediated knockdown of HPK1 blunts TCR-mediated NFkappaB activation and increases cell death. We also demonstrate proteolytic processing of HPK1 into HPK1-C, specifically in AICD-sensitive primary T cells. The cleavage product HPK1-C sequesters the inactive IKK complex and suppresses NFkappaB upon TCR restimulation by binding to IKKalpha and IKKbeta. T cells of HPK1-C transgenic mice are sensitized towards TCR-mediated AICD. Consequently, preventing HPK1-C generation in primary T cells by siRNA-mediated knockdown results in decreased AICD. Thus, these results show a novel mechanism of sensitization of T lymphocytes towards AICD by suppression of NFkappaB, and propose that HPK1 is a life/death switch in T lymphocytes.     

The function of gamma-glutamyl transpeptidase as a determinant in cell sensitivity to azaserine toxicity

The enzyme gamma-glutamyl transpeptidase (GGT) is characteristically present at high levels in mammalian cells that are vulnerable in vivo to the selectively toxic and carcinogenic effects of the naturally occurring diazo amino acid L-azaserine. The possible role of GGT as a determinant of cellular sensitivity to azaserine toxicity was investigated. No correlation was found between GGT activity and the abilities of different cell lines or GGT-deficient cell strains of TuWi, a human nephroblastoma-derived line high in GGT, to accumulate azaserine. However, the thiols glutathione and cysteine were found to inhibit the toxicity of azaserine in cultures of TuWi. In addition, maleate lowered both intracellular and extracellular glutathione levels and enhanced sensitivity of TuWi cells to azaserine, while serine-borate, a potent inhibitor of GGT, increased extracellular glutathione levels and inhibited azaserine toxicity. Since extracellular glutathione accumulation, which may reflect the rate of cellular glutathione turnover, is increased in cultures of azaserine-resistant, GGT-deficient strains of TuWi, we propose that GGT enhances cellular sensitivity to azaserine primarily by increasing the rate of glutathione turnover, thus removing the glutathione from detoxification pathways.