UNLABELLED: Comparing the gene expression profiles of metastatic and nonmetastatic cells has the power to reveal candidate metastasis-associated genes, whose involvement in metastasis can be experimentally tested. In this study, differentially expressed genes were explored in the v-src-transformed metastatic cell line PR9692 and its nonmetastatic subclone PR9692-E9. First, the contribution of homeodomain only protein X (HOPX) in metastasis formation and development was assessed. HOPX-specific knockdown decreased HOPX expression in the nonmetastatic subclone and displayed reduced cell motility in vitro. Critically, HOPX knockdown decreased the in vivo metastatic capacity in a syngeneic animal model system. Genomic analyses identified a cadre of genes affected by HOPX knockdown that intersected significantly with genes previously found to be differentially expressed in metastatic versus nonmetastatic cells. Furthermore, 232 genes were found in both screens with at least a two-fold change in gene expression, and a number of high-confidence targets were validated for differential expression. Importantly, significant changes were demonstrated in the protein expression level of three metastatic-associated genes (NCAM, FOXG1, and ITGA4), and knockdown of one of the identified HOPX-regulated metastatic genes, ITGA4, showed marked inhibition of cell motility and metastasis formation. These data demonstrate that HOPX is a metastasis-associated gene and that its knockdown decreases the metastatic activity of v-src-transformed cells through altered gene expression patterns. IMPLICATIONS: This study provides new mechanistic insight into a HOPX-regulated metastatic dissemination signature.

• MeSH
• buněčný cyklus MeSH
• down regulace MeSH
• experimentální sarkom genetika   patologie   sekundární   MeSH
• forkhead transkripční faktory genetika   metabolismus   MeSH
• genový knockdown MeSH
• geny src MeSH
• homeodoménové proteiny genetika   metabolismus   MeSH
• kur domácí MeSH
• metastázy nádorů genetika   MeSH
• molekuly buněčné adheze nervové genetika   metabolismus   MeSH
• nádorová transformace buněk genetika   MeSH
• nádorové buněčné linie MeSH
• pohyb buněk MeSH
• ptačí proteiny genetika   metabolismus   MeSH
• regulace genové exprese u nádorů MeSH
• sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
• stanovení celkové genové exprese MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• forkhead transkripční faktory MeSH
• homeodoménové proteiny MeSH
• molekuly buněčné adheze nervové MeSH
• ptačí proteiny MeSH

CpG islands are important in the protection of adjacent housekeeping genes from de novo DNA methylation and for keeping them in a transcriptionally active state. However, little is known about their capacity to protect heterologous genes and assure position-independent transcription of adjacent transgenes or retroviral vectors. To tackle this question, we have used the mouse aprt CpG island to flank a Rous sarcoma virus (RSV)-derived reporter vector and followed the transcriptional activity of integrated vectors. RSV is an avian retrovirus which does not replicate in mammalian cells because of several blocks at all levels of the replication cycle. Here we show that our RSV-derived reporter proviruses linked to the mouse aprt gene CpG island remain undermethylated and keep their transcriptional activity after stable transfection into both avian and nonpermissive mammalian cells. This effect is most likely caused by the protection from de novo methylation provided by the CpG island and not by enhancement of the promoter strength. Our results are consistent with previous finding of CpG islands in proximity to active but not inactive proviruses and support further investigation of the protection of the gene transfer vectors from DNA methylation.

• MeSH
• adeninfosforibosyltransferasa genetika   MeSH
• buněčné linie virologie   MeSH
• CpG ostrůvky * MeSH
• defektní viry genetika   MeSH
• DNA virů chemie   genetika   MeSH
• DNA-(cytosin-5-)methyltransferasa metabolismus   MeSH
• experimentální sarkom genetika   virologie   MeSH
• fibroblasty virologie   MeSH
• genetická transkripce * MeSH
• genetické vektory genetika   fyziologie   MeSH
• integrace viru MeSH
• koncové repetice MeSH
• křečci praví MeSH
• křeček rodu Mesocricetus MeSH
• kuřecí embryo MeSH
• metylace DNA MeSH
• myši MeSH
• proviry genetika   MeSH
• regulace exprese virových genů * MeSH
• replikace viru MeSH
• reportérové geny MeSH
• umlčování genů * MeSH
• viry ptačího sarkomu genetika   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• kuřecí embryo MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• adeninfosforibosyltransferasa MeSH
• DNA virů MeSH
• DNA-(cytosin-5-)methyltransferasa MeSH

Interleukin-2 and CD80 transfectants of a methylcholanthrene-induced murine sarcoma Mc12 (Mc12-IL-2 and Mc12-CD80 cells) with similar tumorigenicity in euthymic mice were utilized for experiments designed to investigate a co-stimulatory role of the CD80 molecule in allogeneic, congenitally athymic (nu/nu) mice. The CD80-transfected cells were as tumorigenic in nu/nu mice as the parental Mc12 sarcoma. The IL-2-transfected cells grew only transiently and regressed in all nu/nu recipients during four weeks after challenge with doses up to 5x10(7) cells. The 1:1 mixture of parental Mc12 with Mc12-CD80 cells grew progressively in all inoculated nu/nu mice; in a 1:1 mixture with parental Mc12 cells, Mc12-IL-2 cells were able to cause regressions in approximately 50% of nu/nu mice; the 1:1 mixture of Mc12-IL-2 and Mc12-CD80 transfectants showed only transient growth and regressed during four weeks in all inoculated nu/nu mice. Adoptive transfer of cell-mediated immunity revealed that spleen cells from tumor regressors were capable of transferring the resistance to Mc12 tumor in nu/nu mice. The spleen cells from tumor regressors were not cytolytic when allowed to react in vitro with Mc12, Mc12-IL-2, or Mc12-CD80 target cells. However, when grown in IL-2-containing medium, splenocytes from tumor regressors, but not the splenocytes from tumor progressors, could develop cytolytic activity directed against Mc12 target cells that was comparable to that of the splenocytes from tumor-free controls. These results suggest that the rejection of tumors in nu/nu mice was mediated by IL-2-dependent mechanisms in which the CD80 molecule played a co-stimulatory role; the results also indicate that the ability to be activated by IL-2 and to give rise to cytolytic activity of nu/nu splenocytes from tumor progressors is decreased.

• MeSH
• antigeny CD80 genetika   imunologie   fyziologie   MeSH
• buněčná imunita imunologie   MeSH
• experimentální sarkom genetika   imunologie   terapie   MeSH
• imunoterapie adoptivní * MeSH
• interleukin-2 genetika   imunologie   fyziologie   MeSH
• karcinogeny MeSH
• methylcholanthren MeSH
• myši inbrední C57BL MeSH
• myši nahé MeSH
• myši MeSH
• signální transdukce fyziologie   MeSH
• slezina cytologie   imunologie   MeSH
• transfekce MeSH
• transplantace nádorů MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny CD80 MeSH
• interleukin-2 MeSH
• karcinogeny MeSH
• methylcholanthren MeSH

Spleen cells from mice bearing progressively growing syngeneic sarcomas are immunologically hyporeactive and respond by significantly decreased proliferative response to stimulation with mitogens and cytokines. Here we show that these hyporeactive cells synthesize, after mitogen stimulation, comparable amount of mRNA for tumor necrosis factor (TNF)-alpha as do cells from control mice. However, stimulated spleen cells from the same tumor-bearing mice produce considerably less mRNA for TNF-beta than cells from control mice. These observations were further confirmed using purified peritoneal macrophages and enriched splenic T cells. The results thus demonstrate a distinct regulation of expression of genes for TNF-alpha and TNF-beta, two functionally very similar cytokines, and simultaneously a selective impairment of T-cell function in the course of growth of syngeneic tumors in mice.

• MeSH
• experimentální sarkom genetika   metabolismus   MeSH
• exprese genu MeSH
• lymfotoxin-alfa genetika   MeSH
• messenger RNA biosyntéza   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• peritoneální makrofágy metabolismus   MeSH
• slezina metabolismus   MeSH
• T-lymfocyty metabolismus   MeSH
• TNF-alfa genetika   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• lymfotoxin-alfa MeSH
• messenger RNA MeSH
• TNF-alfa MeSH

Spleen cells from mice bearing progressive growing methylcholanthrene-induced syngenic tumours were deeply hyporeactive in response to T-cell mitogens. This hyporeactivity was associated with decreased ability to synthesize mRNA for the inducible 55,000 MW interleukin-2 receptor (IL-2R). Since the expression of functional IL-2R represents one of the early and pivotal events in lymphoid-cell activation, it is suggested that the defect in effective IL-2R expression may be one of the primary factors responsible for the immunological hyporeactivity of tumour-bearing hosts.

• MeSH
• aktivace lymfocytů MeSH
• experimentální sarkom genetika   imunologie   MeSH
• messenger RNA biosyntéza   MeSH
• molekulová hmotnost MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• receptory interleukinu-2 genetika   MeSH
• RNA nádorová biosyntéza   MeSH
• T-lymfocyty imunologie   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• messenger RNA MeSH
• receptory interleukinu-2 MeSH
• RNA nádorová MeSH

The cysteine proteinase cathepsin B (EC 3.4.22.1) is believed to take part in biochemical processes underlying tumor metastasis. In the present study, the cellular localization, intracellular levels and extracellular release of cathepsin B activity were examined in vitro in cells of two rat sarcoma variants, LW13K2 and RPS, differing in their capacity to metastasize spontaneously to the lung of syngeneic LEW/CUB rats. The LW13K2 sarcoma metastasizes rarely, whereas the LW13K2-derived RPS variant produces a metastasis incidence of above 50%. Using fluorescent cytochemical staining, microgranular reaction centers of cathepsin B were observed in the cell cytoplasm, in some cellular processes, with apical localization in some of them, as well as at the extreme cell periphery in cells of both sarcoma variants. The appearance of this distribution of cathepsin B activity was delayed in the RPS variant. Biochemically, the intracellular level of cathepsin B activity was significantly higher in homogenates of LW13K2 cells than RPS cells. In contrast to the intracellular enzyme activity, RPS cells cultured in serum-free medium at pH 6.5 released a substantially higher amount of cathepsin B activity than LW13K2 cells into the extracellular environment; at pH 7.4 the initially higher release of cathepsin B activity from RPS cells later equalized with that from LW13K2 cells. Taken together, the results indicate that changes of pericellular pH can modulate the extracellular release of cathepsin B in both sarcoma cell variants and suggest that the rate of cathepsin B release under conditions of mildly acid pericellular pH could be related to the incidence of metastases observed in these rat sarcoma variants. Total intracellular cathepsin B activity did not exhibit positive correlation with the metastatic potential of the studied rat sarcoma variants.

• MeSH
• buněčné klony MeSH
• cykloheximid farmakologie   MeSH
• cysteinové endopeptidasy * MeSH
• cytoplazma enzymologie   MeSH
• experimentální sarkom enzymologie   genetika   sekundární   MeSH
• histocytochemie MeSH
• kathepsin B metabolismus   MeSH
• kathepsin H MeSH
• kathepsiny metabolismus   MeSH
• koncentrace vodíkových iontů MeSH
• krysa rodu Rattus MeSH
• kultivační média analýza   MeSH
• metastázy nádorů MeSH
• nádorové buňky kultivované účinky léků   MeSH
• nádory plic enzymologie   sekundární   MeSH
• potkani inbrední LEW genetika   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• Ctsh protein, rat MeSH Prohlížeč
• cykloheximid MeSH
• cysteinové endopeptidasy * MeSH
• kathepsin B MeSH
• kathepsin H MeSH
• kathepsiny MeSH
• kultivační média MeSH

Populations of LW13K2 sarcoma derivatives were compared for their malignancy, patterns of cell behaviour in vitro (dynamic morphology and migration) and karyological pattern. The following tumour cell populations were used: the original LW13K2 sarcoma from inbred LEW/CUB rats, RPS sarcoma derived from it by neoplastic progression, four cell populations isolated in vitro from metastases of a syngeneic LEW-CUB strain rat with RPS tumour and four neoplastic cell populations isolated from spontaneous metastases shed by RPS sarcoma in allogeneic rats, differing from LEW/CUB in weak alloantigenic loci. Although RPS tumour did not grow progressively in MHC-different recipients (while the original LW13K2 tumour did), it grew progressively and metastasized in all groups of non-MHC allogeneic recipients. Parallel with the metastatic potential patterns of in vitro behaviour, such as an increased incidence of the quasi-stellate morphotype at slightly acid culture conditions endowed with enhanced changeability of the cell shape and migrational activity, were found. Cytogenetic analysis demonstrated rather stable chromosomal patterns over the cascade of neoplastic progression from LW13K2 sarcoma over RPS sarcoma to freshly isolated metastases. This indicated that the apparent neoplastic progression observed in the cell populations derived from LW13K2 sarcoma is with high probability not due to the selection at the chromosomal level.

• MeSH
• buněčné dělení MeSH
• chromozomální aberace MeSH
• experimentální sarkom genetika   patologie   MeSH
• krysa rodu Rattus MeSH
• metastázy nádorů MeSH
• nádorové buňky kultivované MeSH
• pohyb buněk MeSH
• potkani inbrední LEW MeSH
• staging nádorů MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

The DNA as isolated from duck fibroblasts transformed by a duck-adapted Prague strain of Rous sarcoma virus and used for transfection. Transformed recipient BLEF and DEF cultures exhibited considerable morphological variability. The virus designated daPR-RSV-C morphf was obtained from the culture with fusiform transformation and cloned. The virus retained the ability to induce fusiform transformation, even after 20 passages on chicken fibroblasts. There was a good correlation between focus forming activity of the virus and its tumorigenicity in chickens. The frequency of morphf mutation to another phenotype was less than 10(-3) in cloned virus. Foreign avian embryonic cells transformed by this virus clone had a similar morphological appearance as transformed chicken cells. The clone also retained two additional non-conditional markers - subgroup C specificity and the ability to replicate efficiently in duck cells ("duck adaptation"). Freshly obtained cloned virus was found not to contain a transformation-defective mutant. Such a mutant occurred in the second passage of the virus of DEF where the mutant was isolated. Inoculation of the td mutant into Brown Leghorn embryos gave rise to a sarcoma in one of the 36 examined chickens. However, no transforming virus was detected in the sarcoma. SDS-polyacrylamide gel electrophoresis showed that cloned daPR-RSV-C morphf contained only genomic RNA; its molecular weight 3.08 X 10(6) daltons corresponded to the molecular weight of a non-defective PR-RSV-C used as control.

• MeSH
• experimentální sarkom genetika   MeSH
• kachny MeSH
• kur domácí MeSH
• molekulová hmotnost MeSH
• mutace MeSH
• replikace viru MeSH
• RNA virová genetika   MeSH
• virová transformace buněk * MeSH
• virové geny MeSH
• viry ptačího sarkomu genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• RNA virová MeSH

• MeSH
• experimentální sarkom genetika   MeSH
• karyotypizace MeSH
• myši MeSH
• pohlavní chromozomy * MeSH
• sexuální faktory MeSH
• transplantace nádorů MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH