Tetsuya Yamagata

A novel interferon regulatory factor family transcription factor, ICSAT/Pip/LSIRF, that negatively regulates the activity of interferon-regulated genes.

We have isolated a novel cDNA clone encoding interferon (IFN) consensus sequence-binding protein in adult T-cell leukemia cell line or activated T cells (ICSAT); this protein is the human homolog of the recently cloned Pip/LSIRF. ICSAT is structurally most closely related to the previously cloned ICSBP, a member of the IFN regulatory factor (IRF) family of proteins that binds to interferon consensus sequences (ICSs) found in many promoters of the IFN-regulated genes. Among T-cell lines investigated, ICSAT was abundantly expressed in human T-cell leukemia virus type 1 (HTLV-1)-infected T cells. When the HTLV-1 tax gene was expressed or phorbol myristake acetate-A23187 stimulation was used, ICSAT expression was induced in Jurkat cells which otherwise do not express ICSAT. When the binding of ICSAT to four different ICSs was tested, the relative differences in binding affinities for those ICSs were determined. To study the functional role of ICSAT, we performed cotransfection experiments with the human embryonal carcinoma cell line N-Tera2. ICSAT was demonstrated to possess repressive function over the gene activation induced by IFN stimulation or by IRF-1 cotransfection. Such repressive function is similar to that seen in IRF-2 or ICSBP. However, we have found that ICSAT has a different repressive effect from that of IRF-2 or ICSBP in some IFN-responsive reporter constructs. These results suggest that a novel mechanism of gene regulation by differential repression is used by multiple members of repressor proteins with different repressive effects on the IFN-responsive genes.

Acetylation of GATA‐3 affects T‐cell survival and homing to secondary lymphoid organs

Acetylation of a transcription factor has recently been shown to play a significant role in gene regulation. Here we show that GATA‐3 is acetylated in T cells and that a mutation introduced into amino acids 305–307 (KRR‐GATA3) creates local hypoacetylation in GATA‐3. Remarkably, KRR‐GATA3 possesses the most potent suppressive effect when compared with other mutants that are disrupted in putative acetylation targets. Expressing this mutant in peripheral T cells results in defective T‐cell homing to systemic lymphnodes, and prolonged T‐cell survival after activation. These findings have significant implications in that the acetylation state of GATA‐3 affects its physiological function in the immune system and, more importantly, provides evidence for the novel role of GATA‐3 in T‐cell survival and homing to secondary lymphoid organs.

The evi-1 oncoprotein inhibits c-Jun N-terminal kinase and prevents stress-induced cell death

Evi‐1 encodes a nuclear protein involved in leukemic transformation of hematopoietic cells. Evi‐1 possesses two sets of zinc finger motifs separated into two domains, and its characteristics as a transcriptional regulator have been described. Here we show that Evi‐1 acts as an inhibitor of c‐Jun N‐terminal kinase (JNK), a class of mitogen‐activated protein kinases implicated in stress responses of cells. Evi‐1 physically interacts with JNK, although it does not affect its phosphorylation. This interaction is required for inhibition of JNK. Evi‐1 protects cells from stress‐induced cell death with dependence on the ability to inhibit JNK. These results reveal a novel function of Evi‐1, which provides evidence for inhibition of JNK by a nuclear oncogene product. Evi‐1 blocks cell death by selectively inhibiting JNK, thereby contributing to oncogenic transformation of cells.

Of the GATA-binding proteins, only GATA-4 selectively regulates the human interleukin-5 gene promoter in interleukin-5-producing cells which express multiple GATA-binding proteins.

Interleukin-5 (IL-5) is produced by T lymphocytes and known to support B-cell growth and eosinophilic differentiation of the progenitor cells. Using ATL-16T cells which express IL-5 mRNA, we have identified a region within the human IL-5 gene promoter that regulates IL-5 gene transcription. This cis-acting sequence contains the core binding motif, (A/T)GATA(A/G), for GATA-binding family proteins and thus suggests the involvement of this family members. In this report, we describe the cloning of human GATA-4 (hGATA-4) and show that hGATA-4 selectively interacts with the -70 GATA site within the IL-5 proximal promoter region. By promoter deletion and mutation analyses, we established this region as a positive regulatory element. Cotransfection experiments revealed that both hGATA-4 and phorbol-12-myristate-13-acetate (PMA)-A23187 stimulation are necessary for IL-5 promoter activation. The requirement for another regulatory element called CLE0, which lies downstream of the -70 GATA site, was also demonstrated. ATL-16T cells express mRNAs of three GATA-binding proteins, hGATA-2, hGATA-3, and hGATA-4, and each of them has a potential to bind to the consensus (A/T)GATA(G/A) motif. However, using ATL-16T nuclear extract, we demonstrated that GATA-4 is the only GATA-binding protein that forms a specific DNA-protein complex with the -70 GATA site. An electrophoretic mobility shift assay with extracts of COS cells expressing GATA-binding proteins showed that GATA-4 has the highest binding affinity for the -70 GATA site among the three GATA-binding proteins. When the transactivation abilities were compared among the three, GATA-4 showed the highest activity. These results demonstrate the selective role of GATA-4 in the transcriptional regulation of the IL-5 gene in a circumstance where multiple members of the GATA-binding proteins are expressed.

Triple Synergism of Human T-Lymphotropic Virus Type 1-Encoded Tax, GATA-Binding Protein, and AP-1 Is Required for Constitutive Expression of the Interleukin-5 Gene in Adult T-Cell Leukemia Cells

Accumulated evidence demonstrates that adult T-cell leukemia (ATL) is frequently associated with eosinophilia, and human T-lymphotropic virus type 1 (HTLV-1)-infected cells frequently express interleukin-5 (IL-5). However, the molecular mechanism of constitutive IL-5 expression in HTLV-1-infected cells remains unclear. To clarify the mechanism of aberrant IL-5 expression in HTLV-1-infected cells, we investigated the response of the human IL-5 promoter to the HTLV-1-encoded protein Tax. Cotransfection experiments using Jurkat cells revealed that Tax is incapable of activating the IL-5 promoter by itself but that it synergistically transactivates the promoter with GATA-binding protein (GATA-4) and 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulation. By introducing a series of mutations within the IL-5 promoter, we found that conserved lymphokine element 0 (CLE0) is responsible for mediating the signal induced by Tax-TPA. A deletion construct of the promoter indicated that the -75 GATA element and CLE0 are sufficient to mediate synergistic activation of the IL-5 promoter. Electrophoretic mobility shift assays using Jurkat cell nuclear extracts demonstrated that TPA induces a transcription factor to bind CLE0, and an experiment using JPX-9 cell nuclear extracts showed that Tax enhances this binding activity. An antibody supershift experiment revealed that this band consists of c-Jun and JunD. However, among the Jun family members, only c-Jun is able to cooperate with Tax and GATA-4 to activate the IL-5 promoter. We have determined the minimum factors required for IL-5 gene activation by reconstituting the IL-5 promoter activity in F9 cells. This is the first report to demonstrate the functional involvement of Tax protein in IL-5 gene regulation and to suggest the functional triple synergism among Tax, GATA-4, and AP-1, which disrupts regulated control of the gene and leads to constitutive expression of the IL-5 gene.

Elevated platelet count features the variant type of BCR/ABL junction in chronic myelogenous leukaemia

A variant form of BCR/ABL junction was identified in a patient with chronic myelogenous leukaemia (CML). The BCR/ABL fusion mRNA of this patient showed in‐frame junction between BCR exon c3 and ABL exon 2. Although the diagnosis of CML was made, the patient showed clinical features of essential thrombocythaemia (ET) rather than that of typical CML. Treatment with interferon‐α showed no cytogenetic response. The c3‐a2 type of BCR/ABL junction seems to be associated with elevated platelet count and thus could form a novel clinical entity different from typical CML.

Impaired spermatogenesis and male fertility defects in CIZ/Nmp4‐disrupted mice

CIZ (Cas interacting zinc finger protein), also called Nmp4 (nuclear matrix protein 4), is a nucleo‐cytoplasmic shuttling transcription factor that regulates the expression of collagen and matrix metalloproteinases. CIZ/Nmp4 was originally cloned by its binding to p130Cas, a focal adhesion protein, and was recently shown to suppress BMP2 (bone mophogenetic protein 2) signalling. To explore the physiological role of CIZ/Nmp4, we disrupted CIZ/Nmp4‐gene by inserting beta‐galactosidase and neomycin resistance genes into the 2nd exon of CIZ/Nmp4‐gene, which is utilized by all the sequenced alternative forms. CIZ−/− mice were born and grew to adulthood. Although they tend to be smaller than wild‐type mice, no pathological abnormality was observed except in the testis. Histological analysis of the testes revealed variable degrees of spermatogenic cell degeneration within the seminiferous tubules of CIZ−/− mice, resembling the histology of the ‘Germinal‐cell aplasia with focal spermatogenesis’. Some of the CIZ−/− male mice developed infertility. TUNEL assay on testis sections revealed an increased occurrence of apoptosis of spermatogenic cells in the testes of CIZ−/− mice. CIZ/Nmp4 was co‐localized with Smad1 in the testis, suggesting that a disregulation of BMP signalling could cause these phenotypes. These results suggest that CIZ/Nmp4 plays roles in the progress and the maintenance of spermatogenesis.

NATURAL INTERFERON‐ALFA FOR BASOPHILIA

bin was 8.8 g/dl. WBC 2 . 3 x 10y/l with 48% neutrophils and 40% lymphocytes and platelets were 3 0 x 1 Oy/l . The tests of liver function returned to normal and metonolone ( 1 5 0 mg daily) was then prescribed for 12 months. and stopped when platelets were 120 x 10y/l, WBC 3 x 1OY/1 with 35% neutrophils and haemoglobin 14.6 gidl. Aplastic anaemia, because of its severe prognosis. is one of the most dreaded adverse drug reactions. In our two cases supportive therapy associated with metenolone were necessary to reverse bone marrow failure. Furthermore, in case 2. intrahepatic cholestasis was associated with aplastic anaemia. The length ofprevious exposure to the drug (respectively 3 and 5 months). the association and the favourable evolution of hepatitis in case 2 , the normalization of blood counts after ticlopidine was stopped. and finally the lack of any other cause of aplastic anaemia suggest in these cases the haematopoietic (Gramont ct al. 1982) and non-haematopoietic toxicity of this drug (Anonymous, 1988). Monitoring of the blood counts during ticlopidine therapy should be performed in order to recognize the first signs of haematological toxicity and alert the physician to withdraw this treatment. Moreover. we think that the benefit-risk ratio of