ectopic expression
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The first step in the development of human colorectal cancer is aberrant activation of the Wnt signaling pathway. Wnt signaling hyperactivation is predominantly caused by loss-of-function mutations in the adenomatous polyposis coli (APC) gene that encodes the pathway negative regulator. In order to identify genes affected by the Apc loss, we performed expression profiling of intestinal epithelium isolated from mice harboring a conditional Apc allele. The gene encoding transcriptional factor msh homeobox 1 (Msx1) displayed robust upregulation upon Apc inactivation. Histological analysis of the Apc-deficient epithelium revealed that in the small intestine, the Msx1 protein was localized exclusively in ectopic crypts, i.e., in pockets of proliferating cells abnormally positioned on the villi. Ablation of the Msx1 gene leads to the disappearance of ectopic crypts and loss of differentiated cells. Moreover, tumors arising from Msx1-deficient cells display altered morphology reminiscent of villous adenomas. In human tumor specimens, MSX1 displayed significantly increased expression in colonic neoplasia with a descending tendency during the lesion progression towards colorectal carcinoma. In summary, the results indicate that Msx1 represents a novel marker of intestinal tumorigenesis. In addition, we described the previously unknown relationship between the Msx1-dependent formation of ectopic crypts and cell differentiation.
- MeSH
- beta-katenin metabolismus MeSH
- buněčná diferenciace MeSH
- kolorektální nádory genetika patologie MeSH
- lidé MeSH
- myši knockoutované MeSH
- nádory tračníku genetika patologie MeSH
- protein familiární adenomatózní polypózy genetika metabolismus MeSH
- regulace genové exprese u nádorů MeSH
- signální dráha Wnt MeSH
- stanovení celkové genové exprese MeSH
- střevní sliznice metabolismus patologie MeSH
- tenké střevo patologie MeSH
- transkripční faktor MSX1 genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Drought stress conditions modify source-sink relations, thereby influencing plant growth, adaptive responses, and consequently crop yield. Invertases are key metabolic enzymes regulating sink activity through the hydrolytic cleavage of sucrose into hexose monomers, thus playing a crucial role in plant growth and development. However, the physiological role of invertases during adaptation to abiotic stress conditions is not yet fully understood. Here it is shown that plant adaptation to drought stress can be markedly improved in tomato (Solanum lycopersicum L.) by overexpression of the cell wall invertase (cwInv) gene CIN1 from Chenopodium rubrum. CIN1 overexpression limited stomatal conductance under normal watering regimes, leading to reduced water consumption during the drought period, while photosynthetic activity was maintained. This caused a strong increase in water use efficiency (up to 50%), markedly improving water stress adaptation through an efficient physiological strategy of dehydration avoidance. Drought stress strongly reduced cwInv activity and induced its proteinaceous inhibitor in the leaves of the wild-type plants. However, the CIN1-overexpressing plants registered 3- to 6-fold higher cwInv activity in all analysed conditions. Surprisingly, the enhanced invertase activity did not result in increased hexose concentrations due to the activation of the metabolic carbohydrate fluxes, as reflected by the maintenance of the activity of key enzymes of primary metabolism and increased levels of sugar-phosphate intermediates under water deprivation. The induced sink metabolism in the leaves explained the maintenance of photosynthetic activity, delayed senescence, and increased source activity under drought stress. Moreover, CIN1 plants also presented a better control of production of reactive oxygen species and sustained membrane protection. Those metabolic changes conferred by CIN1 overexpression were accompanied by increases in the concentrations of the senescence-delaying hormone trans-zeatin and decreases in the senescence-inducing ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) in the leaves. Thus, cwInv critically functions at the integration point of metabolic, hormonal, and stress signals, providing a novel strategy to overcome drought-induced limitations to crop yield, without negatively affecting plant fitness under optimal growth conditions.
- MeSH
- buněčná stěna enzymologie MeSH
- Chenopodium genetika metabolismus MeSH
- ektopická exprese * MeSH
- fotosyntéza MeSH
- geneticky modifikované rostliny genetika metabolismus MeSH
- invertasa genetika metabolismus MeSH
- listy rostlin metabolismus MeSH
- období sucha * MeSH
- regulace genové exprese u rostlin * MeSH
- rostlinné proteiny genetika metabolismus MeSH
- Solanum lycopersicum enzymologie genetika fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Responses to drought, heat, and combined stress were compared in tobacco (Nicotiana tabacum L.) plants ectopically expressing the cytokinin oxidase/dehydrogenase CKX1 gene of Arabidopsis thaliana L. under the control of either the predominantly root-expressed WRKY6 promoter or the constitutive 35S promoter, and in the wild type. WRKY6:CKX1 plants exhibited high CKX activity in the roots under control conditions. Under stress, the activity of the WRKY6 promoter was down-regulated and the concomitantly reduced cytokinin degradation coincided with raised bioactive cytokinin levels during the early phase of the stress response, which might contribute to enhanced stress tolerance of this genotype. Constitutive expression of CKX1 resulted in an enlarged root system, a stunted, dwarf shoot phenotype, and a low basal level of expression of the dehydration marker gene ERD10B. The high drought tolerance of this genotype was associated with a relatively moderate drop in leaf water potential and a significant decrease in leaf osmotic potential. Basal expression of the proline biosynthetic gene P5CSA was raised. Both wild-type and WRKY6:CKX1 plants responded to heat stress by transient elevation of stomatal conductance, which correlated with an enhanced abscisic acid catabolism. 35S:CKX1 transgenic plants exhibited a small and delayed stomatal response. Nevertheless, they maintained a lower leaf temperature than the other genotypes. Heat shock applied to drought-stressed plants exaggerated the negative stress effects, probably due to the additional water loss caused by a transient stimulation of transpiration. The results indicate that modulation of cytokinin levels may positively affect plant responses to abiotic stress through a variety of physiological mechanisms.
- MeSH
- Arabidopsis enzymologie genetika MeSH
- cytokininy metabolismus MeSH
- exprese genu MeSH
- geneticky modifikované rostliny chemie genetika fyziologie MeSH
- období sucha MeSH
- oxidoreduktasy genetika metabolismus MeSH
- promotorové oblasti (genetika) MeSH
- proteiny huseníčku genetika metabolismus MeSH
- regulace genové exprese u rostlin MeSH
- tabák chemie genetika fyziologie MeSH
- vysoká teplota MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The placental DNA methylation landscape is unique, with widespread partially methylated domains (PMDs). The placental "methylome" is conserved across mammals, a shared feature of many cancers, and extensively studied for links with pregnancy complications. Human trophoblast stem cells (hTSCs) offer exciting potential for functional studies to better understand this epigenetic feature; however, whether the hTSC epigenome recapitulates primary trophoblast remains unclear. We find that hTSCs exhibit an atypical methylome compared with trophectoderm and 1st trimester cytotrophoblast. Regardless of cell origin, oxygen levels, or culture conditions, hTSCs show localized DNA methylation within transcribed gene bodies and a complete loss of PMDs. Unlike early human trophoblasts, hTSCs display a notable absence of DNMT3L expression, which is necessary for PMD establishment in mouse trophoblasts. Remarkably, we demonstrate that ectopic expression of DNMT3L in hTSCs restores placental PMDs, supporting a conserved role for DNMT3L in de novo methylation in trophoblast development in human embryogenesis.
- MeSH
- DNA-(cytosin-5-)methyltransferasa * metabolismus genetika MeSH
- epigenom MeSH
- kmenové buňky metabolismus cytologie MeSH
- lidé MeSH
- metylace DNA * genetika MeSH
- myši MeSH
- placenta * metabolismus cytologie MeSH
- těhotenství MeSH
- trofoblasty * metabolismus cytologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The activity of the phytohormone cytokinin depends on a complex interplay of factors such as its metabolism, transport, stability, and cellular/tissue localization. O-glucosides of zeatin-type cytokinins are postulated to be storage and/or transport forms, and are readily deglucosylated. Transgenic tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) plants were constructed over-expressing Zm-p60.1, a maize beta-glucosidase capable of releasing active cytokinins from O- and N3-glucosides, to analyse its potential to perturb zeatin metabolism in planta. Zm-p60.1 in chloroplasts isolated from transgenic leaves has an apparent K(m) more than 10-fold lower than the purified enzyme in vitro. Adult transgenic plants grown in the absence of exogenous zeatin were morphologically indistinguishable from the wild type although differences in phytohormone levels were observed. When grown on medium containing zeatin, inhibition of root elongation was apparent in all seedlings 14 d after sowing (DAS). Between 14 and 21 DAS, the transgenic seedlings accumulated fresh weight leading later (28-32 DAS) to ectopic growths at the base of the hypocotyl. The development of ectopic structures correlated with the presence of the enzyme as demonstrated by histochemical staining. Cytokinin quantification showed that transgenic seedlings grown on medium containing zeatin accumulate active metabolites like zeatin riboside and zeatin riboside phosphate and this might lead to the observed changes. The presence of the enzyme around the base of the hypocotyl and later, in the ectopic structures themselves, suggests that the development of these structures is due to the perturbance in zeatin metabolism caused by the ectopic presence of Zm-p60.1.
- MeSH
- beta-glukosidasa fyziologie metabolismus MeSH
- cytokininy metabolismus MeSH
- financování organizované MeSH
- geneticky modifikované rostliny anatomie a histologie enzymologie účinky léků MeSH
- homeostáza MeSH
- kinetika MeSH
- kukuřice setá enzymologie genetika MeSH
- kultivační média MeSH
- kyselina abscisová metabolismus MeSH
- kyseliny indoloctové metabolismus MeSH
- tabák genetika MeSH
- zeatin farmakologie metabolismus MeSH
BACKGROUND: Although many genetic manipulations of crops providing biofortified or safer food have been done, the acceptance of biotechnology crops still remains limited. We report on a transgenic barley expressing the multi-functional protein osmotin that improves plant defense under stress conditions. METHODS: An Agrobacterium-mediated technique was used to transform immature embryos of the spring barley cultivar Golden Promise. Transgenic barley plants of the T0 and T1 generation were evaluated by molecular methods. Transgenic barley tolerance to stress was determined by chlorophyll, total protein, malondialdehyde and ascorbate peroxidase content. Methanol extracts of i) Fusarium oxysporum infected or ii) salt-stressed plants, were characterized by their acute toxicity effect on human dermal fibroblasts (HDF), genotoxicity and affection of biodiversity interactions, which was tested through monitoring barley natural virus pathogen-host interactions-the BYDV and WDV viruses transmitted to the plants by aphids and leafhoppers. RESULTS: Transgenic plants maintained the same level of chlorophyll and protein, which significantly declined in wild-type barley under the same stressful conditions. Salt stress evoked higher ascorbate peroxidase level and correspondingly less malondialdehyde. Osmotin expressing barley extracts exhibited a lower cytotoxicity effect of statistical significance than that of wild-type plants under both types of stress tested on human dermal fibroblasts. Extract of Fusarium oxysporum infected transgenic barley was not able to damage DNA in the Comet assay, which is in opposite to control plants. Moreover, this particular barley did not affect the local biodiversity. CONCLUSION: Our findings provide a new perspective that could help to evaluate the safety of products from genetically modified crops.
- MeSH
- bezpečnost potravin * MeSH
- biologická adaptace MeSH
- ektopická exprese * MeSH
- fyziologický stres genetika MeSH
- geneticky modifikované rostliny MeSH
- interakce hostitele a patogenu genetika MeSH
- ječmen (rod) genetika MeSH
- lidé MeSH
- rekombinantní proteiny * MeSH
- rostlinné proteiny genetika MeSH
- tabák genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
OBJECTIVE: We recently determined that the ectopic over-expression of miR-429 and other members of the miR-200 family of microRNAs in ovarian cancer (OC) mesenchymal-like cell lines induces mesenchymal-to-epithelial transition (MET) with a concomitant increase in sensitivity to platinum drugs. We sought to determine if metastasizing OC cells isolated from an OC patient could also be induced by miR-429 to undergo MET and become sensitized to established first-line platinum-based therapies. METHODS: We established and characterized a new primary cell line (OCI-984) from free-floating OC cells isolated from the ascites fluid of an advanced stage OC patient. miR-429 was ectopically over-expressed in these cells. RESULTS: The over-expression of miR-429 in OCI-984 cells induced morphological, functional and molecular changes consistent with MET and a concomitant significant increase in the sensitivity of the converted cells to cisplatin. CONCLUSIONS: Our findings indicate that the miR-200 family of microRNAs, and miR-429 in particular, play a critical role in the functioning of OC metastasizing cells and that targeted delivery of miR-429, and perhaps other miR-200 family members, in combination with platinum-based chemotherapies may be an effective strategy in reducing OC metastasis and tumor recurrence.
- MeSH
- cisplatina farmakologie MeSH
- epitelo-mezenchymální tranzice genetika MeSH
- lidé MeSH
- mikro RNA biosyntéza MeSH
- myši nahé MeSH
- myši MeSH
- nádorové buněčné linie * MeSH
- nádory vaječníků farmakoterapie genetika patologie MeSH
- protinádorové látky farmakologie MeSH
- screeningové testy protinádorových léčiv MeSH
- senioři MeSH
- xenogenní modely - testy protinádorové aktivity MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- senioři MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- kazuistiky MeSH
- práce podpořená grantem MeSH
The product of the retinoblastoma susceptibility gene, pRb, is a negative regulator of cell growth. It functions by regulating the activity of transcription factors. Rb represses some genes by sequestering or inactivating the positive transcription factor E2F and seems to activate some others by interacting with factors like Sp1 or ATF-2. However, there are only a few examples of genes which are positively regulated by pRb. In order to find out if there are common mechanisms for promoter regulation by pRb, we were interested to identify more genes which are either stimulated or repressed by pRb. Using the method of differential display (DDRT-PCR) in combination with nuclear run-on analyses we were able to detect a number of genes which are upregulated by ectopic expression of the Rb gene in Rb-deficient mammary carcinoma cells. We could demonstrate not only stimulation of the endogenous mutant Rb gene but also positive regulation of genes coding for diverse classes of proteins, including the endothelial growth regulator endothelin-1 and the proteoglycans versican and PG40. As a second approach, we investigated gene expression in cell lines established from Rb deficient heterozygous and homozygous knockout mouse embryos and normal mice. We have identified several genes the expression of which correlates positively or negatively with the presence of Rb. These data provide further evidence for pRb being a master regulator of a complex network of gene activities defining the difference between dividing and resting or differentiated cells.
- MeSH
- endoteliny biosyntéza genetika MeSH
- geny retinoblastomu fyziologie MeSH
- lidé MeSH
- molekulární sekvence - údaje MeSH
- myši knockoutované MeSH
- myši MeSH
- nádorové buňky kultivované MeSH
- polymerázová řetězová reakce metody MeSH
- proteoglykany biosyntéza genetika MeSH
- regulace genové exprese * MeSH
- retinoblastomový protein genetika fyziologie MeSH
- sekvence nukleotidů MeSH
- transfekce MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Galectin-7 (Gal-7) is negatively regulated in cervical cancer, and appears to be a link between the apoptotic response triggered by cancer and the anti-tumoral activity of the immune system. Our understanding of how cervical cancer cells and their molecular networks adapt in response to the expression of Gal-7 remains limited. METHODS: Meta-analysis of Gal-7 expression was conducted in three cervical cancer cohort studies and TCGA. In silico prediction and bisulfite sequencing were performed to inquire epigenetic alterations. To study the effect of Gal-7 on cervical cancer, we ectopically re-expressed it in the HeLa and SiHa cervical cancer cell lines, and analyzed their transcriptome and SILAC-based proteome. We also examined the tumor and microenvironment host cell transcriptomes after xenotransplantation into immunocompromised mice. Differences between samples were assessed with the Kruskall-Wallis, Dunn's Multiple Comparison and T tests. Kaplan-Meier and log-rank tests were used to determine overall survival. RESULTS: Gal-7 was constantly downregulated in our meta-analysis (p < 0.0001). Tumors with combined high Gal-7 and low galectin-1 expression (p = 0.0001) presented significantly better prognoses (p = 0.005). In silico and bisulfite sequencing assays showed de novo methylation in the Gal-7 promoter and first intron. Cells re-expressing Gal-7 showed a high apoptosis ratio (p < 0.05) and their xenografts displayed strong growth retardation (p < 0.001). Multiple gene modules and transcriptional regulators were modulated in response to Gal-7 reconstitution, both in cervical cancer cells and their microenvironments (FDR < 0.05 %). Most of these genes and modules were associated with tissue morphogenesis, metabolism, transport, chemokine activity, and immune response. These functional modules could exert the same effects in vitro and in vivo, even despite different compositions between HeLa and SiHa samples. CONCLUSIONS: Gal-7 re-expression affects the regulation of molecular networks in cervical cancer that are involved in diverse cancer hallmarks, such as metabolism, growth control, invasion and evasion of apoptosis. The effect of Gal-7 extends to the microenvironment, where networks involved in its configuration and in immune surveillance are particularly affected.
