Cytotoxicity In Human Cells Research Articles

Functional Regions of thePseudomonas aeruginosaCytotoxin ExoU

ExoU, a potent patatin-like phospholipase, causes rapid cell death following its injection into host cells by the Pseudomonas aeruginosa type III secretion system. To better define regions of ExoU required for cytotoxicity, transposon-based linker insertion mutagenesis followed by site-directed mutagenesis of individual residues was employed by using a Saccharomyces cerevisiae model system. Random insertion of five amino acids identified multiple regions within ExoU that are required for cell killing. Five regions were chosen for further characterization: three corresponded to the oxyanion hole, hydrolase motif, and catalytic aspartate motif of the patatin-like domain within the N-terminal half of ExoU; one corresponded to an uncharacterized part of the patatin-like domain; and one corresponded to a region near the C terminus. Specific individual amino acid substitutions in each of the four N-terminal regions prevented killing of yeast and significantly reduced phospholipase activity. Whereas five amino acid insertions in the fifth region near the C terminus markedly reduced cytotoxicity and phospholipase activity, substitution of individual amino acids did not abolish either activity. To determine whether each of the five identified regions of ExoU was also essential for cytotoxicity in human cells, representative mutant forms of ExoU fused to green fluorescent protein were expressed in HeLa cells. These variants of ExoU were readily visualized and caused minimal cytotoxicity to HeLa cells, while wild-type ExoU fused to green fluorescent protein induced significant cell lysis and no detectable fluorescence. Thus, a minimum of five regions, including one which is well removed from the patatin-like domain, are required for the cytotoxicity and phospholipase activity of ExoU.

Nucleocytoplasmic distribution of the ovalbumin serpin PI-9 requires a nonconventional nuclear import pathway and the export factor Crm1.

Proteinase inhibitor 9 (PI-9) is a human serpin present in the cytoplasm of cytotoxic lymphocytes and epithelial cells. It inhibits the cytotoxic lymphocyte granule proteinase granzyme B (graB) and is thought to protect cytotoxic lymphocytes and bystander cells from graB-mediated apoptosis. Following uptake into cells, graB promotes DNA degradation, rapidly translocating to the nucleus, where it binds a nuclear component. PI-9 should therefore be found in cytotoxic lymphocyte and bystander cell nuclei to ensure complete protection against graB. Here we demonstrate by microscopy and subcellular fractionation experiments that PI-9 is present in the nuclei of human cytotoxic cells, endothelial cells, and epithelial cells. We also show that the related serpins, PI-6, monocyte neutrophil elastase inhibitor (MNEI), PI-8, plasminogen activator inhibitor 2 (PAI-2), and the viral serpin CrmA exhibit similar nucleocytoplasmic distributions. Because these serpins lack classical nuclear localization signals and are small enough to diffuse through nuclear pores, we investigated whether import occurs actively or passively. Large (approximately 70 kDa) chimeric proteins comprising PI-9, PI-6, PI-8, MNEI, or PAI-2 fused to green fluorescent protein (GFP) show similar nucleocytoplasmic distributions to the parent proteins, indicating that nuclear import is active. By contrast, CrmA-GFP is excluded from nuclei, indicating that CrmA is not actively imported. In vitro nuclear transport assays show that PI-9 accumulates at a rate above that of passive diffusion, that it requires cytosolic factors but not ATP, and that it does not bind an intranuclear component. Furthermore, PI-9 is exported from nuclei via a leptomycin B-sensitive pathway, implying involvement of the export factor Crm1p. We conclude that the nucleocytoplasmic distribution of PI-9 and related serpins involves a nonconventional nuclear import pathway and Crm1p.

Ex vivo purging of stem cell autografts using cytotoxic cells.

Autologous stem cell transplantation (SCT) is the treatment alternative offered to patients that cannot benefit from allogeneic transplantation due to lack of suitable donor or age limitations. However, the outcome of autologous SCT is largely hindered by the high relapse rate. Two major factors can account for relapse after autologous SCT: the persistence of residual malignant cells resistant to chemo/radiotherapy left either in the body or in the autograft. Therefore, the rationale for purging autografts of residual malignant cells comes from the limitations of conventional high-dose chemo/radiotherapy in achieving a complete eradication of residual tumor cells in the marrow. To date, different purging modalities have been exploited. Immunological methods of purging present the advantage of being non-cross-reactive with conventional chemotherapy. These immunologic methods include depletion using antibody targeting of the malignant cells, ex vivo activation/generation of the autologous cytotoxic cells, in particular that of natural killer/lymphokine-activated killer (NK/LAK) and cytokine-induced killer (CIK) cells, and ex vivo purging of autografts using cytotoxic cell lines. The generation of ex vivo-expanded and activated autologous cytotoxic cells (CTL or NK) has generated increasing interest for the treatment of different malignancies. Unfortunately, the isolation and expansion of these cells have proven to be technically difficult. As an alternative, the use of cytotoxic cell lines as immune effectors has been proposed. The two available human cytotoxic cell lines TALL104 and NK-92 are currently in clinical trials and a number of studies have suggested their effectiveness as an immunotherapeutic agent including for ex vivo purging of autografts.

Protein precipitation In Vitro as a measure of chemical-induced cytotoxicity: an EDIT sub-programme.

As a priority area of the Evaluation-Guided Development of In Vitro Toxicity and Toxicokinetic Tests (EDIT) programme, an in vitro protein precipitation (PP) assay was used on the 50 reference chemicals of the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) project, to confirm and extend the MEIC results. Dose-response curves were generated for only 30 of the chemicals, and the concentrations causing 10% (EC10) and 50% (EC50) protein precipitation versus the positive control were chosen as endpoints. The number of chemicals with a positive response increased to 46 when a new endpoint, the minimum effect concentration (MEC) that induces protein precipitation with respect to the negative control, was used. When the results were correlated with in vitro cytotoxicity in human cell lines, a similarly good correlation was found between the various endpoints of the PP assay at 5 hours and the 24-hour IC50 average cytotoxicity in human cell lines, even though the number of chemicals included in the correlation was larger for the MEC. Using the prediction error, the endpoint that gave the best correlation between the PP assay and human cell cytotoxicity was once more found to be the 5-hour MEC, and this was chosen for the PP assay. The sensitivity of the PP assay is lower than that of the in vitro cell-line cytotoxicity assay, possibly due to its shorter exposure period and because precipitation is the ultimate event in the sequence of a protein disturbance. It is expected that earlier denaturation steps would give better sensitivity. However, this simple, inexpensive and rapid assay could be useful in the early stages of testing chemicals.

Sex Steroids Do Not Prevent Amylin-Induced Apoptosis in Human Cells

Formation of amylin-containing islet amyloid deposits may contribute to the progressive deterioration of β cell function in non-insulin-dependent diabetes mellitus. As diabetes mellitus occurs in male, but rarely in female transgenic mice expressing human amylin in their pancreatic β cells, it is of interest to study the influence of estradiol (E2) and testosterone (T) on amylin-induced cytotoxicity in human cells. The insulinoma cell line CM, thyroid epithelial cells (TEC) in primary culture, and nontransformed fibroblast lines were used. The occurrence of apoptotic cell death was assessed by nuclear labeling with propidium iodide. Amylin was cytotoxic on all cell types tested, but had the most pronounced effect on TEC and the weakest on the CM cell line. Although both E2and T decreased the proportion of apoptotic cells in cultures kept in the absence of amylin, neither of the two hormones was able to counteract amylin-induced cytotoxicity. β cell death and hyperglycemia can thus presumably not be prevented by the neutralization of amylin effects by sex steroids.

Tumor necrosis factor α augments amyloid β protein (25–35) induced apoptosis in human cells

No information is yet available on the effect of tumor necrosis factor α (TNF α) on amyloid β protein (A β)-induced cytotoxicity in human cells. For this reason the induction of apoptosis by TNF α and A β (25–35) was studied in primary cultures of human thyroid and kidney cells as well as in the neuroblastoma line SK-N-SH and in DU-145 cells. Apoptosis occurred in all cell types after A β (25–35) treatment, but was markedly enhanced when TNF α was additionally present. This effect was less pronounced in transformed cell lines than in primary cultures, in which TNF α on its own was not cytotoxic. Apoptosis was still more prevalent under serum free culture conditions. The results demonstrate that TNF α may support the occurrence of A β-mediated cell death and thus contribute to the development of pathological changes in Alzheimer's disease (AD).

The effect of dietary flavonoids on DNA damage (strand breaks and oxidised pyrimdines) and growth in human cells

The effects of the flavonoids quercetin, myricetin and silymarin on DNA damage and cytotoxicity in human cells were investigated. DNA strand breaks and oxidised pyrimidines were determined using alkaline single cell gel electrophoresis (the comet assay). Inhibition of cell growth was also measured. Caco-2 (colon), HepG2 (liver), HeLa (epithelial) cells and normal human lymphocytes showed different, dose-dependent susceptibilities (in terms of strand breakage) to the various flavonoids, quercetin being the most damaging. This agreed well with the ability of the flavonoids to inhibit cell growth. None of the flavonoids induced DNA base oxidation above background levels. All of the flavonoids under investigation caused depletion of reduced glutathione, which, in the case of quercetin, occurred prior to cell death. Neither cytotoxicity nor genotoxicity was associated with the antioxidant enzyme capacity (glutathione, glutathione reductase, glutathione peroxidase and catalase) of the cells.

Fused pyrazine mono-n-oxides as bioreductive drugs. II cytotoxicity in human cells and oncogenicity in a rodent transformation assay

Purpose :To determine what structural moieties of the fused pyrazine mono-N-oxides are determining factors in their in vitro cytotoxicity and oncogenicity. Methods and Materials :A new series of experimental bioreductive drugs, fused pyrazine mono-N-oxides, was evaluated in vitro for aerobic and hypoxic cytoxicity in the HT29 human colon adenocarcinoma cell line by using clonogenic assays. The relative oncogenecities of these compounds were also determined in aerobic cultures of C3H 10T1/2 mouse embryo fibroblasts by using a standard transformation assay. Results : Removal of the 4-methyl piperazine side chain from the parent compound, RB 90740, reduced the potency of the hypoxic cytotoxin. Reduction of the N-oxide function increased the aerobic cytotoxicity and eliminated most of the hypoxic/aerobic cytotoxic differential. The reduced N-oxide also had significant oncogeneity, consistent with a mechanism of genotoxicity following bioreduction of RB 90740. Conclusion : This new series of bioreductive compounds may be effective in cancer therapy, particularly the lead compound RB 90740. The oncogenic potential of these compounds is similar to that for other cancer therapies. Further studies should include evaluation of these compounds in vivo and the development of analogs with reduced oncogenic potential and retention of the hypoxic/aerobic cytoxicity differential.

Selective recognition of the m 5 CpG dinucleotide sequence in DNA by mitomycin C for alkylation and cross-linking

The clinically used natural antitumor agent mitomycin C (MC) is known to alkylate DNA monofunctionally and bifunctionally, resulting in the cross-linking of DNA. These reactions occur selectively with guanines at the CpG sequence. We show, confirming a previous report (Millard, J. T.; Beachy, T. M. Biochemistry 1993, 32, 12850) that cross-linking in oligonucleotides is further enhanced when the cytosines in CpG·CpG are 5-methylated to m 5CpG·m 5CpG. It is shown, furthermore, that guanines in m 5CpG are monoalkylated two- to three-times faster than in CpG indicating that the m 5C-induced rate enhancement occurs at the first, monoalkylation step of the two-step cross-linking process. The same MC-DNA adducts are formed in methylated as in non-methylated DNA. The basepaired but not the 5′-flanking, m 5C residue is responsible for the enhanced alkylation of guanine. Enzymatically activated or Na 2S 2O 4-activated MC shows identical rate-enhancement of alkylation at m 5CpG. pBR322 DNA methylated by CpG-methylase was cross-linked two- to three-times more efficiently by MC than non-methylated DNA, indicating that the m 5C effect is not an artifact of oligonucleotides. An electronic effect of the 5-methyl group of cytosine transmitted via G·C H-bonding to N 2 of guanine is suggested as responsible for increased reactivity with MC. CpG is severely depleted in mammalian DNA and it is speculated that this factor attenuates MC cytotoxicity in human cells.

Production and characterization of monoclonal antibodies raised against recombinant human granzymes A and B and showing cross reactions with the natural proteins

The human serine proteases granzymes A and B are expressed in cytotoplasmic granules of activated cytotoxic T lymphocytes and natural killer cells. Recombinant granzyme A and granzyme B proteins were produced in bacteria, purified and then used to raise specific mouse monoclonal antibodies. Seven monoclonal antibodies (mAb) were raised against granzyme A, which all recognized the same or overlapping epitopes. They reacted specifically in an immunoblot of interleukin-2 (IL-2) stimulated PBMNC with a disulfide-linked homodimer of 43 kDa consisting of 28 kDa subunits. Seven mAb against granzyme B were obtained, which could be divided into two groups, each recognizing a different epitope. On an immunoblot, all mAb reacted with a monomer of 33 kDa protein. By immunohistochemistry, these mAb could be used to detect granzymes A and B expression in activated CTL and NK cells. The availability of these mAb may facilitate studies on the role of human cytotoxic cells in various immune reactions and may contribute to a better understanding of the role of granzmes A and B in the cytotoxic response in vivo.

β-endorphin blunts phosphatidylinositol formation during in vitro activation of isolated human lymphocytes: Preliminary report

We have previously described the regulatory effect of β-endorphin on three human cytotoxic cell populations. We confirmed the variable nature of these effects on human natural killer cell (NK) activity, showed mixed effects on the generation of cytotoxic T lymphocyte (CTL) activity, and demonstrated the reproducible suppression of lymphokine-activated killer cell (LAK) activity. We and others also observed mixed effects of β-endorphin on the proliferative response to mitogens and in mixed leukocyte reactions. In the study reported here, we test the effects of β-endorphin on the formation of phosphatidylinositol during cell activation. 32P-radiolabeled peripheral blood mononuclear cells obtained from normal adult donors and CD2-depleted subpopulations were activated with phytohemagglutinin or in a NK, LAK, or CTL protocol in the absence or presence of recombinant β-endorphin. The total lipidic extract was analyzed by thinlayer chromatography and autoradiography. The results of these studies indicate that β-endorphin blunts the formation of phosphatidylinositol by about 20% in the four systems studied and in all the donors tested. This effect is dose-dependent and is blocked in part by the opioid antagonist, naltrexone, suggesting involvement of the opioid receptor.

Differential effect of beta-endorphin on three human cytotoxic cell populations

We have examined in vitro the effect of the proopiomelanocortin gene product, beta-endorphin (bE), on the cytoxic activity of natural killer (NK) cells, lymphokine activated killer (LAK) cells and cytotoxic T-lymphocytes (CTL). Our studies show that bE reproducibly suppressed LAK cytotoxic activity in all donors tested. The effect of bE on the generation of CTL varied, and was negligible on CTL cytotoxic function. Our study also confirms the variable nature of the effects of bE on NK cytotoxicity. In all instances, the effects of bE were generally small, but could be blocked by opioid receptor antagonists, or by prior heat-inactivation of the peptide. The magnitude of the effects was greatest at low effector:target ratios in all of the three systems studied. These results support the emerging body of evidence that the neuroendocrine system may influence host defense mechanisms mediated by cytotoxic cells.

In vitro human cytotoxic T cell responses against influenza A virus can be induced and selected by synthetic peptides

Studies on human anti-influenza cytolytic activities have demonstrated that cytotoxic T lymphocytes (CTL) from HLA-B37 individuals react preferentially with the peptide corresponding to residues 335-349 of the nucleoprotein, whereas CTL from HLA-A2 donors recognize peptide 57-68 from the viral matrix as a dominant epitope. We studied the secondary CTL response, obtained from peripheral blood mononuclear cells, of an HLA-A2+,B37+ individual stimulated either by infectious virus or by synthetic peptides. Only an HLA-B37-restricted response was detected after stimulation by the whole virus, showing an immunodominance of this activity over that restricted by HLA-A2. Moreover, human cytotoxic cell lines were successfully obtained after stimulation of peripheral blood mononuclear cells with synthetic peptides. Under these conditions, it was possible to selectively reveal the existence of an HLA-A2-restricted activity directed against the matrix peptide. These results demonstrate that, at least in vitro, it is possible to stimulate a latent repertoire by using synthetic peptides. Nevertheless, we could not induce a response against the matrix or the nucleoprotein peptides in HLA-A2- or B37- individuals, suggesting that a finer selection of synthetic peptides would be necessary for their possible utilization to induce CTL during vaccination.

Cloning of a gene that encodes a new member of the human cytotoxic cell protease family

A family of unusual serine proteases that are believed to be involved in the effector mechanism of cell-mediated cytotoxicity have previously been described in the mouse. However, in the human only one gene encoding a member has been isolated. By use of a mixture of murine cDNAs as probes, a second human gene has now been isolated. The primary structures of the gene and the predicted protein are very similar to those of the mouse. In addition, in keeping with the postulated involvement in cytolysis, transcripts were detected only in cytotoxic cells. The organization of the coding and noncoding regions of the gene, the clustering of family members, and the chromosomal location, close to the alpha chain of the T cell antigen receptor, are all conserved between human and mouse.

IL-2 activated human cytotoxic cells conjugation to their targets closely correlates with the killing phenomenon

IL-2 activated human cytotoxic cells conjugation to their targets closely correlates with the killing phenomenon

Molecular cloning of an inducible serine esterase gene from human cytotoxic lymphocytes.

A cDNA clone encoding a human serine esterase gene was isolated from a library constructed from poly(A)+ RNA of allogeneically stimulated, interleukin 2-expanded peripheral blood mononuclear cells. The clone, designated HSE26.1, represents a full-length copy of a 0.9-kilobase mRNA present in human cytotoxic cells but absent from a wide variety of noncytotoxic cell lines. Clone HSE26.1 contains an 892-base-pair sequence, including a single 741-base-pair open reading frame encoding a putative 247-residue polypeptide. The first 20 amino acids of the polypeptide form a leader sequence. The mature protein is predicted to have an unglycosylated Mr of approximately equal to 26,000 and contains a single potential site for N-linked glycosylation. The nucleotide and predicted amino acid sequences of clone HSE26.1 are homologous with all murine and human serine esterases cloned thus far but are most similar to mouse granzyme B (70% nucleotide and 68% amino acid identity). HSE26.1 protein is expressed weakly in unstimulated peripheral blood mononuclear cells but is strongly induced within 6-hr incubation in medium containing phytohemagglutinin. The data suggest that the protein encoded by HSE26.1 plays a role in cell-mediated cytotoxicity.

Humans express natural cytotoxic (NC) cell activity that is similar to murine NC cell activity.

The expression of natural cytotoxic (NC) activity is well defined in mice, but poorly defined in humans. In this paper we report that humans express naturally occurring cytotoxic cell activity that recognizes and lyses murine targets that are sensitive to lysis mediated by murine NC cells, but not murine targets that are resistant to lysis by murine NC cells. We present data showing that these naturally occurring human cytotoxic cells and murine NC cells have similar lytic mechanisms. Both the human cytotoxic cells described here, and murine NC cells, use tumor necrosis factor (TNF) to mediate the lysis of sensitive targets. Moreover, targets that resist murine NC-mediated lysis by a protein synthesis-dependent post-recognitive mechanism use a similar mechanism to prevent lysis mediated by naturally occurring human cytotoxic cells. In addition to the similarity of naturally occurring human cytotoxic cells and murine NC cells in their specificity and lytic mechanism, naturally occurring human cytotoxic cells and murine NC cells are also similar in that their activity is both associated with a monocyte lineage and age independent. Taken together, these data indicate that humans express NC activity.

Differential Effects of Radical Scavengers on X-Ray-Induced Mutation and Cytotoxicity in Human Cells

The cytotoxic and mutagenic effects of X irradiation on a human lymphoblast cell line were examined in the presence of two radioprotective agents which modulate damage to DNA. The cells were treated with X rays alone or in the presence of either dimethyl sulfoxide or cysteamine. Surviving fraction and mutation to trifluorothymidine resistance (tk locus) and to 6-thioguanine resistance (hgprt locus) were measured. Survival was enhanced when the cells were irradiated in the presence of dimethyl sulfoxide; the D0 rose from 58 to 107 rad. However, at both genetic loci the induced mutant fractions were identical in the presence or absence of dimethyl sulfoxide. Survival was enhanced to a greater degree when the cells were irradiated in the presence of cysteamine; the D0 rose from 58 to 200 rad. Cysteamine also protected the cells from X-ray-induced mutation; the frequencies of X-ray-induced mutation at both the tk and hgprt loci were reduced by 50-75%. No protective effects were observed unless dimethyl sulfoxide or cysteamine was present during irradiation. These findings are discussed in terms of the hypothesis that, unlike for cell killing, radiation-induced mutagenesis in human lymphoblast cells is not mediated by the actions of aqueous free radicals, but rather by the direct effects of ionizing radiation.

Protection of antiproliferative effect of cis-diamminedichloroplatinum (II) by sodium thiosulfate.

Utilizing the phytohemagglutinin (PHA) stimulation assay of human peripheral blood mononuclear cells (PBM), the protective effect of sodium thiosulfate (STS) on the antiproliferative action of cis-diamminedichloroplatinum (II) (DDP) against human cells was investigated. DDP alone significantly inhibited the proliferation of PBM over the concentration range of 10(-7) to 5 X 10(-5) M. The antiproliferative effect of DDP was significantly blocked when STS was added to the stimulation culture at the time of exposure to DDP at molar STS/DDP ratios of more than 500. However, STS at molar ratios of less than 100 induced minimal protection. Then, STS was added at various times after DDP exposure with a molar STS/DDP ratio of 1000. The protection was effective within 10 min after exposure to DDP at a concentration of 5 X 10(-5) M, whereas it was not effective beyond 30 min after the exposure. The results indicate that effective protection against DDP cytotoxicity in human cells can be achieved by the concurrent presence of STS with molar STS/DDP ratios of more than 500, but not when a molar STS/DDP ratio of less than 100 is used.

Studies on the cytolytic attack mechanism of the cytotoxic T lymphocyte (CTL): preparation of antisera against cellfree cytosolic extracts of a CTL clone capable of blocking the lethal hit stage of CTL cytolysis and analysis of the cytolytic structure.

Rat antiserum (as well as purified IgG and F(ab')2 fragments) raised against cellfree cytosolic extracts (CFE) of an alloimmune cytotoxic T lymphocyte (CTL) clone (B6.1.SF.1) is a potent inhibitor of CTL-mediated cytotoxicity. Inhibition by this antiserum (termed alpha CTLL) occurred during the postbinding lethal hit stages of cytolysis, because it did not inhibit target cell binding, nor did it prematurely dissociate CTL-target cell conjugates; inhibition was observed regardless of the H-2 haplotype of the target cell or CTL employed; inhibition was reversible when pretreated, and washed CTL were used as effectors; and in Ca++ pulse experiments alpha CTLL inhibited cytolysis beyond the Ca++-dependent (lethal hit) stage of cytolysis. This antiserum did not inhibit lysis of P815 cells by activated murine macrophages or by human cytotoxic cells, and extensive absorption of the antiserum on viable thymocytes, normal spleen cells, or CTL did not reduce its blocking activity. CFE prepared from several sources of CTL, including in vivo elicited peritoneal exudate lymphocytes (PEL), secondary MLC-generated CTL, alloimmune splenic T cells, and CTL clones, contained material(s) that inhibited the ability of alpha CTLL to block CTL-mediated cytolysis. The inhibitory activity was not detected in CFE from a variety of noncytotoxic cell sources, including thymocytes, normal C57BL/6 spleen cells, EL4 or P815 tumor cells, macrophages, and helper T cell clones. It was also absent in CFE prepared from human CTL cells. Furthermore, although alpha CTLL neutralizing activity was not detectable in CFE prepared from memory CTL, it rapidly appeared in CTL parallel to the development of cytolytic activity during secondary MLC cultures. The inhibitory material in CTL-CFE appeared to be specific for alpha CTLL antibody, as it did not affect the CTL blocking activity of anti-Lyt-2 or anti-target cell antisera. Finally, CTL-CFE did not contain proteases that degraded the alpha CTLL antibody. By the use of a soluble-phase immunoabsorbent assay, the biochemical properties of materials present CFE derived from CTL and reactive with alpha CTLL antibody were examined. CTL cytosolic material(s) reactive with alpha CTLL IgG was unstable to brief heating (50 degrees C) or acidic pH, but not to high ionic strength buffers. The material was inactivated by treatment with pronase but not by DNase, collagenase, or trypsin. Gel filtration chromatography of CTL-CFE revealed multiple peaks of alpha CTLL neutralizing activity.(ABSTRACT TRUNCATED AT 400 WORDS)