Quantitative studies of naturally occurring murine leukemia virus infection of AKR mice.

Quantitative studies were made of the organ distribution of murine leukemia virus in AKR mice of various ages. Infectious virus first appeared shortly before or after birth and was continuously present in all mice thereafter. Highest infectivity titers were found in uterus and bone, with spleen slightly lower. Virus titers in normal thymus were relatively low, but increased significantly with the development of thymic lymphoma. The level of viremia decreased after the 1st month of life, but increased sharply in lymphomatous mice.

Thymic lymphoma induction by the AKT8 murine retrovirus.

The directly transforming murine retrovirus, AKT8, was isolated from a spontaneous AKR thymoma and carries the cell-derived viral oncogene, akt. We have now shown that this virus produces thymic lymphomas after inoculation of susceptible mouse strains. The presence of the AKT8 genome in the DNA of the virus-induced tumors was demonstrated by Southern blotting using an akt-specific probe. These results establish the in vivo pathogenicity of the AKT8 virus and its akt oncogene, and imply a potential role for the cellular akt proto-oncogene in tumor development.

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.

Bovine leukemia virus genes in the DNA of leukemic cattle.

Reverse transcripts of the rna genome of the bovine leukemia virus (BLV) as well as 125I-labeled BLV RNA hybridize to the DNA of tissues from leukemic cattle with the adult form of the disease but not to bovine thymic lymphoma or normal bovine tissues.

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.

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.

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.

Cellular DNA region involved in induction of thymic lymphomas (Mlvi-2) maps to mouse chromosome 15.

Two cellular DNA regions representing common domains for proviral DNA integration ( Mlvi -1 and Mlvi -2) have been identified in Moloney murine leukemia virus-induced rat thymic lymphomas. Cellular sequences which were free of repeated DNA derived from a clone that defines the Mlvi -2 integration domain (lambda Cl228 ) were found to be highly conserved in a variety of vertebrate species that we examined, including mice, hamsters, cats, and humans. In this study, we identified the chromosomal map location of the Mlvi -2 homologous sequences in mice by using hamster-mouse somatic cell hybrids. The results show that Mlvi -2 is present on mouse chromosome 15 but is unrelated to the c-myc and c-sis proto-oncogenes, which map on the same chromosome. Since aberrations on chromosome 15 have been observed reproducibly in mouse thymomas, our data suggest that Mlvi -2 may define a novel sequence involved in the induction or progression of murine thymic lymphomas.

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.

Use of transgenic mice reveals cell-specific transformation by a simian virus 40 T-antigen amino-terminal mutant.

We have used the multifunctional transforming protein, simian virus 40 T antigen, as a probe to study the mechanisms of cell growth regulation in the intact organism. T antigen appears to perturb cell growth, at least in part, by stably interacting with specific cellular proteins that function to maintain normal cell growth properties. Experiments in cultured cells indicate that at least three distinct regions of simian virus 40 T antigen have roles in transformation. Two regions correlate with the binding of known cellular proteins, p53, pRB, and p107. A third activity, located near the amino terminus, has been defined genetically but not biochemically. By targeting expression of wild-type and mutant forms of T antigen to distinct cell types in transgenic mice, we have begun to systematically determine which activities play a role in tumorigenesis of each cell type. In this study, we sought to determine the role of the amino-terminal transformation function with such an analysis of the T-antigen mutant dl1135. This protein, which lacks amino acids 17 to 27, retains the p53-, pRB-, and p107-binding activities yet fails to transform cells in culture. To direct expression in transgenic mice, we used the lymphotropic papovavirus transcriptional signals that are specific for B and T lymphocytes and the choroid plexus epithelium of the brain. We show here that although defective in cell culture, dl1135 specifically induced the development of thymic lymphomas in the mouse. Expression of the protein was routinely observed in B- and T-lymphoid cells, although B-cell abnormalities were not observed. Choroid plexus tumors were observed only infrequently; however, dl1135 was not consistently expressed in this tissue. Within a given transgenic line, the penetrance of T-cell tumorigenesis was 100% but appeared to require secondary events, as judged from the clonal nature of the tumors. These experiments suggest that the amino-terminal region of T antigen has a role in the transformation of certain cell types (such as fibroblasts in culture and B lymphocytes) but is dispensable for the transformation of T lymphocytes.

Spontaneous leukemia viruses: lymphomagenic ecotropic viruses of AKR mice.

The spontaneous leukemia (SL) viruses are ecotropic lymphomagenic viruses isolated from AKR spontaneous lymphomas. These viruses are produced stably by continuous cell lines from spontaneous lymphomas and by a cell line derived from the bone marrow stroma of an AKR mouse neonatally inoculated with an SL virus. All cell lines cloned from the parent lymphoma cell lines consistently produce SL viruses. These viruses can be passaged in vivo and maintain their leukemogenic properties. Cloned isolates of SL viruses accelerate lymphoma in AKR mice and induce thymic lymphoma in mice of other strains. Thus their lymphomagenic properties are conclusively shown. In a study with the use of a sensitive host range assay, xenotropic and/or dual-host range viruses are consistently found in spontaneous lymphoma and cell lines derived from them. However, viruses able to replicate in mink lung cells are not expressed in SL virus-induced lymphomas or their derived cell lines.

Development of thymic lymphomas by oral administration of N-nitroso-N-propylurea and establishment of transplantable lines of thymic lymphoma in F344 rats.

Forty-eight female F344/DuCrj rats were given a 400-ppm solution of N-nitroso-N-propylurea (CAS: 816-57-9) continuously in their drinking water. Thymic lymphomas were induced most frequently (85%) followed by duodenal tumors (48%). Sixteen tumors were examined by the immunofluorescence inhibition test for murine leukemia virus-related antigens; 15 were negative and the other was weakly positive. Twenty-six tumors were intraperitoneally and subcutaneously transplanted syngeneically; 25 (96%) were successfully transplanted intraperitoneally and 24 (92%) subcutaneously. The serial intraperitoneal transplantation was continued, and 22 lines of transplantable lymphoma in an ascites form were established. In almost all tumor lines, tumor cells took in a high percentage of recipient rats and caused the death of the recipients within 12-23 days. The thymus, liver, spleen, greater omentum, and lymph nodes were frequently invaded by transplanted tumor cells. The tumor lines were considered to be of T-cell lineage.

Induction of lymphoma and associated xenotropic type C virus in C57L mice by whole-body irradiation.

Adult C57L mice received sublethal whole-body X-irradiation. Between 3 and 11 months later, 5 of the 7 exposed mice developed histopathologically confirmed malignant lymphomas (lymphocytic type) that primarily involved the thymus. The lymphomas were readily transplantable to other C57L mice of any age, which developed fetal lymphomatous involvement; the tumor cells could also be propagated in tissue culture. A xenotropic murine type C virus (MuX) was isolated from the cultured lymphoma cells after cocultivation with a permissive dog line. MuX activity was demonstrated by electron microscopy, complement fixation, indirect fluorescent antibody, infectivity, and genome rescue.

A discrepancy in XC and oncogenicity assays for murine leukemia virus in AKR mice.

Studies of murine leukemia virus expression in AKR mice are presented. Material from in vivo and in vitro sources of normal tissues and lymphomas was assayed for in vitro infectivity, using the XC plaque assay, and for oncogenicity, by assessing lymphoma-accelerating capacity after inoculation into newborn animals. Normal tissues from healthy young AKR mice up to 7 months of age were found to have XC but not oncogenic activity. XC activity persisted, and weak oncogenic activity appeared in older mice. Cocultivation of normal young cells with NIH Swiss mouse embryo cells did not result in the appearance of oncogenic activity, although XC virus increased in titer. A cell-free filtrate of a virus-accelerated lymphoma was studied for host range. Virus as measured by polymerase and gs antigen was found to be propagated on NIH Swiss mouse embryo and wild mouse embryo cells, but not on human rhabdomyosarcoma, normal rat kidney, rabbit corneal, and BALB/c embryo cells. Virus as measured by the XC assay grew better on NIH Swiss mouse than on BALB/c embryo cells. Both of these cell lines propagated virus as measured by the oncogenicity assay. Supernatants from an in vitro cell line from a virus-accelerated lymphoma did not produce XC plaques but were oncogenic. Those from two cell lines of spontaneous lymphomas were negative with both assays. Cultivation of supernatants from these cultured lymphoma cells with NIH Swiss mouse embryo cells resulted in material which produced small plaques on the XC assay. These findings are interpreted as showing the presence of two viruses in AKR mice. One is XC positive and present throughout life. The other is oncogenic, appears later in life, and could be a separate virus or a variant of the first one.

Lymphomagenesis in AKR.Fv-1b congenic mice.

In the AKR.Fv-1b congenic strain the Fv-1n allele of the AKR/J mice was substituted with the Fv-1b allele, thereby limiting viral replication and spread of the endogenous N-tropic murine leukemia virus. As a result of this genetic change AKR.Fv-1b mice develop a low spontaneous incidence (7%) of T-cell lymphomas and about 28% of Ly-1+ B-cell lymphomas are observed in old mice. Characteristic changes in thymus subpopulations of AKR/J mice (related to the formation of the dual tropic mink cell focus inducing (MCF) type virus in the thymus) were not observed in the thymus of AKR.Fv-1b mice. In contrast to the low susceptibility to spontaneous T-cell lymphoma development, these mice were highly sensitive to fractionated irradiation or to radiation leukemia virus (a mixture of N- and B-tropic viruses) induced T-cell lymphoma. Potential lymphoma cells (that would ultimately develop into Ly-1+ B-cell lymphomas) were demonstrated in bone marrow and spleens of 16-24-month-old mice. Analysis of the Ly-1+ IgM+ B-cell population in spleens of 18-month-old mice revealed a significant increase in this population (35% versus 2% in young spleens). The spontaneous Ly-1+ B-cell lymphoma incidence could be enhanced (up to 77%) by in vivo administration of anti-CD8 monoclonal antibody or IL-4 to 18-month-old mice. Virological analysis of T/B-cell lymphomas for class I MCF viruses indicated that Class I MCF development was tightly correlated with T-lymphoma development (except radiation induced tumors that showed no MCF provirus involvement). In contrast, Ly-1+ B-cell lymphoma development was independent of Class I MCF pathogenic virus involvement.

Presence of prelymphoma cells in the bone marrow of the lymphomagenic virus-treated AKR mouse.

Prelymphoma cells (PLC) are defined as cells which give rise to lymphoma cells but are not in themselves autonomous. They are present in the bone marrow of young AKR mice, a strain with a high natural incidence of thymic lymphoma. PLC are identified by transfer of AKR bone marrow into 400-rad-treated F1 recipients, one of the parents being AKR. Normal 1-month-old AKR bone marrow cells result in thymic lymphomas of AKR type in the hybrid recipients after latent periods of 6 to 16 months. In the present study, PLC resulting in lymphoma of AKR type 3 to 4 months after inoculation to irradiated hybrids are described. They are consistently found in the bone marrow of 21- to 28-day-old AKR mice treated at 3 to 5 days of age with a lymphomagenic virus, SL3-3. Long-term culture of these bone marrow cells allows the survival of totipotent hemopoietic stem cells but not of PLC. Thymic stromal remnants prepared from the 21- to 28-day-old virus-treated mice efficiently replicate oncogenic virus; however, they do not contain PLC. This was determined by grafting the remnants to irradiated and nonirradiated hybrid recipients. No AKR type lymphomas developed in the grafted mice. We conclude that the bone marrow of young oncogenic virus-treated AKR mice contains PLC modified by oncogenic virus so that they can produce thymic lymphomas after a shorter latent period than can PLC found in 1-month-old normal AKR mice. The modified PLC are not derived from totipotent marrow stem cells or sequestered in the thymic stroma of virus-treated mice.

Frequency of Synaptic Autoantibody Accompaniments and Neurological Manifestations of Thymoma.

IMPORTANCE: Thymoma is commonly recognized in association with paraneoplastic autoimmune myasthenia gravis (MG), an IgG-mediated impairment of synaptic transmission targeting the nicotinic acetylcholine receptor of muscle. Newly identified synaptic autoantibodies may expand the serological profile of thymoma. OBJECTIVE: To investigate the frequency of potentially pathogenic neural synaptic autoantibodies in patients with thymoma. DESIGN, SETTING, AND PARTICIPANTS: We retrospectively identified patients with histopathologically confirmed thymoma and serum available to test for synaptic autoantibodies (collected 1986-2014) at the Mayo Clinic Neuroimmunology Laboratory. We identified and classified 193 patients with thymoma into 4 groups: (1) lacking neurological autoimmunity (n = 43); (2) isolated MG (n = 98); (3) MG plus additional autoimmune neurological manifestations (n = 26); and (4) neurological autoimmunity other than MG (n = 26). MAIN OUTCOMES AND MEASURES: Clinical presentation and serum profile of autoantibodies reactive with molecularly defined synaptic plasma membrane proteins of muscle, peripheral, and central nervous systems. RESULTS: Of the 193 patients with thymoma, mean patient age was 52 years and did not significantly differ by sex (106 women) or group. Myasthenia gravis was the most prevalent clinical manifestation (64%) followed by dysautonomia (16 patients [8%]) and encephalopathy (15 patients [8%]); 164 patients (85%) had at least 1 synaptic autoantibody, and 63 of these patients (38%) had at least 1 more. Muscle acetylcholine receptor was most frequent (78%), followed by ganglionic acetylcholine receptor (20%), voltage-gated Kv1 potassium channel-complex (13%), and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (5%). Less frequent were aquaporin-4, voltage-gated Kv1 potassium channel-complex related proteins (leucine-rich glioma-inactivated 1 and contactin-associated protein-like 2), glycine receptor, and γ-aminobutyric acid-A receptor. Synaptic autoantibodies were significantly more frequent in patients with neurological autoimmunity than in those without and were most frequent in patients with neurological manifestations other than or in addition to MG. CONCLUSIONS AND RELEVANCE: Synaptic autoantibodies, particularly those reactive with ion channels of the ligand-gated nicotinic acetylcholine receptor superfamily (namely α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, glycine, and γ-aminobutyric acid-A receptors), were prevalent in patients with thymoma. Autoantibodies of this extended spectrum may enhance autoimmune serological testing as an aid to preoperative thymoma diagnosis. Detection of currently known synaptic autoantibody specificities absent from this profile have potential algorithmic usefulness as negative predictors for thymoma (as recognized for neuronal voltage-gated calcium channel autoantibodies).

flvi-2, a target of retroviral insertional mutagenesis in feline thymic lymphosarcomas, encodes bmi-1.

LC-FeLV is a myc-containing strain of feline leukemia virus which induces thymic lymphosarcoma in the domestic cat with short latency. A locus in feline DNA, termed flvi-2, is commonly interrupted in naturally occurring and experimentally induced thymic lymphosarcomas containing LC-FeLV; thus, interruption of a gene encoded by flvi-2 may cooperate with the myc oncogene in the induction of T-cell tumors by LC-FeLV. Clones homologous to flvi-2 have been isolated from a normal human thymus cDNA library. Nucleotide sequence analysis of the cDNA clones demonstrates that flvi-2 encodes bmi-1, a gene previously identified as a target for MoMuLV integration and as a myc-collaborator in retrovirally-induced B-cell lymphomas in E mu-myc transgenic mice. In feline thymic lymphomas, retroviral integrations occur downstream of the gene, and result in enhanced expression of a bmi-1 transcript of normal size. These findings demonstrate the interruption of bmi-1 in natural as well as experimentally induced tumors, implicate the activation of bmi-1 in the induction of T-cell as well as B-cell lymphoma, and support the premise that bmi-1 functions as a myc collaborator.

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.

Identification of malignant cell clones in radio-induced murine thymic lymphomas by viral and cellular probes.

The role of B ecotropic recombinant retroviruses in the emergence and the progression of radio-induced thymic lymphomas was evaluated by analyzing the cell populations present in nine primary and in in vivo propagated tumors. For this, tumor DNAs were analyzed by the Southern method using probes specific for newly acquired proviral sequences, T-cell receptor beta-chain, and immunoglobulin heavy chain genes. Our results show that primary radio-induced tumors are composed of several tumoral cell clones but do not support that malignant cell transformation and proliferation are conferred, solely, by the newly acquired ecotropic recombinant retroviral sequences.