Properties of a sarcoma-growth-factor-like peptide from cells transformed by a temperature-sensitive sarcoma virus

Serum-free conditioned media was collected from three sarcoma virus-transformed cell lines and an untransformed cell line. All three virally transformed lines produced and released growth factors into their serum-free media. The major activity in all cases, whether the cells were transformed by Moloney sarcoma virus (MSV) or Kirsten sarcoma virus (KiSV), or whether they were mouse or rat, was a sarcoma-growth-factor (SGF)-like activity with an apparent molecular weight of 10,000. The SGF-like pools from a Moloney sarcoma virus-transformed mouse 3T3 cell and a Kirsten sarcoma virus-transformed NRK cell were further purified by carboxymethyl cellulose chromatography. The elution profiles of these peptides were very similar. The serum-free conditioned media from the untransformed cells showed no detectable growth stimulating activity. The temperature sensitivity of an SGF-like growth factor from the supernate of a NRK cell transformed by a wild-type Kirsten sarcoma virus (KiSV) was compared with that of the SGF-like activity from the supernates of a NRK cell transformed by a ts-mutant of KiSV that is temperature sensitive with respect to transformation (ts-371 Cl 5). Neither the cells transformed by the wild-type sarcoma virus nor those transformed by the temperature sensitive virus released a SGF-like activity that was temperature sensitive under the conditions of the assays.     

Properties of Noninfectious and Transforming Viruses Released by Murine Sarcoma Virus-Induced Hamster Tumor Cells

The cell culture lines HTG2 and HTG3 were established from a transplantable hamster tumor induced by a murine sarcoma virus (strain Gz-MSV) after 17 and 60 in vivo passages, respectively. The viruses released by these two cell lines markedly differ in morphology, antigenic composition, infectivity, transforming ability, and enzymatic activity. HTG2 virions contained the sarcoma genome but were noninfectious for mouse and hamster cells (S+H-virus). HTG3 virions transformed hamster but not mouse cells. Whereas HTG2 cells and its virus contained murine type C virus gs-1 antigen, all HTG3 clonal lines expressed both murine and hamster type C virus gs-1 antigens. The RNA-dependent DNA polymerase activity of HTG2 virus was very low, whereas that of HTG3 virus was relatively high. HTG2 virions contained electron-lucent centers only. HTG3 virus consisted of the expected mixture of virions with electron-dense and electron-lucent centers. Many broken or incomplete virions were present in both viruses. HTG2 virus is a noninfectious "defective" sarcoma virus without detectable helper virus. Data obtained in these experiments suggest that HTG3 virus is a hamster type C virus pseudotype of Gz-MSV (Gz-MSV [HaLV]). The genome of Gz-MSV is capable of antigenic expression in heterologous cells and in the presence of heterologous viruses. Attempts to chemically activate hamster type C virus (HaLV) from HTG2 cells were unsuccessful. The HTG1 cell culture line, established from another Gz-MSV-induced hamster tumor, initially released a virus indistinguishable from the HTG2 virus. After in vitro passage, spontaneous activation of HaLV occurred in HTG1 cells, and the resultant Gz-MSV (HaLV) had properties similar to those of the HTG3 virus.     

Transformation by v-H-ras does not restore proliferation of a set of temperature-sensitive cell-cycle mutants of rat 3Y1 fibroblasts

Three temperature-sensitive cell-cycle mutants of rat 3Y1 fibroblasts (3Y1tsD123, 3Y1tsG125, and 3Y1tsH203, each belonging to distinct complementation groups) were transformed with plasmid DNA carrying Harvey murine sarcoma virus cDNA. The criteria for transformation were increase in saturation cell density, capability to clone in soft agar, and alteration in the cellular morphology. At 39.8 degrees C (restrictive temperature of the parental cell lines), all the transformed sublines of each mutant ceased to proliferate and were arrested reversibly in the G1 phase of the cell cycle like the parental lines. At both 39.8 degrees C and 33.8 degrees C (permissive temperature for the parental lines), all the untransformed parental lines synthesized p21ras at low rate. At 33.8 degrees C, all the transformed sublines synthesized p21ras at much higher rate and expressed the morphological phenotype characteristic to v-H-ras-induced transformation. At 39.8 degrees C, the rate of p21ras synthesis was not changed in the transformed sublines of 3Y1tsD123 and 3Y1tsG125, and the morphology of transformed phenotype also remained intact. In the transformed subline of 3Y1tsH203, the rate of p21ras synthesis was lowered at 39.8 degrees C to that seen in the untransformed parental line, and the transformed phenotype in morphology disappeared. In all of the transformed sublines, the amount of v-H-ras mRNA markedly expressed at both 33.8 degrees C and 39.8 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Recombinant retroviral DNA yielding high expression of hepatitis B surface antigen

A genomic fragment of hepatitis B virus encoding the surface antigen (HBsAg) was inserted into the proviral genome of Moloney mouse sarcoma virus (MSV), obtained from the mouse cell line G8 -124. The recombinant DNA was introduced into NIH 3T3 mouse fibroblasts. Cells, morphologically transformed by the oncogene of MSV (v-mosM) were selected, established as cell lines and tested for expression of HBsAg. An expression level of up to 4.5 micrograms/10(7) cells/day was detected.     

Cells transformed by a wide variety of agents express higher abundance levels of some cellular RNA species

A cDNA-cloned library was prepared from mRNA synthesized by SV40-transformed mouse cells. Eleven cDNA clones were selected based on their ability to hybridize higher levels of mRNA in SV40-transformed 3T3 cells than in 3T3 cells. These cDNA clones were employed to screen the steady-state levels of cytoplasmic RNAs in a wide variety of viral (SV40, polyoma, adenovirus, and Rous sarcoma virus) and nonviral (methylcholanthrene, embryonal carcinoma) transformed cell lines. Two of the cDNA clones—A17 and 104—detected greater than 40–100-fold higher levels of mRNA in all the transformed cell lines tested when compared to nontransformed cells (3T3, C3HEF). The levels of mRNA complementary to these two cDNAs were regulated in a temperature-sensitive fashion (87–100-fold) in both SV40tsA- and RSV ts-src-transformed murine cell lines. These two cDNA clones detected greater than 100-fold, higher levels of complementary RNA derived from SV40 tumor tissue than in normal mouse liver. RNA species complementary to cDNA clones A17 or 104 were not detected in either actively growing nontransformed cells or in serum-stimulated 3T3 cells. The abundance levels of mRNAs detected by these two cDNA clones appear to be regulated 100-fold or greater by the transformed state, independent of the transforming agent. The higher levels of these RNA species detected in transformed mouse cells appear not to be solely regulated by the state of growth of nontransformed cells.     

FBJ osteosarcoma virus in tissue culture. III. Isolation and characterization of non-virus-producing FBJ-transformed cells.

Hamster and rat cell lines have been established that have been transformed by FBJ murine sarcoma virus (FBJ-MuSV) but that do not produce virus. The hamster cell line originated from an osteosarcoma that appeared in a hamster inoculated at birth with an extract of a CFNo1 mouse FBJ-osteosarcoma. The rat cell line was obtained by transferring the FBJ-MuSV genome to normal rat kidney cells in the absence of the FBJ type C virus (FBJ-MuLV), which, usually in high concentration, accompanies the FBJ-MuSV. Both transformed hamster and rat cell lines contain the FBJ-MuSV genome, which can be rescued by ecotropic and xenotropic murine type C viruses. This rescued genome produces characteristic FBJ-MuSV foci in tissue culture and, in appropriate animal hosts, induces osteosarcomas typical of those induced by FBJ-MuSV. FBJ-MuSV was isolated originally from a parosteal osteosarcoma that occurred naturally in a mouse. Since there was no previous history of passage of the agent through any other animal species, these non-virus-producing hamster and rat cells transformed by FBJ-MuSV should be very helpful in molecular studies examining the origin of spontaneous sarcoma genomes in mice.     

Four different classes of retroviruses induce phosphorylation of tyrosines present in similar cellular proteins.

Chicken embryo cells transformed by the related avian sarcoma viruses PRC II and Fujinami sarcoma virus, or by the unrelated virus Y73, contain three phosphoproteins not observed in untransformed cells and increased levels of up to four other phosphoproteins. These same phosphoproteins are present in increased levels in cells transformed by Rous sarcoma virus, a virus which is apparently unrelated to the three aforementioned viruses. In all cases, the phosphoproteins contain phosphotyrosine and thus may be substrates for the tyrosine-specific protein kinases encoded by these viruses. In one case, the site(s) of tyrosine phosphorylation within the protein is the same for all four viruses. A homologous protein is also phosphorylated, at the same major site, in mouse 3T3 cells transformed by Rous sarcoma virus or by the further unrelated virus Abelson murine leukemia virus. A second phosphotyrosine-containing protein has been detected in both Rous sarcoma virus and Abelson murine leukemia virus-transformed 3T3 cells, but was absent from normal 3T3 cells and 3T3 cells transformed by various other viruses. We conclude that representatives of four apparently unrelated classes of transforming retroviruses all induce the phosphorylation of tyrosines present in the same set of cellular proteins.     

Transformation of rat cells by fusion-infection with Rous sarcoma virus

The transformation of a rat cell line, 3Y1, by nonmammalian tropic strains of avian sarcoma virus was tested using cell-virus fusion mediated by Sendai virus or polyethylene glycol. Furthermore, the establishment of several transformed 3Y1 cell clones induced by the Schmidt-Ruppin strain of Rous sarcoma virus (RSV), its derivative mutants, and the Bryan high-titer strain of RSV is reported. The presence and expression of the viral genomes in these cells were examined, and all transformed cell clones tested were found to contain rescuable RSV genomes when they had been fused with normal chicken embryo fibroblast cells or those preinfected with Rous-associated virus type 1. However, the gag gene product, pr76, was barely detectable in wild-type RSV-transformed cells, whereas it was produced in considerable amounts in cells transformed by env-deleted mutants, the Bryan high-titer strain of RSV and NY8 derived from the Schmidt-Ruppin strain of RSV.     

Nonlethal G0-ts mutant tsJT60 becomes lethal at the nonpermissive temperature after transformation: a hint for new cancer chemotherapeutics

tsJT60 is a nonlethal temperature-sensitive (ts) mutant of a Fischer rat cell line (3Y1) classified as a G0 mutant; i.e., the ts defect is not expressed within the cell growth cycle but is expressed only between the G0 and S phase. tsJT60 clones transformed with oncogenes such as adenovirus E1A, polyoma large T, polyoma middle T, v-Ki-ras, and LTR activated c-myc, or with a chemical carcinogen N-methyl-N'-nitro-N-nitrosoguanidine, grew well at 34 degrees C. However, most of these clones grew slowly at 40 degrees C, producing many floating dead cells, and some clones were killed at 40 degrees C. When they were cultured under conditions inadequate for growth of untransformed cells, such as high cell density or serum restriction, they were killed at 40 degrees C. These and previous results from SV40- and adenovirus-transformed tsJT60 clones favour the idea that transformed tsJT60 cells occasionally enter the G0 phase and are metabolically imbalanced at 40 degrees C during self-stimulation from the G0 to S phase. We propose that a drug which exclusively block, G0-G1 transition would be cytocidal to transformed cells but cytostatic to normal cells.     

31P NMR spectra of rodent and avian fibroblasts transformed by Rous or Kirsten sarcoma viruses

31P NMR spectra of normal rodent and avian fibroblasts were compared to those of the same cells transformed either by the Rous sarcoma virus (RSV) or by the Kirsten sarcoma virus (Ki-MSV). Under physiological conditions, the spectra of living or perchloric acid extracted chicken embryo fibroblasts, rat cell line FR3T3 and mouse cell line C127 did not differ from those of their counterparts transformed by RSV or Ki-MSV. However, in the case of FR3T3 cells, on shifting from 37 degrees C to 20 degrees C, and particularly if PBS replaced serum growth medium, a different, though transitory, response of the transformed cells was detected. They then showed, within few minutes, a more rapid ATP depletion with accumulation of fructose 1,6-diphosphate (FDP), as compared to normal control cells.     

Human epidermal keratinocytes retain radiation resistance following in vitro immortalization and malignant transformation

The radiobiology of human tumors suggests that multiple factors are involved in clinical radioresponsiveness. To date, no direct experimental evidence is available to correlate intrinsic cellular radiosensitivity with the steps of malignant transformation. We developed an in vitro multistage model of epithelial neoplasia using human epidermal keratinocytes to examine the effects of malignant transformation on radiation response. These cells were first immortalized as a result of infection with a hybrid virus (adenovirus 12 and simian virus 40) and subsequently transformed either by infection with a second virus (Kirsten murine sarcoma virus) or by treatment with a chemical carcinogen (N-methyl-N'-nitro-N-nitrosoguanidine or 4-nitroquinoline-1-oxide). We demonstrate that primary human epidermal keratinocytes were radiation resistant (D0 = 2.24 Gy) as compared with human fibroblasts (D0 = 1.45 Gy) and that this resistance was retained in the immortalized as well as the transformed cell lines. These findings present direct experimental evidence that radiation sensitivity of malignant human keratinocytes is an intrinsic property of the precursor cell that may be conserved through the stages of neoplastic transformation.     

Coordinate regulation of the levels of type III and type I collagen mRNA in most but not all mouse fibroblasts

A mouse genomic clone was isolated by cross-hybridization with a DNA fragment which codes for the NH2-propeptide of chick alpha1(III) collagen. The region of cross-hybridization within the mouse clone was localized, its sequence determined, and an exon coding for the NH2-propeptide of mouse alpha1(III) collagen was identified. This DNA fragment hybridizes to an RNA species of approximately 5300 nucleotides, slightly larger than the major alpha2(I) collagen RNA species. The mouse type III collagen probe was used to examine the effect of transformation on alpha1(III) collagen RNA levels in mouse fibroblasts. The levels of type III and type I collagen mRNA levels were compared in control and sarcoma virus-transformed murine cell lines, as well as in NIH 3T3 cells transformed by members of the human ras oncogenes. The levels of type III RNA decreased about 10-15-fold in Moloney sarcoma virus-transformed cells and in a cell line transformed with a v-mos-containing plasmid, but showed only a 50% decrease in a Kirsten murine sarcoma virus-transformed BALB 3T3 cell line, and increased 4-fold in a Rous sarcoma virus (RSV)-transformed BALB 3T3 cell line. In contrast, the levels of alpha2(I) collagen mRNA are 8- to 10-fold lower in all these cell lines when compared to untransformed cells. NIH 3T3 cells transformed with two human ras oncogenes showed decreased levels of alpha2(I) and alpha1(III) mRNAs. In contrast to the RSV-transformed mouse cell line, RSV-transformed chick embryo fibroblasts contained much smaller amounts of type III RNA than control chick embryo fibroblasts. We conclude that the levels of alpha1(III) and alpha2(I) collagen mRNA are often but not necessarily coordinately regulated by transformation in mouse cells.     

Transformation of Rat Liver Cells with Chicken Sarcoma Virus B77 and Murine Sarcoma Virus

Rat liver cells in vitro were transformed with chicken sarcoma virus B77, giving RL(B77) cells, and with murine sarcoma virus (Harvey), giving RL(MSV) cells. Rat liver cells transformed spontaneously in vitro were designated RL cells. In addition, the RL(MSV) cell line was adapted for growth in culture fluid containing 25 mug of 5-bromodeoxyuridine per ml. All cell lines were tumorigenic in 1-wk-old rats. The number of cells needed for induction of tumor growth was 1,000-fold higher in the case of RL(B77) cells in comparison with RL(MSV) cells and RL cells. No production of viral particles from any of the cell lines investigated was detected by plating concentrated supernatant fluid of the cultures on different secondary embryo cells with and without fusion by Sendai virus, by labeling with uridine-5-(3)H, or by assay for deoxyribonucleic acid polymerase activity. The viral genome was rescued by fusion of RL(B77) cells with chicken cells. Chicken sarcoma virus rescued from (RL(B77) cells differed in plating efficiency on duck cells from B77 virus rescued from transformed rat embryo cells. No virus was rescued after fusion of RL(MSV) and RL cells with mouse, rat, or chicken embryo cells. Infectious murine sarcoma virus can be induced by 5-bromodeoxyuridine from RL(MSV) cells.     

Arrangement of Integrated Avian Sarcoma Virus DNA Sequences Within the Cellular Genomes of Transformed and Revertant Mammalian Cells

We have examined the arrangement of integrated avian sarcoma virus (ASV) DNA sequences in several different avian sarcoma virus transformed mammalian cell lines, in independently isolated clones of avian sarcoma virus transformed rat liver cells, and in morphologically normal revertants of avian sarcoma virus transformed rat embryo cells. By using restriction endonuclease digestion, agarose gel electrophoresis, Southern blotting, and hybridization with labeled avian sarcoma virus complementary DNA probes, we have compared the restriction enzyme cleavage maps of integrated viral DNA and adjacent cellular DNA sequences in four different mouse and rat cell lines transformed with either Bratislava 77 or Schmidt-Ruppin strains of avian sarcoma virus. The results of these experiments indicated that the integrated viral DNA resided at a different site within the host cell genome in each transformed cell line. A similar analysis of several independently derived clones of Schmidt-Ruppin transformed rat liver cells also revealed that each clone contained a unique cellular site for the integration of proviral DNA. Examination of several morphologically normal revertants and spontaneous retransformants of Schmidt-Ruppin transformed rat embryo cells revealed that the internal arrangement and cellular integration site of viral DNA sequences was identical with that of the transformed parent cell line. The loss of the transformed phenotype in these revertant cell lines, therefore, does not appear to be the result of rearrangement or deletions either within the viral genome or in adjacent cellular DNA sequences. The data presented support a model for ASV proviral DNA integration in which recombination can occur at multiple sites within the mammalian cell genome. The integration and maintenance of at least one complete copy of the viral genome appear to be required for continuous expression of the transformed phenotype in mammalian cells.     

Abortive and transforming infection of rat cell line 3Y1 by adenovirus type 12.

Human adenovirus type 12 underwent an abortive and transforming infection in clonal cultures of rat cell line 3Y1. The frequency of transformation was proportional to the dose of input virus. The ratio of PFU to transforming units was 1.1 X 10(5).     

Construction and biological analysis of deletion mutants of Fujinami sarcoma virus: 5''-fps sequence has a role in the transforming activity.

Fujinami sarcoma virus (FSV) genome codes for the gag-fps fusion protein FSV-P130. The amino acid sequence of the 3' one-third portion in v-fps is partially homologous to the 3' half of pp60src, or the kinase domain, but the sequence of the 5' portion is unique to v-fps. To identify a possible domain structure in the v-fps sequence responsible for cell transformation, we constructed various deletion mutants of FSV with molecularly cloned viral DNA. Their transforming activities were assayed by measuring focus formation on chicken embryo fibroblasts and rat 3Y1 cells and tumor formation in chickens. The mutants carrying a deletion at the 3' portion in v-fps, the kinase domain, lost transforming activity. The mutants carrying an approximately 1-kilobase deletion within the 5' portion of the v-fps sequence retained focus-forming activity and tumorigenicity in the chicken system, but the efficiency of focus formation was about 10 times lower than that of the wild type. The morphology of these transformed cells was distinct from that observed in cells infected with wild-type FSV. Furthermore, these mutants could not transform rat 3Y1 cells, although wild-type FSV DNA transformed rat 3Y1 cells at a high frequency. The mutants carrying a larger deletion in the 5' portion of fps completely lacked the transforming activity. These results suggest that the 3' portion of the v-fps sequence is necessary but not sufficient for cell transformation and that the 5' portion of v-fps has a role in the transforming activity.     

Epstein-Barr virus nuclear protein 2 is a critical determinant for tumor growth in SCID mice and for transformation in vitro.

Injection of Epstein-Barr virus (EBV)-transformed human lymphoblastoid B cells into immunodeficient SCID mice results in the appearance of rapidly growing, fatal human B-cell tumors. To evaluate the role of EBV nuclear protein 2 (EBNA-2) in this process, we generated lymphoblastoid cell lines transformed by several EBV mutants which were identical except for deletions in the EBNA-2 gene (J. I. Cohen, F. Wang, and E. Kieff, J. Virol. 65:2545-2554, 1991). These cell lines were injected intraperitoneally into SCID mice, and the interval until tumor detection was determined. Cell lines transformed with EBV type 1 (strain W91) or with EBV type 2 (strain P3HR-1) with an inserted type 1 EBNA-2 gene grew at the same rapid rate, indicating the potential importance of EBNA-2 for tumor formation in vivo. Cell lines derived from three different EBV mutants with deletions in the amino half of EBNA-2 produced tumors more slowly than cell lines transformed by wild-type W91 virus. In contrast, a cell line transformed with an EBV mutant with a deletion in the carboxy terminus of EBNA-2 grew more rapidly than cell lines transformed by wild-type virus. EBV mutants with deletions in the amino half of EBNA-2 had had reduced transforming activity in vitro, while the carboxy-terminal EBNA-2 mutant had had transforming activity greater than or equal to that of the wild type. These data indicate that EBNA-2 plays a critical role both for B-cell tumor growth in SCID mice and for B-lymphocyte transformation in vitro.     

Stimulation of cell proliferation by vitamin A derivatives on murine sarcoma virus-transformed mouse cells in serum-free culture

The effect of retinoids (Rds) on cell proliferation was studied in serum-free culture condition, using non-transformed and transformed derivatives of BALB 3T3. Cell proliferation of an SV40-transformed line was inhibited significantly by Rd treatment. However, proliferation of two cell lines that were transformed by a Kirsten and Moloney strain of murine sarcoma virus (MSV) and produced growth factor into culture medium, was remarkably stimulated by Rds. Addition of serum masked both the inhibitory and stimulatory effects of Rds.     

Unstable resistance of G mouse fibroblasts to ecotropic murine leukemia virus infection.

G mouse cells were resistant to N- and NB-tropic Friend leukemia viruses and to B-tropic WN 1802B. Though the cells were resistant to focus formation by the Moloney isolate of murine sarcoma virus, they were relatively sensitive to helper component murine leukemia virus. To amphotropic murine leukemia virus and to focus formation by amphotropic murine sarcoma virus, G mouse cells were fully permissive. When the cell lines were established starting from the individual embryos, most cell lines were not resistant to the murine leukemia viruses. Only one resistant line was established. Cloning of this cell line indicated that the resistant cells constantly segregated sensitive cells during the culture; i.e., the G mouse cell cultures were probably always mixtures of sensitive and resistant cells. Among the sensitive cell clones, some were devoid of Fv-1 restriction. Such dually permissive cells, and also feral mouse-derived SC-1 cells, retained glucose-6-phosphate dehydrogenase-1 and apparently normal number 4 chromosomes. The loss of Fv-1 restriction in these mouse cells was not brought about by any gross structural changes in the vicinity of Fv-1 on number 4 chromosomes.     

In vitro tumoricidal activity of macrophages against virus-transformed lines with temperature-dependent transformed phenotypic characteristics.

The susceptibility of targets to destruction by tumoricidal rat and mouse macrophages was studied with virus-transformed cell lines in which various elements of the transformed phenotype are only expressed at specific temperatures. BHK cells transformed by the ts3 mutant of polyoma virus, rat embryo 3Y1 cells transformed by a temperature-sensitive A cistron mutant of simian virus 40 (SV40) and the ts-H6-15 temperature-sensitive line of SV40-transformed mouse 3T3 cells were killed in vitro by macrophages at both the permissive (33 °C) or nonpermissive (39 °C) temperatures for expression of the transformed phenotype. 3T3, 3Y1 and BHK cells transformed by wild-type SV40 or polyoma virus were also destroyed by tumoricidal macrophages at both 33 and 39 °C, but untransformed 3T3, 3Y1, and BHK cells were not. Thus, transformed cells are killed by macrophages regardless of whether or not they express cell surface LETS protein or Forssman antigen, display surface changes which permit agglutination by low doses of plant lectins, express SV40 T antigen, have a low saturation density, or exhibit density-dependent inhibition of DNA synthesis.