Transformation Of Chicken Embryo Fibroblasts Research Articles

Expression of 9E3 mRNA is associated with mitogenicity, phosphorylation, and morphological alteration in chicken embryo fibroblasts.

Transformation of chicken embryo fibroblasts (CEF) with viruses encoding src, ros, yes, and fps as well as ras, mos, middle T, erbA and erbB, myc, and crk stimulated 9E3 mRNA expression. Treatment of CEF with agents that modulate cell shape or attachment to the substratum caused an increase in 9E3 mRNA without an increase in tyrosine phosphorylation. 9E3 mRNA was also increased in CEF in response to several agents which modulate phosphorylation, including phorbol myristic acetate, vanadate, and okadaic acid, which suggests that the rapid induction of 9E3 mRNA expression in CEF by the src protein occurs downstream of morphological or phosphorylation events.

Expression of 9E3 mRNA Is Associated with Mitogenicity, Phosphorylation, and Morphological Alteration in Chicken Embryo Fibroblasts

Transformation of chicken embryo fibroblasts (CEF) with viruses encoding src, ros, yes, and fps as well as ras, mos, middle T, erbA and erbB, myc, and crk stimulated 9E3 mRNA expression. Treatment of CEF with agents that modulate cell shape or attachment to the substratum caused an increase in 9E3 mRNA without an increase in tyrosine phosphorylation. 9E3 mRNA was also increased in CEF in response to several agents which modulate phosphorylation, including phorbol myristic acetate, vanadate, and okadaic acid, which suggests that the rapid induction of 9E3 mRNA expression in CEF by the src protein occurs downstream of morphological or phosphorylation events.

Monoclonal antibodies to individual tyrosine-phosphorylated protein substrates of oncogene-encoded tyrosine kinases.

Cellular transformation by oncogenic retroviruses encoding protein tyrosine kinases coincides with the tyrosine-specific phosphorylation of multiple protein substrates. Previous studies have shown that tyrosine phosphorylation of a protein of 120 kDa, p120, correlated with src transformation in chicken embryo fibroblasts. Additionally, we previously identified two phosphotyrosine-containing cellular proteins, p130 and p110, that formed stable complexes with activated variants of pp60src, the src-encoded tyrosine kinase. To study transformation-relevant tyrosine kinase substrates, we have generated monoclonal antibodies to individual tyrosine phosphoproteins, including p130, p120, p110, and five additional phosphoproteins (p210, p125, p118, p85, and p185/p64). These antibodies detected several of the same tyrosine phosphoproteins in chicken embryo fibroblasts transformed by avian retroviruses Y73 and CT10, encoding the yes and crk oncogenes, respectively. Protein substrates in mouse, rat, hamster, and human cells overexpressing activated variants of chicken pp60src were also detected by several of the monoclonal antibodies.

Isolation and characterization of fra-2, an additional member of the fos gene family.

Antiserum raised against a Fos peptide (amino acids Lys-127 to Arg-140 of chicken c-Fos) recognizes a 46-kDa Fos-related protein in cell lysates of growth-stimulated chicken embryo fibroblasts. Induction of the 46-kDa protein is transient but is slightly prolonged relative to c-Fos following growth stimulation. Using a mixed oligonucleotide probe encoding the peptide antigen, we have cloned the chicken genomic locus that encodes this protein and have determined its gene structure. This locus consists of four exons, each of which has some homology with the corresponding exons of the chicken c-fos gene, and it expresses a 6-kilobase mRNA after growth stimulation. The deduced amino acid sequence of the gene (323 amino acids) contains a "leucine zipper" and includes five distinct regions that exhibit strong sequence homology to the recently identified fos-related antigens Fra-1 (rat) and FosB (mouse) as well as c-Fos. Since the other regions of the gene have little homology to any of these three proteins, this gene was named "fra-2." When the fra-2 gene was overexpressed by an avian retrovirus vector, it caused transformation of chicken embryo fibroblasts. The fra-2 gene product formed a complex in cells with the c-Jun protein, indicating that c-fos and fra-2 share biological and biochemical functions.

Transformation of chicken embryo fibroblasts by direct DNA transfection of single oncogenes: comparative analyses of src, erbB, myc, and ras

Chicken embryo fibroblasts (CEF) have been used extensively to study the transformation parameters of a number of avian sarcoma-leukemia viruses. Previously, oncogene transformation of CEF has been conducted almost exclusively with replicating viruses, because of perceived difficulties with direct DNA transfection. Here, we show that CEF can be efficiently and stably transfected by selection for the neomycin resistance gene (neo). Cotransfection of neo with various oncogenes resulted in CEF transformation in vitro and, in several instances, sarcoma formation in vivo. Transfection of src, myc, erbB, and ras, either singly or in combination, resulted in soft-agar colonies with unique morphologies. Transfection of a family of v-src, c-src, and v/c-src chimeric constructs demonstrated the ability of the assay to discriminate between transforming and nontransforming genes. Transfection of a number of erbB variants showed that internal mutations, primarily in the kinase domain, contribute significantly to the ability to transform fibroblasts. The tumorigenic potential detected by transfection of oncogenes faithfully reproduced those previously reported by using viral infections. Our studies establish the utility of CEF transformation by direct DNA transfection. This method should prove useful in analyzing oncogenes, (e.g., myc) that do not readily transform rodent cell lines and in studying host-range mutants of oncogenes, such as those recently identified for src and erbB.

Transformation of BALB/3T3 cells by simian virus 40 causes a decreased synthesis of a collagen-binding heat-shock protein (hsp47).

The synthesis of a major collagen-binding heat-shock protein of molecular weight 47,000 (hsp47) was shown previously to be decreased after transformation of chick embryo fibroblasts by Rous sarcoma virus (Nagata, K., and Yamada, K. M. (1986) J. Biol. Chem. 261, 7531-7536; and Nagata, K., Saga, S., and Yamada, K. M. (1986) J. Cell Biol. 103, 223-229). In this paper, further study demonstrated that the total amount and the synthesis of hsp47 are also decreased by a factor of three in BALB/3T3 cells transformed by simian virus 40 (SV40). Higher synthesis was observed for BALB/3T3 cells in the resting state compared to those in the proliferating state. The synthesis of hsp47 in SV40-transformed cells, however, was consistently lower than that in normal cells irrespective of the cell density. Pulse label and chase experiments revealed that hsp47 was stable in the cells for at least 6 h and that there was no difference between normal and transformed BALB/3T3 cells in terms of the half-life. Decreases in the amount and the synthesis of hsp47 by transformation apparently correlate with the decreased synthesis of collagen in transformed cells. Immunoprecipitation using rat monoclonal antibody against hsp47 demonstrated the association of hsp47 with intracellular procollagen. Immunofluorescence studies using anti-hsp47 monoclonal antibody and anti-collagen antibody confirmed the co-localization of hsp47 and procollagen in both nonshocked and heat-shocked cells. Furthermore, we determined the biochemical characteristics of hsp47 of heat-shocked cells.

Suppression of Rous sarcoma virus-induced tumor formation by preinfection with viruses encoding src protein with novel N termini

Two recovered avian sarcoma viruses (rASVs), rASV157 and rASV1702, encode src products which contain novel, nonmyristoylated N-terminal amino acids. These viruses transform chicken embryo fibroblasts and cause tumors in chicks. However, the tumors rASVs induce are small and regress within 2 weeks. To determine whether this regression results from weak tumorigenicity or from the active immunity of the host, we injected 1-week-old chicks with rASV and several days later injected the chicks with challenge virus of a different subgroup. Of the rASV1702-preinfected chicks challenged 5 days later with Rous sarcoma virus (RSV), 40% showed no subsequent tumor formation and 60% formed tumors which regressed within 1 week. The potency of this protective effect depended on the dosage of preinfection virus used and increased as the interval between preinfection and challenge infection was lengthened (when the interval was 9 days, none of the challenged chicks formed tumors). rASV157-preinfected chicks challenged with RSV after 9 days showed only partial protection: 42% formed tumors which regressed, whereas 58% formed tumors which continued to grow. Challenging rASV-preinfected chicks with Fujinami sarcoma virus or a RSV vector encoding the v-fps oncogene or polyomavirus middle T resulted in no suppression of tumor formation. Preinfection with src mutants or a RSV vector encoding polyomavirus middle T antigen, both of which induce slow-growing tumors, failed to elicit the protective effect. Finally, a novel N-terminal domain encoded by rASV1702 src was shown to be involved in but not sufficient for full protection. These data indicate that determinants on or induced by rASV157 and rASV1702 can elicit a potent protection against the tumorigenic potential of RSV-encoded p60v-src.

Linker insertion-deletion mutagenesis of the v-src gene: isolation of host- and temperature-dependent mutants

The host cell regulators and substrates of the Rous sarcoma virus transforming protein pp60v-src remain largely unknown. Viral mutants which induce a host-dependent phenotype may result from mutations which affect the interaction of pp60v-src with host cell components. To isolate such mutants and to examine the role of different regions of src in regulating pp60v-src function, we generated 46 linker insertion and 5 deletion mutations within src. The mutant src genes were expressed in chicken embryo fibroblasts and in rat-2 cells by using retrovirus expression vectors. Most linker insertions within the kinase domain (residues 260 to 512) inactivated kinase activity and transforming capacity, while most insertions in the N-terminal domain and at the extreme C terminus were tolerated. A number of mutations generated a host-dependent phenotype. Insertions after residues 225 and 227, within the N-terminal regulatory domain (SH2), produced a fusiform transformation in chicken embryo fibroblasts and abolished transformation in rat-2 cells; a similar phenotype also resulted from two deletions affecting SH2 (residues 149 to 174 and residues 77 to 225). Insertions immediately C terminal to Lys-295, which is involved in ATP binding, also produced a conditional phenotype. Insertions after residues 299 and 300 produced a temperature-sensitive phenotype, while insertions after residues 304 and 306 produced a host cell-dependent phenotype. An insertion which removed the major tyrosine autophosphorylation site (Tyr-416) greatly reduced transformation of rat-2 cells, a property not previously observed with other mutations at this site. We conclude that mutations at certain sites within src result in conditional phenotypes. These sites may represent regions important in interactions with host cell components.

A host-dependent temperature-sensitive mutant of rous sarcoma virus: Evidence for host factors affecting transformation

We have characterized a host range mutant of Rous sarcoma virus in order to identify host cell factors involved in transformation. This mutant, tsLA33-1, which was isolated from a stock of the temperature-sensitive mutant tsLA33, is not temperature-sensitive for transformation of chicken embryo fibroblasts, as judged by its ability to induce morphological changes and agar colony formation at both 36 and 41.5°. In Rat-3 cells, however, this mutant induced a temperature-dependent transformation: infected Rat-3 cells were transformed at 34° but not at 39.5°. Retransformants were isolated from tsLA33-1-infected Rat-3 cells by growth in agar suspension at 39.5°. Virus rescued from these retransformants induced a temperature-dependent transformation when reintroduced into rat cells. The level of expression of pp60 v- src at 39.5° was unchanged in the retransformants. When the retransformants were treated with herbimycin, an antibiotic which induces turnover of certain protein-tyrosine kinases, they reverted to a normal phenotype, indicating that the transformed phenotype of the retransformants was dependent on continued expression of pp v- src . The re-transformants are therefore pseudorevertants in which a cellular alteration has occurred that allows transformation at 39.5° by the mutant pp60 v- src . Thus the temperature-dependence of transformation by tsLA33-1 is affected by the cellular environment, and is suppressed or complemented both in chicken cells and in the rat cell pseudorevertants. No clear correlation between levels of phosphorylation at tyrosine and transformation was observed. In Rat-3 cells the pp60 v- src encoded by tsLA33-1 may be defective in its interaction with low abundance substrates that are critical for transformation; alternatively the nonpermissive cells may require a higher threshold dose of pp60 v- src for transformation.

Mutation of a protein kinase C phosphorylation site in the erbB protein of avian erythroblastosis virus.

Tumor promoter-stimulated phosphorylation of threonine 98 of the erbB protein of avian erythroblastosis virus (AEV) correlates with inhibition of erbB-dependent mitogenesis. To more clearly define the role of phosphorylation of this residue in regulation of the activity of the erbB protein, we have constructed erbB mutations which encode alanine (Ala-98), tyrosine (Tyr-98), or serine (Ser-98) at position 98. The biosynthesis and stability of the three mutant proteins were similar to those of the wild-type erbB protein, and all three retained the ability to transform chicken embryo fibroblasts. Treatment of transformed CEF with 12-tetradecanoylphorbol-13-acetate (TPA) stimulated incorporation of 32Pi into wild-type and mutant erbB proteins and resulted in a slight decrease in the electrophoretic mobilities of all the erbB proteins. Tryptic maps of erbB phosphopeptides showed no endogenous or TPA-stimulated phosphorylation of alanine 98 or tyrosine 98 in cells transformed by the Ala-98 and Tyr-98 mutants. Analysis of tryptic phosphopeptides by high-pressure liquid chromatography revealed that TPA treatment of cells stimulated phosphorylation of other sites of the erbB protein in addition to threonine 98. A high endogenous level of phosphorylation of serine 98 of the Ser-98 mutant protein was found, and TPA treatment of cells did not result in further phosphorylation of this residue. Cells transformed by wild-type and mutant AEV were equally sensitive to TPA-dependent inhibition of growth in soft agar and TPA-dependent inhibition of [3H]thymidine incorporation. TPA treatment inhibited tyrosine phosphorylation to a similar extent in cells transformed by wild-type or Ala-98 AEV. These data indicate that phosphorylation of threonine 98 of the erbB protein is not responsible for TPA-dependent inhibition of growth of AEV-transformed cells or TPA-induced inhibition of erbB-dependent tyrosine phosphorylation. TPA-stimulated phosphorylation of the erbB protein at other sites may mediate these effects. The data also show that subtle changes in a phosphorylation site (i.e., changing threonine to serine) can drastically alter recognition by protein kinases.

Immunological characterization of proteins detected by phosphotyrosine antibodies in cells transformed by Rous sarcoma virus

Phosphotyrosine antibodies were used to identify tyrosine-phosphorylated proteins in Rous sarcoma virus (RSV)-transformed chicken embryo fibroblasts. A large number of tyrosine phosphoproteins were detected. A similar set of proteins was observed in RSV-transformed murine cells. An 85,000-dalton protein, however, was present in transformed avian cells but missing in transformed murine cells. Neither the 85,000-dalton protein nor any of the other tyrosine phosphoproteins appeared to be viral structural proteins. Use of RSV mutants encoding partially deleted src gene products enabled us to identify a 60,000-dalton cellular tyrosine phosphoprotein that comigrated with wild-type pp60v-src. With the exception of calpactin I, the major tyrosine phosphoproteins detected in immunoblots appeared to be different from several previously characterized substrates of pp60v-src with similar molecular masses (ezrin, vinculin, and the fibronectin receptor).

Isolation of a chicken thioredoxin cDNA clone. Thioredoxin mRNA is differentially expressed in normal and Rous sarcoma virus-transformed chicken embryo fibroblasts.

We have isolated a cDNA clone for chicken thioredoxin by differential screening of a cDNA library. The protein product which this clone encodes is very similar to other thioredoxins, and it displays thioredoxin activity when expressed in Escherichia coli. This clone represents the first metazoan thioredoxin for which the protein or nucleic acid sequence is known. Comparison of the chicken thioredoxin protein sequence with those from bacteria and plants indicates structural features that appear to be essential for activity. Transformation of chicken embryo fibroblasts by Rous sarcoma virus elevates the level of thioredoxin mRNA whereas the level of thioredoxin mRNA in a nonproliferative tissue (brain) is much lower than in chicken embryo fibroblasts.

Proviral-activated c-erbB is leukemogenic but not sarcomagenic: characterization of a replication-competent retrovirus containing the activated c-erbB.

Avian leukosis virus (ALV) induces erythroblastosis in chickens by integrating its DNA into the host c-erbB locus and by activating expression of truncated c-erbB transcripts. Although there is a 100% correlation of c-erbB activation with ALV-induced erythroblastosis, direct evidence that the activated c-erbB is oncogenic has not been established. We have constructed a replication-competent retrovirus containing the activated c-erbB to investigate its transforming potential. The Rous c-erbB virus (REB-c) was constructed by inserting the activated c-erbB cDNA into a Rous sarcoma virus vector in place of src. When transfected into transformed quail fibroblasts (QT6), the REB-c construct stably integrates and expresses c-erbB-specific transcripts and produces infectious virus. The REB-c retrovirus produces short-latency polyclonal erythroblastosis in chickens. However, in contrast to avian erythroblastosis virus which contains v-erbB, the REB-c construct does not transform chicken embryo fibroblasts in vitro, nor does the REB-c virus produce sarcomas when injected into the wing web of chickens. Our results provide the first direct evidence that the activated c-erbB which lacks the amino-terminal extracellular domain but which retains the entire carboxy-terminal sequences is leukemogenic but not sarcomagenic.

Nonmyristoylated p60v-src fails to phosphorylate proteins of 115-120 kDa in chicken embryo fibroblasts.

We have used anti-phosphotyrosine antibodies to identify a large number of tyrosine phosphoproteins in Rous sarcoma virus (RSV)-transformed chicken embryo fibroblasts. Most of these proteins were found in the 100,000 X g supernatant when cells were separated into soluble and particulate fractions; however, one group of proteins, of 115-120 kDa, was found in the particulate fraction. The phosphotyrosine content of the 115- to 120-kDa proteins was greatly reduced in chicken embryo fibroblasts infected with mutants of RSV (NY314 and SD10) encoding nonmyristoylated forms of the viral src gene product that do not associate with cellular membranes. Since RSV mutants NY314 and SD10 do not transform cells, phosphorylation of this group of 115- to 120-kDa membrane proteins may be related to the process of transformation.

Substitution of Ser-17 of pp60c-src: Biological and Biochemical Characterization in Chicken Embryo Fibroblasts

pp60c-src is phosphorylated mainly on Ser-17 and Tyr-527 in vivo. In this study, we examined the effect of the phosphorylation of Ser-17 on the properties of pp60c-src by introducing Rous sarcoma virus variants carrying pp60c-src in which Ser-17 had been substituted, into chicken embryo fibroblasts. The Ala-17 substitution in wild-type pp60c-src and pp60c-src carrying Phe-527 caused a two- to threefold elevation in the kinase activity in vitro of these proteins; the former variant resulted in no morphological changes of infected cells, whereas the latter variant transformed chicken embryo fibroblasts. Since the substitution of Tyr-527 per se has been reported to activate pp60c-src, these results suggest that the abolishment of the phosphorylation of Ser-17 does not affect noticeably the properties of pp60c-src in chicken embryo fibroblasts.

Substitution of Ser-17 of pp60c-src: biological and biochemical characterization in chicken embryo fibroblasts.

pp60c-src is phosphorylated mainly on Ser-17 and Tyr-527 in vivo. In this study, we examined the effect of the phosphorylation of Ser-17 on the properties of pp60c-src by introducing Rous sarcoma virus variants carrying pp60c-src in which Ser-17 had been substituted, into chicken embryo fibroblasts. The Ala-17 substitution in wild-type pp60c-src and pp60c-src carrying Phe-527 caused a two- to threefold elevation in the kinase activity in vitro of these proteins; the former variant resulted in no morphological changes of infected cells, whereas the latter variant transformed chicken embryo fibroblasts. Since the substitution of Tyr-527 per se has been reported to activate pp60c-src, these results suggest that the abolishment of the phosphorylation of Ser-17 does not affect noticeably the properties of pp60c-src in chicken embryo fibroblasts.

Efficient transformation by Prague A Rous sarcoma virus plasmid DNA requires the presence of cis-acting regions within the gag gene.

A region in addition to and outside the long terminal repeats (LTRs) in the gag gene of the Prague A strain of Rous sarcoma virus was found to be essential in cis for efficient cell transformation by cloned viral DNA. Transformation in chicken embryo fibroblasts, which requires infectious virus production and reinfection, was facilitated in cis by sequences between nucleotides 630 and 1659. Efficient transformation of NIH 3T3 cells in which secondary spread of virus is not necessary (as it is in chicken embryo fibroblasts) required sequences between nucleotides 630 and 1149. A src cDNA clone which also lacks this region demonstrated low transformation efficiency, indicating that the role of the cis element cannot be attributed to interference with RNA splicing. The gag gene segment required in cis for transformation, between nucleotides 630 and 1149, could substitute for the simian virus 40 enhancer in either orientation, and cells transfected with Rous sarcoma virus LTR-driven plasmids containing the gag cis element had a two- to threefold increase in steady-state viral RNA levels compared with plasmids lacking this region. Thus, additional cis-acting regulatory elements located outside the viral LTRs may modulate viral gene expression and contribute to the efficiency of cell transformation.

Infection of neuroretinal cells in vitro by avian sarcoma viruses UR 1 and UR 2: Transformation, cell growth stimulation, and changes in transducin levels

Infection in vitro of differentiating chick embryo neuroretinal cells with avian sarcoma viruses UR 1 and UR 2 results in mitogenic stimulation and morphologic conversion of both support and neuronal cells. This was shown by the continuous propagation of transformed cells for over 4 months and growth of reaggregated colonies in liquid medium as well as in soft agar. Production of the transforming proteins p150 gag-fps and p68 gag-ros of UR 1 and UR 2, respectively, was similar to that of transformed chick embryo fibroblasts, as judged from in vitro kinase activity assays. The two protein subunits, T β and T γ, but not T α of the GTP binding protein transducin, found in the retina of many animal species, were present in control neuroretinal cells. Infection with Rous sarcoma virus or UR 2 resulted in an inhibition of T γ synthesis and enhancement of T β-like protein production.

Characterisation of fibronectin fragments and plasminogen activators released by RSV-transformed cells

In the present study, we followed qualitatively the release of fibronectin/s (FNs) and of plasminogen activators (PAs) into the serum-free medium of normal and Rous sarcoma virus (RSV) transformed chicken embryo fibroblasts (CEF). It has been found by Western blotting analysis that RSV transformed cells release into the medium intact FN and FN peptides with a Mr ranging between 230 and I10 kDa while uninfected cells and cells infected by NY68 and PAI (temperature sensitive, ts, mutants for transformation), grown at the restrictive temperature for transformation (41°C), release mainly intact FN. The zymographic analysis of the PAs released by normal and transformed cells showed that transformed cells release into the medium a set of PA forms with Mr ranging between 180 and 43kDa while uninfected cells or cells infected with NY68 or PA1 grown at 41°C do not release or release only the 43 kDa form respectively. The analysis of the PAs present in the extracts of uninfected or PAI and NY68 infected cells, grown at 35° or 41°C, revealed mainly the presence of the 43 kDa form.

Cell transformation by a virus containing a molecularly constructed gag-erbB fused gene.

A provirus DNA that contains a gag-erbB fused gene as the sole and transforming gene was molecularly constructed from plasmid pSRA2 containing the entire genome of Rous sarcoma virus and pAE7.7 containing the entire genome of an avian erythroblastosis virus (AEV), AEV-H. A virus containing the gag-erbB fused gene (GEV) was recovered from chicken embryo fibroblasts transfected with the proviral DNA and a helper virus DNA. GEV could transform chicken embryo fibroblasts as efficiently as could AEV-H. Anti-erbB and anti-gag sera immunoprecipitated a protein with a molecular weight of about 110,000 from GEV-transformed cells. The erbB and gag-erbB fused-gene products in AEV-H- and GEV-transformed cells were analyzed.