This article summarizes the essential steps in understanding the chicken Rous sarcoma virus (RSV) genome association with a nonpermissive rodent host cell genome. This insight was made possible by in-depth study of RSV-transformed rat XC cells, which were called virogenic because they indefinitely carry virus genetic information in the absence of any infectious virus production. However, the virus was rescued by association of XC cells with chicken fibroblasts, allowing cell fusion between both partners. This and additional studies led to the interpretation that the RSV genome gets integrated into the host cell genome as a provirus. Study of additional rodent virogenic cell lines provided evidence that the transcript of oncogene v-src can be transmitted to other retroviruses and produce cell transformation by itself. As discussed in the text, two main questions related to nonpermissiveness to retrovirus infection remain to be solved. The first is changes in the retrovirus envelope gene allowing virus entry into a nonpermissive cell. The second is the nature of the permissive cell functions required by the nonpermissive cell to ensure infectious virus production. Both lines of investigation are being pursued.

• Klíčová slova
• cell transformation, nonpermissiveness to virus infection, virus integration, virus rescue,
• MeSH
• buněčné linie MeSH
• fúze buněk * MeSH
• genom virový genetika   MeSH
• genové produkty env genetika   MeSH
• krysa rodu Rattus MeSH
• kur domácí virologie   MeSH
• onkogenní protein pp60(v-src) genetika   MeSH
• proviry genetika   růst a vývoj   MeSH
• virová transformace buněk MeSH
• virus Rousova sarkomu genetika   růst a vývoj   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• genové produkty env MeSH
• onkogenní protein pp60(v-src) MeSH

Metastatic spreading of cancer cells is a highly complex process directed primarily by the interplay between tumor microenvironment, cell surface receptors, and actin cytoskeleton dynamics. To advance our understanding of metastatic cancer dissemination, we have developed a model system that is based on two v-src transformed chicken sarcoma cell lines-the highly metastatic parental PR9692 and a non-metastasizing but fully tumorigenic clonal derivative PR9692-E9. Oligonucleotide microarray analysis of both cell lines revealed that the gene encoding the transcription factor EGR1 was downregulated in the non-metastatic PR9692-E9 cells. Further investigation demonstrated that the introduction of exogenous EGR1 into PR9692-E9 cells restored their metastatic potential to a level indistinguishable from parental PR9692 cells. Microarray analysis of EGR1 reconstituted cells revealed the activation of genes that are crucial for actin cytoskeleton contractility (MYL9), filopodia formation (MYO10), the production of specific extracellular matrix components (HAS2, COL6A1-3) and other essential pro-metastatic abilities.

• MeSH
• buněčná adheze MeSH
• buněčné linie MeSH
• cytoskelet metabolismus   MeSH
• fenotyp MeSH
• kinetika MeSH
• kur domácí MeSH
• metastázy nádorů genetika   MeSH
• nádorová transformace buněk genetika   patologie   MeSH
• onkogenní protein pp60(v-src) genetika   metabolismus   MeSH
• pohyb buněk MeSH
• proliferace buněk MeSH
• protein 1 časné růstové odpovědi genetika   metabolismus   MeSH
• regulace genové exprese u nádorů MeSH
• sarkom genetika   patologie   MeSH
• stanovení celkové genové exprese MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• onkogenní protein pp60(v-src) MeSH
• protein 1 časné růstové odpovědi MeSH

Active, wild-type v-Src and its kinase-dead double Y416F-K295N mutant were expressed in hamster fibroblasts. Expression of the active v-Src induced activation of endogenous c-Src and increased general protein-tyrosine phosphorylation in the infected cells. Expression of the kinase-dead mutant induced hypophosphorylation of Tyr416 of the endogenous c-Src. The inactivation of c-Src was reversible, as confirmed by in vitro kinase activity of c-Src immunoprecipitated from the kinase-dead v-Src-expressing cells. Both activation and inactivation of c-Src may be explained by direct interaction of the v-Src and c-Src that may either facilitate transphosphorylation of the regulatory Tyr416 in the activation loop, or prevent it by formation of transient dead-end complexes of the Y416F-K295N mutant with c-Src. The interaction was also indicated by co-localization of v- and c-Src proteins in immunofluorescent images of the infected cells. These results suggest that dimerization of Src plays an important role in the regulation of Src tyrosine kinase activity.

• MeSH
• aktivace enzymů MeSH
• buněčné linie MeSH
• C-terminální Src kinasa MeSH
• fibroblasty metabolismus   MeSH
• lidé MeSH
• mutace MeSH
• mutageneze cílená MeSH
• onkogenní protein pp60(v-src) genetika   metabolismus   MeSH
• regulace genové exprese enzymů fyziologie   MeSH
• skupina kinas odvozených od src-genu MeSH
• tyrosinkinasy metabolismus   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• C-terminální Src kinasa MeSH
• CSK protein, human MeSH Prohlížeč
• onkogenní protein pp60(v-src) MeSH
• skupina kinas odvozených od src-genu MeSH
• tyrosinkinasy MeSH

In the cells transformed by Rous sarcoma virus (RSV), two Src proteins are expressed: the ubiquitous tyrosine kinase c-Src and the v-Src, the product of the transforming gene of the virus. Using three synthetic peptide substrates widely used for testing Src kinase activity, we show that they are phosphorylated with different efficiencies by the v-Src and c-Src tyrosine kinases immunoprecipitated from the tumor cell line H19. The v-Src displays higher efficiency (Vmax/Km ratio) toward all three peptides used, but the Vmax of v-Src is much lower than Vmax of c-Src with two peptides out of three. This difference in substrate specificity, if ignored, may cause misestimation of the amounts of active c-Src and v-Src in RSV-transformed cells. On the other hand, the different peptide substrate specificities may also reflect different protein substrate specificities of the v-Src and c-Src kinases in vivo.

• MeSH
• C-terminální Src kinasa MeSH
• kinetika MeSH
• křečci praví MeSH
• nádorové buňky kultivované MeSH
• onkogenní protein pp60(v-src) metabolismus   MeSH
• peptidy metabolismus   MeSH
• precipitinové testy MeSH
• ptačí sarkom enzymologie   MeSH
• skupina kinas odvozených od src-genu MeSH
• substrátová specifita MeSH
• tyrosinkinasy metabolismus   MeSH
• virová transformace buněk MeSH
• viry ptačího sarkomu * enzymologie   MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• C-terminální Src kinasa MeSH
• onkogenní protein pp60(v-src) MeSH
• peptidy MeSH
• skupina kinas odvozených od src-genu MeSH
• tyrosinkinasy MeSH

Our data show that in hamster fibroblasts transformed by Rous sarcoma virus (RSV), the phosphoinositide 3'-kinase (PI-3K)/Akt/glycogen synthase kinase 3 antiapoptotic pathway is upregulated and involved in increased protein synthesis through activation of initiation factor eIF2B. Upon inhibition of PI-3K by wortmannin, phosphorylation of 70-kDa ribosomal protein S6 kinase (p70 S6k) and its physiological substrate, ribosomal protein S6, decreased in the non-transformed cells but not in RSV-transformed cells. Thus PI-3K, which is thought to be involved in regulation of p70 S6k, signals to p70 S6k in normal fibroblasts, but it does not appear to be an upstream effector of p70 S6k in fibroblasts transformed by v-src oncogene, suggesting that changes in the PI-3K signalling pathway upstream of p70 S6k are induced by RSV transformation.

• MeSH
• aktivace enzymů MeSH
• androstadieny farmakologie   MeSH
• buněčné linie MeSH
• eukaryotický iniciační faktor 2B fyziologie   MeSH
• fosfatidylinositol-3-kinasy metabolismus   MeSH
• inhibitory enzymů farmakologie   MeSH
• inhibitory fosfoinositid-3-kinasy MeSH
• kinasa 3 glykogensynthasy metabolismus   MeSH
• kinasy ribozomálního proteinu S6, 70-kDa fyziologie   MeSH
• křečci praví MeSH
• onkogenní protein pp60(v-src) genetika   MeSH
• protein-serin-threoninkinasy * MeSH
• protoonkogenní proteiny c-akt MeSH
• protoonkogenní proteiny metabolismus   MeSH
• signální transdukce * MeSH
• transformované buněčné linie MeSH
• virová transformace buněk MeSH
• viry ptačího sarkomu genetika   patogenita   MeSH
• wortmannin MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• androstadieny MeSH
• eukaryotický iniciační faktor 2B MeSH
• inhibitory enzymů MeSH
• inhibitory fosfoinositid-3-kinasy MeSH
• kinasa 3 glykogensynthasy MeSH
• kinasy ribozomálního proteinu S6, 70-kDa MeSH
• onkogenní protein pp60(v-src) MeSH
• protein-serin-threoninkinasy * MeSH
• protoonkogenní proteiny c-akt MeSH
• protoonkogenní proteiny MeSH
• wortmannin MeSH

We achieved production of v-Src of the low-oncogenic PRC and its variant proviral structure H19 in Dictyostelium discoideum, an emerging host system suitable for synthesis of heterologous proteins. To accomplish their expression, the first six codons of the N-terminus of v-src had to be changed according to the D. discoideum codon preference. Alternatively, N-terminal fusions of 6xHis-tag or GFP were sufficient to overcome the incompatibility in codon usage. D. discoideum-expressed v-Src kinases of the expected molecular weight were recognized by Src-specific antibodies; GFP-PRC was distributed uniformly in the cytosol. In contrast to other lower eukaryotes, where the accumulation of v-Src leads to growth inhibition, D. discoideum cells silenced the kinase activity of PRC-derived v-Src and showed no developmental or growth defects.

• MeSH
• chromatografie afinitní MeSH
• Dictyostelium genetika   MeSH
• exprese genu * MeSH
• fluorescenční mikroskopie MeSH
• genetické vektory MeSH
• geny src genetika   MeSH
• onkogenní protein pp60(v-src) biosyntéza   genetika   izolace a purifikace   MeSH
• viry ptačího sarkomu genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• onkogenní protein pp60(v-src) MeSH

Existing variants of the oncogene v-src differ in their transforming potential as well as in the range of their hosts. We compared the protein kinase activities of two Prague C v-Src variants (PRC and H19), reported to be of low oncogenic potential (Plachý et al., 1995), with the highly oncogenic Schmidt-Ruppin A v-Src (SRA). We employed in vitro kinase assays of affinity-purified proteins expressed in rabbit reticulocyte lysate and in S. cerevisiae. In both systems used, the specific kinase activity of the Prague C v-Src kinases amounted to only ca 20% of the activity of SRA. This positions the PRC Src close to activated c-Src, despite the lack of the regulatory C-terminal tail in PRC. We constructed chimeras between PRC and SRA v-Src and tested them for specific kinase activity in S. cerevisiae. Remarkably, the regulatory N-terminal part of PRC, when fused to the SRA-derived kinase domain, lowered the chimeras' PK activity to ca 20%, suggesting that it is the regulatory part of PRC that is responsible for its low phosphotransferase activity.

• MeSH
• druhová specificita MeSH
• fosforylace MeSH
• geny src MeSH
• katalytická doména MeSH
• onkogenní protein pp60(v-src) antagonisté a inhibitory   chemie   farmakologie   MeSH
• posttranslační úpravy proteinů MeSH
• rekombinantní fúzní proteiny genetika   farmakologie   MeSH
• Saccharomyces cerevisiae MeSH
• terciární struktura proteinů MeSH
• viry ptačího sarkomu klasifikace   enzymologie   genetika   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• onkogenní protein pp60(v-src) MeSH
• rekombinantní fúzní proteiny MeSH

The contact between the SH2 domain and the C-terminal tail of c-Src inhibits its kinase activity via a complex network of interactions, including the SH3 domain. We examined the role of the SH3 domain in v-Src, where the C-terminal tail is mutated and unbound. We used the v-Src variants Prague C (PRC) and Schmidt-Ruppin A (SRA), which are of low and high kinase activities, respectively, to measure phosphorylation in vitro by immunoprecipitated kinases produced in Saccharomyces cerevisiae. Swapping the regulatory domains between SRA and PRC revealed that N117D, I96T, and V124L mutations in the n-src- and RT-loops of the SH3 domain of PRC are responsible for the low kinase activity of PRC. Moreover, introducing D117N, R95W, T96I, and L124V into activated c-Src(Y527F) caused a 2.5-fold increase in its activity. The mutations in the CD linker KP249,250DG and L255A, which were shown to activate c-Src, had no effect on the activity of the "SH2-activated" Src kinases. Together our data suggest that in the "SH2-activated" forms of Src, the SH3 domain continues to influence the kinase activity via the direct contacts of the n-src- and RT-loops with the kinase N-terminal lobe.

• MeSH
• down regulace MeSH
• fosfotyrosin metabolismus   MeSH
• kur domácí MeSH
• molekulární modely MeSH
• mutace MeSH
• onkogenní protein pp60(v-src) chemie   genetika   metabolismus   MeSH
• protoonkogenní proteiny pp60(c-src) chemie   metabolismus   MeSH
• rekombinantní fúzní proteiny chemie   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   MeSH
• src homologní domény MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fosfotyrosin MeSH
• onkogenní protein pp60(v-src) MeSH
• protoonkogenní proteiny pp60(c-src) MeSH
• rekombinantní fúzní proteiny MeSH

When c-Src and v-Src were immunoprecipitated together from hamster fibroblasts transformed by Rous sarcoma virus containing v-src oncogene, the total Src activity was almost threefold higher compared to c-Src activity in the control cells. The activity of v-Src immunoprecipitated separately, however, accounting for only 40% of the total Src activity, indicating that c-Src is activated upon transformation. An increased activity of Csk was also found in RSV-transformed cells. It decreased upon serum stimulation in parallel with an increase in Src kinase activity. In nontransformed cells, serum stimulation induced an enhanced Csk activity, but no changes in c-Src activity were observed. This may suggest that Csk may have more functions in hamster fibroblasts, in addition to its inhibitory effect on c-Src.

• MeSH
• aktivace enzymů účinky léků   MeSH
• androstadieny farmakologie   MeSH
• buněčné linie MeSH
• C-terminální Src kinasa MeSH
• fibroblasty cytologie   účinky léků   metabolismus   MeSH
• inhibitory enzymů farmakologie   MeSH
• křečci praví MeSH
• krevní proteiny farmakologie   MeSH
• onkogenní protein pp60(v-src) genetika   metabolismus   MeSH
• protinádorová antibiotika farmakologie   MeSH
• protoonkogenní proteiny pp60(c-src) metabolismus   MeSH
• signální transdukce fyziologie   MeSH
• sirolimus farmakologie   MeSH
• skupina kinas odvozených od src-genu MeSH
• tyrosinkinasy metabolismus   MeSH
• virová transformace buněk fyziologie   MeSH
• viry ptačího sarkomu genetika   MeSH
• wortmannin MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• androstadieny MeSH
• C-terminální Src kinasa MeSH
• inhibitory enzymů MeSH
• krevní proteiny MeSH
• onkogenní protein pp60(v-src) MeSH
• protinádorová antibiotika MeSH
• protoonkogenní proteiny pp60(c-src) MeSH
• sirolimus MeSH
• skupina kinas odvozených od src-genu MeSH
• tyrosinkinasy MeSH
• wortmannin MeSH

DNA vaccination is particularly efficient for induction of cytotoxic T-lymphocyte (CTL) response. In our experiments, we used MHC(B) congenic chicken lines CB and CC (regressors and progressors of v-src-induced tumours, respectively) and a mutated, non-oncogenic v-src gene construct as the DNA vaccine. A high degree of vaccine protection against oncogenic v-src challenge was achieved in the CB line chickens. CTL response was demonstrated in vitro and by adoptive transfer of immune cells to the syngeneic host and to the CC line chickens rendered tolerant to CB cells. In the CC line chickens we observed tumour growth retardation after a low-dose DNA vaccination administered to immature recipients while higher amounts of DNA vaccine in immunocompetent chickens exerted an enhancing effect.

• MeSH
• DNA vakcíny terapeutické užití   MeSH
• geny src genetika   imunologie   MeSH
• imunologická odpověď na dávku MeSH
• kur domácí MeSH
• kuřecí embryo MeSH
• onkogenní protein pp60(v-src) imunologie   MeSH
• převzatá imunita metody   MeSH
• ptačí sarkom imunologie   prevence a kontrola   MeSH
• věkové faktory MeSH
• virová transformace buněk MeSH
• virové vakcíny genetika   terapeutické užití   MeSH
• viry ptačího sarkomu genetika   imunologie   MeSH
• zvířata kongenní MeSH
• zvířata MeSH
• Check Tag
• kuřecí embryo MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA vakcíny MeSH
• onkogenní protein pp60(v-src) MeSH
• virové vakcíny MeSH