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The organization of RSV genes present in the mouse ascites sarcoma cells was analysed. The transformed cells kept in vivo and in vitro for a long time continued to contain intact RSV genome and the cellular integration site was unchanged. RSV was recovered by transfection of chick embryo cells with these cellular DNA. As these cells, especially in ascites form, grow very rapidly and it is easy to obtain a large amount of cells at a time, they are useful for further study as RSV-transformants derived from mouse cells.

The polymerase chain reaction was used for Moloney murine sarcoma virus (MoMuSV) detection in frozen and formalin-fixed, paraffin-embedded tissue sections and cultured cells isolated from MoMuSV-induced tumors. Rapid DNA extraction by proteinase K digestion, followed by CHROMA SPIN + TE-100 column purification proved to be satisfactory. Two pairs of overlapping primers, flanking 1026 base pair (bp) to 221 bp, allowed to choose among four different length of DNA-amplified segments. Although net amplification was obtained for frozen tissue and tumor cultured cells in all combinations of primers, the maximum specificity and sensitivity resulted with 602 bp fragment. This product was fully and adequately digestible using Apa I and Sau3A I restriction endonucleases. DNA extracted from paraffin-embedded sections yielded an amplification product only when the primer pair which delineated a 221-bp segment was used. This reproducible method could be useful for diagnostic and for pathogenetic investigations of MoMuSV infections.

PSK, a protein-bound polysaccharide derived from basidiomycetes, is a biological response modifier that exhibits a variety of activities following oral administration, including prevention of infection. In this study, the effects of oral administration of this drug on microbial flora of tumor-bearing mice were examined. Numbers of Staphylococcus, Streptococcus and Pseudomonas were increased, and those of Bifidobacterium and Lactobacillus were reduced in fresh feces of mice from later than 9 weeks after inoculation of sarcoma 180. However, these changes were prevented by oral administration of PSK. In mice bearing sarcoma 180, the increases in numbers of Staphylococcus and Pseudomonas and the decrease in those of Bifidobacterium were further enhanced by intraperitoneal administration of anticancer agent mitomycin C, but such changes were suppressed by oral administration of PSK. These results suggest that PSK has a preventive effect against abnormal conditions of the intestinal flora induced by tumor inoculation or the administration of anticancer agents.

Primary mouse keratinocytes transformed with an activated ras oncogene transduced by helper-free Harvey sarcoma virus (HaSV) form predominantly benign tumors. In contrast, keratinocytes transformed with helper-associated HaSV form malignant tumors. We report here that this different tumorigenic behavior correlated with a much higher level of v-Ha-ras p21 protein in cells transformed with the helper-associated virus. The high level of v-ras expression in these cells was due to viral spread beyond the initial infection. The low level of v-ras p21 expression that resulted from single-hit infection with helper-free virus, together with the intrinsic heterogeneity of primary keratinocytes, explains, at least in part, the different tumorigenic behavior of keratinocytes transformed by the two types of viruses.

The malignant cell, in vivo, metabolizes and respires anaerobically. For a good many years scientists have been aware of anaerobia being present in malignant growths. The prevailing opinion is that the malignant cell is able to remain viable in an anaerobic medium. This assumption is incorrect and is actually misleading. My investigative studies indicates that the malignant cell metabolizes and respires intracellularly so that the consequence of such a physiology is anaerobic and the basis for such anaerobia is a metabolic phenomenon. There is a reason for this anaerobiosis. Studies demonstrated the fact that there are present within the malignant cell and in the immediate area bacterial spores arising from one of several varieties of plant bacteria. And with an adequate circulating flow of blood, by the animal (human) host, to that particular site, provides the metabolites, enzymes, and the exchange of gases to enhance this abnormal physiopathological anaerobiosis. It is this intracellular anaerobic metabolism within an animal cell that becomes the basis for its malignant transformation.

Stress accelerates the growth of certain types of tumors. Here we report a possible metabolic mechanism underlying this phenomenon. Some early features of transformation include increased number of glucose transporters and greatly enhanced rates of glucose uptake; this adaptation accommodates the vast energy demands needed for neoplastic growth. In contrast, glucocorticoids, a class of steroid hormones secreted during stress, inhibit glucose transport in various tissues; this is one route by which circulating glucose concentrations are raised during stress. We reasoned that should transformed cells become resistant to this inhibitory action of glucocorticoids, such cells would gain preferential access to these elevated concentrations of glucose. In agreement with this, we observed that Fujinami sarcoma virus-transformed fibroblasts became resistant to this glucocorticoid action both in vitro and in the rat. As a result, under conditions where glucocorticoids exerted catabolic effects upon nontransformed fibroblasts (inhibition of metabolism and ATP concentrations), the opposite occurred in the virally transformed cells. We observe that this glucocorticoid resistance upon transformation cannot be explained by depletion of glucocorticoid receptors; previous studies have suggested that transformation causes an alteration in trafficking of such receptors. Because of this resistance of transformed fibroblasts to the inhibitory effects of glucocorticoids upon glucose transport, glucose stores throughout the body are, in effect, preferentially shunted to such tumors during stress.

The Schmidt-Ruppin or the B77 strain of Rous sarcoma virus (RSV) was inoculated into limb buds of 4.5-days-old avian embryos. No sarcoma but blister formation was observed in those RSV-inoculated embryos. Protein kinase activity of pp60v-src in RSV-inoculated embryos, even in the site of virus inoculation, was the same as that in mock-infected embryos. This indicated that the expression of the v-src gene did not attain superiority over that of the c-src gene in RSV-inoculated embryos. The v-src gene was detected in every DNA from tissues of RSV-inoculated embryos but not in DNAs from tissues of RSV-inoculated chicken except for the DNA from Rous sarcoma. Those results confirmed that the lack of sarcoma induction in early avian embryos by RSV was due to the lack of the expression of the v-src gene which was present in the target cells.

Rous sarcoma virus-33 (RSV-33) was obtained from a sample of chicken Rous sarcoma which had been dried and stored in 1933. RSV-33, like the RSV-29, has the minimal number of passages beyond its isolation from chicken tumour No. 1. Our experiments demonstrated that the Rous sarcoma virus-33 was replication non-defective and was pathogenic for rats. Established rat tumorigenic cell lines express the viral genome. All three species of viral RNA were detected and v-src proteins and gag polyproteins were identified as well in cells of R9 and R74 lines. The virus can be rescued from cells of R9 and R74 lines, thus indicating that the cells are virogenic. The cells of a permanent tumorigenic line RT1 are infected but not transformed by RSV-33. Although they contain a complete proviral genome, they do not express detectable virus-specific RNA. The virus is not rescuable from RT1 cells under in vivo conditions. Proviral DNA analysis showed that the RSV-33 contained a full-length genome, including the env gene, in contrast to the RSV-29 which was found replication defective.

We have investigated the in vivo role of 2 different adhesion molecules, LFA-1 and LECAM-1, in the immune reaction to Moloney-murine-sarcoma-virus(M-MSV)-induced tumors, which undergo a peculiar spontaneous regression due to generation of a strong virus-specific cytotoxic-T-lymphocyte(CTL) response. Repeated administration of anti-LFA-1 monoclonal antibody (FD441.8 MAb), i.p. or at the site of virus inoculation, enhanced tumor growth and delayed regression, while i.p. administration of anti-LECAM-1 MEL-14 MAb gave rise to tumors that grew progressively and caused host death. Evaluation of the immunological response in MAb-treated mice showed reduced generation of virus-specific CTL precursors (p) in the spleen of animals given FD441.8 MAb i.p.; CTLp frequency in locally treated mice overlapped with that of control mice injected with virus only. FD441.8 MAb treatment did not interfere with CTL homing in the tumor, since the frequency of M-MSV-specific CTLps in sarcomas was similar in treated and control mice. Cytofluorimetric analysis indicated that the majority of tumor-infiltrating lymphocytes (TIL) from MAb-treated mice were covered by anti-LFA-1 MAb, and lacked cytotoxic activity when assayed against target cells bearing relevant tumor antigens. Instead, in mice injected i.p. with MEL-14 MAb, a very low frequency of CTLps was detected in lymph nodes draining the tumor area, and within the tumor. Our results indicate that enhanced tumor growth, depending on the MAb used, is the resultant of an inhibitory effect on different T-lymphocyte functions. Tumor progression in anti-LFA-1 MAb-injected mice is explained mostly by blockage of CTL lytic activity at the tumor site; in mice receiving i.p. MEL-14 MAb treatment, by the failure of naive T lymphocytes to enter peripheral lymph nodes and subsequently by the lack of generation of tumor-specific CTLs.

Previous studies have indicated that the regression versus progression of v-src-DNA-induced sarcomas is dependent on chicken line. As a first step in analyzing the role of tumor immunity as a determinant of this line dependence, experiments were undertaken to ascertain whether an antisarcoma immune response is generated in the course of sarcoma growth in TK chickens, a regressor line. To assay for this response, test TK chickens in which v-src-induced wing web sarcomas had regressed, as well as control TK chickens that had not been exposed to v-src, were challenged in protocols known to yield v-src-dependent sarcoma formation and monitored for challenge sarcoma growth. Compared with the control chickens, the test chickens showed a significant resistance to the sarcomagenic challenge. These results raise the possibility that the antisarcoma response that is inducible in regressor lines, as demonstrated here in terms of a protective effect against a subsequent sarcomagenic challenge, may also underlie the regression of v-src-induced primary sarcomas.

The v-erbB oncogene isolated from the R (or ES4) strain of avian erythroblastosis virus is capable of inducing erythroleukemia and fibrosarcomas. This oncogene differs from the proto-oncogene c-erbB, the avian homolog of the epidermal growth factor receptor, by its lack of an intact ligand-binding domain as well as additional alterations in its cytoplasmic coding sequences. By contrast, the insertionally activated c-erbB, a variant oncogene, which encodes a product that also lacks the ligand-binding domain but is otherwise unaltered in its cytoplasmic coding sequences, is capable of inducing leukemia but cannot induce sarcomas. In this report, we show that the critical changes for activating the sarcomagenic potential displayed by v-erbB R are two point mutations within the tyrosine kinase domain and an internal deletion of 21 amino acids in the carboxyl-terminal regulatory domain. The removal of the carboxyl-terminal autophosphorylation sites is not obligatory. These activating mutations (Arg-263 to His, Ile-384 to Ser, and the deletion of residues 494 to 514), when introduced singly into the insertionally activated c-erbB, all dramatically increase fibroblast-transforming potential. Arg-263 resides near the highly conserved HRD motif of the kinase domain, and its mutation to His increases the autophosphorylation activity. The other two mutations do not alter the intrinsic kinase activity and presumably affect other aspects of the receptor involved in growth signaling. Therefore, the high transforming potential of v-erbB R is a consequence of synergism among multiple activating mutations.

Herpesvirus saimiri is an oncogenic herpesvirus that induces rapidly progressing lymphomas in New World primates. Using retrovirus vectors for gene transfer, specific open reading frames of H. saimiri were tested for their ability to transform rodent cells in culture. One open reading frame, designated STP-C488 (for saimiri-transformation-associated protein of the subgroup C strain 488), phenotypically transformed Rat-1 cells, resulting in formation of foci, growth at reduced serum concentration, and growth to higher cell densities. Cells transformed by STP-C488 formed invasive tumors in nude mice. The STP-A11 reading frame of strain 11 (subgroup A) was much less potent in its transforming ability than STP-C488. These results demonstrate the oncogene nature of these two open reading frames and provide a means for studying their transforming functions independent of the rest of the H. saimiri genome.

There are bacteria present within the malignant cell, but they are present only as spores. Although their origin stems from a branching or budding plant type of microorganism, the latter is not physically present and is not involved, per se, in the malignant cell's etiology. It is the primitive but now the asexual reproductive, conidial unicellular, ovular, or spherical shaped bodies, arising from the adult plant microorganism, in consequence to a duressing environment, but also the ones with the genetic capability of surviving within a sac or cell, that are present within the malignant cell. It is these spores, reproducing as spores within the malignant cell and within the immediate surrounding area, as long as there exists an adequate circulating flow of blood by the animal (human) host, to account for the anaerobic or preferably 'deoxygenating' metabolism that occurs in the malignant cell and is the reason for the cellular accumulation and tumor growth. This type of metabolism, respiration, and reproductive pattern is similar to that of the plant type of microorganism activity. In comparison, the healthy non-tumor tissue of animal (human) cells are genetically and primarily aerobic in respiration and metabolism. There are substantial suggestive findings and structural facts uncovered experimentally to definitely establish credence to the presence of those spores within the malignant cell and to align their presence to the pathway of activity, resulting in the etiology and physiopathology of the malignant cell and growth.

Long-term culturing of viral Kirsten-ras-oncogene (v-Ki-ras)-transformed BALB/c 3T3 cells (KiMSV) selectively enriches for cells which have deleted the viral oncogene. In contrast, v-Ki-ras in vivo is amplified and expression increased in all late-stage tumors and lung metastases relative to late-passage KiMSV cells being injected. The nature and significance of these selection processes, in terms of the v-Ki-ras gene, have been explored using genetically-tagged cells, as have the properties of v-Ki-ras- revertant subclones. Inoculation of KiMSV late-passage cells (containing less than 5% v-Ki-ras+ cells) into nude mice, generated primary and lung metastatic tumors with the v-Ki-ras gene at increased dosage in all tumors and their single-cell clones, isolated at both early and late stages of tumor development; this demonstrates early and specific in vivo selection for v-Ki-ras+ cells in both induction and progression of tumors in this system. v-Ki-ras- revertant subclones, isolated from late-passage KiMSV cells and inoculated individually into athymic nude mice, yielded tumors for only 1 of the 4 revertants, with no evidence for v-Ki-ras sequences in these tumor cells, thereby revealing a v-Ki-ras-independent mechanism for tumor formation in a small subset of revertant cells. Mixtures of the 4 subclones yielded tumors in all animals, although at a much longer latent period than observed with v-Ki-ras+ cells. Experiments with mixtures of v-Ki-ras- revertant cells and pSV2neo0tagged/v-Ki-ras+ cells (both complex NeoR cell mixtures and individual NeoR clones tested) at various cell ratios revealed clonal variability among v-Ki-ras+ cells for dominance during tumor formation. Moreover, the complex NeoR cell mixtures yielded both primary and metastatic tumors with simplified patterns of pSV2neo integration sites, suggesting that secondary genetic or epigenetic events, in addition to v-Ki-ras, contribute to the tumor-progressing phenotype. These experiments taken together demonstrate (a) clone-specific early selection of distinct v-Ki-ras+ cells amongst themselves and over v-Ki-ras- cells in both induction and progression of tumors, (b) reduced tumorigenic competence of v-Ki-ras- revertant cells, with a small subset displaying a v-Ki-ras-independent mechanism for tumor formation in this BALB/c 3T3 system, and (c) the significance of additional genetic or epigenetic events for tumor-progressing competence in unique subsets of v-Ki-ras+ cells.

Myeloproliferative syndrome was induced in adult DBA/2 mice by inoculation with myeloproliferative sarcoma virus (MPSV) and Friend murine leukemia virus (F-MuLV) as a helper virus. On day 26 after infection, the spleen weighed a maximum of 2.0 g (about 30 times the control weight). Assay of multipotent stem cells in vitro showed that the more enlarged spleens contained an increased number and concentration of mixed colony-forming units (CFU-mix) (at maximum, 11 times higher than the control). When the supernatant of cultured spleen cells was added to a serum-free bone marrow cell culture with or without erythropoietin (Epo) for detection of burst-promoting activity (BPA), it enhanced erythroid mixed colony (E-mix) formation only in the presence of Epo (p less than 0.05). Even when addition of Epo was delayed, it still induced a significant number of E-mix (p less than 0.05). These findings rule out a mimic effect of Epo resembling BPA and indicate the presence of BPA in the spleen. The culture supernatant also supported the proliferation of interleukin 3 (IL-3)-dependent 32Dcl cells. Therefore, although purification of the BPA substance has not yet been accomplished, BPA in the supernatant seems to depend on the presence of IL-3, which is known to be one of the factors stimulating multipotent hemopoietic stem cells. The presence of BPA- or CFU-mix-stimulating activity in the spleen after infection might be responsible for the development of panmyelosis, which is a characteristic of MPSV-induced myeloproliferative syndrome.

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.

Previous results have established that subcutaneous inoculation of chickens (line SC) with a v-src(+) subviral DNA fragment induces the formation of progressor sarcomas at the wing web site of inoculation. Because the sarcoma cells are incompetent for production of exogenous progeny virus, this system is a useful model of tumor expansion by sarcoma cell division, in the absence of infection-mediated recruitment of new tumor cells. The present study was undertaken to define conditions that modulate the pattern of growth (regression vs progression) of v-src DNA-induced sarcomas. These conditions were found to include the line of chicken or the presence on the subviral v-src(+) DNA fragment of a viral replication-specific sequence that includes env.

We investigated DNA damage caused by carcinogenic metals in a murine sarcoma virus (MuSV)-based mutagenicity assay in which mutations targeted to v-mos expression can be selected. Nickel chloride treatment of NRK cells (termed 6m2 cells) infected with MuSVts110, a retrovirus conditionally defective in viral RNA splicing and cell transformation, caused the outgrowth of transformed "revertants" with changes in the MuSVts110 RNA splicing phenotype. Cadmium and chromium treatment of 6m2 cells resulted in the selection of a second class of revertants with what appeared to be frameshift mutations allowing the translation of a readthrough gag-mos protein. In both classes of metal-induced revertants, viral gene expression was distinct from that observed in revertants arising in untreated 6m2 cultures, arguing that metal treatment did not simply enhance the rate of spontaneous reversion. In one representative nickel revertant line the operative nickel-induced mutation affecting MuSVts110 RNA splicing was a duplication of 70 bases surrounding the 3' splice site. The effect of this mutation was to direct splicing to the most downstream of the duplicated 3' sites and concomitantly relax its characteristic thermosensitivity. These data establish the mutagenic potential of nickel and provide the first example of a defined nickel-induced mutation in a mammalian gene.

N-tropic murine leukemia viruses have been observed in connection with radiation-induced osteosarcomagenesis in BALB/c mice. We have investigated the bone disease-inducing potential of molecularly cloned, BALB/c-derived N-tropic viruses in the random-bred NMRI mouse strain. The germ-line virus and an exogenous virus isolate were found to induce high incidences of osteopetrosis and lymphomas and a lower incidence of osteomas. Two viruses derived from somatically acquired proviruses of independent radiation-induced osteosarcomas induced lower incidence of osteopetrosis and lymphomas. Nucleotide sequence analysis of the long terminal repeat regions and RNase T1 fingerprint analysis revealed only few differences between the isolates. The possible involvement of N-tropic murine leukemia viruses in radiation-induced osteosarcomagenesis in the BALB/c mouse strain is discussed.

Permanent alterations of the epithelial differentiation pattern were investigated after infection of HH-16 cl. 4 adenocarcinoma cells with Moloney murine sarcoma virus (MoMuSV). Transformed cell clones with fibroblastoid morphology were isolated and compared with clones of unchanged epithelioid phenotype. Southern blot analyses showed intact MoMuSV proviral genomes in copy numbers between 4 and 9 in the DNA of the morphologically transformed cell clones as well as in the clones with unaltered morphology. The fibroblastoid cells produced sarcomas after inoculation of newborn rats, whereas MoMuSV-infected cell clones with unaltered epithelioid morphology yielded adenocarcinomas. Immunocytochemical analyses revealed that morphological transformation into the fibroblastoid phenotype was accompanied by loss of cytokeratin expression and appearance of the mesenchymal marker protein vimentin. Proviral DNA was transcribed in the infected cell clones irrespective of their phenotype; however, transcription was significantly higher in cells with fibroblastoid morphology than in epithelioid cells.

Adhesion has been evaluated for tumor cell populations derived from Kirsten murine sarcoma virus (KiMSV)-transformed BALB/c 3T3 cells responding to substrata coated with intact plasma fibronectin (pFN), a family of related proteolytic fragments from pFN or cellular fibronectins (FNs), and the heparan sulfate-binding platelet factor-4 (PF4). Both early-passage KiMSV cells, harboring the viral Kirsten ras oncogene (v-Ki-ras+), and late-passage KiMSV cells, in which most cells have lost the oncogene (v-Ki-ras-), are compared with primary tumor and lung metastatic tumor cells after three routes of injection into nude mice; nontumorigenic v-Ki-ras- revertant cells have been cloned from the late-passage KiMSV population. Attachment of early-passage KiMSV, primary tumor, and lung metastatic tumor cells was optimal and resistant to soluble RGDS peptide in the medium on intact pFN, on fragment F-155 from pFN containing the RGDS cell-binding domain and the heparinII domain, and on PF4 but decreased for metastatic cells on F110 containing only the RGDS domain (and sensitive to RGDS peptide). Cytoplasmic spreading of early-passage KiMSV and all tumor cells was good to excellent in polygonal patterns on pFN and on F155, while most cells remained round on F110. Responses for KiMSV and tumor cells varied on different heparin-binding proteins; cells remained rounded or detached on F38 derived from pFN or on PF4 but spread effectively with long linear process extension on cellular FN-derived fragments F44 + 47 harboring the extra domaina sequence. That F44 + 47 may contain a new cell-binding site for v-Ki-ras+ cells was also indicated by resistance to bacterial heparitanase in cell responses on F44 + 47 but not on PF4 and extensive catabolism of proteoglycans in the substratum-attached material of these cells. v-Ki-ras- revertant cells, nontumorigenic in nude mice, have reacquired 3T3-like responses to proteolytic fragments, including much more effective spreading on PF4 or on F38 substrata, and have reverted in generating microfilament stress fibers on pFN, a competence lacking in all v-Ki-ras+ cells. These results indicate that (a) v-Ki-ras+ primary and metastatic tumor cells respond similarly to most proteolytic fragments of FNs harboring known binding domains, with a few exceptions; (b) v-Ki-ras gene expression correlates with a new cell surface receptor activity recognized by extra domaina-containing fragments from cellular FNs; and (c) loss of the viral oncogene to generate v-Ki-ras- revertant cells reverts their FN-mediated adhesion responses.

Dermal sarcoma is a benign skin tumor of adult walleyes (Stizostedion vitreum vitreum) with a suspected viral etiology. A laboratory study was initiated to determine if the tumor could be experimentally transmitted by inoculating young walleyes with materials prepared from tumors from adult fish. Eighty walleye fingerlings were divided into four groups of 20 fish each. Two groups were inoculated intramuscularly at 4 months of age either with live tumor cells or with cell-free filtrates of sonicated tumor cells. The two other groups were used as controls and were inoculated either with cultured cells from normal walleye fry or with tissue culture media. Neoplasms, similar to the dermal sarcoma affecting adult walleyes, were observed after 4 months only in fingerlings inoculated with cell-free filtrates of sonicated tumor cells. Like the tumor affecting wild adult walleyes, the transmitted tumors were restricted to the dermis and originated from the superficial surface of scales. They never invaded locally and never metastasized. The transmitted tumors differed from tumors of adult walleyes in their severity and the absence of osteoid. The multicentric origin of transmitted walleye dermal sarcoma suggests that the virus spreads systemically and that tumor cells are polyclonal. This successful transmission of the lesion, along with the presence of C-type virus particles budding from tumor cells in two of seven tumor-bearing fingerlings, supports a retroviral etiology.

Murine bone-marrow-culture-derived-macrophages can be differentially activated to lyse either vesicular stomatitis virus infected BALB/c3T3 cells or the tumor target P815. Macrophages were activated in a manner so that they could lyse both targets. The ability of this activated population to lyse either target type was differentially inhibited by varying the assay conditions. The lysis of P815 targets was more sensitive to inhibition by the proteinase inhibitor N-p-tosyl-L-lysine chloromethyl ketone than was the lysis of virally infected cells. On the other hand, reduction of the concentration of glucose in the assay medium, which inhibits the production of oxygen metabolites by the hexose monophosphate shunt, or the addition of anti-tumor necrosis factor (anti-TNF) serum were able to decrease the lysis of virally infected targets but not P815 targets. Thus, the observed differences in the lysis of these two targets were due to both the activation state of the macrophages and the differential susceptibility of the targets to different effector mechanisms.

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.

Previous studies have shown that increased expression of oncogenes from the myc-family can down-regulate the level of MHC class I antigens in tumor cells. This has suggested a mechanism by which amplification/overexpression of myc-genes may contribute to the malignancy development of certain tumors. Earlier published data from the murine SEWA tumor, a polyomavirus-induced osteosarcoma, have correlated the degree of tumorigenicity of different sublines to their level of c-myc amplification. Here I present a quantitative and qualitative analysis of MHC class I antigens from five sublines of this tumor system. No differences could be found, between sublines with different degrees of tumorigenicity, regarding MHC class I antigen expression. Thus, in the SEWA tumor, the enhancement of the tumorigenicity caused by c-myc amplification is not mediated through down-regulation of MHC class I antigens.

RSV-transformed rat sarcoma (XC) cells were characterized as to their ability to anchorage-dependent and anchorage-independent growth in various culture conditions. The proliferation of XC cells was dependent on serum concentration and initial cell density. The relationship between seeding density and the growth rate of XC cells indicated that the investigated cells produce factors with growth stimulating and growth inhibiting activity. The anchorage-independent growth inhibiting activity was found in ultrafiltrate (500 Mr 10,000) of XC cells conditioned medium. The obtained results suggests that anchorage-independent growth of XC cells may be regulated by two types of autocrine factors, which are antagonistic in their biological affects.

The amount of Forssman glycolipid (GL-5) was investigated in two clones of Nil hamster cells grown either in monolayer or in spinner culture. GL-5 assayed by the complement-fixation inhibition test increased with increasing cell density in the monolayer. However, cells grown in spinner cultures failed to show the density-dependent response in both clones examined. Cells transformed by hamster sarcoma virus did not show the density-dependent increase of GL-5, even in cells grown in monolayer. The effect of transfer from confluent to sparse cultures on the amount and the synthesis of GL-5 was also examined. It is suggested that the GL-5 that accumulates in cells during confluency is diluted into the daughter cells and that the decrease of the Forssman lipid does not precede cell division.

Two types of virus particles, intracisternal type A and extracellular type C with budding, were detected in the same cells of BF osteosarcoma, its cultured cell lines, and their BFO tumors in CBA mice. The type C particles were approximately 100 microns in diameter. The buoyant density of the virions was 1.16g/ml in sucrose and 1.07 g/ml in Ficoll. A 72S RNA was demonstrated by gel electrophoresis, but no DNA was detected. Reverse transcriptase activity was also demonstrated in detergent-treated virions. Thus, the particles seem to be RNA virus. Cellular transformation and focus formation were observed after rat and mouse embryo cell monolayers were infected with the virus. The same kind of osteosarcoma was produced by inoculation of cloned transformed cells (BFOSV) of CBA embryo cells into CBA mice. Thus, the virus seems to be an oncornavirus.

Fifteen spontaneous immunocytomas originating in the ileocecal lymph nodes of Lou/C/Wsl rats were studied by means of electron microscopy. The histology was characteristic, the tumor being formed by an accumulation of large, rounded cells with slightly eccentric ovoid nuclei, large nucleoli, and finely condensed chromatin along the nuclear walls; the cytoplasma was rich in polyribosomes. The appearance of the rough endoplasmic reticulum was apparently the same whether or not the tumor was secretory. Its development varied from one cell to another, and in only a small proportion of cells did it attain any considerable volume. In all the tumors examined, we noted the presence of intracisternal A-particles. In its morphology, the rat immunocytoma resembled the plasmacytomas induced in mice, and it also resembled certain human tumors such as Burkitt's lymphoma.

Rasheed rat sarcoma virus, derived by in vitro cocultivation of two rat cell lines (Rasheed et al., Proc. Natl. Acad. Sci. U.S.A. 75:2972-2976, 1978), has been reported to code for a protein of 29,000 Mr, immunologically related to the 21,000 Mr src gene product of Harvey and Kirsten sarcoma viruses. Rat sarcoma virus p29 was thought to contain at least part of a rat type C virus structural protein, since antiserum prepared against whole rat virus was able to immunoprecipitate rat sarcoma virus p29 but not Harvey or Kirsten sarcoma virus p21 (Young et al., Proc. Natl. Acad. Sci. U.S.A. 76:3523-3527, 1979). We now report that antiserum directed against rat type C virus p15, but not viral p12, p10, or p27, immunoprecipitated rat sarcoma virus p29. The p15 antiserum was also able to immunoprecipitate both denatured p29 and a peptide derived by V-8 protease cleavage of p29, indicating that this antiserum contains antibodies directed against primary amino acid determinants. Finally, five separate isolates of rat sarcoma virus were found to code for p29, which indicates that a highly specific site of recombination is involved in the generation of sarcoma viruses in rat cells.

A clone of the Moloney murine sarcoma virus mos gene derived by in vitro reverse transcription was characterized. When assayed for focus formation by DNA transfection on NIH/3T3 cells, this clone was biologically inactive, presumably due to the absence of a long terminal repeat sequence. Therefore, a long terminal repeat was inserted into the clone by in vitro recombination, after which the most gene was able to transform NIH/3T3 cells efficiently. The nucleotide sequence encompassing the transforming region of this clone was determined. A single long open reading frame was observed, which potentially encodes a polypeptide of 41,000 daltons. This open reading frame initiates with the first five amino acids of the murine leukemia virus env gene, after which it enters the mos sequence, where it terminates. The nucleotide sequence described in this paper was compared with other sequences of mos in an effort to resolve discrepancies in the position of the long open reading frame. Although Moloney murine sarcoma virus retains the 3' splicing site of the murine leukemia virus env gene, a mos-specific mRNA which corresponds structurally to the murine leukemia virus env mRNA was not identified. The sequence described here revealed a single nucleotide change in the proposed env gene 3' splicing site which was retained in Moloney murine sarcoma virus. This deviation from the consensus 3' splicing sequence may underlie the observed absence of mos expression via the env gene splicing pathway.

A novel method for the isolation of both macromolecular DNA and RNA from solid tissues based upon the disruption by vibration of deep-frozen material in a mechanical device termed Mikro-dismembrator, is described. This technique reveals a yield of, on the average, 1 to 3 mg of either DNA or RNA per gram of tissue. The quality of the purified nucleic acids permits the detailed analysis of integrated tumor virus DNA sequences and their mRNA transcripts. Furthermore, the efficient isolation of papilloma virions from keratinized wart tissue is facilitated by the application of the Mikro-dismembrator.

A BALB/c mouse-passaged methylcholanthrene-induced fibrosarcoma tumor caused severe impairment of resistance to systemic listeriosis. Depressed resistance expressed immediately after tumor implantation was traced to inadvertent association of tumor with lactic dehydrogenase virus. Tumor cured of virus was totally inactive.

Adult AKR and C58 mice injected intramuscularly with murine sarcoma virus, Moloney isolate (M-MSV), developed high incidence of nonregressing local tumors. Histologically, these tumors revealed the typical pleomorphism of M-MSV sarcomas; in some cases, however, neoplastic tissue showed a nodular or diffuse growth of monomorphic myoblastlike cells, reminiscent of clonal aggregates. No depression of immune reactivity was found in M-MSV-injected mice as evaluated by direct hemolytic plaque-forming cells against SRBC and by virus-neutralizing antibody production. The MSV recovered from the induced tumors proved to be, by neutralization assay, a Gross (G)-MSV pseudotype. Moreover, tumor cell suspensions absorbed out cytotoxic antibody directed against G-cell surface antigens. Therefore, the conclusion was drawn that MSV with envelope characteristics of endogenous G leukemia virus had formed in vivo through a phenotypic mixing phenomenon. The failure of tumors to regress has been interpreted as mainly due to the partial unresponsiveness of host immune reactivity towards G-MuLV specified antigens. Since MSV-tumors arose in AKR mice after a very long latent period, the possibility was considered that this relative resistance might depend on immunologic mechanisms. In fact, M-MSV-injected AKR mice immunodepressed by goat antimouse lymphocyte serum or rendered partially tolerant by neonatal M-MuLV inoculation developed sarcomas with higher incidence and with a shorter latency. Furthermore, the MSV recovered from these early tumors proved to be the original Moloney pseudotype.

Groups of F-344 rats were inoculated with the Bratislava-77 strain of avian sarcoma virus (B-77 ASV) within 24 hours of birth, at 9 days of age, or between 97 and 119 days of age. Intracranial tumors developed in each age group. Multiple tumors with mixed histologic patterns developed in rats inoculated at 1 or 9 days of age. Solitary tumors with a uniform histologic pattern developed in rats inoculated as adults. On the basis of light and electron microscopic study, the majority of tumors in each age group were classified as astrocytomas and divided into either poorly differentiated, gemistocytic, pilocytic, or polymorphic varieties. The polymorphic astrocytomas were most common among neonatally inoculated rats, while the pilocytic astrocytomas were most common among rats inoculated as adults. Ultrastructural characteristics of astrocytes, including gap junctions and 7- to 9-nm filaments, were present in the majority of tumors in each age groups. Astrocytomas induced in adult rats were remarkable for the presence of extensive basement membrane alone the astrocytic cell surfaces. Intracytoplasmic virus-like particles (R particles) were common in the tumor cells. These virus-like particles are morphologically distinct from C-type B-77 ASV, and no morphologic evidence of C-type virus replication was observed in any of the tumors.

Four subclones of the originally cloned Harvey murine sarcoma virus-transformed Madin Darby canine kidney (MDCK) cells have been isolated. These subclones fall into two general classes. Two subclones have a fibroblastic morphology, have lost the growth requirement for prostaglandin E1 (PGE1), do not respond to glucagon or vasopressin, and, in general, appear transformed. Two other subclones have epithelioid morphologies, are growth-stimulated by PGE1, respond to vasopressin with an increase in intracellular cAMP. We propose that these cells represent revertants to a more non-transformed phenotype. Unlike normal cells, however, these revertants grow under anchorage-independent conditions, express detectable but reduced amounts of the transforming gene product, p21, and grow in nude mice. The appearance of such revertants may be one cause of the observed heterogeneity of tumor cells.

In contrast to the B77 virus, its B77(RBI) rat variant continuously produced by the rat RBI sarcoma cells in vivo and in vitro, efficiently induced progressively growing tumors in hamsters and transformed hamster embryo cells in vitro. Both, induced tumors and transformed cell cultures showed production of infectious virus and presence of the avian gs-antigen. Clonal analysis of the established hamster sarcoma cell line RBHtc brought evidence that despite the quantitative differences in the virus production and gs-antigen content among various clonal populations, full expression of the B77(RBI) virus genome does occur in each cell of the virus-induced hamster sarcoma. The permissive relationship of the B77(RBI) virus with rat and hamster cells as well as changed antigenic composititon implies that a genetic change of the virus occured, which is reflected in its increased mammalian tropism and its genetically stable firus-productive interaction with mammalian cells. The possible explanations of these phenomena are discussed.

Analysis of endogenous X-tropic BALB/c retrovirus-specific RNAs in a BALB/c mouse tissue containing mostly non-driving cells (liver), in two normal tissues having significant proportions of dividing cells (20-h regenerating liver and 12-day embryo) and in cells of a lymphosarcoma of BALB/c mice were carried out by determining the extent to which the RNAs from these tissues hybridized to the virus 3H-cDNA probe and by their relative sedimentation values in a sucrose gradient. RNAs from 20-h regenerating liver, 12-day embryo and lymphosarcoma, each containing a significant proportion of proliferating cells, showed 8 to 21 % higher hybridization values than normal liver RNA. Differences in the exact size classes of virus-specific RNAs and, in their relative proportions were found to exist in the four different tissue types examined and no correlation between a specific RNA size-profile and the proliferating activity of a tissue could be detected.

Type C virus produced by dog thymus cells (A7573) that were infected with virus (HL-23V), isolated from cultured leukocytes of an acute myelogenous leukemia patient, transformed marmoset and horse cells in vitro and induced virus-producing fibromas in marmosets. The tumors and transformed foci were indistinguishable morphologically from those induced by simian sarcoma virus, type 1 (SSV-1/SSAV-1). HL-23V was indistinguishable from SSV-1/SSAV-1 by immunofluorescence and neutralization tests, and the nontransforming virus associated with HL-23V completely inhibited SSV-1 focus induction in interference tests. Cell cultures established from a marmoset fibroma produced transforming and nontransforming virus biologically and antigenically indistinguishable from HL-23V and SSV-1/SSAV-1.

RNA tumor virus-specific DNA in cells can be detected by its capacity to 1) alter the reassociation kinetics of labeled double-stranded product of viral RNA-directed DNA polymerase; 2) anneal single-stranded DNA (cDNA) synthesized by viral polymerase; or 3) hybridize labeled viral 70S (genomic) RNA. Duplexes formed with these procedures can be analyzed for fidelity of base pairing, and the integration of viral DNA into the host genome can be established with a simple but stringent technique. We illustrate this methodology as applied to detection of Rous sarcoma virus (RSV)-specific DNA in XC cells and of mouse mammary tumor virus (MMTV)-specific DNA in murine and human tissues.

9-(2-Phosphonylmethoxyethyl)adenine (PMEA) is a potent and selective inhibitor of the replication of human immunodeficiency virus (HIV) in vitro in human T-lymphocyte MT-4, H9, and ATH8 cells. PMEA also inhibits Moloney murine sarcoma virus (Mo-MSV)-induced transformation of murine C3H embryo fibroblasts. Moreover, PMEA causes a dose-dependent suppression of tumor formation and associated mortality in mice inoculated with Mo-MSV. At a dose of 50 or 20 mg/kg per day PMEA effected a 90-100% protection of the mice against Mo-MSV-induced tumor formation and mortality. Even with a PMEA dose as low as 1 to 5 mg/kg per day, tumor formation was significantly delayed and the survival rate was significantly enhanced. In parallel experiments, azidothymidine exhibited a comparable inhibitory effect on Mo-MSV-induced tumor formation and associated death only at a 25-fold higher dose than PMEA. Because PMEA has stronger in vivo antiretrovirus potency and selectivity than azidothymidine and various other compounds currently being subjected to clinical trials, PMEA studies should be pursued to assess the potential of this compound in the treatment of acquired immunodeficiency syndrome (AIDS) and other retrovirus infections in humans.

Tumorigenic clonal lines derived from soft agar colonies induced by DMBA-transformed feline embryo cells were isolated and characterized. The morphologically altered clonal cells formed large aggregates, growing in this aggregate form when suspended in liquid growth medium above an agar base and forming colonies in soft agar with high efficiency. When inoculated into athymic nude mice, chemically altered clonal cells produced progressively growing sarcomas. Cells established from the tumors morphologically resembled the DMBA-transformed feline embryo cells and were characterized as cat cells by karyological analysis. The tumorigenic lines were negative for feline oncornavirus-associated cell membrane antigen (FOCMA), and for "gag-X" the transformation-related polyprotein which is encoded by the replication defective feline sarcoma virus.

Eight cell lines were established from murine osteosarcomas induced in vivo with the radionuclides 224Ra and 227Th. They have been compared by light and electron microscopy, by karyology, and by their growth properties. The morphology, the growth pattern, and the ability to induce tumors in mice indicate that five of them are tumor cell lines. Chromosome studies demonstrated that the five cell lines have marker chromosomes. The other cell lines only showed some criteria generally used to score for transformation of fibroblasts and they may be derived from stromal cells. All cell lines release virus particles in the culture fluid which have the typical properties of RNA tumor viruses. They possess C-type morphology, a density of 1.16--1.18 g/cm3, a 60--70 S RNA, a RNA dependent DNA polymerase and they induce syncytia in rat XC cells. The possible significance of these virus particles in radiation osteosarcomagenesis is discussed.

The effects of different sources of protein (milk, soy, wheat, fish and beef), fat (corn oil and butter), and carbohydrate (dextrin and sucrose) on tumor development and on spleen characteristics were investigated in BALB/c mice injected subcutaneously with 5 X 10(5) herpes simplex virus Type 2-transformed cells (H238 cells). Low or high levels of protein and fat were used. Several weeks post-injection results indicated that a high level of fat significantly enhanced tumor incidence. A high fat level was also associated with a lower spleen weight and a smaller proportion of mature granulocytes in the spleen. Butter, compared to corn oil, significantly restricted tumor volume. Among the most highly significant findings was the low tumor incidence in mice fed protein from either a milk or a fish source.