GRIM-19 associates with the serine protease HtrA2 for promoting cell death.

We have isolated a novel interferon (IFN)-retinoid regulated cell death regulatory protein genes associated with retinoid-IFN-induced mortality (GRIM)-19 earlier. To understand its mechanism of action, we have employed a yeast-two-hybrid screen and identified serine protease HtrA2 as its binding partner. GRIM-19 physically interacts with HtrA2 and augments cell death in an IFN/all-trans retinoic acid (RA)-dependent manner. In the presence of GRIM-19, the HtrA2-driven destruction of the antiapoptotic protein X-linked inhibitor of apoptosis (XIAP) is augmented. These interactions were disrupted by an human herpes virus-8 (HHV-8)-coded oncoprotein, vIRF1, and conferred resistance to IFN/RA-induced cell death. These data show a critical role of HtrA2 in a cytokine-induced cell death response for the first time and its inhibition by a viral protein.

A proteomic analysis reveals the loss of expression of the cell death regulatory gene GRIM-19 in human renal cell carcinomas.

Gene associated with retinoid interferon-induced mortality (GRIM)-19, an inhibitor of transcription factor STAT3, was originally identified as a critical regulatory protein in a genetic screen that was designed to identify the gene products necessary for Interferon (IFN)-beta- and retinoic acid-induced cell death. Over expression of GRIM-19 activates cell death. Conversely, inactivation of its expression promotes cell growth. STAT3 is a transcription factor that regulates gene expression in response to multiple extra cellular growth factors. In contrast to its normal feedback inhibition, a constitutive activation of STAT3 has been documented in several tumors. Although many STAT3-inhibitors are described, their relevance to human cancer is unclear. In an attempt to define the molecular alterations associated with human renal cell carcinoma (RCC) using mass spectrometry, we have discovered that expression of GRIM-19 is lost or severely depressed in a number of primary RCC and in some urinogenital tumors. Using an RCC cell line, we show that down regulation of GRIM-19 promotes tumor growth via an augmentation of STAT3-dependent gene expression. These studies for the first time show a tumor-suppressor like activity of GRIM-19.

Transcription factor GATA-4 regulates cardiac muscle-specific expression of the alpha-myosin heavy-chain gene.

The alpha-myosin heavy-chain (alpha-MHC) gene is the major structural protein in the adult rodent myocardium. Its expression is restricted to the heart by a complex interplay of trans-acting factors and their cis-acting sites. However, to date, the factors that have been shown to regulate expression of this gene have also been found in skeletal muscle cells. Recently, transcription factor GATA-4, which has a tissue distribution limited to the heart and endodermally derived tissues, was identified. We recently found two putative GATA-binding sites within the proximal enhancer of the alpha-MHC gene, suggesting that GATA-4 might regulate its expression. In this study, we establish that GATA-4 interacts with the alpha-MHC GATA sites to stimulate cardiac muscle-specific expression. Mutation of the GATA-4-binding sites either individually or together decreased activity by 50 and 88% in the adult myocardium, respectively. GATA-4-dependent enhancement of activity from a heterologous promoter was mediated through the alpha-MHC GATA sites. Coinjection of an alpha-MHC promoter construct with a GATA-4 expression vector permitted ectopic expression in skeletal muscle but not in fibroblasts. Thus, the lack of alpha-MHC expression in skeletal muscle correlates with a lack of GATA-4. GATA-4 DNA binding activity was significantly up-regulated in triiodothyronine- or retinoic acid-treated cardiomyocytes. Putative GATA-4-binding sites are also found in the regulatory regions of other cardiac muscle-expressed structural genes. This indicates a mechanism whereby triiodothyronine and retinoic acid can exert coordinate control of the cardiac phenotype through a trans-acting regulatory factor.

Gene induction by interferons: functional complementation between trans-acting factors induced by alpha interferon and gamma interferon.

HeLaM is a variant cell line in which the transcriptional induction of many genes by alpha interferon has special characteristics (Tiwari et al., Mol. Cell. Biol. 8:4289-4294, 1988). The same characteristics were also displayed for induced transcription of a permanently transfected chimeric gene containing the interferon-stimulated response element of gene 561. For understanding the molecular basis of the special requirements of HeLaM cells, an analysis of the interferon-stimulated gene factors (ISGF) was undertaken. By using gel shift assays, it was shown that the activation of ISGF3 by alpha interferon treatment of HeLaM cells had characteristics identical to those of induced transcription: inhibition by 2-aminopurine and the need for ongoing protein synthesis which was obviated by pretreating the cells with gamma interferon. Upon mixing in vitro the cytoplasmic fraction of gamma interferon-treated HeLaM cells with that of cells treated with alpha interferon and cycloheximide, active ISGF3 was reconstituted, presumably through complementation of two components, ISGF3 gamma and ISGF3 alpha, present in the two respective fractions. Because, unlike other cells, untreated HeLaM cells did not contain detectable levels of either component, we could induce them individually and study their independent properties. Induction of ISGF3 gamma but not of ISGF3 alpha needed ongoing protein synthesis and was blocked by 2-aminopurine. Once induced, ISGF3 gamma was active for 24 h and was present in both the nuclear and cytoplasmic fractions. Activated ISGF3 alpha, on the other hand, did not translocate to the nucleus in the absence of ISGF3 gamma, and in the cytoplasm its activity decayed within 2 h of its activation. In reference to our working model, all of the above observations indicate that ISGF3 gamma is the product of signal 1 and ISGF3 alpha is the product of signal 2.

Thioredoxin reductase mediates cell death effects of the combination of beta interferon and retinoic acid.

Interferons (IFNs) and retinoids are potent biological response modifiers. By using JAK-STAT pathways, IFNs regulate the expression of genes involved in antiviral, antitumor, and immunomodulatory actions. Retinoids exert their cell growth-regulatory effects via nuclear receptors, which also function as transcription factors. Although these ligands act through distinct mechanisms, several studies have shown that the combination of IFNs and retinoids synergistically inhibits cell growth. We have previously reported that IFN-beta-all-trans-retinoic acid (RA) combination is a more potent growth suppressor of human tumor xenografts in vivo than either agent alone. Furthermore, the IFN-RA combination causes cell death in several tumor cell lines in vitro. However, the molecular basis for these growth-suppressive actions is unknown. It has been suggested that certain gene products, which mediate the antiviral actions of IFNs, are also responsible for the antitumor actions of the IFN-RA combination. However, we did not find a correlation between their activities and cell death. Therefore, we have used an antisense knockout approach to directly identify the gene products that mediate cell death and have isolated several genes associated with retinoid-IFN-induced mortality (GRIM). In this investigation, we characterized one of the GRIM cDNAs, GRIM-12. Sequence analysis suggests that the GRIM-12 product is identical to human thioredoxin reductase (TR). TR is posttranscriptionally induced by the IFN-RA combination in human breast carcinoma cells. Overexpression of GRIM-12 causes a small amount of cell death and further enhances the susceptibility of cells to IFN-RA-induced death. Dominant negative inhibitors directed against TR inhibit its cell death-inducing functions. Interference with TR enzymatic activity led to growth promotion in the presence of the IFN-RA combination. Thus, these studies identify a novel function for TR in cell growth regulation.

Mechanism of repression of squamous differentiation marker, SPRR1B, in malignant bronchial epithelial cells: role of critical TRE-sites and its transacting factors.

The overexpression of SPRR1B in bronchial epithelium is a marker for early metaplastic changes and the loss of its expression is associated with an irreversible malignant transformation. In the present study, we have used a model system consisting of normal and malignant bronchial epithelial (BE) cells to elucidate the differential transcriptional control of SPRR1B. SPRR1B expression is either detectable or PMA (phorbol 13-myristate 12-acetate) -inducible in several malignant BE cells including squamous, adeno, small and large cell carcinomas. Loss of SPRR1B expression is correlated well with the lack of strong in vivo protein-DNA interactions at the -152 bp promoter, which contains two functional TRE sites. Even though the basal level AP-1 protein DNA binding pattern is different between normal and malignant cells, PMA significantly enhances Jun and Fos binding to the consensus TRE site in both cell types. Intriguingly, the composition of AP-1 protein binding to the -152 to -86 bp SPRR1B promoter is quite different. In untreated cells, SPRR1B promoter is predominantly occupied by JunD and Fra2. PMA significantly induced binding of JunB and Fra1 in normal cells, while JunB and Fra2 bound to TREs in the malignant cells. Overexpression of fra1 in malignant cells significantly enhanced SPRR1B promoter activity. In contrast, overexpression of fra2, but not fra1, strongly reduced both basal and PMA-inducible promoter activities in normal cells. Together, these results indicate that either temporal expression and/or differential activation of AP-1 proteins, especially Fra1 and Fra2, might contribute to the dysregulation of terminal differentiation marker, SPRR1B, expression in various BE cells.

Thioredoxin participates in a cell death pathway induced by interferon and retinoid combination.

Interferons (IFNs) and retinoids are potent tumor growth suppressors. We have shown earlier that the IFN-beta and all-trans retinoic acid combination, but not the single agents, induces death in several tumor cell lines. Employing a genetic approach we have recently identified several Genes associated with Retinoid-IFN induced Mortality (GRIM) that mediate the cell death effect of IFN/RA combination. One of the GRIMs, GRIM-12, was identical to human thioredoxin reductase (TR), an enzyme that controls intracellular redox state. To define the participants of TR mediated death pathway we have examined the role of thioredoxin (Trx), its downstream substrate, and its influence on IFN/RA-induced death regulation. Inhibition of the thioredoxin expression by antisense RNA suppressed cell death. Similarly, a mutant Trx1 lacking the critical cysteine residues blocked cell death. In contrast, overexpression of wildtype thioredoxin augmented cell death. This effect of Trx1 was in part due to its ability to augment cell death via caspase-8. The redox inactive Trx1 mutant inhibits the cell death induced by caspase-8 but not caspase-3. These studies identify a novel mechanism of cell death regulation by IFN/RA combination involving redox enzymes.

Modulation of p53 dependent gene expression and cell death through thioredoxin-thioredoxin reductase by the Interferon-Retinoid combination.

We have shown earlier that the IFN-beta and all-trans retinoic acid (RA) combination, but not the single agents, induces death in several tumor cell lines. Employing a genetic technique we have identified several Genes associated with Retinoid-IFN induced Mortality (GRIM). One of the GRIMs was human thioredoxin reductase (TR), a redox enzyme. Since the overexpressed TR augments IFN/RA stimulated cell death, we explored the mechanisms of TR-mediated death. Here we show that TR augments cell death by upregulating the transcriptional activity of p53 tumor suppressor. This process does not involve a physical increase in levels of p53. Using redox inactive mutants of TR and its substrate, thioredoxin (Trx), we demonstrate that IFN/RA-induced regulation of p53 dependent gene expression requires TR and Trx. In contrast-over-expression of wildtype TR or Trx augment the p53 dependent gene expression in response to IFN/RA treatment. Consistent with these results an increased DNA binding activity of p53 was noted in the presence of TR. These studies identify a novel mechanism of p53 mediated cell death regulation involving redox enzymes.

Virus interception of cytokine-regulated pathways.

Cytokines regulate host antiviral, immune and antitumor responses. Viruses combat the host-imposed inhibitory pathways to survive and spread the infection. Some viruses have evolved molecules that override apoptotic programs to promote cell survival until virus assembly is complete, persistence is established or cellular transformation occurs.

The interferon-beta and tamoxifen combination induces apoptosis using thioredoxin reductase.

Interferons (IFNs) suppress cell growth by inducing cellular genes. The anti-estrogen tamoxifen (Tam), binds to estrogen receptor and inhibits transcription of estrogen stimulated genes. In cells resistant to IFN-induced growth suppression, IFN/Tam combination causes cell death. We previously reported that the combination of IFN-beta and Tam was a more potent growth suppressor of human tumor xenografts than either agent alone. The IFN/Tam combination acts in a manner similar to the IFN/retinoic acid combination. Using a genetic technique, we have recently identified several genes associated with retinoid-IFN-induced mortality (GRIM). One such gene, GRIM-12, was identical to human thioredoxin reductase (TR). In the present study we have examined whether the IFN/Tam combination also requires GRIM-12 for inducing cell death. We report here that GRIM-12 is necessary for mediating the cell death effects of IFN/Tam, and its expression is induced by IFN/Tam at a post-transcriptional stage. Repression of GRIM-12 levels either by antisense expression or by dominant negative inhibitors caused resistance to IFN/Tam induced death and promoted cell growth. Overexpression of GRIM-12 increased IFN/Tam induced apoptosis. Thus, these studies have identified a critical role for GRIM-12 (TR) in apoptosis.

Interleukin-6 modulates interferon-regulated gene expression by inducing the ISGF3 gamma gene using CCAAT/enhancer binding protein-beta(C/EBP-beta).

Although interleukin-6 (IL-6) alone does not induce the expression of IFN stimulated genes (ISG), a low dose priming of cells with IL-6 strongly enhances the cellular responses to interferon-alpha (IFN-alpha). This effect of IL-6 is not due to superstimulation of the JAK-STAT pathway. Rather, IL-6 induces expression of ISGF3 gamma (p48), a subunit of the multimeric transcription factor ISGF3. As a result IFN-alpha robustly activates gene transcription in IL-6 primed cells. We have shown earlier that the transcription of ISGF3 gamma gene is regulated through a novel element GATE (gamma-IFN activated transcriptional element). We show here IL-6 induces the ISGF3 gamma gene through GATE. Transcription factor C/EBP-beta is required for inducing ISGF3 gamma gene expression through GATE. A mutant C/EBP-beta inhibits the IL-6 inducible ISGF3 gamma gene expression through GATE. Together, these results establish a molecular basis for the synergy between IFNs and IL-6.

Synergistic antitumor effects of a combination of interferons and retinoic acid on human tumor cells in vitro and in vivo.

Solid tumors are relatively resistant to growth inhibition by IFNs. To enhance sensitivity, we assessed combinations of IFNs with all-trans-retinoic acid (RA). Antiproliferative studies in vitro suggested that the growth of three human breast carcinomas (MCF-7, MDA-MB-231, and MDA-MB-468), an ovarian carcinoma (NIH-OVCAR-3), and a malignant melanoma (SK-MEL-1) was inhibited to a greater degree by combination treatment with human IFN-beta and RA compared to single agents. Some of these cell lines were resistant to 10-100 IU/ml human IFN-alpha2b or IFN-beta or to 0.1-1.0 microM RA. Growth was inhibited significantly by combinations of IFNs and RA in all cell lines tested, and in some cases, cytotoxicity was observed. Sequential treatment of MCF-7 cells with RA followed by IFN-beta was more effective at inhibiting growth than treatment with IFN-beta followed by RA, suggesting that RA modulated the anticellular response of IFN-beta rather than the converse. In nude mice, the growth of MCF-7 and NIH-OVCAR-3 tumors was suppressed completely when combination treatment was started 2 days after tumor inoculation. Established, 6-week-old NIH-OVCAR-3 tumors underwent regression when treated with the combination of IFN-beta and RA but not with single-agent therapy. Together with our recent studies that demonstrated enhancement of IFN-stimulated gene expression by RA pretreatment in IFN-resistant cells, these data suggest that combination treatment with RA and IFNs may increase IFN-stimulated gene expression in IFN-resistant tumors, leading to augmented antitumor effects.

Retinoic acid and interferon in human cancer: mechanistic and clinical studies.

Various combinations of retinoids, metabolic and synthetic derivatives of vitamin A, and interferons (IFNs) have demonstrated synergistic antiproliferative, differentiating, and antiangiogenic activity in some human hematologic and solid-tumor systems. This synergistic antitumor activity may be due to enhanced gene expression. In several cell systems, the actions of IFNs are enhanced by differentiation of cells with retinoic acid (RA). Combined RA-IFN effects have been correlated with the induction of higher levels of IFN-stimulated genes than the levels induced by either agent alone. Natural and synthetic retinoids have been found to augment the antiproliferative activity of IFNs in several squamous cell carcinoma (SCC) and breast tumor cell lines. Results of recent clinical trials indicate substantial activity of 13-cis-RA (13cRA) combined with IFN against advanced SCC of the skin and cervix, and possibly against other solid tumors. Two phase II trials have confirmed activity against locally advanced SCC of the cervix. Successful integration of this regimen with radiotherapy appears to be the most probable means of optimizing clinical outcome. Further studies are needed to determine the mechanistic details of the RA-IFN interaction.

Induction of interferon synthesis and activation of interferon-stimulated genes by liposomal transfection reagents.

Liposome-mediated transfection is a widely used technique for the introduction of exogenous DNA into mammalian cells. We observed a significant induction of endogenous interferon (IFN)-stimulated genes (ISGs) in cells treated with the liposomal reagents, lipofectamine and DOSPER, in the absence of DNA. Liposome treatment induced expression of reporter constructs driven by IFN-responsive promoter elements, demonstrating a generalized effect on ISG expression. The kinetics of ISG induction were markedly delayed in response to liposome as compared with IFN or double-stranded RNA. ISG induction could be transferred to naive cells with conditioned medium from liposome-treated cells, suggesting that a secreted factor was responsible for this activity. A cell line defective in IFN signaling was refractory to liposome-induced ISG expression, indicating a role for IFN in this induction. Indeed, liposome treatment directly induced IFN-beta gene expression and, thus, represents a novel IFN inducer. IFN induction by liposomal reagents and its potential effects on transgene expression should be considered in the choice of transfection reagent. The ability of liposomal gene delivery reagents to induce IFN synthesis in the host may prove useful in gene therapy approaches to viral and neoplastic diseases.

Chromosomal localization of human GRIM-19, a novel IFN-beta and retinoic acid-activated regulator of cell death.

Apoptosis is a tightly regulated mechanism that controls the proliferation of cells in metazoans. In mammals, multiple genes are required to regulate cell death. We have employed a gene expression knockout technique to isolate cell death-related genes. One such gene, gene associated with retinoid-interferon-induced mortality-19 (GRIM-19), is essential for tumor cell death induced by interferon-beta (IFN-beta) and retinoic acid (RA). Here, we describe the localization of GRIM-19 to human chromosome 19p13.2. This region is essential for prostate tumor suppression. Together with its death-inductive role in the IFN-retinoid-regulated pathways and the tumor-suppressive function of this locus, the data suggest that GRIM-19 may be a novel tumor suppressor.

IFN-gamma inhibits lipopolysaccharide-induced interleukin-1 beta in primary murine macrophages via a Stat1-dependent pathway.

Interleukin-1 (IL-1) plays an important role in host defenses against microbial pathogens. Excessive production of this cytokine, however, may be responsible in part for the lethality observed during sepsis. Our studies show that interferon-gamma (IFN-gamma) downregulates lipopolysaccharide (LPS)-induced interleukin-1beta (IL-1beta) transcription in primary macrophages. This phenomenon does not occur in splenocytes or bone marrow-derived macrophages from signal transducer and activator of transcription (Stat1)-deficient mice, suggesting that Stat1, a transcription factor involved in IFN signaling, plays a critical role in this process. Moreover, nitric oxide (NO) was also involved in the downregulation of LPS-induced IL-1 by IFN, as addition of the inducible nitric oxide synthase (iNOS) inhibitor L-N(6)-(1-iminoethyl)lysine (NIL) negated the effect. Kinetic analysis of IL-1 and IFN levels in LPS-treated mice in vivo suggests that IFN-mediated inhibition of IL-1 might be an important negative feedback mechanism for limiting IL-1 generation in vivo.

Increasing effectiveness of interferon-alpha for malignancies.

Alpha-interferons (IFN-alphas) have been shown to be effective agents in inducing the regression of various malignancies, including leukemias and lymphomas as well as solid tumors, and are the first human therapeutic proteins to result in increased survival in cancer patients. Based on their pleiotropic activities, IFNs have the potential for interacting synergistically with other anticancer agents. For example, preclinical in vitro and animal data suggest a synergistic antitumor interaction between IFN-alpha2 and the antiestrogens toremifene and tamoxifen. Further studies are required to elucidate the molecular basis for such synergistic interactions, particularly with respect to IFN-stimulated genes.

Preparation and characterization of immunoliposomes for targeting of antiviral agents.

Antibodies specific to avian myeloblastosis virus envelope glycoprotein gp80 were raised. Immunoliposomes were prepared using anti-avian myeloblastosis virus envelope glycoprotein gp80 antibody. The antibody was palmitoylated to facilitate its incorporation into lipid bilayers of liposomes. The fluorescence emission spectra of palmitoylated IgG have exhibited a shift in emission maximum from 330 to 370 nm when it was incorporated into the liposomes. At least 50% of the incorporated antibody molecules were found to be oriented towards the outside in the liposomes. The average size of the liposome was found to be 300 A, and on an average, 15 antibody molecules were shown to be present in a liposome. When adriamycin encapsulated in immunoliposomes was incubated in a medium containing serum for 72 h, about 75% of the drug was retained in liposomes. In vivo localization studies, revealed an enhanced delivery of drug encapsulated in immunoliposomes to the target tissue, as compared to free drug or drug encapsulated in free liposomes. These data suggest a possible use of the drugs encapsulated in immunoliposomes to deliver the drugs in target areas, thereby reducing side effects caused by antiviral agents.

Regulation of interferon-alpha/beta-stimulated gene expression through the gamma-activated transcriptional element.

Interferons (IFNs) stimulate gene expression to mediate their biological actions. A multimeric transcription factor consisting of STAT1, STAT2 and p48, a DNA binding protein, regulates IFN-alpha/beta stimulated gene expression. Since the cellular level of p48 is also increased by pre-treatment of cells with IFN-gamma, it is also known as ISGF3gamma. To understand how IFN-gamma regulates the expression of the p48 gene, we have previously isolated and characterized the promoter of murine p48 gene and identified a novel gamma-IFN activated transcriptional element (GATE). In this study using several mutant constructs of p48 promoter we have determined that the same element responds to IFN-alpha/beta treatment. Relatively high doses of IFN-alpha/beta compared to IFN-gamma are required for the induction of p48 promoter. This ability of IFN-alpha/beta to regulate GATE dependent gene expression is linked to the activation of a factor induced by IFN-alpha. However, IFN-gamma induces the binding of two gamma-IFN inducible factors (GIFs) to GATE. The IFN-alpha inducible GATE binding factor is not recognized by specific antibodies raised against the known IFN-regulated factors. It is likely IFN-gamma is a stronger inducer of this gene because it activates two GIFs. GATE-like elements present in hither to undefined IFN-stimulated genes may control IFN-responses in a unique manner.

Transcriptional induction of genes by IFN-beta in mouse cells is regulated by a transcription factor similar to human ISGF-3.

Previous studies of IFN-stimulated transcription factors in murine cells have identified a variety of trans-acting factors that bind to the IFN-stimulated response element (ISRE) whose role in gene expression remain unclear. The present investigation was undertaken to delineate the signal transduction pathway as well as to identify the transcription factors regulated by murine IFN-beta in L929 cells. Tyrosine kinase inhibitor, Genistein, abrogated gene induction and activation of transcription factors by IFN-beta. As early as 5 min after IFN-beta treatment, a transcription factor was activated in the cytoplasm which subsequently migrated into the nucleus. Anti-phosphotyrosine antibodies detected a specific transcription factor induced by mIFN-beta. Antibodies raised against human ISGF-3 subunit proteins p48, p84, p91 and p113 recognized this factor in the cytoplasm as well as in the nucleus of IFN-beta-treated L929 cells. An antibody raised against an oligopeptide of human p113 (residues 435-450) recognized the ISGF-3 complexes both in human and murine cells. However, a different antibody against the C-terminus of human p113 (residues 671-806) did not recognize the ISGF-3 like complex in mouse cells, indicating differences in the primary sequence of these proteins.