Biological activity and separation of a leukaemogenic virus from murine sarcoma virus-Harvey (MSV-H)

Twelve different preparations of Murine Sarcoma Virus-Harvey (MSV-H) had leukaemogenic activity when tested in BALB/c and C₃H/Bi mice or Sprague-Dawley rats, some of which ultimately developed generalized lymphocytic leukaemia instead of reacting rapidly and characteristically with sarcoma formation and/or erythroblastic splenomegaly. Inactivation of MSV-H by ether treatment, reducing its titre by dilution, and using the intramuscular route of injection, all biased towards leukaemogenesis. The leukaemogenic component could be separated by vertical transmission from three MSV-H-infected female mice, which themselves showed only sarcomas and/or splenic erythroblastosis, to as many as six successive generations of their untreated offspring. Presence of a leukaemogenic virus, probably MLV, was confirmed by serial passage of plasma filtrates from leukaemic animals, including leukaemic progeny, in newborn mice and rats. Lymphocytic leukaemia developed exclusively in these recipients and in those given a plasma concentrate. Moreover, pre-treatment with the leukaemogenic virus enhanced the oncogenicity of MSV-H in 8- and 14-day-old mice, as judged by an increased incidence of sarcomas. Pathological changes in the kidneys of the leukaemic mice are also described, which probably represent an immune deposit lesion related to the deposition of circulating complexes of viral antigen and anti-viral antibody on the basement membranes of the glomerular capillaries.     

The differential action of neonatal thymectomy in mice infected with Murine Sarcoma Virus-Harvey (MSV-H)

Intact C3H/Bi and BALB/c mice infected with Murine Sarcoma Virus-Harvey (MSV-H) at 3–16 days developed either ?early”? erythroblastic splenomegaly with concomitant severe anaemia and sarcomas at sites related to the route of inoculation or, after a perceptible delay, ?late”? lymphocytic leukaemia. These effects were dependent on the dilution of the virus and the age of the recipients. Neonatal thymectomy both accelerated the erythroblastosis and increased the tumour incidence in older 14–16 day recipients but prevented (or indefinitely postponed) the lymphocytic leukaemia. This differential action of neonatal thymectomy is discussed in the light of current immunological theory. A virus causing only lymphocytic leukaemia was separated by passage from the plasma of leukaemic intact or non-leukaemic thymectomized mice. Thus, in these experiments, MSV-H behaved as a complex of two viruses, one causing the early changes and a second, always present in higher concentration but slower acting, responsible for the late changes.     

Studies on Murine Sarcoma Virus; a morphological comparison of tumorigenesis by the Harvey and Moloney strains in mice, and the establishment of tumor cell lines

Anatomic lesions produced by the Moloney and Harvey strains of Murine Sarcoma Virus (MSV-M and MSV-H) in mice have been compared. Erythroblastic splenomegaly is a distinctive feature of disease produced by MSV-H. Solid tumors induced by both viruses were quite similar in morphology, although some consistent differences were noted. The tumors appear to arise by massive recruitment of primitive mesenchymal cells rather than by clonal proliferation. The morphological features of these lesions would suggest that, although MSV-M and MSV-H are similar entities, they are not quite identical. Four transplant lines have been established from the mouse tumors; three MSV-H and one MSV-M. The MSV-M line (CBA strain) was initiated with difficulty and did not release sarcoma virus. The MSV-H lines (two CBA and one BALB/c) were easier to initiate and virus release was demonstrated both in vivo and in vitro. In addition to tumor growth at the site of implantation, the BALB/c line also produced splenic erythroblastosis. The MSV-M and one of the MSV-H CBA lines underwent a distinctive alteration during the course of in vivo passage in which the original spindle-cell lesion evolved into an undifferentiated small-cell tumor.     

Susceptibility of Praomys (Mastomys) natalensis to the Murine Sarcoma Virus-Harvey (MSV-H)

Praomys (Mastomys) natalensis, which are African rodents intermediate in size between rats and mice, were tested for susceptibility to the Murine Sarcoma Virus-Harvey (MSV-H) and to the Friend Leukaemia Virus (FLV). Newborn Mastomys, like rats and mice, reacted within 28–127 days to MSV-H injected intraperitoneally. The majority (14/16) developed gross splenomegaly (with concomitant anaemia) due partly to proliferation of erythroblasts and partly to the formation of large sarcomas. This tumour formation was much more regular, rapid and extensive than that seen in the spleens of rats and mice, while the erythroblastosis was equally intense. Sarcomas were found at other sites, namely, in the diaphragm, peritoneal wall and splenic mesentery of only 4/16 infected Mastomys, suggesting that the splenic tumours were primary lesions. One Mastomys developed reticulum cell leukaemia and another a leukaemia characterized by a mixed population of lymphoblasts and erythroblasts. In line with findings in the rat and mouse, but not the hamster, there was no change in the species specificity of MSV-H recovered from the plasma of infected Mastomys which was fully infective when tested in newborn Mastomys and BALB/c mice. By contrast, newborn or weanling Mastomys injected intravenously or intraperitoneally with FLV remained healthy during an observation period of 5 months.     

Leukemogenic activity of murine type C viruses after long-term passage in vitro.

Cloned stocks of several murine leukemia viruses (MuLVs) were shown to be leukemogenic for susceptible mice after more than nine years of in vitro passaging in mouse embryo fibroblasts. Tissue culture-grown Rauscher (R-) MuLVs injected into newborn or young adult BALB/c mice induced lymphocytic leukemias in 100% of the animals beginning 80 days post-inoculation. No erythroblastic leukemia was observed even after passaging the tissue-culture-grown R-MuLVs twice through mice, indicating that the component responsible for that disease had been lost or attenuated during growth in fibroblasts. The tissue-culture-grown stock of Moloney (M-) MuLVs likewise induced lymphocytic leukemias in 94% of injected newborn BALB/c mice, and the tissue culture-grown Gross (G-) MuLVs induced lymphocytic leukemias in 42% of injected newborn C3Hf mice. The host range and neutralization characteristics of viruses recovered from animals that became leukemic after injection with the tissue-culture-maintained MuLVs were found to be identical with those of the injected viruses. These data implicate the injected MuLVs in the induction of the leukemias and suggest that the capacity to induce the disease is stably inherited as part of the viral genome even in the absence of expression.     

The effect of cyclophosphamide on MSV-H oncogenesis.

The effect of cyclophosphamide on MSV-H oncogensis and the immune response of young mice has been investigated. A single, sublethal dose (100 and 50 mg/kg of cyclophosphamide) in 8-day-old mice given 24 h before or after MSV-H infection led to an earlier and lower incidence of tumours in comparison with controls infected only with MSV-H. The protective effect of cyclophosphamide, and the mechanism of action of both cyclophosphamide and MSV-H on the target cells, mesenchymal cells in rapid replication, as well the immunological implications of the findings are discussed.     

Antitumor effect of OK-432 (3)--mechanisms of tumor growth inhibition by OK-432 induced activated macrophages

Spleen cells and peritoneal exudate cells obtained from BALB/c mice which had received an i.p. injection of 0.1 mg of OK-432 4 days previous to sacrifice, were examined by Winn's neutralization assay for their antitumor activity against Meth-A sarcoma cells in BALB/c mice. Both of the cell preparations clearly inhibited the growth of admixed Meth-A cells, but when these same cell populations were treated on a Sephadex G-10 column, the effector activity seen in Winn's assay disappeared. The effector cells responsible for tumor inhibition were therefore considered to be cytotoxic macrophages. However, the inhibitory effect of these cytotoxic macrophages in Winn's assay was not evident in either X ray (300 rad)-irradiated BALB/c mice or in nu/nu BALB/c mice. These results indicate that the antitumor activity of cytotoxic macrophages is associated with a sequential immune mechanism in which T cells may play an important role.     

Distribution, covalent binding, and DNA adduct formation of 7,12-dimethylbenz(a)anthracene in SENCAR and BALB/c mice following topical and oral administration

SENCAR mice are much more susceptible to tumor initiation by 7,12-dimethylbenz(a)anthracene (DMBA) administered topically than p.o. and are also more susceptible to initiation by topically applied DMBA than are BALB/c mice. To determine how the distribution and metabolic activation of DMBA differed in these strains and with route of administration, BALB/c and SENCAR mice were exposed to [3H]DMBA topically and p.o., and the distribution and DNA binding of DMBA were analyzed. Both the amount of DMBA in skin and the covalent binding of DMBA to epidermal DNA were greater following topical administration than after p.o. administration in both strains. Differences in DMBA distribution and macromolecular binding were found between SENCAR and BALB/c mice, with the binding of DMBA to DNA in epidermis tending to be greater in BALB/c mice than in SENCAR mice when differences were observed. The formation of individual DMBA:DNA adducts in epidermis was also examined in SENCAR and BALB/c mice following topical administration of DMBA. No substantial qualitative or quantitative differences in DMBA:DNA adducts were found between SENCAR and BALB/c mice. The anti/syn-DMBA-diol-epoxide-DNA adduct ratios calculated from the three major DMBA:deoxyribonucleoside adducts increased with time in both SENCAR and BALB/c mice. The data suggest that differences in the distribution and macromolecular binding of DMBA are responsible for the much greater skin tumor initiating activity of DMBA applied topically than p.o. but do not account for the greater sensitivity of the SENCAR mouse to DMBA-induced epidermal tumorigenesis.     

Genetic unresponsiveness to a murine fibrosarcoma determined by the host genetic environment but not by lymphocyte precursor genotype

(BALB/c X C3Hf) (H-2d X H-2k)F1 hybrid mice but not parental BALB/c or other BALB/c X H-2k F1 hybrids, were unresponsive in transplantation and in neutralization (Winn) assay against a 3-methylcholanthrene-induced BALB/c fibrosarcoma. In BALB/c mice the antitumor activity revealed by Winn assay with antitumor immune lymphoid cells was shown to be tumor specific and mediated by Thy 1+ cells. Mouse chimeras were constructed by injecting fetal liver cells into irradiated recipients. BALB/c----(BALB/c X C3Hf)F1 and control (BALB/c X C3Hf)F1----(BALB/c X C3Hf)F1 chimeras were unable to develop a transplantation immunity against the immunizing tumor, whereas (BALB/c X C3Hf)F1----BALB/c chimeras were able to respond to the immunizing tumor. Thus, unresponsiveness was shown to be due to a defect in the maturation of precursor stem cells both of parental and F1 hybrid origin in the body of the (BALB/c X C3Hf)F1 animals.     

Immune-mediated arrest and reversal of established visceral metastases in athymic mice.

The metastasizing MDAY-D2 tumor of DBA/2 mice disseminates in BALB/c allogeneic athymic nude (nu/nu) mice in a manner identical to that observed in the syngeneic host. Both the kinetics and organ distribution pattern of metastases from s.c. implants of MDAY-D2 are routinely predictable at any given tumor dose. BALB/c heterozygote (nu/+) litter-mates reject MDAY-D2 grafts on the basis of the multiple minor histocompatibility differences that exist between DBA/2 and BALB/c mice. The in vitro cell-mediated cytotoxic response detected in tumor-bearing BALB/c nu/+ mice is "low grade" (isotope release is approximately 40 to 50% by 24-hr 111-indium-8-hydroxyquinoline assay and approximately 6 to 8% by 6-hr 51Cr assay) and yet correlates directly with tumor rejection. BALB/c nu/nu mice can be protected against MDAY-D2 by previous reconstitution with lymphoid cells from normal or MDAY-D2-sensitized BALB/c nu/+ mice. In addition, surgically documented, established visceral metastases in BALB/c nu/nu mice can be arrested and regressed by the adoptive transfer of MDAY-D2-sensitized BALB/c nu/+ spleen cells. This represents one of the few models where established metastases have been immunotherapeutically regressed. As such, the MDAY-D2 BALB/c nu/nu mouse model offers unique advantages for studying the role of the immune system in regulating the metastatic process.     

Different weekly changes in immune response in virus-transformed cell-injected mice fed two different diets

Changes in tumor development and in certain immune responses were investigated at 7-weekly intervals after subcutaneous injection of 5 X 10(5) herpes simplex virus Type 2-transformed cells (H238 cells) into male BALB/c mice fed 2 different diets. One diet contained 11% casein and 5% fat while the other had 11% supplemented wheat gluten and 30% fat. Weanling mice (140/group) were fed one or the other of the diets for 12 weeks before injection and subsequent testing of 15 injected and 5 non-injected mice from each diet group each week. In mice fed the low-fat diet containing casein both tumor incidence and tumor volume were significantly lower (P less than or equal to 0.05) than in the group fed the 30% fat diet containing supplemented wheat protein. The casein-fed mice also had less splenomegaly and a higher proportion of mature lymphocytes in the spleen during tumor growth. The proliferative capability of the spleen cells after phytohemagglutinin stimulation was enhanced 2 weeks after H238 cell injection only in the casein-fed mice.     

Different hematological diseases induced by type C viruses chemically activated from embryo cells of different mouse strains.

Type C RNA viruses can be induced by certain chemicals from cells of many mouse strains. Both C58 and BALB/c cells have been shown to contain endogenous viruses that are designated N-tropic because they grow preferentially in cells of NIH Swiss mouse origin. While demonstrating many similar biological and immunological properties, the C58-induced virus is around 10-fold more infectious per physical particle than the N-tropic virus of BALB/c cells. In the present studies, inoculation of these viruses into newborn NIH Swiss mice led to the development of diseases associated with splenomegaly and lymphadenopathy at similar frequency in each group. The disease induced by C58-MuLV was histophathologically diagnosed as lymphoblastic leukemia and was highly malignant following transplantation into newborn mice. The histopathological appearance of spleens from BALB/c virus-affected animals was distinguishable, demonstrating instead myeloid metaplasia or myelogenous leukemia. These findings provide evidence that different endogenous mouse type C viruses can induce distinct diseases in the same mouse strain. Furthermore, they implicate the N-tropic virus endogenous to C58 cells as a major factor in the development of lymphoblastic leukemia that occurs at high frequency in that strain.     

The importance of p53-independent apoptosis in the intestinal toxicity induced by raltitrexed (ZD1694, Tomudex): genetic differences between BALB/c and DBA/2 mice.

The thymidylate synthase inhibitor raltitrexed (ZD1694, Tomudex) induces greater intestinal toxicity, manifested as diarrhea and weight loss, in BALB/c than in DBA/2 mice. No convincing pharmacokinetic or pharmacodynamic reason for this strain difference has been established. We have investigated whether this strain difference in response to raltitrexed is related to differential susceptibilities of intestinal mucosae to undergo apoptosis and also whether p53 expression, a critical factor in 5-fluorouracil-induced intestinal apoptosis and toxicity, modulates this response. Ten mg/kg or 100 mg/kg raltitrexed were administered as single or double i.p. injections 24 h apart to BALB/c, DBA/2, and p53-/- mice. Apoptosis, mitosis, and tissue damage were assessed in intestinal epithelium, and animal weight was recorded. BALB/c mice developed diarrhea and weight loss following 100 mg/kg x2 raltitrexed, whereas DBA/2 mice did not. BALB/c mice were more sensitive than DBA/2 to induction of small-intestinal and colonic apoptosis 24 h following 100 mg/kg raltitrexed. Inhibition of mitosis was equivalent in both strains. Both strains showed histopathological damage to the small intestine after 100 mg/kg x2 raltitrexed, but only BALB/c mice demonstrated colonic damage. p53-null mice showed the same level of small intestinal apoptosis as their wild-type counterparts 24 h after 100 mg/kg x1 raltitrexed and also the same levels of intestinal toxicity 3, 5, and 7 days after 100 mg/kg x2 raltitrexed. Thus, BALB/c mice were more susceptible to induction of intestinal apoptosis by raltitrexed than DBA/2 mice and also demonstrated more histopathological damage in the colon correlating with the induction of diarrhea and weight loss. In contrast to 5-fluorouracil, the intestinal apoptosis and toxicity induced by raltitrexed were p53-independent.     

Decreased pathogenicity of murine leukemia virus-Moloney in gnotobiotic mice

Newborn germ-free (GF) and conventional (CV) BALB/c mice were infected with murine leukemia virus-Moloney (MuLV-M) and subsequently monitored for virus expression and leukemia development. GF mice expressed more than 10-fold less virus in peripheral blood compared with CV mice, despite equivalent numbers of infected cells in the spleens, lymph nodes, thymi, and bone marrow of both groups. In addition to lower levels of virus expression, the latency period before the onset of fatal leukemias was greatly extended in GF mice; the first and last fatalities were recorded at 25 and 43 weeks postinfection, respectively, with a mean survival time of approximately 36 weeks. In CV mice, the first and last fatalities occurred at 8 and 17 weeks, respectively, with a mean survival time of approximately 13.5 weeks. Finally, the gross pathology of involved lymphoid organs varied in the two groups. GF mice experienced severe splenomegaly with or without lymphadenopathy but without thymoma; CV mice, in contrast, developed splenomegaly, lymphadenopathy, and severe thymoma. Collectively, these results indicate a marked resistance of GF animals to MuLV-M and suggest that the level of immune system activation may influence the pathogenicity of nontransforming retroviruses.     

Effect of antithymocytic serum on viral leukemia, erythroblastosis, and sarcoma in mice

The effect of heterologous antilymphocytic sera (ALS) on the growth of a number of virus-induced erythroblastoses, leukemias and sarcomas in mice has been investigated. Erythroblastosis is markedly stimulated by ALS, given either before or several days after virus inoculation. The stimulating effect of ALS administration on the outgrowth of murine sarcoma is strong if ALS is given before MSV inoculation, and weak when given no more than 24 h after the virus. Conversely, a stimulating effect of ALS on the development of leukemias was barely detectable, suggesting that immune reactions are less effective in the studied leukemia than in sarcoma and erythroblastic disease. An erythroblastic disease similar to the Friend-Rauscher virus induced splenomegaly was observed after inoculation of Moloney leukemia virus or sarcoma virus and ALS.     

Leukemoid reaction in BALB/c mice bearing primary tumors induced by 3-methylcholanthrene

During the growth of five of ten primary tumors that were induced by 3-methylcholanthrene in BALB/cMk and BALB/cMk X C57BL/6F1 mice, leukemoid reactions, characterized by increase in number of granulocytes in peripheral blood, and splenomegaly were observed. However, no such reactions occurred in five C57BL/6 mice bearing primary tumors induced by 3-methylcholanthrene. The results of leukemoid reactions in mice bearing 3-methylcholanthrene-induced primary tumors are compared with the reactions found in mice bearing transplanted tumors.     

Growth of human melanoma in nude mice: suppression by T-lymphocytes from the tumor donor: brief communication.

Human malignant melanoma cell lines established in tissue culture were successfully transplanted sc into BALB/c nude mice. The growth rate of the resulting tumors was significantly suppressed when lymphocytes from the patient in whom the tumor arose were injected iv into BALB/c nude mice at the same time as sc tumor transplantation, but inoculation with lymphocytes from a person without a tumor was ineffective. Cell separation identified T-lymphocytes as the active subpopulation. Growth of the tumors was also significantly suppressed by reconstitution of the mice with normal BALB/c lymphocytes; lymphocytes from BALB/c mice previously immunized against the melanoma line were not more effective than nonimmune lymphocytes in preventing tumor growth. Sera from normal BALB/c mice or BALB/c mice immunized against a human melanoma cell line effectively suppressed growth of that cell line in BALB/c nude mice if given at the time of tumor transplant. The results suggested that, whereas murine lymphocytes reconstitute the ability of nude mice to react to xenogeneic antigens on the human tumor, human lymphocytes showed greater specificity to autologous human melanoma-associated antigens.     

Development and characterization of the BALB/cNIV mouse strain

The strain BALB/cNIV/Crgl was developed by infecting BALB/c/Crgl mice with mouse mammary tumor virus from C3Hf mice. A BALB/c normal mammary duct was transplanted into the gland-free fat pad of a hormone-stimulated female C3Hf X BALB/c F1 mouse. A hyperplastic alveolar nodule was found in the BALB/c ductal outgrowth and was transplanted into another hybrid gland-free fat pad. The resultant hyperplastic alveolar outgrowth was finally transplanted to female BALB/c mice. The hyperplastic alveolar outgrowth contained an exogenous, infectious mouse mammary tumor virus named the nodule-inducing virus, which was thought to be derived from the endogenous low oncogenic mouse mammary tumor virus found in C3Hf mice. The hyperplastic alveolar outgrowth-bearing BALB/c mice were inbred for four generations, and one family was selected as the strain BALB/cNIV/Crgl. It was found that (a) the mouse mammary tumor virus found in the BALB/cNIV strain was milk transmitted, but not transmitted by infected males; (b) the BALB/cNIV breeding females had a low tumor incidence (40%) and a longer latent period (14 months) than did female BALB/cfC3H mice (92% at 8 months); (c) the BALB/cNIV nodule outgrowths had low tumor-producing capabilities (50%) and longer latent periods (13.4 months) than did nodule outgrowths derived from female BALB/cfC3H mice (100% at 7.7 months).     

Genetic control of antinuclear antibodies in mice infected with Rauscher leukemia virus.

The incidence of antinuclear antibodies after Rauscher leukemia virus inoculation was found to be significantly higher in C57BL/6 than in BALB/c mice and still greater in their F1 hybrids. The relationships among antinuclear antibody incidence, erythroblastic disease, Rauscher leukemia virus production, and the H-2 genotypes were studied in the F1 generation and backcrosses using different virus inocula. The results observed suggest that (a) at least two genes are involved in the control of susceptibility to Rauscher leukemia virus-induced erythroblastosis, one of them probably being H-2 linked, and that (b) a non-H-2-linked gene seems to control, at the same time, induction of antinuclear antibodies, focus-forming virus production in the spleen, and susceptibility to the disease. It can be concluded that C-type viruses play an active role in antinuclear antibody induction.