Spi-1/PU.1: an oncogene of the Ets family.
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Amino Acid Sequence↗
Biomedical subjects
F Moreau-Gachelin
Publications and source records attributed to F Moreau-Gachelin.
The Spi-1/PU.1 and Spi-B ets family transcription factors and the recombination signal binding protein RBP-J kappa interact with an Epstein-Barr virus nuclear antigen 2 responsive cis-element.
Epstein-Barr virus (EBV) immortalizes resting human B cells very efficiently in vitro. The EBV nuclear protein EBNA2 is absolutely required for this process. It also activates transcription of cellular, as well as viral, genes. It is assumed that EBNA2 contributes to B cell immortalization by its transactivating potential, since its transforming and transactivating functions could not be separated. Mutational analysis of the 80 bp EBNA2 responsive cis-element within the viral bidirectional LMP/TP2 promoter region identified two sequence elements, which are both essential for transactivation by EBNA2. These sequences harbour putative consensus binding sites for Spi-1 oncoprotein and recombination signal binding protein RBP-J kappa, the homologue of Drosophila Suppressor of Hairless. Electrophoretic mobility shift assays demonstrated the high affinity binding of Spi-1 and Spi-B, both members of the Ets family of transcription factors, to one sequence element. The other element bound RBP-J kappa with low affinity. In addition, co-transfections showed that the replacement of the Spi-1/Spi-B binding site in the bi-directional LMP/TP2 promoter by the analogous SV40 Spi-1 responsive element did not impair its function on EBNA2-mediated transactivation. It is concluded that the transcriptional regulators Spi-1 and Spi-B as well as RBP-J kappa play an essential role in transactivating the LMP/TP2 promoter by EBNA2 and therefore in the immortalization of B cells by EBV.
Antigens, Viral↗
Putative oncogenic role of the erythropoietin receptor in murine and human erythroleukemia cells.
To determine whether the erythropoietin receptor (Epo-R) plays a role in the course of malignant erythropoietic disorders, this gene was studied in murine and human erythroleukemia cells. An altered Epo-R gene was found in a murine Friend erythroleukemia cell line, FCL1, due to a spleen focus-forming virus (SFFV) long terminal repeat insertion within the noncoding region of the first exon, leading to Epo-R mRNA overexpression. A similar mechanism of Epo-R activation has previously been described in the T3CL-2 Friend erythroleukemia cell line. An elevated number of Epo-binding sites has been observed in two human erythroleukemia cell lines, TF-1 and UT7. In UT7 cells, homogeneously staining region of the short arm of chromosome 19 [hsr (19)] was evidenced, which contained an amplification of the Epo-R gene. This Epo-R gene amplification was confirmed by the quantification of Southern blots in which the intensity of the Epo-R signal was compared in UT7 DNA and in DNA from normal cells. The Epo-R gene was present in UT7 at a mean number of seven to eight copies per cell. Interestingly, the Epo-R gene was rearranged; the breakpoint region was located near the 3' end of the gene, 3 kb downstream from the end of the last exon. Taken together, these results suggest that, in both murine and human systems, genetic alterations of the Epo-R gene are not rare events and could be involved in the occurrence of the erythroleukemic process.
Animals↗
Involvement of the transcription factor PU.1/Spi-1 in myeloid cell-restricted expression of an interferon-inducible gene encoding the human high-affinity Fc gamma receptor.
Induction by gamma interferon (IFN-gamma) of the gene encoding the human high-affinity Fc gamma receptor (Fc gamma R1) in myeloid cells requires an IFN-gamma response region (GRR) and a myeloid cell-activating transcription element (MATE). GRR and MATE interact with factors to form, respectively, an IFN-gamma-activating complex (GIRE-BP), depending on the phosphorylation of the 91-kDa protein (subunit of ISGF3), and a cell-type-specific complex (MATE-BP). Although GIRE-BP is detected in cells of different origins after IFN-gamma treatment, the presence of MATE-BP was found to be restricted to B- and myeloid cell lines. Sequence analysis of a cDNA encoding a polypeptide recognizing specifically the MATE motif led to the identification of this product as the proto-oncogene PU.1/Spi-1, a transcriptional activator expressed in myeloid and B cells. Expression of this factor in nonhematopoietic cells allowed IFN-gamma-induced expression of a reporter gene under control of the GRR and MATE sequences. The presence of these motifs in other gene promoters indicates that the binding of PU.1/Spi-1 and IFN regulatory proteins to their respective motifs could be part of a general mechanism leading to cell-type-restricted and IFN-induced gene expression.
B-Lymphocytes↗
Inhibition of Friend cells proliferation by spi-1 antisense oligodeoxynucleotides.
The spi-1 proto-oncogene encodes the transcription factor PU.1 which is normally expressed in all hematopoietic cell lineages except in T cell lines. During the murine acute erythroleukemia induced by the Friend retrovirus, SFFV, spi-1 deregulation by insertional mutagenesis results in the overexpression of Spi-1/PU.1 in the malignant proerythroblastic cell. To assess the Spi-1 role in the proliferation and the differentiation arrest of the Friend tumor cells we inhibited spi-1 gene expression in two Friend cell lines by using antisense oligodeoxyribonucleotides. Proliferation and cloning efficiency of both cell lines were significantly inhibited by spi1 antisense. This antiproliferative effect was not related to an apparent maturation of erythroleukemic cells demonstrating that repression of spi-1 expression is not sufficient per se to restore the ability of the proerythroblastic cells to spontaneously differentiate in mature erythroblasts. These data suggest that the spi-1 gene would be involved in the Friend leukemic process by promoting the proerythroblast to proliferate.
Base Sequence↗
Functional interference between the Spi-1/PU.1 oncoprotein and steroid hormone or vitamin receptors.
The Spi-1/PU.1 protein is an Ets-related transcription factor, whose overexpression is a consequence of SFFV integration in Friend erythroleukemic cells. We present evidence that Spi-1/PU.1 can specifically repress the glucocorticoid-induced activation of promoters carrying a glucocorticoid response element (GRE). Conversely, the glucocorticoid receptor (GR) represses Spi-1/PU.1-mediated transcriptional activation in the presence of hormone. Spi-1/PU.1 also antagonized activation by other nuclear receptors, such as the thyroid hormone or the retinoic acid receptors, in several cell lines, including K562 erythroleukemic cells. These observations suggest that accumulation of the Spi-1/PU.1 protein may interfere with the action of hormones in the erythrocyte differentiation pathway.
Base Sequence↗
Characterization of Spi-B, a transcription factor related to the putative oncoprotein Spi-1/PU.1.
We have cloned a human cDNA from a new gene, spi-B, on the basis of its homology with the DNA-binding domain of the Spi-1/PU.1 putative oncogene product. spi-B codes for a protein of 262 amino acids presenting 43% overall identity with Spi-1. Its highly basic carboxy-terminal region exhibits 34% sequence identity with the DNA-binding domain of the Ets-1 protein. We showed that the Spi-B protein is able to bind the purine-rich sequence (PU box) recognized by Spi-1/PU.1 and to activate transcription of a reporter plasmid containing PU boxes. Chromosome in situ hybridization allowed us to map spi-B to the 19q13.3-19q13.4 region of the human genome. spi-B, like spi-1, was found to be expressed in various murine and human hematopoietic cell lines except T lymphoid cell lines.
Amino Acid Sequence↗
Localization of the human oncogene SPI1 on chromosome 11, region p11.22.
Spi1 is an oncogene specifically activated in acute murine erythroleukemias induced by the Friend spleen focus forming virus (SFFV). Three probes were used for the chromosomal assignment of the human SPI1 oncogene: cDb1 and RaB2 correspond respectively to murine Spi1 and human SPI1 cDNA probes; C45a6B probe is a murine genomic DNA sequence located in the Spi1 5' region and is known as a major SFFV integration site in murine erythroleukemia cells. Somatic hybrid cells enabled cDb1 and RaB2 to be assigned to chromosome 11. The murine C45a6B probe, which is not included in the Spi1 gene, detected a homologous sequence on human chromosome 11. RaB2 was assigned to 11p11.22 by in situ hybridization. Three human genes known between 11p11 and 11p13 (FSHB, CAT, ACP2) were on murine chromosome 2. Therefore, the localization of human SPI1 on 11p11.22 was consistent with the assignment of the Spi1 oncogene to murine chromosome 2.
Animals↗
The human homologue of the putative proto-oncogene Spi-1: characterization and expression in tumors.
Spi-1 is a putative proto-oncogene involved in murine virus-induced acute erythroleukemias. We report here the identification of the human homologue of Spi-1 and its expression in normal and tumorigenic human tissues. Characterization of cDNA clones revealed that the human Spi-1 gene encodes a 216 amino acids protein showing 85% identity with the murine counterpart. By sequencing genomic clones, five exons were identified. To investigate the possible role of Spi-1 gene in human cancers, we studied its expression in a panel of human tumors by Northern blot analysis. Spi-1 expression was detected in all the tumors examined. There was no noticeable evidence of messenger RNA alteration as compared to normal tissues.
Adenocarcinoma↗
Spi-1 oncogene activation in Rauscher and Friend murine virus-induced acute erythroleukemias.
The Friend viruses, like the Rauscher virus, cause murine acute erythroleukemias which evolve in a similar multistep process. In previous studies it has been described that the late malignant proerythroblastic transformation induced by the polycythemia-inducing strain of Friend spleen focus-forming virus (SFFVP) is correlated with Spi-1 oncogene activation by insertional mutagenesis. In this paper we report that Spi-1 genomic rearrangements were also observed in 90% of tumors induced by the anemia-inducing strain of Friend spleen focus-forming virus (SFFVA) and in all Rauscher-induced tumors analyzed. SFFVA and Rauscher proviral insertions occurred in the viral integration cluster previously characterized in SFFVP-induced tumors. The Spi-1 1.4-Kb messenger RNA was found highly expressed in all SFFVA and Rauscher-induced malignant cells as compared to normal tissues. The nucleotide sequence of Spi-1 cDNA isolated from a library constructed from SFFVA-induced tumor cells revealed no difference between the Spi-1 gene transcripts expressed in both SFFVP and SFFVA-induced leukemic cells. These results indicate that Spi-1 gene activation is a general feature in the malignant proerythroblastic transformation which occurs in mice infected with Friend and Rauscher viruses.
Acute Disease↗
The putative oncogene Spi-1: murine chromosomal localization and transcriptional activation in murine acute erythroleukemias.
We have shown previously that spleen focus forming virus integration near the Spi-1 putative oncogene is observed in 95% of erythroid Friend tumors (Moreau-Gachelin et al., 1988). Here we describe how the proviral insertion in the Spi-1 domain is associated with the enhanced transcription of a 1.4 kb mRNA normally expressed at a low level in normal cells. The gene is localized on murine chromosome 2, band E3. The structure of the Spi-1 gene was determined by sequencing genomic and cDNA clones. The gene has an open reading frame encoding a protein of 218 amino acids, extending over five exons. Proviruses always integrate outside, upstream and in the opposite orientation of the protein-encoding domain. This suggests that SFFV integration activates the expression of Spi-2 gene that may contribute to the erythroleukemic process.
Amino Acid Sequence↗
Protooncogene expression in normal, preleukemic, and leukemic murine erythroid cells and its relationship to differentiation and proliferation.
The expression of 18 protooncogenes was examined by Northern blot analysis in preleukemic and leukemic stages of murine erythroleukemias induced by Friend viruses. As controls, erythropoietically stimulated spleens from phenylhydrazine-treated mice were studied. Expression of 10 protooncogenes (c-erb-A, c-erb-B, c-ets, c-sis, c-mos, c-rel, c-src, c-fes, c-fms, N-myc [corrected] was not detectable in Friend erythroleukemias. One protooncogene (c-src) was found expressed in normal erythroid cells but not in erythroleukemias. Four protooncogenes (c-fos, c-abl, N-ras, and c-raf) were expressed at low levels in both steps of erythroleukemia. c-fos and c-abl RNAs were barely detectable in normal erythroid cells. High levels of four protooncogene transcripts (c-H-ras, c-K-ras, c-myc, and c-myb) were detected in preleukemic and leukemic tissues. While c-H-ras RNA was found at similar levels in normal and leukemic erythroid cells, c-myc, c-myb, and c-K-ras were not expressed in normal erythroid cells. To determine whether the elevated levels of c-myc, c-myb, and c-K-ras RNAs in erythroleukemic cells are related to the proliferative state or the undifferentiated state of the cells, the effect of dimethyl sulfoxide-induced differentiation on oncogene expression in two erythroleukemia cell lines was examined. Terminal differentiation was associated with lack of c-myb expression while c-myc and c-K-ras expression was essentially unaffected. These results suggest that the high levels of c-myb transcripts in erythroleukemias may reflect the undifferentiated state of the leukemic cells. In contrast, the elevated expression of c-myc and c-K-ras at both stages of the Friend diseases is probably not related to the stage of differentiation but rather to the uncontrolled proliferation of the cells. Finally among 18 protooncogenes surveyed, only the accumulation of c-myc and c-K-ras RNAs appears to be associated with the Friend erythroleukemic process before the late leukemic phase develops.
Animals↗
Spi-1 is a putative oncogene in virally induced murine erythroleukaemias.
Retroviral insertional mutagenesis has been proposed as an efficient mechanism to turn on or to increase the expression of oncogenes in several avian or mammal models. Integration site studies of avian leukosis virus, murine leukaemia and murine mammary tumour viruses led to the coleutification of highly conserved genes whose expression is induced or increased during leukaemogenesis, probably through enhancer elements present in the retroviral long terminal repeats. This is reminiscent of the activation of cellular proto-oncogenes or putative oncogenes in numerous human tumours and leukaemias as a result of chromosomal translocations or DNA rearrangements. Here we report the characterization of a new putative oncogene isolated from a murine erythroleukaemia induced by the acute leukaemogenic retrovirus spleen focus forming virus (SFFV). An important and unusual feature of this genomic locus Spi-1 (for SFFV proviral integration) is that rearrangements due to SFFV integration were found in 95% of the erythroid tumours studied. A 4.0-kilobase messenger RNA was detected in rearranged tumours. No Spi-1 rearrangement was detected in other virally induced myeloid, lymphoid or erythroid tumours tested.
Animals↗
Retroviral endogenous transcripts related to the envelope gene of Friend spleen focus-forming virus in normal mouse tissues.
Retroviral endogenous sequences related to the envelope (env) gene of Friend spleen focus forming virus (SFFV) and of mink cell focus forming viruses (MCF) are present in the genome of various mouse strains. We have examined the transcription of these SFFV/MCF-related sequences in normal tissues of two mouse strains, ICFW and DBA/2. Cytoplasmic Poly A+ RNAs of normal mouse tissues were analyzed by dot-blot and Northern blot hybridizations with a subcloned env SFFV DNA fragment (0.4 kbp BamH I-Sma I). In both mice, the level of SFFV/MCF env related transcripts was very low in bone marrows and spleens whereas it was high in kidneys. Intermediate levels of transcripts were observed in other tissues (thymus, liver and brain). In both mouse strains, the size of SFFV/MCF env related transcripts varied from one tissue to another. Some transcripts in DBA/2 mice were reminiscent of full-size viral message indicating an occasional expression of xenotropic/MCF endogenous virus in this low-leukemic strain. Sizes of the other SFFV/MCF related env transcripts were unusual, but were similar in both strains for each tissue studied. This last result suggests a tissue-specific transcription of endogenous sequences related to the SFFV/MCF env gene. A 1.8 kb SFFV/MCF env RNA was the major transcript in the tissues which expressed a high level of these env transcripts. Treatment of mice with phenylhydrazine which greatly stimulates erythroid differentiation in spleens increased the level of SFFV/MCF related env RNAs only in the spleens, suggesting a possible correlation between the SFFV/MCF env transcription and the stimulation of the erythroid spleen cells.
Animals↗
Analysis of integrated proviral DNA sequences with an octadecanucleotide probe designed for specific identification of spleen focus-forming virus in the mouse genome.
The Friend spleen focus-forming virus (SFFV) is an envelope gene recombinant between the ecotropic Friend murine leukemia virus and the endogenous xenotropic mink cell focus-forming retroviral sequences. We synthesized an octadecanucleotide complementary to the 3' end of the SFFV env gene designed for discriminating the SFFV proviruses from the xenotropic mink cell focus-forming virus and ecotropic exogenous or endogenous viral sequences. Under appropriate hybridization conditions this probe allowed the identification, in addition to few endogenous DNA fragments, of multiple SFFV proviruses integrated in the genome of Friend malignant cells. Therefore this probe should be of interest in further characterizing the SFFV integration sites and possibly the SFFV ancestor gene.
Animals↗
Integration of spleen focus-forming virus proviruses in Friend tumor cells.
Using the Southern blot procedure, we studied the presumed spleen focus-forming virus (SFFV) provirus integration sites in the genome of the premalignant and the malignant cells isolated during the course of Friend erythroleukemia. Two restriction endonucleases, PstI and BamHI, discriminated the presumed integrated SFFV proviruses from the endogenous xenotropic-mink cell focus-forming viral sequences. No SFFV integration sites were detectable in the premalignant cells, suggesting a random integration of SFFV proviruses in the genome of these cells. In contrast, SFFV proviruses were detected at a single or very few sites in the genome of all malignant cells we analyzed. These results indicate that the event leading to the malignant transformation in acute Friend leukemia is clonal. In two of the six animals examined, tumors cells isolated from the spleens and the livers of individual mice showed identical SFFV integration patterns. This last result suggests that in some cases different tumors in a same leukemic animal could be derived from a unique clonal event.
Animals↗
Expression of c-ras and c-myc oncogenes in murine erythroleukemias induced by Friend viruses.
We have examined the expression of three cellular oncogenes (c-Ki-ras, c-Ha-ras and c-myc) in different stages of murine erythroleukemias induced in ICFW mice by Friend viruses (F-MuLV and SFFV) in comparison to normal mice spleens and bone marrows, including erythropoietically-stimulated spleens from phenylhydrazine-treated mice. There is no evidence of c-Ki-ras RNA expression in any of the tissues tested. c-Ha-ras RNA was found at similar levels in erythroleukemic cells and normal erythroid cells. In contrast, increased levels of normal 2.3 Kb and short 1.8 Kb c-myc RNA transcripts were detected in both early preleukemic and late leukemic phases of the diseases, as compared to normal erythroid cells. Apparently, neither myc gene amplification nor myc gene rearrangement was observed in erythroleukemias. Our results suggest a possible involvement of c-myc gene either in erythroid cell differentiation or in an early proliferation step of the erythroleukemic process.
Animals↗
Genesis of a new spleen focus-forming virus: possible role of MCF viruses.
A Friend mink cell focus-inducing (Fr-MCF) virus isolated from a Friend tumor cell line was able to induce acute erythroleukemia associated with polycythemia when injected as a Friend murine ecotropic leukemia virus (F-MuLV) pseudotype into adult Swiss and ICFW mice. One virus isolate recovered from leukemic cells and designated as FV-F3 presented the following properties: (i) persistence of the same leukemogenic power when propagated in vivo and in vitro; (ii) in vivo spleen focus-forming (SFFV) capacity; (iii) presence of erythropoietin (EPO)-independent CFU-E in leukemic animals; (iv) expression of a 32 RNA specifically recognized by a SFFV probe, in FV-F3 infected cells; and (v) expression in FV-F3-infected cell of polypeptides in the range of gp52 SFFV. Peptide analysis of these products revealed close similarities with the parental MCF virus. These data suggest that a SFFV genome arose by genetic recombinational events involving MCF virus.
Animals↗