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C M Zacharchuk

Publications and source records attributed to C M Zacharchuk.

At least 19 recordsLinked to original sources
PubMed1998

Apoptosis induced by a chimeric Fas/FLICE receptor: lack of requirement for Fas- or FADD-binding proteins.

Current models for Fas (CD95)-mediated apoptosis suggest that FLICE/caspase-8 is recruited and activated, which results in cell death. However, the role of additional molecules in Fas signaling and FLICE activation is not clear. A chimeric Fas/FLICE (F/F) receptor, containing the extracellular/transmembrane portion of Fas and the caspase region of FLICE, mediated anti-Fas apoptosis. FLICE protease subunits were generated from the F/F precursor. Killing induced by Fas, but not F/F, was blocked by a dominant negative FADD. Apoptosis triggered through Fas and F/F was inhibited by coexpression of CrmA and p35, but not Bcl-xL. F/F bypassed Fas resistance in COS-7 cells and blocking by the death effector domain (DED)-containing viral protein MC159. These results show that: 1) F/F induces cell death, indicating that FLICE activation is sufficient for apoptosis and does not require additional Fas- or FADD-binding proteins; and 2) F/F bypasses proximal defects in Fas signaling that prevent FLICE recruitment or activation.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Do CTL kill target cells by inducing apoptosis?

Inhibitors of ICE-family proteases (caspases) block many examples of apoptotic cell death in vivo and in vitro, including multiple apoptotic stimuli for T lymphocytes. We have tested whether cell death induced by cytotoxic T lymphocytes was also blocked by caspase inhibitors. We found that the rapid apoptotic target cell death induced by Fas ligand-bearing CTL using the target Fas death pathway was efficiently blocked by caspase inhibitors. In contrast, target lysis induced by the CTL granule exocytosis pathway is not detectably blocked by such inhibitors, although the accompanying apoptotic nuclear damage is efficiently blocked. Thus caspase inhibitors prevent the hallmark phenotype of apoptosis without measurably affecting target cell death as evidenced by lysis.

Animals↗
PubMed1997

Target cell lysis by CTL granule exocytosis is independent of ICE/Ced-3 family proteases.

Activation of ICE/Ced-3 family proteases (caspases) has been proposed to mediate both the granule exocytosis and Fas-Fas ligand pathways of rapid target cell death by cytotoxic T lymphocytes. In agreement with this model, two peptide fluoromethyl ketone caspase inhibitors and baculovirus p35 blocked apoptotic nuclear damage and target cell lysis by the CTL-mediated Fas-Fas ligand pathway. The peptide caspase inhibitors also blocked drug-induced apoptotic cell death in tumor cells. In contrast, the caspase inhibitors blocked CTL granule exocytosis-induced target apoptotic nuclear damage, but did not inhibit target lysis. These results are consistent with recent demonstrations that granzyme B can activate caspases leading to apoptotic nuclear damage, but show that target cell lysis by CTL granule exocytosis occurs by a caspase-independent pathway.

Animals↗
PubMed1997

Lack of a role for Jun kinase and AP-1 in Fas-induced apoptosis.

Cross-linking of Fas (CD95) induces apoptosis, a response that has been reported to depend upon the Ras activation pathway. Since many examples of apoptosis have been reported to involve AP-1 and/or the AP-1-activation pathway. Since many examples of apoptosis have been reported to involve AP-1 and/or the AP-1-activating enzyme Jun kinase (JNK), downstream effectors of Ras or Ras-like small GTP-binding proteins, we evaluated the role of these molecules in Fas-mediated apoptosis. Although cross-linking of Fas on Jurkat T cells did result in JNK activation, increased activity was observed relatively late, being detectable only after 60 min of stimulation. Expression of a dominant negative form of SEK1 that blocked Fas-mediated induction of JNK activity had no effect on Fas-mediated apoptosis. Furthermore, maximally effective concentrations of anti-Fas did not cause JNK activation if apoptosis was blocked by a cysteine protease inhibitor, suggesting that under these conditions, activation of JNK may be secondary to the stress of apoptosis rather than a direct result of Fas engagement. Despite the activation of JNK, there was no induction of AP-1 activity as determined by gel shift assay or induction of an AP-1-responsive reporter. The lack of a requirement for AP-1 induction in Fas-mediated death was further substantiated with Jurkat cells that were stably transfected with a dominant negative cJun, TAM-67. While TAM-67 effectively prevented AP-1-dependent transcription of both the interleukin-2 and cJun genes, it had no effect on Fas-induced cell death, even at limiting levels of Fas signaling. Thus, induction of JNK activity in Jurkat cells by ligation of Fas at levels sufficient to cause cell death is likely a result, rather than a cause, of the apoptotic response, and AP-1 function is not required for Fas-induced apoptosis.

Amino Acid Chloromethyl Ketones↗
PubMed1996

Recombinant mouse Bcl-2(1-203). Two domains connected by a long protease-sensitive linker.

Bcl-2 is a cytoplasmic integral membrane protein with potent anti-apoptotic activity but whose mechanism of action is poorly understood. The purpose of this paper was to obtain large amounts of soluble Bcl-2 protein for structural and functional studies. Mouse Bcl-2(1-203) (missing the COOH-terminal hydrophobic tail) was produced in bacterial inclusion bodies, solubilized in guanidine, and refolded by dialysis. The resulting protein was monomeric in nondenaturing solution and was active in protecting mouse T hybridoma cells from glucocorticoid-induced apoptosis. Refolded Bcl-2(1-203) showed no tendency to homodimerize by gel filtration or analytical ultracentrifugation. Limited proteolysis experiments identified a region between the BH3 and BH4 homology domains of Bcl-2(1-203) which was extremely susceptible to digestion by several common proteases, but not by a cell extract known to contain CPP-32-like (interleukin-1beta-converting enzyme family) protease activity. The protease-sensitive sites were located within a 50-residue stretch that contained most of the nonconserved and proline residues of Bcl-2(1-203). Trypsin-cleaved Bcl-2(1-203) eluted in the same position as the undigested protein on gel filtration in nondenaturing solution, indicating that the two portions of the molecule connected by the protease-sensitive region associate stably and noncovalently. The solution properties of Bcl-2(1-203) suggest that it consists of two noncovalently associated domains connected by a long protease-sensitive linker and that its structure is similar to that of Bcl-xL, which has been determined by x-ray and NMR analysis.

Amino Acid Sequence↗
PubMed1996

Apoptosis signaling pathways in normal T cells: differential activity of Bcl-2 and IL-1beta-converting enzyme family protease inhibitors on glucocorticoid- and Fas-mediated cytotoxicity.

Fas-mediated apoptosis plays an important role in regulating the immune response in peripheral T cells. Restimulation of T cell blasts up-regulates Fas and Fas ligand expression, with subsequent interaction leading to cell death. Overexpression of Bcl-2 in tumor cells blocks apoptosis induced by many stimuli, but inhibition of Fas-mediated killing has not been consistently observed. To examine the behavior of Bcl-2 in normal cells, T cell blasts were transiently transfected with Bcl-2 and related gene products to determine the effect on apoptotic signaling. Transient overexpression of Bcl-2 in mouse and human T cell blasts did not block Fas-mediated apoptosis, whereas etoposide- and glucocorticoid-induced cytotoxicity was potently inhibited. Expression of Bcl-xL and adenovirus E1B 19K did not interfere with anti-Fas killing. In contrast, interleukin-1beta-converting enzyme family protease inhibitors Ac-DEVD-CHO and CrmA blocked Fas-mediated apoptosis. These results suggest that peripheral T cells use distinct apoptosis signaling pathways with differential sensitivity to Bcl-2 and interleukin-1beta-converting enzyme family protease inhibitors. Since T cells normally express Bcl-2 and Bcl-xL following activation, their inability to block Fas-mediated apoptosis may allow for the elimination of self-reactive cells and the appropriate regulation of immune responses.

Adenovirus E1B Proteins↗
PubMed1995

Bcl-2 blocks glucocorticoid- but not Fas- or activation-induced apoptosis in a T cell hybridoma.

Overexpression of Bcl-2 can prevent or markedly delay cell death induced by a variety of apoptotic stimuli. Although Fas and Fas ligand (FasL) interactions play a major role in the elimination of self-reactive T cells in the periphery, inhibition of Fas-mediated killing by Bcl-2 has not been consistently observed. The mouse T hybridoma 2B4.11 (2B4) has been a useful model to study glucocorticoid- and activation-induced apoptosis, which is mediated through Fas and FasL. Using both stable transfectants and transient transfections, overexpression of Bcl-2 or Bcl-xL readily blocked glucocorticoid-induced but not activation-induced apoptosis of 2B4 cells. Bcl-2 expression did not inhibit Fas-mediated cytotoxicity triggered by cells expressing FasL or by the transient transfection of human Fas. Similarly, overexpression of Bcl-2 in the mouse T hybridoma A1.1 did not block activation-induced/Fas-mediated apoptosis. In Jurkat cells, however, expression of Bcl-2 partially inhibited anti-Fas-induced cell death. A Bcl-2-related protein that can interfere with anti-Fas killing, the adenoviral E1B 19K, also did not block activation-induced/Fas-mediated apoptosis in 2B4 cells. In contrast, expression of CrmA, a cowpox virus protein that inhibits ICE-like protease activity, blocked activation-induced apoptosis in 2B4 cells but had little effect on Dex-mediated cytotoxicity. These results show that: 1) Bcl-2 can have strikingly different anti-cell death activity in the same cell depending upon the apoptotic stimulus, 2) distinct apoptosis signaling pathways may exist with differential sensitivity to Bcl-2 and ICE-like protease inhibitors.

Animals↗
PubMed1995

A simple assay for examining the effect of transiently expressed genes on programmed cell death.

Programmed cell death (PCD) has been observed in a wide variety of cell types in response to physiologic signals or types of stress. How these stimuli trigger PCD, and whether there is a common PCD signal transduction pathway, is not clear. As more genes are described that may participate in or regulate PCD, an assay system in which gene products can easily be introduced and/or modulated would be of great value. To avoid the generation and screening of multiple individual stable cell transfectants, a simple transient transfection death assay has been developed. 2B4.11, a murine T cell hybridoma, was transfected by electroporation with a constitutively active beta-galactosidase reporter gene and the cells were incubated in culture medium or with a PCD-inducing stimulus. The amount of beta-galactosidase activity remaining in the intact cells at the end of the culture period represented only viable transfected cells. Bcl-2 was chosen to examine whether this system would be useful to study the effect of transiently transfected genes since it blocks PCD in a number of experimental systems. Consistent with data obtained using stable transfectants, transient expression of Bcl-2 in 2B4.11 completely protected cells from glucocorticoid- and cytotoxic agent-induced PCD. This protection from death was confirmed at the individual cell level by the transient co-expression of a class I Ld surface antigen and flow cytometric analysis. Some of the advantages of the transient transfection death assay described here are; (1) the simple and sensitive beta-galactosidase assay, (2) the rapidity of the assay, (3) the ability to perform conventional viability assays to monitor treatment-induced cytotoxicity, (4) multiple gene products can be tested alone, and in combination, (5) antisense or dominant negative approaches can be used, and (6) the adaptability of this assay system to other cell types, transfection techniques, or reporter and expression vectors. The transient transfection death assay should make it easier to identify and order important steps in the PCD signal transduction pathways.

Animals↗
PubMed1995

Bispecific antibodies retarget murine T cell cytotoxicity against syngeneic breast cancer in vitro and in vivo.

Bispecific antibodies with specificity for CD3 and a tumor antigen can redirect cytolytic T cells to kill tumor targets, regardless of their natural specificity. To assess the clinical potential of bispecific antibodies for treatment of human cancers we have, in the present study, adapted a totally synergeic mouse model to the targeting of mouse T cells against mouse tumors in immunocompetent mice. We show that gp52 of the mouse mammary tumor virus (MTV) can serve as a tumor-specific antigen for redirected cellular cytotoxicity. Chemically crosslinked and genetically engineered bispecific antibodies with specificities for gp52 and murine CD3 epsilon-chain induced activated mouse T cells to specifically lyse mouse mammary tumor cells from cultured lines and primary tumors from C3H-MTV+ mice. Retargeted T cells also blocked the growth of mammary tumors in vitro as well as their growth in syngeneic mice. These findings identify murine MTV-induced mammary adenocarcinomas as a solid-tumor, animal model for retargeting T cells with bispecific antibodies against syngeneic breast cancer.

Adenocarcinoma↗
PubMed1995

A bispecific antibody prolongs survival in mice bearing lung metastases of syngeneic mammary adenocarcinoma.

In the present study we tested whether T cells retargeted with a bispecific antibody (bsAb) could block the growth of lung metastases of syngeneic mammary adenocarcinoma in immunocompetent mice. BALB/c mice were injected i.v. with tumor and i.p. with a genetically engineered bispecific F(ab')2 [bs(Fab')2] having specificity for murine CD3 epsilon chain and for the gp52 mouse mammary tumor viral glycoprotein, which is expressed on the tumor cells. The bs(Fab')2 was physically stable in blood and serum, was removed from the body with a half-time of 12-15 h, and accumulated in lymphoid tissue where it bound to T cells. We show that treatment of tumor bearing mice with the bs(Fab')2 significantly prolonged their survival relative to untreated controls. Two other genetically engineered bs(Fab')2s having specificity for murine CD3 epsilon chain and irrelevant antigens did not inhibit tumor growth. In addition, survival was not affected by bsAb therapy using a variant tumor cell line that expressed low levels of the gp52 target antigen. Inhibition of tumor growth was even more evident by histologic analysis. Treatment with the relevant bs(Fab')2 resulted in a marked reduction of tumor burden in lung sections taken on days 7, 9 and 11. This is the first report demonstrating that a bsAb can inhibit the growth of syngeneic solid tumor metastases in mice without addition of T cell activators.

Adenocarcinoma↗
PubMed1994

Dominant negative mutant of c-Jun inhibits NF-AT transcriptional activity and prevents IL-2 gene transcription.

Expression of the transcription complex AP-1, composed of Jun and Fos family members, can be induced by a variety of stimuli. In lymphocytes, AP-1 transcriptional activity increases after TCR ligation and plays an important role in T cell activation events such as lymphokine secretion. To explore the requirements for AP-1 in IL-2 production, the AP-1 complex was targeted with a dominant negative mutant c-Jun protein, TAM-67, from which the transactivation domain has been deleted. In transient transfections of Jurkat cells, TAM-67 efficiently inhibited endogenous AP-1 transcriptional activity and blocked the activity of a reporter construct containing the 5' regulatory region of the IL-2 gene. TAM-67 also inhibited the transcriptional activity of nuclear factor-AT (NF-AT), whereas the NF-kappa B, NF-IL-2A, and the proximal TRE-like sites were relatively unaffected. The use of this dominant negative transcription factor suggests that: 1) transactivation-defective nuclear factors represent a novel approach to study the functional consequences of nuclear protein interactions on gene transcription; 2) the proximal TRE-like site from the IL-2 promoter is different from the consensus TRE; and 3) AP-1 plays an important role in the transcriptional activation mediated by the NF-AT binding complex.

Base Sequence↗
PubMed1992

Role of Ly-6A/E and T cell receptor-zeta for IL-2 production. Phosphatidylinositol-anchored Ly-6A/E antagonizes T cell receptor-mediated IL-2 production by a zeta-independent pathway.

Ly-6A/E molecules were originally implicated in regulation of T cell activation because anti-Ly-6A/E mAb induce IL-2 production. More recently we have shown that anti-Ly-6A/E also inhibits IL-2 production induced by anti-CD3. In the present study we used mutant and transfected cell lines that varied in expression of Ly-6A/E or TCR-zeta to test whether the positive and negative modulations of IL-2 production by anti-Ly-6A/E occur by distinct mechanisms. Anti-Ly-6A/E inhibited anti-CD3-induced IL-2 production for Ly-6E.1-transfected EL4J cells, but did not affect IL-2 production of the parental Ly-6A/E-negative EL4J cells. These results indicate that TCR-mediated IL-2 production can occur in the absence of Ly-6A/E expression and establish that anti-Ly-6A/E-induced inhibition of IL-2 production was the result of antibody binding to Ly-6A/E. As expected, MA5.8 (zeta-negative) or CT108 (zeta-truncated) variants of the 2B4.11 T cell hybridoma did not produce IL-2 when stimulated with anti-Thy-1 or anti-Ly-6A/E mAb. In contrast, anti-Ly-6A/E inhibited anti-CD3-induced IL-2 production by MA5.8 and CT108. Furthermore, anti-Ly-6A/E-induced IL-2 production was restored for zeta-transfected MA5.8. Thus, although induction of IL-2 by anti-Ly-6A/E depends on zeta expression, inhibition of IL-2 by anti-Ly-6A/E occurs by a zeta-independent mechanism. Interestingly, anti-Ly-6A/E, but not anti-Thy-1, inhibited anti-CD3-induced IL-2 production by MA5.8 and Ly-6E.1-transfected EL4J. Therefore, inhibition of IL-2 production by anti-Ly-6A/E was not a general property of a mAb binding to a phosphatidylinositol-linked molecule, as has been suggested for induction of IL-2 production. Taken together these data suggest that the molecular mechanisms of induction and inhibition of IL-2 production by anti-Ly-6A/E are separable and expression of TCR-zeta is one variable that distinguishes these two pathways.

Animals↗
PubMed1991

Thymocyte activation and death: a mechanism for molding the T cell repertoire.

The programmed death of thymocytes and T cells was studied. Injection of anti-TCR antibodies into adult mice caused the specific deletion of CD4+ CD8+ thymocytes, an effect that was largely reversed by cyclosporin A. Surprisingly, using either anti-TCR antibodies or superantigens, it was found that the susceptibility of these thymocytes to clonal deletion changed during ontogeny. Double positive thymocytes from newborn and young (3 week old) mice were readily depleted, whereas thymocytes from 1 week old mice were relatively refractory. The differences between these groups could not be accounted for by cell surface TCR expression, TCR-mediated early signal transduction pathways such as phosphoinositide hydrolysis or Ca2+ mobilization, or differences in susceptibility to Dex- or ionomycin-induced programmed cell death. These results suggest that there is a relatively synchronous wave of maturing thymocytes that are susceptible to deletional signals during fetal life and shortly after birth, but not 7 days after birth. By 3 weeks of age, the next wave (or waves) of susceptible cells have populated the thymus. These observations closely follow the experimental model known as "neonatal tolerance," and we suggest that the failure to tolerize 1 week old mice in that system reflects an alteration in the cells' susceptibility to clonal deletion. In a separate set of experiments exploring the mechanisms of PCD, it was found that although the activation- and glucocorticoid-induced PCD pathways were distinct (being distinguishable by their sensitivity to CsA and the glucocorticoid antagonist RU-486), they were mutually antagonistic. Attempts to identify the level of the antagonism failed to demonstrate any direct interference between the two stimuli, up to and including the transcription and translation of a GRE-controlled reporter gene. Based upon these observations, we propose the following model of thymocyte development: glucocorticoids eliminate thymocytes with little or no avidity for self; antagonism between glucocorticoids and cellular activation allows thymocytes that recognize self with low or moderate avidity to survive (positive selection); activation of thymocytes that recognize self with high avidity dominates the antagonistic effect of glucocorticoids, leading to PCD (negative selection).

Animals↗
PubMed1991

Variations in thymocyte susceptibility to clonal deletion during ontogeny. Implications for neonatal tolerance.

Activation of immature thymocytes via the TCR results in programmed cell death and clonal deletion. We have examined thymocytes from mice of different ages and observed that, whereas TCR-mediated signaling caused deletion of thymocytes from newborn and 3-week-old mice, it failed to delete thymocytes from mice of 1 week of age. This could not be attributed to differences in cell surface TCR expression, TCR-mediated phosphoinositide hydrolysis or Ca2+ mobilization, or total cellular levels of TCR zeta- and eta-chains. Moreover, thymocytes of all ages were equally susceptible to corticosteroid- and Ca2+ ionophore-induced programmed cell death. These data are consistent with the notion that fetal and neonatal thymocytes represent a relatively synchronous wave of cells passing through phases in which they are susceptible and then resistant to TCR-induced programmed cell death. They also support the notion that the classical phenomenon of neonatal tolerance is due to clonal deletion and that the inability of allogeneic cells to tolerize mice at 1 week of age is because the thymocytes are refractory to TCR-alpha beta-mediated clonal deletion.

Age Factors↗
PubMed1990

Programmed T lymphocyte death. Cell activation- and steroid-induced pathways are mutually antagonistic.

Both cellular activation signals and exposure to glucocorticoids such as dexamethasone (Dex) cause programmed cell death in T cell hybridomas. When cells were activated in the presence of Dex, however, the degree of killing that was achieved by either stimulus alone was markedly reduced. Dex-induced programmed cell death of normal T cell clones was also prevented by cellular activation. Cyclosporin A (CsA) completely blocked the activation-induced death of T cell hybridomas, but actually enhanced the killing caused by Dex. The addition of CsA to activated T cell hybridomas in the presence of Dex allowed killing to proceed, consistent with ability of CsA to block activation-induced nuclear gene transcription. A number of independent approaches were used to explore the effect of activation on the glucocorticoid signaling/effector pathway. First, RU-486, which binds the glucocorticoid receptor and is a potent competitive antagonist of Dex, did not inhibit activation-induced cell killing. Second, activation of T cell hybridomas did not cause the translocation of the glucocorticoid receptor from the cytoplasm to the nucleus, nor did it prevent the receptor translocation induced by treatment with Dex. Finally, T cell hybridomas were transfected with a plasmid containing the chloramphenicol acetyltransferase (CAT) gene under the control of two tandemly arranged glucocorticoid-responsive elements. Activation of these cells did not induce CAT activity, and did not inhibit the CAT activity induced by Dex. In fact, there was a paradoxical increase in CAT activity when cells were treated with both stimuli. We conclude that cellular activation does not directly utilize the glucocorticoid receptor nor the glucocorticoid pathway when inducing programmed cell death. Furthermore, the ability of activation to inhibit Dex-mediated killing is not due to interference with the classical glucocorticoid signaling pathway, up to and including the initiation of gene transcription. Alternative mechanisms of antagonism, as well as the possible relevance of this phenomenon to the positive selection of self-recognizing thymocytes, are discussed.

Animals↗
PubMed1983

Macrophage-mediated cytotoxicity: role of a soluble macrophage cytotoxic factor similar to lymphotoxin and tumor necrosis factor.

Guinea pig peritoneal macrophages, when activated for cytotoxicity by the calcium ionophore A23187 or lipopolysaccharide, produce a cytotoxic factor [macrophage cytotoxic factor (M phi-CF)] that is not blocked by catalase or protease inhibitors. Fractionation of culture supernates containing M phi-CF by gel filtration revealed one peak of cytotoxic activity of Mr approximately 45,000, the same as guinea pig lymphotoxin (LT). Antiserum prepared against purified guinea pig LT completely neutralized the cytotoxic activity of M phi-CF. In addition, the cytotoxic factor in guinea pig tumor necrosis serum was found to have a Mr of 45,000 and was neutralized by anti-LT. Thus, M phi-CF is physicochemically and immunochemically similar to LT and tumor necrosis factor, if not identical. To investigate the role of M phi-CF in macrophage-mediated cytotoxicity, anti-LT was added to A23187- or lipopolysaccharide-activated macrophages before addition of L-929 target cells. In 10 of 16 experiments, the inhibition of macrophage-mediated cytotoxicity was 100%. In the others, cytotoxicity was blocked partially, the lowest inhibition being 49%. The effectiveness of inhibition appeared to be inversely related to the intensity of macrophage activation. These results indicate that M phi-CF plays a significant role in macrophage-mediated cytotoxicity but involvement of another mechanism cannot be excluded.

Animals↗