Maternally transmitted resistance to lymphoma development in mice of reciprocal crosses of the RF/J and AKR/J strains

Females but not males of the low-lymphoma RF/J strain transmit a non-Mendelian factor which suppresses the development of lymphoma in F1 crosses with mice of the high-lymphoma AKR/J strain. Suppression of lymphoma was also evident in the first backcross generation to the parental AKR strain, but only when (RF female x AKR male)F1 mice had been the female parent. This "maternal resistance factor" was transmitted independently of the dominant, lymphoma-suppressing Fv-1n allele transmitted by both males and females of the RF strain, but the suppressive capacities of the two factors appeared to be additive. In this cross, F1 progeny of RF females also showed marked suppression of ecotropic murine leukemia virus expression by comparison with mice of the reciprocal F1 cross, but this suppression of virus expression was not detected in the lymphoma-suppressed AKR backcross population. The observation of lymphoma suppression in the absence of ectropic virus suppression in mice of the (RF X AKR)F1 female x AKR male backross generation indicates a qualitative or quantitative difference in the determination of these two effects.     

Role of recombinant ecotropic and polytropic viruses in the development of spontaneous thymic lymphomas in HRS/J mice.

The biological and genetic characteristics of murine leukemia viruses (MuLV) derived from leukemic and normal HRS/J mice were studied. T1-oligonucleotide fingerprinting and mapping of viral RNAs from unpassaged isolates revealed the presence of complex mixtures of viral genomes. MuLV that were purified by endpoint dilution were genetically heterogeneous. Thus, endogenous retroviral sequences expressed in the tissues of HRS/J mice readily recombined with one another. Furthermore, the regular recovery of recombinant ecotropic MuLV suggested reciprocal in vivo complementation of a genetic defect(s) in each of the endogenous ecotropic proviruses Emv-1 and Emv-3. Some recombinant ecotropic viruses contained sequences in the p15E-U3 region that were not derived from Emv-1 and Emv-3 but were found in recombinant polytropic HRS/J viruses. Finally, comparison of the genetic structures of leukemogenic and nonleukemogenic MuLV of this strain implied that the oncogenic phenotype of these MuLV is encoded within env or the U3 region of the genome or both. Our results are consistent with a stepwise convergent evolution of recombinant MuLV in vivo in individual HRS/J mice. Ultimately, this process of selection results in formation of leukemogenic polytropic viruses.     

50 Hz magnetic fields of 1 mT do not promote lymphoma development in AKR/J mice

Some epidemiological studies suggest that exposure to power-frequency magnetic fields increases the risk of leukemia, especially in children with high residential exposures. In contrast, most animal studies did not find a correlation between magnetic-field exposure and hematopoietic diseases. The present study was performed to investigate whether chronic, high-level (1 mT) magnetic-field exposure had an influence on lymphoma development in a mouse strain that is genetically predisposed to thymic lymphoblastic lymphoma. Three groups of 160 unrestrained female AKR/J mice were sham-exposed or exposed to sinusoidal 50 Hz magnetic fields beginning at the age of 12 weeks for 32 weeks, 7 days per week, either for 24 h per day or only during nighttime (12 h). Exposure was carried out in a blind design. Exposure did not affect survival time, body weight, lymphoma development or hematological parameters. The resulting data do not support the hypothesis that exposure to sinusoidal 50 Hz magnetic fields is a significant risk factor for hematopoietic diseases, even at this relatively high exposure level.     

Genetic alterations of RNA leukemia viruses associated with the development of spontaneous thymic leukemia in AKR/J mice.

T1-oligonucleotide fingerprinting and mapping were used to study the expression of RNA leukemia viruses in leukemic and preleukemic AKR/J mice, with techniques designed to minimize the loss or inadvertent selection of viruses in vitro before biochemical analysis. In leukemic animals, complex mixtures of ecotropic and mink-tropic viruses were expressed. Unique but similar polytropic virus-like genomes were present in each tumor isolate. In preleukemic mice, viral isolates from the thymus that were grown on NIH3T3 fibroblasts contained genomes with non-Akv polytropic virus-related oligonucleotides. This phenomenon was not evident in fingerprints of viruses from the spleen and bone marrow of the same animals. Remarkably, the non-Akv oligonucleotides located in the 3' portion of the P15E gene, the U3 noncoding region, and the 5' part of the gp70 gene were often expressed independently. Our results suggest the following. (i) Recombinant viruses can be detected in the thymuses of young preleukemic AKR mice and increase in relative abundance with age. (ii) During in vivo generation of the recombinant leukemogenic viruses, the selection of polytropic virus-related sequences in the 3' part of p15E and the U3 region and the 5' portion of gp70 occurs independently. (iii) Independent biological properties encoded in the gp70 and p15E regions of env of the recombinant viruses may mediate viral selection or leukemogenicity. (iv) The leukemogenic polytropic viruses of AKR/J mice arise via genetic recombination involving at least three endogenous viral sequences.     

Somatically acquired recombinant murine leukemia proviruses in thymic leukemias of AKR/J mice.

We have probed the structure and arrangement of murine leukemia virus genomes in eight spontaneous AKR thymic leukemias by Southern hybridization with one ecotropic pol and four ecotropic env probes. These probes revealed many (in 2 cases over 15) somatically acquired proviruses that had undergone complex patterns of recombination. The large majority were not deleted and were structurally analogous to the oncogenic mink cell focus-inducing murine leukemia viruses isolated from AKR tumors in that the amino-terminal p15E-coding region derived from ecotropic AKR murine leukemia virus sequences, whereas certain gp70-coding sequences were nonecotropic. Nevertheless, we observed a few proviruses which did not appear to be gp70 recombinants; however, these proviruses were in general clearly recombinant within the p15E-coding sequences. Although the proviral recombination patterns were quite variable, in general the large majority of recombinant proviruses within each tumor appeared structurally identical, indicating that they originate from a common parent. Each tumor contained a unique pattern of provirus integrations; densitometer tracings of the Southern hybridizations indicated that many of the integrated proviruses were present at one copy per cell, suggesting that the tumors derive from a single cell which contained multiple integrated copies of a unique recombinant virus structurally similar to the mink cell focus-inducing viruses.     

Development of donor-derived thymic lymphomas after allogeneic bone marrow transplantation in AKR/J mice

The transplantation of bone marrow cells from BALB/c (but not C57BL/6 and C3H/HeN) mice was observed to lead to the development of thymic lymphomas (leukemias) in AKR/J mice. Two leukemic cell lines, CAK1.3 and CAK4.4, were established from the primary culture of two thymic lymphoma, and surface phenotypes of these cell lines found to be H-2d and Thy-1.2+, indicating that these lymphoma cells are derived from BALB/c donor bone marrow cells. Further analyses of surface markers revealed that CAK1.3 is L3T4+ Lyt2+ IL2R-, whereas CAK4.4 is L3T4- Lyt2- IL2R+. Both CAK1.3 and CAK4.4 were transplantable into BALB/c but not AKR/J mice, further indicating that these cells are of BALB/c bone marrow donor origin. The cells were found to produce XC+-ecotropic viruses, but xenotropic and mink cell focus-forming viruses were undetectable. Inasmuch as thymic lymphomas are derived from bone marrow cells of leukemia-resistant BALB/c strain of mice under the allogeneic environment of leukemia-prone AKR/J mice, this animal model may serve as a useful tool not only for the analysis of leukemic relapse after bone marrow transplantation but also for elucidation of the mechanism of leukemogenesis.     

Unstable heart rate and temperature regulation predict mortality in AKR/J mice

Elderly populations face greater risks of mortality when exposed to changes in environmental stress. The purpose of the following study was to develop an age-dependent susceptibility model that achieved the following three goals: 1) to operationally define homeostasis by assessing the stability and periodicity in physical activity, heart rate (HR), and deep body temperature (T(db)), 2) to specify alterations in activity, HR, and T(db) regulation that signal imminent death, and 3) to test the hypothesis that the decay in homeostasis associated with imminent death incorporates the coincident disintegration of multiple physiological systems. To achieve these goals, the circadian regulation of activity, HR, and T(db) was assessed using radiotelemeters implanted in AKR/J (n = 17) inbred mice at approximately 190 days of age. During a 12:12-h light-dark cycle, weekly measurements were obtained at 30-min intervals for 48-h periods until each animal's natural death. The average (+/-SE) life span of surgically treated animals did not differ from untreated controls (319 +/- 12 vs. 319 +/- 14 days). Cardiac and thermal stability were characterized by a circadian periodicity, which oscillated around stable daily averages of 640 +/- 14 beats/min in HR and 36.6 +/- 0.1 degrees C in T(db). Stable HR and T(db) responses were compared with extreme conditions 3 days before death, during which a disintegration of circadian periodicity was coincident with a fall in the daily average HR and T(db) of approximately 29 and approximately 13% lower (i.e., 456 +/- 22 beats/min and 31.7 +/- 0.6 degrees C), respectively. The results further suggested that multiple predictors of cardiac and thermal instability in AK mice, including significant bradycardia, hypothermia, and a loss of circadian periodicity, forecast life span 5-6 wk before expiration.     

Emv-13 (Akv-3): a noninducible endogenous ecotropic provirus of AKR/J mice

All AKR/J mice carry at least three endogenous ecotropic viral loci which have been designated Emv-11 (Akv-1), Emv-13 (Akv-3), and Emv-14 (Akv-4) (Jenkins et al., J. Virol. 43:26-36, 1982.) Using two independent AKR/J-derived sets of recombinant inbred mouse strains, AKXL (AKR/J x C57L/J) and AKXD (AKR/J x DBA/2J), as well as the HP/EiTy strain (an Emv-13-carrying inbred strain partially related to AKR/J mice) (Taylor et al., J. Virol. 23:106-109, 1977), we have examined the association of these endogenous viral loci with virus expression. Strains which transmit Emv-11 or Emv-14 or both were found to produce virus spontaneously, whereas strains that transmit Emv-13 alone were negative for virus expression. Restriction endonuclease digestion and hybridization with an ecotropic virus-specific hybridization probe of DNAs from strains which transmit only Emv-13 yielded enzyme cleavage patterns identical to those observed with DNAs from strains transmitting Emv-11 or Emv-14 or both. These findings indicate the absence of any gross rearrangement of Emv-13 proviral sequences. Cell cultures derived from recombinant inbred strains that carry only Emv-13 failed to express detectable infectious virus, viral proteins, or cytoplasmic ecotropic virus-specific RNA even after treatment with 5-iodo-2-deoxyuridine or 5-azacytidine, an inhibitor of DNA methylation. Our results indicate that a mechanism(s) other than methylation of Emv-13 proviral DNA is responsible for inhibition of Emv-13 expression.     

Linkage of the Fv-2 gene to a newly reinserted ecotropic retrovirus in Fv-2 congenic mice

Restriction enzyme and Southern gel analyses were used to determine the number and location of endogenous ecotropic retroviruses in the germ line of several mouse strains congenic at the Fv-2 gene locus. A new endogenous ecotropic provirus was observed in the germ line of B6.S (Fv-2ss) mice, in addition to the resident provirus found in its congenic partner C57BL/6 (Fv-2rr). This new provirus was similar in structure to the C57BL provirus. The SIM strain of mice, the donors of the Fv-2s allele in B6.S mice, does not contain ecotropic proviruses, suggesting that the new provirus in the B6.S mouse strain arose by germ-line reintegration during the construction of this strain. Mendelian segregation analysis indicated that this new provirus was linked to the Fv-2 gene locus on chromosome 9. In three other Fv-2s congenic mouse strains--B10.C (47N), B6.C (H-7b), and C57BL/6J Trfa, Bgsd--no additional ecotropic endogenous viruses were detected, suggesting that the reinsertion event that occurred during the construction of B6.S is not essential for the acquisition of the Fv-2s phenotype in the C57BL genetic background. Although numerous reports of germ-line reinsertions of ecotropic virus in high-virus mouse strains have been received, the present results provide definitive evidence that similar germ-line amplifications of endogenous ecotropic virus can occur in a low-virus mouse strain.     

Fv-1 determinants in xenotropic murine leukemia viruses studied with biological assay systems: Isolation of xenotropic virus with N-tropic Fv-1 activity in the cryptic form.

By a biological assay system using phenotypically mixed ecotropic and xenotropic murine leukemia viruses, we investigated whether in the virions of a xenotropic virus there is N- or B-tropic Fv-1 determinant in active form. The existence of N-tropic Fv-1 determinant was demonstrated in SL-XT-1 xenotropic virus isolated from the spleen of a 3-month-old SL mouse, and the N-tropic Fv-1 tropism was confirmed by analysis of the phenotypically mixed viruses harvested from clonal SC-1 cells doubly infected with the SL-XT-1 and B-tropic ecotropic viruses. However, neither N- nor B-tropic Fv-1 determinant was demonstrated in any xenotropic viruses isolated from embryo cells of BALB/c, NZB, or DBA/2 mice, or Cas E #1-IU, and xenotropic-like virus isolated from a wild mouse.     

Genetic and nongenetic factors in expression of infectious murine leukemia viruses in mice of the DBA/2 x RF cross

Mice of the RF and DBA/2 strains possess endogenous ecotropic murine leukemia virus (E-MuLV) genomes but express only low to undetectable levels of infectious virus in their lymphoid tissues. F1 mice of this cross showed high levels of infectious E-MuLV if DBA/2 was the maternal parent but very low levels if RF was the maternal parent. E-MuLV expression, if present, was always higher in the spleen than in the thymus. Studies of reciprocal backcross generations with both parental strains indicated that the presence of the virus was governed by a single dominant autosomal locus present in the RF strain, and that RF females, but neither RF males nor DBA/2 females or males, transmitted a non-Mendelian factor which powerfully suppressed virus expression in their progeny. Some but not all (DBA/2♀ × RF♂)F1 females also possessed the capacity to transmit this maternal suppression to their progeny. Xenotropic murine leukemia virus (X-MuLV) showed a different pattern of expression in this cross. In the thymus it was detected in a minority of DBA/2 and in no RF mice; in crosses the presence of X-MuLV in this organ was independent of the presence of E-MuLV. In the spleen, X-MuLV was detected only in a percentage of E-MuLV-positive mice. The maternal factor from RF mothers which suppressed E-MuLV did not suppress thymic expression of X-MuLV. Skin painting with 3-methylcholanthrene induced a high incidence of thymic lymphoma in mice of both parental strains and in F1 hybrids, all of which normally show only low incidences of the diseases; the treatment did not induce markedly increased expression of E-MuLV or X-MuLV in mice of either parental strain, although it did abrogate the diminution of E-MuLV titers seen with age in (DBA/2♀ × RF♂)F1 mice beyond the age of three months.     

Comparison of endogenous murine leukemia virus proviral organization and RNA expression in 3-methylcholanthrene-induced and spontaneous thymic lymphomas in RF and AKR mice.

3-Methylcholanthrene-induced T-cell thymic lymphomas in RF mice were examined for involvement of murine leukemia virus (MuLV)-related sequences in leukemogenesis. Both the expression of MuLV-related RNA species and the organization of endogenous MuLV proviral DNA were analyzed. Of 27 primary tumors examined, only 5 exhibited elevated MuLV-related RNA species homologous to xenotropic specific env DNA. None of these RNA species hybridized with ecotropic p15E DNA sequences. Only two of these five tumors contained MuLV-like RNA species that hybridized with ecotropic MuLV long terminal repeat sequences, despite the probe's ability to detect both ecotropic MuLV and mink cell focus-inducing viral RNA. No muLV resembling mink cell focus-inducing virus whose expression could be correlated with lymphomagenesis was detected in either preleukemic thymocytes, primary 3-methylcholanthrene-induced thymic tumors, tumors passaged in vivo, or cell lines derived from tumors. Restriction endonuclease analysis of DNA from both primary tumors and cell lines failed to reveal either proviral DNA with recombinant env genes or rearrangement of endogenous MuLV proviruses. Therefore, chemically induced lymphomagenesis in RF mice appears different from the spontaneous lymphomagenic process in AKR mice with respect to the involvement of endogenous MuLV sequences.     

Scrapie infection activates the replication of ecotropic, xenotropic, and polytropic murine leukemia virus (MuLV) in brains and spinal cords of senescence-accelerated mice: implication of MuLV in progression of scrapie pathogenesis

Senescence-accelerated mice (SAMP8) have a short life span, whereas SAMR1 mice are resistant to accelerated senescence. Previously it has been reported that the Akv strain of ecotropic murine leukemia virus (E-MuLV) was detected in brains of SAMP8 mice but not in brains of SAMR1 mice. In order to determine the change of MuLV levels following scrapie infection, we analyzed the E-MuLV titer and the RNA expression levels of E-MuLV, xenotropic MuLV, and polytropic MuLV in brains and spinal cords of scrapie-infected SAM mice. The expression levels of the 3 types of MuLV were increased in scrapie-infected mice compared to control mice; E-MuLV expression was detected in infected SAMR1 mice, but only in the terminal stage of scrapie disease. We also examined incubation periods and the levels of PrPSc in scrapie-infected SAMR1 (sR1) and SAMP8 (sP8) mice. We confirmed that the incubation period was shorter in sP8 (210+/-5 days) compared to sR1 (235+/-10 days) after intraperitoneal injection. The levels of PrPSc in sP8 were significantly greater than sR1 at 210+/-5 days, but levels of PrPSc at the terminal stage of scrapie in both SAM strains were virtually identical. These results show the activation of MuLV expression by scrapie infection and suggest acceleration of the progression of scrapie pathogenesis by MuLV.     

AKR leukemogenesis: identification and biological significance of thymic lymphoma receptors for AKR retroviruses.

We have previously demonstrated that in vitro cell lines of mouse thymic lymphomas express surface receptors specific for the retrovirus that induced them. This study extends these observations to an analysis of receptor-bearing cells in the preleukemic and leukemic phases of spontaneous AKR thymic lymphomagenesis. AKR mice regularly begin expressing N-tropic retroviruses (as assayed on NIH fibroblasts by the XC plaque assay) in several tissues early in life; thymic lymphocytes also express these viruses, but are not autonomously transformed. Later thymic lymphomas emerge which are capable of metastasizing in the host of origin or transplanting leukemias into syngeneic hosts. Just prior to the appearance of thymic lymphomas, these mice also begin producing xenotropic retroviruses [as assayed in xenogeneic (For example, mink) fibroblasts], and concomitant with the appearance of the leukemias is the appearance of "recombinant" retroviruses which cause mink fibroblast foci (MCF); these viruses express elements of both N- and X-tropic virus envelopes and N-tropic viral gene products in their cores. Spontaneous AKR leukemias also produce other retroviruses which do not cause XC plaques or mink fibroblast foci; these are called SL viruses. The subject of this study was to test whether in vivo thymocytes in the preleukemic and leukemic periods also bear receptors specific for N-tropic, recombinant MCF and SL AKR retroviruses. We demonstrated that each spontaneous thymic lymphoma does bear receptors that bind viruses produced by the lymphomas and MCF-247 to a high degree and that bind N-ecotropic AKR retroviruses less well. Thymic lymphocytes predominating in the preleukemic period do not express detectable levels of receptors for either of the viruses. In some mice, receptor-positive cells co-exist with receptor-negative cells; only the receptor-positive cells are capable of transplanting leukemia to syngeneic hosts. We conclude that the presence of specific cell surface receptors for lymphoma cell-produced and recombinant AKR retroviruses is a marker for leukemia in these hosts.     

Chromosomal localization of Emv-16 and Emv-17, two closely linked ecotropic proviruses of RF/J mice.

Emv-16 and Emv-17, the two closely linked ecotropic proviral loci of RF/J mice, have been mapped to chromosome 1 between leaden, ln, and the mouse engrailed homeo-box locus, En-1, by using recombinant inbred strains and conventional backcross analysis.     

Pattern of expression of ecotropic murine leukemia virus in gonads of inoculated SWR/J mice.

An ecotropic murine leukemia virus (MuLV) isolate has recently been shown to be able to infect the germ line or the early embryo or both when inoculated at birth to SWR/J females (J. J. Panthier, H. Condamine, and F. Jacob, Proc. Natl. Acad. Sci. USA 85:1156-1160, 1988). We have used this isolate to further study this phenomenon. By using the techniques of RNA-RNA in situ hybridization, immunocytochemistry, and transmission electron microscopy, the identities of two important cell types that are infected by ecotropic MuLV in the gonads of inoculated mice were determined. These cells are the thecal cells surrounding the follicles in the ovary and the Leydig cells in the testis. Both types actively synthesize viral RNA and express a viral antigen. Furthermore, we documented the production of viral particles by the thecal cells. The expression of ecotropic MuLV by nonlymphoid cells in vivo may play a key role in the vertical transmission of these viruses by females as well as in their horizontal transmission.     

Chromosomal assignment of two endogenous ecotropic murine leukemia virus proviruses of the AKR/J mouse strain.

The AKR/J mouse strain is genetically fixed for three different ecotropic murine leukemia virus genomes, designated Akv-1, Akv-3, and Akv-4 (Emv-11, Emv-13, and Emv-14). With recombinant inbred strains and crosses with linkage-testing stocks, Akv-3 and Akv-4 were placed on the mouse chromosome map. Akv-3, which encodes a replication-defective provirus, maps near the agouti coat color locus, a, on chromosome 2. Akv-4, which is replication competent, maps near the neurological mutant gene locus trembler, Tr, on chromosome 11. Akv-1 and Akv-2 (Emv-12), an ecotropic provirus carried by AKR/N but not AKR/J, have previously been mapped to chromosome 7 and 16, respectively. Thus, the four Akv proviruses mapped to date are on four different chromosomes. Akv-3 is the second ecotropic murine leukemia virus provirus to be mapped near the agouti locus. The results are discussed in relation to possible nonrandomness of viral integration.