Sin3 corepressor function in Myc-induced transcription and transformation.

Many basic-helix-loop-helix-leucine zipper (b-HLH-LZ) proteins, including the Myc family and non-Myc family, bind a common DNA sequence CACGTG, yet have quite different biological actions. Myc binds this sequence as a heterodimer with Max in the activation of both transcription and transformation. The Myc family members Mad and Mxi1 are known to suppress Myc-induced transcription and transformation and to dimerize with Max to form ternary complexes with the mammalian Sin3 transcriptional corepressor (mSin3). The b-HLH-LZ domain of TFEB, which cannot heterodimerize within the Myc family, does not suppress Myc-induced transcription or transformation. However, transfer of a 25- to 36-aa region from Mad or Mxi1, which interacts with mSin3, to the b-HLH-LZ of TFEB, mediated profound suppression of Myc-induced transcription and transformation. These results suggest that the DNA binding specificities of the Myc family and non-Myc family b-HLH-LZ proteins, in the context of the cellular genes involved in Myc-induced transformation, are shared. The results also demonstrate that targeting mSin3 to CACGTG sites via a non-Myc family DNA binding domain is sufficient to oppose Myc activity in growth regulation.

Inhibition of tumor progression by suppression of stress protein GRP78/BiP induction in fibrosarcoma B/C10ME.

Stress protein GRP78/BiP is highly induced in progressively growing tumors and has recently been shown to exert a protective role against lysis by cytotoxic T cells and tumor necrosis factor in vitro. This raises the question whether the in vitro observed protective function of GRP78/BiP translates into the in vivo situation in which tumors grow progressively, killing the host. Herein we report that molecular inhibition of GRP78/BiP induction in the fibrosarcoma B/C10ME, while not affecting in vitro cell proliferation, causes a dramatic increase in apoptotic cell death upon Ca2+ depletion of the endoplasmic reticulum. When B/C10ME cells incapable of inducing GRP78/BiP are injected into mice, tumors are initially formed that, however, regress presumably due to a cytotoxic T-cell response demonstrable by a strong in vitro response to the tumor with spleen cells of regressor mice. Since sensitivity to apoptosis is key to tumor rejection, these results may point to new approaches to the therapy of cancer via regulation of stress protein GRP78/BiP.

A potential pathogenetic mechanism for multiple endocrine neoplasia type 2 syndromes involves ret-induced impairment of terminal differentiation of neuroepithelial cells.

Germ-line missense mutations of the receptor-like tyrosine kinase ret are the causative genetic event of the multiple endocrine neoplasia (MEN) type 2A and type 2B syndromes and of the familial medullary thyroid carcinoma. We have used the rat pheochromocytoma cell line, PC12, as a model system to investigate the mechanism or mechanisms by which expression of activated ret alleles contributes to the neoplastic phenotype in neuroendocrine cells. Here we show that stable expression of ret mutants (MEN2A and MEN2B alleles) in PC12 cells causes a dramatic conversion from a round to a flat morphology, accompanied by the induction of genes belonging to the early as well as the delayed response to nerve growth factor. However, in the transfected PC12 cells, the continuous expression of neuronal specific genes is not associated with the suppression of cell proliferation. Furthermore, expression of ret mutants renders PC12 cells unresponsive to nerve growth factor-induced inhibition of proliferation. These results suggest that induction of an aberrant pattern of differentiation, accompanied by unresponsiveness to growth-inhibitory physiological signals, may be part of the mechanism of action of activated ret alleles in the pathogenesis of neuroendocrine tumors associated with MEN2 syndromes.

Isolation of 10 differentially expressed cDNAs in p53-induced apoptosis: activation of the vertebrate homologue of the drosophila seven in absentia gene.

We report the isolation of 10 differentially expressed cDNAs in the process of apoptosis induced by the p53 tamor suppressor. As a global analytical method, we performed a differential display of mRNA between mouse M1 myeloid leukemia cells and derived clone LTR6 cells, which contain a stably transfected temperature-sensitive mutant of p53. At 32 degrees C wild-type p53 function is activated in LTR6 cells, resulting in programmed cell death. Eight genes are activated (TSAP; tumor suppressor activated pathway), and two are inhibited (TSIP, tumor suppressor inhibited pathway) in their expression. None of the 10 sequences has hitherto been recognized as part of the p53 signaling pathway. Three TSAPs are homologous to known genes. TSAP1 corresponds to phospholipase C beta 4. TSAP2 has a conserved domain homologous to a multiple endocrine neoplasia I (ZFM1) candidate gene. TSAP3 is the mouse homologue of the Drosophila seven in absentia gene. These data provide novel molecules involved in the pathway of wild-type p53 activation. They establish a functional link between a homologue of a conserved developmental Drosophila gene and signal transduction in tumor suppression leading to programmed cell death.

CNI-1493 inhibits monocyte/macrophage tumor necrosis factor by suppression of translation efficiency.

Tumor necrosis factor (TNF) mediates a wide variety of disease states including septic shock, acute and chronic inflammation, and cachexia. Recently, a multivalent guanylhydrazone (CNI-1493) developed as an inhibitor of macrophage activation was shown to suppress TNF production and protect against tissue inflammation and endotoxin lethality [Bianchi, M., Ulrich, P., Bloom, O., Meistrell, M., Zimmerman, G. A., Schmidtmayerova, H., Bukrinsky, M., Donnelley, T., Bucala, R., Sherry, B., Manogue, K. R., Tortolani, A. J., Cerami, A. & Tracey, K. J. (1995) Mol. Med. 1, 254-266, and Bianchi, M., Bloom, O., Raabe, T., Cohen, P. S., Chesney, J., Sherry, B., Schmidtmayerova, H., Zhang, X., Bukrinsky, M., Ulrich, P., Cerami, A. & Tracey, J. (1996) J. Exp. Med., in press]. We have now elucidated the mechanism by which CNI-1493 inhibits macrophage TNF synthesis and show here that it acts through suppression of TNF translation efficiency. CNI-1493 blocked neither the lipopolysaccharide (LPS)-induced increases in the expression of TNF mRNA nor the translocation of nuclear factor NF-kappa B to the nucleus in macrophages activated by 15 min of LPS stimulation, indicating that CNI-1493 does not interfere with early NF-kappa B-mediated transcriptional regulation of TNF. However, synthesis of the 26-kDa membrane form of TNF was effectively blocked by CNI-1493. Further evidence for the translational suppression of TNF is given by experiments using chloram-phenicol acetyltransferase (CAT) constructs containing elements of the TNF gene that are involved in TNF translational regulation. Both the 5' and 3' untranslated regions of the TNF gene were required to elicit maximal translational suppression by CNI-1493. Identification of the molecular target through which CNI-1493 inhibits TNF translation should provide insight into the regulation of macrophage activation and mechanisms of inflammation.

Cross-modulation by transforming growth factor beta in human tuberculosis: suppression of antigen-driven blastogenesis and interferon gamma production.

In tuberculosis, Mycobacterium tuberculosis (MTB)-stimulated T-cell responses are depressed transiently, whereas antibody levels are increased. Lymphoproliferative responses of peripheral blood mononuclear cells (PBMCs) from Pakistani tuberculosis (TB) patients to both mycobacterial and candidal antigens were suppressed by approximately 50% when compared to healthy purified protein derivative (PPD)-positive household contacts. Production of interferon gamma (IFN-gamma) in response to PPD also was depressed by 78%. Stimulation with PPD and the 30-kDa alpha antigen of MTB (30-kDa antigen) induced greater secretion of transforming growth factor beta (TGF-beta), but not interleukin 10 (IL-10) or tumor necrosis factor alpha (TNF-alpha), by PBMCs from TB patients compared to healthy contacts. The degree of suppression correlated with the duration of treatment; patients treated for <1 month had significantly lower T-cell blastogenesis and IFN-gamma production and higher levels of TGF-beta than did patients treated for >1 month. Neutralizing antibody to TGF-beta normalized lymphocyte proliferation in response to PPD, partially restored blastogenesis to candidal antigen, and significantly increased PPD-stimulated production of IFN-gamma in TB patients but not in contacts. Neutralizing antibody to IL-10 augmented, but did not normalize, T-cell responses to both PPD and candida in TB patients and candidal antigen in contacts. TGF-beta, produced in response to MTB antigens, therefore plays a prominent role in down-regulating potentially protective host effector mechanisms and looms as an important mediator of immunosuppression in TB.

Suppression of apoptosis by basement membrane requires three-dimensional tissue organization and withdrawal from the cell cycle.

The basement membrane (BM) extracellular matrix induces differentiation and suppresses apoptosis in mammary epithelial cells, whereas cells lacking BM lose their differentiated phenotype and undergo apoptosis. Addition of purified BM components, which are known to induce beta-casein expression, did not prevent apoptosis, indicating that a more complex BM was necessary. A comparison of culture conditions where apoptosis would or would not occur allowed us to relate inhibition of apoptosis to a complete withdrawal from the cell cycle, which was observed only when cells acquired a three-dimensional alveolar structure in response to BM. In the absence of this morphology, both the GI cyclin kinase inhibitor p21/WAF-1 and positive proliferative signals including c-myc and cyclin DI were expressed and the retinoblastoma protein (Rb) continued to be hyperphosphorylated. When we overexpressed either c-myc in quiescent cells or p21 when cells were still cycling, apoptosis was induced. In the absence of three-dimensional alveolar structures, mammary epithelial cells secrete a number of factors including transforming growth factor alpha and tenascin, which when added exogenously to quiescent cells induced expression of c-myc and interleukin-beta1-converting enzyme (ICE) mRNA and led to apoptosis. These experiments demonstrate that a correct tissue architecture is crucial for long-range homeostasis, suppression of apoptosis, and maintenance of differentiated phenotype.

Pathogenesis of influenza virus-induced pneumonia: involvement of both nitric oxide and oxygen radicals.

The role of nitric oxide (NO) in the pathogenesis of influenza virus-induced pneumonia in mice was investigated. Experimental influenza virus pneumonia was produced with influenza virus A/Kumamoto/Y5/67(H2N2). Both the enzyme activity of NO synthase (NOS) and mRNA expression of the inducible NOS were greatly increased in the mouse lungs; increases were mediated by interferon gamma. Excessive production of NO in the virus-infected lung was studied further by using electron spin resonance (ESR) spectroscopy. In vivo spin trapping with dithiocarbamate-iron complexes indicated that a significant amount of NO was generated in the virus-infected lung. Furthermore, an NO-hemoglobin ESR signal appeared in the virus-infected lung, and formation of NO-hemoglobin was significantly increased by treatment with superoxide dismutase and was inhibited by N(omega)-monomethyl-L-arginine (L-NMMA) administration. Immunohistochemistry with a specific anti-nitrotyrosine antibody showed intense staining of alveolar phagocytic cells such as macrophages and neutrophils and of intraalveolar exudate in the virus-infected lung. These results strongly suggest formation of peroxynitrite in the lung through the reaction of NO with O2-, which is generated by alveolar phagocytic cells and xanthine oxidase. In addition, administration of L-NMMA resulted in significant improvement in the survival rate of virus-infected mice without appreciable suppression of their antiviral defenses. On the basis of these data, we conclude that NO together with O2- which forms more reactive peroxynitrite may be the most important pathogenic factors in influenza virus-induced pneumonia in mice.

Oral tolerance in myelin basic protein T-cell receptor transgenic mice: suppression of autoimmune encephalomyelitis and dose-dependent induction of regulatory cells.

Orally administered antigens induce a state of immunologic hyporesponsiveness termed oral tolerance. Different mechanisms are involved in mediating oral tolerance depending on the dose fed. Low doses of antigen generate cytokine-secreting regulatory cells, whereas high doses induce anergy or deletion. We used mice transgenic for a T-cell receptor (TCR) derived from an encephalitogenic T-cell clone specific for the acetylated N-terminal peptide of myelin basic protein (MBP) Ac-1-11 plus I-Au to test whether a regulatory T cell could be generated from the same precursor cell as that of an encephalitogenic Th1 cell and whether the induction was dose dependent. The MBP TCR transgenic mice primarily have T cells of a precursor phenotype that produce interleukin 2 (IL-2) with little interferon gamma (IFN-gamma), IL-4, or transforming growth factor beta (TGF-beta). We fed transgenic animals a low-dose (1 mg x 5) or high-dose (25 mg x 1) regimen of mouse MBP and without further immunization spleen cells were tested for cytokine production. Low-dose feeding induced prominent secretion of IL-4, IL-10, and TGF-beta, whereas minimal secretion of these cytokines was observed with high-dose feeding. Little or no change was seen in proliferation or IL-2/IFN-gamma secretion in fed animals irrespective of the dose. To demonstrate in vivo functional activity of the cytokine-secreting cells generated by oral antigen, spleen cells from low-dose-fed animals were adoptively transferred into naive (PLJ x SJL)F1 mice that were then immunized for the development of experimental autoimmune encephalomyelitis (EAE). Marked suppression of EAE was observed when T cells were transferred from MBP-fed transgenic animals but not from animals that were not fed. In contrast to oral tolerization, s.c. immunization of transgenic animals with MBP in complete Freund's adjuvant induced IFN-gamma-secreting Th1 cells in vitro and experimental encephalomyelitis in vivo. Despite the large number of cells reactive to MBP in the transgenic animals, EAE was also suppressed by low-dose feeding of MBP prior to immunization. These results demonstrate that MBP-specific T cells can differentiate in vivo into encephalitogenic or regulatory T cells depending upon the context by which they are exposed to antigen.

Suppression of experimental arthritis by gene transfer of interleukin 1 receptor antagonist cDNA.

Restoration of the impaired balance between pro- and antiinflammatory cytokines should provide effective treatment of rheumatoid arthritis. Gene therapy has been proposed as an approach for delivery of therapeutic proteins to arthritic joints. Here, we examined the efficacy of antiinflammatory gene therapy in bacterial cell wall-induced arthritis in rats. Human secreted interleukin 1 receptor antagonist (sIL-1ra) was expressed in joints of rats with recurrent bacterial cell wall-induced arthritis by using ex vivo gene transfer. To achieve this, primary synoviocytes were transduced in culture with a retroviral vector carrying the sIL-1ra cDNA. Transduced cells were engrafted in ankle joints of animals prior to reactivation of arthritis. Animals in control groups were engrafted with synoviocytes transduced with lacZ and neo marker genes. Cells continued to express transferred genes for at least 9 days after engraftment. We found that gene transfer of sIL-1ra significantly suppressed the severity of recurrence of arthritis, as assessed by measuring joint swelling and by the gross-observation score, and attenuated but did not abolish erosion of cartilage and bone. The effect of intraarticularly expressed sIL-1ra was essentially local, as there was no significant difference in severity of recurrence between unengrafted contralateral joints in control and experimental groups. We estimate that locally expressed sIL-1ra was about four orders of magnitude more therapeutically efficient than systemically administered recombinant sIL-1ra protein. These findings provide experimental evidence for the feasibility of antiinflammatory gene therapy for arthritis.

In vitro activation of the interferon-induced, double-stranded RNA-dependent protein kinase PKR by RNA from the 3' untranslated regions of human alpha-tropomyosin.

The cellular kinase known as PKR (protein kinase RNA-activated) is induced by interferon and activated by RNA. PKR is known to have antiviral properties due to its role in translational control. Active PKR phosphorylates eukaryotic initiation factor 2 alpha and leads to inhibition of translation, including viral translation. PKR is also known to function as a tumor suppressor, presumably by limiting the rate of tumor-cell translation and growth. Recent research has shown that RNA from the 3' untranslated region (3'UTR) of human alpha-tropomyosin has tumor-suppressor properties in vivo [Rastinejad, F., Conboy, M. J., Rando, T. A. & Blau, H. M. (1993) Cell 75, 1107-1117]. Here we report that purified RNA from the 3'UTR of human alpha-tropomyosin can inhibit in vitro translation in a manner consistent with activation of PKR. Inhibition of translation by tropomyosin 3'UTR RNA was observed in a rabbit reticulocyte lysate system, which is known to contain endogenous PKR but was not seen in wheat germ lysate, which is not responsive to a known activator of PKR. A control RNA purified in the same manner as the 3'UTR RNA did not inhibit translation in either system. The inhibition of translation observed in reticulocyte lysates was prevented by the addition of adenovirus virus-associated RNA1 (VA RNAI), an inhibitor of PKR activation. Tropomyosin 3'UTR RNA was bound by immunoprecipitated PKR and activated the enzyme in an in vitro kinase assay. These data suggest that activation of PKR could be the mechanism by which tropomyosin 3'UTR RNA exerts its tumor-suppression activity in vivo.

Interleukin 2 induces CD8+ T cell-mediated suppression of human immunodeficiency virus replication in CD4+ T cells and this effect overrides its ability to stimulate virus expression.

The nonlytic suppression of human immunodeficiency virus (HIV) production from infected CD4+ T cells by CD8+ lymphocytes from HIV-infected individuals is one of the most potent host-mediated antiviral activities observed in vitro. We demonstrate that the pleiotropic cytokine interleukin 2 (IL-2), but not IL-12, is a potent inducer of the CD8+ HIV suppressor phenomenon. IL-2 induces HIV expression in peripheral blood or lymph node mononuclear cells from HIV-infected individuals in the absence of CD8+ T cells. However, IL-2 induces CD8+ T cells to suppress HIV expression when added back to these cultures, and this effect dramatically supersedes the ability to IL-2 to induce HIV expression. Five to 25 times fewer CD8+ cells were required to obtain comparable levels of inhibition of viral production if they were activated in the presence of IL-2 as compared with IL-12 or no exogenous cytokine. Furthermore, IL-2 appeared either to induce a qualitative increase in HIV suppressor cell activity or to increase the relative frequency of suppressor cells in the activated (CD25+) CD8+ populations. Analyses of proviral levels in peripheral blood mononuclear cells suggest that CD8+ T cell-mediated lysis of in vivo infected cells is not induced by IL-2. These results have implications for our understanding of the effects of impaired IL-2 production during HIV disease as well as the overall effects of IL-2-based immunotherapy on HIV replication in vivo.

Suppression of retinal neovascularization in vivo by inhibition of vascular endothelial growth factor (VEGF) using soluble VEGF-receptor chimeric proteins.

The majority of severe visual loss in the United States results from complications associated with retinal neovascularization in patients with ischemic ocular diseases such as diabetic retinopathy, retinal vein occlusion, and retinopathy of prematurity. Intraocular expression of the angiogenic protein vascular endothelial growth factor (VEGF) is closely correlated with neovascularization in these human disorders and with ischemia-induced retinal neovascularization in mice. In this study, we evaluated whether in vivo inhibition of VEGF action could suppress retinal neovascularization in a murine model of ischemic retinopathy. VEGF-neutralizing chimeric proteins were constructed by joining the extracellular domain of either human (Flt) or mouse (Flk) high-affinity VEGF receptors with IgG. Control chimeric proteins that did not bind VEGF were also used. VEGF-receptor chimeric proteins eliminated in vitro retinal endothelial cell growth stimulation by either VEGF (P < 0.006) or hypoxic conditioned medium (P < 0.005) without affecting growth under nonstimulated conditions. Control proteins had no effect. To assess in vivo response, animals with bilateral retinal ischemia received intravitreal injections of VEGF antagonist in one eye and control protein in the contralateral eye. Retinal neovascularization was quantitated histologically by a masked protocol. Retinal neovascularization in the eye injected with human Flt or murine Flk chimeric protein was reduced in 100% (25/25; P < 0.0001) and 95% (21/22; P < 0.0001) 0.0001) of animals, respectively, compared to the control treated eye. This response was evident after only a single intravitreal injection and was dose dependent with suppression of neovascularization noted after total delivery of 200 ng of protein (P < 0.002). Reduction of histologically evident neovascular nuclei per 6-microns section averaged 47% +/- 4% (P < 0.001) and 37% +/- 2% (P < 0.001) for Flt and Flk chimeric proteins with maximal inhibitory effects of 77% and 66%, respectively. No retinal toxicity was observed by light microscopy. These data demonstrate VEGF's causal role in retinal angiogenesis and prove the potential of VEGF inhibition as a specific therapy for ischemic retinal disease.

A heme-protein-based oxygen-sensing mechanism controls the expression and suppression of multiple proteins in anoxia-tolerant turtle hepatocytes.

The O2 sensitivity of protein expression was assessed in hepatocytes from the western painted turtle. Anoxic cells consistently expressed proteins of 83.0, 70.4, 42.5, 35.3, and 16.1 kDa and suppressed proteins of 63.7, 48.2, 36.9, 29.5, and 17.7 kDa. Except for the 70.4-kDa protein, this pattern was absent during aerobic incubation with 2 mM NaCN, suggesting a specific requirement for O2. Aerobic incubation with Co2+ or Ni2+ increased expression of the 42.5-, 35.3-, and 16.1-kDa protein bands which was diminished with the heme synthesis inhibitor 4,6-dioxoheptanoic acid. Proteins suppressed in anoxia were also suppressed during aerobic incubation with Co2+ or Ni2+ but this was not relieved by 4,6-dioxoheptanoic acid. The anoxia- and Co2+/Ni2+-induced expression of the 42.5-, 35.3-, and 16.1-kDa protein bands was antagonized by 10% CO; however, with the exception of the 17.7-kDa protein, this was not found for any of the O2- or Co2+/Ni2+-suppressed proteins. Anoxia-induced proteins were compared with proteins expressed during heat shock. Heat shock proteins appeared at 90.2, 74.8, 63.4, 25, and 15.5 kDa and were of distinct molecular masses compared with the anoxia-induced proteins. These results suggest that O2-sensing mechanisms are active in the control of protein expression and suppression during anoxia and that, in the case of the 42.5-, 35.3-, 17.7-, and 16.1-kDa proteins, a conformational change in a ferro-heme protein is involved in transducing the O2 signal.

CT-2576, an inhibitor of phospholipid signaling, suppresses constitutive and induced expression of human immunodeficiency virus.

Viruses such as human immunodeficiency virus (HIV) require cellular activation for expression. Cellular activation in lymphoid cells is associated with augmented accumulation of certain phosphatidic acid (PA) species derived from the hydrolysis of glycan phosphatidylinositol (GPI). This suggests that activation of a phospholipid pathway may play a role in initiation of viral replication. To test this hypothesis, we examined the effect of tat gene expression on the production of cellular PA species, as the Tat protein is essential for HIV expression and has been implicated in activating the expression of multiple host cellular genes. Expression of tat increased the expression of PA. We then tested whether synthetic inhibitors of PA metabolism would inhibit activation of the HIV long terminal repeat by Tat and tumor necrosis factor alpha (TNF-alpha). CT-2576 suppressed both PA generation induced by Tat and HIV long terminal repeat-directed gene expression in response to Tat or TNF-alpha at a posttranscriptional step. CT-2576 also inhibited constitutive as well as TNF-alpha- and interleukin 6-induced expression of HIV p24 antigen in chronically infected U1 cells and in peripheral blood lymphocytes acutely infected with a clinical isolate of HIV. Pharmacological inhibition of synthesis of selected PA species may therefore provide a therapeutic approach to suppression of HIV replication.