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PubMed1984

Expression of RSV-specific proteins in RSV-transformed mammalian cells.

Expression of RSV-specific proteins was analysed in two virogenic and three helper-dependent RSV-transformed mammalian cell lines by the radioimmunoprecipitation technique. In all cell lines the only product of the gag gene was represented by the precursor Pr76gag without its further processing. The env gene was expressed in several lines in the form of 85K protein, but the product of this gene was absent in TWERC cells. The src gene product (pp60src) was identified by the protein kinase assay in all cells. In the kinase reaction in vitro, this protein can also phosphorylate some cellular proteins (130K, 34-36K and others) and the heavy chain of IgG.

Animals↗
PubMed1992

Identification and developmental expression of Src+ mRNAs in Xenopus laevis.

Alternative splicing of src mRNA has been demonstrated in several vertebrate species to yield a neuron-specific form of src protein termed pp60+. The function of pp60+ is unknown. The early developmental expression pattern of src+ RNA has not been previously examined. We have identified and characterized src+ transcripts corresponding to the two src genes in Xenopus laevis using a reverse transcription/polymerase chain reaction (RT/PCR) method. Both Xenopus pp60+ proteins have a 5-amino-acid insert in contrast to the 6-amino-acid insert in fish, birds, and mammals. Src+ mRNA first appears in neural plate stage Xenopus embryos, after neural induction signaling events but prior to neural differentiation. Analysis of dissected neural plate stage embryos showed that src+ mRNAs are localized to the neural plate. These findings suggest that pp60+ may play a role in elaboration of neuron structure.

Amino Acid Sequence↗
PubMed2006

Functional development of Src tyrosine kinases during evolution from a unicellular ancestor to multicellular animals.

The Src family of tyrosine kinases play pivotal roles in regulating cellular functions characteristic of multicellular animals, including cell-cell interactions, cell-substrate adhesion, and cell migration. To investigate the functional alteration of Src kinases during evolution from a unicellular ancestor to multicellular animals, we characterized Src orthologs from the unicellular choanoflagellate Monosiga ovata and the primitive multicellular sponge Ephydatia fluviatilis. Here, we show that the src gene family and its C-terminal Src kinase (Csk)-mediated regulatory system already were established in the unicellular M. ovata and that unicellular Src has unique features relative to multicellular Src: It can be phosphorylated by Csk at the negative regulatory site but still exhibits substantial activity even in the phosphorylated form. Analyses of chimera molecules between M. ovata and E. fluviatilis Src orthologs reveal that structural alterations in the kinase domain are responsible for the unstable negative regulation of M. ovata Src. When expressed in vertebrate fibroblasts, M. ovata Src can induce cell transformation irrespective of the presence of Csk. These findings suggest that a structure of Src required for the stable Csk-mediated negative regulation still is immature in the unicellular M. ovata and that the development of stable negative regulation of Src may correlate with the evolution of multicellularity in animals.

Amino Acid Sequence↗
PubMed1994

Endothelial cell transformation by polyomavirus middle T antigen in mice lacking Src-related kinases.

BACKGROUND: Expression of polyomavirus middle T antigen (PymT) rapidly induces endothelial tumors (hemangiomas) in mice, with an apparent single rate-limiting step. Because activation of Src-like kinases is thought to be an important component of PymT-induced transformation, we have analyzed the functional requirement for individual kinases in this process. This type of analysis has only recently become possible, with the generation of 'gene knock-out' mice lacking each of the kinase genes src, fyn and yes. RESULTS: Hemangiomas develop efficiently in newborn mice lacking either src, fyn or yes after inoculation with a PymT-transducing retrovirus. In src- and fyn-deficient mice, the kinetics of induction and the histological properties of the tumors were indistinguishable from those in wild-type mice. In contrast, a reduced number of tumors arose in yes-deficient mice, with a significantly longer latency period. Transformed endothelial cell lines derived from the induced hemangiomas, however, did not differ in their morphological and tumorigenic properties from cell lines established previously from wild-type mice. Biochemical analysis of complexes between PymT and the Src-related kinases in these cell lines suggests that the Yes kinase is responsible for a significant amount of the PymT-associated kinase activity in transformed endothelial cells. CONCLUSION: We have demonstrated that inactivation of a single tyrosine kinase of the Src family in endothelial cells does not abrogate PymT-induced hemangioma formation. As the remaining kinases do not compensate for the absence of a family member by elevated kinase activity, the loss--which affects the transformation process to varying degrees--can be studied in this model system. Our studies suggest that the PymT-Yes kinase complex plays a major role in the tumor-initiating action of PymT.

Animals↗
PubMed1993

"Macrophage" nitric oxide synthase is a glucocorticoid-inhibitable primary response gene in 3T3 cells.

Both nitric oxide and prostaglandins are potent paracrine mediators of intercellular communication. An endotoxin-lipopolysaccharide (LPS) inducible form of nitric oxide synthase (mac-NOS) has recently been cloned from murine macrophages. An inducible prostaglandin synthase (TIS10/PGS-2), cloned from 3T3 cells, is also induced in LPS-activated macrophage. Because of the wide range of ligands that induce primary response genes in 3T3 cells, the ease of studying chimeric promoter constructs in 3T3 cells, and the importance of both nitric oxide and prostaglandins as paracrine mediators, we examined expression of mac-NOS in 3T3 cells. Tetradecanoyl phorbol-13-acetate (TPA), forskolin, platelet-derived growth factor, fibroblast growth factor, and serum all induce mac-NOS expression in Swiss 3T3 cells. Thus the mac-NOS gene can respond to a far wider range of inducers than previously suspected. mac-NOS is a primary response gene; cycloheximide does not block induction. TPA-induced mac-NOS and TIS10/PGS-2 mRNA accumulation patterns are similar. LPS is a potent inducer of mac-NOS in Swiss 3T3 cells but cannot induce TIS10/PGS-2. In contrast, v-src expression induces TIS10/PGS-2 message, but not iNOS message in a BALB/c 3T3 cell line containing a temperature-sensitive v-src gene. Dexamethasone (DEX) prevents induction of TIS10/PGS-2, but not most other primary response genes. DEX also blocks mac-NOS induction in Swiss 3T3 cells. The inducible TIS10/PGS-2 and mac-NOS genes, responsible for the production of two distinct paracrine agents, appear to share many regulatory features in 3T3 cells.

3T3 Cells↗
PubMed1976

Mapping oligonucleotides of Rous sarcoma virus RNA that segregate with polymerase and group-specific antigen markers in recombinants.

The RNase-T1-resistant oligonucleotides of two Prague Rous sarcoma viruses with temperature-sensitive (ts) DNA polymerases (DNA nucleotidyltransferases), termed ts LA 337 and 335 of one leukosis virus, RAV-6, and 20 of their recombinant progeny have been mapped relative to the 3' poly (A) terminus of the viral RNA. The resulting oligonucleotide maps have been ocrrelated with markers of the four known viral genetic elements encoded in the RNA of 10,000 nucleotides. In accord with previous results recombinant RNAs contained (i) oligonucleotides characteristic of the src gene, coding for sarcoma formation, between the poly(A) end and 2000 nucleotides and (ii) olignucleotides characteristic of the env gene, coding for the envelope glycoprotein, between 2500 and 5000 nucleo tides from the poly(A) end. (iii) A cluster of four oligonucleotides that mapped between 6000 and 8000 nucleotides from the 3' poly(A) end of each RNA was shared by both parental viruses and all recombinants. Since all other map segments of our recombinants failed to segregate with the ts- or wild-type markers of the parental DNA polymerase gene (pol), it was concluded that the ts pol lesion maps in this RNA segment. (iv) The 5' segment of each recombinant RNA contained a cluster of four to five oligonucleotides whose parental origin correlated with an electrophoretic marker of one of the parental virion proteins, p27, a major product of the viral gag gene. The gene order 5'-gag-pol-env-src-poly(A) is consistent with our data.

Antigens, Viral↗
PubMed2000

Increased expression of N-myristoyltransferase in gallbladder carcinomas.

BACKGROUND: Activated Src, which has intrinsic protein tyrosine kinase activity, has been found in human solid tumors such as colorectal and breast carcinomas. The Src gene encodes a cytoplasmic tyrosine kinase p60src, which attaches to the inner surface of the membrane after N-terminal myristoylation and is implicated in transduction of signals to the nucleus. N-myristoyltransferase (NMT) catalyzes the biochemical modification process called N-myristoylation. To investigate whether, through Src, NMT contributes to the pathogenesis of gallbladder carcinoma, the authors investigated expression of NMT and p53 in in situ and invasive carcinomas. METHODS: One hundred cases of documented gallbladder carcinoma were reviewed, and 30 cases were selected randomly to evaluate expression of NMT and p53 by immunohistochemistry in both in situ and in invasive tumor components. RESULTS: Eighteen cases (60%) of gallbladder carcinoma showed moderate to strong cytoplasmic positivity for NMT with increased intensity in the invasive component, and 12 cases (40%) were negative. The in situ component revealed mild to moderate cytoplasmic staining in 20 cases (67%), whereas the normal gallbladder mucosa showed weak to negative cytoplasmic staining. Moderate to strong p53 staining was observed in 17 in situ cases (63%) and 24 invasive cases (80%). The in situ staining patterns of p53 were unrelated to the clinical outcome of the tumor. However, moderate to strong staining of the invasive component as observed in 15 cases (50%) was associated with a mean survival of 8.8 months. Amplification of intron-8 in normal gallbladder mucosa and invasive carcinoma were similar in intensity, suggesting the absence of NMT gene amplification in these tumors. CONCLUSIONS: The increased expression of NMT in these tumors could be due to transcriptional activation. Tumors with increased expression of NMT and p53 were associated with poor clinical outcomes as evidenced by their mean survival times. NMT is likely to play a pathogenic role in gallbladder carcinoma.

Acyltransferases↗
PubMed1988

A selectable temperature-sensitive v-src Moloney retrovirus.

We have cloned the v-src gene of ts339B77 RSV into a new Moloney-based retroviral expression vector (YN). In the resulting construct (ts339YNsrc), the ts339 gene is transcribed from the viral LTR, while a selectable resitance marker, the Tn5 neomycin phosphotransferase (neor) gene, is transcribed from an internal thymidine kinase (Tk) promoter. G418 resistance and focus formation were induced in NIH3T3 cells at comparable efficiencies within the permissive temperature range (33-37 degrees C). At 39 degrees, on the other hand, focus induction was reduced 15-fold with no corresponding decrease in expression of G418 resistance. In cells infected with ts339YNsrc, phosphoproteins were elevated and similar in pattern on SDS PAGE regardless of whether the cells were grown at the permissive or restrictive temperature. The ts339YNsrc virus will be useful for the study of effects of v-src expression, and may also be of help in identifying relevant substrates of the v-src product.

3T3 Cells↗
PubMed1979

Initiation of DNA synthesis by the avian retrovirus reverse transcriptase in vitro: nature and location of the oligodeoxycytidylic acid primer binding site.

We have investigated the use of oligodeoxycytidylic acid [oligo(dC)] as a primer for the initiation of DNA synthesis by the avian retrovirus reverse transcriptase in vitro, employing the viral RNA genome as template. The addition of oligo(dC)(12-18) to viral 35S RNA results in a stimulation of DNA synthesis by the viral RNA-directed DNA polymerase comparable to that observed when oligo(dT) is employed as a primer. Under similar conditions neither oligo(dA)(12-18) nor oligo(dG)(12-18) was active as primer for transcription of the avian retrovirus genome. Several different approaches have been employed to localize the oligo(dC)(12-18) binding site on the viral genome, including isolation of poly(A)-containing fragments, competition hybridization, and RNase H hydrolysis. These analyses indicate that oligo(dC)(12-18) binds to a site approximately 2,000 to 3,000 nucleotides from the 3' terminus of the genome of transforming strains of avian sarcoma viruses and approximately 700 to 1,000 nucleotides from the 3' terminus of nontransforming avian retroviruses. Therefore, the major site of initiation of DNA synthesis by oligo(dC)(12-18) appears to be in the vicinity of the 3' end of the env gene and the 5' end of the src gene, although the presence of minor initiation sites located elsewhere on the viral genome cannot be excluded by these data. Characterization of oligonucleotides after pancreatic RNase hydrolysis and poly(C)-Sepharose chromatography of viral RNA directly demonstrates the presence of oligoguanylic acid residues in the avian sarcoma virus genome. DNA sequences transcribed from the oligo(dC) primer appear to be conserved in all of the avian leukosis-sarcoma viruses tested. The use of oligo(dC) as a tool for the production of specific complementary DNA probes is discussed.

Alpharetrovirus↗
PubMed1983

Highly purified pp60src induces the actin transformation in microinjected cells and phosphorylates selected cytoskeletal proteins in vitro.

The src gene product of Rous sarcoma virus (pp60(src)) was highly purified from a rat tumor cell line and shown to have physiological actin transformation activity in a cellular microinjection assay that measures the dissolution of actin microfilament bundles in vivo. The purified pp60(src) fraction consisted of two major proteins, seen on silver-stained sodium dodecyl sulfate-polyacrylamide gels: a 60,000-dalton (60K) protein, identified as pp60(src) by immunoprecipitation with tumor-bearing rabbit immunoglobulin G (IgG) and peptide mapping, and an unrelated 65K protein. There was no evidence for proteolytic cleavage of pp60(src). A 7,000-fold purification of the tyrosine-specific protein kinase activity of pp60(src) was achieved by this procedure. Purified pp60(src) phosphorylated tumor-bearing rabbit IgG heavy chains, casein, histones H1 and H2B, tubulin, and microtubule-associated proteins when assayed in vitro. When incubated with [gamma-(32)P]ATP in the absence of exogenous phosphoacceptor substrates, purified pp60(src) became labeled with (32)P at the tyrosine residues exclusively. Phosphatase and cyclic AMP-dependent protein kinase activities were undetectable in the purified fraction. Microinjection of highly purified pp60(src) into the cytoplasm of normal Swiss 3T3 mouse fibroblasts caused rapid and reversible dissolution of actin stress fibers, as visualized by indirect immunofluorescence with actin antibodies. The actin-disrupting activity was thermolabile and sensitive to inhibition by coinjection of tumor-bearing rabbit IgG, and purified to about the same extent (8,000-fold) as did the IgG kinase activity of pp60(src), thus implicating pp60(src) as the active agent. Examination of actin-associated proteins as substrates for the pp60(src) kinase in vitro showed that vinculin was phosphorylated directly by pp60(src), although to a small extent. Actin, myosin, and tropomyosin were not phosphorylated. Thus, pp60(src) purified by this procedure retains native functional properties and provides a useful probe for analyzing transformation-dependent changes in actin cytoarchitecture.

Actins↗
PubMed1991

The v-src oncogene blocks the differentiation of a murine myeloid progenitor cell line and induces a tumorigenic phenotype.

We have investigated the ability of the v-src oncogene to block the differentiation of the murine myeloid progenitor cell line 32D cl3. In response to granulocyte-colony stimulating factor (G-CSF), 32D cl3 cells are induced to differentiate into mature granulocytes (Valtieri et al., 1987). In contrast, no differentiation was observed following G-CSF treatment of 32D cl3 cells infected with a murine retrovirus carrying the wild-type v-src oncogene. Furthermore, cells infected with a v-src temperature-sensitive (ts) mutant did not differentiate at the permissive temperature, however, at the nonpermissive temperature G-CSF induced granulocytic differentiation. Differentiation of 32D cl3 cells infected with ts WP31A (ts LA31A src gene inserted into amphotropic murine leukemia virus 4070A; Anderson et al., 1987) occurred with the same kinetics as uninfected 32D cl3 cells. Temperature-shift experiments indicate that after 72 hours of treatment with G-CSF at the nonpermissive temperature, approximately half of the 32D cl3 cells infected with ts WP31A virus become committed to differentiation. Prior to that time, activation of v-src by shifting the cells to the permissive temperature resulted in the presence of only undifferentiated blast cells after six days in culture. In contrast to normal 32D cl3 cells, cells infected with the wild-type v-src were tumorigenic when injected into nu/nu Swiss mice. Lesions appeared in the spleen, liver, kidney, lungs and lymph nodes following subcutaneous injection. Growth factor-independent cells were recovered from the tumor, spleen, bone marrow and a lymph node of tumor-bearing nude mouse. Analysis of the proviral integration site by inverse polymerase chain reaction (PCR) demonstrated that the tumor cells were of donor cell origin.

Animals↗
PubMed1978

Evidence that the transforming gene of avian sarcoma virus encodes a protein kinase associated with a phosphoprotein.

Avian sarcoma virus (ASV) induces sarcomas in animals and transforms fibroblasts to a neoplastic state in cell culture. A single viral gene (src) is responsible for both the induction and maintenance of neoplastic transformation. Recent work has identified a protein with a molecular weight of 60,000 daltons that is apparently encoded in src and may be the effector molecule for the gene (Brugge and Erikson, 1977; Purchio et al, 1978). The putative product of src can be immunoprecipitated by antisera obtained from rabbits bearing tumors induced by ASV. We have used this approach to isolate the protein to characterize further its genetic origins and possible function. Our rabbit tumor antisera precipitated a protein with a molecular weight of 60,000 daltons; according to serological, biochemical and genetic criteria, this protein is encoded in src. We found that this protein is phosphorylated and therefore denoted it pp60. Phosphorylation of pp60 could be accomplished in vitro with extracts of ASV-infected cells. A temperature-sensitive conditional mutation in src had no demonstrable effect on either the production or stability of pp60 in the infected cell, but phosphorylation of the protein was temperature-sensitive. Since the mutant src is not expressed at the restrictive temperature, our findings raise the possibility that phosphorylation of pp60 is required for its function as the putative effector of src. Immunoprecipitates prepared with extracts of ASV-infected cells and the rabbit tumor antisera contained a protein kinase activity that catalyzed phosphorylation of the heavy chains of immunoglobulin molecules, using either ATP or GTP as phosphate donor. The kinase activity immunoprecipitated in parallel with pp60 was obtained only from cells that contained a functioning product of src and could not be precipitated with antisera directed against structural proteins of ASV. A temperature-sensitive conditional mutation in src caused the kinase activity to be thermally inactivated in vitro far more rapidly than the activity from cells infected with wild-type virus. We conclude that both the protein kinase and pp60 are encoded in src, and that the enzymatic activity may be an intrinsic property of pp60. Phosphorylation of pp60 in cellular extracts was inhibited by calcium ion, whereas the immunoprecipitable kinase activity was not, suggesting that the kinase responsible for pp60 phosphorylation may be distinct from that encoded in src. Collett and Erikson (1978) have also identified a protein kinase activity associated with pp60. These findings raise the possibility that phosphorylation of specific cellular targets might account for transformation of the host cell by src.

Adenosine Triphosphate↗
PubMed1994

Origin and evolution of the c-src-transducing avian sarcoma virus PR2257.

Avian sarcoma virus PR2257 transduced de novo the c-src gene and about 900 bp of 3' non-coding sequences belonging to the src locus. This virus contains only one mutation in the c-src coding sequence causing a reading frame shift after Pro-525. The molecular clone studied was derived from a cell line of transformed quail fibroblasts, C7. It contains endogenous virus (ev) derived sequences in the U5 and 3' non-coding regions, indicating that multiple recombination occurred with endogenous virus. Here we investigated the possible evolution of PR2257 when the original tumour was repeatedly passaged in vivo. After 16 passages a new virus, designated PR2257/16, appeared with a tenfold higher titre. The sequence of PR2257/16 was determined and showed that PR2257/16 resulted from recombination of PR2257 with the env gene of the helper virus (td daPR-C). This recombination expanded the env gene content in PR2257/16 and, in addition, five point mutations occurred in its genome. Because we thought that an endogenous virus might be involved in the mechanism of c-src transduction, we also reinvestigated the presence of ev sequences in PR2257 proviruses from several early passages of the original tumour. We found that in contrast with the first isolate from the C7 cell line, the provirus in these tumours did not contain such sequences. These results do not support the hypothesis that endogenous sequences were involved in the transduction process.

Animals↗
PubMed2003

An increase in the expression and total activity of endogenous p60(c-Src) in several factor-independent mutants of a human GM-CSF-dependent leukemia cell line (TF-1).

Growth factor independence of hematopoietic cells can be induced by ectopic expression of a variety of oncogenes encoding receptor or cytoplasmic tyrosine kinases. To examine whether the activation of tyrosine kinases occurs in factor-independent mutants in vivo, the tyrosine-phosphorylated proteins from 14 factor-independent mutants of a GM-CSF-dependent cell line (TF-1) were analysed. These mutants did not secrete any growth-stimulating activity for TF-1 cells, suggesting that activation of intracellular signaling rather than an autocrine stimulation by secreted growth factors is responsible for their factor-independent growth. In 11 out of 14 GM-CSF-independent mutants analysed, a constitutively tyrosine-phosphorylated protein of 60 kDa was detected, which was subsequently identified as p60(c-Src). The kinase activity of p60(c-Src) was increased up to 12-fold in these mutants, which was at least in part due to overexpression of the c-src gene on the RNA and protein level. The Src substrate Sam68 showed an increased phosphorylation in mutants with high Src activity, suggesting that p60(c-Src) triggers downstream signaling in these cells. Treatment of the factor-independent mutants with the Src kinase inhibitor PP2 resulted in a reduced proliferation, demonstrating that Src kinases are essential for these cells for maximal proliferation. Further analysis of factor-independent mutants with low or undetectable Src activity revealed a constitutive phosphorylation of the common beta chain of the GM-CSF receptor and STAT5. Our data indicate an increase in the expression and total activity of endogenous p60(c-Src) in several GM-CSF-independent TF-1 mutants, further underlining the role of Src in the process of autonomous growth of hematopoietic cells.

Base Sequence↗
PubMed2001

[The role of beta-catenin in the repair of the damaged airway epithelium in smoking mice].

OBJECTIVE: To evaluate the role of beta-catenin(beta-cat) in the repair of the damaged airway epithelium by comparing their expressions of beta-cat in different stages of smoking in murine model. METHODS: With HE staining, morphological changes of airway epithelium were observed microscopically in mice model at 5th week (S5 w), 10th week (S10 w) and 15th week (S15 w) of smoking. Immunohistochemistry and hybridization in situ were performed to investigate the expressions of beta-cat mRNA, beta-cat, c-Src gene and phospho-rytyrosine-protein (p-Tyr). RESULTS: Morphological changes of the airway epithelium showed predominant damage(S5 w), repair(S10 w) and excessive proliferation (S15 w) Mean beta-cat OD values at S5 w, S10 w and S15 w were 0.097 +/- 0.007, 0.160 +/- 0.003 and 0.107 +/- 0.008, being significantly lower than the control value(0.204 +/- 0.012, all P < 0.01); The mean beta-cat mRNA OD value of S5 w, S10 w and S15 w were (0.123 +/- 0.011, 0.173 +/- 0.008 and 0.116 +/- 0.004) were also lower than that in control(0.223 +/- 0.008, all P < 0.01), but the OD value of S10 w was higher than those of S5 w and S15 w. OD value of S15 w was the lowest with only traced beta-cat mRNA expression in the nuclei. The expressions of c-Src and p-Tyr in normal airway epithelium(OD values: 0.048 +/- 0.015, 0.075 +/- 0.011) were significantly lower than those in S5 w(0.127 +/- 0.018, 0.112 +/- 0.012), S10 w (0.147 +/- 0.010, 0.127 +/- 0.018) and S15 w(0.147 +/- 0.010, 0.127 +/- 0.018, all P < 0.001). CONCLUSION: beta-cat may play an important role in the process of repair of airway epithelium. Downregulation of beta-cat may be caused by c-Src which phosphorylates tyrosin residues of beta-cat, leading to its degradation; Intracellular level of p-Tyr may, to some extent, reflect the functional condition of beta-cat.

Animals↗
PubMed1994

v-src transformation of rat embryo fibroblasts. Inefficient conversion to anchorage-independent growth involves heterogeneity of primary cultures.

To clarify whether a single oncogene can transform primary cells in culture, we compared the transforming effect of a recombinant retrovirus (ZSV) containing the v-src gene in rat embryo fibroblasts (REFs) to that in the rat cell line 3Y1. In the focus assay, REFs exhibited resistance to transformation as only six foci were observed in the primary cultures as opposed to 98 in 3Y1 cells. After G418 selection, efficiency of transformation was again somewhat lower with REFs compared to that with 3Y1 cells, but the number of G418-resistant REF colonies was much greater than the number of foci in REF cultures. Furthermore, while 98% of G418-resistant colonies of ZSV-infected REFs were morphologically transformed, only 25% were converted to anchorage-independent growth, as opposed to 100% conversion seen in ZSV-infected 3Y1 cells. The poor susceptibility of REFs to anchorage-independent transformation did not involve differences in expression and subcellular distribution of p60v-src, or its kinase activity in vitro and in vivo. It rather reflected a property of the primary cultures, as cloning of REFs before ZSV infection demonstrated that only 2 out of 6 REF clones tested were permissive for anchorage-independent growth. The nonpermissive phenotype was dominant over the permissive one in somatic hybrid cells, and associated with organized actin filament bundles and a lower growth rate, both before and after ZSV infection. These results indicate that the poor susceptibility of REFs to anchorage-independent transformation by p60v-src reflects the heterogeneity of the primary cultures. REFs can be morphologically transformed by p60v-src with high efficiency but only a small fraction is convertible to anchorage-independent growth. REF resistance seems to involve the presence of a suppressor factor which may emerge from REF differentiation during embryonic development.

Actins↗
PubMed1993

Retroviral infection can abrogate the factor-dependency of hematopoietic cells by autocrine and non-autocrine mechanisms depending on the presence of a functional viral oncogene.

The mechanisms responsible for abrogation of the growth factor-dependency of a hematopoietic cell line were investigated. FDC-P1 cells were infected with retroviral constructs containing the neo gene and either a wild-type or a temperature-sensitive v-src oncogene. v-srcwt abrogated the factor-dependency of these cells since each G418r colony gave rise to factor-independent cells and no autocrine growth factor activity was detected. Moreover, the vast majority (< 99%) of cells infected with the v-srcts mutant gave rise to conditional factor-independent cells. Therefore a functional v-src gene product was required for growth factor-independence which occurred by a non-autocrine mechanism. A minority of factor-independent cells which arose after v-srcts infection, grew at the non-permissive temperature and one-half secreted granulocyte/macrophage-colony stimulating factor (GM-CSF) which supports the growth of the parental cells. Since the v-srcts viral stock contained a helper virus, Murine Leukemia Virus (MuLV), the ability of this virus to relieve factor-dependency was examined. A low frequency of factor-independent transformants was recovered after MuLV infection and one-half secreted GM-CSF. Therefore, retroviruses such as MuLV which lack an oncogene, can transform cells by stimulating autocrine growth factor secretion. Subsequent experiments performed with helper-free v-src preparations indicated that they could abrogate factor-dependency directly by a non-autocrine mechanism. These results demonstrate that a hematopoietic cell line can be transformed by two different mechanisms after retroviral infection and may be relevant for understanding hematopoietic cell transformation after persistent viral infection.

Animals↗
PubMed1985

c-src is consistently conserved in the chromosomal deletion (20q) observed in myeloid disorders.

The proto-oncogene c-src has been mapped to two bands in human chromosomes, 1p36 and 20q13, both of which are involved in rearrangements in human tumors. In particular, deletions (loss of part of a chromosome) of the long arm of chromosome 20, del(20q), are commonly observed in hematologic malignant diseases. By using in situ chromosomal hybridization of a c-src probe to metaphase cells prepared from leukemic bone marrow cells of three patients with a del(20q), we observed specific labeling on the deleted chromosome in each patient, indicating that the c-src locus was conserved. The presence on the rearranged chromosomes of c-src, which is normally located on the most distal band of 20q, indicated that the deletions were not terminal as they appeared to be on the basis of chromosome morphology, but rather that they were interstitial. The location of c-src relative to the distal breakpoint in these deletions is unknown. By using the v-src probe in Southern blot analysis of genomic DNA from three patients with a del(20q), we found that no major genomic rearrangements or amplification of the c-src genes had occurred within the regions homologous to v-src. Our observation that c-src is consistently preserved in these rearranged chromosomes suggests that this gene may play a role in the pathogenesis of some myeloid disorders.

Aged↗