Radiation leukemia in C57BL/6 mice. II. Lack of ecotropic virus expression in the majority of lymphomas.

The expression of endogenous ecotropic viruses in radiation-induced thymomas of C57BL/6 mice was examined. Competition radioimmunoassays for AKR MuLV gp71, p30, and p12 were used for viral antigen expression. 3 of 40 lymphomas had readily detectable ecotropic gp71 at levels of 95-689 ng/mg protein; the remainder of the tumors had no detectable gp71 (less than 1.0 ng/mg protein). 30 thymomas were characterized by the presence of MuLV p30 at levels of 1-10 ng/mg protein, levels that were comparable to those found in thymus extracts from age-matched, nonirradiated control. 10 tumors were characterized by having p30 levels of 10-30 ng/mg protein. In one tumor significant levels of AKR MuLV p12 were detectable. Since B-tropic and N-tropic viruses from C57BL/6 mice have glycoproteins (gp71) indistinguishable from AKR MuLV gp71 and the N-tropic virus had a p12 serologically identical to AKR MuLV p12, these results demonstrate that overt endogenous B-tropic virus was detectable in 2 of 40 thymomas and endogenous N-tropic virus was detectable in 1 of 40 thymomas. The lack of overt expression of gp71 or p12 was also confirmed by cytotoxicity assays using monospecific antisera to these viral proteins. Radiation-induced lymphomas were also examined for the presence of reverse transcriptase after chromatography of tissue extracts on poly G-Sepharose. One tumor, which was characterized by the lack of gp71, also had no detectable reverse transcriptase; whereas one tumor with gp71 was characterized by readily detectable levels of reverse transcriptase in cellular extracts. The presence of viral RNA was examined using AKR cDNA. Low levels of RNA capable of hybridizing with AKR cDNA were found in age-matched, nonirradiated mice; these hybrids had Tm's of 72 degrees C, while hybrids with AKR MuLV 70S RNA had Tm's of 80 degrees C. In 1 of 12 thymomas the concentration of hybridizable RNA and the Tm of the hybrids were identical to control values. In 9 of 12 thymomas the concentration of hybridizable sequences increased approximately three-to fivefold and the Tm of these hybrids varied from 73 to 75 degrees C. In 1 of 12 thymomas the concentration of hybridizable sequences increased over 100-fold, hybridized completely with AKR MuLV cDNA, and the hybrids had Tm's of 79 degrees C. This thymoma was also characterized by the presence of the AKR MuLV type of gp71 and p12. One tumor was characterized by a 10-to 100-fold increase in hybridizable sequences, which only partially hybridized with AKR MuLV cDNA, and hybrids had a Tm of 73 degrees C. This tumor was characterized by the presence of AKR MuLV gp71 but not AKR MuLV p12. The results taken together demonstrate that overt endogenous ecotropic virus expression is only rarely detectable in radiation-induced thymomas of C57BL/6 mice.

Proviral integration site Mis-1 in rat thymomas corresponds to the pvt-1 translocation breakpoint in murine plasmacytomas.

Two loci independently implicated in T-and B-lymphocyte neoplasia are shown to be equivalent. The Mis-1 locus is a common proviral integration site in retrovirally induced rat T lymphomas, while the pvt-1 locus on murine chromosome 15 frequently translocates to the kappa locus in plasmacytomas bearing 6;15 translocations. By comparing cloned sequences, we show that pvt-1 is the murine homolog of Mis-1.

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.

Thymocyte subsets transformed by Abelson murine leukemia virus.

The infectious complex of Abelson murine leukemia virus was altered by replacing its usual helper virus, Moloney leukemia virus, with radiation leukemia virus (RadLV). After intrathymic injection of the Abelson-RadLV complex, thymomas arose rapidly, as described previously for injection of the Abelson-Moloney complex. Cell lines were derived from thymomas induced by each Abelson virus complex and were classified according to normal thymus cell phenotypes. Each virus complex induced some cell lines which were like a 0.7% subpopulation of murine thymocytes in that they failed to express the Thy-1 cell-surface antigen. These lines are thus far indistinguishable from some Abelson-derived bone marrow transformants classified as pre-B cells. However, the Abelson-Moloney complex induced some cell lines which expressed low levels of Thy-1 and which shared most markers with immature blast cells of the thymic medulla, whereas the Abelson-RadLV complex induced some lines which were clearly like thymic cortex blast cells. Thus, Abelson virus can induce thymoma cell lines of at least two, and possibly three, distinct phenotypes corresponding to normal thymocyte blast subsets, the determination of which can be influenced by helper virus sequences.

Strong selection for cells containing new ecotropic recombinant murine leukemia virus provirus after propagation of C57BL/6 radiation-induced thymoma cells in vitro or in vivo.

Using the Southern procedure, we have studied the presence of ecotropic-specific murine leukemia viral sequences in genomic DNA isolated from primary X-ray-induced thymomas, from lymphoid cell lines established from them, or from secondary tumors passaged in vivo. We found that primary radiation-induced thymomas and infiltrated spleens do not harbor newly acquired ecotropic provirus. However, additional ecotropic proviruses (which appear recombinant in the gagpol region) could be detected in most of the tumorigenic cell lines established in vitro from them and in tumors arising from subcutaneous transplantation of the primary thymomas. These results suggest that primary radiation-induced thymomas may not be clonal. They also indicate a strong correlation between the presence of ecotropic recombinant proviruses in the genome and the growth ability, both in vitro and in vivo, of specific cells within these thymomas, suggesting a possible mitogenic function for murine leukemia virus.

Deregulation of the c-myc oncogene in virus-induced thymic lymphomas of AKR/J mice.

A high frequency (greater than or equal to 65%) of thymomas induced by mink cell focus-forming virus 69L1 in AKR/J mice contain proviral integrations which are clustered 0.7-kilobase upstream of the c-myc oncogene predominantly in the opposite transcriptional orientation. Blot hybridization experiments showed that on the average there was only a twofold elevation of steady-state c-myc RNA in the thymomas as compared with levels in normal AKR/J thymocytes. Such an increase would not appear to be sufficient as a mechanism of oncogene activation in this system. In contrast, S1 nuclease analysis of transcripts initiated from the two known c-myc promoters indicated a strong shift in promoter usage in virtually all thymomas tested. In normal thymus the ratio of transcripts initiated from the proximal promoter P1 to the distal promoter P2 was 0.2 to 0.3. In contrast, most of the thymomas tested (18 of 23) showed an average P1/P2 ratio of 1.2 regardless of whether or not proviral integrations could be detected within a 21-kilobase EcoRI fragment containing the three c-myc exons. We conclude that alterations in P1/P2 ratios are good indicators of c-myc deregulation in thymic lymphomas.

Ultrastructure of spontaneous and urethan-induced thymomas in buffalo rats.

Normal thymuses from Buffalo and Long-Evans rats of various ages, and spontaneous and urethan-induced thymomas in Buffalo rats, were examined by electron microscopy. Histological variabilities among thymomas of the lymphoid, mixed, and epithelial cell types were a reflection of the number of lymphoid cells within the network composed of neoplastic epithelial reticular cells. In the cytoplasm of these cells, development of tonofilaments and membrane-bound bodies and inverse development of the rough-surfaced endoplasmic reticulum were recognized in the sequential process from the lymphoid cell type to the epithelial cell type. An important role of the development of the rough-surfaced endoplasmic reticulum for thymic function was suggested. Phagocytic activity of the neoplastic epithelial reticular cells was revealed, and some of the membrane-bound bodies in these cells, especially those with moniliform structures, were regarded as remnants of damaged lymphocytes. Evidence for neoplastic epithelial reticular cell-lymphoid cell transformation could not be established from study of the thymoma tissue. No virus-like structures were observed in these thymomas.

Measurement of binding specificity between T cell lymphomas and murine leukemia viruses.

We have previously reported the presence of receptors on radiation leukemia virus (RadLV)-induced thymomas and malignant thymocytes from AKR mice which specifically bind retrovirus produced by these T cell clones. These receptors have been shown to have specificity for virus reminiscent of an immune-specific receptor. Previous studies on T cell lymphoma binding to retroviruses have involved measurement of the interaction of labelled virus with cells using fluorescence-activated cell sorter (FACS) analysis (McGrath et al., J. Virol. (1978) 25, 923; McGrath and Weissman, Cell (1979) 17, 65; Weissman and McGrath, Curr. Top. Microbiol. Immunol. (1982) 98, 103). Here we report development of an assay for measuring lymphoma binding to virus, prepared as an immunoabsorbent adhered to a microtiter plate. Using this assay, we have shown that only T and not B cell lymphomas can bind to T cell-tropic viruses, and some cell lines have greatest specificity for homologous virus. The AKR-derived T cell lymphomas, SL3 and KKT-2, show greater specificity for leukemogenic AKR viruses, than for an AKR xenotropic virus or the recombinant AKR virus, MCF247. The RadLV-induced T cell lines, C6VL/1 and BL/VL3, have been found to bind cross-reactively to several different murine leukemia viruses (MuLVs). RadLV-induced T cell lymphomas do have greater specificity for their cognate retroviruses since free, homologous retrovirus can best block the interaction between cells and virus adhered to the wells of a microtiter plate. Cross-reactive interactions are more easily demonstrated by this assay, probably because low avidity interactions are stabilized as a result of the mode of virus presentation. Binding specificity for retroviral envelope determinants has been demonstrated using a rat anti-retroviral antiserum prepared as an F(ab)1 fragment. This antiserum can inhibit the interaction between the C6VL/1 thymoma and its RadLV virus. Specificity of this antibody for a gp70-like protein was confirmed by NaDodSO4-polyacrylamide gel electrophoresis (PAGE) and by loss of this activity after absorption of antibody on virus. Antibodies specific for RadLV/VL3 gp70 determinants can inhibit the interaction of C6VL/1 with RadLV/VL3 suggesting that cross-reactive binding to heterologous virus is also specific for a gp70 viral env determinant.

Molecular analysis of emerging radiation leukemia virus variants of C57BL/Ka mice.

Molecular cloning of several primary or passaged RadLV variants and their biological characterization has allowed us to propose a model of their emergence following X-ray irradiation of C57BL/6 mouse.

Pathogenesis and treatment of myasthenia gravis.

Lymphocyte subsets and viruses were examined in the thymuses and thymomas of patients with myasthenia gravis (MG). Most of the lymphocytes were considered to be from cortical thymocytes since they were positive for OKT4, 6, 8 and 10. Retrovirus-like particles were detected in the thymuses and thymomas from patients with MG. These data suggest contributions of lymphocytes and viruses in the pathogenesis of MG. HLA antigen frequencies in 49 patients with MG were studied to investigate a genetic background. Although the association of HLA-B8, DR3, DRw52 haplotype with Caucasian MG has been reported, Japanese MG seems to be associated with a different haplotype, HLA-B39, Cw7, DRw8. To achieve a better understanding of the association of HLA with MG, analysis of restriction fragment length polymorphism (RFLP) of DNA from MG patients was performed. When DQ beta cDNA probe was used, the increased frequency of PstI 12 kilobase fragment was observed in HLA-DR2 positive patients compared to healthy DR2 positives. For treating MG patients, we have been utilizing low-dose prednisolone (PSL) or high-dose gammaglobulin. Our results so far indicate that low-dose PSL seems to be as effective as high-dose PSL therapy, however, we could not observe any favorable effect of high-dose gammaglobulin therapy on MG.

Retrovirus-like particles in human thymomas.

The pathogenesis of thymoma is unclear. In this study retrovirus-like particles in human thymomas were detected by electron microscopy. Forty-two thymomas; 25 without complications and 17 associated with autoimmune disorders such as myasthenia gravis (13), systemic lupus erythematosus (1), polymyositis (1), Sjögren's syndrome (1), and pure red cell anemia (1), were examined. Thymic tissues from 9 infants suffering from congenital heart diseases and 7 hyperplastic thymuses obtained from myasthenic patients served as controls. The retrovirus-like particles were observed in 37.0% of thymomas without complications; 50.0% of thymomas associated with myasthenia gravis and other autoimmune disorders; 62.5% of thymuses associated with myasthenia gravis; and 33.3% of thymuses from infants with heart disease. The envelopes, including the central cores of the retrovirus-like particles, had diameters ranging from 70 to 460 nm, depending on the source of the specimen. The retrovirus-like particles were located in the cytoplasm, vacuoles, vesicles and lumens of the endoplasmic reticula of epithelial and/or plasma cells. Some retrovirus-like particles were seen budding from plasma membranes. These observations suggest that the retrovirus-like particles in thymomas might be an activated form of retrovirus originating in normal thymic tissue.

Specific integration of recombinant proviral sequences in ecotropic Gross virus-accelerated AKR thymomas.

Integration and amplification of ecotropic and recombinant proviral sequences in high-molecular-weight cellular DNAs from ecotropic Gross virus-accelerated AKR thymomas were analyzed using an ecotropic-specific probe, p400, and an envelope-specific probe, pAKV-5. New ecotropic proviral sequences were detected at three sites in the DNAs from eight Gross virus-accelerated thymomas following EcoRI restriction endonuclease digestion and at six sites following PvuII restriction endonuclease digestion. The integration of these new ecotropic proviral sequences appeared to be random. Recombinant 3' proviral-cellular DNA junction fragments were detected at 30 sites following digestion with EcoRI. These new recombinant fragments ranged in size from 9.0 to 2.5 kb with 6/8 thymoma DNAs containing a fragment of 2.7 kb. PvuII generated new recombinant 3' proviral-cellular junction fragments that ranged in size from 12.5 to 2.1 kb with 5/8 thymoma DNAs containing a fragment of 2.5 kb. It appears that the leukemia-accelerating ecotropic Gross virus is responsible for the generation of a unique 3' recombinant proviral-cellular junction fragment. This fragment can be detected against a background of randomly integrated ecotropic and recombinant proviruses.

Isolation of transforming murine leukemia viruses from mice with a high incidence of spontaneous lymphoma.

Murine leukemia viruses capable of malignant transformation of mink tissue culture cells have been isolated from an AKR thymoma cell line and from a spontaneous reticulum cell sarcoma in an NIH Swiss mouse partially congenic for the AKR ecotropic virus-inducing locus Akv-2. In contrast to the recently described mink cell focus-inducing strains of murine leukemia virus, at least one of the two transforming strains is replication defective. Nonproducer mink cells carrying the genome of the transforming virus of AKR origin have been isolated, and pseudotype transforming viruses generated.

Distinct helper virus requirements for Abelson murine leukemia virus-induced pre-B- and T-cell lymphomas.

Abelson murine leukemia virus (A-MuLV) can induce pre-B- or T-cell lymphomas (thymomas) in mice depending on the route and time of injection. Previous studies have shown that the choice of the helper virus used to rescue A-MuLV greatly influences its ability to induce pre-B-cell lymphomas. In this study, we investigated the role of the helper virus in A-MuLV-induced thymomas. A-MuLV rescued with the helper Moloney MuLV, BALB/c endogenous N-tropic MuLV, and two chimeric MuLVs derived from these two parents were injected intrathymically in young adult NIH Swiss mice. All four A-MuLV pseudotypes were found to be equally efficient in the induction of thymomas, whereas drastic differences were observed in their pre-B-cell lymphomagenic potential. Thymoma induction by A-MuLV was independent of the replication potential of the helper virus in the thymus, and no helper proviral sequences could be detected in the majority of thymomas induced by A-MuLV rescued with parental BALB/c endogenous or chimeric MuLVs. In the thymomas in which helper proviruses were present, none of them were found integrated in the Ahi-1 region, a common proviral integration site found in A-MuLV-induced pre-B-cell lymphomas (Y. Poirer, C. Kozak, and P. Jolicoeur, J. Virol. 62:3985-3992, 1988). In addition, helper-free stocks of A-MuLV were found to be as lymphomagneic as other pseudotypes in inducing thymomas after intrathymic inoculation, in contrast to their inability to induce pre-B-cell lymphomas when injected intraperitoneally in newborn mice. Restriction enzyme analysis revealed one to three A-MuLV proviruses in each thymoma, indicating the oligoclonality of these tumors. Analysis of the immunoglobulin and T-cell receptor loci confirmed that the major population of cells of these primary thymomas belongs to the T-cell lineage. Together, these results indicate that the helper virus has no effect in the induction of A-MuLV-induced T-cell lymphomas, in contrast to its important role in the induction of A-MuLV-induced pre-B-cell lymphomas. Our data also revealed distinct biological requirements for transformation of these two target cells by v-abl.

Increased incidence of thymoma in Chinese myasthenia gravis: possible relationship with Epstein-Barr virus.

Thymectomy was carried out for treatment of myasthenia gravis in 27 unselected Chinese patients and thymoma was found in 13 of them. This 48% incidence of thymomas is two to three times greater than in Japanese and European patients, respectively. The reason for the higher incidence of thymomas observed in Chinese patients may be related to the presence of the Epstein-Barr virus genome in thymoma. Furthermore, all of the thymomas in our patients were lymphoepithelial and histologically resemble nasopharyngeal carcinoma and undifferentiated carcinoma of the salivary gland. Both these tumours are closely linked to the Epstein-Barr virus and in Hong Kong, nasopharyngeal carcinoma is the third commonest cause of death from malignancy. We recommend early thymectomy for patients with myasthenia gravis particularly in geographical areas where there is a high incidence of nasopharyngeal carcinoma and undifferentiated carcinoma of the salivary gland.

A comparative analysis of radiation- and virus-induced leukemias in BALB/c mice.

Endogenous murine leukemia virus (MuLV) proviral copies were analyzed in thymomas induced in normal BALB/c (Fv-1b) and in Fv-1n congenic mice by X-irradiation. Both strains of mice developed leukemia with similar kinetics, indicating that N-tropism of endogenous MuLV was not a rate-limiting factor in development of disease. Southern blot analysis, using a probe specific for ecotropic virus and for ecotropic-specific sequences retained in pathogenic, env-recombinant viruses, showed that the majority of radiation leukemias lacked newly acquired, clonally integrated, proviruses. This was in contrast to virus-induced leukemias, which routinely exhibited several new proviral integration sites. When an internal proviral DNA restriction fragment was monitored, some radiation leukemias showed evidence of nonclonal infection, accounting for more frequent isolation of infectious virus from such leukemias. Differences in expression of T-cell surface antigens were found in X-ray-induced and virus-induced leukemias. All radiation leukemias were TL positive, whereas virus-induced leukemias were primarily negative for TL. Some differences were also found in Lyt-1 and Lyt-2 expression. The data as a whole suggest that, in the majority of cases, radiation leukemogenesis is not initiated by a viral route--that is, the sort of viral mechanism for which exogenous infection by known pathogenic MuLV is the paradigm.

Rearrangement of a DNA sequence homologous to a cell-virus junction fragment in several Moloney murine leukemia virus-induced rat thymomas.

We have studied the integrated proviruses in Moloney murine leukemia virus-induced rat thymomas. By Southern blot analysis, we found several complete integrated proviruses in each tumor. In most tumors, we could not detect defective or recombinant proviruses. Several of these integrated proviruses (with their flanking cellular sequences) from a single tumor were cloned in Charon 4A. Their flanking cellular fragments were subcloned into pBR322 and used as a probe to screen other thymoma DNAs. With one clone (pMo-lC) used as a probe, we could detect novel fragments in 11 out of 20 thymoma DNAs analyzed. In three of these thymomas we could analyze in more detail, it appears that these novel fragments are generated by the insertion of a provirus. This specific integration of Moloney provirus in the host genome may represent an important genetic event leading to tumor formation.

Unique role for the T cell receptor in retrovirus binding by the C6VL thymoma.

Binding of cognate Radiation leukemia virus (RadLV) by the C6VL/1 thymoma involves a subset of TCR molecules in association with CD4 molecules expressed by that cell line. A CD4- variant of C6VL/1 has now been isolated which also has RadLV binding capacity. Stable expression of the TCR, class I, and CD5 molecules but not Thy1.2 and CD4 molecules has been demonstrated, and the C6VL/1 origin of this cell has been confirmed by Southern blot analysis using probes specific for the TCR beta chain gene. This cell line has maintained binding capacity for RadLV/C6VL prepared as an immunoabsorbent matrix, but unlike the parent C6VL/1 cell line, binds significantly less well to the related RadLV/VL3 isolate. Binding of the variant cell line to RadLV/C6VL can be completely inhibited by anti-clonotypic antibody to the TCR but only weakly by anti-H-2Kb antibody used at the same concentration. These data suggest that the TCR on C6VL/1 can interact with RadLV in the absence of any co-receptor function of CD4 and implicates the TCR as a sufficient receptor for retrovirus.