Junji Nishida

An acute myeloid leukemia gene, AML1, regulates hemopoietic myeloid cell differentiation and transcriptional activation antagonistically by two alternative spliced forms.

The AML1 gene on chromosome 21 is disrupted in the (8;21)(q22;q22) and (3;21)(q26;q22) translocations associated with myelogenous leukemias and encodes a DNA binding protein. From the AML1 gene, two representative forms of proteins, AML1a and AML1b, are produced by alternative splicing. Both forms have a DNA binding domain but, unlike AML1b, AML1a lacks a putative transcriptional activation domain. Here we demonstrate that overexpressed AML1a totally suppresses granulocytic differentiation and stimulates cell proliferation in 32Dcl3 murine myeloid cells treated with granulocyte colony‐stimulating factor. These effects of AML1a were canceled by the concomitant overexpression of AML1b. Such biological phenomena could be explained by our observations that (i) AML1a, which on its own has no effects as a transcriptional regulator, dominantly suppresses transcriptional activation by AML1b, and (ii) AML1a exhibits the higher affinity for DNA binding compared with AML1b. These antagonistic actions could be important in leukemogenesis and/or myeloid cell differentiation because more than half of myelogenous leukemia patients showed an increase in the relative amounts of AML1a.

Dual functions of the AML1/Evi-1 chimeric protein in the mechanism of leukemogenesis in t(3;21) leukemias.

The chromosomal translocation t(3;21)(q26;q22), which is found in blastic crisis in chronic myelogenous leukemias and myelodysplastic syndrome-derived leukemias, produces AML1/Evi-1 chimeric transcription factor and is thought to play important roles in acute leukemic transformation of hemopoietic stem cells. We report here the functional analyses of AML1/Evi-1. It was revealed that AML1/Evi-1 itself does not alter the transactivation level through mouse polyomavirus enhancer-binding protein 2 (PEBP2; PEA2) sites (binding site of AML1) but dominantly suppresses the transactivation by intact AML1, which is assumed to be a stimulator of myeloid cell differentiation. DNA-binding competition is a putative mechanism of such dominant negative effects of AML1/Evi-1 because it binds to PEBP2 sites with higher affinity than AML1 does. Furthermore, AML1/Evi-1 stimulated c-fos promoter transactivation and increased AP-1 activity, as Evi-1 (which is not normally expressed in hemopoietic cells) did. Experiments using deletion mutants of AML1/Evi-1 showed that these two functions are mutually independent because the dominant negative effects on intact AML1 and the stimulation of AP-1 activity are dependent on the runt domain (DNA-binding domain of AML1) and the zinc finger domain near the C terminus, respectively. Furthermore, we showed that AML1/Evi-1 blocks granulocytic differentiation, otherwise induced by granulocyte colony-stimulating factor, of 32Dcl3 myeloid cells. It was also suggested that both AML1-derived and Evi-1-derived portions of the fusion protein play crucial roles in this differentiation block. We conclude that the leukemic cell transformation in t(3;21) leukemias is probably caused by these dual functions of AML1/Evi-1 chimeric protein.

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.

Molecular cloning of a novel human cDNA encoding a zinc finger protein that binds to the interleukin-3 promoter.

The CT/GC-rich region (-76 to -47) is one transcriptional regulatory region of the interleukin-3 (IL-3) gene which confers basic transcriptional activity and responds to trans-activation by human T-cell leukemia virus type I-encoded Tax. We isolated three types of cDNAs encoding Cys2/His2-type zinc finger proteins that bind to this region. Two were identical to known transcription factors, EGR1 and EGR2, and the other clone, named DB1, encoded a novel protein of 516 amino acids with six zinc finger motifs. DB1 mRNA was present in human tissues, ubiquitously. Two constitutive transcripts of 4.0 and 4.8 kb in length were present in Jurkat cells. Electrophoretic mobility shift assay, with specific antibodies, showed that DB1 constitutively binds to this region whereas EGR1 binds in a T-cell activation-dependent manner. Overexpression of DB1 in Jurkat cells had no detectable effect on the transcription activity of the IL-3 promoter, in a transient-transfection assay. EGR1 and EGR2 increased IL-3 promoter activity when the transfected cells were stimulated with phorbol-12-myristate-13-acetate and A23187. When DB1 was cotransfected with a Tax expression vector, transcription activity of the IL-3 promoter induced by Tax was significantly increased, while EGR1 and EGR2 were without effect. These results suggest that EGR1 has a role in inducible transcription of the IL-3 gene, while DB1 sustains basal transcriptional activity and also cooperates with Tax to activate the IL-3 promoter.

Molecular cloning of the closed circular provirus of human T cell leukaemia virus type I: a new open reading frame in the gag-pol region.

A DNA clone of human T cell leukaemia virus type I (HTLV-I) was isolated from extrachromosomal closed circular copies in chronically infected promyelocytic leukaemia HL60 cells. The new HTLV-I isolate had an intact reading frame in the gag-pol region which could encode protein of 234 amino acids. This open reading frame has not been observed in previous HTLV-I isolates, although similar open reading frames have been reported in the corresponding locations in the related bovine leukaemia virus and HTLV type II. We consider that this open reading frame codes for the virus-encoded protease, on the basis of the homology of the predicted amino acid sequence with those of previously identified retrovirus proteases.

A point mutation at codon 13 of the N-ras oncogene in a human stomach cancer

A surgically removed human stomach cancer with the histological diagnosis of poorly differentiated adenocarcinoma contained an activated N-ras oncogene detected by an in vivo selection assay in nude mice using transfected NIH3T3 cells. Analysis using synthetic 20-mer oligonucleotide probes revealed a point mutation from G to C at the first letter of codon 13 of the N-ras gene resulting in the substitution of arginine for glycine. This is the first observation of an activated N-ras oncogene in human stomach cancers.

Highly frequent detection of transforming genes in acute leukemias by transfection using in vivo selection assays.

DNAs from nine out of ten acute leukemia cases that were negative by in vitro focus forming assays exhibited transforming activity tested by in vivo selection assays in nude mice using transfected NIH3T3 cells. Of the nine cases, six cases contained activated N-ras genes, and one case exhibited activation of the c-K-ras gene. None of the ras gene family showed homology with the transforming genes derived from the other two cases. Our observations indicate that in vivo selection assays detect transforming genes including ras oncogenes at high frequency, and that activated N-ras genes are frequently detected in human acute leukemias.

Activation mechanism of the N-ras oncogene in human leukemias detected by synthetic oligonucleotide probes

The synthetic oligonucleotide probes were used for the analysis of N-ras oncogenes detected in human acute leukemias. The mutations of N-ras genes were observed to occur randomly among the subtypes of myeloid leukemias, whereas the N-ras mutations at codon 12 are more likely to occur in lymphoid leukemias than other mutations. The mutations at codon 13 of the N-ras gene were not detected in acute leukemias although they were found in myelodysplastic syndrome that is considered to be a preleukemic state.

T Cell Activation Signals and Regulation of Lymphokine Gene by Viral and Cellular Transactivators

T cells activated by antigen stimulation produce a set of lymphokines. By employing protein kinase C (PKC) which is active without stimulation or viral transactivator HTLV-I p40tax or BPV E2 protein, we characterized the T cell antigen receptor signal transduction pathway downstream of PKC. Consistent with the earlier observations that activation of PKC and Ca2+ influx are necessary for T cell activation, the IL-2 promoter is activated by actions of constitutively active PKC and Ca2+ ionophore in the human T cell leukemia line Jurkat. We found that p40tax or E2 protein activate transfected GM-CSF gene as well as SV40 and HIV promoters without external stimuli. The sequence of GM-CSF promoter required for stimulation by PMA/A23187 is localized between positions -95 and -73 (CLE2). The same region responds to p40tax or E2 protein. Another sequence, located between -113 and -96 (CLE1), mediates inducible response to p40tax but not to E2 protein or PMA/A23187 stimulation. Activation of the SV40 promoter by p40tax or E2 protein is dependent on SV40 enhancer sequences. Only one copy of the segment carrying the NF-κ B binding site is sufficient to mediate the induction by E2 protein, p40tax or PMA/A23187 stimulation. HIV LTR promoter also responds to E2 protein or p40tax through the same DNA element. These results indicate that p40tax or E2 protein activate GM-CSF and viral promoters by interacting with cellular component(s) in the T cell activation signal transduction pathway.