Genetic and immunological parameters governing in vivo susceptibility/resistance to retrovirally induced murine malignant histiocytosis.

Malignant histiocytosis sarcoma virus (MHSV) arose as a recombinant of c-Harvey-ras murine sarcoma virus (Ha-MuSV) and Friend mink cell focus-forming virus (F-MCFV). It is a defective acute transforming retrovirus that, along with Friend murine leukemia helper virus (F-MuLV), induces malignant histiocytosis (MH) in susceptible adult mice. We have assessed the in vivo susceptibility to MHSV in inbred homozygous, F1 hybrid, congenic, and recombinant inbred (RI) mice. We have shown that: (1) in vivo resistance to MHSV is multigenic, regulated by MHC and non-MHC genes in a different fashion than with F-MCFV, F-MuLV, or Ha-MuSV; (2) using BXD RI mice, the resistance phenotype is linked with 95.8% probability to two linked loci, Pmv-9 and Iapls3-14, on chromosome 13 (homologous to the area of human chromosome 5 for which a chromosomal break point at position 5q35 is associated with human MH); and (3) CD4+ T cells are critical for MHSV resistance.

Perturbations of multiple hemopoietic lineages in retrovirus-induced murine malignant histiocytosis.

A fatal systemic proliferation of malignant histiocytes resembling human malignant histiocytosis was induced in susceptible mice following infection with the murine retrovirus malignant histiocytosis sarcoma virus (MHSV). It is shown that MHSV additionally caused profound alterations of erythropoiesis, granulocytopoiesis and thrombocytopoiesis, and in the hemopoietic stem cell compartment. In the erythroid lineage, MHSV induced a normocytic peripheral anemia, which was paralleled by an unphysiologic, multifocal clonal expansion of erythroid blasts in the spleen. These cells were not transformed and appeared to have a maturation defect since blood reticulocytes did not increase above control values. Moreover, MHSV exerted cytopathic effects on neutrophilic granulocytes and megakaryocytes, since their numbers transiently decreased in the spleen, and agranulocytosis and thrombocytopenia was observed in the blood. Nonetheless, regeneration was found in both lineages at later stages of the infection, which was accompanied by a terminal granulocytosis. The number of lineage-committed and multipotential colony-forming cells in the CFU-S assay increased transiently, but decreased to very low levels in the final stages of the disease. Thus, the studies demonstrate that the same etiologic agent, MHSV, had different effects on hemopoietic cells, which included malignant transformation, hyperproliferative and cytopathic effects.

The specificity of the disease induced by defective murine retroviruses containing abl, fos, or Ha-ras is usually not determined by their LTR.

The long terminal repeats (LTR) of the defective murine sarcoma viruses (MSV) containing v-abl, v-Ha-ras, or v-fos were exchanged for LTRs from other retroviruses having different tissue tropism. The new chimeric MSV were found to induce the same diseases as the parental viruses, indicating that sequences outside the LTR, most likely those of the oncogene, are responsible for the disease specificity of these defective MSV.

Altered glycosylation in Madin-Darby canine kidney (MDCK) cells after transformation by murine sarcoma virus.

The changes in glycosylation of an immortalized epithelial cell line (MDCK) before and after progression towards a more malignant phenotype have been studied. The parental MDCK-3 cells were immortalized after long-term passage in vitro and have shown no tendency for spontaneous acquisition of malignancy-related phenotypes such as tumorigenicity, invasion and metastasis. They conserved morphological and functional characteristics of the epithelial tissue of origin. The ras-MDCK cells acquired the fully malignant phenotype after transformation with a Harvey murine sarcoma virus; they were immortalized, invasive in vitro and produced invasive and also metastatic tumors after subcutaneous injection into nude mice. Using immobilized lectins and gel chromatography, before and after liberation of O-linked glycans from the peptide moieties and also after removal of terminal sialic acid, we have found differences in the glycosylpeptides of both whole cells and cell surface trypsinates from ras-MDCK cultures as compared to the parental MDCK-3 cultures: (i) more sialic acid in the N-linked tri- and tetra-antennary structures; (ii) more fucosylation in the N-glycosylpeptides; (iii) more bi-antennary N-glycosylpeptides and less O-linked glycans; and (iv) a lower molecular weight of the O-linked glycans probably due to a decreased sialylation. It is concluded that alterations in sialylation and fucosylation of the cell surface exposed glycans accompanied progression of MDCK-3 cells towards a more malignant phenotype.

Moloney murine sarcoma virus 349 induces Kaposi's sarcomalike lesions in Balb/c mice.

Moloney murine sarcoma virus (MoMuSV349) is produced by MuSV349 cells in at least eight-fold excess over the replication-competent helper virus. Less than 48-hours-old Balb/c mice inoculated intraperitoneally with supernatant from MuSV349 cells containing approximately 10(4) MuSV349 infectious units developed clinical symptoms, including severe generalized wasting, 15 to 20 days after inoculation. These infected mice became moribund 35 to 45 days after inoculation. Gross examination of the bodies revealed the presence of cutaneous and subcutaneous 0.2-cm to 1.5-cm macules, plaques, or nodules located predominantly on the ventral abdomen and legs. Nodules also were found in the spleen, liver, ovaries, testes, meninges, nerves, and skin. The nodules were semisoft, cystic, or solid and some expressed variable amounts of blood. Histologic examination of the macules, plaques, and nodules showed spindlelike cells intermingled with tortous, jagged vascular channels lined by plump and normal endothelial cells and unlined slitlike spaces filled with erythrocytes. These angiomatous lesions were infiltrated extensively with neutrophils, lymphocytes, macrophages, and some plasma cells. In some cases the lesions also included foci of densely packed eosinophils. These angiomatous lesions are clearly distinguishable from the fibrosarcomas induced by the myeloproliferative sarcoma virus (MPSV), and resemble the sarcomas induced in mice by Gz-MSV and Balb MSV, the sarcomas induced in rats by MPSV and Ha-MSV, and the acute generalized form of Kaposi's sarcoma (KS) associated with acquired immune deficiency syndrome (AIDS) in humans. Electron microscopy also revealed the presence of numerous extracellular type C virions and virions budding from the plasma membrane of endothelial and spindlelike cells. Erythrophagocytosis by the endothelial and spindlelike cells was demonstrated by light and electron microscopy. The widely disseminated lesions appear to have developed simultaneously as a consequence of viremia rather than metastasis.

[Hematopoietic stem cells (CFU-S) in retrovirus-induced murine malignant histiocytosis]. / Hämatopoietische Stammzellen (CFU-S) bei retrovirus-induzierter, muriner maligner Histiozytose.

The murine malignant histiocytosis sarcoma virus (MHSV) is a replication-defective murine retrovirus which contains a ras-oncogen. Upon intravenous infection of normal adult NIH-mice a rapidly fatal systemic proliferation of transformed mononuclear phagocytes develops. We have investigated the involvement of other mature hematopoietic lineages and of hematopoietic stem cells during the course of the disease. The erythroid lineage is affected by a profound anemia which is temporarily accompanied by an increase of erythroid precursors in bone marrow and spleen and a final ablation of erythroid stem cells. A granulopenia occurs with a concurrent decrease of granulocytic precursors and stem cells. The platelets fall to very low levels early during the disease despite of an increase of megakaryocytes and megakaryocytic stem cells. Multipotential stem cells show temporarily a slight increase followed by an almost total terminal ablation. The results show that MHSV has distinct effects on other hematopoietic lineages apart from its transforming properties.

Susceptibility to raf and raf/myc retroviruses is governed by different genetic loci.

The susceptibility to tumors induced by raf and raf/myc retroviruses was investigated in BALB/c, C57BL/6, (BALB/c x C57BL/6)F1 and (BALB/c x C57BL/6) backcross mice. Newborn mice were susceptible to neoplasms generated by both viruses, but resistance to raf-induced leukemia developed rapidly in all mice as they matured. Older C57BL/6 mice were also resistant to raf/myc lymphomas, whereas BALB/c mice remained susceptible to the virus at all ages, indicating that different genes control susceptibility to raf and raf/myc tumors. From these data and the susceptibility of C x B recombinant inbred strains, it appears that very few genes (perhaps even a single gene) may govern susceptibility to raf/myc lymphomas and that resistance is the dominant trait.

Recombinant BALB and Harvey sarcoma viruses with normal proto-ras-coding regions transform embryo cells in culture and cause tumors in mice.

The ras genes of BALB and Harvey sarcoma viruses contain point mutations in codon 12 or codons 12 and 59, relative to proto-ras from normal animal and human cells. By in vitro recombination between cloned rat proto-ras and cloned BALB and Harvey sarcoma proviruses, we constructed recombinant proviruses with normal proto-ras-coding regions. These recombinant proviruses transformed mouse 3T3 cells upon transfection. However, when the transforming efficiencies of proviral DNAs were compared after transfection with helper provirus, recombinant proviruses were 2 to 30 times less efficient than the corresponding wild-type proviruses. Recombinant sarcoma viruses isolated from cells transformed by cloned proviral DNA contained the expected normal ras-coding region. They transformed rat embryo cells and induced erythroblastosis and sarcomas in newborn mice as efficiently as wild-type viruses did. We conclude that conversion of normal proto-ras genes to viral ras genes depends on truncation of normal proto-ras regulatory elements and substitution by retroviral (long terminal repeat) promoters and that the transforming function of long terminal repeat-ras genes is enhanced by point mutations.

Murine retrovirus-induced malignant histiocytosis, an experimental model for the disease in humans.

The hematopoietic disregulation in adult mice induced by the malignant histiocytosis sarcoma virus (MHSV) and the Harvey murine sarcoma virus (Ha-MuSV), which both possess c-Ha-ras-related oncogenic sequences, was investigated. Spleen focus formation induced by MHSV and Ha-MuSV was not restricted by the Fv-2 resistance locus in congenic DDD and C57BL/6 mice, unlike leukemogenesis induced by Friend virus, Rauscher virus, and the myeloproliferative sarcoma virus (MPSV). C57BL/6 mice were much more resistant to MHSV and Ha-MuSV-induced spleen focus formation than DDD mice regardless of their Fv-2 state. Infection of DDD mice with MHSV caused a systemic histiocytic neoplasia, best described as murine malignant histiocytosis. Transformed histiocytic cells proliferated excessively in the bone marrow, spleen, and lymph nodes and, in the final stages of the disease, in all major parenchymal organs. The Ha-MuSV caused a strikingly different benign histiocytic tumor in DDD mice and, unlike MHSV, did not induce a rapid, progressive splenomegaly in C57BL/6 mice. Infection of DDD mice with MHSV induced a rapid and synchronized depletion of early and late erythroid precursor cell pools. In MHSV-infected C57BL/6 mice comparable changes were observed with dissimilar kinetics. Macrophage colony-forming cells of MHSV-infected mice were increased in number and proliferated independently of stimulating growth factors. The disease induced by MHSV in mice can thus serve as a model for malignant histiocytosis in humans.

v-ras genes from Harvey and BALB murine sarcoma viruses can act as initiators of two-stage mouse skin carcinogenesis.

Activated Harvey murine sarcoma virus ras genes were introduced into epidermal cells in vivo by direct application of retroviruses to mouse skin. Subsequent treatment with the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) induced benign papillomas, some of which progressed to invasive carcinomas. Initiation with virus was irreversible for at least 4 months, since TPA treatment after this latency period produced papillomas within 4 weeks. Analysis of viral integration sites showed that carcinomas are clonal in origin. Both papillomas and carcinomas express virus-specific ras mRNA and the viral form of ras P21 protein. The results show that activated ras genes can replace chemical carcinogens in initiation of mouse skin carcinogenesis. This system presents a novel approach to in vivo analysis of the biological role of oncogenes in epithelial tumorigenesis.

Induction of tumorigenesis and chromosomal abnormalities in human amniocytes infected with simian virus 40 and Kirsten sarcoma virus.

Cell cultures of epithelial-like human amniocytes were infected with simian virus 40 (SV40) and Kirsten sarcoma virus (KSV) in various sequential orders, and tested for agar growth, chromosome abnormalities, and tumorigenesis in the nude mouse assay. We observed that regardless of the order in which the viruses were introduced, the doubly infected cells always exhibited the typical SV40 premalignantly transformed phenotype before changing to the malignant phenotype. All doubly transformed cells from different cell donors produced tumors in adult and suckling nude athymic mice, classified as poorly differentiated sarcomas. Infection with SV40 alone conferred extended life span and accelerated growth without the malignant capability of tumor production. Kirsten sarcoma virus alone produced only focal cell alterations with no change in cell longevity or tumorigenesis. Chromosome studies of the premalignant and malignant cells from one cell donor did not reveal any significant clonal development for marker chromosomes in either cell line. Chromosome 12, which carries the homologous cellular oncogene to KSV, had no increase in aberrations in the malignant cells. Chromosome 8 was most often involved in aberrations, and the most frequent aberration for both series was dicentric chromosomes due to telomere fusion. For other translocations the breakpoints were almost exclusively in the centromere regions. The vulnerability of telomere and centromere regions to the free virus present in these precrisis cells is discussed, and similarities in regard to types of aberrations in transfection experiments are noted.

Brain tumors in hamsters induced by murine sarcoma virus (Moloney).

Murine sarcoma virus, CS-Moloney substrain, was inoculated intracranially into 2 litters of newborn Syrian hamsters within 24 h of birth. Seven of 12 hamsters which survived more than 30 days developed brain tumors in the cerebral cortex 104 to 153 days, 139 days on the average, after the virus inoculation. The tumors consisted of spindle-shaped, round or polygonal astrocytes which showed a positive reaction for glial fibrillary acidic protein by the immunoperoxidase method.

Induction of rat brain tumor with xenotropic pseudotype murine sarcoma virus.

The oncogenicity of xenotropic pseudotype Kirsten murine sarcoma virus (MSV) was investigated in Sprague-Dawley rats. When fetal or newborn rats were inoculated intracerebrally with xenotropic pseudotype MSV, brain tumors developed after about one month. Tumors were induced both in the cerebrum and the cerebellum. Histologically, the tumors were predominantly glioblastoma multiforme and hemangioendotheliomas. In cerebellar lesions, malignant transformation of vascular endothelial cells, polycystic areas and numerous giant cells were noted. Proliferation of Purkinje cells was also observed in some of the cerebellar tumors. Inoculation of the same virus by other routes, such as s.c., i.p. and i.m., also caused cerebral and cerebellar tumors. Brain tumors thus induced were transplantable subcutaneously into suckling rats.

Murine xenotropic type C viruses. IV. Replication and pathogenesis of ducks.

The xenotropic (X-tropic) mouse type C virus (MuLV) and its pseudotype of murine sarcoma virus (MSV) were inoculated into several fertilized developing Pekin duck eggs. The development of the duck embryos was substantially reduced in those receiving the X-tropic viruses compared to eggs inoculated only with tissue culture medium. Infections virus was isolated from some of the adult animals; in others, evidence for integrated virus sequences in the tissues was noted. No specific pathology was found in the ducks that received X-trophic MuLV alone, but one duck developed multiple fibrosarcomas when inoculated at birth with the X-tropic virus pseudotype of MSV. Two ducks receiving X-tropic MuLv had signs of haematopoietic disorders. In addition, more virus-inoculated animals had evidence of hepatitis and encephalitis than control ducks. Antibody production to X-tropic MuLv was present in several ducks inoculated with virus either in embryo or at birth. Absence of antiviral antibodies was noted in those animals whose tissue contained replicating virus. These studies confirm the observations with X-tropic virus in tissue culture. They demonstrate in vivo that avian species are susceptible to infection by the mouse X-tropic virus and that their fibroblasts can be transformed by the X-tropic MuLV pseudotype of MSV.

Viral-versus non-viral-induced yolk sac tumors in the rat.

Yolk sac tumors were induced in fetectomized rats by displacement of the visceral yolk sac outside the uterus with or without injection of murine sarcoma virus (MSV) into the placenta. Some of the animals were immunized with MSV prior to surgery. The biological, morphological and immunological characteristics of the tumors which develop in animals either injected or not with virus are compared. The results indicate that MSV inoculation into the placenta increases the tumor incidence, shortens the latency period, and induces, apart from yolk sac carcinoma (YSC), less differentiated tumors (embryonal cell carcinoma, EC). The transplanted and/or in vitro cultured YSC derived from primary tumors obtained in animals either injected or not with MSV show the same morphology, are not immunogenic, secrete alphafetoprotein and express endodermal antigen.