Common Virus Integration Site Research Articles

Common Viral Integration Sites Identified in Avian Leukosis Virus-Induced B-Cell Lymphomas.

ABSTRACTAvian leukosis virus (ALV) induces B-cell lymphoma and other neoplasms in chickens by integrating within or near cancer genes and perturbing their expression. Four genes—MYC, MYB, Mir-155, and TERT—have previously been identified as common integration sites in these virus-induced lymphomas and are thought to play a causal role in tumorigenesis. In this study, we employ high-throughput sequencing to identify additional genes driving tumorigenesis in ALV-induced B-cell lymphomas. In addition to the four genes implicated previously, we identify other genes as common integration sites, including TNFRSF1A, MEF2C, CTDSPL, TAB2, RUNX1, MLL5, CXorf57, and BACH2. We also analyze the genome-wide ALV integration landscape in vivo and find increased frequency of ALV integration near transcriptional start sites and within transcripts. Previous work has shown ALV prefers a weak consensus sequence for integration in cultured human cells. We confirm this consensus sequence for ALV integration in vivo in the chicken genome.

Clonality of mouse and human cardiomyogenesis in vivo

An analysis of the clonality of cardiac progenitor cells (CPCs) and myocyte turnover in vivo requires genetic tagging of the undifferentiated cells so that the clonal marker of individual mother cells is traced in the specialized progeny. CPC niches in the atria and apex of the mouse heart were infected with a lentivirus carrying EGFP, and the destiny of the tagged cells was determined 1-5 months later. A common integration site was identified in isolated CPCs, cardiomyocytes, endothelial cells (ECs), and fibroblasts, documenting CPC self-renewal and multipotentiality and the clonal origin of the differentiated cell populations. Subsequently, the degree of EGFP-lentiviral infection of CPCs was evaluated 2-4 days after injection, and the number of myocytes expressing the reporter gene was measured 6 months later. A BrdU pulse-chasing protocol was also introduced as an additional assay for the analysis of myocyte turnover. Over a period of 6 months, each EGFP-positive CPC divided approximately eight times generating 230 cardiomyocytes; this value was consistent with the number of newly formed cells labeled by BrdU. To determine whether, human CPCs (hCPCs) are self-renewing and multipotent, these cells were transduced with the EGFP-lentivirus and injected after acute myocardial infarction in immunosuppressed rats. hCPCs, myocytes, ECs, and fibroblasts collected from the regenerated myocardium showed common viral integration sites in the human genome. Thus, our results indicate that the adult heart contains a pool of resident stem cells that regulate cardiac homeostasis and repair.

Enhanced expression of p210BCR/ABL and aberrant expression of Zfp423/ZNF423 induce blast crisis of chronic myelogenous leukemia

AbstractChronic myelogenous leukemia (CML) is a hematopoietic disorder originating from p210BCR/ABL-transformed stem cells, which begins as indolent chronic phase (CP) but progresses into fatal blast crisis (BC). To investigate molecular mechanism(s) underlying disease evolution, CML-exhibiting p210BCR/ABL transgenic mice were crossed with BXH2 mice that transmit a replication-competent retrovirus. Whereas nontransgenic mice in the BXH2 background exclusively developed acute myeloid leukemia, p210BCR/ABL transgenic littermates developed nonmyeloid leukemias, in which inverse polymerase chain reaction detected 2 common viral integration sites (CISs). Interestingly, one CIS was transgene’s own promoter, which up-regulated p210BCR/ABL expression. The other was the 5′ noncoding region of a transcription factor, Zfp423, which induced aberrant Zfp423 expression. The cooperative activities of Zfp423 and p210BCR/ABL were demonstrated as follows: (1) introduction of Zfp423 in p210BCR/ABL transgenic bone marrow (BM) cells increased colony-forming ability, (2) suppression of ZNF423 (human homologue of Zfp423) in ZNF423-expressing, p210BCR/ABL-positive hematopoietic cells retarded cell growth, (3) mice that received a transplant of BM cells transduced with Zfp423 and p210BCR/ABL developed acute leukemia, and (4) expression of ZNF423 was found in human BCR/ABL-positive cell lines and CML BC samples. These results demonstrate that enhanced expression of p210BCR/ABL and deregulated expression of Zfp423/ZNF423 contribute to CML BC.

Identification of Potential Human Oncogenes by Mapping the Common Viral Integration Sites in Avian Nephroblastoma

Gene deregulation is a frequent cause of malignant transformation. Alteration of the gene structure and/or expression leading to cellular transformation and tumor growth can be experimentally achieved by insertion of the retroviral genome into the host DNA. Retrovirus-containing host loci found repeatedly in clonal tumors are called common viral integration sites (cVIS). cVIS are located in genes or chromosomal regions whose alterations participate in cellular transformation. Here, we present the chicken model for the identification of oncogenes and tumor suppressor genes in solid tumors by mapping the cVIS. Using the combination of inverse PCR and long terminal repeat-rapid amplification of cDNA ends technique, we have analyzed 93 myeloblastosis-associated virus type 2-induced clonal nephroblastoma tumors in detail, and mapped >500 independent retroviral integration sites. Eighteen genomic loci were hit repeatedly and thus classified as cVIS, five of these genomic loci have previously been shown to be involved in malignant transformation of different human cell types. The expression levels of selected genes and their human orthologues have been assayed in chicken and selected human renal tumor samples, and their possible correlation with tumor development, has been suggested. We have found that genes associated with cVIS are frequently, but not in all cases, deregulated at the mRNA level as a result of proviral integration. Furthermore, the deregulation of their human orthologues has been observed in the samples of human pediatric renal tumors. Thus, the avian nephroblastoma is a valid source of cancer-associated genes. Moreover, the results bring deeper insight into the molecular background of tumorigenesis in distant species.

A Novel Subgroup of Poor Prognostic AML with Low CEBPA Expression, CEBPA Promoter Hypermethylation and DNMT3b Overexpression.

The CCAAT/Enhancer Binding Protein a (CEBPA) is a 37kDa transcription factor essential for granulocytic differentiation. The C-terminal part is composed of a basic region that binds to DNA and a leucine zipper that mediates dimerization (bZIP). Recent studies reported N and C-terminal mutations in approximately 5 – 10% of patients with acute myeloid leukemia (AML). In case a C-terminal mutation is observed in one CEBPA allele, the other allele most frequently harbors an N-terminal mutation. N-terminal mutations cause the expression of a 30kDa protein, whereas C-terminal mutations usually represent in-frame insertions or deletions in the bZIP-domain resulting in deficient DNA-binding of CEBPA. Gene array analysis carried out on 285 AML cases, revealed a group of AML patients with a unique gene expression profile, which contained most of AML cases with biallelic, CEBPA mutations (Valk et al. N Engl J Med. 2004, 350: pp1617–28). Interestingly, among this cluster 50% of the cases did not harbor any CEBPA mutation, while presenting exactly the same gene expression profile. Instead of carrying CEBPA mutations, these particular cases did not express CEBPA mRNA at all. Moreover, the patients with low CEBPA expression showed a poor response to anti leukemic treatment, whereas the CEBPA mutant AML cases respond favorably to therapy. To study the mechanism by which CEBPA expression might have been down regulated, methylation-specific PCR was applied using the bisulfite method. This analysis revealed that in most of the cases with low CEBPA mRNA levels, the CEBPA promoter was silenced by hypermethylation. None of the other AML cases that we analyzed showed CEBPA hyper methylation. To verify whether the hypermethylation correlated with the expression levels of any of the distinct DNA-methyl transferase (DNMTs), the expression of these genes was studies in all 285 AML cases. Intriguingly, AML blasts in which CEBPA was methylated showed significant overexpression of the DNA-methyl transferase DNMT3B, whereas the expression this gene was low in the cases with CEBPA mutations. The fact that AML cases with mutated CEBPA show the same gene expression profile as the cases that harbored methylated CEBPA, indicate that the same pathways are affected within those two subclasses of AML. Intriguingly, the majority of the genes within this profile are strongly down regulated, suggesting that they may represent CEBPA target genes whose expression is negatively affected by the absence of wt-CEBPA or the presence of mutated CEBPA. The gene that was most significantly down regulated in AML cases with either CEBPA mutations or CEBPA methylation is alpha-catenin (CTNNA1) (10 fold downregulation), a gene that we previously identified as a common virus integration site in retrovirally induced myeloid leukemias in mice (Erkeland et al., J Virol. 2004; 78, pp1971–80). We suggest that down regulation of CTNNA1 in CEBPA defective AML may be a critical step in leukemic transformation. Here we report the identification of a novel subgroup of AML with poor-prognosis, which overexpress DNMT3B and in which CEBPA expression is turned off, frequently as the result of hypermethylation of the CEBPA promoter.

The peripheral cannabinoid receptor Cb2, frequently expressed on AML blasts, either induces a neutrophilic differentiation block or confers abnormal migration properties in a ligand-dependent manner

AbstractCb2, the gene encoding the peripheral cannabinoid receptor, is located in a common virus integration site and is overex-pressed in retrovirally induced murine myeloid leukemias. Here we show that this G protein-coupled receptor (GPCR) is also aberrantly expressed in a high percentage of human acute myeloid leukemias. We investigated the mechanism of transformation by Cb2 and demonstrate that aberrant expression of this receptor on hematopoietic precursor cells results in distinct effects depending on the ligand used. Cb2-expressing myeloid precursors migrate upon stimulation by the endocannabinoid 2-arachidonoylglycerol and are blocked in neutrophilic differentiation upon exposure to another ligand, CP55940. Both effects depend on the activation of Gαi proteins and require the mitogen-induced extracellular kinase/extracellular signal-regulated kinase (MEK/ERK) pathway. Down-regulation of cyclic adenosine monophosphate (cAMP) levels upon Gαi activation is important for migration induction but is irrelevant for the maturation arrest. Moreover, the highly conserved G protein-interacting DRY motif, present in the second intracellular loop of GPCRs, is critical for migration but unimportant for the differentiation block. This suggests that the Cb2-mediated differentiation block requires interaction of Gαi proteins with other currently unknown motifs. This indicates a unique mechanism by which a transforming GPCR, in a ligand-dependent manner, causes 2 distinct oncogenic effects: altered migration and block of neutrophilic development. (Blood. 2004;104:526-534)

Cyclin D1 is necessary but not sufficient for anchorage-independent growth of rat mammary tumor cells and is associated with resistance of the Copenhagen rat to mammary carcinogenesis.

To identify genes associated with the resistance of Copenhagen (Cop) rats to mammary carcinogenesis, we infused a retrovirus harboring v-Ha-ras directly into the main mammary ducts of resistant F1 rats from a cross between Cop and susceptible Wistar Furth (WF) rats. Adenocarcinomas formed in approximately 50% of infused glands. Cell lines derived from these tumors were clonal, but did not share a common viral integration site, suggesting that a high level of v-Ha-ras expression was able to overcome resistance in the F1 rats. Some of the cell lines were able to grow in soft agar, but a significant number did not display anchorage-independent growth. These growth characteristics were independent of v-Ha-ras expression levels. The ability to grow in soft agar was associated with the size of tumors induced by injecting the cells into nude mice, and showed a striking positive association with the expression of cyclin D1. Furthermore, while resistance to anchorage-independent growth was fully overcome by transfection of cyclin D1 in some clones, in the others the effect was partial. A similar pattern of cyclin D1 upregulation and growth in soft agar was also observed when the cells were transfected with an active form of beta-catenin. Hybrid cells from the somatic fusion of an anchorage-dependent to an anchorage-independent clone did not grow in soft agar. These results suggest that while a high expression level of cyclin D1 is necessary for anchorage-independent growth in all clones, it is not sufficient for full growth capacity in soft agar, raising the possibility that the loss of a tumor suppressor gene in the cell lines is required to fully confer anchorage-independent growth. Our anchorage-dependent and -independent rat mammary tumor-derived cell lines may recapitulate the resistance and susceptibility of Cop and WF rats, respectively, to mammary carcinogenesis that could facilitate the identification of breast cancer susceptibility genes.

Identification, characterization, and function of a novel oncogene: the peripheral cannabinoid receptor Cb2.

By means of retroviral insertional mutagenesis, we identified a novel common virus integration site (cVIS) (Evi11) in murine leukemias, and demonstrated that Cb2, encoding the peripheral cannabinoid receptor, is the potential proto-oncogene located in that region. Cb2 is a 7-transmembrane G protein-coupled receptor (GPCR), which is normally expressed on B lymphocytes. Using transwell assays we observed strong migration of Cb2-expressing cells upon stimulation with 2-arachidonoylglycerol (2-AG), a potent endocannabinoid. Overexpression of Cb2 receptor on murine myeloid precursor cells causes a block of neutrophilic differentiation, a major characteristic of myeloid leukemia. Intriguingly, we could not detect functional Cb2 receptors on normal murine bone marrow precursor cells. Furthermore, analysis of human acute myeloid leukemia (AML) samples revealed the presence of CB2 mRNA transcripts in several cases. Furthermore, migration could be induced by 2-AG when analyzed in one of the patient samples. Our data suggest that the initially identified cVIS, Evi11, encodes for a murine onco-protein and that human CB2 may be involved in certain cases of human AML as well.

The peripheral cannabinoid receptor Cb2, a novel oncoprotein, induces a reversible block in neutrophilic differentiation

We previously identified a novel common virus integration site, Evi11, by means of retroviral insertional mutagenesis. We demonstrated that the gene encoding the peripheral cannabinoid receptor (Cb2) is the potential target, suggesting that Cb2 is a proto-oncogene. To elucidate a role for this G protein-coupled receptor (GPCR) in leukemic transformation we generated a Cb2-EGFP cDNA construct that was introduced into 32D/G-CSF-R cells. These cells require interleukin 3 (IL-3) to proliferate in vitro, whereas in the presence of granulocyte-colony-stimulating factor (G-CSF) they differentiate toward mature neutrophils. We demonstrate that 32D/G-CSF-R/Cb2-EGFP cells migrate in a transwell assay in reponse to the Cb2 ligand 2-arachidonoylglycerol (2-AG), indicating that the fusion protein was functional. When cultured in the presence of G-CSF neutrophilic differentiation of Cb2-EGFP-expressing 32D/G-CSF-R cells was completely blocked. Moreover, a Cb2-specific antagonist fully recovered the G-CSF-induced neutrophilic differentiation of 32D/G-CSF-R/Cb2-EGFP cells. To investigate which signal transduction pathway(s) may be involved in the block of neutrophilic maturation, differentiation experiments were carried out using specific inhibitors of signaling routes. Interestingly, full rescue of G-CSF-induced neutrophilic differentiation was observed when cells were cultured with the mitogen-induced extracellular kinase (MEK) inhibitors, PD98059 or U0126, and partial recovery was detected with the phosphoinositide 3-kinase (PI3-K) inhibitor LY-294002. These studies demonstrate that the Cb2 receptor is an oncoprotein that blocks neutrophilic differentiation when overexpressed in myeloid precursor cells. Cb2 appears to mediate its activity through MEK/extracellular signal-related kinase (ERK) and PI3-K pathways.

Leukemic predisposition of pSca-1/Cb2 transgenic mice.

The gene encoding the peripheral cannabinoid receptor Cb2 is located in the common virus integration site Evi11 and is associated with hematopoietic malignancies in mice. To determine the effect of Cb2 overexpression on hematopoietic development in vivo, Cb2 transgenic mice were generated. A Cb2 expression vector was constructed containing a Cb2 cDNA fragment cloned into the 14kb Sca-1 (Ly-6E.1) gene. Two transgenic lines in which Cb2 expression is controlled by the Sca-1 promoter were generated, and the effect on hematopoietic development was studied. Expression of Cb2 mRNA or protein was studied by RNase protection analysis and ligand binding assays, respectively. Leukemic predisposition was investigated by injecting newborn transgenic as well as control animals with Cas-Br-M murine leukemia virus (Cas-Br-M MuLV). Although increased expression of the Cb2 gene was observed in hematopoietic tissues, follow-up of more than 1 year did not reveal any hematologic defect. Interestingly, infection of newborn pSca-1/Cb2 transgenic mice with Cas-Br-M MuLV revealed that significantly more transgenic mice developed leukemia than virus-treated control littermates. Because these studies provide evidence for the cooperative potential of Cb2 in leukemia progression, we wished to identify genes that may collaborate with Cb2 in leukemic transformation. Our study suggests that Evi1, another common target for proviral integration in mouse leukemias, may be overexpressed in virus-induced leukemias in pSca-1/Cb2 transgenic mice. The data indicate that hematopoietic precursor cells that express high levels of Cb2 possess increased susceptibility for leukemia development and that Cb2 and Evi1 might collaborate in leukemogenesis.

Retroviral insertions in Evi12, a novel common virus integration site upstream of Tra1/Grp94, frequently coincide with insertions in the gene encoding the peripheral cannabinoid receptor Cnr2.

The common virus integration site (VIS) Evi11 was recently identified within the gene encoding the hematopoietic G-protein-coupled peripheral cannabinoid receptor Cnr2 (also referred to as Cb2). Here we show that Cnr2 is a frequent target (12%) for insertion of Cas-Br-M murine leukemia virus (MuLV) in primary tumors in NIH/Swiss mice. Multiple provirus insertions in Evi11 were cloned and shown to be located within the 3' untranslated region of the candidate proto-oncogene Cnr2. These results suggest that proviral insertion in the Cnr2 gene is an important step in Cas-Br-M MuLV-induced leukemogenesis in NIH/Swiss mice. To isolate Evi11/Cnr2 collaborating proto-oncogenes, we searched for novel common VISs in the Cas-Br-M MuLV-induced primary tumors and identified a novel frequent common VIS, Evi12 (14%). Interestingly, 54% of the Evi11/Cnr2-rearranged primary tumors contained insertions in Evi12 as well, which suggests cooperative action of the target genes in these two common VISs in leukemogenesis. By interspecific backcross analysis it was shown that Evi12 resides on mouse chromosome 10 in a region that shares homology with human chromosomes 12q and 19p. Sequence analysis demonstrated that Evi12 is located upstream of the gene encoding the molecular chaperone Tra1/Grp94, which was previously mapped to mouse chromosome 10 and human chromosome 12q22-24. Thus, Tra1/Grp94 is a candidate target gene for retroviral activation or inactivation in Evi12. However, Northern and Western blot analyses did not provide evidence that proviral insertion had altered the expression of Tra1/Grp94. Additional studies are required to determine whether Tra1/Grp94 or another candidate proto-oncogene in Evi12 is involved in leukemogenesis.

Proviral integrations at the Evi5 locus disrupt a novel 90 kDa protein with homology to the Tre2 oncogene and cell-cycle regulatory proteins.

Evi5 is a common site of retroviral integration in T-cell lymphomas of AKXD mice. Mapping studies have localized Evi5 to a region approximately 18 kb upstream of another common viral integration site, Gfi1, on mouse chromosome 5 (Liao X, Jenkins NA and Copeland NG, (1995a). J. Virol., 69, 7132-7137). Gfi1 encodes a zinc finger transcription factor involved in interleukin-2 signaling. To determine if Evi5 encodes a gene separate from Gfi1 that might also be involved in T-cell disease, we have searched within the Evi5 locus for novel transcripts. A 6.0 kb transcript was identified in these studies that spans the Evi5 locus and is disrupted by viral integration at Evi5. This transcript is expressed in all embryonic and adult mouse tissues examined. While blast searches indicated that Evi5 is a novel gene, homologies were detected between Evi5 and a known oncogene, Tre2, as well as mammalian and yeast cell cycle regulatory proteins. Evi5 thus encodes a gene separate from Gfi1 that may also be involved in T-cell disease.

The genes encoding the peripheral cannabinoid receptor and alpha-L-fucosidase are located near a newly identified common virus integration site, Evi11.

A new common region of virus integration, Evi11, has been identified in two retrovirally induced murine myeloid leukemia cell lines, NFS107 and NFS78. By interspecific backcross analysis, it was shown that Evi11 is located at the distal end of mouse chromosome 4, in a region that shows homology with human 1p36. The genes encoding the peripheral cannabinoid receptor (Cnr2) and alpha-L-fucosidase (Fuca1) were identified near the integration site by using a novel exon trapping system. Cnr2 is suggested to be the target gene for viral interference in Evi11, since proviruses are integrated in the first intron of Cnr2 and retroviral integrations alter mRNA expression of Cnr2 in NFS107 and NFS78. In addition, proviral integrations were demonstrated within the 3' untranslated region of Cnr2 in five independent newly derived CasBrM-MuLV (mouse murine leukemia virus) tumors, CSL13, CSL14, CSL16, CSL27, and CSL97. The Cnr2 gene encodes a seven-transmembrane G-protein-coupled receptor which is normally expressed in hematopoietic tissues. Our data suggest that the peripheral cannabinoid receptor gene might be involved in leukemogenesis as a result of aberrant expression of Cnr2 due to retroviral integration in Evi11.

Cooperative activation of Hoxa and Pbx1-related genes in murine myeloid leukaemias

Retroviruses induce myeloid leukaemia in BXH-2 mice by the insertional mutation of cellular proto-oncogenes or tumour suppressor genes. Disease genes can thus be identified by proviral tagging through the identification of common viral integration sites in BXH-2 leukaemia. Here, we describe a new approach for proviral tagging that greatly facilitates the identification of BXH-2 leukaemia genes. Using this approach, we identify three genes whose expression is activated by proviral integration in BXH-2 leukaemias; Hoxa7, Hoxa9, and a Pbx1-related homeobox gene, Meis1. Proviral activation of Hoxa7 or Hoxa9 is strongly correlated with proviral activation of Meis1 implying that Hoxa7 and Hoxa9 cooperate with Meis1 in leukaemia formation. These studies provide the first genetic evidence that Pbx1-related genes cooperate with Hox genes in leukaemia formation and identify a number of new murine myeloid leukaemia genes.

Evi-5, a common site of retroviral integration in AKXD T-cell lymphomas, maps near Gfi-1 on mouse chromosome 5.

We have identified a novel common site of retroviral integration, Evi-5, in AKXD T-cell lymphomas. All proviruses located at Evi-5 are clustered within a 7-kb genomic region and, where determined, are oriented in the same transcriptional direction. Interspecific backcross analysis localized Evi-5 to mouse chromosome 5, where it cosegregated with another common viral integration site, Gfi-1. Gfi-1 encodes a novel zinc finger transcription factor whose expression is thought to be important for interleukin-2 signaling. Physical mapping studies showed that Evi-5 is located approximately 18 kb upstream of Gfi-1, and Southern analysis showed that Gfi-1, like Evi-5, is a common integration site in AKXD T-cell tumors. With one exception, Evi-5 and Gfi-1 integrations were mutually exclusive. Ten of the tumors with Evi-5 or Gfi-1 integrations also harbored viral integrations at other common integration sites causally associated with T-cell disease. These results are consistent with the hypothesis that T-cell lymphomagenesis is a multistep disease and that viral integration at Evi-5 or Gfi-1 is causally associated with this disease process.

Retroviral integration at the Evi-2 locus in BXH-2 myeloid leukemia cell lines disrupts Nf1 expression without changes in steady-state Ras-GTP levels

Approximately 15% of BXH-2 myeloid leukemias harbor proviral integrations at the Evi-2 common viral integration site. Evi-2 is located within a large intron of the Nf1 tumor suppressor gene, raising the possibility that proviral integration at Evi-2 predisposes mice to myeloid tumor development by disrupting Nf1 expression. This hypothesis is supported by data suggesting that mutations in the human NF1 gene are causally associated with the development of juvenile chronic myelogenous leukemia (K. M. Shannon, P. O'Connell, G. A. Martin, D. Paderanga, K. Olson, P. Dinndorf, and F. McCormick, N. Engl. J. Med. 330:597-601, 1994) and mouse studies showing that aged mice, heterozygous for a germ line Nf1 mutation, develop myeloid leukemia with loss of the wild-type Nf1 allele (T. Jacks, T. S. Shih, E. M. Schmitt, R. T. Bronson, A. Bernards, and R. A. Weinberg, Nat. Genet. 7:353-361, 1994). To determine if viral integration at Evi-2 disrupts Nf1 expression, we derived a series of BXH-2 myeloid leukemia cell lines with or without viral integrations at Evi-2. In all cell lines examined, viral integration at Evi-2 resulted in the production of only truncated Nf1 transcripts and no stable, full-length neurofibromin. Although neurofibromin is a GTPase-activating protein (GAP) for p21ras proteins, its loss in the BXH-2 leukemic cell lines was not correlated with an increased steady-state level of p21ras bound to GTP. These data suggest that neurofibromin is not the sole mediator of Ras-GAP activity in myeloid cells and may have a GAP-independent function in myeloid cells.

Evi-1 raises AP-1 activity and stimulates c-fos promoter transactivation with dependence on the second zinc finger domain.

Evi-1 is a gene, encoding a zinc finger protein, associated with a common viral integration site in murine leukemias. It is suggested that Evi-1 plays important roles in embryogenesis and transformation of myeloid cells. To elucidate mechanisms by which Evi-1 induces such biological effects, we analyzed the relationship between Evi-1 and AP-1 which could regulate cellular proliferation and differentiation. When Evi-1 was expressed, transactivation through a 12-O-tetradecanoylphorbol 13-acetate-responsive element was observed in NIH3T3 and P19 cells. Evi-1-transfected P19 cells showed some differentiated phenotypes and increased expression of endogenous c-Jun and c-Fos. These results indicate that Evi-1 raises AP-1 activity. Evi-1 caused stimulation of the c-fos promoter transactivation, which seems to be a main mechanism of AP-1 activation, through at least two portions of the promoter. Evi-1 has the first zinc finger domain at the N terminus and the second zinc finger domain near the C-terminus. We constructed deletion mutants of Evi-1 and investigated the functions of these domains. It was shown that the second zinc finger domain is essential for the activation of AP-1 and transactivation of the c-fos promoter.

Identification of a common viral integration region in Cas-Br-E murine leukemia virus-induced non-T-, non-B-cell lymphomas

The Cas-Br-E murine leukemia virus is a nondefective retrovirus that induces non-T-, non-B-cell lymphomas in susceptible NIH/Swiss mice. By using a DNA probe derived from Cas-Br-E provirus-flanking sequences, we identified a DNA region, originally called Sic-1, rearranged in 16 of 24 tumors analyzed (67%). All proviruses were integrated in a DNA segment smaller than 100 bp and were in the same 5'-to-3' orientation. Ecotropic as well as mink cell focus-forming virus types were found integrated in that specific DNA region. On the basis of Southern blot analysis of somatic cell hybrids and progeny of an interspecies backcross, the Sic-1 region was localized on mouse chromosome 9 near the previously described proto-oncogenes or common viral integration sites: Ets-1, Cbl-2, Tpl-1, and Fli-1. Restriction map analysis shows that this region is identical to the Fli-1 locus identified in Friend murine leukemia virus-induced erythroleukemia cell lines and thus may contain sequences also responsible for the development of mouse non-T-, non-B-cell lymphomas.

Evi-2, a Common Integration Site Involved in Murine Myeloid Leukemogenesis

BXH-2 mice have the highest incidence of spontaneous retrovirally induced myeloid leukemia of any known inbred strain and, as such, represent a valuable model system for identifying cellular proto-oncogenes involved in myeloid disease. Chronic murine leukemia viruses often induce disease by insertional activation or mutation of cellular proto-oncogenes. These loci are identified as common viral integration sites in tumor DNAs. Here we report on the characterization of a novel common viral integration site in BXH-2 myeloid leukemias, designated Evi-2. Within the cluster of viral integration sites that define Evi-2, we identified a gene that has the potential for encoding a novel protein of 223 amino acids. This putative proto-oncogene possesses all of the structural features of a transmembrane protein. Within the transmembrane domain is a "leucine zipper," suggesting that Evi-2 is involved in either homopolymer or heteropolymer formation, which may play an important role in the normal functioning of Evi-2. Interestingly, the human homolog of Evi-2 has recently been shown to be tightly linked to the von Recklinghausen neurofibromatosis locus, suggesting a role for Evi-2 in human disease as well.

Evi-2, a common integration site involved in murine myeloid leukemogenesis.

BXH-2 mice have the highest incidence of spontaneous retrovirally induced myeloid leukemia of any known inbred strain and, as such, represent a valuable model system for identifying cellular proto-oncogenes involved in myeloid disease. Chronic murine leukemia viruses often induce disease by insertional activation or mutation of cellular proto-oncogenes. These loci are identified as common viral integration sites in tumor DNAs. Here we report on the characterization of a novel common viral integration site in BXH-2 myeloid leukemias, designated Evi-2. Within the cluster of viral integration sites that define Evi-2, we identified a gene that has the potential for encoding a novel protein of 223 amino acids. This putative proto-oncogene possesses all of the structural features of a transmembrane protein. Within the transmembrane domain is a "leucine zipper," suggesting that Evi-2 is involved in either homopolymer or heteropolymer formation, which may play an important role in the normal functioning of Evi-2. Interestingly, the human homolog of Evi-2 has recently been shown to be tightly linked to the von Recklinghausen neurofibromatosis locus, suggesting a role for Evi-2 in human disease as well.