242 research outputs found

    Chondrogenic differentiation of growth factor-stimulated precursor cells in cartilage repair tissue is associated with increased HIF-1Ξ± activity

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    SummaryObjectiveTo investigate the chondrogenic potential of growth factor-stimulated periosteal cells with respect to the activity of Hypoxia-inducible Factor 1Ξ± (HIF-1Ξ±).MethodsScaffold-bound autologous periosteal cells, which had been activated by Insulin-like Growth Factor 1 (IGF-1) or Bone Morphogenetic Protein 2 (BMP-2) gene transfer using both adeno-associated virus (AAV) and adenoviral (Ad) vectors, were applied to chondral lesions in the knee joints of miniature pigs. Six weeks after transplantation, the repair tissues were investigated for collagen type I and type II content as well as for HIF-1Ξ± expression. The functional role of phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) and mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling on BMP-2/IGF-1-induced HIF-1Ξ± expression was assessed in vitro by employing specific inhibitors.ResultsUnstimulated periosteal cells formed a fibrous extracellular matrix in the superficial zone and a fibrocartilaginous matrix in deep zones of the repair tissue. This zonal difference was reflected by the absence of HIF-1Ξ± staining in superficial areas, but moderate HIF-1Ξ± expression in deep zones. In contrast, Ad/AAVBMP-2-stimulated periosteal cells, and to a lesser degree Ad/AAVIGF-1-infected cells, adopted a chondrocyte-like phenotype with strong intracellular HIF-1Ξ± staining throughout all zones of the repair tissue and formed a hyaline-like matrix. In vitro, BMP-2 and IGF-1 supplementation increased HIF-1Ξ± protein levels in periosteal cells, which was based on posttranscriptional mechanisms rather than de novo mRNA synthesis, involving predominantly the MEK/ERK pathway.ConclusionThis pilot experimental study on a relatively small number of animals indicated that chondrogenesis by precursor cells is facilitated in deeper hypoxic zones of cartilage repair tissue and is stimulated by growth factors which enhance HIF-1Ξ± activity

    Renal tubular HIF-2Ξ± expression requires VHL inactivation and causes fibrosis and cysts.

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    The Hypoxia-inducible transcription Factor (HIF) represents an important adaptive mechanism under hypoxia, whereas sustained activation may also have deleterious effects. HIF activity is determined by the oxygen regulated Ξ±-subunits HIF-1Ξ± or HIF-2Ξ±. Both are regulated by oxygen dependent degradation, which is controlled by the tumor suppressor "von Hippel-Lindau" (VHL), the gatekeeper of renal tubular growth control. HIF appears to play a particular role for the kidney, where renal EPO production, organ preservation from ischemia-reperfusion injury and renal tumorigenesis are prominent examples. Whereas HIF-1Ξ± is inducible in physiological renal mouse, rat and human tubular epithelia, HIF-2Ξ± is never detected in these cells, in any species. In contrast, distinct early lesions of biallelic VHL inactivation in kidneys of the hereditary VHL syndrome show strong HIF-2Ξ± expression. Furthermore, knockout of VHL in the mouse tubular apparatus enables HIF-2Ξ± expression. Continuous transgenic expression of HIF-2Ξ± by the Ksp-Cadherin promotor leads to renal fibrosis and insufficiency, next to multiple renal cysts. In conclusion, VHL appears to specifically repress HIF-2Ξ± in renal epithelia. Unphysiological expression of HIF-2Ξ± in tubular epithelia has deleterious effects. Our data are compatible with dedifferentiation of renal epithelial cells by sustained HIF-2Ξ± expression. However, HIF-2Ξ± overexpression alone is insufficient to induce tumors. Thus, our data bear implications for renal tumorigenesis, epithelial differentiation and renal repair mechanisms

    Regulation of a rat VL30 element in human breast cancer cells in hypoxia and anoxia: role of HIF-1

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    Novel approaches to cancer gene therapy currently exploit tumour hypoxia to achieve transcriptional targeting using oxygen-regulated enhancer elements called hypoxia response elements. The activity of such elements in hypoxic cells is directly dependent on upregulation of the hypoxia-inducible transcription factor-1 However tumours also contain areas of anoxia, which may be considered a more tumour-selective transcriptional stimulus than hypoxia for targeting gene therapy to tumours. Another element, from the rat virus-like retrotransposon, VL30 (termed the β€˜secondary anoxia response element’) has been reported to be more highly inducible in rat fibroblasts under anoxia than hypoxia. To investigate anoxia as a potential transcriptional target in human tumours, we have examined secondary anoxia response element inducibility in two human breast cancer cell lines, MCF-7 and T47D, under anoxia, hypoxia and normoxia. In both cell types, the trimerised secondary anoxia response element showed greater inducibility in anoxia than hypoxia (1% and 0.5% O2). The anoxic response of the secondary anoxia response element was shown to be dependent on hypoxia-inducible transcription factor-1 and the presence of a hypoxia-inducible transcription binding site consensus (5β€²-ACGTG-3β€²). Mutational analysis demonstrated that the base immediately 5β€² to this modulates the anoxic/hypoxic induction of the secondary anoxia response element, such that TACGTG>GACGTG>>CACGTG. A similar correlation was found for erythropoietin, phosphoglycerate kinase 1, and aldolase hypoxia response elements, which contain these respective 5β€² flanking bases

    The antiapoptotic gene survivin is highly expressed in human chondrosarcoma and promotes drug resistance in chondrosarcoma cells in vitro

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    Background Chondrosarcoma is virtually resistant to chemotherapy and radiation therapy. Survivin, the smallest member of the inhibitor of apoptosis protein family, is a critical factor for tumor progression and resistance to conventional therapeutic approaches in a wide range of malignancies. However, the role of survivin in chondrosarcoma has not been well studied. We examined the importance of survivin gene expression in chondrosarcoma and analysed its influences on proliferation, apoptosis and resistance to chemotherapy in vitro. Methods Resected chondrosarcoma specimens from which paraffin-embedded tissues could be extracted were available from 12 patients. In vitro experiments were performed in human chondrosarcoma cell lines SW1353 and Hs819.T. Immunohistochemistry, immunoblot, quantitative PCR, RNA interference, gene-overexpression and analyses of cell proliferation and apoptosis were performed. Results Expression of survivin protein was detected in all chondrosarcoma specimens analyzed, while undetectable in adult human cartilage. RNA interference targeting survivin resulted in a G2/M-arrest of the cell cycle and led to increased rates of apoptosis in chondrosarcoma cells in vitro. Overexpression of survivin resulted in pronounced resistance to doxorubicin treatment. Conclusions These findings indicate that survivin plays a role in the pathogenesis and pronounced chemoresistance of high grade chondrosarcoma. Survivin antagonizing therapeutic strategies may lead to new treatment options in unresectable and metastasized chondrosarcoma

    In vivo Identification and Specificity assessment of mRNA markers of hypoxia in human and mouse tumors

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    <p>Abstract</p> <p>Background</p> <p>Tumor hypoxia is linked to poor prognosis, but identification and quantification of tissue hypoxia remains a challenge. The hypoxia-specificity of HIF-1Ξ± target genes in vivo has been questioned due to the confounding influence of other microenvironmental abnormalities known to affect gene expression (e.g., low pH). Here we describe a new technique that by exploiting intratumoral oxygenation heterogeneity allows us to identify and objectively rank the most robust mRNA hypoxia biomarkers.</p> <p>Methods</p> <p>Mice carrying human (FaDu<sub>dd</sub>) or murine (SCCVII) tumors were injected with the PET hypoxia tracer FAZA. Four hours post-injection tumors were removed, frozen, and crushed into milligram-sized fragments, which were transferred individually to pre-weighed tubes containing RNAlater and then weighed. For each fragment radioactivity per tissue mass and expression patterns of selected mRNA biomarkers were analyzed and compared.</p> <p>Results</p> <p>In both tumour models, fragmentation into pieces weighing 10 to 60 mg resulted in tissue fragments with highly variable relative content of hypoxic cells as evidenced by an up to 13-fold variation in FAZA radioactivity per mass of tissue. Linear regression analysis comparing FAZA retention with patterns of gene expression in individual tissue fragments revealed that CA9, GLUT1 and LOX mRNA levels were equally and strongly correlated to hypoxic extent in FaDu<sub>dd</sub>. The same link between hypoxia and gene expression profile was observed for CA9 and GLUT1, but not LOX, in SCCVII tumors. Apparent in vivo hypoxia-specificity for other putative molecular markers of tissue hypoxia was considerably weaker.</p> <p>Conclusions</p> <p>The portrayed technique allows multiple pairwise measurements of mRNA transcript levels and extent of hypoxia in individual tumors at a smallest possible volumetric scale which (by limiting averaging effects inherent to whole-tumor analysis) strengthen the conclusiveness on true hypoxia-specificity of candidate genes while limiting the required number of tumors. Among tested genes, our study identified CA9, GLUT1 and possibly LOX as highly specific biomarkers of tumor hypoxia in vivo.</p

    Differentiation in Neuroblastoma: Diffusion-Limited Hypoxia Induces Neuro-Endocrine Secretory Protein 55 and Other Markers of a Chromaffin Phenotype

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    Background: Neuroblastoma is a childhood malignancy of sympathetic embryonal origin. A high potential for differentiation is a hallmark of neuroblastoma cells. We have previously presented data to suggest that in situ differentiation in tumors frequently proceeds along the chromaffin lineage and that decreased oxygen ( hypoxia) plays a role in this. Here we explore the utility of Neuro-Endocrine Secretory Protein 55 ( NESP55), a novel member of the chromogranin family, as a marker for this process.Methodology/Principal Findings: Immunohistochemical analyses and in situ hybridizations were performed on human fetal tissues, mouse xenografts of human neuroblastoma cell lines, and on specimens of human neuroblastoma/ganglioneuroma. Effects of anaerobic exposure on gene expression by cultured neuroblastoma cells was analyzed with quantitative real-time PCR. Fetal sympathetic nervous system expression of NESP55 was shown to be specific for chromaffin cell types. In experimental and clinical neuroblastoma NESP55 immunoreactivity was specific for regions of chronic hypoxia. NESP55 expression also correlated strikingly with morphological evidence of differentiation and with other chromaffin-specific patterns of gene expression, including IGF2 and HIF2 alpha. Anaerobic culture of five neuroblastoma cell lines resulted in an 18.9-fold mean up-regulation of NESP55.Conclusions/Significance: The data confirms that chronic tumor hypoxia is a key microenvironmental factor for neuroblastoma cell differentiation, causing induction of chromaffin features and NESP55 provides a reliable marker for this neuronal to neuroendocrine transition. The hypoxia-induced phenotype is the predominant form of differentiation in stroma-poor tumors, while in stroma-rich tumors the chromaffin phenotype coexists with ganglion cell-like differentiation. The findings provide new insights into the biological diversity which is a striking feature of this group of tumors

    Relation of hypoxia inducible factor 1Ξ± and 2Ξ± in operable non-small cell lung cancer to angiogenic/molecular profile of tumours and survival

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    Hypoxia inducible factors HIF1Ξ± and HIF2Ξ± are important proteins involved in the regulation of the transcription of a variety of genes related to erythropoiesis, glycolysis and angiogenesis. Hypoxic stimulation results in rapid increase of the HIF1Ξ± and 2Ξ± protein levels, as a consequence of a redox-sensitive stabilization. The HIFΞ±s enter the nucleus, heterodimerize with the HIF1Ξ² protein, and bind to DNA at the hypoxia response elements (HREs) of target genes. In this study we evaluated the immunohistochemical expression of these proteins in 108 tissue samples from non-small-cell lung cancer (NSCLC) and in normal lung tissues. Both proteins showed a mixed cytoplasmic/nuclear pattern of expression in cancer cells, tumoural vessels and tumour-infiltrating macrophages, as well as in areas of metaplasia, while normal lung components showed negative or very weak cytoplasmic staining. Positive HIF1Ξ± and HIF2Ξ± expression was noted in 68/108 (62%) and in 54/108 (50%) of cases respectively. Correlation analysis of HIF2Ξ± expression with HIF1Ξ± expression showed a significant association (P < 0.0001, r = 0.44). A strong association of the expression of both proteins with the angiogenic factors VEGF (P < 0.004), PD-ECGF (P < 0.003) and bFGF (P < 0.04) was noted. HIF1Ξ± correlated with the expression of bek-bFGF receptor expression (P = 0.01), while HIF2Ξ± was associated with intense VEGF/KDR-activated vascularization (P = 0.002). HIF2Ξ± protein was less frequently expressed in cases with a medium microvessel density (MVD); a high rate of expression was noted in cases with both low and high MVD (P = 0.006). Analysis of overall survival showed that HIF2Ξ± expression was related to poor outcome (P = 0.008), even in the group of patients with low MVD (P = 0.009). HIF1Ξ± expression was marginally associated with poor prognosis (P = 0.08). In multivariate analysis HIF2Ξ± expression was an independent prognostic indicator (P = 0.006, t-ratio 2.7). We conclude that HIF1Ξ± and HIF2Ξ± overexpression is a common event in NSCLC, which is related to the up-regulation of various angiogenic factors and with poor prognosis. Targeting the HIF pathway may prove of importance in the treatment of NSCLC. Β© 2001 Cancer Research Campaignhttp://www.bjcancer.co

    Clinicopathologic significance of HIF-1Ξ±, p53, and VEGF expression and preoperative serum VEGF level in gastric cancer

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    <p>Abstract</p> <p>Background</p> <p>Hypoxia influences tumor growth by inducing angiogenesis and genetic alterations. Hypoxia-inducible factor 1Ξ± (HIF-1Ξ±), p53, and vascular endothelial growth factor (VEGF) are all important factors in the mechanisms inherent to tumor progression. In this work, we have investigated the clinicopathologic significance of HIF-1Ξ±, p53, and VEGF expression and preoperative serum VEGF (sVEGF) level in gastric cancer.</p> <p>We immunohistochemically assessed the HIF-1Ξ±, p53, and VEGF expression patterns in 114 specimens of gastric cancer. Additionally, we determined the levels of preoperative serum VEGF (sVEGF).</p> <p>Results</p> <p>The positive rates of p53 and HIF-1Ξ± (diffuse, deep, intravascular pattern) were 38.6% and 15.8%, respectively. The VEGF overexpression rate was 57.9%. p53 and HIF-1Ξ± were correlated positively with the depth of invasion (<it>P </it>= 0.015, <it>P </it>= 0.001, respectively). Preoperative sVEGF and p53 levels were correlated significantly with lymph node involvement (<it>P </it>= 0.010, <it>P </it>= 0.040, respectively). VEGF overexpression was more frequently observed in the old age group (β‰₯ 60 years old) and the intestinal type (<it>P </it>= 0.013, <it>P </it>= 0.014, respectively). However, correlations between preoperative sVEGF level and tissue HIF-1Ξ±, VEGF, and p53 were not observed. The median follow-up duration after operation was 24.5 months. HIF-1Ξ± was observed to be a poor prognostic factor of disease recurrence or progression (<it>P </it>= 0.002).</p> <p>Conclusion</p> <p>p53, HIF-1Ξ± and preoperative sVEGF might be markers of depth of invasion or lymph node involvement. HIF-1Ξ± expression was a poor prognostic factor of disease recurrence or progression in patients with gastric cancers.</p

    Hypoxia Impairs Primordial Germ Cell Migration in Zebrafish (Danio rerio) Embryos

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    Background: As a global environmental concern, hypoxia is known to be associated with many biological and physiological impairments in aquatic ecosystems. Previous studies have mainly focused on the effect of hypoxia in adult animals. However, the effect of hypoxia and the underlying mechanism of how hypoxia affects embryonic development of aquatic animals remain unclear. Methodology/Principal Findings: In the current study, the effect of hypoxia on primordial germ cell (PGC) migration in zebrafish embryos was investigated. Hypoxic embryos showed PGC migration defect as indicated by the presence of mis-migrated ectopic PGCs. Insulin-like growth factor (IGF) signaling is required for embryonic germ line development. Using real-time PCR, we found that the mRNA expression levels of insulin-like growth factor binding protein (IGFBP-1), an inhibitor of IGF bioactivity, were significantly increased in hypoxic embryos. Morpholino knockdown of IGFBP-1 rescued the PGC migration defect phenotype in hypoxic embryos, suggesting the role of IGFBP-1 in inducing PGC mis-migration. Conclusions/Significance: This study provides novel evidence that hypoxia disrupts PGC migration during embryonic development in fish. IGF signaling is shown to be one of the possible mechanisms for the causal link between hypoxia and PGC migration. We propose that hypoxia causes PGC migration defect by inhibiting IGF signaling through the induction of IGFBP-1

    Expression of delta-like ligand 4 (Dll4) and markers of hypoxia in colon cancer

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    BACKGROUND: Delta-like ligand 4 (Dll4) is a Notch ligand that is upregulated by hypoxia and vascular endothelial growth factor-A (VEGF-A) and is reported to have a role in tumor angiogenesis. Evidence from xenograft studies suggests that inhibiting Dll4-Notch signalling may overcome resistance to anti-VEGF therapy. The aim of this study was to characterise the expression of Dll4 in colon cancer and to assess whether it is associated with markers of hypoxia and prognosis. METHOD: In all, 177 colon cancers were represented in tissue microarrays. Immunohistochemistry was performed using validated antibodies against Dll4, VEGF, hypoxia-inducible factor (HIF)-1alpha, HIF-2alpha, prolyl hydroxylase (PHD)1, PHD2, PHD3 and carbonic anhydrase 9 (CA9). RESULTS: The expression of Dll4 was observed preferentially in the endothelium of 71% (125 out of 175) of colon cancers, but not in the endothelium adjacent to normal mucosa (none out of 107, P&lt;0.0001). The expression of VEGF was significantly associated with HIF-2alpha (P&lt;0.0001) and Dll4 (P=0.010). Only HIF-2alpha had a significant multivariate prognostic effect (hazard ratio 1.61, 95% confidence interval 1.01-2.57). Delta-like ligand 4 was also expressed by neoplastic cells, particularly neoplastic goblet cells. CONCLUSION: Endothelial expression of Dll4 is not a prognostic factor, but is significantly associated with VEGF. Assessing endothelial Dll4 expression may be critical in predicting response to anti-VEGF therapies
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