Sarcoma Virus-transformed Chicken Embryo Research Articles

Loss of Caveolin-1 Polarity Impedes Endothelial Cell Polarization and Directional Movement

The ability of a cell to move requires the asymmetrical organization of cellular activities. To investigate polarized cellular activity in moving endothelial cells, human endothelial cells were incubated in a Dunn chamber to allow migration toward vascular endothelial growth factor. Immunofluorescent staining with a specific antibody against caveolin-1 revealed that caveolin-1 was concentrated at the rear of moving cells. Similarly, monolayer scraping to induce random cell walk resulted in relocation of caveolin-1 to the cell rear. These results suggest that posterior polarization of caveolin-1 is a common feature both for chemotaxis and chemokinesis. Dual immunofluorescent labeling showed that, during cell spreading, caveolin-1 was compacted in the cell center and excluded from nascent focal contacts along the circular lamellipodium, as revealed by integrin beta1 and FAK staining. When cells were migrating, integrin beta1 and FAK appeared at polarized lamellipodia, whereas caveolin-1 was found at the posterior of moving cells. Notably, wherever caveolin-1 was polarized, there was a conspicuous absence of lamellipod protrusion. Transmission electron microscopy showed that caveolae, similar to their marker caveolin-1, were located at the cell center during cell spreading or at the cell rear during cell migration. In contrast to its unphosphorylated form, tyrosine-phosphorylated caveolin-1, upon fibronectin stimulation, was associated with the focal complex molecule phosphopaxillin along the lamellipodia of moving cells. Thus, unphosphorylated and phosphorylated caveolin-1 were located at opposite poles during cell migration. Importantly, loss of caveolin-1 polarity by targeted down-regulation of the protein prevented cell polarization and directional movement. Our present results suggest a potential role of caveolin polarity in lamellipod extension and cell migration.

Assignment of GRP78 gene to swine chromosome 1q2.12-->q2.13 by fluorescent in situ hybridization.

HereditasVolume 121, Issue 2 p. 203-205 Open Access Assignment of GRP78 Gene to Swine Chromosome lq2.12→q2.13 by Fluorescent in Situ Hybridization Hiroshi Yasue, Corresponding Author Hiroshi Yasue Animal Genome Research Group, National Institute of Animal Industry, Norin Kenkyu–danchi kyoku, P.O. Box 5, Tsukuba, Ibaraki 305, Japan*To whom all correspondence should he addressed.Search for more papers by this authorTakashi Harumi, Takashi Harumi Animal Genome Research Group, National Institute of Animal Industry, Norin Kenkyu–danchi kyoku, P.O. Box 5, Tsukuba, Ibaraki 305, JapanSearch for more papers by this authorTakashi Awata, Takashi Awata Animal Genome Research Group, National Institute of Animal Industry, Norin Kenkyu–danchi kyoku, P.O. Box 5, Tsukuba, Ibaraki 305, Japan Laboratory of Animal Breeding, Tohoku National Agricultural Experiment Station, Morioka 020–01, JapanSearch for more papers by this author Hiroshi Yasue, Corresponding Author Hiroshi Yasue Animal Genome Research Group, National Institute of Animal Industry, Norin Kenkyu–danchi kyoku, P.O. Box 5, Tsukuba, Ibaraki 305, Japan*To whom all correspondence should he addressed.Search for more papers by this authorTakashi Harumi, Takashi Harumi Animal Genome Research Group, National Institute of Animal Industry, Norin Kenkyu–danchi kyoku, P.O. Box 5, Tsukuba, Ibaraki 305, JapanSearch for more papers by this authorTakashi Awata, Takashi Awata Animal Genome Research Group, National Institute of Animal Industry, Norin Kenkyu–danchi kyoku, P.O. Box 5, Tsukuba, Ibaraki 305, Japan Laboratory of Animal Breeding, Tohoku National Agricultural Experiment Station, Morioka 020–01, JapanSearch for more papers by this author First published: November 1994 https://doi.org/10.1111/j.1601-5223.1994.t01-1-00203.xCitations: 1 AboutPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat References Awata, T., Yamakuchi, H., Kijmagai, M. and Yasue, H. 1994. Assignment of the tenascin gene (HXB) to swine chromosome lq21.1→q21.3 by fluorescence in situ hybridization. Cytogenet. Cell Genet. In press Day, A. R. and Lee, A. S. 1989. Transcriptional regulation of the gene encoding the 78–kd glucose–regulated protein GRP78 in mouse Sertoli cells binding of specific factors to the GRP78 promoter. DNA 8: 301– 310 Kozutsumi, Y., Segal, M., Normington, K., Gethinig, M–J. and Sambrook, J. 1988. The presence of malfolded proteins in the endoplasmatic reticulum signals the induction of glucoseregulated proteins. Nature 332: 462– 464 Lee, A. S. 1987. Coordinated regulation of a set of genes by glucose and calcium ionophores in mammalian cells. Trends Biochem. Sci. 12: 20– 23 Lee, A. S., Wells, S., Kim, K. S. and Scheffler, I. E. 1986. Enhanced synthesis of the glucose/calcium regulated proteins in a hamster cell mutant deficient in transfer of oligosaccharide core to polypeptides. J. Cell Physiol. 129: 277– 282 Mellink, C. H. M., Lahbib-Mansais, Y., Yrlb, E. M. and Gellin, J. 1993. Localization of four new markers to pig chromosomes 1, 6 and 14 by radioactive in situ hybridization. Cytogener. Cell Genet. 64: 256– 260 Pelham, H. R. B. 1986. Speculations on the functions of the major heat shock and glucose–regulated proteins. Cell 46: 959– 961 Resendez, E. Jr., Attenello, J. W., Grafsky, A., Chang, S. S. and Lee, A. S. 1985. Calcium ionophore A23187 induces expression of glucose–regulated genes and their heterologous fusion genes. Mol. Cell Biol. 5: 1212– 1219 Rønne, M., Siefanoval, E., Di Berardino, D. and Poulsen, B. S. 1987. The R–banded karyotype of the domestic pig (Sus scrofa domestica L.). Hereditas 106: 219– 231 Shiu, R. P. C., Pouyssegur, J. and Pastan, I. 1977. Glucose depletion accounts for the induction of two transformationsensitive membrane proteins in Rous sarcoma virus-transformed chick embryo fibroblasts. Proc. Natl. Acad. Sci. USA 74: 3840– 3844 Ziegler, A., Hagmann, J., Kiefer, B. and Nagamine, Y. 1990. Ca2+ potentiates CAMP–dependent expression of urokinase–type plasminogen activator gene through a almodulinand protein kinase C–independent mechanism. J. Biol. Chem. 265: 21194– 21201 Citing Literature Volume121, Issue2November 1994Pages 203-205 ReferencesRelatedInformation

Chapter 29 Invadopodia: Unique methods for measurement of extracellular matrix degradation in vitro

Publisher Summary Immunofluorescence, Nomarski illumination, and electron microscopy allow detection of invadopodia on the basis of visualization of membrane or invadopodia associated antigens. Using these methods it was observed that when invasive cells such as Rous sarcoma virus-transformed chicken embryo fibroblasts were cultured on extracellular matrix (ECM), invadopodia extended into the matrix. The chapter develops a quantitative assay to measure invadopodia activity. First, fluorescein isothiocyanate is coupled to an ECM substrate, for example, gelatin. Then a thin coat of ftuorescently conjugated matrix is coated and crosslinked on a glass coverslip. When cells are cultured on this labeled matrix, they extend invadopodia that degrade the matrix and leave dark areas in a bright field of fluorescence. Areas of invadopodia activity can be identified using conventional immunofluorescence microscopy. The area of matrix degradation in each field of view can be quantified after digital image capture using an image analysis program. Thus, there are two aspects to the assay: first, the visual, qualitative determination of the ability of a cell line to make invadopodia and, second, the quantitative measurement of this activity.

Regulatory role of adenosine deaminase complexing protein (dipeptidyl peptidase IV = CD26) on the malignancy marker adenosine deaminase: Effect of membrane cholesterol and phase-transition

While adenosine deaminase (ADA) is an established malignancy marker and its clinical involvement in severe combined immunodeficiency (SCID) is understood, the biological significance of its binding to adenosine deaminase complexing protein (CP = DPPIV = CD26) remains enigmatic. The role of lipid-protein interactions in the modulation of ADA activity by membrane-bound CP was sought. ADA bound specifically to CP reconstituted in L-a-dimyristoylphosphatidylcholine (DMPC) or asolectin vesicles. Without CP, the binding and specific activity of ADA were negligible. In the presence of CP, the specific activity recovered to values similar to those in buffer. The addition of cholesterol increased the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH)-labeled vesicles and resulted in a bell-shaped dependence of the specific activity of ADA on cholesterol concentration. Vesicles with small cholesterol ratios mimicked Rouse sarcoma virus-transformed chick embryo fibroblasts (CEF) in that both had reduced ADA activity and increased membrane fluidity. In contrast to continuous Arrhenius plots of ADA activity in solution, free or bound to CP, ADA bound to CP reconstituted in DMPC vesicles exhibited two breaks, around 25 and 13°C, yielding three lines with similar apparent activation energies (E a ). Increased ADA activity of ∼30% was observed at each of these discontinuities. A model in which active site accessibility is dependent on membrane fluidity led to a successful simulation of the phase transitions. This model could also account for ADA reduced activity associated with increased membrane fluidity in transformed vs. normal fibroblasts.

Cloning, expression, and characterization of chicken tissue inhibitor of metalloproteinase‐2 (TIMP‐2) in normal and transformed chicken embryo fibroblasts

Rous sarcoma virus-transformed chicken embryo fibroblasts (RSVCEF), when compared to normal CEF, produce elevated levels of matrix metalloproteinase-2 (MMP-2) that exists in a form free of complexed tissue inhibitor of metalloproteinase-2 (TIMP-2). In order to ascertain whether the increased levels of TIMP-free MMP-2 in RSVCEF cultures are due to diminished expression of TIMP-2 or alterations in TIMP-2 that diminish its MMP-2 binding ability, it was necessary to clone, characterize, and express chicken TIMP-2 cDNA. The TIMP-2 cDNA was cloned from a chick embryo λgt11 library by RT-PCR using primers based on amino-acid sequences determined from isolated TIMP-2. The deduced amino acid sequence for chicken TIMP-2 is 81% identical to human TIMP-2; most of the sequence differences lie in the carboxyl terminal portion of chicken TIMP-2. Northern analysis of mRNA levels in CEF and RSVCEF demonstrates that TIMP-2 mRNA levels are increased in RSVCEF. However, TIMP-2 protein levels, relative to proMMP-2 levels, appear to decrease upon transformation and suggest additional control of TIMP-2 at the post-transcriptional level. Addition of recombinantly expressed TIMP-2 to RSVCEF cultures causes a disappearance of TIMP-free (TF) proMMP-2 with a corresponding increase in the TIMP-complexed (TC) proMMP-2 levels, demonstrating that TF proMMP-2 is capable of converting to TC pro-MMP-2 when free TIMP-2 is available. Surprisingly, RSVCEF cultures manifest a TIMP-2 population that is not complexed to MMP-2, despite the coexistence of TIMP-free proMMP-2. Gel-filtration analysis indicates that this uncomplexed TIMP-2 exhibits an apparent molecular weight of 50 kDa, indicating it is not free TIMP-2 and that it exists in transformed cultures in a noncovalent complex with an undefined molecule. Thus transformed cells can alter the TIMP-2/MMP-2 balance by transcriptional and post-translational modifications, yielding a population of inhibitor-free, proteolytically active MMP2. J. Cell. Physiol. 174:342–352, 1998. © 1998 Wiley-Liss, Inc.

Cloning, expression, and characterization of chicken tissue inhibitor of metalloproteinase-2 (TIMP-2) in normal and transformed chicken embryo fibroblasts

Rous sarcoma virus-transformed chicken embryo fibroblasts (RSVCEF), when compared to normal CEF, produce elevated levels of matrix metalloproteinase-2 (MMP-2) that exists in a form free of complexed tissue inhibitor of metalloproteinase-2 (TIMP-2). In order to ascertain whether the increased levels of TIMP-free MMP-2 in RSVCEF cultures are due to diminished expression of TIMP-2 or alterations in TIMP-2 that diminish its MMP-2 binding ability, it was necessary to clone, characterize, and express chicken TIMP-2 cDNA. The TIMP-2 cDNA was cloned from a chick embryo lambda gt11 library by RT-PCR using primers based on amino-acid sequences determined from isolated TIMP-2. The deduced amino acid sequence for chicken TIMP-2 is 81% identical to human TIMP-2; most of the sequence differences lie in the carboxyl terminal portion of chicken TIMP-2. Northern analysis of mRNA levels in CEF and RSVCEF demonstrates that TIMP-2 mRNA levels are increased in RSVCEF. However, TIMP-2 protein levels, relative to proMMP-2 levels, appear to decrease upon transformation and suggest additional control of TIMP-2 at the post-transcriptional level. Addition of recombinantly expressed TIMP-2 to RSVCEF cultures causes a disappearance of TIMP-free (TF) proMMP-2 with a corresponding increase in the TIMP-complexed (TC) proMMP-2 levels, demonstrating that TF proMMP-2 is capable of converting to TC pro-MMP-2 when free TIMP-2 is available. Surprisingly, RSVCEF cultures manifest a TIMP-2 population that is not complexed to MMP-2, despite the coexistence of TIMP-free proMMP-2. Gel-filtration analysis indicates that this uncomplexed TIMP-2 exhibits an apparent molecular weight of 50 kDa, indicating it is not free TIMP-2 and that it exists in transformed cultures in a noncovalent complex with an undefined molecule. Thus transformed cells can alter the TIMP-2/MMP-2 balance by transcriptional and post-translational modifications, yielding a population of inhibitor-free, proteolytically active MMP2.

NSAID-Induced Apoptosis in Rous Sarcoma Virus-Transformed Chicken Embryo Fibroblasts is Dependent on v-src and c-myc and is Inhibited by bcl-2

Mounting epidemiological and experimental evidence implicates nonsteroidal antiinflammatory drugs as anti-tumorigenic agents. Our previous work showed that nonsteroidal antiinflammatory drug treatment of src-transformed chicken embryo fibroblasts caused apoptosis -- a mechanism by which these drugs might exert their anti-tumorigenic effect. The present studies employ a sensitive technique for detecting single- and double-stranded DNA cleavage (the comet assay) to quantitate apoptosis. By this method pp60 v-src, which antagonizes apoptosis in many cell systems, was found to induce apoptosis in 11–23% of serum-starved fibroblasts. However, treatment with diclofenac following pp60 v-src activation produced a much stronger response beginning within 6 hours of treatment that resulted in 100% lethality. During cell death, cyclooxygenase-2 but not cyclooxygenase-1 mRNA was found to be uniformly increased by all apoptotic drugs tested. Examination of the expression of apoptosis-associated genes showed that c-rel and p53 (found in normal or v-src-transformed chicken embryo fibroblasts at moderate levels), and bcl-2 (present at an extremely low level) were largely unchanged by treatment with eight different nonsteroidal antiinflammatory drugs. However, over-expression of human bcl-2 inhibited diclofenac-mediated apoptosis by 90%, demonstrating directly that bcl-2 expression can regulate nonsteroidal antiinflammatory drug induction of cell death. The proto-oncogene c-myc is known to cause apoptosis in chicken embryo fibroblasts when artificially overexpressed in cells deprived of trophic factors. We found that nonsteroidal antiinflammatory drug treatment following pp60 v-src activation persistently induced myc protein and mRNA by more than 20-fold above that evoked by pp60 v-src activation alone. Moreover, transfection of antisense c-myc oligonucleotides reduced drug-induced myc expression by 80% and caused a concomitant 50% reduction in cell death. These findings suggest that nonsteroidal antiinflammatory drug-induced apoptosis proceeds through a src/myc dependent pathway which is negatively regulated by bcl-2.

What structure and function of avian plasminogen activator and matrix metalloproteinase-2 reveal about their counterpart mammalian enzymes, their regulation and their role in tumor invasion.

Rous sarcoma virus-transformed chick embryo fibroblasts (RSVCEF) constitute a well-characterized model system for oncogenic transformation, matrix degradation, and cancer invasion. As RSVCEF cultures employ both serine protease and metalloprotease cascades in the process of matrix degradation, they have contributed significantly to understanding the nature and regulation of these molecules involved in invasive cell behavior. RSVCEF produce elevated levels of a matrix metalloprotease-2 (MMP-2) whose hemopexin domain differs from mammalian MMP-2. The majority of MMP-2 produced by RSVCEF is present in a TIMP-free form which enhances its activation, catalytic activity and substrate specificity and therefore its matrix-degrading ability. RSVCEFs also exhibit high levels of urokinase-type plasminogen activator (uPA), which is found in active form in their conditioned medium in complete absence of plasminogen. Recombinantly expressed avian uPA is also in active form, while an active-site mutant of the same maintains its zymogen form, indicating the mechanism of activation of chicken uPA is autocatalytic. A domain and sequence comparison between chicken and human uPA attempts to identify motifs potentially responsible for the zymogen instability of avian uPA and its capability to autoactivate.

Crosslinked gelatin beads: A culture method for the study of cell adhesion and invasion

A method is described for culturing invasive cell lines on crosslinked gelatin beads and preparing them for immunocytochemical and morphological observation. Very invasive cells such as Rous sarcoma virus-transformed chicken embryo fibroblasts, and human melanoma LOX and RPMI7951 and breast carcinoma MDA-MB-231 cells will actively degrade this matrix, extending cellular protrusions, called invadopodia, into the sites of degradation. Normal chicken embryo fibroblasts and other non-invasive cell lines do not disrupt the surface of these beads and do not form invadopodia. Invadopodia extending into the bead can be visualized by electron microscopy. Cellular removal of fluorescent fibronectin that has been covalently coupled to the bead surface can be monitored using fluorescence microscopy of frozen-thin-sections. In double label experiments, immunocytochemistry is used to localize antigens in invadopodia at sites of membrane invasion. The materials for bead preparation are inexpensive, and this method has the advantage that many cell types will attach and spread readily on the beads, while only highly invasive cells will invade into the bead.

Localization of a 215-kDa tyrosine-phosphorylated protein that cross-reacts with tensin antibodies

Tyrosine phosphorylation of cytoskeletal proteins at adhesive junctions has been speculated to play a role in the regulation of cell signaling at these sites. Previously, monoclonal antibodies were generated against phosphotyrosine-containing proteins from Rous sarcoma virus-transformed chick embryo fibroblasts, resulting in two antibodies which recognized antigens of 76 and 215 kDa that localized to focal contacts. We have now localized the 215-kDa antigen to a number of adhesive junctions in vivo, including the zonula adherens, intercalated discs, and myotendinous and neuromuscular junctions. In sections of skeletal muscle and in isolated myofibrils, the 215-kDa protein was localized to the I-band. By immunoprecipitation and immunoblot analysis, we determined that the 215-kDa antigen cross-reacts with a polyclonal anti-tensin antibody.

Protein and lipid lateral diffusion in normal and Rous sarcoma virus transformed chick embryo fibroblasts

We measured the lateral diffusion of the fluorescent lipid analogue dioctadecylindocarbocynanine iodide (DiI) and of membrane glycoproteins labeled with tetramethylrhodamine (TRITC) succinyl concanavalin A (SConA) via fluorescence photobleaching recovery (FPR) at selected times during a temperature downshift experiment on transformation-defective temperature sensitive (td-ts) Rous sarcoma virus (RSV) NY68-transformed chicken embryo fibroblasts (CEF) and on identically treated CEF and RSV-transformed CEF. There were no significant differences in the lateral diffusion of DiI at any of the times measured. The lateral diffusion of TRITC-SConA on the RSV-transformed CEF, (1.32 ± 0.12) · 10 −10 cm 2 s −1, was approximately two times faster than that observed in normal CEF, (0.61 ± 0.06) · 10 −10 cm 2 s −1. In the cells undergoing RSV NY68-mediated transformation, TRITC-SConA diffusion increased over a 24-h period from a value comparable to that observed in normal CEF, (0.72 ± 0.13) · 10 −10 cm 2 s −1 to a value comparable to the RSV-CEF transformed cells, (1.74 ± 0.20) · 10 −10 cm 2 s −1. All diffusion measurements reported were made at the permissive temperature for RSV-NY68 (35°C) unless stated otherwise. The changes in the lateral diffusion of TRICT-SConA occurred between the fifth and twelfth hour of the downshift course and could be associated with cytoskeletal disruption and/or fibronectin degradation, both known to occur at this time in RSV-transformed cells. To assess the contribution of extracellular matrix (ECM) degradation, SConA mobility was measured in normal and RSV-transformed cells treated with trypsin. This treatment increased SConA mobility approximately 4-fold in the normal cells relative to untreated controls and only 2-fold in the RSV-CEF transformed cells. No significant difference in SConA mobility between trypsinized spherical normal and transformed cells was apparent.

Transformation-dependent activation of urokinase-type plasminogen activator by a plasmin-independent mechanism: involvement of cell surface membranes.

Transformed cells produce elevated levels of the urokinase-type plasminogen activator (u-PA), which has been linked with the invasive or migratory phenotype of these cells. The u-PA is secreted and normally maintained in the inactive, single-chain form (scu-PA) and it has been assumed that natural activation occurs via a plasmin-mediated cleavage converting scu-PA to the active, two-chain form (tcu-PA). We now demonstrate that secreted scu-PA in Rous sarcoma virus-transformed chicken embryo fibroblast (RSVCEF) cultures is activated by an endogenous, plasmin-independent mechanism. Normal CEFs and CEFs infected with a temperature-sensitive RSV mutant and incubated at the nonpermissive temperature do not activate scu-PA. Conditioned medium harvested from plasmin-free cultures of RSVCEFs contains active tcu-PA as determined by two independent methods. The scu-PA is progressively converted with time in culture and requires the presence of intact cells or a plasma membrane-enriched fraction. When added to RSVCEF cultures, a synthetic peptide corresponding to residues 20-41 of the growth factor domain of chicken u-PA blocks the conversion to tcu-PA, and scu-PA accumulates in the cultures. These results suggest that scu-PA is secreted by cells, becomes bound to a u-PA receptor, and is proteolytically converted to active tcu-PA by a catalytic mechanism on the surface of RSV-transformed fibroblasts.

Isolation and characterization of a 70-kDa metalloprotease (gelatinase) that is elevated in Rous sarcoma virus-transformed chicken embryo fibroblasts.

Chicken embryo fibroblasts (CEF) transformed by Rous sarcoma virus (RSVCEF) secrete a 70-kDa metallo-gelatinase at elevated levels over that of normal CEF. The 70-kDa enzyme has been purified from RSVCEF conditioned medium and represents 1-3% of the total protein in the RSVCEF conditioned medium. A 22-kDa protein, which appears to be the avian form of the tissue inhibitor of metalloproteases (TIMP), is co-isolated in association with the 70-kDa enzyme and can be separated from the enzyme by gel filtration carried out under denaturing conditions. The isolated 70-kDa species is in the zymogen form. It can be activated by treatment with the organomercurial, p-aminophenylmercuric acetate (APMA), yielding a 62-kDa active species derived by an apparent autoproteolytic cleavage from the 70-kDa proenzyme as determined by both substrate gel analysis and immunoblots using a monospecific antibody to the 70-kDa proenzyme. The proenzyme is poorly activated by trypsin and not activated by plasmin. The APMA-activated enzyme rapidly degrades denatured collagens but under identical conditions is unable to degrade native collagens, including basement membrane type IV collagen. Only at very high enzyme to substrate ratios (1:2) will native type IV collagen be hydrolyzed. Partial N-terminal amino acid sequencing of both the 70-kDa proenzyme and the 62-kDa active enzyme indicates that the avian enzyme is a member of the matrix metalloprotease family (MMP-2). When CEF cultures, infected with a temperature sensitive mutant of RSV, conditional for the expression of the transforming src oncogene, were incubated at the permissive and nonpermissive temperatures, differential levels of the 70-kDa enzyme were produced in direct proportion to the functioning of the src oncogene.

The v-src inducible gene 9E3/pCEF4 is regulated by both its promoter upstream sequence and its 3' untranslated region.

The 9E3/pCEF4 mRNA is strongly induced in Rous sarcoma virus-transformed chicken embryo fibroblasts when compared to untransformed cells. To identify cis-acting transcriptional elements that confer inducibility by v-src, we isolated the 9E3 promoter upstream region. We found that 1.53 kilobases upstream of the transcriptional start site, when placed in front of a reporter gene, conferred a small degree of inducibility by v-src, in both transient and stable transfections. Two potential AP-1 sites were identified in the 9E3 promoter. AP-1 elements have been implicated previously in mediating a transcriptional response to v-src in fibroblast cell lines. These elements alone do not confer a significant inducibility by v-src in primary chicken embryo fibroblasts. Since the 9E3 mRNA is stabilized in transformed cells, we replaced the 3' untranslated region of the reporter gene with the 9E3 3' untranslated region and found this construct to be strongly responsive to stimulation by v-src. In addition, the 9E3 3' untranslated region increased the response to serum and the tumor promoter phorbol 12-myristate 13-acetate. This suggests that a posttranscriptional mechanism plays a major role in the induction of 9E3 expression.

Serine protease and metallo protease cascade systems involved in pericellular proteolysis

Cultures of transformed fibroblasts actively involved in extracellular matrix degradation have been examined for initial activation of serine and metallo protease cascade systems. Rous sarcoma virus transformed chick embryo fibroblasts (RSVCEF), in contrast to transformed mammalian cells, produce active, two chain urokinase-type plasminogen activator (tcu-PA). Active tcu-PA is found in serum-free, plasmin-free conditioned medium from RSVCEF cultures as determined by two independent methods, immunoprecipitation and differential DFP sensitivity. RSVCEF cultures synthesize and secrete inactive, single chain uPA (scu-PA) which is converted to tcu-PA in a time dependent manner by a catalytic mechanism that appears to involve a functioning uPA receptor on the surface of intact cells. The enzyme activity responsible for this conversion may represent the initiating catalytic event in the PA/plasminogen serine protease cascade system. A 70 kDa prometalloprotease capable of degrading denatured collagen following its activation also is significantly elevated in RSVCEF cultures over that of normal CEF. Trace amounts of the active 62 kDa form of the metalloprotease (gelatinase) is found in the transformed RSVCEF cultures indicating that these cultures produce a natural activator of the prometalloprotease. Plasmin and/or PA do not appear to be the activator of this enzyme as determined by indirect inhibition assays and direct assays employing purified enzymes. The possible central position of pro PA and the 70 kDa prometalloprotease in an interacting, complex protease cascade system involved in extracellular matrix degradation is discussed.

The podosomes of Rous sarcoma virus transformed chondrocytes show a peculiar ultrastructural organization

The ultrastructure of F-actin-containing punctate adhesion structures (podosomes) and of their rosette-like clusters has been studied by transmission electron microscopy in Rous sarcoma virus transformed chick embryo chondrocytes. Peculiar “glove finger” invaginations were found to take origin from the ventral membrane at sites of close contact; they were directed toward the center of the cell perpendicularly from the substratum. These new structures may be the sites where the local release of proteases takes place at the side of cell-to-substratum adhesion in podosome-bearing cells. The cytoplasmic face of glove finger invaginations and of the plasma membrane at cell-to-cell contact is lined by thick accumulations of microfilamentous material.

Modulation of arachidonic acid metabolism by Rous sarcoma virus

Arachidonic acid (C20:4) metabolites were released constitutively from wild-type Rous sarcoma virus-transformed chicken embryo fibroblasts (CEF). 3H-labeled C20:4 and its metabolites were released from unstimulated and uninfected CEF only in response to stimuli such as serum, phorbol ester, or the calcium ionophore A23187. High-pressure liquid chromatography analysis showed that the radioactivity released from [3H]arachidonate-labeled transformed cells was contained in free arachidonate and in the cyclooxygenase products prostaglandin E2 and prostaglandin F2 alpha; no lipoxygenase products were identified. The release of C20:4 and its metabolites from CEF infected with pp60src deletion mutants was correlated with serum-independent DNA synthesis and with the expression of the mRNA for 9E3, a gene expressed in Rous sarcoma virus-transformed cells which has homology with several mitogenic and inflammatory peptides. 3H-labeled C20:4 release was not correlated with p36 phosphorylation, which argues against a role for this protein as a phospholipase A2 inhibitor. CEF infected with other oncogenic viruses encoding a tyrosine kinase also released C20:4, as did CEF infected with viruses that contained mos and ras; however, infection with a crk-containing virus did not result in stimulation of 3H-labeled C20:4 release, suggesting that utilization of this signaling pathway is specific for particular transformation stimuli.

Novel tyrosine kinase substrates from Rous sarcoma virus-transformed cells are present in the membrane skeleton.

We have previously reported the production of monoclonal antibodies directed against phosphotyrosine, which is the modification induced by many oncogene products and growth factor receptors. One of these antiphosphotyrosine antibodies (py20) was used in affinity chromatography to isolate phosphotyrosine (PY)-containing proteins from Rous sarcoma virus-transformed chick embryo fibroblasts (RSV-CEFs). Mice were immunized with these PY-proteins for the production of monoclonal antibodies to individual substrates. Fifteen antibodies were generated in this way to antigens with molecular masses of 215, 76, 60, and 22 kD. Antibodies to individual substrates were used to analyze the subcellular location in normal and RSV-CEFs. Antibodies to the 215- and 76-kD antigen stained focal contacts when used in immunofluorescence microscopy while anti-22-kD protein antibodies resulted in punctate staining concentrated in the margins of cells and in parallel arrays. Both distributions were altered in transformed cells. When cells were extracted with nonionic detergent under conditions that stabilize the cytoskeleton, 50% of the 76-kD protein and greater than 90% of the 22-kD protein fractionated with the cytoskeleton. This study offers a new approach to both the identification of membrane skeletal proteins in fibroblasts and changes that occur upon transformation by an activated tyrosine kinase.

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.

The binding of a monoclonal antibody reactive with pp60 v-src to the rat CNS both in vitro and in vivo: evidence that the epitope is present intracellularly as well as being associated with a number of antigenically related polypeptides located externally in the plasma membrane only in the synapic region

A monoclonal antibody, F4, has been produced which reacts with an epitope possessing an unusual subcellular distribution. It binds to the external surface of the neuronal plasma membrane only in the region of the synapse. This is evidenced by binding of F4 to presynaptic terminals in unfixed cultures of rat cerebellum and to preparations of unfixed synaptosomes. In addition, much larger amounts of the epitope are present intracellularly. In fixed nervous tissue, the epitope is found in many neurons, and is associated mainly with presynaptic plasma membranes, synaptic vesicles, postsynaptic densities (cerebral cortex and hippocampus, but not cerebellum), rough endoplasmic reticulum, and the Golgi apparatus. The epitope is especially abundant in large neurons (e.g. pyramidal cells). Similar amounts of epitope are present in the chromaffin cells of the adrenal medulla. It is also expressed in ependymal cells in the brain, and in epithelial cells present in ducts of the medulla, but not cortex, of the kidney. However, the epitope is not found in glial cells in the brain, or in either liver, spleen, skeletal muscle, or testes. F4 is not species specific, as it binds to postmortem adult human cerebral cortex and neonatal cerebellum in a manner as described for the rat. It also binds to homogenates of brains of fish, chicken and mouse. The appearance of the epitope during development of the cerebellum in vivo and in vitro occurs in parallel with the differentiation of neurons and formation of synapses, though small amounts are also present in neuronal precursor cells. The F4 antibody can detect nanogram amounts of pp60 v-src on immunodots. The strength of this reaction is high enough that F4 can be used to demonstrate pp60 v-src-like immunoreactivity in Rous Sarcoma virus-transformed chick embryo fibroblasts. However, present evidence suggests that it may be premature to assign the immunocytochemical reactivity of F4 in the brain exclusively to pp60 c-src. This conclusion is based on the fact that F4 reacts with several polypeptides from synaptic plasma membranes on Western blots of renaturing, two-dimensional gels that are dissimilar in size to pp60 c-src, and from the fact that it can cross-react, albeit weakly, with several other serine protein kinases in an immunodot assay. Appreciation of this cross-reactivity, and of the evolutionary conservation of the epitope, as well as its sensitivity to denaturation, has led to our working hypothesis that F4 binds to a conformational epitope present on several polypeptides that may be most perfectly represented by some aspect of the catalytic domain of tyrosine protein kinases.