Common proviral integration sites in C57BL mouse lymphomas induced by radiation leukemia virus and absence of novel virus-related sequences in radiogenic lymphoma DNA

Three C57BL/Ka mice were inoculated with RadLV/VL3, a thymotropic and leukemogenic virus population released by the permanent BL/VL3 cell line, which was derived from a C57BL/Ka lymphoma induced by radiation leukemia virus (RadLV). The neoplastic thymus and bone marrow cells from these mice were grown until the cultures became permanently established, and their DNAs were examined for the presence of virus-related sequences by restriction enzyme analysis. All six cell lines displayed identical EcoRI and BamHI restriction fragments, not found in control C57BL/Ka DNA and accounting for the presence of more than one novel provirus. The primary and secondary tumors were thus clonal and, even though they were obtained from different animals, possessed identical integration sites. The BL/VL3 cell line also displayed the clonal appearance of novel proviral sequences, partly identical, with respect to location and BamHI restriction pattern, to those found in the RadL/VL3-induced tumor cell lines. Neither radiation-induced tumors, nor cloned cell lines derived therefrom, whether producing leukemogenic virus (BL/RL12-P) or not (BL/RL12-NP), displayed the presence of novel virus-related sequences when compared with control tissues. Our results strongly suggest not only that, as described in the case of avian leukosis virus-induced tumors of the chicken, RadLV-induced leukemogenesis might be a consequence of cellular gene activation by promotor insertion, but also that more than one integration site might be involved. Radiation-induced tumorigenesis might be initiated by a comparable mechanism, not requiring the participation of a virus.     

Characterization of env gene recombination in x-ray--induced thymomas of C57BL/6 mice

A role for specific recombination events at the env region of endogenous murine leukemia virus (MuLV) sequences in radiation carcinogenesis in C57BL mice has been suggested by a number of studies. We characterized env-related cell surface antigens from a primary, x-ray--induced, and several transplanted C57BL/6 thymomas of viral and radiation etiologies by immunoprecipitation and two-dimensional peptide mapping. DNA from lymphoma cells was also analyzed by Southern blotting for evidence of mink cell focus-forming (MCF) type env recombination events. Although gp70 molecules with novel structural determinants were found on all transplanted lymphomas examined, expression of novel env antigens was variable among these lymphomas, and there was a lack of correlation of characteristic MCF-type env recombination events in endogenous retrovirus DNA sequences with novel env antigens on lymphoma cell surfaces. Neither novel gp70 antigens nor MCF-type env provirus recombinant structures were consistent features of the C57BL/6 thymomas of radiation etiology examined in this study, even though MCF-type env recombination events have been suggested as etiologically significant in MuLV-mediated lymphomagenesis in both RadLV and x-ray--induced tumors in C57BL/6 mice.     

Radiation leukemia virus-induced T-cell lymphomas with common T-cell receptor variable region structure and similar binding specificity for retrovirus

The 5C2 cell line was derived following culture of mouse spleen cells exposed in vivo and in vitro to radiation leukemia virus (RadLV) containing supernatants from the C6VL/1 T cell lymphoma. This cell line has been found to express an alpha beta T-cell receptor (TCR) identifiable with the Mab124-40 anti-clonotypic antibody which is specific for C6VL/1. It has been shown to be genetically and phenotypically distinct from C6VL/1 with a unique phenotype, i.e. CD4-, CD8-, CD3+, TCR-alpha beta. 5C2 has been shown to express high levels of alpha and beta chain mRNA and to utilize the same or similar V alpha and V beta region genes as C6VL/1. Whereas C6VL/1 binds cross-reactively to both RadLV/C6VL and an unrelated isolate RadLV/VL3, 5C2 has binding specificity for only RadLV/C6VL, which induced its proliferation. The anti-clonotypic antibody Mab124-40 specifically and completely inhibits binding of 5C2 to RadLV/C6VL at concentrations as low as 300 ng/ml. The 5C2 cell line can also be stimulated to increased proliferation by RadLV/C6VL. All of these data are consistent with the role of a TCR alpha beta heterodimer in binding and stimulation by RadLV and satisfy one prediction of the receptor-mediated leukemogenesis hypothesis that T-cell clones identifiable by their T-cell receptor clonotype may be targets for transformation by a particular retrovirus.     

A mouse mammary tumor virus-like long terminal repeat superantigen in human breast cancer

We previously reported a 660-bp mouse mammary tumor virus (MMTV)-like env gene sequence in approximately 38% of human breast cancer DNA, but not in normal breasts or other tumors. This MMTV-like env gene sequence was expressed in 66% of the env gene-positive human breast cancers. An entire proviral structure was identified in human breast cancer DNA with high homology to MMTV and low homology to known human endogenous retrovirus. MMTV-like long terminal repeat (LTR) sequences were also detected in 41.5% of human breast cancers. They contain hormone-responsive elements, TEF-1 family elements, and the open reading frame for the superantigen (SAg). We have now amplified and sequenced MMTV-like sag sequences from 10 human breast cancers, and we found that they are highly homologous to those of MMTV. However, deletions and insertions at the COOH-terminal of sag were observed. The immune function of the human MMTV-like LTR SAg was also investigated. The sag gene was cloned and expressed in a human B-cell line (Ramos). T-cell proliferation and cytokine releasing assays were performed after cocultivation of T cells with irradiated Ramos SAg-expressing cells. The results indicate that expression of the human SAg stimulates T-cell activation in vitro, as the mouse SAg does. Because the T-cell responses in vitro are considered similar to those in vivo, these results suggest that the human LTR SAg might also play a role in human breast carcinogenesis.     

Requirement for reverse immune surveillance for the growth of germinal center-derived murine lymphomas

The concept of reverse immune surveillance, first conceived over 12 years ago, described the relationship that existed between germinal center-derived B cell lymphoma cells and the host immune system in SjL/J mice. According to reverse immune surveillance, recognition of tumor cell antigens and a response by the host immune system is required for tumor growth. The phenomenon of reverse immune surveillance related to B cell lymphomas has recently also been characterized in another inbred mouse strain, C57L/J. Moreover, elements of reverse immune surveillance have been observed in several other mouse strains that develop B cell lymphomas, suggesting that this lymphomagenic mechanism may be more common than first envisioned. In SJL and C57L mice, the B lymphoma cells express an MMTV-encoded superantigen (vSAg29) that stimulates syngeneic CD4+ T cells bearing Vbeta16 in their TCR. In contrast to the mRNAs for other MMTVs in normal mouse B cells, vSAg29 mRNA initiates in the env (META) region, undergoes splicing in the 3' env region, and continues through the 3' LTR. Copious cytokine production, including IFN-gamma, IL-4 and IL-5 accompanies the response of the T cells to this vSAg. In addition to cytokines produced by vSAg-responsive T cells, more recent evidence indicates that another cytokine, LTalphabeta2, which is expressed on the lymphoma cell surface, also plays a role in the promotion of the B cell lymphoma growth. It is possible that interaction with LTbeta-R on follicular dendritic cells or other stromal elements facilitates tumor growth by preventing apoptosis of the malignant B cells. To what degree these findings in the mouse are relevant to the development and/or growth of human B lymphoma cells remains to be determined. However, endogenous retroviral sequences do exist in the human genome. Interestingly, some of these sequences are homologous to MMTV, and are transcribed in B lymphoblastoid cells. Moreover microorganisms that are infectious for human B cells, such as EBV and Herpes Virus 8, may also produce superantigens.     

Differential Expression of Subspecies of Polyomavirus and Murine Leukemia Virus Enhancer Core Binding Protein, PEBP2, in Various Hematopoietic Cells

The core sequence of the enhancer of murine leukemia virus (MuLV) long terminal repeat is highly conserved in a large number of MuLV strains and appears to play an essential role when SL3-3 or Moloney strains induce T cell lymphoma in mice. We found by using the electrophoretic mobility shift assay that a polyomavirus enhancer core-binding protein, PEBP2, bound to this core motif of MuLV. We also noted that PEBP2 in several hematopoietic cell lines derived from B lymphocyte, macrophage and myelocyte lineages migrated significantly faster than the authentic PEBP2 detected in NIH3T3 (ibroblasts. Interestingly, PEBP2 detected in the cell lines of T lymphocyte lineage appeared to contain both types, which were indistinguishable in electrophoretic mobility from those of NIH3T3 and of B lymphocyte, macrophage and myelocyte lineages. The treatment of the nuclear extract containing PEBP2 with phosphatase generated PEBP3, which is a subcomponent of PEBP2 and retained the same DNA-binding specificity as PEBP2. The altered mobility of hematopoietic cell-derived or T lymphocyte-derived PEBP2 was found to be due to the alteration of the mobility of PEBP3. Based on the distinct mobility of PEBP2/3 of T lymphocytes from those of other hematopoietic cells, we discuss the implication of PEBP2 in MuLV-induced T cell leukemia and T cell-specific gene expression.     

Differential expression of subspecies of polyomavirus and murine leukemia virus enhancer core binding protein, PEBP2, in various hematopoietic cells.

The core sequence of the enhancer of murine leukemia virus (MuLV) long terminal repeat is highly conserved in a large number of MuLV strains and appears to play an essential role when SL3-3 or Moloney strains induce T cell lymphoma in mice. We found by using the electrophoretic mobility shift assay that a polyomavirus enhancer core-binding protein, PEBP2, bound to this core motif of MuLV. We also noted that PEBP2 in several hematopoietic cell lines derived from B lymphocyte, macrophage and myelocyte lineages migrated significantly faster than the authentic PEBP2 detected in NIH3T3 fibroblasts. Interestingly, PEBP2 detected in the cell lines of T lymphocyte lineage appeared to contain both types, which were indistinguishable in electrophoretic mobility from those of NIH3T3 and of B lymphocyte, macrophage and myelocyte lineages. The treatment of the nuclear extract containing PEBP2 with phosphatase generated PEBP3, which is a subcomponent of PEBP2 and retained the same DNA-binding specificity as PEBP2. The altered mobility of hematopoietic cell-derived or T lymphocyte-derived PEBP2 was found to be due to the alteration of the mobility of PEBP3. Based on the distinct mobility of PEBP2/3 of T lymphocytes from those of other hematopoietic cells, we discuss the implication of PEBP2 in MuLV-induced T cell leukemia and T cell-specific gene expression.     

In vivo antigenic modification of tumor cells. III. Metastatic thymic lymphoma specifically infected by thymotropic retrovirus

Tissue distribution of radiation leukemia virus (RadLV) was examined after its inoculation into normal C57BL/6J (B6) mice, B6 mice bearing a transplantable, non-virus-producing thymic lymphoma (RL12-NP), and B6 mice bearing a transplanted non-virus producing, Harvey murine sarcoma virus-transformed fibrosarcoma. Virus expression was determined by competition radioimmunoassay for murine leukemia virus (MuLV) p30 (predominant group-reactive antigen of MuLV) and for RadLV p12 (a highly type-specific MuLV polypeptide) and by membrane immunofluorescence for cell surface gp71 (predominant envelope glycoprotein of MuLV). Normal adult B6 mice were given three sequential iv injections of RadLV and were examined several times up to 200 days later for the appearance of neoplastic disease or expression of virion antigens. No clinical abnormalities were noted, and animals remained healthy for greater than 200 days. Significant levels of MuLV p30 and RadLV p12 were detected only in the thymuses. Organs and tumors from RL12-NP-inoculated animals contained low or nondetectable levels of virion antigens. Inoculation of mice with RL12-Rad, a cell line derived by in vitro infection of RL12-NP cells with RadLV, produced widespread, discrete metastatic tumors and infiltrated the lymphoid organs of B6 mice in a pattern identical to that observed after administration of RL12-NP cells. Lymphoid organs of RL12-Rad-inoculated animals expressed variable levels of virion antigens reflecting differences in the extent of tumor cell infiltration as opposed to virus spread from tumor to host cells. Administration of infectious RadLV systemically into RL12-NP tumor-bearing animals converted these tumors to viron antigen expressors with levels in superinfected tumors equivalent to those found in RL12-Rad-induced tumors. Infection was highly selective, and host tissues were minimally contaminated by the inoculated virus. Part of this selectivity was explained by the thymotropic property of RadLV. A rapidly dividing murine fibrosarcoma was not infected by RadLV, but this same non-virus-expressing tumor could be infected by common fibrotropic MuLV isolates.     

Establishment, characterization and virus expression of cell lines derived from radiation- and virus-induced lymphomas of C57BL/Ka mice.

Permanent cell lines have been established in vitro from lymphoid tumors induced in C57BH/Ka mice by fractionated X-irradiation or by inoculation of the radiation leukemia virus (RadOV). The cultured cells are lymphoblastic, replicate rapidly in vitro, and are tumorigenic in vivo. The cell surface markers Thy 1, Ly 1, Ly 2,3 and GIX are expressed by the lymphoid tumor cells in the mouse and persist in the corresponding cell lines; expression of the H-2 and TL antigens is greatly reduced during in vitro passage, but is restored on in vivo transplantation. The cell lines derived from RadLV-induced tumors (BL/VL lines) produce a virus population (RadLV/LTC) with the thymotropic and leukemogenic attributes of RadLV. Those derived from radiation-induced, virus-negative lymphomas (BL/RL lines) are initially devoid of MuLV expression, but frequently become spontaneous virus producers during in vitro cultivation.     

Characteristics of potential lymphoma-inducing cells in mice sensitive or resistant to lymphomagenesis by radiation leukemia virus variants

The relationship between the H-2-associated responsiveness of mice to radiation leukemia virus variants (A-RadLV and D-RadLV) lymphomagenesis and the characteristics of early occurring potential lymphoma-inducing cells (PLC) among thymus and bone marrow cells of these virus-infected mice was investigated. Sensitivity to virus-induced T-cell lymphomagenesis was shown to involve early occurrence of Thy-positive PLC, found predominantly among thymocytes, whereas resistance was rather related with early identification of PLC-Thy-negative cells mostly among bone marrow cells. PLC were further characterized in the sensitive (BL/6 + A-RadLV) and resistant (BL/6 + D-RadLV) situations by testing in parallel the tumorigenic potential (using the transplantation bioassay method) and type of thymus and bone marrow cell populations separated by different methods such as size fractionation by centrifugal elutriation, cytotoxic elimination of lymphocytes, or panning. The early occurring PLC among thymocytes of BL/6 mice 10-20 days following infection with A-RadLV were shown to be cortisone-resistant, Thy+, CD4+, and/or CD8+ medium size dividing thymocytes. PLC among thymocytes of BL/6 mice + D-RadLV, identified among the medium and large cell fractions, were shown to be cortisone-resistant Thy-, CD4-CD8- lymphocytes. High tumorigenic potential of PLC was demonstrated only among unseparated or separated (on size basis) bone marrow cells of BL/6 + D-RadLV (72-84%), whereas unseparated or separated fractions of bone marrow from BL/6 + A-RadLV had a low lymphomagenic potential (15-20%). The parallelism between the bone marrow fractions that induced optimal thymus cellularity following reconstitution of lethally irradiated mice and optimal lymphomagenicity stress the prothymocyte characteristics of PLC among bone marrow cells of BL/6 mice infected with D-RadLV. It is suggested that in resistant and sensitive haplotypes RadLV variants infect different cell populations and thereby induce PLC which differ in their capacity to present associative MuLV antigens with self H-2.     

Human prolactin regulates transfected MMTV LTR-directed gene expression in a human breast-carcinoma cell line through synergistic interaction with steroid hormones.

Prolactin plays a key role in the regulation and growth of mammary cells, and influences tumor promotion. We have shown that chronic energy restriction intake depresses prolactin levels, inhibits production of MMTV proviral DNA and proto-oncogene expression in mammary glands and prevents development of mammary tumors. Since the expression and proto-oncogene activation of MMTV are regulated by promoter/enhancer elements within its long terminal repeat (LTR), in the present study we used a chloramphenicol acetyl transferase (CAT) reporter gene system and gene transfection methods to study the effect of prolactin on MMTV LTR using a human ductal carcinoma cell line T47D stably or transiently transfected with a plasmid consisting of the LTR upstream of CAT gene. Human prolactin or dexamethasone induced, respectively, a 2-fold or 6-fold increase in CAT activity compared with background CAT activity in the absence of hormones. However, the combination of human prolactin and dexamethasone strongly enhanced (20-fold) induction of the LTR compared with the control. Human prolactin also showed a synergistic effect with progesterone on LTR induction. Both LTR and CAT genes needed to be linked for induction of CAT activity by prolactin and dexamethasone. Our results indicate that human prolactin can act synergistically with steroid hormones to regulate MMTV LTR-directed gene expression in transfected T47D cells.     

Induction of IL-4 secretion by the radiation leukemia virus (RadLV): role in autocrine growth stimulation of RadLV infected pre-leukemic cells.

Intrathymic inoculation of radiation-leukemia virus (RadLV) into C57BL/6 mice induces a population of pre-leukemic (PL) T cells which progress into clonal, mature thymic lymphomas after a latency period of 3 to 5 months. In order to understand how PL cells are retained in the thymus for a prolonged period of time we determined whether RadLV infected cells secrete and/or respond to a T-cell growth factor that may be involved in the long-term maintenance of a thymic PL-cell pool. We have previously found that in vitro proliferation of RadLV-infected PL cells is IL-4-dependent. Here we show that RadLV induces IL-4 secretion and IL-4 receptor (IL-4R) expression in normal thymic lymphocytes. RadLV-infected PL thymocytes express IL-4R and secrete IL-4. Their IL-4 secretion could be enhanced if incubated in the presence of RadLV and this enhancement was inhibited by anti-RadLV antibodies. Several RadLV-induced lymphoma lines secreted IL-4 and/or expressed IL-4R, but these features were not essential for their continuous growth. The results suggest that RadLV induces IL-4-dependent autocrine growth which maintains a population of PL T cells in the thymus. Transition from a PL state to overt thymic lymphoma involves emancipation of a PL cell from IL-4 dependency.     

Retroviral src gene expression in continuous marrow culture increases the self-renewal capacity of multilineage hematopoietic stem cells

To define the action of the retroviral src gene on hematopoietic stem cells, C57BL/6 x DBA/2 (B6D2F1) mouse long-term marrow cultures were infected at initiation with Moloney murine leukemia virus (MuLV) pseudotypes of src-recombinant retroviruses with the src gene inserted in the env region of an amphotropic MuLV (src-Ampho), or in the gag region of Moloney MuLV (src-Mo). Other cultures were infected with Friend spleen focus-forming virus polycythemia-inducing strain (SFFVp), Moloney MuLV, or amphotropic MuLV, or were uninfected controls. Harvested nonadherent cells were tested weekly for multilineage, granulocyte-erythroid-megakaryocyte macrophage (CFU-GEMM) colony formation in vitro in recombinant murine IL-3 and erythropoietin, and individual colonies were removed, split 1:2, with half of each replated for in vitro self-renewal and the other half examined morphologically for number of hematopoietic cellular lineages, or tested for release of MuLV and src virus. Cultures infected with src-Ampho, src-Mo, or SFFVp demonstrated a significant increase in cumulative nonadherent cell and CFU-GEMM production. There was prolonged self-renewal over seven serial transfers of individual CFU-GEMM from src virus-infected cultures over seven serial transfers, and five of 61 individual colonies from the second or third generations contained detectable v-src gene sequences, but none released detectable src virus. Self-renewal of CFU-GEMM was similar to that with permanent IL-3-dependent cell line B6SUtA. In contrast, MuLV-infected or control uninfected cultures produced fewer cells, and self-renewal of CFU-GEMM did not exceed three generations. IL-3-dependent clonal hematopoietic progenitor cell lines, derived from each culture group, formed no detectable tumors in vivo; however, each released the original helper and/or transforming virus. Adherent cell lines, derived from src-Ampho-infected cultures released src virus and formed fibro-sarcomas in vivo. The data support the conclusion that src-recombinant virus expression in long-term marrow cultures increases the self-renewal capacity of multilineage hematopoietic stem cells.