Identification of a common ecotropic viral integration site, Evi-1, in the DNA of AKXD murine myeloid tumors.

AKXD-23 recombinant inbred mice develop myeloid tumors at a high frequency, unlike other AKXD recombinant inbred strains which develop B-cell lymphomas, T-cell lymphomas, or both. AKXD-23 myeloid tumors are monoclonal, and their DNA contains somatically acquired proviruses, suggesting that they are retrovirally induced. We identified a common site of ecotropic proviral integration that is present in the DNA of all AKXD-23 myeloid tumors that were analyzed and in the DNA of all myeloid tumors that occur in AKXD strains other than AKXD-23. We designated this locus Evi-1 (ecotropic viral integration site 1). Rearrangements in the Evi-1 locus were also detected in the DNA of a number of myeloid tumors and myeloid cell lines isolated from strains other than AKXD. In contrast, few Evi-1 rearrangements were detected in the DNA of T- or B-cell tumors. Evi-1 may thus identify a new proto-oncogene locus that is involved in myeloid disease.     

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.     

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.     

Identification of a new common provirus integration site in gross passage A murine leukemia virus-induced mouse thymoma DNA.

The Gross passage A murine leukemia virus (MuLV) induced T-cell leukemia of clonal (or oligoclonal) origin in inoculated mice. To study the role of the integrated proviruses in these tumor cells, we cloned several newly integrated proviruses (with their flanking cellular sequences) from a single tumor in procaryotic vectors. With each of the five clones obtained, a probe was prepared from the cellular sequences flanking the provirus. With one such probe (SS8), we screened several Gross passage A MuLV-induced SIM.S mouse tumor DNAs and found that, in 11 of 40 tumors, a provirus was integrated into a common region designated Gin-1. A 26-kilobase-pair sequence of Gin-1 was cloned from two lambda libraries, and a restriction map was derived. All proviruses were integrated as a cluster in the same orientation within a 5-kilobase-pair region of Gin-1, and most of them had a recombinant structure of the mink cell focus-forming virus type. The frequency of Gin-1 occupancy by provirus was much lower in thymoma induced by other strains of MuLV in other mouse strains. Using somatic-cell hybrid DNAs, we mapped Gin-1 on mouse chromosome 19. Gin-1 was not homologous to 16 known oncogenes and was distinct from the other common regions for provirus integration previously described. Therefore, Gin-1 appears to represent a new common provirus integration region. The integration of a provirus within Gin-1 might be an important event leading to T-cell transformation, and the Gin-1 region might harbor sequences which are involved in tumor development.     

The c-myc locus is a common integration site in type B retrovirus-induced T-cell lymphomas

Type B leukemogenic virus (TBLV) induces rapidly appearing T-cell leukemias. TBLV insertions near the c-myc gene were detectable in 2 of 30 tumors tested, whereas 80% of the tumors showed c-myc overexpression. TBLV insertions on chromosome 15 (including a newly identified locus, Pad7) may cause c-myc overexpression by cis-acting effects at a distance.     

Sint1, a common integration site in SL3-3-induced T-cell lymphomas, harbors a putative proto-oncogene with homology to the septin gene family

The murine retrovirus SL3-3 is a potent inducer of T-cell lymphomas when inoculated into susceptible newborn mice. Previously, DNAs from twenty SL3-3-induced tumors were screened by PCR for provirus integration sites. Two out of 20 tumors demonstrated clonal provirus insertion into a common region. This region has now been isolated and characterized. The region, named SL3-3 integration site 1 (Sint1), maps to the distal end of mouse chromosome 11, corresponding to human chromosome 17q25, and may be identical to a mouse mammary tumor virus integration site in a T-cell lymphoma, Pad3. Two overlapping genomic lambda clones spanning about 35 kb were isolated and used as a starting point for a search for genes in the neighborhood of the virus integration sites. A genomic fragment was used as a hybridization probe to isolate a 3-kb cDNA clone, the expression of which was upregulated in one of two tumors harboring a provirus in Sint1. The cDNA clone is predicted to encode a protein which shows 97.0% identity to a human septin-like protein encoded by a gene which has been found as a fusion partner gene of MLL in an acute myeloid leukemia with a t(11;17)(q23;q25). Together these findings raise the possibility that a proto-oncogene belonging to the septin family, and located about 15 kb upstream of the provirus integration sites, is involved in murine leukemia virus-induced T-cell lymphomagenesis.     

Rorgamma (Rorc) is a common integration site in type B leukemogenic virus-induced T-cell lymphomas

The retrovirus type B leukemogenic virus (TBLV) causes T-cell lymphomas in mice. We have identified the Rorgamma locus as an integration site in 19% of TBLV-induced tumors. Overexpression of one or more Rorgamma isoforms in >77% of the tumors tested may complement apoptotic effects of c-myc overexpression.     

Identification of a common helper provirus integration site in Abelson murine leukemia virus-induced lymphoma DNA.

Abelson murine leukemia virus induces oligoclonal pre-B lymphoma in mice. The expression of the v-abl oncogene in target cells does not appear to be sufficient for tumor induction in several mouse strains, and additional genetic events are thought to be required. We postulated that the helper Moloney murine leukemia virus might induce these events, and its potential role as an insertional mutagen was assessed by the search of a common helper provirus integration site in Abelson murine leukemia virus lymphomas. Molecular cloning of cellular sequences adjacent to Moloney proviruses enabled us to identify a cellular region, designated Ahi-1, which was found occupied by the helper proviruses in 16% of Abelson pre-B-cell lymphomas. All proviruses for which the precise integration site within Ahi-1 could be mapped were found to be in the same orientation. Ahi-1 has been mapped to mouse chromosome 10 and represents a new common proviral integration site. These data suggest that the helper virus contributes to the induction of secondary genetic events which may be important for the development of Abelson murine leukemia virus-induced pre-B-cell lymphoma.     

Nucleosomes, DNA-binding proteins, and DNA sequence modulate retroviral integration target site selection.

Integration of retroviral DNA can serve as a paradigm for cellular functions that are affected by the packaging of DNA into chromatin. We have used a novel polymerase chain reaction-based assay to survey DNA and chromatin for the precise distribution of many integration sites. Integration into naked DNA targets is non-uniform, implying a nucleotide sequence bias. In chromatin, integration occurs preferentially at positions where the major groove is on the exposed face of the nucleosomal DNA helix, generating a 10 bp periodic spacing of preferred sites. Chromatin assembly enhances the reactivity of many sites, so that integration occurs most frequently at sites in nucleosomal, rather than nucleosome-free, regions of minichromosomes. In contrast, integration is prevented in a region occupied by a site-specific DNA-binding protein. Comparisons of integration events mediated by viral nucleoprotein complexes or by two different retroviral integrases show that the integration machinery also affects target site selection.     

Avian bic, a gene isolated from a common retroviral site in avian leukosis virus-induced lymphomas that encodes a noncoding RNA, cooperates with c-myc in lymphomagenesis and erythroleukemogenesis

bic is a novel gene identified at a common retroviral integration site in avian leukosis virus-induced lymphomas and has been implicated as a collaborator with c-myc in B lymphomagenesis. It lacks an extensive open reading frame and is believed to function as an untranslated RNA (W. Tam, Gene 274:157-167, 2001; W. Tam, D. Ben-Yehuda, and W. S. Hayward, Mol. Cell. Biol. 17:1490-1502, 1997). The oncogenic potential of bic, particularly its ability to cooperate with c-myc in oncogenesis, was tested directly by expressing c-myc and bic, either singly or in pairwise combination, in cultured chicken embryo fibroblasts (CEFs) and in chickens using replication-competent retrovirus vectors. Coexpression of c-myc and bic in CEFs caused growth enhancement of cells. Most importantly, chick oncogenicity assays demonstrated that bic can cooperate with c-myc in lymphomagenesis and erythroleukemogenesis. The present study provides direct evidence for the involvement of untranslated RNAs in oncogenesis and provides further support for the role of noncoding RNAs as riboregulators.     

Evidence for the involvement of pim-2, a new common proviral insertion site, in progression of lymphomas.

We have compared proviral integrations near (putative) proto-oncogenes in Moloney murine leukemia virus-induced primary and transplanted T cell lymphomas. We previously found proviruses integrated near c-myc, pim-1, and N-myc in primary tumors (Selten et al., 1984; Van Lohuizen et al., 1989a; Van Lohuizen et al., 1989b). We have now identified an additional common proviral integration site, called pim-2, that carries somatically acquired proviruses in the majority of transplanted tumors. In primary tumors integration near pim-2 is usually undetectable or present in only a minor fraction of the tumor cells. This subpopulation selectively grows out upon transplantation. Insertion near pim-2 is a relatively late event in tumorigenesis and is often preceded by proviral insertions in other common insertion sites, yielding tumor clones which carry proviruses in up to three different common insertion sites within the same cell (c-myc, pim-1 and pim-2). The data suggest that pim-2 plays an important role in tumor progression.     

Activation of a novel proto-oncogene, Frat1, contributes to progression of mouse T-cell lymphomas.

Acceleration of lymphomagenesis in oncogene-bearing transgenic mice by slow-transforming retroviruses has proven a valuable tool in identifying cooperating oncogenes. We have modified this protocol to search for genes that can collaborate effectively with the transgene in later stages of tumor development. Propagation of tumors induced by Moloney murine leukemia virus (M-MuLV) in E mu-Pim1 or H2-K-myc transgenic mice by transplantation to syngeneic hosts permitted proviral tagging of 'progression' genes. Molecular cloning of common proviral insertion sites that were detected preferentially in transplanted tumors led to the identification of a novel gene, designated Frat1. The initial selection for integrations near Frat1 occurs in primary tumor cells that have already acquired proviruses in other common insertion sites, yielding primary lymphomas that contain only a minor fraction of tumor cells with an activated Frat1 allele. Transplantation of such primary lymphomas allows for a further expansion of tumor cell clones carrying a proviral insertion near Frat1, resulting in detectable Frat1 rearrangements in 17% of the transplanted E mu-Pim1 tumors and 30% of the transplanted H2-K-myc tumors, respectively. We have cloned and sequenced both the mouse Frat1 gene and its human counterpart. The proteins encoded by Frat1 and FRAT1 are highly homologous and their functions are thus far unknown. Tumor cell lines with high expression of Myc and Pim1 acquired an additional selective advantage in vivo upon infection with a Frat1-IRES-lacZ retrovirus, thus underscoring the role of Frat1 in tumor progression, and the ability of Frat1 to collaborate with Pim1 and Myc in lymphomagenesis.     

Identification of a novel tumor-specific CTL epitope presented by RMA, EL-4, and MBL-2 lymphomas reveals their common origin

C57BL/6 mice generate a vigorous H-2Db-restricted CTL response against murine leukemia virus (MuLV)-induced tumors. For many years it has been suggested that this response is directed to an MuLV-encoded peptide as well as to a nonviral tumor-associated peptide. Recently, a peptide from the leader sequence of gag was demonstrated to be the MuLV-derived epitope. Here we describe the molecular identification of the tumor-associated epitope. Furthermore, we show that the CTL response against this epitope can restrict the outgrowth of MuLV-induced tumors in vivo. The epitope is selectively presented by the MuLV-induced T cell tumors RBL-5, RMA, and MBL-2 as well as by the chemically induced T cell lymphoma EL-4. Intriguingly, these tumors share expression of the newly identified epitope because they represent variants of the same clonal tumor cell line, as evident from sequencing of the TCR alpha- and beta-chains, which proved to be identical. Our research shows that all sources of RBL-5, RMA, RMA-S, MBL-2, and EL-4 tumors are derived from a single tumor line, most likely EL-4.     

Identification of a novel mouse membrane-bound family 1 glycosidase-like protein,which carries an atypical active site structure

We have identified a novel mouse gene klph (Klotho-LPH related protein; where LPH stands for lactase-phlorizin hydrolase) that encodes a novel mammalian family 1 glycosidase-like protein. KLPH was a putative type I membrane protein that consists of N-terminal signal sequence, glycosidase domain, transmembrane region and short cytoplasmic tail. Despite its overall structural similarity to other family 1 glycosidases, the glutamic acid for the acid-base catalyst was not conserved in this protein. klph mRNA was predominantly expressed in the kidney and skin. Epitope-tagged KLPH was localized to the perinuclear tubular network structure of the endoplasmic reticulum in cultured cells.     

Allocation of a transgenic c-myc integration site to mouse chromosome 8B3-C1

A recombinant plasmid containing the mouse c-myc gene was injected into mouse pronuclei. The transgenic line 478 contains about 100 copies of the transgene integrated into one chromosome site. By in situ hybridization, the integration site was localized to chromosome 8B3-C1.     

Identification of a novel cDC2-committed progenitor within mouse common dendritic cell progenitor population

Dear Editor, Dendritic cells(DCs)are the most efficient professional antigen presenting cells that act as sentinels of the immune system and conduct vital funct...