Isolation of clonal strains of chicken embryo fibroblasts

A method for cloning of chicken embryo fibroblasts (CEF) was developed, yielding a cloning efficiency of up to 50% without use of feeder cells or conditioned medium. An analysis of the growth potential of over 200 randomly selected clones showed that only approx. 4% of the clones were capable of doubling more than 35 times before undergoing cellular senescence. A positive correlation between initial growth rate and in vitro lifespan was observed. This served as a basis for a simple selection procedure for fibroblast strains suitable for long-term culturing. None of over 200 clones thus isolated could be established into a line. Subclones from clonal CEF strains were more homogeneous than uncloned CEF cultures with respect to morphology and growth behaviour, but still heterogeneous in their in vitro life span. All fibroblast strains tested could be effectively infected and transformed by a variety of avian sarcoma and leukosis viruses.     

Comparison between the viral transforming gene (src) of recovered avian sarcoma virus and its cellular homolog.

Recovered avian sarcoma viruses are recombinants between transformation-defective mutants of Rous sarcoma virus and the chicken cellular gene homologous to the src gene of Rous sarcoma virus. We have constructed and analyzed molecular clones of viral deoxyribonucleic acid from recovered avian sarcoma virus and its transformation-competent progenitor, the Schmidt-Ruppin A strain of Rous sarcoma virus. A 2.0-megadalton EcoRI fragment containing the entire src gene from each of these clones was subcloned and characterized. These fragments were also used as probes to isolate recombinant phage clones containing the cellular counterpart of the viral src gene, termed cellular src, from a lambda library of chicken deoxyribonucleic acid. The structure of cellular src was analyzed by restriction endonuclease mapping and electron microscopy. Restriction endonuclease mapping revealed extensive similarity between the src regions of Rous sarcoma virus and recovered avian sarcoma virus, but striking differences between the viral src's and cellular src. Electron microscopic analysis of heteroduplexes between recovered virus src and cellular src revealed a 1.8-kilobase region of homology. In the cellular gene, the homologous region was interrupted by seven nonhomologous regions which we interpret to be intervening sequences. We estimate the minimum length of cellular src to be about 7.2 kilobases. These findings have implications concerning the mechanism of formation of recovered virus src and possibly other cell-derived retrovirus transforming genes.     

Epithelioid and fibroblastic rat kidney cell clones: epidermal growth factor (EGF) receptors and the effect of mouse sarcoma virus transformation.

Fibroblastic and epithelioid clones have been isolated from the normal rat kidney line, NRK. These clones were studied for their ability to bind epidermal growth factor (EGF), susceptibility to transformation by mouse sarcoma virus (MSV), and alteration in EGF binding upon sarcoma virus transformation. The epithelioid clones bound much more EGF than the fibroblastic clones; Scatchard plots on two of these clones, one epithelioid and one fibroblastic, showed that the higher EGF binding (1.3 x 10(5) molecules per cell for the epithelioid clone and 1.3 x 10(4) molecules per cell for the fibroblastic clone) was due to a greater number or receptors on the epithelioid cells rather than to a difference in the apparent affinity constant. When the clones were transformed by Moloney murine sarcoma virus the EGF binding decreased, the effect being greater with the fibroblastic clones. In 20 out of 20 independently isolated sarcoma virus transformed fibroblastic clones, the level of EGF binding was either greatly reduced or completely eliminated. In contrast to EGF, another growth factor, multiplication stimulating activity (MSA), bound to a greater extent to the fibroblastic clones than the epithelioid clones, and its binding was not decreased by sarcoma virus transformation. The results show that loss of EGF binding ability correlates with expression of the murine sarcoma virus transformation.     

Establishment and proteomic characterization of a novel synovial sarcoma cell line,NCC-SS2-C1

Synovial sarcoma is an aggressive mesenchymal tumor, characterized by the presence of unique transfusion gene, SS18–SSX. Cell lines enable researchers to investigate the molecular backgrounds of disease and the significance of SS18–SSX in relevant cellular contexts. We report the establishment and proteomic characterization of a novel synovial sarcoma cell line. Primary tissue culture was performed using tumor tissue of synovial sarcoma. The established cell line was authenticated by assessing its DNA microsatellite short tandem repeat analysis and characterized by in vitro assay. Proteomic study was achieved by mass spectrometry, and the results were analyzed by treemap. The cell line NCC-SS2-C1 was established from a primary tumor tissue of a synovial sarcoma patient. The cell line has grown well for 11 mo and has been subcultured more than 15 times. The established cells were authenticated by assessing their short tandem repeat pattern comparing with that of original tumor tissue. The cells showed polygonal in shape and formed spheroid when seeded on the low-attachment dish. Proteomic analysis revealed the molecular pathways which are unique to the original tumor tissue or the established cell line. In conclusion, a novel synovial sarcoma cell line NCC-SS2-C1 was successfully established from the primary tumor tissue. The cell line has characteristic transfusion SS18–SSX and poses aggressive in vitro growth and capability of spheroid formation. Thus, NCC-SS2-C1 cell line will be a useful tool for investigation of the mechanisms of disease and the biological role of fusion gene.     

Establishment of a novel patient-derived Ewing’s sarcoma cell line,NCC-ES1-C1

Ewing’s sarcoma is an aggressive mesenchymal tumor characterized by the presence of a unique EWSR1-FLI1 translocation. Ewing’s sarcoma primarily occurs in the bone and soft tissues. Cell lines enable researchers to investigate the molecular backgrounds of disease and the significance of genetic alterations in relevant cellular contexts. Here, we report the establishment and characterization of a novel Ewing’s sarcoma cell line following primary Ewing’s sarcoma tumor tissue culture. The established cell line was authenticated by DNA microsatellite short tandem repeat analysis, characterized by in vitro assays, and named NCC-ES1-C1. The NCC-ES1-C1 cell line grew well for 15 mo and was subcultured more than 50 times during this period. Characterization of the cells revealed that they were not adherent and showed floating features. In conclusion, we successfully established a novel Ewing’s sarcoma cell line, NCC-ES1-C1, from primary tumor tissue. The cell line has the characteristic EWSR1-FLI1 gene fusion and exhibits aggressive growth in vitro. Thus, the NCC-ES1-C1 cell line will be a useful tool for investigating the mechanisms of disease and the biological role of the EWSR1-FLI1 fusion gene.     

Telomeric profiles and telomerase activity in turkey satellite cell clones with different in vitro growth characteristics

The satellite cell population in postnatal skeletal muscle is heterogeneous because individual satellite cells isolated from a single muscle have differing abilities to proliferate under the same in vitro conditions. Telomeres are structures found at the ends of all eukaryotic chromosomes that are characterized by repetitive DNA sequences, and they are important in determining cellular proliferation potential. The relationship between satellite cell proliferative heterogeneity and telomeric DNA was examined by digesting genomic DNA from large-colony-forming and small-colony-forming turkey satellite cell clones with HinfI, separating the restriction fragments on an agarose gel, and hybridizing the gels with an oligonucleotide probe specific for telomeric DNA. Turkey satellite cells generated telomeric restriction fragments up to approximately 180 kB. The large-colony-forming satellite cell clones had a larger proportion (P<0.05) of total telomeric restriction fragments below 33 kB than the small-colony-forming satellite cell clones. However, telomerase expression was detected in cultures from large-colony-forming and small-colony-forming turkey satellite cells suggesting that the differences in telomeric restriction fragments may not be related to the differences in in vitro proliferative behavior and that telomerase may contribute to the high in vitro growth capacity of turkey satellite cells.     

Independence from the substrate of multiplication in nonclonal and clonal tumor cell populations

Experiments were made with sarcoma PS-103 cells cultured in vitro. The sarcoma was induced by implantation of a plastic plate into CBA mice. Clonal analysis of the cell culture demonstrated that 1) all 3 clones isolated from substrate (SB) grew in 1.2% methyl cellulose (MC) at the same efficiency as parental cells; 2) all 5 clones isolated from MC formed in a semi-solid medium 10-100-fold more colonies than PS-103. During the subcloning of one of PS-103 clones in solid substrate and in MC, it turned out that the majority of MC and SB subclones had the plating efficiency in MC similar to that in PS-103. Apparently, the PS-103 population contains clones with different degrees of anchorage independence.     

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.     

Cloning and characterization of an envelope-specific probe from xenotropic murine leukemia proviral DNA.

UR2 is a newly characterized avian sarcoma virus whose genome contains a unique sequence that is not related to the sequences of other avian sarcoma virus transforming genes thus far identified. This unique sequence, termed ros, is fused to part of the viral gag gene. The product of the fused gag-ros gene of UR2 is a protein of 68,000 daltons (P68) immunoprecipitable by antiserum against viral gag proteins. In vitro translation of viral RNA and in vivo pulse-chase experiments showed that P68 is not synthesized as a large precursor and that it is the only protein product encoded in the UR2 genome, suggesting that it is involved in cell transformation by UR2. In vivo, P68 was phosphorylated at both serine and tyrosine residues. Immunoprecipitates of P68 with anti-gag antisera had a cyclic nucleotide-independent protein kinase activity that phosphorylated P68, rabbit immunoglobulin G in the immune complex, and alpha-casein. The phosphorylation by P68 was specific to tyrosine of the substrate proteins. P68 was phosphorylated in vitro at only one tyrosine site, and the tryptic phosphopeptide of in vitro-labeled P68 was different from those of Fujinami sarcoma virus P140 and avian sarcoma virus Y73-P90. A comparison of the protein kinases encoded by UR2, Rous sarcoma virus, Fujinami sarcoma virus, and avian sarcoma virus Y73 revealed that UR2-P68 protein kinase is distinct from the protein kinases encoded by those viruses by several criteria. Our results suggest that several different protein kinases encoded by viral transforming genes have the same functional specificity and cause essentially the same cellular alterations.     

EGF induces differentiation of an IL-3-dependent cell line expressing the EGF receptor.

Interleukin 3 (IL-3) is a T cell-derived lymphokine that supports the growth and development of hematopoietic cells. Tyrosine phosphorylation has been suggested to play an important role in IL-3-dependent cell proliferation. To test whether a growth factor receptor carrying a tyrosine kinase can be functional in IL-3 dependent cells, we used a retroviral vector to introduce the human EGF receptor into a murine IL-3-dependent pre-mast cell line, IC2. The EGF receptors expressed on the infected clones bind EGF with both high and low affinities. EGF stimulates the infected cells for a short term growth response. In the presence of IL-3 and EGF, infected clones differentiate into more mature mast cells characterized by increases in intracellular granulation and histamine content. This differentiation is reversible when EGF is removed. EGF induces tyrosine phosphorylation of several cellular proteins and the expression of oncogenes c-fos and c-myc, in a manner analogous to IL-3 stimulation. These results indicate that the EGF receptor is functional in the pre-mast IC2 cells; EGF can support short-term proliferation and activates the signals that induce cell differentiation. Thus, EGF receptor-expressing IC2 cells provide a unique cellular system for in vitro study of mast cell differentiation.     

Genetic transmission of retroviral genes and cellular oncogenes

Several different families of retrovirus genome have been found to exist, each in multiple copies, in the cellular DNA of rodents and primates. There are at least four distinct families of genome in rodents: two type C families, the MTV family and another related to mouse type A particles. In primates there are also at least two families of endogenous type C virogenes and a third type D virogene family. Both in rodents and in primates, the virus-related sequences constitute almost 0.1% of the cellular genome. We have been able to generate transforming viruses, starting with endogenous mouse 'helper' type C viruses by passing them through chemically transformed mouse cells and selecting for variant viruses that have acquired the ability to induce normal cells to display anchorage-independent growth. These viruses produce both sarcomas and carcinomas in the animal; clones that produce only pulmonary carcinomas have also been selected. These presumably have arisen by recombination between the helper and 'transforming' sequences derived from the cells. Moloney sarcoma virus-transformed cells produce a new peptide, called sarcoma growth factor (SGF), that makes normal cells take on some of the properties of transformed cells. Studies with temperature-sensitive viral mutants show that the production of SGF is under the control of the transforming genes of the sarcoma virus.     

Analysis of recombinant DNA clones specific for the murine p53 cellular tumor antigen.

Three cDNA clones, corresponding to two non-overlapping regions of the mRNA coding for the mouse p53 cellular tumor antigen, were isolated and characterized. In hybridization-selection assays, these clones were capable of selectively binding p53 mRNA, as demonstrated by in vitro translation and immunoprecipitation with anti-p53 monoclonal antibodies. The p53 mRNA appeared to be the only messenger species specifically selected by these clones. The size of the p53 mRNA was found to be approximately 2 kb, and its levels to vary substantially among different types of transformed cells. Evidence was found for the existence of two distinct p53-specific genes in mouse genomic DNA. Two partially overlapping recombinant phage clones were obtained, both derived from the same p53-specific genomic DNA region. The orientation of the various cDNA clones relative to that of the p53 mRNA was established by S1 analysis and the relationship between the cDNA clones and the genomic ones was determined by comparative restriction enzyme mapping and nucleic acid hybridization.     

Isolation of monoclonal antibodies that recognize the transforming proteins of avian sarcoma viruses.

Thirteen clones of hybrid cells which synthesize antibodies directed against the Rous sarcoma virus (RSV) transforming protein, pp60src, were isolated. Mouse myeloma cells were fused with spleen cells from mice that had been immunized with purified pp60src from bacterial recombinants which direct the synthesis of the RSV src gene. The hybridomas which survived the selection medium were screened by immunoprecipitation of pp60src from 32P-labeled lysates of RSV-transformed cells. Monoclonal antibodies produced by subclones derived from 13 hybridomas recognized pp60src encoded by the Schmidt-Ruppin and Prague strains of RSV and the cellular homolog of pp60src. Antibody from clone 261 had a high affinity for the viral yes gene product, and antibodies from clones 443 and 463 recognized the transforming proteins encoded by viruses containing the related transforming genes fps and ros. Several other clones had a low affinity for the viral yes, fps, and ros gene products which could be detected by in vitro phosphorylation of the transforming proteins after immunoprecipitation with the monoclonal antibody. All of the monoclonal antibodies allowed phosphorylation of pp60src and casein in an immune complex-bound reaction.     

Characterization of unintegrated retroviral DNA with long terminal repeat-associated cell-derived inserts.

We have used a replication-competent shuttle vector based on the genome of Rous sarcoma virus to characterize genomic rearrangements that occur during retrovirus replication. The strategy involved cloning circular DNA that was generated during an acute infection. While analyzing a class of retroviral DNA clones that are greater than full length, we found several clones which had acquired nonviral inserts in positions adjacent to the long terminal repeats (LTRs). There appear to be two distinct mechanisms leading to the incorporation of cellular sequences into these clones. Three of the molecules contain a cell-derived insert at the circle junction site between two LTR units. Two of these molecules appear to be the results of abortive integration attempts, because of which, in each case, one of the LTRs is missing 2 bases at its junction with the cell-derived insert. In the third clone, pNO220, the cellular sequences are flanked by an inappropriately placed copy of the tRNA primer-binding site on one side and a partial copy of the U3 sequence as part of the LTR on the other side. A fourth molecule we characterized, pMD96, has a single LTR with a U5-bounded deletion of viral sequences spanning gag and pol, with cell-derived sequences inserted at the site of the deletion; its origin may be related mechanistically to pNO220. Sequence analysis indicates that all of the cellular inserts were derived from the cell line used for the acute infection rather than from sequences carried into the cell as part of the virus particle. Northern (RNA) analysis of cellular RNA demonstrated that the cell-derived sequences of two clones, pNO220 and pMD96, were expressed as polyadenylated RNA in uninfected cells. One mechanism for the joining of viral and cellular sequences suggested by the structures of pNO220 and pMD96 is recombination occurring during viral DNA synthesis, with cellular RNA serving as the template for the acquisition of cellular sequences.     

Mathematical models and computer simulations of proliferation of human diploid fibroblast clones.

A mathematical model based on the simple concept of an oscillatory mechanism for regulation of cellular proliferation has been developed that describes the growth of clones of human diploid fibroblasts in vitro. Lineages of these cells have been obtained from time-lapse cinematographic sequences of proliferating clones. Computer simulations based upon the oscillator model have generated genealogies that behave as the experimentally-derived genealogies.     

Progression of the transformed phenotype in clonal lines of Abelson virus-infected lymphocytes.

Some molecular changes which correlate with the tumorigenic progression of neoplastic cells can best be studied with in vitro cell lines that represent each stage in the progression. Lymphoid cells infected by Abelson murine leukemia virus exhibit a wide range of growth potential in vitro and in vivo. Uncloned populations that are poorly oncogenic early after infection become progressively more oncogenic with successive passages of the cells in culture. In such mass cultures, it is difficult to evaluate whether a rare subpopulation of highly oncogenic cells becomes dominant in the culture or whether the individual cells progress in oncogenic phenotype. To examine this latter possibility, Abelson virus-infected lymphoid cells were cloned by limiting-dilution culture 10 days postinfection. We isolated two clones that grew poorly in agar, required feeder layers of adherent bone marrow cells for growth in liquid culture, and were extremely slow to form tumors in syngeneic animals. Both clones, after passage in the presence of adherent feeder layers for 3 months, grew well in liquid and agar-containing cultures in the absence of feeder layers and formed tumors in animals at a rapid rate. The progression of these clonal cell lines to a more malignant growth phenotype occurred in the absence of detectable changes in the concentration, half-life, phosphorylation, in vitro kinase activity, or cell localization of the Abelson virus-encoded transforming protein. No change in the concentration or arrangement of integrated Abelson viral DNA sequences was detected in either clone. Thus, perhaps changes in the expression of cellular genes would appear to alter the growth properties of lymphoid cells after their initial transformation by Abelson virus. Such cellular changes could complement the activity of the Abelson virus transforming protein in producing the fully malignant growth phenotype.     

Transfer of defective avian tumor virus genomes by a Rous sarcoma virus RNA packaging mutant.

SE21Q1b, a Rous sarcoma virus mutant which packages cellular rather than viral RNA, is competent for infection of quail cells and can transmit defective transforming retrovirus genes. Stably transformed recipient clones have been obtained by using this mutant.