Absence of GAPDH regulation in tumor-cells of different origin under hypoxic conditions in – vitro

<p>Abstract</p> <p>Background</p> <p>Gene expression studies related to cancer diagnosis and treatment are important. In order to conduct such experiment accurately, absolutely reliable housekeeping genes are essential to normalize cancer related gene expression. The most important characteristics of such genes are their presence in all cells and their expression levels remain relatively constant under different experimental conditions. However, no single gene of this group of genes manifests always stable expression levels under all experimental conditions. Incorrect choice of housekeeping genes leads to interpretation errors of experimental results including evaluation and quantification of pathological gene expression. Here, we examined (a) the degree of GAPDH expression regulation in Hep-1-6 mouse hepatoma and Hep-3-B and HepG2 human hepatocellular carcinoma cell lines as well as in human lung adenocarcinoma epithelial cell line (A-549) in addition to both HT-29, and HCT-116 colon cancer cell lines, under hypoxic conditions <it>in vitro </it>in comparison to other housekeeping genes like β-actin, serving as experimental loading controls, (b) the potential use of GAPDH as a target for tumor therapeutic approaches was comparatively examined <it>in vitro </it>on both protein and mRNA level, by western blot and semi quantitative RT-PCR, respectively.</p> <p>Findings</p> <p>No hypoxia-induced regulatory effect on GAPDH expression was observed in the cell lines studied <it>in vitro </it>that were; Hep-1-6 mouse hepatoma and Hep-3-B and HepG2 human hepatocellular carcinoma cell lines, Human lung adenocarcinoma epithelial cell line (A-549), both colon cancer cell lines HT-29, and HCT-116.</p> <p>Conclusion</p> <p>As it is the case for human hepatocellular carcinoma, mouse hepatoma, human colon cancer, and human lung adenocarcinoma, GAPDH represents an optimal choice of a housekeeping gene and/(or) loading control to determine the expression of hypoxia induced genes in tumors of different origin. The results confirm our previous findings in human glioblastoma that this gene is not an attractive target for tumor therapeutic approaches because of the lack of GAPDH regulation under hypoxia.</p

GAPDH is not regulated in human glioblastoma under hypoxic conditions

<p>Abstract</p> <p>Background</p> <p>Gene expression studies related to cancer diagnosis and treatment are becoming more important. Housekeeping genes that are absolutely reliable are essential for these studies to normalize gene expression. An incorrect choice of housekeeping genes leads to interpretation errors of experimental results including evaluation and quantification of pathological gene expression. Here, we examined (a) the degree of regulation of GAPDH expression in human glioblastoma cells under hypoxic conditions <it>in vitro </it>in comparison to other housekeeping genes like β-actin, serving as experimental loading controls, (b) the potential use of GAPDH as a target for tumor therapeutic approaches and (c) differences in GAPDH expression between low-grade astrocytomas and glioblastomas, for which modest and severe hypoxia, respectively, have been previously demonstrated. GAPDH and β-actin expression was comparatively examined <it>in vivo </it>in human low-grade astrocytoma and glioblastoma on both protein and mRNA level, by Western blot and semiquantitative RT-PCR, respectively. Furthermore, the same proteins were determined <it>in vitro </it>in U373, U251 and GaMG human glioblastoma cells using the same methods. HIF-1α protein regulation under hypoxia was also determined on mRNA level <it>in vitro </it>in GaMG and on protein level in U251, U373 and GaMG cells.</p> <p>Results</p> <p>We observed no hypoxia-induced regulatory effect on GAPDH expression in the three glioblastoma cell lines studied <it>in vitro</it>. In addition, GAPDH expression was similar in patient tumor samples of low-grade astrocytoma and glioblastoma, suggesting a lack of hypoxic regulation <it>in vivo</it>.</p> <p>Conclusion</p> <p>GAPDH represents an optimal choice of a housekeeping gene and/or loading control to determine the expression of hypoxia induced genes at least in glioblastoma. Because of the lack of GAPDH regulation under hypoxia, this gene is not an attractive target for tumor therapeutic approaches in human glioblastoma.</p

Inhibition of N-Myc down regulated gene 1 in in vitro cultured human glioblastoma cells

AIM: To study short dsRNA oligonucleotides (siRNA) as a potent tool for artificially modulating gene expression of N-Myc down regulated gene 1 (NDRG1) gene induced under different physiological conditions (Normoxia and hypoxia) modulating NDRG1 transcription, mRNA stability and translation. METHODS: A cell line established from a patient with glioblastoma multiforme. Plasmid DNA for transfections was prepared with the Endofree Plasmid Maxi kit. From plates containing 5 x 10(7) cells, nuclear extracts were prepared according to previous protocols. The pSUPER-NDRG1 vectors were designed, two sequences were selected from the human NDRG1 cDNA (5'-GCATTATTGGCATGGGAAC-3' and 5'-ATGCAGAGTAACGTGGAAG-3'. reverse transcription polymerase chain reaction was performed using primers designed using published information on -actin and hypoxia-inducible factor (HIF)-1 mRNA sequences in GenBank. NDRG1 mRNA and protein level expression results under different conditions of hypoxia or reoxygenation were compared to aerobic control conditions using the Mann-Whitney U test. Reoxygenation values were also compared to the NDRG1 levels after 24 h of hypoxia (P < 0.05 was considered significant). RESULTS: siRNA- and iodoacetate (IAA)-mediated downregulation of NDRG1 mRNA and protein expression in vitro in human glioblastoma cell lines showed a nearly complete inhibition of NDRG1 expression when compared to the results obtained due to the inhibitory role of glycolysis inhibitor IAA. Hypoxia responsive elements bound by nuclear HIF-1 in human glioblastoma cells in vitro under different oxygenation conditions and the clearly enhanced binding of nuclear extracts from glioblastoma cell samples exposed to extreme hypoxic conditions confirmed the HIF-1 Western blotting results. CONCLUSION: NDRG1 represents an additional diagnostic marker for brain tumor detection, due to the role of hypoxia in regulating this gene, and it can represent a potential target for tumor treatment in human glioblastoma. The siRNA method can represent an elegant alternative to modulate the expression of the hypoxia induced NDRG1 gene and can help to monitor the development of the cancer disease treatment outcome through monitoring the expression of this gene in the patients undergoing the different therapeutic treatment alternatives available nowadays

Effects of HIF-1 inhibition by chetomin on hypoxia-related transcription and radiosensitivity in HT 1080 human fibrosarcoma cells

<p>Abstract</p> <p>Background</p> <p>Hypoxia-inducible factor-1 (HIF-1) overexpression has been linked to tumor progression and poor prognosis. We investigated whether targeting of HIF-1 using chetomin, a disrupter of the interaction of HIF-1 with the transcriptional coactivator p300, influences the radiosensitivity of hypoxic HT 1080 human fibrosarcoma cells.</p> <p>Methods</p> <p>Optimal dose of chetomin was determined by EGFP-HRE gene reporter assay in stably transfected HT 1080 cells. Cells were assayed for expression of the hypoxia-inducible genes carbonic anhydrase 9 (CA9) and vascular endothelial growth factor (VEGF) by RT-PCR and for clonogenic survival after irradiation with 2, 5 or 10 Gy, under normoxic or hypoxic (0.1% O₂, 12 h) conditions in the presence or absence of chetomin (150 nM, 12 h, pre-treatment of 4 h).</p> <p>Results</p> <p>Chetomin treatment significantly reduced CA9 and VEGF mRNA expression in hypoxic cells to 44.4 ± 7.2% and 39.6 ± 16.0%, respectively, of untreated hypoxic controls. Chetomin clearly reduced the modified oxygen enhancement ratio (OER') compared to untreated cells, from 2.02 to 1.27, from 1.86 to 1.22 and from 1.49 to 1.06 at the 50%, 37% and 10% clonogenic survival levels, respectively.</p> <p>Conclusion</p> <p>HIF-1 inhibition by chetomin effectively reduces hypoxia-dependent transcription and radiosensitizes hypoxic HT 1080 human fibrosarcoma cells <it>in vitro</it>.</p

Stocking and species composition of second growth forests in Peninsular Malaysia.

Management of the production forests in Malaysia is currently undergoing a major change as the total extent of undisturbed forest being harvested is diminishing. Currently most of the harvesting operations are being conducted in second growth (rotation) forests and in the near future all production forest will solely consist of only logged forests. This is expected to result in a significant reduction of the supply of raw materials to the industry because second growth forest stands are generally poorer and not so well-stocked with quality timber species. According to the forest management systems applied to these forests, namely the Selective Management System (SMS) and the Malayan Uniform System (MUS), the residual forests should be able to recover in the specified rotation cycle and there should be sufficient quality crop for the second and subsequent harvests. To understand the situation, a study was carried to assess the stocking and species composition of second rotation forests in two production forests located in Tekam Forests Reserve, Pahang and Cherul Forest Reserve, Terengganu. The study results indicated that the second rotation forests are not as productive as predicted but still able to produce an economic harvest in terms of total timber yield within the specified rotation cycle. However, based on inventory projections of existing stocks, it was found that in general the forests have not fully recovered in terms of stocking of commercial species. Species composition has been altered favouring higher dominance of non-dipterocarp species. Some of the major factors that could have contributed to this phenomenon are slower recovery of the forest after the first cut, higher mortality due to logging damage, and implementation of cutting limit prescriptions that favour high removal of dipterocarps as they are dominant in the upper diameter classes. It must be noted that the second growth forest assessed were those that were more than 20 years old. Currently, forest management practices have improved significantly and thus the recent second growth forests are expected to be in a much better condition. The information generated from this project on the status of the stocking and species composition of second growth forest will be essential for improving planning and management of the resource with the aim of enhancing future productivity

Формирование сварного шва при сварке с раздельным токоподводом к изделию

Объектом исследования является процесс дуговой сварки неплавящимся электродом в защитной среде аргона с раздельным токоподвод к изделию. Цель работы – исследование и разработка технологии сварки неплавящимся электродом в аргоне с управлением собственным магнитным дутьем, при раздельном токоподводе к изделию. В процессе исследования проводились анализ методов повышения эффективности и способы сварки неплавящимся электродом с применением импульсного питания сварочной дуги, проведен анализ сварки модулированным током и предложен наиболее перспективный способ. В результате исследования разработан процесс сварки неплавящимся электродом с управлением магнитным дутьем, при раздельном токоподводе к изделию.The object of research is the process of arc welding with a non-melting electrode in a protective argon environment. The purpose of this work is to research and develop a technology for welding with a non-melting electrode in argon with its own magnetic blast control. In the process of investigation the analysis of methods of improving efficiency and methods of consumable electrode welding using pulsed arc power, the analysis welding of the modulated current and proposed the most promising method. As a result of the research, the process of welding with a non-melting electrode with magnetic blast control was developed

Design and learning strategies applied in MOOC: a meta-analysis

The purpose of the study is to examines the dominant design and learning strategy used by various MOOCs platforms to foster students’ Self Directed Learning. Method used in the study was based on the search of relevant literature through online database such as IEEE Explore, ProQuest, ScienceDirect and ResearchGate. The keywords in the search for the relevant literature include MOOCs and learning strategy, MOOCs and design strategy, MOOCs and Self Directed Learnig. The result of the meta-analysis revealed that the most frequently used learning strtegies by the various MOOCs platforms are the social construcvist and peer-to-peer approach to learning. These two strategies are found to be related to cMOOCs and xMOOCs. Similarly, of all the designs the dominant design strategy use by MOOCs providers is cMOOC and partially the blended or hybrid MOOCs. The study revealed the dominant learning strategies employed by MOOCs platforms. This may help other MOOCs designers to give emphasis to the use of best learning strategies and perhaps improve on the existing ones. The findings may also have implication to students willling to acquire knowledge through MOOCs to choose the appropriate instruction strategy that will Foster SDL

Elevated tumor and serum levels of the hypoxia-associated protein osteopontin are associated with prognosis for soft tissue sarcoma patients

<p>Abstract</p> <p>Background</p> <p>Osteopontin (OPN) overexpression is correlated with a poor prognosis for tumor patients. However, only a few studies investigated the prognostic impact of expression of OPN in soft tissue sarcomas (STS) yet.</p> <p>Methods</p> <p>This study is based on tumor and serum samples from 93 adult STS patients. We investigated OPN protein levels in serum (n = 86) and tumor tissue (n = 80) by ELISA and OPN mRNA levels in tumor tissue (n = 68) by quantitative real-time PCR.</p> <p>Results</p> <p>No correlation was found between OPN levels in serum and tumor tissue. Moreover, an elevated OPN protein level in the serum was significantly associated with clinical parameters such as higher stage (p = 0.004), higher grade (p = 0.003), subtype (p = 0.002) and larger tumor size (p = 0.03). OPN protein levels in the tumor tissue were associated with higher stage (p = 0.06), higher grade (p = 0.003), subtype (p = 0.07) and an increased rate of relapse (p = 0.02). In addition, using a Cox's proportional hazards regression model, we found that an elevated OPN protein level in the serum and tumor tissue extracts is a significant negative prognostic factor for patients with STS. The relative risks of tumor-related death were 2.2 (p < 0.05) and 3.7 (p = 0.01), respectively.</p> <p>Conclusion</p> <p>Our data suggest OPN protein in serum as well as in tumor tissue extracts is an important prognostic factor for soft tissue sarcoma patients.</p