L-GILZ binds p53 and MDM2 and suppresses tumor growth through p53 activation in human cancer cells.

The transcription factor p53 regulates the expression of genes crucial for biological processes such as cell proliferation, metabolism, cell repair, senescence and apoptosis. Activation of p53 also suppresses neoplastic transformations, thereby inhibiting the growth of mutated and/or damaged cells. p53-binding proteins, such as mouse double minute 2 homolog (MDM2), inhibit p53 activation and thus regulate p53-mediated stress responses. Here, we found that long glucocorticoid-induced leucine zipper (L-GILZ), a recently identified isoform of GILZ, activates p53 and that the overexpression of L-GILZ in p53(+/+) HCT116 human colorectal carcinoma cells suppresses the growth of xenografts in mice. In the presence of both p53 and MDM2, L-GILZ binds preferentially to MDM2 and interferes with p53/MDM2 complex formation, making p53 available for downstream gene activation. Consistent with this finding, L-GILZ induced p21 and p53 upregulated modulator of apoptosis (PUMA) expression only in p53(+/+) cells, while L-GILZ silencing reversed the anti-proliferative activity of dexamethasone as well as expression of p53, p21 and PUMA. Furthermore, L-GILZ stabilizes p53 proteins by decreasing p53 ubiquitination and increasing MDM2 ubiquitination. These findings reveal L-GILZ as a regulator of p53 and a candidate for new therapeutic anti-cancer strategies for tumors associated with p53 deregulation.

IL-2 induces and altered CD4/CD8 ratio of splenic T lymphocytes from transgenic mice overexpressing the glucocorticoid-induced protein GILZ.

We used transgenic mice to investigate the effect of IL-2 stimulation on T lymphocyte functions of GILZ-overexpressing splenic T cells. When compared to their controls, T cells from transgenic mice underwent normal activation after stimulation with anti-CD3 plus anti-CD28 monoclonal antibodies, as evaluated by CD25 expression, CD2 up-regulation and proliferation. IL-10, IL-13 and IFN-gamma increased more consistently in CD3/CD28-triggered TG compared to WT splenic CD4(+)cells. Analysis of the CD4(+)and CD8(+)T cells demonstrated a decreased CD4(+)/CD8(+)T-cell ratio (1:1 instead of 1:2) in response to IL-2 stimulation, possibly due to an unresponsiveness of IL-2 receptor beta and/or gamma chains. Finally, the total number of T cells was significantly increased in aged mice and this was due to the augmentation of CD4(+)T cells. These results support the hypothesis that GILZ regulates, at least in part, peripheral T-cell functions by influencing their responsiveness to IL-2.

Modulation of T-cell activation by the glucocorticoid-induced leucine zipper factor via inhibition of nuclear factor kappaB.

Previously a novel gene was identified that encodes a glucocorticoid-induced leucine zipper (GILZ) whose expression is up-regulated by dexamethasone. This study analyzed the role of GILZ in the control of T-cell activation and its possible interaction with nuclear factor kappaB (NF-kappaB). Results indicate that GILZ inhibits both T-cell receptor (TCR)-induced interleukin-2/interleukin-2 receptor expression and NF-kappaB activity. In particular, GILZ inhibits NF-kappaB nuclear translocation and DNA binding due to a direct protein-to-protein interaction of GILZ with the NF-kappaB subunits. Moreover, GILZ-mediated modulation of TCR-induced responses is part of a circuit because TCR triggering down-regulates GILZ expression. These results identify a new molecular mechanism involved in the dexamethasone-induced regulation of NF-kappaB activity and T-cell activation. (Blood. 2001;98:743-753)

Glucocorticoid hormones in the regulation of cell death.

The immune T-cell compartment maintains the capability to respond to a wide variety of antigens (Ag). This whole process is regulated by lymphocyte apoptosis (programmed cell death, PCD) and involves the coordinated expression of a great number of genes including those coding for cytokines and their receptors, such as for example IL-2/IL-2R and the Fas/FasL systems and those coding for transcription factors, including the NF-kB complex, involved in T-cell activation and apoptosis in that they simultaneously activate cell suicide and an anti-death programme. This binary effect, PCD activation and inhibition, is due on one hand to GCH-induced activation of the caspases cascade and on the other to the induction of expression of a new gene that we have named GILZ. In fact, GILZ over-expression in transfected cells inhibits the sequential increase of NF-kB/DNA-binding activity, IL-2 production and IL-2R expression, and transcription of the Fas/FasL complex that follows TCR triggering and plays an important role in the control of T-lymphocyte apoptosis. These results indicate a new mechanism responsible for the GCH-mediated inhibition of T-cell death and activation that could contribute to anti-inflammatory and immunosuppressive efficacy.

Cloning and expression of a short Fas ligand: A new alternatively spliced product of the mouse Fas ligand gene.

The Fas/FasL system mediates apoptosis in several different cell types, including T lymphocytes. Fas ligand (FasL), a 40-kD type II membrane protein also expressed in activated T cells, belongs to the tumor necrosis factor ligand family. We describe a new alternative splicing of mouse FasL, named FasL short (FasLs), cloned by reverse transcriptase-polymerase chain reaction. FasLs is encoded by part of exon 1 and part of exon 4 of FasL gene. The protein encoded by FasLs mRNA has a putative initiation code at position 756 and preserves the same reading frame as FasL, resulting in a short molecule lacking the intracellular, the transmembrane, and part of the extracellular domains. RNase protection and immunoprecipitation analysis showed that FasLs is expressed in nonactivated normal spleen cells and in hybridoma T cells and that it is upregulated upon activation by anti-CD3 monoclonal antibody (MoAb). Moreover, FasLs-transfected cells expressed soluble FasLs in the supernatant and became resistant to apoptosis induced by agonist anti-Fas MoAb. Thus, FasLs, a new alternative splicing of FasL, is involved in the regulation of Fas/FasL-mediated cell death.

Interleukin-6 (IL-6) prevents activation-induced cell death: IL-2-independent inhibition of Fas/fasL expression and cell death.

Triggering of the TCR/CD3 complex with specific antigen or anti-CD3 monoclonal antibody initiates activation-induced cell death (AICD) in mature T cells, an effect also mediated by the Fas/FasL system. We have previously shown that CD2 stimulation rescues T cells from TCR/CD3-induced apoptosis by decreasing the expression of Fas and FasL. In the present study, we examined whether the endogenous production of IL-2 plays a role in the effects mediated by CD2 triggering. The results indicated that transcription of Fas/FasL is controlled by interleukin-2 (IL-2) production and that CD2 triggering rescues a T-cell hybridoma from AICD via decreased production of IL-2. To ascertain whether modulation of IL-2 may be a general mechanism of AICD control, we examined other stimuli, capable of modulating the expression of the Fas/FasL system and the ensuing AICD, for ability to affect production of IL-2. We found that IL-6 reduced the level of TCR/CD3-induced apoptosis and the expression of Fas/FasL, yet failed to inhibit IL-2 production. Because IL-2 is involved in both apoptosis and activation events, these results indicate that, in contrast to CD2, which inhibits apoptosis and T cell activation, IL-6 inhibits apoptosis but not IL-2-induced activation. These observations may provide the basis for differential control of T-cell activation and apoptosis.

CD2 rescues T cells from T-cell receptor/CD3 apoptosis: a role for the Fas/Fas-L system.

Anti-CD3 monoclonal antibodies (MoAbs) and glucocorticoid hormones induce apoptosis in immature thymocytes and peripheral T lymphocytes. This process is inhibited by a number of growth factors, including interleukin-2 (IL-2), IL-3, and IL-4, as well as by triggering of the adhesion molecule CD44, which would indicate that signals generated by membrane receptors can modulate the survival of lymphoid cells. To investigate whether triggering of CD2 may also affect apoptosis in lymphoid cells, we analyzed the effect of stimulation with anti-CD2 MoAbs on T-cell apoptosis induced by two stimuli, anti-CD3 MoAbs and dexamethasone (DEX), using a hybridoma T-cell line and a T-helper cell clone. The results show that CD2 engagement decreased anti-CD3 MoAb-induced apoptosis, but did not influence DEX-induced cell death. Furthermore, the decrease appeared to be related to the expression of Fas/APO-1 (CD95) and Fas-ligand (Fas-L). In fact, we show that CD2 stimulation inhibits apoptosis by preventing the CD3-induced upregulation of Fas and Fas-L in a Fas-dependent experimental system. These data suggest that a costimulatory molecule may control a deletion pathway and may therefore contribute to the regulation of peripheral tolerance.

A new dexamethasone-induced gene of the leucine zipper family protects T lymphocytes from TCR/CD3-activated cell death.

By comparing mRNA species expressed in dexamethasone (DEX)-treated and untreated murine thymocytes, we have identified a gene, glucocorticoid-induced leucine zipper (GILZ), encoding a new member of the leucine zipper family. GILZ was found expressed in normal lymphocytes from thymus, spleen, and lymph nodes, whereas low or no expression was detected in other nonlymphoid tissues, including brain, kidney, and liver. In thymocytes and peripheral T cells, GILZ gene expression is induced by DEX. Furthermore, GILZ expression selectively protects T cells from apoptosis induced by treatment with anti-CD3 monoclonal antibody but not by treatment with other apoptotic stimuli. This antiapoptotic effect correlates with inhibition of Fas and Fas ligand expression. Thus, GILZ is a candidate transcription factor involved in the regulation of apoptosis of T cells.

Modulation of superantigen-induced T-cell deletion by antibody anti-Pgp-1 (CD44).

We examined the effects of anti-Pgp-1 (CD44) antibody on the in vitro deletion of murine CD4 and CD8 single positive T cells induced by Staphylococcal enterotoxin B (SEB). Soluble anti-Pgp-1 antibody enhanced the apoptosis and decreased the proliferation of SEB-responding T cells. In contrast, cross-linked anti-Pgp-1 antibody provided costimulatory signals for the T-cell activation induced by anti-CD3 antibody. Hyaluronic acid (HA), a ligand of Pgp-1, did not affect proliferation and deletion induced by SEB, whereas it mimicked the effects of the cross-linked antibody in anti-CD3-driven proliferation. T-cell Pgp-1 surface expression after 48 hr incubation with SEB was unchanged as compared to unstimulated cells. However, when the memory T cells were established, some V beta 8+ (SEB-specific) T cells Pgp-1low became Pgp-1high, displaying a bimodal character. Moreover, the Pgp-1 increased expression correlated with an increase of Pgp-1 soluble form in the supernatant. These findings suggested that signals following the triggering of the Pgp-1 molecule are important in controlling T-cell survival.

CD44 (Pgp-1) inhibits CD3 and dexamethasone-induced apoptosis.

Anti-CD3 monoclonal antibodies (MoAbs) and glucocorticoid hormones (GCH) induce apoptosis in immature thymocytes and peripheral T lymphocytes. This process is inhibited by a number of growth factors, including interleukin-2 (IL-2), IL-3, and IL-4, indicating that signals generated by membrane receptors can modulate the survival of lymphoid cells. To investigate whether signals activated by adhesion receptors have a similar activity, we analyzed the effect of CD44 (Pgp-1) adhesion molecule receptor stimulation on T-cell apoptosis induced by three stimuli (anti-CD3 MoAbs, dexamethasone [DEX] treatment, and exposure to ultraviolet irradiation [UV]) on a 3DO T-cell line. The results show that CD44 engagement, either by hyaluronic acid (HA) or anti-CD44 MoAbs, inhibits DNA fragmentation and apoptosis induced by DEX and anti-CD3 MoAbs, whereas that induced by UV, a p53-dependent phenomenon, was not inhibited. Furthermore, the antiapoptotic effect exerted through CD44 activation does not seem related to overexpression of bcl-2 or to have appreciable effects on cell proliferation. Our results indicate that adhesion molecules modulate T-cell survival by counteracting apoptosis induced by DEX or anti-CD3 MoAbs.

Superantigen-induced peripheral T-cell deletion: the effects of chemical modification of antigen-presenting cells, interleukin-4 and glucocorticoid hormones.

Experiments were performed to evaluate the role of antigen-presenting cells (APC) and the effect of interleukin-4 (IL-4) and glucocorticoid hormone (GCH) exposure on the in vitro deletion of CD4+ CD8- and CD8+ CD4- T cells by staphylococcal enterotoxin B (SEB). APC fixation with the chemical cross-linker 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide (ECDI) inhibited their capacity to induce SEB-specific deletion of mature T lymphocytes. Deletion was not influenced by treatment with anti-CD28 antibodies, which modulate T-cell activation. However, it was augmented by IL-4, known to counteract anti-CD3- and GCH-induced thymocyte apoptosis, and was inhibited by dexamethasone (DEX). These results indicate that metabolically active APC are required for deletion of antigen-specific mature T cells and suggest that IL-4 and GCH can modulate this phenomenon in vitro.

PMA inhibits NK cell generation, cytotoxic activity and NK-1.1 expression.

We investigated the role of protein kinase C activator phorbol 12-myristate 13-acetate (PMA) on IL-2-driven NK cell differentiation, by using an in vitro model previously set up by our laboratory. Bone marrow precursor cells, from mice treated with 5-fluorouracil (FUBM), when cultured with IL-2, generated mature NK cells. The biochemical system involved in this process has not yet been defined. We investigated the possible mechanism by analyzing the effect of PCK activator PMA on NK cell differentiation and lytic activity of mature NK cells. We now report that: (1) PMA inhibited the IL-2-induced NK cell differentiation and induced development of cells which lyse the NK-resistant target P815. (2) PMA inhibited the lytic ability of mature NK cells against NK-sensitive target YAC-1. We evaluated the effects of PMA using the expression of NK-associated antigen NK-1.1 and the ability to lyse YAC target as parameters of NK cell differentiation. PMA down-regulated both these parameters, reducing their expression during the differentiation process of NK cells and inducing down-modulation of these in mature NK cells. The results suggest that PKC regulatory control could be under the process of differentiation and activation of NK cells.

Long-term cultures of mouse bone marrow cells: a model for studying the generation of natural killer cells.

We investigated the generation of natural killer (NK) cells, using a long-term bone marrow culture (LTBMC) system. Mouse bone marrow cells were cultured for 2 weeks in complete medium without growth factors to obtain an enriched population of NK precursor cells. When these cells were recultured in the presence of interleukin-2 (IL-2) and conditioned medium (CM) from LTBMC, lytic NK cells were generated within 4 days. Replacing CM with fresh medium, before adding IL-2, decreased NK cell generation markedly, suggesting that endogenous factors present in CM were necessary for IL-2 induction of NK cells. NK cell precursors were also cultured with a combination of IL-2 and interferon-gamma (IFN-gamma) or IL-2 and tumor necrosis factor-alpha (TNF-alpha), but no CM. Results show that IFN-gamma and TNF-alpha were able to substitute CM. The addition of anti-IFN-gamma or anti-TNF-alpha antibodies to LTBMC cells, cultured in the presence of IL-2 and CM, inhibited cytotoxicity induction in a dose-dependent manner. The data indicate that IFN-gamma and TNF-alpha production may be required for IL-2 induction of NK activity, and are consistent with the hypothesis that NK generation involves collaboration between IL-2 and other bone marrow microenvironmental growth factors.

Effect of recombinant murine tumor necrosis factor on the generation of natural killer cells in bone marrow cultures.

We investigated the possible role of tumor necrosis factor-alpha (TNF-alpha) in the interleukin-2 (IL-2)-dependent generation of natural killer (NK) cells from bone marrow precursors. TNF-alpha synergistically augmented both cytotoxic activity against NK-sensitive targets and cell number at the end of the 7-day incubation period. After this time, NK activity was not induced by TNF-alpha in the absence of IL-2. The cytotoxic cells generated by IL-2 + TNF-alpha had the phenotype of mature NK cells, including expression of NK-1.1, asialo-GM1, Ly-5, LFA-1 and Thy-1. TNF-alpha was also able to up-regulate the mRNA expression for the IL-2 receptor alpha-chain (P55) as well as the mRNA expression of c-myc protooncogene. Blocking studies with monoclonal antibodies against the alpha-chain P55 of the IL-2 receptor confirmed the functional role ascribed to IL-2 in the in vitro generation of NK cells from bone marrow cultures. Additional proliferation studies demonstrated that the up-regulation of c-myc protooncogene was associated with an increased uptake of thymidine. These data indicate that the TNF-alpha-induced increase of IL-2-dependent NK cell generation from bone marrow precursors was associated with an augmented proliferation and an up-regulation of mRNA expression for IL-2 receptor and c-myc protooncogene.

Natural killer (NK) cell generation in bone marrow cultures: role of IL-1 alpha.

Previous studies have demonstrated that IL-2 is able to induce the development of NK cells from bone marrow (BM) cultures, and that other cytokines acted synergistically with IL-2 in determining an increase of NK cells development. The addition of TNF alpha greatly enhanced the IL-2-mediated induction of NK effector. However, the effect of IL-2 and TNF alpha could be due to direct stimulation of NK progenitors, or to the endogenous production of other factors, which are then responsible of the development of NK cells. As results show that the mRNA specific for IL-1 alpha could be detected in BM cells cultured with IL-2, but not in that supplemented with IL-2 + TNF alpha, it would seem that this lymphokine plays a role only in IL-2-dependent development of NK cells. Studies with Ab anti-IL-1 alpha, showed that the antibody abrograted the IL-2-driven generation of NK cells, but did not affect the NK differentiation induced by IL-2 + TNF alpha. The cytotoxic cells generated by IL-2 or by IL-2 + TNF alpha had the phenotype of mature NK cells including expression of NK 1.1, asialo GM1, Lyt-5, LFA-1, and Thy-1. These data suggest that in spite of phenotypical and morphological similarity of the cells generated with IL-2 or IL-2 + TNF alpha, the endogenous production of IL-1 alpha, appears functionally important only for the differentiation of NK cells induced by IL-2 alone.

Effect of a new peptidyl-hypoxanthine derivative on natural killer cells and antitumor activity.

A new immunomodifier, [omega-(hypoxanthin-9-yl) pentoxy-carbonyl-leucyl-methionine] (RM06), was synthesized and its effect was evaluated on the activity of Natural Killer (NK) cells. Results indicate that RM06 is able to boost the NK activity of normal mice as well as to augment the regeneration of NK activity of lethally irradiated mice transplanted with syngeneic bone marrow (BM). This later effect also correlated with a significant increase in anti-tumor activity as evaluated by the resistance to metastasis in mice injected with syngeneic melanoma cells. These data indicate that RM06 is able to modulate the NK cell activity as well as the antitumor resistance.

In vivo effects of cytokines on development of natural killer cells and antitumor activity in lethally irradiated bone marrow transplanted recipients.

We have evaluated the effects of combinations of various cytokines on the reconstitution of natural killer (NK) cell activity and resistance to metastases from B16 melanoma, in lethally irradiated mice transplanted with syngeneic bone marrow. Treatment with some combinations of interleukin-2 (IL-2) and other cytokines (IL-2 + IL-1 + TNF alpha or IL-2 + IL-1 + LT) induced appreciably greater and more rapid augmentation of NK cell regeneration than IL-2 alone, as measured in vitro in the 4-h 51Cr release assay against YAC-1 or in vivo in an assay of lung clearance of 125IUdR-labeled tumor cells. The same treatments also induced significant augmentation of in vivo resistance against pulmonary metastases in C57BL/6 mice injected with B16 melanoma cells. These data indicate that stimulation of NK activity in tumor-bearing bone marrow transplanted recipients may be of value in the control of metastatic disease.