Background: Exposure to intermittent hypoxia (IH) may play a role in the development of metabolic impairments in the context of obstructive sleep apnea syndrome, probably by elevated plasma levels of free fatty acids. Employing gas-permeable cultureware to grow differentiated human and mouse adipocytes in vitro, we directly studied the effects of pericellular oxygen fluctuations on key adipocyte metabolic functions-spontaneous lipolytic rates, triglyceride accumulation, de novo lipogenesis, and expression of adipocyte-specific marker genes. Materials and Methods: 3T3-L1 fibroblasts and human subcutaneous preadipocytes were differentiated under conditions that induced repetitive pericellular-oxygen cycles IH between 1% O2 (5 min) and 16% O2 (5 min), continuously for 14 days or under control conditions. Chemicals were used to inhibit the flux of acetyl-CoA from glycolysis (alfa-cyano-4-hydroxy cinnamate) or the tricarboxylic acid cycle (SB204990), or to stimulate the flux of acetyl-CoA from pyruvate to the lipogenic pool. Lipolytic rate, intracellular lipids, and expression of adipocyte differentiation markers were assessed and t-test or ANOVA were used to find significant differences. Results: The rate of lipolysis increased by 211% in 3T3-L1 cells and by 39% in obese human adipocytes. Exposure to IH reduced intracellular lipid stores by 37% and reduced the expression of adipocyte differentiation markers. Pharmacological stimulation or inhibition of de novo lipogenesis did not modify the intracellular lipid content under IH. Conclusions: Pericellular oxygen fluctuations directly stimulated lipolysis, but did not increase de novo lipogenesis from endogenous substrates. Similarly, IH hampered adipocyte differentiation from precursors.

• MeSH
• acetylkoenzym A metabolismus   MeSH
• buněčná diferenciace genetika   fyziologie   MeSH
• buňky 3T3-L1 MeSH
• citrátový cyklus MeSH
• glykolýza MeSH
• hypoxie buňky genetika   fyziologie   MeSH
• kinetika MeSH
• lidé MeSH
• lipogeneze genetika   fyziologie   MeSH
• lipolýza genetika   fyziologie   MeSH
• myši MeSH
• spotřeba kyslíku genetika   MeSH
• stanovení celkové genové exprese MeSH
• triglyceridy metabolismus   MeSH
• tukové buňky metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

This paper aims to identify and describe new genetic markers involved in the processes of protein expression and modification reflected in the change of mitochondrial activity before and after in vitro maturation of the oocyte. Porcine oocytes collected from the ovaries of slaughtered landrace gilts were subjected to the process of in vitro maturation. Transcriptomic changes in the expression profile of oocyte genes involved in response to hypoxia, the transmembrane protein receptor serine threonine kinase signaling pathway, the "transforming growth factor β receptor signaling pathway", "response to protein stimulus", and "response to organic substance" were investigated using microarrays. The expression values of these genes in oocytes was analyzed before (immature) and after (mature) in vitro maturation, with significant differences found. All the significantly altered genes showed downregulation after the maturation process. The most changed genes from these gene ontologies, FOS, ID2, VEGFA, BTG2, CYR61, ESR1, AR, TACR3, CCND2, CHRDL1, were chosen to be further validated, described and related to the literature. Additionally, the mitochondrial activity of the analyzed oocytes was measured using specific dyes. We found that the mitochondrial activity was higher before the maturation process. The analysis of these results and the available literature provides a novel insight on the processes that occur during in vitro oocyte maturation. While this knowledge may prove to be useful in further research of the procedures commonly associated with in vitro fertilization procedures, it serves mostly as a basic reference for further proteomic, in vivo, and clinical studies that are necessary to translate it into practical applications.

• MeSH
• hypoxie buňky genetika   MeSH
• IVM techniky MeSH
• kultivované buňky MeSH
• mitochondrie genetika   metabolismus   MeSH
• oocyty cytologie   metabolismus   MeSH
• oogeneze genetika   MeSH
• prasata MeSH
• signální transdukce MeSH
• transformující růstový faktor beta metabolismus   MeSH
• transkriptom * MeSH
• tyrosinkinasové receptory metabolismus   MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Long non-coding RNAs (lncRNAs) are DNA transcripts longer than 200 nucleotides without protein-coding potential. As they are key regulators of gene expression at chromatic, transcriptional and posttranscriptional level, they play important role in various biological and pathological processes. Dysregulation of lncRNAs has been observed in several diseases including cancer. Breast cancer is heterogeneous disease with many molecular subtypes specific in different prognosis and treatment responses. Hypoxia, a common micro-environmental feature of rapidly growing tumour is associated with metastases, recurrences and resistance to therapy. Aberrant expression of hypoxia related lncRNAs significantly correlates with poor outcomes in cancer patients, as the lncRNAs play an important regulatory role in the breast cancer-cell survival. Thus, a better understanding of lncRNAs role in the hypoxic conditions of breast cancer is crucial for precise understanding of the tumorigenesis, disease features and poor clinical outcome, especially in highly aggressive breast cancer subtypes (HER2-positive and triple-negative types). Moreover, lncRNAs may represent tumour marker predicting prognosis and therapeutic targets improving precise and personalized therapy for better patient´s survival. In this review, we summarize the recent information on lncRNAs in breast cancer with special focus on the hypoxia-responsive lncRNAs and their potential impact on the prognosis, therapy algorithms and individual outcomes. Presented data helps in better understanding of the specific mechanisms predicting new therapeutic agents and strategies for the pharmacological intervention.

• MeSH
• hypoxie buňky genetika   MeSH
• klinické zkoušky jako téma MeSH
• lidé MeSH
• nádory prsu genetika   patologie   terapie   MeSH
• RNA dlouhá nekódující genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

BACKGROUND: Carbonic anhydrase IX (CA IX) is a tumor-associated, highly active, transmembrane carbonic anhydrase isoform regulated by hypoxia and implicated in pH control and adhesion-migration-invasion. CA IX ectodomain (ECD) is shed from the tumor cell surface to serum/plasma of patients, where it can signify cancer prognosis. We previously showed that the CA IX ECD release is mediated by disintegrin and metalloproteinase ADAM17. Here we investigated the CA IX ECD shedding in tumor cells undergoing apoptosis in response to cytotoxic drugs, including cycloheximide and doxorubicin. METHODS: Presence of cell surface CA IX was correlated to the extent of apoptosis by flow cytometry in cell lines with natural or ectopic CA IX expression. CA IX ECD level was assessed by ELISA using CA IX-specific monoclonal antibodies. Effect of recombinant CA IX ECD on the activation of molecular pathways was evaluated using the cell-based dual-luciferase reporter assay. RESULTS: We found a significantly lower occurrence of apoptosis in the CA IX-positive cell subpopulation than in the CA IX-negative one. We also demonstrated that the cell-surface CA IX level dropped during the death progress due to an increased ECD shedding, which required a functional ADAM17. Inhibitors of metalloproteinases reduced CA IX ECD shedding, but not apoptosis. The CA IX ECD release induced by cytotoxic drugs was connected to elevated expression of CA IX in the surviving fraction of cells. Moreover, an externally added recombinant CA IX ECD activated a pathway driven by the Nanog transcription factor implicated in epithelial-mesenchymal transition and stemness. CONCLUSIONS: These findings imply that the increased level of the circulating CA IX ECD might be useful as an indicator of an effective antitumor chemotherapy. Conversely, elevated CA IX ECD might generate unwanted effects through autocrine/paracrine signaling potentially contributing to resistance and tumor progression.

• MeSH
• apoptóza účinky léků   genetika   MeSH
• cykloheximid aplikace a dávkování   MeSH
• epitelo-mezenchymální tranzice genetika   MeSH
• HeLa buňky MeSH
• hypoxie buňky genetika   MeSH
• karboanhydrasa IX aplikace a dávkování   genetika   metabolismus   MeSH
• lidé MeSH
• monoklonální protilátky aplikace a dávkování   MeSH
• nádory genetika   patologie   MeSH
• protein ADAM17 genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Hypoxia can halt cell cycle progression of several cell types at the G1/S interface. The arrest needs to be overcome by cancer cells. We have previously shown that the hypoxia-inducible cellular oxygen sensor PHD3/EGLN3 enhances hypoxic cell cycle entry at the G1/S boundary. METHODS: We used PHD3 knockdown by siRNA and shRNA in HeLa and 786-0 renal cancer cells. Flow cytometry with cell synchronization was used to study cell growth at different cell cycle phases. Total and phosphospecific antibodies together with cycloheximide chase were used to study p27/CDKN1B expression and fractionations for subcellular protein localization. RESULTS: Here we show that PHD3 enhances cell cycle by decreasing the expression of the CDK inhibitor p27/CDKN1B. PHD3 reduction led to increased p27 expression under hypoxia or VHL mutation. p27 was both required and sufficient for the PHD3 knockdown induced cell cycle block. PHD3 knockdown did not affect p27 transcription and the effect was HIF-independent. In contrast, PHD3 depletion increased the p27 half-life from G0 to S-phase. PHD3 depletion led to an increase in p27 phosphorylation at serine 10 without affecting threonine phosphorylation. Intact serine 10 was required for normal hypoxic and PHD3-mediated degradation of p27. CONCLUSIONS: The data demonstrates that PHD3 can drive cell cycle entry at the G1/S transition through decreasing the half-life of p27 that occurs by attenuating p27S10 phosphorylation.

• MeSH
• buněčný cyklus genetika   MeSH
• fosforylace MeSH
• genový knockdown MeSH
• HeLa buňky MeSH
• hypoxie buňky genetika   MeSH
• inhibitor p27 cyklin-dependentní kinasy biosyntéza   genetika   MeSH
• karcinom genetika   patologie   MeSH
• kontrolní body fáze G1 buněčného cyklu genetika   MeSH
• lidé MeSH
• malá interferující RNA MeSH
• prolyl-4-hydroxylasy HIF antagonisté a inhibitory   genetika   MeSH
• regulace genové exprese u nádorů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Cellular hypoxia is a hallmark of cancer. Hypoxia-inducible factor-1alpha (HIF-1alpha) and von Hippel-Lindau protein (pVHL) are the key mediators of cellular response to hypoxia. Little is known about their role in germ cell tumors of the testis. We therefore examined their status in a cohort of germ cell tumors of the testis. Thirty-six primary germ cell tumors of the testis (11 seminomas, 24 mixed germ cell tumors, and 1 case of pure intratubular germ cell neoplasia) were included in the study. HIF-1alpha and pVHL expression were studied using immunohistochemical (IHC) methods in the tumor and adjacent benign tissue. Selected cases with a low pVHL expression were further tested for genetic alterations using polymerase chain reaction. HIF-1alpha protein expression was not detectable in adjacent atrophic seminiferous tubules. In contrast, HIF-1alpha was expressed in one third of the malignancies, but in a low percentage of cells (mean, 3%; range, 0% to 20%). No difference in HIF-1alpha expression was observed between seminomas and nonseminomas (P=0.71). pVHL was expressed in atrophic tubular epithelium and in the Leydig cells, whereas a substantial loss of pVHL expression was observed in germ cell tumors regardless of the histologic type (mean, 45.6%; range, 0% to 100%). No genetic alterations of the VHL gene were observed in the cases with low pVHL expression. No significant correlation between HIF-1alpha and pVHL expression was observed (P=0.16). Germ cell tumors of the testis, regardless of the histologic type, are characterized by consistently low HIF-1alpha protein overexpression and a partial loss of pVHL without underlying VHL gene alterations. Further studies are necessary to clarify the functional importance of such alterations in testicular germ cell tumors.

• MeSH
• dospělí MeSH
• exony MeSH
• exprese genu * MeSH
• faktor 1 indukovatelný hypoxií - podjednotka alfa * genetika   metabolismus   MeSH
• germinální a embryonální nádory * MeSH
• hypoxie buňky genetika   MeSH
• imunohistochemie MeSH
• Leydigovy buňky metabolismus   patologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• nádorové mikroprostředí MeSH
• nádorový supresorový protein VHL * genetika   metabolismus   MeSH
• polymerázová řetězová reakce MeSH
• semenotvorné kanálky metabolismus   patologie   MeSH
• seminom * MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH

• MeSH
• diferenciální diagnóza MeSH
• erytropoéza fyziologie   genetika   imunologie   MeSH
• financování organizované MeSH
• genetické nemoci vrozené diagnóza   etiologie   MeSH
• genetické techniky využití   MeSH
• hypoxie buňky genetika   imunologie   MeSH
• lidé MeSH
• molekulární biologie MeSH
• polycytemie diagnóza   etiologie   genetika   MeSH
• polymerázová řetězová reakce metody   využití   MeSH
• receptory erythropoetinu krev   MeSH
• transkripční faktory genetika   krev   MeSH
• Check Tag
• lidé MeSH

Erythropoietin (EPO) is a factor essential for erythroid cell proliferation, differentiation, and survival. The production of EPO by the kidneys in response to hypoxia and anemia is well documented. To determine whether EPO is also produced by hematopoietic cells, we analyzed the expression of EPO in normal human hematopoietic progenitors and in their progeny. Undifferentiated CD34(+)lin- hematopoietic progenitors do not have detectable EPO mRNA. Differentiating CD34(+) cells that are stimulated with recombinant human EPO in serum-free liquid cultures express both EPO and EPO receptor (EPOR). Because CD34(+) cells represent a heterogeneous cell population, we analyzed individual burst-forming units-erythroid (BFU-E) and nonerythroid colony-forming unit-granulocyte-macrophage colonies for EPO mRNA. Only BFU-E colonies were positive for EPO mRNA. Lysates from pooled BFU-E colonies stained positively for EPO by immunoblotting. To further confirm the intrinsic nature of erythroid EPO, we replaced extrinsic EPO in erythroid colony cultures with EPO-mimicking peptide (EMP). We show EPO expression in the EMP-stimulated BFU-Es at both mRNA and protein levels. Stimulation of bone marrow mononuclear cells (BMMCs) with EMP upregulated EPO expression. Furthermore, we found EPO and EPOR mRNAs as well as EPO protein in K562 cells, a human erythroleukemia cell line. Stimulation of K562 cells with EMP upregulated EPO expression. We suggest that EPO of erythroid origin may have a role in the regulation of erythropoiesis.

• MeSH
• akutní erytroblastická leukemie patologie   MeSH
• antigeny CD34 analýza   MeSH
• chronická myeloidní leukemie patologie   MeSH
• dospělí MeSH
• ELISA MeSH
• erythropoetin biosyntéza   genetika   MeSH
• erytroidní prekurzorové buňky metabolismus   účinky léků   MeSH
• erytropoéza genetika   účinky léků   MeSH
• hematopoetické kmenové buňky metabolismus   účinky léků   MeSH
• hepatocelulární karcinom patologie   MeSH
• hypoxie buňky genetika   MeSH
• kobalt farmakologie   MeSH
• kultivační média bez séra MeSH
• kultivované buňky MeSH
• lidé MeSH
• messenger RNA biosyntéza   MeSH
• molekulární sekvence - údaje MeSH
• nádorové buňky kultivované MeSH
• nádory jater patologie   MeSH
• peptidy farmakologie   MeSH
• receptory erythropoetinu biosyntéza   genetika   MeSH
• regulace genové exprese MeSH
• sekvence nukleotidů MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH