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.     

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.     

Effects of iodoacetamide on cells of Rauscher virus leukemia

Iodoacetamide reduces the leukaemogenic properties of the Rauscher leukaemia virus, by direct contact in vitro. Murine leukaemic spleen cells incubated for 1 hour at 37° C in vitro can immunize isogeneic mice against the Rauscher virus. The mechanism of action of this product is discussed: 1) action by direct contact on the leukaemogenic properties of the virus; 2) cellular toxicity of the product which modifies the viral reproduction by treated cells; 3) action on the antigenicity of the treated cells.     

In vitro interference between low and high leukaemogenic variants of Rauscher virus

Cultures of the virus-free P4bis line, originating from a C57B1 mouse, were infected in vitro with a “low leukaemogenic” variant (RC) of the Rauscher leukaemia virus (RV), prepared from long-term cultures of this virus in syngeneic cells. Later, after different delays, these infected cultures (P4bis + RC) were superinfected with a highly pathogenic variant (RR) of the RV, obtained from short-term cultures of RV extracted from spleens of leukaemic mice. Eighteen days after these superinfections with RR, supernatants of the doubly-infected cell cultures (P4bis+RC+RR) and their corresponding controls were assayed for leukaemogenic potential by intraperitoneal inoculation into young adult BALB/c mice. The proportion of mice which had developed leukaemia by the 120th day after inoculation served for the evaluation of in vitro interference between the RC and RR virus variants. Even when the in vitro infection of cells with RC virus was followed only 1 h later by the RR superinfection, there was a significant difference between the proportion of leukaemic animals in the group inoculated with P4bis+RC+RR supernatant (56%) and that in the P4bis+RR positive control group (88%). With intervals of 12 days or 21 days between the two in vitro viral infections, the drop in leukaemogenicity of the doubly-infected cultures became more accentuated; the longer the time interval, the greater was the interference effect. Indirect immunofluorescence tests made at intervals after infection of P4bis with RC showed a steady increase in the percentage of RV antigen positive cells up to the 3rd week after infection. The mechanism of the observed interference is discussed, and it is suggested that a straight relationship exists between the spread, horizontal and/or vertical, of the RC virus infection in the cultures and the blocking of their superinfection by the highly pathogenic RR virus.     

Studies on mouse Moloney virus induced tumours: II. Detection of p30 in the serum of mice with Moloney leukaemia by in vitro blocking of complement dependent antibody mediated cytotoxicity.

Sera from Balb/c mice bearing Moloney leukaemia block complement dependent antibody mediated cytotoxicity of an antiserum prepared in rats against syngeneic Moloney virus induced lymphomata when either spleen cells from mice bearing Moloney leukaemia (M) or an in vitro line of Moloney virus transformed cells (MSC) are used as targets. This antiserum has been shown to recognize p30, the major internal virion protein, as a cytotoxic target on these cells. Viral particles were identified by electron microscopic examination of pelleted material obtained from leukaemic sera after high speed centrifugation. However, removal of virus did not affect the capacity of the leukaemic sera to absorb cytotoxicity of rat ILR-3 for MSC targets, and only depressed somewhat its ability to absorb activity of the same antisera against M targets. Virus-free leukaemic sera also blocks complement dependent antibody mediated cytotoxicity of an antiserum prepared in goats against the gs3 determinant of p30. This indicates that the material in leukaemic sera responsible for the in vitro block of antibody mediated cytotoxicity was p30. A lesser degree of block was observed with sera obtained from normal Balb/c mice, but the nature of material responsible is as yet undefined.     

Leukaemias in BN/a and BN/b mice after prolonged treatment with antilymphocyte globulin

The influence of prolonged treatment with anti-lymphocyte globulin on tumour incidence in BN/a and BN/b mice was studied. One ALG sample appeared to be highly leukaemogenic in two independent experiments. All treated mice developed, after a short latency period, non-thymic lymphomas with involvement of abdominal lymph nodes and spleen resulting in ascites. Three established ascites leukaemia cell lines were examined for the presence of MuLV. In all lines viral C-type particles, soluble viral gs-1 antigen and cell surface antigen related to Gross virus were demonstrated. The mechanism of induction of these tumours remains obscure. The hypothetical role of immunosuppression, stimulation of lymphoid cells and activation of leukaemogenic virus is discussed.     

An E mu-BCL-2 transgene facilitates leukaemogenesis by ionizing radiation.

Clonogenic murine B cell precursors are normally ultrasensitive to apoptosis following genotoxic exposure in vitro but can be protected by expression of an E mu-BCL-2 transgene. Such exposures are likely to be mutagenic. This in turn suggests that a level of in vivo genotoxic exposure that usually has minimal pathological consequences might become leukaemogenic when damaged cells fail to abort by apoptosis. If this were to be the case, then the cell type that becomes leukaemic and the chromosomal/molecular changes that occur would also be of considerable interest. We tested this possibility by exposing E mu-BCL-2 and wild-type mice of differing ages to a single dose of X-irradiation of 1-4 Gy. Young (approximately 4-6 weeks) transgenic mice developed leukaemia at a high rate following exposure to 2 Gy but adult mice (4-6 months) did not. Exposure to 4 Gy produced leukaemia in both young and adult transgenic mice but at a higher frequency in the former. Leukaemic cell populations showed clonal rearrangements of the IGH gene but in most cases analysed had immunophenotypic features of an early B lympho-myeloid progenitor population which has not previously been recorded in radiation leukaemogenesis. Molecular cytogenetic analysis of leukaemic cells by banded karyotype and FISH revealed a consistent double abnormality: trisomy 15 plus an interstitial deletion of chromosome 4 that was confirmed by LOH analysis.     

Influence of cyclosporin A on growth of an acute T-cell leukaemia in PVG rats

Our objective was to examine the effect of cyclosporin A (CsA; 25 or 12.5 mg/kg) on growth of an acute (Roser) T-cell leukaemia in male PVG rats. The leukaemic blasts were shown (by immunocytochemical analysis) to have a mature, T-helper-cell phenotype, i.e., OX-19 (CD5) +/- , W3/25 (CD4)+, OX44+, MHC-class I+, OX-26+, corresponding to a population comprising 5% of normal rat medullary thymocytes. Animals received 20 X 10(3) viable tumour cells intramuscularly (day 0) and were given either CsA (25 or 12.5 mg/kg) or drug vehicle by gavage from day 0 or day 14, by which latter time leukaemic blasts normally appeared in the circulation. Administration of the higher dose of CsA from day 0 or day 14 significantly delayed the appearance of leukaemic cells in the peripheral circulation, whereas treatment with 12.5 mg/kg was without significant effect. CsA whole blood levels on day 17 were twice as high in leukaemic rats as in normal controls. Leukaemic infiltration of the spleen and the liver was reduced on day 17 after 25 mg/kg CsA, but no such effect was observed in lymph nodes or kidneys. A heterogeneous, host "reactive" cell population, which developed in response to the leukaemia, was inhibited by CsA, indicating that the effect of the drug was probably not mediated by host defence mechanisms. In CsA-treated leukaemic animals, there was biochemical evidence of synergistic impairment of glomerular and tubular function.     

Antigens of tumours induced by naturally occurring murine sarcoma virus (MSV-FBJ). II. Detection of cell-surface antigens by indirect membrane immunofluorescence

Cell surface antigens expressed by cells transformed in vivo by FBJ virus, a wild type murine sarcoma virus (MSV) complex derived from a spontaneously arising sarcoma in a CF1 mouse, have been studied by indirect membrane immunofluorescence (MIF). Using mouse antisera raised by immunization of syngeneic CBA mice with transplanted FBJ sarcomata an antigen common to all FBJ tumours was detected which was also present on Gross (G) antigen positive tissues, viz. leukaemic and preleukaemic AKR lymphoid cells, but absent from the tissues of mice of G negative strains. Failure to demonstrate antigenic cross-reactivity in reciprocal MIF tests using FBJ immune sera and antisera to MSV-H (Harvey), an MSV isolate of Friend-Moloney-Rauscher (FMR) sub-group specificity, established the virus type-specificity of antigens expressed by sarcoma cells transformed by the respective MSV.     

Activation of murine leukaemia virus under different physiological conditions.

The leukaemic lesions in intact and ovariectomized mice of strain ICRC, induced with 20-methylcholanthrene (20-MCA) in combination with or without hormones were investigated for the presence of mouse leukaemia virus (MuLV) by (i) bioassays and (ii) electron microscopy. The different experimental groups treated with 20-MCA were (i) intact females, (ii) ovariectomized females, (iii) ovariectomized females with pituitary graft, (iv) ovariectomized females with 10 mug oestradiol/day for 30 days and (v) ovariectomized females with 1 mug oestradiol together with 1 mg progesteron/day for 30 days. It was possible to transmit nearly all these experimentally induced leukaemias to syngeneic mice through acellular extracts, compared with very poor transmissibility of spontaneous leukaemias in the ICRC strain, indicating functional activation of viral agents on combined treatment with carcinogen and hormones. Potency of the acellular leukaemic extract from the mice of group (ii) without the ovarian hormones was much weaker than that from mice of the other experimental groups. The leukaemogenic activity of MuLV was enhanced on serial transmission in syngeneic hosts. Leukaemic lesions of ovariectomized mice treated with 20-MCA and oestradiol were also transmissible to the sucklings of allogeneic mice of strain C3H-MTV, C57-BL and Dba-MTV. The cell-free supernatant medium of the cultures of these leukaemic lesions induced leukaemias on back inoculation into syngeneic mice. Electron microscopic studies of lesions induced with carcinogen and oestradiol consistently showed abundant intracytoplasmic type A particles. Numerous intracytoplasmic type A particles as well as some type B particles were found in the leukaemic tissues of ovariectomized females treated with MCA and oestradiol combined with progesterone. Type C particles, characteristic of MuLV were seen in the leukaemic tissues of all other experimental groups. These findings indicate a significant influence of the physiological condition of the host, particularly the hormonal make up, on expression and activity of specific viral agents.     

Clonality analysis suggests that early-onset acute lymphoblastic leukaemia is of single-cell origin and implies no major role for germ cell mutations in parents.

Childhood leukaemia presenting at a young age has been suspected of resulting from a leukaemogenic mutation in parental germ cells, either spontaneously or due to the exposure of a parent to leukaemogenic environmental hazards, particularly ionizing radiation. Mathematical modelling of leukaemogenesis suggests that any such patient would be especially prone to multiple independent leukaemogenic events leading to multiclonality in terms of cell of origin (analogous to bilaterality in familial retinoblastoma). To test this hypothesis we have carried out a search for multiclonal leukaemogenesis in infant and childhood acute lymphoblastic leukaemia (ALL). We used a polymerase chain reaction-based analysis of the X-linked monoamine oxidase A (MAOA) gene locus to study the clonality of marrow samples obtained from female paediatric ALL patients at the time of disease presentation. We obtained presentation samples from 102 patients of whom 72 were found to be informative at the MAOA locus. These included 20 infant leukaemias (< 1 year at diagnosis). Sixty-six samples were found to be unequivocally monoclonal while the remaining six could not, with certainty, be assigned a clonal origin. We also obtained bone marrow aspirates at first relapse as well as at presentation from eight patients. In each case the same pattern of X-linked allelic inactivation was observed at both time points of the course of the disease. No evidence was found for leukaemic multiclonality in any age group at presentation or for leukaemic 'clone-switching' in relapse. These findings suggest that both infant and childhood ALL is of single-cell origin and implies that leukaemic predisposition resulting from germ cell mutation is unlikely to have a major role in their pathogenesis.     

Vitamin B12 levels in whole blood,plasma, and blood cells in transplantable rat leukaemias

Vitamin B₁₂ levels in whole blood, plasma, and red and white cells were determined by microbiological assay in normal and leukaemic A × C inbred rats. The concentration of B₁₂ in white cells was 200 to 300 times higher than in red cells in both groups. A marked increase in the B₁₂ content of whole blood was observed in the leukaemic animals which was of the same order of magnitude as the increase in the number of white cells. The concentration of B₁₂ in red cells was the same in normal and leukaemic blood. That in plasma and white cells was only slightly increased in leukaemia. Therefore, the overall increase in blood B₁₂ is attributable to the increase in the number of white cells in the leukaemic animals.     

A spleen weight assay for Murine Sarcoma Virus Harvey (MSV-H)

A spleen weight assay has been developed for titrating murine sarcoma virus-Harvey (MSV-H) 3 weeks after intraperitoneal infection of newborn BALB/c and random-bred albino mice. Induction of splenomegaly corresponded in time with development of sarcomas and anaemia. The assay was also used to evaluate sera for neutralizing antibodies to MSV-H. Weanling mice were much less susceptible to erythroblastic splenomegaly and tumour induction than newborns. Random-bred mice were found more satisfactory for the assays than BALB/c mice. Newborn Sprague-Dawley rats also responded to MSV-H inoculation with a dose-dependent erythroblastic splenomegaly.     

Azathioprine-induced lymphocytic neoplasms of NZB mice lack ecotropic murine leukaemia virus.

NZB mice injected intramuscularly throughout a 6-month period with the immunosuppressant azathioprine (Imuran) developed lymphocytic lymphomas 6--7 months after treatment was initiated. These malignancies were quite distinct from the reticulum-cell neoplasia which occurs spontaneously in the strain, and were readily transplantable to NZB or histocompatible BALB/c recipients. Xenotropic, but not ecotropic murine leukaemia virus (MuLV) was detected in leukaemic tissues of some donor and recipient NZBs when tested in vitro by co-cultivation with permissive cell lines, genome rescue, XC and viral polymerase assays. Virus filtrates prepared from donor leukaemic tissues were non-pathogenic when injected into newborn C3H mice. These results are evidence against a mandatory ecotropic MuLV genome in lymphocytic neoplasia.     

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.     

Antigens of tumours induced by naturally occurring murine sarcoma virus (MSV-FBJ). I. Detection of group and type specific antigens by complement fixation

Antigens associated with cells transformed in vivo by FBJ virus, a wild type murine sarcoma virus (MSV) complex originating from a spontaneously arising osteosarcoma in a CF1 mouse, have been partially characterized by complement fixation (CF). Using rat antisera against antigens specified by Gross leukaemia virus (GLV) the group specific (gs) antigen of C-type RNA murine tumour viruses was demonstrated in FBJ tumours as well as in GLV rat leukaemias, AKR lymphomata and sarcomata induced by MSV-H (Harvey), an MSV isolate of Friend-Moloney-Rauscher (FMR) subgroup specificity. Using mouse antisera against antigens present in FBJ cells the Gross (G) or wild type specificity of FBJ tumours was demonstrated by cross reactivity with antigens expressed on normal AKR lymphoid tissues and leukaemias. These antigens were absent from MSV-H induced sarcomata and in reciprocal tests mouse antisera to MSV-H failed to react with antigens present in FBJ tumour cells. No distinction between cellular and virion antigens expressed by FBJ cells was possible by CF although evidence for a cellular antigen with G specificity was obtained in tests using aged C57B1 antiserum containing a naturally occurring G antibody lacking significant virus neutralizing capacity. However, the likelihood that mouse FBJ antisera contain antibodies to type specific viral envelope antigens (VEA) as well as cellular antigen is discussed.     

Survival and characteristics of murine leukaemic and normal stem cells after hyperthermia: a murine model for human bone marrow purging

The effect of hyperthermia in vitro on the survival and leukaemogenic effectiveness of WEHI 3-B cells and on the survival and transplantation efficiency of bone marrow cells was compared in a murine model system. Normal murine clonogenic haemopoietic cells (day 9 CFU-S and CFU-GM) proved to be significantly less sensitive to 42.5 degrees C hyperthermia (Do values: 54.3 and 41.1 min, respectively) than leukaemic clonogenic cells (CFU-L) derived from suspension culture or from bone marrow of leukaemic mice (Do: 17.8 min). Exposure for 120 min to 42.5 degrees C reduced the surviving fraction of CFU-L to 0.002 and that of CFU-S to 0.2. If comparable graft sizes were transplanted from normal or heat exposed bone marrow, 60-day survival of supralethally irradiated mice was similar. Surviving WEHI 3-B cells were capable of inducing leukaemia in vivo. The two log difference in the surviving fraction of CFU-L and CFU-S after 120 min exposure to 42.5 degrees C suggests that hyperthermia ex vivo may be a suitable purging method for autologous bone marrow transplantation.     

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.     

Sustained leukaemic phenotype after inactivation of BCR-ABLp190 in mice

Pharmacological inactivation of cancer genes or products is being used as a strategy for therapy in oncology. To investigate the potential role of BCR-ABLp190 cessation in leukaemia development, we generated mice carrying a tetracycline-repressible BCR-ABLp190 transgene. These mice were morphologically normal at birth, and developed leukaemias. Disease was characterized by the presence of B-cell blasts co-expressing myeloid markers, reminiscent of the human counterpart. BCR-ABLp190 activation can initiate leukaemia in both young and adult mice. Transitory expression of BCR-ABLp190 is enough to develop leukaemia. Suppression of the BCR-ABLp190 transgene in leukaemic CombitTA-p190 mice did not rescue the malignant phenotype, indicating that BCR-ABLp190 is not required to maintain the disease in mice. Similar results were obtained by inactivation of BCR-ABLp190 with STI571 (Gleevec; Novartis, East Hanover, NJ, USA) in leukaemic CombitTA-p190 mice. However, gradual suppression of BCR-ABLp190 in leukaemic CombitTA-p190 mice identified a minimum level of BCR-ABLp190 expression necessary to revert the specific block in B-cell differentiation in the leukaemic cells. Overall, the findings indicate that BCR-ABLp190 appears to cause epigenetic and/or genetic changes in tumour-maintaining cells that render them insensitive to BCR-ABLp190 inactivation.