Biomedical subjects
Publications and source records attributed to F Lilly.
Reciprocal bone marrow transfers were performed between mouse strains sensitive or resistant to 3-methylcholanthrene-induced thymic lymphoma. Sensitivity and resistance are properties inherent in bone marrow, and cannot be altered by maturation of marrow in an environment of the opposite phenotype.
Friend virus was used to infect and transform Fv-2rr erythroprogenitor cells in vivo. The RB (Fv-2rr) cell line was characteristic of Friend virus-induced cell lines in Fv-2ss mice, i.e., it produced infectious Friend virus and synthesized hemoglogin. The RB (Fv-2rr) cell line expressed the envelope protein of the spleen focus-forming virus (gp52) and a novel, related envelope protein (gp48). The results demonstrate that Fv-2rr erythroprogenitor cells can be infected and transformed in vivo.
BSB is a variant strain of Friend virus selected for pathogenicity in C57BL/6 mice that are resistant to parental Friend virus strains by virtue of their homozygosity for the recessive Fv-2r allele (Steeves et al., 1970, Int. J. Cancer 5, 346-356). Lines and clones of erythroleukemia cells could readily be established in culture from the enlarged spleens of BSB-infected Fv-2r homozygotes. All lines expressed viral gene products and could be induced to express hemoglobin. Some lines produced infectious virus. In addition to the viral envelope-related proteins (gPr85, gp70, and gp52) detected by precipitation with goat anti-Rauscher gp70 antiserum from tumor cell lines induced by parental Friend virus strains, BSB-induced cell lines also expressed gp80, p52, and gp45 products precipitable with the same antiserum. A rat monoclonal antibody that recognizes an epitope of an amino-terminal region of gp52 (Wolff et al., 1982, J. Virol. 43, 472-481) also precipitated the gp80 and gp45 viral proteins. The data indicate that the BSB strain of Friend virus is oncogenic in Fv-2r homozygotes. Transformation is correlated with the expression of an altered SFFV env-gene product.
BALB/c mice injected with seven 0.15-ml samples of whole rabbit serum over a 2-week period developed nonneoplastic proliferation of the extrahepatic bile duct epithelium and glandular components. Sera from other animals, including bovines, humans, pigs, goats, and chickens as well as non-serum-containing secretions such as human breast milk and bile also produced this effect. Partial purification utilizing gel filtration and affinity chromatography of the active 33-65% saturated ammonium sulfate precipitate of whole serum indicated that the distribution and characteristics of this glycoprotein showed some similarities with those of IgA. Chromatographically purified human IgA was administered to BALB/c mice and was found to induce bile duct proliferation identical to that seen with whole human serum. Purified human IgG and IgM had no activity. Since IgA-containing serum from BALB/c mice was inactive, it appears that heterologous IgA functions as a specific extrahepatic bile duct growth factor (BDGF) in BALB/c mice. Murine susceptibility to the growth-stimulating effect of serum was strain specific; genetic studies utilizing crosses of susceptible (BALB/c) and resistant (C57BL/10) strains of mice revealed that the ability to respond to the infusion of BDGF is inherited in a polygenic fashion.
We have constructed a series of "synthetic" target cell lines for an analysis of the specificity of anti-Friend virus (FV) CTL. Our results show that murine H-2 genes and individual retroviral genes can be stable expressed in Fisher rat embryo (FRE) cells, and that their products have the potential to form target structures recognized by mouse CTL. Cells expressing H-2Db and either the env or gag genes of one component of FV, helper Friend murine leukemia virus (FMuLV), were lysed by anti-FV CTL and by CTL generated against FMuLV alone. Experiments with Db-transfected FRE clones infected only with the replication-defective spleen focus-forming virus (SFFV) component of FV indicate that the SFFV genome also provides specificities recognized by both anti-FV and anti-FMuLV CTL, thus demonstrating the existence of a crossreactive CTL population. An unexpected finding was that anti-FMuLV CTL, but not anti-FV CTL were also able to lyse FRE clones that expressed H-2Kb in either the presence or absence of FV. The use of heterologous cell lines for the construction of synthetic target cells thus offers a useful approach for the analysis of T cell specificity.
AKR mice, which spontaneously develop greater than 90% incidence of lymphocytic leukemia (LL), crossed with SJL mice, which show greater than 80% incidence of Hodgkin's-like reticulum-cell sarcoma (RCS), produced F1 progeny showing incidences of 30% LL and 0% RCS. Thus, each strain possesses one or more dominant genes capable of interfering with the emergence of the tumor type typical of the other strain. Although mice of reciprocal F1 crosses showed a profound difference in expression of endogenous ecotropic murine leukemia virus (E-MuLV) due to a maternal resistance factor transmitted by SJL females but not males, the two populations did not differ detectably in LL incidence. Like AKR mice, mice of 5 other strains studied (C58, DBA/2, PL, RF and ST/b) possessed one or more genes conferring resistance to RCS in F1 crosses with SJL. Analysis of LL incidences in F1 generations of all possible crosses among these 7 strains revealed 4 different categories of strains with respect to susceptibility/resistance to LL; only ST/b mice, which show no significant incidence of spontaneous LL, lacked genes that could suppress the disease in crosses with high- or moderate-incidence strains. SJL mice treated topically with 3-methylcholanthrene (MCA) developed a 50% incidence of LL, mostly before one year of age; treated mice surviving after one year of age developed a high incidence of RCS.
Trisomy 15 is a frequent chromosomal abnormality found in murine thymic tumors that develop spontaneously or by induction with irradiation, chemical carcinogens, or leukemogenic retroviruses. The results of our studies demonstrate that T-cell lymphomas induced in RF mice with the chemical carcinogen 3-methylcholanthrene predominantly exhibit a normal diploid complement of chromosomes with no evidence of chromosome 15 trisomy after in vivo and in vitro passage. We conclude that accumulation of aneuploidy is not necessary for maintenance of the tumor phenotype and that chemically induced lymphomas can develop in a defined subpopulation of T cells in the absence of trisomy 15.
Expression of 11 cellular oncogenes was determined in normal vs. lymphomatous thymic tissues of RF and AKR mice; only c-myc and c-myb transcripts were detected in an age-inappropriate pattern in thymomas. Normal thymocytes from young RF mice contained RNA transcripts of both genes, but the transcripts were no longer detected at 9 or more weeks of age. More than 90% of RF thymomas, occurring at 20-28 weeks of age after skin painting with 3-methylcholanthrene at 12 weeks, contained c-myc transcripts, and 70% of the tumors contained c-myb transcripts. Seven cell lines derived from these 3-methylcholanthrene thymomas expressed both cellular genes, as did 2 rare spontaneous thymomas of 12-month-old RF mice. No indication of rearrangement or amplification of either gene was seen in any of the RF tumors or cell lines. In AKR mice, transcripts of the 2 genes persisted longer in the normal thymus than in RF mice, but they were no longer detected at 26 weeks of age. Of 3 thymomas in 6-month-old 3-methylcholanthrene-treated AKR mice, all expressed c-myb and 2 expressed c-myc. Among 11 spontaneous AKR thymomas, however, only 2 showed detectable levels of both genes, and 2 more expressed c-myc or c-myb but not both.
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.
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3-Methylcholanthrene (MCA) skin painting rapidly induced a 100% incidence of thymic lymphomas in RF/J mice. Tumors developed exclusively in cells of the T-lymphocyte lineage and displayed the surface phenotype: Thy-1+, Lyt-1+, Lyt-2+, Qa-1+, H-2K+, H-2D+, TL-, Ig-, Ia-. This phenotype resembled that of cortisone-resistant, medullary thymocytes, whereas the phenotype of spontaneous AKR thymomas resembles that of cortisone-sensitive, cortical thymocytes. The phenotype differed very little from one tumor to another and was maintained during syngeneic passage in vivo and adaptation to growth in culture. Infectious ecotropic murine leukemia virus (MuLV) was produced in a minority of primary tumors and cell lines, but no xenotropic or mink cell focus-inducing virus (MCF) MuLV production was reliably detected. MuLV-related antigen expression on normal thymocytes increased in an age-dependent manner, and levels on thymoma cells were similar to those on thymocytes from age-matched normal controls. MuLV nonproducer tumor cells expressed few or no epitopes of AKR ecotropic gp70, but they were positive when tested with an antiserum prepared against MCFenv glycoprotein.
Multiple low-dose injections of streptozotocin (STZ) induce a delayed but progressively increasing state of hyperglycemia in mice. Different inbred strains of mice show different susceptibility to this treatment. We examined whether genetic factors associated with the H-2 complex influence the susceptibility or resistance, using a selected group of 12 inbred and 5 congenic resistant strains of mice. We found that different congenic strains differed significantly in their susceptibility to STZ-induced diabetes, suggesting that H-2-associated genes do influence the susceptibility. However, at least some inbred strains sharing the same H-2 haplotype also differed in their susceptibility, indicating that genes outside the H-2 complex may also affect the susceptibility. Therefore, there appear to be at least two genes, one within and one or more outside the H-2 complex, that determine the susceptibility to multiple low doses of STZ.
Females of the RF and SJL inbred mouse strains transmit to their progeny of both sexes a nonmendelian maternal resistance factor (MRF) able to suppress the expression of endogenous ecotropic murine leukemia virus (E-MuLV). This MRF is demonstrable in crosses with AKR mice by comparing E-MuLV expression in the spleens and thymuses of reciprocal F1 generations. DBA/2 and ST/b mice are MRF negative by these criteria. Neonatal inoculation of E-MuLV-containing spleen extracts gives rise to persistent expression of infectious virus in mice of the MRF- but not the MRF+ strains. However, inoculation of the virus in 30-d-old females of the MRF- strains no longer leads to a state of persistent infection; instead, these females become MRF+ and transmit protection against E-MuLV expression to their progeny by AKR and RF males. The MRF appears to be transmitted to the progeny mainly through the milk, since foster-nursing AKR neonates on RF (but not DBA/2) mothers greatly reduces E-MuLV expression in the progeny. These RF-fostered AKR mice also show a reduced and delayed lymphoma incidence, a finding consistent with the idea that maternally transmitted resistance to E-MuLV expression is the basis for the classic maternal resistance to lymphomagenesis seen in the progeny of RF mothers.
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Mouse spleen cells, cultivated in vitro for 6 days in the presence or absence of syngeneic irradiated virus-induced tumor cells, lysed a variety of virus-induced tumor cells and normal cells in a long (19-hr) 51Cr-release cytotoxicity assay. Little or no specificity for viral or H-2 antigens was detected. This anomalous killing (AK) activity contrasted with H-2-restricted cytotoxic T lymphocyte (CTL) activity generated in secondary mixed leukocyte-tumor cell cultures and detected in a short (4- to 6-hr) 51Cr-release assay. Studies of competitive inhibition by unlabeled target cells in the long assay suggested that AK effectors were a broadly heterogeneous mixture of cells representing many clones, each recognizing a different antigenic determinant. AK effector cells shared properties with CTL: they could be generated from spleen cells of natural killer cell-deficient mice bearing the beige mutation but not from those of T cell-deficient nude mice; they expressed Thy-1 and Lyt-2 antigens; and they were nonphagocytic. AK activity could be generated from normal spleen cells of mice of all inbred strains examined, but the levels of activity generated varied in a strain-specific manner and were not significantly influenced by H-2 genotype.
Comparative quantitative experiments were designed to study the expression of H-2Kd and H-2Dd antigens on three different leukemia cell lines induced by Gross murine leukemia virus (MuLV)in BALB/c (H-2d) mice. The H-2 restriction patterns of syngeneic cytolytic T lymphocytes (CTL) directed against Gross MuLV-induced tumors were correlated with these quantitations of H-2Kd and H-2Dd antigens, Our results obtained by quantitative absorption of monospecific antisera indicated that the three BALB/c tumor cell lines expressed different amounts of H-2Kd and H-2Dd antigens, with H-2Dd antigen showing the greatest variability in expression because it ranged from barely detectable levels to one-eighth the amount of H-2Dd antigen expressed on normal BALB/c spleen cells. The H-2 restriction patterns of Gross MuLV-specific CTL were directly affected by these quantitative modulations in the expression of H-2Kd and H-2Dd antigens, as revealed by three independent approaches: (a) inhibition of CTL activity by monospecific anti-H-2 sera in the absence of complement; (b)competitive inhibition of CTL-mediated cytotoxicity by the addition of excess tumor cells into the reaction mixture; and (c) analysis of CTL specificities using cloned CTL populations. Our results thus indicate that H-2 restriction of tumor-specific CTL activity can be directed at the target cell level by variations in the expression of H-2 antigens.
An H-2Db heterozygous tumor cell line and a variant subclone bearing a mutant gene product were used to analyze the H-2Db specificity of cytotoxic T lymphocytes (CTL) generated during a Moloney murine sarcoma virus (MSV) infection. When the mutant cells were used as targets for MSV-specific CTL, the amount of cell lysis, compared with that seen with the nonmutant parental cells, was drastically decreased. However, cells of the mutant clones remained susceptible to allogeneic CTL specific for the nonmutant H-2Db molecule. The mutant cells also did not differ from the parent cells in their level of viral antigen expression. Biochemically the parental and mutant molecules were similar but not identical. The data indicate that minor alterations of the H-2 antigens caused by somatic mutation may prevent virus-infected cells from being recognized as targets by CTL.
Immunization of rats with syngeneic cells infected with spleen focus-forming virus (SFFV) but not with its helper, Friend murine leukemia virus (FMuLV), produces antisera which specifically neutralize SFFV, and not FMuLV, in the Friend virus complex. To determine which SFFV-encoded protein molecule bears the antigen recognized by these neutralizing antibodies, we studied different lots of rat anti-SFFV antiserum by immunoprecipitation and virus neutralization assays. The ability of these sera to neutralize SFFV correlated with the titer of antibodies to p45gag and not with the titer of those to gp52, suggesting that the neutralizing antibodies recognize the p45gag molecule. To verify this specificity for p45gag, we tested antisera to various MuLV gag gene-encoded proteins for neutralization of SFFV. Goat anti-Rauscher murine leukemia virus (RMuLV) p30 and goat anti-RMuLV p10 sera neither precipitated p45gag from SFFV-infected nonproducer cells nor neutralized SFFV. In contrast, goat anti-RMuLV Pr65gag and goat anti-RMuLV p12 sera precipitated p45gag from SFFV-infected cells and also specifically neutralized SFFV in the Friend virus complex. These findings suggest that, unlike the gag proteins coded for by FMuLV, the proteins coded for by defective SFFV are incorporated into the envelope of virions carrying the SFFV genome, but not into the envelope of those carrying the helper FMuLV genome.