Construction and evaluation of a prognostic risk assessment model of gastric cancer by using hypoxia features.

In this study, mRNA expression of gastric cancer tissue and clinical data of patients in TCGA-STAD dataset were used, together with the hypoxia-related gene sets in the MsigDB database, to screen hypoxia-related differentially expressed genes (DEGs) in GC. Thereafter, univariate and multivariate Cox regression analyses were carried out on hypoxia-related DEGs. The optimal feature genes related to prognosis were obtained to construct a prognostic risk assessment model. According to the model, the riskScore of GC patients was measured, and GC samples were assigned into high- and low-risk groups in accordance with the median riskScore. Based on the Kaplan-Meier curve and Receiver operating characteristic curve, validity of the prognostic risk assessment model was measured. Gene set enrichment analysis was performed on the two risk groups through Gene set enrichment analysis software. The results revealed that in the high-risk group, 9 signaling pathways were remarkably activated in several terms, like focal adhesion, extracellular matrix receptor interaction, Cell adhesion molecules cams, Cytokine-cytokine receptor interaction, TGF-beta signaling pathway, NOD-like receptor signaling pathway, JAK-STAT signaling pathway, Toll-like receptor signaling pathway and MAPK signaling pathway. In combination with riskScore and clinical factors, univariate and multivariate Cox regression analyses verified the independence of the model. Meanwhile, a nomogram was constructed to predict the 1-, 3- and 5-year survival of GC patients. The calibration curve indicated that the survival status predicted by the nomogram fitted better with actual survival status. On the whole, the prognostic risk model of GC on the basis of hypoxia-related genes demonstrated good predictive ability. It can provide more powerful technical support for clinicians to make prognostic determination and therapeutic plans.

Bayesian inference of selection in the Wright-Fisher diffusion model.

The increasing availability of population-level allele frequency data across one or more related populations necessitates the development of methods that can efficiently estimate population genetics parameters, such as the strength of selection acting on the population(s), from such data. Existing methods for this problem in the setting of the Wright-Fisher diffusion model are primarily likelihood-based, and rely on numerical approximation for likelihood computation and on bootstrapping for assessment of variability in the resulting estimates, requiring extensive computation. Recent work has provided a method for obtaining exact samples from general Wright-Fisher diffusion processes, enabling the development of methods for Bayesian estimation in this setting. We develop and implement a Bayesian method for estimating the strength of selection based on the Wright-Fisher diffusion for data sampled at a single time point. The method utilizes the latest algorithms for exact sampling to devise a Markov chain Monte Carlo procedure to draw samples from the joint posterior distribution of the selection coefficient and the allele frequencies. We demonstrate that when assumptions about the initial allele frequencies are accurate the method performs well for both simulated data and for an empirical data set on hypoxia in flies, where we find evidence for strong positive selection in a region of chromosome 2L previously identified. We discuss possible extensions of our method to the more general settings commonly encountered in practice, highlighting the advantages of Bayesian approaches to inference in this setting.

Induction and Assessment of Hypoxia in Glioblastoma Cells In Vitro.

To simulate and study the hypoxic microenvironment associated with intracerebral glioma in vivo, simple and reproducible methods are described and discussed for inducing hypoxia or chemical pseudohypoxia in glioma cell cultures and assessing their effects on the expression and nuclear translocation of hypoxia-inducible factor (HIF)-1α, a key transcriptional factor of oxygen homeostasis, by Western blot analysis and immunocytochemistry.

Integrative Analysis of DCE-MRI and Gene Expression Profiles in Construction of a Gene Classifier for Assessment of Hypoxia-Related Risk of Chemoradiotherapy Failure in Cervical Cancer.

PURPOSE: A 31-gene expression signature reflected in dynamic contrast enhanced (DCE)-MR images and correlated with hypoxia-related aggressiveness in cervical cancer was identified in previous work. We here aimed to construct a dichotomous classifier with key signature genes and a predefined classification threshold that separated cervical cancer patients into a more and less hypoxic group with different outcome to chemoradiotherapy. EXPERIMENTAL DESIGN: A training cohort of 42 patients and two independent cohorts of 108 and 131 patients were included. Gene expression data were generated from tumor biopsies by two Illumina array generations (WG-6, HT-12). Technical transfer of the classifier to a reverse transcription quantitative PCR (RT-qPCR) platform was performed for 74 patients. The amplitude ABrix in the Brix pharmacokinetic model was extracted from DCE-MR images of 64 patients and used as an indicator of hypoxia. RESULTS: Classifier candidates were constructed by integrative analysis of ABrix and gene expression profiles in the training cohort and evaluated by a leave-one-out cross-validation approach. On the basis of their ability to separate patients correctly according to hypoxia status, a 6-gene classifier was identified. The classifier separated the patients into two groups with different progression-free survival probability. The robustness of the classifier was demonstrated by successful validation of hypoxia association and prognostic value across cohorts, array generations, and assay platforms. The prognostic value was independent of existing clinical markers, regardless of clinical endpoints. CONCLUSIONS: A robust DCE-MRI-associated gene classifier has been constructed that may be used to achieve an early indication of patients' risk of hypoxia-related chemoradiotherapy failure. Clin Cancer Res; 22(16); 4067-76. ©2016 AACR.

Detailed assessment of gene activation levels by multiple hypoxia-responsive elements under various hypoxic conditions.

OBJECTIVE: HIF-1/HRE pathway is a promising target for the imaging and the treatment of intractable malignancy (HIF-1; hypoxia-inducible factor 1, HRE; hypoxia-responsive element). The purposes of our study are: (1) to assess the gene activation levels resulting from various numbers of HREs under various hypoxic conditions, (2) to evaluate the bidirectional activity of multiple HREs, and (3) to confirm whether multiple HREs can induce gene expression in vivo. METHODS: Human colon carcinoma HCT116 cells were transiently transfected by the constructs containing a firefly luciferase reporter gene and various numbers (2, 4, 6, 8, 10, and 12) of HREs (nHRE+, nHRE-). The relative luciferase activities were measured under various durations of hypoxia (6, 12, 18, and 24 h), O2 concentrations (1, 2, 4, 8, and 16 %), and various concentrations of deferoxamine mesylate (20, 40, 80, 160, and 320 µg/mL growth medium). The bidirectional gene activation levels by HREs were examined in the constructs (dual-luc-nHREs) containing firefly and Renilla luciferase reporter genes at each side of nHREs. Finally, to test whether the construct containing 12HRE and the NIS reporter gene (12HRE-NIS) can induce gene expression in vivo, SPECT imaging was performed in a mouse xenograft model. RESULTS: (1) gene activation levels by HREs tended to increase with increasing HRE copy number, but a saturation effect was observed in constructs with more than 6 or 8 copies of an HRE, (2) gene activation levels by HREs increased remarkably during 6-12 h of hypoxia, but not beyond 12 h, (3) gene activation levels by HREs decreased with increasing O2 concentrations, but could be detected even under mild hypoxia at 16 % O2, (4) the bidirectionally proportional activity of the HRE was confirmed regardless of the hypoxic severity, and (5) NIS expression driven by 12 tandem copies of an HRE in response to hypoxia could be visualized on in vivo SPECT imaging. CONCLUSION: The results of this study will help in the understanding and assessment of the activity of multiple HREs under hypoxia and become the basis for hypoxia-targeted imaging and therapy in the future.

Immunohistochemical assessment of intrinsic and extrinsic markers of hypoxia in reproductive tissue: differential expression of HIF1α and HIF2α in rat oviduct and endometrium.

Hypoxia is thought to be critical in regulating physiological processes within the female reproductive system, including ovulation, composition of the fluid in the oviductal/uterine lumens and ovarian follicle development. This study examined the localisation of exogenous (pimonidazole) and endogenous [hypoxia inducible factor 1α and 2α (HIF1α, -2α), glucose transporter type 1 (GLUT1) and carbonic anhydrase 9 (CAIX)] hypoxia-related antigens within the oviduct and uterus of the rat reproductive tract. The extent to which each endogenous antigen co-compartmentalised with pimonidazole was also assessed. Female Wistar Furth rats (n = 10) were injected intraperitoneally with pimonidazole (60 mg/kg) 1 h prior to death. Reproductive tissues were removed immediately following death and fixed in 4% paraformaldehyde before being embedded in paraffin. Serial sections were cut (6-7 µm thick) and antigens of interest identified using standard immunohistochemical procedures. The mucosal epithelia of the ampulla, isthmus and uterus were immunopositive for pimonidazole in most sections. Co-compartmentalisation of pimonidazole with HIF1α was only expressed in the mucosa of the uterus whilst co-compartmentalisation with HIF2α was observed in the mucosa of the ampulla, isthmus and uterus. Both GLUT1 and CAIX were co-compartmentalised with pimonidazole in mucosa of the isthmus and uterus. This study confirms that mucosal regions of the rat oviduct and uterus frequently experience severe hypoxia and there are compartment specific variations in expression of endogenous hypoxia-related antigens, including the HIF isoforms. The latter observation may relate to target gene specificity of HIF isoforms or perhaps HIF2α's responsiveness to non-hypoxic stimuli such as hypoglycaemia independently of HIF1α.

In vivo identification and specificity assessment of mRNA markers of hypoxia in human and mouse tumors.

BACKGROUND: Tumor hypoxia is linked to poor prognosis, but identification and quantification of tissue hypoxia remains a challenge. The hypoxia-specificity of HIF-1α 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. METHODS: Mice carrying human (FaDudd) 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. RESULTS: 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 FaDudd. 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. CONCLUSIONS: 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.

Application of the comet assay for assessment of oxidative DNA damage in circulating lymphocytes of Tetralogy of Fallot patients.

Tetralogy of Fallot (TOF) is a common and severe cyanotic congenital heart defect characterized by frequent episodes of hypoxia due to cyanosis. The hypoxia of cyanotic heart disease results in a down-regulation of antioxidant defenses, making cells vulnerable to oxidant damage, which subsequently leads to the single strand breaks and oxidative DNA damage. Quantification of DNA damage was performed in circulating lymphocytes of Tetralogy of Fallot patients (n=63) and healthy controls (n=65). The damage of DNA was assessed by alkaline comet assay in lymphocytes isolated from all children followed by silver staining. The DNA migrates out of the nucleus forming a tail, which represents the extent of DNA damage in individual cells. TOF patients exerted a higher percent of comet tails, which are indicative of DNA damage, when compared to control children (p<0.001). The mean comet tail length was significantly higher in TOF patients (2.57+/-0.29) when compared with healthy controls (1.28+/-0.32). The results showed that hypoxia is associated with an increase in the level of oxidants and a simultaneous decrease in the level of antioxidants in patients. Hence, the present study concludes unequivocally that hypoxia causes oxidative DNA damage in TOF patients.

An assessment of the role of the inhibitory gamma subunit of the retinal cyclic GMP phosphodiesterase and its effect on the p42/p44 mitogen-activated protein kinase pathway in animal and cellular models of pulmonary hypertension.

1. We have previously reported that the inhibitory gamma subunit of the rod photoreceptor type 6 cyclic GMP phosphodiesterase (PDEgamma) is expressed in nonretinal tissues and is involved in the stimulation of the p42/p44 mitogen-activated protein kinase (MAPK) pathway by growth factors and G-protein-coupled receptor agonists. We have now investigated whether PDEgamma plays a role in modulating chronic hypoxic-dependent mitogenic signalling pathways in pulmonary smooth muscle from rats with pulmonary hypertension (PHT). 2. We show for the first time that PDEgamma is expressed in rat main, first, intrapulmonary and resistance pulmonary arteries. Moreover, its expression is increased in all the arteries to varying extents by chronic hypoxia. The extent of the increased expression of PDEgamma is correlated with an enhanced activation of p42/p44 MAPK in these vessels. 3. We also report that PDEgamma translation from mRNA transcript is increased in cultured human pulmonary artery smooth muscle cells subjected to chronic hypoxia for 14 days. This was correlated with hypoxic-dependent increase in p42/p44 MAPK activation. 4. In conclusion, our studies identify for the first time a major chronic hypoxic-dependent change in the phenotypic expression of an intermediate protein regulating mitogenic signalling in pulmonary arteries. This may have a significant effect on arterial remodelling in PHT. Future studies will focus on strategies designed to knockout rod PDEgamma to assess whether this rescues rats from chronic hypoxic-dependent changes in arterial remodelling and PHT.