The mechanism of interferon effect on cell transformation by murine sarcoma virus

Mouse interferon (IFN) was found to inhibit murine sarcoma virus (MSV)-induced neoplastic transformation of normal rat kidney (NRK) cells. This effect was observed upon examining the formation of foci of morphologically altered cells and colonies of anchorage-independent cells. IFN had no cytotoxic effect on MSV-transformed NRK cells, nor on their focus or colony-forming ability. It was therefore apparent that its inhibitory effect was directed against the viral role in cell transformation. In attempts to define the mechanism of this effect, we found that IFN delayed the initiation of the cytoplasmic viral DNA synthesis. However, the amount of this DNA eventually formed in IFN-treated cells was the same as in the control cells. Furthermore, the transport of this DNA to the nucleus was slower in IFN-treated cells, although all of it was finally transferred. However, while most of the viral DNA integrated into the genome of the control cells, very little integration occurred in IFN-treated cells. The unintegrated viral DNA of these cells was slowly degraded. Therefore, if the cells recovered from the antiviral effect of IFN when intact viral DNA molecules still existed in their nucleus, they could resume viral DNA integration and cell transformation. IFN was found to block viral DNA supercoiling. Since supercoiled viral DNA is considered to be a precursor to integrated provirus, it seems that the inhibition of both integration and cell transformation is due to this impaired coiling.     

Radiation induction of endogenous type C virus from mouse cells transformed in vitro by murine sarcoma virus.

Cell lines from AKR and BALB/c mouse embryos were compared for their sensitivity to X-ray induction of endogenous type C virus. K-Balb cells, a Balb/3T3 cell line nonproductively transformed by Kirsten murine sarcoma virus, were found to be sensitive to X-irradiation. At a dose as low as 50 R, X-rays induced virus expression in K-Balb cells, and the induction frequency increased with increasing dose of X-rays up to 400 R. Among two classes of inducible endogenous viruses carried by K-Balb cells, only Balb:virus-2 was activated by X-irradiation, whereas both Balb:virus-1 and Balb:virus-2 were activated after the cells were treated with 5-iodo-2'-deoxyuridine. UV light and 4-nitroquinoline 1-oxide were also shown to induce virus expression in K-Balb cells. The virus-induction frequency for these physical and chemical carcinogens was much lower (approximately 3 times 10(-4)) than that for 5-bromo-2'-deoxyuridine (approximately 1 times 10(-1)).     

Characterization of guinea-pig embryo cells transformed by Kirsten mouse sarcoma virus

Guinea-pig embryo cells were transformed in vitro by the Kirsten strain of mouse sarcoma virus (Ki-MSV). The transformed cells were found to release infectious virus continuously and produced high titers of group-specific, complement-fixing antigen characteristic of the murine leukemia—sarcoma virus complex. Foci of transformed cells were similar in appearance to those obtained with Ki-MSV in mouse and rat cells. The transformed cells produced RNA-dependent DNA polymerase and type-C virus particles with a density of approximately 1.15 g/ml in sucrose gradients by ³H-uridine labelling. The transformed cells from one line produced tumors when transplanted into newborn guinea-pigs. A number of focus-derived clonal lines and “normal cells” derived from infected cells were isolated and characterized. All the focus-derived lines were found to be MSV producers. The Ki-MSV grown in guinea-pig cells replicated efficiently in guineapig and NRK cells but very poorly in mouse cells. A non-cytopathic type-C virus-producing line (clone No. 2) was isolated. A non-focus-forming virus grown in guinea-pig embryo cells (clone No. 2) rescued infectious MSV by direct cocultivation with Ki-MSK non-producer NRK cells. The rescued MSV virus was neutralized by Ki-MSV antiserum and produced foci readily in mouse, rat and guinea-pig cells.     

Modification of steroidogenesis in rat adrenocortical cells transformed by Kirsten murine sarcoma virus

Adrenal fibroblasts from adult rats acquire some adrenocortical parenchymal characteristics as a consequence of transformation in early passage with Kirsten murine sarcoma virus. To further define the effects of Kirsten murine sarcoma virus-induced transformation on the steroid enzymes of these cells, we investigated the capacity of Kirsten murine sarcoma virus-transformed and untransformed adrenocortical fibroblasts to convert progesterone to C19 and C21 steroid metabolites. Over 95% of metabolites produced were identified and quantitated, and rates of enzyme activities over 24 h were calculated. The transformed and untransformed cells exhibited 5 alpha- and 5 beta-reductase, 3 alpha-, 3 beta-, and 20 alpha-hydroxysteroid dehydrogenase; 11 beta-, 17-, and 21-hydroxylase (HY); and C17-20-lyase activities. Viral transformation resulted in several metabolites not found in untransformed cells, significantly increased 5 beta-reductase, 3 beta-hydroxysteroid dehydrogenase, and C17-20-lyase activities, and significantly decreased 5 alpha-reductase, 3 alpha- and 20 alpha-hydroxysteroid dehydrogenase, and 21-HY activities. The 11 beta-HY and 17-HY activities remained unchanged. The results support previous data suggesting that adrenocortical fibroblasts express some characteristics of adrenocortical parenchymal stem cells. In contrast to other experimental systems, viral transformation of adrenocortical fibroblasts did not cause a generalized reduction of differentiated functions. Instead, specific increases and decreases in individual enzyme activities, with persisting synthesis of fetal and adult adrenocortico-specific steroids, resulted in an altered steroid profile that may have unique effects on the biology of the malignant cells.     

Specific increase in polyamine levels in chick embryo cells transformed by Rous sarcoma virus.

Chick embryo fibroblasts and chorioallantoic membranes of chick embryos infected with oncogenic or nononcogenic viruses were analyzed for polyamines. Nononcogenic viruses (influenze, Newcastle disease, or vaccinia virus) had no effect on the polyamine content of chorioallantoic membranes. Transformation of chorioallantoic membranes by a wild-type or temperature-sensitive mutant strain of Rous sarcoma virus under permissive conditions (37 degrees) caused a 2- to 4-fold increase in cellular spermidine and putrescine content. Only putrescine accumulated in chick embryo fibroblasts transformed by Rous sarcoma virus at 37 degrees. At the nonpermissive temperature (42 degrees), the temperature-sensitive mutant, unlike the wild-type strain, did not alter cellular morphology or polyamine content.     

Association of the glycolipid pattern with antigenic alterations in mouse fibroblasts transformed by murine sarcoma virus.

The level of the neutral glycolipid, Galnac beta 1 leads to 4 Gal beta 1 leads to 4Glc leads to Cer (asialo GM2), in BALB/c 3T3 mouse fibroblasts transformed by Kirsten murine sarcoma virus (3T3KIMSV) was greatly increased compared to the nontransformed parental cells (3T3). This elevated chemical quantity was found to be localized on the surface of intact cells and accessible to external reagents, as detected by immunofluorescence and labeling with galactose oxidase: NaB3H4. Furthermore, immunization of rabbits with 3T3KiMSV cells but not with 3T3 cells resulted in antibody production against asialo GM2. These results demonstrate the potential usefulness of glycolipids as tumor-associated cell surface markers.     

Temperature-dependent alterations in 2-deoxyglucose uptake in rat cells transformed by a cold-sensitive murine sarcoma virus mutant

Cells transformed by Moloney sarcoma virus (MSV) take up 2-deoxyglucose at a faster rate at 39° C than uninfected or Moloney leukemia virus (MoLV)—infected normal rat kidney (NRK) cells. In a sarcoma-virus-transformed cell line, NRK (MSV-lb), whose transformed phenotype is expressed at 39° C (permissive) but not at 33° C (non-permissive), the stimulation of 2-deoxyglucose uptake at 39° C is temperature dependent; the increase is observed when cells are grown at 39° C but not at 33 C. When these cells were shifted from the non-permissive to the permissive temperature, the uptake increased from a rate near that of uninfected cells to a rate half that of NRK cells infected with Moloney sarcoma-leukemia complex (MSV-MoLV). The reverse change occurred when the cells were shifted from the permissive to the non-permissive temperature. Thus in a sarcoma virus-infected rat cell line where the maintenance of the transformed state is dependent on a cold-sensitive viral function, the uptake of 2-deoxyglucose is reduced in the cold-sensitive cells and correlated with the expression of the transformed phenotype.     

Properties of flat variants of murine sarcoma virus transformed non-producer cells isolated after high-temperature passage

High-temperature passage (40.5°C) increased the incidence of flat variants obtained from Kirsten sarcoma virus transformed BALB/3T3 non-producer cells from less than 0.1% to 4%. The flat variants had growth properties similar to those of the BALB/3T3 line. The variant lines did not release detectable type C virus, but some contained intracisternal type-A particles. Super-infection of the variants with murine leukemia virus (MuLV) resulted in retransformation and release of sarcoma virus. Some of the variant lines may be suitable indicator cells for the assay of MuLV. Modal chromosome numbers of the variant lines were considerably higher than those of their progenitor cell line.     

Effect of mouse interferon on cell transformation and virus production in rat cells exogenously infected with moloney murine sarcoma and leukemia viruses

Foci of transformed cells, produced by MSV(124), appeared to result only from the primary infection, since this virus stock yielded a virus-nonproducing infection. On the other hand, the majority of foci scored in MSV/MLV-infected cultures, were generated by multiple secondary infections with the progenies of the primary infection. Mouse interferon (IF) was highly inhibitory for cell transformation by both virus stocks. However, this inhibition was apparent in MSV(124) infected cultures only if IF was added at least 12 h before infection, whereas in MSV/MLV-infected cultures IF was highly effective even if added 24 h after infection. The inhibition of focus formation by MSV(124) was irreversible after removal of IF, suggesting that IF inhibited an early step before provirus integration into the host genome. By contrast, in MSV/MLV-infected cultures focus formation was almost completely restored after recovery from the IF effect Nevertheless, examination of virus production after IF removal proved that in MSV/MLV infection, too IF exerted and inhibitory effect before provirus integration.     

Non-producer human cells induced by murine sarcoma virus.

Non-produces (NP) human cells were isolated from transformed foci induced by the Kirsten mouse sarcoma virus. These morphologically altered NP cells produced neither infectious virus nor complement-fixing antigens of the murine sarcoma-leukemia virus complex. However, the sarcoma virus genome could be rescued from these NF cells by co-cultivation with cells carrying "helper" Kirsten mouse leukemia virus or Woolly Monkey leukemia virus. The possible usefulness of these cells in efforts designed to detect covert or repressed RNA tumor viruses in various animal and human tissues is discussed.     

Decreased alkaline phosphatase in cells transformed by rous sarcoma virus.

Chick embryo cells transformed by either of two strains of Rous sarcoma virus (Bryan high titer or Schmidt-Ruppin) have low levels of alkaline phosphatase activity compared with nontransformed chick embryo cells. Essentially no differences in acid phosphatase activity were observed between these transformed and nontransformed cells. A virus mutant, RSV-BH-Ta, induces temperature-dependent transformation in infected cells. At 41 degrees, the transformation-nonpermissive temperature, alkaline phosphatase activities were similar to those of chick embryo cells. Shifting these cells to 37 degrees resulted in a change to transformed morphology and a progressive loss of enzyme activity, requiring 18 to 24 hr to reach the level of transformed cells. Rat embryo cells transformed by murine sarcoma virus also contained lower alkaline phosphatase levels than did nontransformed cells. These observations suggest that decreased alkaline phosphatase activities may be a general property of transformed cells.     

A nonviral, virus strain-specific antigen expressed on rat cells transformed by avian sarcoma virus

Five of six rat sarcomas, induced by the Schmidt-Ruppin (SR) strain of avian tumor virus, expressed a Mr 60,000 tumor cell surface antigen (TSA), immunoprecipitable from non-ionic detergent extracts. Expression of the antigen was exclusive to rat cells transformed by the SR virus strain. Moreover, expression of TSA appeared restricted by cell type. The five TSA-positive SR-transformed rat cell lines tested were apparently of fibroblastic origin, but cultured rat cerebral endothelial cells (RCE-T1), transformed by SR virus, showed no expression of TSA. However, the antigen emerged on cultured tumors obtained after histoincompatible transplantation of these cells into newborn rats of another strain (tumor digest cells). Investigation of TSA for immunological relationship to viral structural antigens and the src gene product indicated that the TSA is distinct from any of these and more probably derives from a virus-directed alteration in a host molecule.     

Altered microfilament structure in cells transformed with a temperature-sensitive transformation mutant of murine sarcoma virus.

The structure and distribution of microfilaments were examined by electron microscopy in uninfected normal rat kidney (NRK) cells, murine sarcoma virus (MSV)-transformed NRK cells, and NRK cells infected with a cold-sensitive transformation mutant of MSV, i.e., NRK (MSV-1b) cells, grown at both permissive (39 degrees) and nonpermissive (33 degrees) temperature. The uninfected cells contained numerous microfilaments which were especially prominent at sites of intercellular adherens junctions. In contrast, the MSV-transformed cells contained few microfilaments and did not form adherens junctions. At 33 degrees, the NRK (MSV-1b) cells appeared normal but formed an altered form of adherens junction with disorganized microfilaments. At 39 degrees, these cells resembled NRK cells transformed by wild-type MSV but still formed a few of the altered type of adherens junctions. Disorganized adherens junction microfilaments were also found in cells newly infected with wild-type MSV. These results suggest that the perturbed assembly of microfilaments at adherens junctions may be an intermediate stage in the loss of adherens junctions during viral transformation.     

Synthesis and secretion of plasminogen activators and collagenases in human cells transformed by Kirsten murine sarcoma virus and N-methyl-N'-nitro-N-nitrosoguanidine

The secretion of elevated levels of proteinases is considered to be a distinct property of most transformed cells. The cellular and secreted levels of plasminogen activators and collagenases have been examined in the nonmalignant human osteosarcoma (HOS), the malignant Kirsten murine sarcoma virus transformed (KHOS/NP), the temperature sensitive revertant of virus transformed HOS (KHOS-240S) and N-methyl-N'-nitro-N-nitrosoguanidine transformed HOS (MNNG/HOS) clones. Virus and MNNG transformed clones exhibit 100- and 7-fold higher cellular and and 270- and 30-fold higher extracellular plasminogen activator (PA) activity as compared with untransformed HOS controls. The cellular PA activity of the revertant clone is similar to but the secreted level is slightly higher than the HOS controls. SDS-PAGE in the presence of casein and plasminogen is consistent with the major PA species of urinary type (u-PA) and with the absence of PA inhibitor in the parent and revertant clones. The cellular levels of active collagenase are low in all the clones. However, on activation by trypsin, the two active collagenase bands of similar intensity are observed for all the lines in SDS-PAGE in the presence of gelatin. While there appears to be some elevation of secreted collagenase prior to trypsin activation, the activated collagenases appear to have the same size and activity in all of the clones.     

Transformation of rat thyroid epithelial cells by Kirsten murine sarcoma virus

Fischer rat thyroid epithelial cella (FRT) growing continuously in culture were infected with the Kirsten murine sarcoma virus KiMSV(KiMuLV) and found to produce this virus constitutively. Although the morphology of the FRT cells did not change appreciably, the cells became malignant after infection with KiMSV(KiMuLV) as shown by the growth of infected cels in semi-solid media (uninfected FRT cells did not grow) and by the tumorigenicity of infected when injected into syngeneic animals (uninfected FRT cells or FRT cells infected with non-transforming retro-viruses were not tumorigenic). The induced tumors morphologically resembled moderately differentiated carcinomas. Two markers of thyroid epithelial differentiation were absent in the original FRT clone and remained unexpressed after transformation. Fully differentiated rat thyroid epithelial cells (FRT-L cells) infected with another strain of the Kirsten murine sarcoma virus, the KiMSV(MolMuLV), were also transformed as demonstrated by the ability also of these cells to grow as carcinomas (after in vitro transformation) in syngeneic animals. Our results clearly demonstrate that the Kirsten murine sarcoma virus can transform in vitro cells of epithelial as well as of fibroblastic origin.     

Stimulatory effect of immunization on tumor induction by Moloney murine sarcoma virus.

Adult mice were immunized with varying doses of inactivated Moloney murine leukemia virus (M-MuLV). Eight weeks after immunization, mice were challenged with a dose of Moloney murine sarcoma virus (M-MuSV) that could induce tumors in approximately 50% of normal animals. Mice immunized with high doses of M-MuLV (10(10) particles) had significantly decreased tumor incidences, whereas mice immunized with low doses of M-MuLV (10(2) particles) had significnatly increased tumor incidences compared to those in nonimmunized controls. The stimulatory effect could be abrogated by the irradiation of mice with 450 rads 24 hours prior to M-MuSV challenge, whereas the inhibitory effect was resistant to this irradiation procedure. The results suggested that immunization with virus can either stimulate or inhibit virus-induced tumorigenesis, depending on the dose of virus used for immunization.