Effects of time of initial exposure to MSV sarcoma on bone induction by dentine matrix implants and on orthotopic femora.

HCl-demineralized murine lower incisors were implanted intramuscularly into syngeneic BALB/c mice to induce heterotopic osteogenesis. Implants were exposed at the early, preosteogenic stage (4), or at the later, osteogenic stage (12) to the Moloney sarcoma virus (MSV), which within 3-4 days results in a sarcoma. The yield of bone induction was determined by weight of dry bone mass following NaOH hydrolysis of soft tissues. To verify the effect of this sarcoma on orthotopic local femoral bone, the dry mass of the tumor-exposed femora was measured and compared with the weight of MSV-unexposed contralateral controls. MSV-sarcoma or cells involved with their spontaneous rejection have a stimulatory effect on the periosteal membrane of the tumor-adjacent femoral bones, increasing their dry mass on average by 18%. No stimulatory effect on heterotopic bone induction was observed when the MSV sarcoma grew during the early, preosteogenic stage (4 onward), but when the tooth matrix had been exposed to such tumor at the already bone-forming stage, (12 onward), the yield of bone induction was enhanced. Thus, it is postulated that lesions induced by MSV during the early, preosteogenic stage inhibit recruitment of osteoprogenitor cells or degrade Bone Morphogenetic Proteins (BMPs) released by matrix resorbing inflammatory cells, whereas when acting on already existing bone they have a stimulatory effect.

A single Glu(62)-to-Lys(62) mutation in the Mos residues of the R7Delta447Gag-tMos protein causes the mutant virus to induce brain lesions.

We previously reported that R7Delta447, a 2954-base-pair (bp) laboratory-generated Moloney murine sarcoma virus, induced subcutaneous tumors in about 14% of infected mice but did not induce brain lesions. We now report that R7Delta447K, a spontaneous mutant of R7Delta447, induced brain lesions as well as subcutaneous tumors in all injected mice. The genomes of the two viruses differ in a single base pair: the deduced Glu(62) of the Mos residue of the R7Delta447 Gag-tMos protein is changed to Lys(62). More R7Delta447 than R7Delta447K focus-forming units were detected in both NIH3T3 and mouse cerebral vascular endothelial (MCVE) cells. However, R7Delta447K transformed NIH3T3 and MCVE cells more acutely than did R7Delta447. A distinctive feature that distinguished the morphologic transformation of R7Delta447- and R7Delta447K-infected MCVE cells is the markedly prolonged spindle-shaped phase exhibited by R7Delta447-infected MCVE cells. In addition, R7Delta447K was more efficient in inducing the phosphorylation of ERK1/2 than R7Delta447 in both MCVE and NIH3T3 cells. Moreover morphologic transformation was inhibited, and levels of phosphorylated ERK1/2 were reduced when R7Delta447- or R7Delta447K-infected NIH3T3 or MCVE cells were grown in the presence of the MEK1/2-specific inhibitor PD98095. Thus, we have identified a key residue in the Gag-tMos protein that profoundly affects activation of the Mos/MEK/ERK pathway, virus and cell replication, morphologic transformation in vitro and pathogenicity in vivo.

Potentiating effect of ribavirin on the anti-retrovirus activity of 3'-azido-2,6-diaminopurine-2',3'-dideoxyriboside in vitro and in vivo.

3'-Azido-2,6-diaminopurine-2',3'-dideoxyriboside (AzddDAPR) is a potent and selective inhibitor of human immunodeficiency virus (HIV) replication in vitro. It also inhibits Moloney murine sarcoma virus (MSV)-induced transformation of murine C3H/3T3 embryo fibroblasts. AzddDAPR causes a marked dose-dependent suppression of MSV-induced tumor formation and mortality therewith associated in newborn mice infected with MSV. Combination of AzddDAPR with ribavirin resulted in a marked potentiation of its anti-retrovirus activity in vitro and a significant enhancement of its inhibitory effect on MSV-induced tumor formation in vivo. A slight increase in the in vivo toxicity of AzddDAPR was noted when combined with ribavirin.

Novel spectral method for the study of viral carcinogenesis in vitro.

Fourier transform infrared (FTIR) spectroscopy is a unique technique for the laboratory diagnosis of cellular variations based on the characteristic molecular vibrational spectra of the cells. Microscopic FTIR was used to investigate spectral differences between normal and malignant fibroblasts transformed by retrovirus infection. A detailed analysis showed significant differences between cancerous and normal cells. The contents of vital cellular metabolites were significantly lower in the transformed cells than in the normal cells. In an attempt to identify the cellular components responsible for the observed spectral differences between normal and cancerous cells, we found significant differences between DNA of normal and cancerous cells.

Sarcoma viruses containing the mos oncogene induce lesions resembling Kaposi's sarcoma.

Kaposi's sarcoma is most accurately described as a multifocal angioproliferative lesion primarily involving the skin, although some studies indicated concomitant and/or exclusive visceral involvement. The mechanism(s) involved in Kaposi's sarcoma development, especially AIDS multiorgan distributed form, is not completely understood. Recent reports have identified a murine retrovirus, MoMuSV-349 that induces multiorgan disseminated angiosarcomatous tumors which resemble Kaposi's sarcoma when virus is inoculated intraperitoneally in newborn BALB/c mice. Performed time point experiment on mice infected with MoMuSV-349 indicated a two stages development of Kaposi's sarcoma-like tumors; sarcomatous and angiosarcomatous. Preliminary studies have demonstrated the pathogenesis of the disease process and the role of angiogenic growth factors released by the spindled cells in the stage development of mouse tumors. It is hypothesized that growth factors such as fibroblast growth factors released by the spindled cells stimulate the proliferation of endothelial cells and neovascularization which is an essential component of Kaposi's sarcoma-like tumors. Understanding the mechanism(s) of endothelial cells proliferation and neovascularization as a response to viral insult and microenvironmental changes will raise the possibilities of developing new therapeutic approaches to block this process.

Local hypertrophic/hyperplastic changes of keratinizing squamous epithelium of pinna induced by concanavalin A and other immunomodulators in mice.

Intradermal administration of concanavalin A, a potent T-cell mitogen, into an ear lap resulted in activation of chondrogenesis and stimulation of epidermis proliferation. This proliferation is sometimes invasive in character (pearls and epidermal nests form in the underlying connective tissue) but never turns into true cancerous lesions. This reaction can be delayed, but not prevented, by the prostaglandin inhibitor indomethacin. Stimulation of epidermis proliferation was also caused by administration of other immunomodulators, such as carrageenan type IV, Moloney sarcoma development, and rarely in the course of GvHr, but to much lesser degree than with concanavalin A. It is suggested that the same growth factors, which are mediators of local chondrocyte stimulation, are also mediators of keratinocyte activation.

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.

Overexpression/enhanced kinase activity of BCR/ABL and altered expression of Notch1 induced acute leukemia in p210BCR/ABL transgenic mice.

Chronic myelogenous leukemia (CML) is a hematopoietic disorder, which begins as indolent chronic phase but inevitably progresses to fatal blast crisis. p210BCR/ABL, a constitutively active tyrosine kinase, is responsible for disease initiation but molecular mechanism(s) underlying disease evolution remains largely unknown. To explore this process, we employed retroviral insertional mutagenesis to CML-exhibiting p210BCR/ABL transgenic mice (Tg). Virus infection induced acute lymphoblastic leukemia (ALL) in p210BCR/ABL Tg with a higher frequency and in a shorter latency than wild-type littermates, and inverse PCR detected two retrovirus common integration sites (CISs) in p210BCR/ABL Tg tumors. Interestingly, one CIS was the transgene itself, where retrovirus integrations induced upregulation of p210BCR/ABL and production of truncated BCR/ABL with an enhanced kinase activity. Another CIS was Notch1 gene, where retrovirus integrations resulted in overexpression of Notch1 and generation of Notch1 lacking the C-terminal region (Notch1DeltaC) associated with stable expression of its activated product, C-terminal-truncated Notch intracellular domain (NICD Delta C). In addition, generation of Tg for both p210BCR/ABL and Notch1DeltaC developed ALL in a shortened period with Stat5 activation, demonstrating the cooperative oncogenicity of Notch1DeltaC/NICD Delta C with p210BCR/ABL involving Stat5-mediated pathway. These results demonstrated that overexpression/enhanced kinase activity of BCR/ABL and altered expression of Notch1 induces acute leukemia in a transgenic model for CML.

A comparative endpoint study of lesions induced by MoMuSV-m1, MoMuSV-HT1, MoMuSV-124 and MoMuSV-349.

Different MoMuSVs produced predictable clinical, gross, and histologic similarities and differences when inoculated into susceptible hosts intraperitoneally. The neoplasms induced by MoMuSV-124 and MoMuSV-349 were histologically indistinguishable. The distribution of the neoplasms was the most widespread in these two groups. Histologically, the neoplasms induced by MoMuSV-124 and MoMuSV-349 were best described as angioproliferative. The neoplasms induced by MoMuSV-HT1 were most accurately described as fibrosarcomas. Histologically, the neoplasms induced by MoMuSV-m1 had characteristics common to neoplasms induced by MoMuSV-124 and MoMuSV-349, but with a less prominent vascular component. All mice inoculated with these MoMuSVs had moderate to severe thymic atrophy. The degree of thymic atrophy associated with both MoMuSV-124 and MoMuSV-349 was histologically more severe than that associated with MoMuSV-m1 and MoMuSV-HT1. Both MoMuSV-HT1 and MoMuSV-m1 had infiltrates of primitive myeloid cells within the spleen. In the case of MoMuSV-m1, there was an apparent leukaemia with infiltrates of similar cells within the bone marrow. With MoMuSV-HT1, these primitive cells were confined to the spleen. In summary, this study demonstrated that some of the different strains of MoMuSV (with the exception of MoMuSV-124 and MoMuSV-349), induced histologically distinct lesions in BALB/c mice.

Point mutations in the U3 region of the long terminal repeat of Moloney murine leukemia virus determine disease specificity of the myeloproliferative sarcoma virus.

The myeloproliferative sarcoma virus (MPSV) is made up entirely of sequences derived from the Moloney murine leukemia virus (Mo-MuLV) and the cellular mos oncogene. As other members of the Moloney murine sarcoma virus (Mo-MuSV) family, MPSV transforms fibroblasts in vitro and causes sarcomas in vivo. In addition, however, MPSV also causes an acute myeloproliferative disease in adult mice. The mos oncogene is essential for its transforming capacity, but sequences specific to the long terminal repeat (LTR) U3 region of MPSV account for its expanded target specificity as compared to Mo-MuSV (C. Stocking, R. Kollek, U. Bergholz, and W. Ostertag, Proc. Natl. Acad. Sci. USA 82, 5746-5750 (1985)). The U3 region of the LTR of MPSV is, however, closely related to that of the Mo-MuLV, and it appeared likely that the difference between MPSV and Mo-MuSV was caused by a divergent evolution of Mo-MuSV LTRs. In this paper, we show that this is not the case. The few nucleotide differences in the LTR between Mo-MuLV and MPSV are crucial for the expanded host range of MPSV. Moreover, Mo-MuLV-related gag sequences retained in MPSV are not essential for the distinctive biological properties of MPSV.

Study of the 78A1 isolate of Moloney murine sarcoma virus. II. Haematopoietic tropism and tumourigenicity.

A new isolate of Moloney murine sarcoma virus (Mo-MuSV), designated 78A1, has been molecularly cloned. The cloned genome, found to be larger than that of other known isolates of the same virus is close in size to that of the myeloproliferative sarcoma virus (MPSV), also a derivative of the original Mo-MuSV/Moloney murine leukaemia virus (Mo-MuLV) complex. Until now, MPSV was the only Mo-MuSV isolate known to be capable of inducing a myeloproliferative disease associated with a tumoural syndrome when injected intravenously into sensitive mice. We compared the biological activity of our cloned virus isolate (78A1) and that of another cloned Mo-MuSV virus (HT1) whose genome is slightly smaller than that of 78A1. The helper virus (Mo-MuLV) associated with the Mo-MuSV isolates was also injected alone as control. After injection into sensitive mice only the isolate 78A1, as well as MPSV caused a tumoural syndrome invading spleen, liver and other haematopoietic organs, and the appearance of granulo-macrophage precursors not requiring exogenous stimulating factors for their proliferation and differentiation. The 78A1 virus has a longer latency period (3 months) than MPSV (several days) and does not induce a typical myeloproliferative disease.

R7, a spontaneous mutant of Moloney murine sarcoma virus 124 with three direct repeats and an in-frame truncated gag-mos gene, induces brain lesions.

We have isolated Recombinant 7 (R7), a spontaneous mutant of SV7, a molecular clone of MoMuSV124. Like SV7, R7 induces subcutaneous fibrosarcomas, spleen tumors, and mesentery tumors infiltrated by proliferating vessels lined by transformed endothelial cells. However, it also induces brain lesions. We have molecularly cloned and sequenced the R7 proviral DNA and shown that the R7 genome consists of 3401 bp. It has three direct repeats in each enhancer. Its coding sequence consists of only 176 bp of p15, 263 bp of p30, a 7-bp insertion, and 853 bp of an N-terminally truncated mos gene. From the sequence of R7 we have deduced that the truncated mos sequence is in-frame with all of the gag sequence and the 7-bp insertion. The incorporation of the 3' end of the p15 sequence further suggests that the R7 Gag-Mos is myristylated. We have also shown that the molecularly cloned R7 virus transformed NIH/3T3 fibroblasts about sevenfold better than the parental SV7. We have also confirmed that molecularly cloned R7 induces the same disease phenotype as that induced by the nonmolecularly cloned R7.

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.

Studies on the pathology, especially brain lesions, induced by R7, a spontaneous mutant of Moloney murine sarcoma virus 124.

We have recently isolated R7, a spontaneous Moloney murine sarcoma virus (MoMuSV) 124 variant. Molecular cloning and sequence analysis showed that, relative to MoMuSV 124, R7 has an extra repeat in each enhancer and a truncated mos gene in frame with the truncated gag coding sequence. This report presents a detailed study on the pathology induced by R7. R7 induced not only sarcomas with well developed angiomatous components but also brain lesions. Brain lesions were observed in all less-than-48-hour-old BALB/c mice inoculated with greater than 2 x 10(5) R7 focus-forming units (FFUs). R7 was detected in all brains examined by day 9 after inoculation, and brain lesions were observed in two of four mice examined by day 14 after inoculation. Light microscopy of brains revealed that approximately 15% of the lesions were unenclosed blood pools of varying sizes containing red blood cells and inflammatory cells spreading into surrounding brain tissues. The remainder of the brain lesions had tumor cells. These lesions ranged from a few enlarged vascular endothelial cells intermixed with blood cells to large circumscribed lesions consisting of well developed tangled masses of vessels surrounded by blood pools. Activated astrocytes surrounded and infiltrated the tumors. In addition, the thymus of R7-infected mice regressed significantly and precipitously due to apoptosis (especially of cortical thymocytes) at the end stage of the disease.

Studies on the pathology, especially brain hemorrhage and angioendotheliomas, induced by two new mos-containing viruses.

Recombinant virus 7 (R7), a spontaneous deletion mutant of SV7, which is itself a molecular clone of Moloney murine sarcoma virus 124 (MoMuSV 124), induces brain lesions and tumors of the subcutaneous tissue and spleen in all infected mice. In contrast, SV7 only induces tumors of the spleen and subcutaneous tissues. One of the genetic differences between R7 and SV7 is that R7 encodes a Gag-Mos protein whereas SV7 encodes an Env-Mos protein. To investigate whether the novel R7 gag-mos oncogene is required for brain lesion induction, two viruses (SV7d1 and SVM1) were constructed in which the R7 gag-mos sequences and the adjacent 53 bp of the 5' noncoding sequence were replaced by either the SV7 or myeloproliferative sarcoma virus (MPSV) env-mos oncogenes, respectively. Like R7, SV7d1 and SVM1 induced brain lesions and tumors in the spleen and subcutaneous tissues. A prominent component of R7-, SV7d1-, and SVM1-induced tumors of the brain, subcutaneous tissues, and spleen was the presence of abnormally enlarged cells with eccentric nuclei lining vessels, scattered singly or in small clusters. Their size, localization to the luminal surface of distended vessels, and binding to Bandeiraea simplicifolia (BS-1) lectin, an endothelial cell (EC) marker, suggest that they are most likely transformed ECs. Our findings therefore indicate that the induction of brain lesions is not limited to the expression of the R7 Gag-Mos protein. However, our findings also indicate that expression of the different forms of the Mos protein results in differences in the relative abundance of ECs in brain angioendotheliomas and subcutaneous and spleen tumors induced by these viruses.