Oncogenic potential of the miR-106-363 cluster and its implication in human T-cell leukemia.

We previously reported the identification of the Kis2 common retrovirus integration site, located on mouse chromosome X, in radiation leukemia virus-induced T-cell leukemias. Tumors with a provirus at the Kis2 locus overexpressed a novel noncoding RNA (ncRNA) with a complex splicing pattern and no polyA tail. Database upgrade revealed the presence of a microRNA (miRNA) cluster, miR-106-363, just downstream of the Kis2 ncRNAs. We found that Kis2 ncRNAs are the pri-miRNA of miR-106-363, and we present evidence that Kis2 ncRNA overexpression in mouse tumors results in miR-106a, miR-19b-2, miR-92-2, and miR-20b accumulation. We show the oncogenic potential of those miRNAs in anchorage independence assay and confirm pri-miR-106-363 overexpression in 46% of human T-cell leukemias tested. This overexpression contributes in rising miR-92 and miR-19 levels, as this is the case for miR-17-92 cluster overexpression. Furthermore, we identified myosin regulatory light chain-interacting protein, retinoblastoma-binding protein 1-like, and possibly homeodomain-interacting protein kinase 3 as target genes of this miRNA cluster, which establishes a link between these genes and T-cell leukemia for the first time.

Radiation leukemia virus common integration at the Kis2 locus: simultaneous overexpression of a novel noncoding RNA and of the proximal Phf6 gene.

Retroviral tagging has been used extensively and successfully to identify genes implicated in cancer pathways. In order to find oncogenes implicated in T-cell leukemia, we used the highly leukemogenic radiation leukemia retrovirus VL3 (RadLV/VL3). We applied the inverted PCR technique to isolate and analyze sequences flanking proviral integrations in RadLV/VL3-induced T lymphomas. We found retroviral integrations in c-myc and Pim1 as already reported but we also identified for the first time Notch1 as a RadLV common integration site. More interestingly, we found a new RadLV common integration site that is situated on mouse chromosome X (XA4 region, bp 45091000). This site has also been reported as an SL3-3 and Moloney murine leukemia virus integration site, which strengthens its implication in murine leukemia virus-induced T lymphomas. This locus, named Kis2 (Kaplan Integration Site 2), was found rearranged in 11% of the tumors analyzed. In this article, we report not only the alteration of the Kis2 gene located nearby in response to RadLV integration but also the induction of the expression of Phf6, situated about 250 kbp from the integration site. The Kis2 gene encodes five different alternatively spliced noncoding RNAs and the Phf6 gene codes for a 365-amino-acid protein which contains two plant homology domain fingers, recently implicated in the Börjeson-Forssman-Lehmann syndrome in humans. With the recent release of the mouse genome sequence, high-throughput retroviral tagging emerges as a powerful tool in the quest for oncogenes. It also allows the analysis of large DNA regions surrounding the integration locus.

Cyclo-oxygenase type 2-dependent prostaglandin E2 secretion is involved in retrovirus-induced T-cell dysfunction in mice.

MAIDS (murine AIDS) is caused by infection with the murine leukaemia retrovirus RadLV-Rs and is characterized by a severe immunodeficiency and T-cell anergy combined with a lymphoproliferative disease affecting both B- and T-cells. Hyperactivation of the cAMP-protein kinase A pathway is involved in the T-cell dysfunction of MAIDS and HIV by inhibiting T-cell activation through the T-cell receptor. In the present study, we show that MAIDS involves a strong and selective up-regulation of cyclo-oxygenase type 2 in the CD11b+ subpopulation of T- and B-cells of the lymph nodes, leading to increased levels of PGE2 (prostaglandin E2). PGE2 activates the cAMP pathway through G-protein-coupled receptors. Treatment with cyclo-oxygenase type 2 inhibitors reduces the level of PGE2 and thereby reverses the T-cell anergy, restores the T-cell immune function and ameliorates the lymphoproliferative disease.

Identification of a common site of provirus integration in radiation leukemia virus-induced T-cell lymphomas in mice.

The BL/VL(3) Kaplan radiation leukemia virus (RadLV-VL(3)) is a nondefective retrovirus that induces T cell lymphomas in several strains of mice. By using DNA probes derived from RadLV/VL(3) provirus-flanking sequences cloned from the BL/VL(3) cell line, we identified a DNA region rearranged in 5 of 19 tumors analysed (25%). All proviruses were integrated in the same 5'-to-3' orientation in a small DNA region called Kis1 (Kaplan integration site 1). This region was localized on distal mouse chromosome 2 in a region not previously identified as important to lymphomagenesis. The cells rearranged at the Kis1 locus represent a clonal subpopulation of the clonal tumor masses examined, indicating a probable role of Kis1 in tumor progression.

The regulation of murine H-2Dd expression by activation transcription factor 1 and cAMP response element binding protein.

Resistance to radiation leukemia virus (RadLV)-induced leukemia is correlated with an increase in H-2Dd expression on the thymocyte surface. It has been shown that elevated H-2Dd expression on infected thymocytes is a result of elevated mRNA transcription and that the transcriptional increase is correlated with elevated levels of a DNA binding activity, H-2 binding factor 1 (H-2 BF1), which recognizes the 5'-flanking sequence (5'-TGACGCG-3') of the H-2Dd gene. Recently, it has been shown that the activation transcription factor 1 (ATF-1) homodimer is one form of the H-2 BF1 complex. Here we demonstrate that the cAMP response element binding protein (CREB) homodimer and the heterodimer of CREB/ATF-1 also recognize the cis regulatory motif and are two additional forms of the H-2 BF1 complex. The levels of mRNA encoding ATF-1 and CREB were both increased in RadLV-infected thymocytes that showed increased levels of H-2 mRNA. Also, all three H-2 BF1 binding activities, ATF-1 homodimer, CREB homodimer, and ATF-1/CREB heterodimer, were increased in RadLV-infected thymocytes that expressed high levels of H-2Dd Ag on the cell surface. Transfection experiments demonstrated that ATF-1 and CREB activated a reporter plasmid containing the H-2 BF1 motif. These observations strongly suggest that both ATF-1 and CREB are involved in the regulation of H-2 gene expression following RadLV infection of mouse thymocytes.

Phylogenetic relationship of the complete Rauscher murine leukemia virus genome with other murine leukemia virus genomes.

We report the complete nucleotide sequence of the genome of Rauscher murine leukemia virus (R-MuLV), the replication-competent helper virus present in the Rauscher virus complex, and its phylogenetic relationship with other murine leukemia virus genomes. An overall sequence identity of 97.6% was found between R-MuLV and the Friend helper virus (F-MuLV), and the two viruses were closely related on the phylogenetic trees constructed from either gag, pol, or env sequences. Moloney murine leukemia virus (Mo-MuLV) was the next closest relative to R-MuLV and F-MuLV on all trees, followed by Akv and radiation leukemia virus (RadLV). The most distantly related helper virus was Hortulanus murine leukemia virus (Ho-MuLV). Interestingly, Cas-Br-E branched with Mo-MuLV on the gag and pol trees, whereas on the env tree, it revealed the highest degree of relatedness to Ho-MuLV, possibly due to an ancient recombination with an Ho-MuLV ancestor. In summary, a phylogenetic analysis involving various MuLVs has been performed, in which the postulated close relationship between R-MuLV and F-MuLV has been confirmed, consistent with the pathobiology of the two viruses.

Prophylactic intervention in RadLV-induced lymphomagenesis with an anti-IL-4 monoclonal antibody.

Intrathymic inoculation of the radiation leukemia virus (RadLV) into C57BL/6 mice induces thymic lymphomas after 4-6 months. During premalignant latency, a population of RadLV-infected prelymphoma (PL) cells (whose survival is dependent on autostimulation with IL-4) persists in the thymus. PL cells explanted from RadLV-inoculated mice can be propagated in cultures containing IL-4, and in vitro growth of PL cells is effectively inhibited by anti-IL-4 antibodies. We subjected RadLV-inoculated mice to prophylactic treatment with anti-IL-4 antibodies and a virus-specific immunotoxin (IT). Administration of IT delayed the onset of lymphoma but was not curative. Anti-IL-4 antibodies had a similar effect when administered at low doses. High doses of anti-IL-4, given 3-5 weeks after virus inoculation, provided complete protection against lymphoma. These results demonstrate the effectiveness of prophylactic intervention during premalignancy by using antagonists that restrain the growth of PL cells.

Activation transcription factor 1 involvement in the regulation of murine H-2Dd expression.

Resistance to radiation leukemia virus-induced leukemia is correlated with an increase in H-2D expression on the thymocyte surface. Recently, it has been shown that elevated H-2Dd expression on the infected thymocyte is a result of elevated mRNA transcription and that the transcriptional increase is correlated with elevated levels of a DNA binding activity, H-2 binding factor 1 (H-2 BF1), which recognizes the 5'-flanking sequences (5'-TGACGCG-3') of the H-2Dd gene. This target for transcription factor binding has been found to be identical in the 5'-regulatory region of 12 rodent class I genes, nine of which have been shown to be functional genes. Furthermore, this cis-element is found 5' of 20 primate class I genes (15 human genes), seven of which are known to be functional. Here, we demonstrate that activation transcription factor 1 (ATF-1) is one component of H-2 BF1. In addition, the levels of ATF-1 mRNA in uninfected and radiation leukemia virus-infected thymocytes parallel those of H-2Dd mRNA, and therefore, it is suggested that ATF-1 up-regulates the transcription of the H-2Dd gene after radiation leukemia virus infection of thymocytes. Transfection experiments also demonstrate that ATF-1 activates a reporter plasmid that contains the H-2 BF1 motif, but not a reporter lacking this motif. This is the first demonstration of the interaction of ATF-1 with 5'-regulatory sequences of major histocompatibility complex class I genes.

Molecular cloning of the genes suppressed in RVC lymphoma cells by topoisomerase inhibitors.

Etoposide is a topoisomerase II inhibitor that induces DNA cleavable complex and has been used as an antitumor drug. We isolated two genes that were transcriptionally suppressed at an early stage of incubation in etoposide-treated RVC lymphoma cells, using modified PCR-based subtractive hybridization. Sequencing revealed that one of these genes, which was approximately 1.7 kb and which encoded a protein of 320 amino acids, was identical to hnRNP A1. The other was a novel gene of about 2.2 kb encoding a protein of 469 amino acids. These genes were also down-regulated in the cells incubated with camptothecin, a topoisomerase I inhibitor that induces DNA single strand breaks, but not in those exposed to ICRF-154, a topoisomerase II inhibitor that does not induce DNA cleavable complex formation. These results suggest that the early down-regulation of these genes contributes to the cytotoxicity of the topoisomerase inhibitors that induce DNA cleavage.

Clonal dominance of RadLV-induced lymphomas.

RadLV-induced leukemogenesis begins with the emergence of a pleioclonal population of preleukemic (PL) cells, which subsequently gives rise to a monoclonal lymphoma. We have recently found that the pleioclonal-->monoclonal transition may occur early during the PL latency and long before the eruption of a full blown lymphoma. We sought to find out what causes one PL clone to become dominant. Our working hypothesis was that a dominant clone(s) at the PL stage has the ability to inhibit the development of other, recessive clones. Since some premalignant characteristics of a progenitor clone are probably maintained in the descending lymphoma, we studied whether tumors that developed after injection of a high dose (HD) of PL cells were dominant over tumors that developed after injection of a limiting dose (LD) of PL cells. To identify dominant clones, HD and LD lymphomas were mixed in a co-culture and the outgrowth of one clone over the other was determined by T beta-TCR rearrangement analysis. A checker-board combination of seven lymphomas revealed a dominance hierarchy scale. Lymphomas induced directly by the virus (without transfer) were dominant over both HD and LD lymphomas. High dose lymphomas were dominant over LD lymphomas and LD lymphomas were always recessive. The speed at which a dominant lymphoma outgrew the co-culture suggested that dominance is acquired through the ability of the prevailing cells to actively suppress the growth of recessive cells.

Prophylactic intervention in radiation-leukemia-virus-induced murine lymphoma by the biological response modifier polysaccharide K.

Polysaccharide K (PSK) is a biological response modifier used for adjuvant immunotherapy of malignant diseases. We studied the potential applicability of PSK for preventing tumor progression using an experimental model of murine lymphoma. Mice inoculated with the radiation leukemia virus (RadLV) develop thymic lymphomas after a latency of 3-6 months. However, 2 weeks after virus inoculation, prelymphoma cells can already be detected in the thymus. We found that PSK treatment induced hyperresponsiveness to concanavalin A and heightened production of interleukin-2 (IL-2) and IL-4 in spleen cells of both control and prelymphoma mice. The response was transient and was accompanied with a dominant usage of T cells expressing V beta 8, but other T cell subsets were also stimulated by PSK. T lymphoma cells expressing V beta 8.2 underwent apoptosis when incubated with PSK. Treatment of RadLV-inoculated mice with PSK delayed the onset of overt lymphoma (and mortality) but could not protect the mice from the disease. Combined treatment with PSK and a RadLV-specific immunotoxin prevented synergistically the progression of the prelymphoma cells to frank lymphoma. The results suggest that PSK contains a superantigen-like component that selectively activates V beta 8+ T cells. Its administration prelymphoma mice interfered with the process of lymphoma progression.

The effects of passive antiviral immunotherapy in AKR mice: I. The susceptibility of AKR mice to spontaneous and induced T cell lymphomagenesis.

The AKR inbred mouse strain displays a high incidence of spontaneous T cell lymphomas that arise predominantly in the thymus of 6 to 12-month-old mice. Heterogenous nonacute transforming retroviruses are associated with the etiology of the disease: the endogenous ecotropic viruses (inherited in AKR mice at two non-linked chromosomal loci, Akv-1 and Akv-2), the xenotropic virus and recombinant viruses. Prevention of spontaneous T cell lymphomagenesis in AKR mice by passive anti-viral immunotherapy was accomplished by suppressing endogenous ecotropic virus release. Treatment with monoclonal antibody Hy-72 reacting only with Akv1 type ecotropic viruses, or with mAb 18-5 with specificity for both ecotropic and MCF recombinant virus envelope glycoprotein (administered from birth for 10 days) inhibited similarly T cell lymphoma development. A reduced thymus cellularity observed in these mAb treated mice coincided with reduced level of earliest intrathymic low CD4 precursor population in their thymus. The role of endogenous viruses (MuLV) and presence of potential lymphoma cells (PLCs) (identified among bone marrow cells of untreated AKR mice) in enhanced T cell lymphomagenesis in AKR mice, triggered by different leukemogenic agents, was evaluated. Intrathymic injection of the radiation leukemia virus variant A-RadLV or administration of methylnitrosourea resulted in a high lymphoma incidence within a short latent period of 80-100 days irrespective of the presence or absence of MuLV or PLCs in these treated mice. Thus, a direct action of these agents on thymocytes seems to occur. The high susceptibility of untreated AKR mice to radiation induced T cell lymphomagenesis was not affected by pretreatment with mAb Hy-72; in contrast to markedly reduced sensitivity following pretreatment with mAb 18-5 (15 vs 100%). The mAb 18-5 induced resistance to radiation lymphomagenesis seems to be related to defects in the bone marrow stem cell pool as well as in the thymus microenvironment of mAb 18-5 treated mice. Thus, different developmental pathways are involved in enhanced T cell lymphomagenesis in AKR mice.

Characterization of mature and immature RadLV-induced thymic T-cell lines for tumorigenesis and MHC-class-I gene expression.

Class-I-MHC molecules are divided into class-Ia molecules, which play major roles in recognition of virus-infected cells, graft rejection and immune responses against tumors, and class-Ib molecules, which are less polymorphic and may be responsible for presenting unique classes of peptides. Our report characterizes RadLV-induced thymic T-cell lines that differ both in their tumorigenic potential and in the level of protein for class-Ia and TL genes. The PD1.1 cell line is CD4-CD8+ and expresses relatively high levels of class-I as compared with the CD4+CD8+ PD1.2 cell line. These class-I-expression levels correlate with thymocytes and splenic T cells of the same phenotype, except that normal cells fail to express TL3b. Interferon-treated PD1.2 cells demonstrate significantly lower levels of class-I expression than do interferon-treated PD1.1 cells, and were shown to contain large amounts of degraded class-I mRNA, at least some of which was TL in origin. These RNA products were not detected in PD1.1 cells, suggesting the existence of a mechanism controlling cell-specific and gene-specific mRNA stability. Such RadLV-induced cell lines provide a means for obtaining stage-specific T cells, which can be used for studying the regulation of class-I gene expression during T-cell differentiation, as well as factors that differentially regulate class-Ia and class-Ib expression and are potentially useful for studying T-cell differentiation in general.

Induction of leukemia in mice using a radiation leukemia virus-induced cell line: a model system for studying oncogenic progression.

Leukemogenesis induced by slowly transforming retroviruses is a multistep process which is difficult to dissect because of its long latency and the problem of distinguishing oncogenic from differentiative events. A method for leukemia induction in mice has been developed using a cell line isolated following in vitro infection with the slowly transforming murine radiation leukemia virus (RadLV). The CI-V13D cell line represents a lymphoid precursor cell type at an early stage in cell transformation and can develop subcutaneous tumors in irradiated syngeneic hosts but not in allogeneic mice even after sublethal irradiation. Selective growth in allogeneic (CBA/H) mouse thymus has been demonstrated, but this requires preirradiation of the recipient. Upon reisolation from CBA/H thymus, C1-V13D progeny clones displayed increased tumorigenic potential in comparison to the 'parental' CI-V13D cell line. Tumorigenicity was shown to increase with serial passage through thymus and electron micrographs of clones also revealed increased production of C-type retroviruses. This new model for oncogenic progression should be more amenable to analysis of early genetic changes occurring during replication of leukemia in the thymus.

A RadLV-induced gamma delta T cell lymphoma displaying an antitumoral cytotoxicity.

We described previously the induction by RadLV infection of a lymphoma (NS8) expressing a cytolytic activity against an MCA-induced fibrosarcoma. We report here that the cytolytic activity of these immortalized CD3+, CD8+ T cells is non-MHC-restricted. We then determined the structure and expression of the TCR chains expressed by these cells. Only partial rearrangement of the beta chain associated to an abnormally short transcript was detected in NS8 cells, whereas the gamma chain is rearranged and normally transcribed. On the opposite, rearrangement and expression of these genes were found in the other RadLV-induced lymphomas analysed. Moreover, gamma delta TCR proteins were detected on the cell surface of NS8 cells only, whereas the alpha beta complex, presents on the other T cell lines, was not expressed by NS8 cells. The ability of NS8 cells or of cells obtained from activated lymph nodes (harvested from mice grafted with the T2 sarcoma used to induce the NS8 line) to lyse the T2 sarcoma cell line was analysed. With both types of lymphocytes, the cytotoxicity was partially inhibited by a preincubation of the effector cells with anti-gamma delta antibodies. These results demonstrate that gamma delta lymphocytes can mediate anti-tumour cytotoxicity and NS8 lymphoma line may be representative of the TCR gamma delta CD8+ T cell subpopulation expressing non MHC-restricted cytotoxicity and displaying antitumoral activity.

Analysis of oncogenic progression in a radiation leukemia virus model.

The mechanism by which non-oncogene-bearing, slowly transforming retroviruses induce leukemia is not well understood, but appears to represent a multi-step process. Cell lines have been isolated following in vitro infection of lymphoid cells with radiation leukemia virus (RadLV) and they have been used to develop a two-step model for leukemia development. Thymic tumors were induced when one of the cell lines, C1-V13D, was inoculated into CBA/H mouse thymus. Upon reisolation of C1-V13D cells after one, two and three passages through thymus, individual cloned cell lines displayed increased tumorigenic potential compared with the non-tumorigenic parental line. Southern analysis has been used to track any genetic changes occurring while cells undergo further transformation and become increasingly tumorigenic. Specifically, retrovirus integration has been monitored in clones derived from C1-V13D at the primary, secondary and tertiary passage through thymus using probes specific for long terminal repeat (LTR), gag, pol and env genes of RadLV. The data indicate multiple ecotropic retrovirus integration sites in C1-V13D cells. Primary thymic tumors also showed the integration of a new recombinant or defective virus. There was no evidence that new ecotropic retrovirus integration had occurred during subsequent passage of primary tumors through the thymus, i.e. during the progression to oncogenesis. All data indicate an important role for the thymic environment in the development of a fully transformed cell.

Radiation leukemia virus-induced thymic lymphomas express a restricted repertoire of T-cell receptor V beta gene products.

We have investigated the phenotypic changes that take place during the process of neoplastic transformation in the thymocytes of C57BL/Ka mice infected by the radiation leukemia virus (RadLV). By the combined use of antibodies against the envelope glycoprotein gp70 of RadLV, the transformation-associated cell surface marker 1C11, and the CD3-T-cell receptor (TCR) complex, we found that in the RadLV-infected thymus, the earliest expression of viral gp70 is in 1C11hi cells; a small but significant percentage of these cells also express CD3. A first wave of viral replication, manifested by the expression of high levels of gp70 in thymocytes (over 70% positive), reaches a peak at 2 weeks; during this period, no significant changes are observed in the expression of 1C11 or CD3. The population of gp70+ cells is drastically reduced at 3 to 4 weeks after infection. However, a second cohort of gp70+ cells appears after 4 weeks, and these cells express high levels of 1C11 and TCR determinants as well. RadLV-induced lymphomas differ from normal thymocytes in their CD4 CD8 phenotype, with domination by one or more subsets. Characterization of TCR gene rearrangements in RadLV-induced lymphomas shows that most of these tumors are clonal or oligoclonal with respect to the J beta 2 TCR gene, while the J beta 1 TCR gene is rearranged in a minority (4 of 11) of lymphomas. TCR V beta repertoire analysis of 12 tumors reveals that 6 (50%) express exclusively the V beta 6 gene product, 2 (17%) are V beta 5+, and 1 (8%) each are V beta 8+ and V beta 9+. In normal C57BL/Ka mice, V beta 6 is expressed on 12%, V beta 5 is expressed on 9%, V beta 8 is expressed on 22%, and V beta 9 is expressed on 4% of TCRhi thymocytes. Thus, it appears that RadLV-induced thymic lymphomas are not randomly selected with respect to expressed TCR V beta type.

Thymus involvement in murine acquired immunodeficiency (MAIDS).

Due to self-renewal of the peripheral pool of T-cells, adult thymectomy has normally little influence on immunocompetence. However, thymus might play a more important role in the setting of viral-induced cytopathic effects on T-cells in the periphery. Therefore, thymus weight, cell numbers, and subset distribution were sequentially analysed after infection with RadLV-Rs, a viral mixture known to induce murine retrovirus induced immunodeficiency (MAIDS). Infection induced thymic atrophy (concerning organ weight as well as total cell number) which culminated seven weeks after inoculation. The atrophic process mostly reflected the depletion of double positive CD4+ CD8+ cells since their proportion sharply decreased around week 6. Single positive T-cells were less affected by the process. The proportion of B-cells progressively increased. Surprisingly, there was a strong correlation between the extent of atrophy and the frequency of B-cells in the thymus. Finally, an abnormal CD4+ T-cell subset lacking Thy-1 and previously described in the periphery also appeared in the thymus and its frequency was strongly correlated with the expansion of B-cells in this organ.