Regulation of redox and DNA repair genes by arsenic : low dose protection against oxidative stress?

We have evaluated the molecular responses of human epithelial cells to low dose arsenic to ascertain how target cells may respond to physiologically relevant concentrations of arsenic. Data gathered in numerous experiments in different cell types all point to the same conclusion: low dose arsenic induces what appears to be a protective response against subsequent exposure to oxidative stress or DNA damage, whereas higher doses often provoke synergistic toxicity. In particular, exposure to low, sub-toxic doses of arsenite, As(III), causes coordinate up-regulation of multiple redox and redox-related genes including thioredoxin (Trx) and glutathione reductase (GR). Glutathione peroxidase (GPx) is down-regulated in fibroblasts, but up-regulated in keratinocytes, as is glutathione S-transferase (GST). The maximum effect on these redox genes occurs after 24 h exposure to 5–10 mM As(III). This is 10-fold higher than the maximum As(III) concentrations required for induction of DNA repair genes, but within the dose region where DNA repair genes are co-ordinately down-regulated. These changes in gene regulation are brought about in part by changes in DNA binding activity of the transcription factors activating protein-1 (AP-1), nuclear factor kappa-B, and cAMP response element binding protein (CREB). Although sub-acute exposure to micromolar As(III) up-regulates transcription factor binding, chronic exposure to submicromolar As(III) causes persistent down-regulation of this response. Similar long-term exposure to micromolar concentrations of arsenate in drinking water results in a decrease in skin tumour formation in dimethylbenzanthracene (DMBA)/phorbol 12-tetradecanoate 13-acetate (TPA) treated mice. Altered response patterns after long exposure to As(III) may play a significant role in As(III) toxicology in ways that may not be predicted by experimental protocols using short-term exposures.

Fasting activates the gene expression of UCP3 independent of genes necessary for lipid transport and oxidation in skeletal muscle

Fasting triggers a complex array of adaptive metabolic and hormonal responses including an augmentation in the capacity for mitochondrial fatty acid (FA) oxidation in skeletal muscle. This study hypothesized that this adaptive response is mediated by increased mRNA of key genes central to the regulation of fat oxidation in human skeletal muscle. Fasting dramatically increased UCP3 gene expression, by 5-fold at 15 h and 10-fold at 40 h. However the expression of key genes responsible for the uptake, transport, oxidation, and re-esterification of FA remained unchanged following 15 and 40 h of fasting. Likewise there was no change in the mRNA abundance of transcription factors. This suggests a unique role for UCP3 in the regulation of FA homeostasis during fasting as adaptation to 40 h of fasting does not require alterations in the expression of other genes necessary for lipid metabolism.

Upregulation of glutathione-related genes and enzyme activities in cultured human cells by sublethgal concentrations of inorganic arsenic

Inorganic arsenic (jAs), a known human carcinogen, acts as a tumor promoter in part by inducing a rapid burst of reactive oxygen species (ROS) in mammalian cells. This causes oxidative stress and a subsequent increase in the level of cellular glutathione (GSH). Glutathione, a ubiquitous reducing sulfhydryl tripeptide, is involved in ROS detoxification and its increase may be part of an adaptive response to the oxidative stress. Glutathione related enzymes including glutathione reductase (GR) and glutathione S-transferase (GST) also play key roles in these processes. In this study the regulatory effects of inorganic arsenite (As111) on the activities of GSH-related enzymes were investigated in cultured human keratinocytes. Substantial increases in GR enzyme activity and mRNA levels were shown in keratinocytes and other human cell lines after exposure to low, subtoxic, micromolar concentrations of As111 for 24 h. Upregulation of GSH synthesis paralleled the upregulation of GR as shown by increases in glutamatecysteine lyase (GeL) enzyme activity and mRNA levels, cystine uptake, and intracellular GSH levels. Glutathione S-transferase activity was also shown to increase slightly in keratinocytes, but not in fibroblasts or breast tumor cells. Overall the results show that sublethal arsenic induces a multicomponent response in human keratinocytes that involves upregulation of parts, but not all of the GSH system and counteracts the acute toxic effects of jAs. The upregulation of GR has not previously been shown to be an integral part of this response, although GR is critical for maintaining levels of reduced GSH.

Effect of creatine supplementation on housekeeping genes in human skeletal muscle using real-time RT-PCR

The present study examined the validity and reliability of measuring the expression of various genes in human skeletal muscle using quantitative real-time RT-PCR on a GeneAmp 5700 sequence detection system with SYBR Green 1 chemistry. In addition, the validity of using some of these genes as endogenous controls (i.e., housekeeping genes) when human skeletal muscle was exposed to elevated total creatine levels and exercise was also examined. For all except 28S, linear relationships between the logarithm of the starting RNA concentrations and the cycle threshold (C_T) values were established for ß-actin, ß2-microglobulin (ß₂M), cyclophilin (CYC), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We found a linear response between C_T values and the logarithm of a given amount of starting cDNA for all the genes tested. The overall intra-assay coefficient of variance for these genes was 1.3% and 21% for raw C_T values and the linear value of 2^-C_T, respectively. Interassay variability was 2.3% for raw C_T values and 34% for the linear value of 2^-C_T. We also examined the expression of various housekeeping genes in human skeletal muscle at days 0, 1, and 5 following oral supplementation with either creatine or a placebo employing a double-blind crossover study design. Treatments were separated by a 5-wk washout period. Immediately following each muscle sampling, subjects performed two 30-s all-out bouts on a cycle ergometer. Creatine supplementation increased (P < 0.05) muscle total creatine content above placebo levels; however, there were no changes (P > 0.05) in C_T values across the supplementation periods for any of the genes. Nevertheless, 95% confidence intervals showed that GAPDH was variable, whereas ß-actin, ß₂M, and CYC were the least varying genes. Normalization of the data to these housekeeping genes revealed variable behavior for ß₂M with more stable expressions for both ß-actin and CYC. We conclude that, using real-time RT-PCR, ß-actin or CYC may be used as housekeeping genes to study gene expression in human muscle in experiments employing short-term creatine supplementation combined with high-intensity exercise.

Identification of hypothalamic genes implicated in the development of obesity in Psammomys obesus using differential display PCR

The hypothalamus is a key central controller of energy homeostasis and is the source and/or site of action of many neuropeptides involved in this process. The aim of this study was to isolate hypothalamic genes differentially expressed between lean and obese Psammomys obesus, a polygenic animal model of obesity and type 2 diabetes. Differential display PCR was used to compare hypothalamic gene expression profiles of lean and healthy, obese and hyperinsulinemic, and obese, diabetic P. obesus in both the fed and fasted states. We conducted differential display with 180 separate primer combinations to amplify approximately 9000 expressed transcripts. Sixty differentially expressed bands were excised. Taqman PCR was performed on 36 of these transcripts to confirm differential gene expression in a larger sample population. Of these 36 transcripts, 9 showed homology to known genes, and 27 were considered to be novel sequences. Gene expression profiles for two of these genes are presented here. In conclusion, differential display PCR was successfully used to isolate several transcripts that may be involved in the central regulation of energy balance. We are currently conducting numerous studies to further investigate the role of these genes in the development of obesity in P. obesus.

Conservation, duplication and divergence of the zebrafish stat5 genes

There are seven mammalian signal transducer and activator of transcription (Stat) proteins that act downstream of cytokine and growth factor receptors to mediate rapid changes in gene expression. The mammalian Stat5a and Stat5b genes show high sequence identity and lie adjacent in a head-to-head configuration next to the Stat3 gene, apparently the result of a relatively recent mammal-specific gene duplication event. We have identified and characterized two stat5 homologues that are expressed in zebrafish, named stat5.1 and stat5.2. The stat5.1 gene shows a high level of conservation with the single stat5 gene found in other teleosts and lies next to the stat3 gene, in the same relative orientation as the mammalian Stat5b gene. In contrast, the stat5.2 gene lies on a different chromosome to stat5.1 and stat3, and has diverged from the stat5 genes of other teleosts, with no apparent orthologue. Together, these data suggest that the ancestral Stat5 gene has undergone two independent gene duplication events to generate a stat5.2 paralogue in zebrafish and a Stat5a paralogue in mammals.

The systematics of freshwater crayfish of the genus Cherax Erichson (Decapoda : Parastacidae) in eastern Australia re-examined using nucleotide sequences from 12S rRNA and 16S rRNA genes

Nucleotide sequence data were used to re-examine systematic relationships and species boundaries within the genus Cherax from eastern Australia. Partial sequences were amplified from the 12S (~365 bp) and 16S (~545 bp) rRNA mitochondrial gene regions. Levels of intra- and inter-specific divergence for Cherax species were very similar between the two gene regions and similar to that reported for other freshwater crayfish for 16S rRNA. Phylogenetic analyses using the combined data provided strong support for a monophyletic group containing 11 eastern Australian species and comprising three well-defined species-groups: the 'C. destructor' group containing three species, the 'C. cairnsensis' group containing four species and the 'C. cuspidatus' group containing two species. Cherax dispar and C. robustus are distinct from all other species and each other. In addition, two northern Australian and a New Guinean species were placed in the 'Astaconephrops' group, which is the sister-group to the eastern Australian Cherax lineage. Several relationships were clarified, including: the status of northern and southern C. cuspidatus as separate species; a close relationship between C. cairnsensis and C. depressus; the validity of C. rotundus and C. setosus as separate species and their close affinities with C. destructor; and the distinctiveness of the northern forms of Cherax. The analysis of the 12S rRNA and 16S rRNA data is highly concordant with the results of previous allozyme studies.

Identification of novel genes expressed during rhabdomyosarcoma differentiation using cDNA microarrays

Rhabdomyosarcomas (RMS) are highly aggressive tumors that are thought to arise as a consequence of the regulatory disruption of the growth and differentiation of skeletal muscle progenitor cells. Normal myogenesis is characterized by the expression of the myogenic regulatory factor gene family but, despite their expression in RMS, these tumor cells fail to complete the latter stages of myogenesis. The RMS cell line RD-A was treated with 12-O-tetradecanoylphorbol-13-acetate to induce differentiation and cultured for 10 days. RNA was extracted on days 1, 3, 6, 8 and 10. A human skeletal muscle cDNA microarray was developed and used to analyze the global gene expression of RMS tumors over the time-course of differentiation. As a comparison, the genes identified were subsequently examined during the differentiated primary human skeletal muscle cultures. Prothymosin alpha (PTMA), and translocase of inner mitochondrial membrane 10 (Tim10), two genes not previously implicated in RMS, showed reduced expression during differentiation. Marked differences in the expression of PTMA and Tim10 were observed during the differentiation of human primary skeletal muscle cells. These results identify several new genes with potential roles in the myogenic arrest present in rhabdomyosarcoma. PTMA expression in RMS biopsy samples might prove to be an effective diagnostic marker for this disease.

Glucose ingestion during exercise blunts exercise-induced gene expression of skeletal muscle fat oxidative genes.

Ingestion of carbohydrate during exercise may blunt the stimulation of fat oxidative pathways by raising plasma insulin and glucose concentrations and lowering plasma free fatty acid (FFA) levels, thereby causing a marked shift in substrate oxidation. We investigated the effects of a single 2-h bout of moderate-intensity exercise on the expression of key genes involved in fat and carbohydrate metabolism with or without glucose ingestion in seven healthy untrained men (22.7 ± 0.6 yr; body mass index: 23.8 ± 1.0 kg/m²; maximal O2 consumption: 3.85 ± 0.21 l/min). Plasma FFA concentration increased during exercise (P < 0.01) in the fasted state but remained unchanged after glucose ingestion, whereas fat oxidation (indirect calorimetry) was higher in the fasted state vs. glucose feeding (P < 0.05). Except for a significant decrease in the expression of pyruvate dehydrogenase kinase-4 (P < 0.05), glucose ingestion during exercise produced minimal effects on the expression of genes involved in carbohydrate utilization. However, glucose ingestion resulted in a decrease in the expression of genes involved in fatty acid transport and oxidation (CD36, carnitine palmitoyltransferase-1, uncoupling protein 3, and 5'-AMP-activated protein kinase-α2; P < 0.05). In conclusion, glucose ingestion during exercise decreases the expression of genes involved in lipid metabolism rather than increasing genes involved in carbohydrate metabolism.

Decreased expression of adipogenic genes in obese subjects with type 2 diabetes

Objective: Our objective was to delineate the potential role of adipogenesis in insulin resistance and type 2 diabetes. Obesity is characterized by an increase in adipose tissue mass resulting from enlargement of existing fat cells (hypertrophy) and/or from increased number of adipocytes (hyperplasia). The inability of the adipose tissue to recruit new fat cells may cause ectopic fat deposition and insulin resistance.

Research Methods and Procedures: We examined the expression of candidate genes involved in adipocyte proliferation and/or differentiation [ CCAAT/enhancer-binding protein (C/EBP) alpha, C/EBPdelta, GATA domain-binding protein 3 (GATA3), C/EBPbeta, peroxisome proliferator-activated receptor (PPAR) gamma2, signal transducer and activator of transcription 5A (STAT5A), Wnt-10b, tumor necrosis factor alpha, sterol regulatory element-binding protein 1c (SREBP1c), 11 beta-hydroxysteroid dehydrogenase, PPARG angiopoietin-related protein (PGAR), insulin-like growth factor 1, PPARitalic gamma coactivator 1alpha, PPARitalic gamma coactivator 1beta, and PPARdelta] in subcutaneous adipose tissue from 42 obese individuals with type 2 diabetes and 25 non-diabetic subjects matched for age and obesity.

Results: Insulin sensitivity was measured by a 3-hour 80 mU/m2 per minute hyperinsulinemic glucose clamp (100 mg/dL). As expected, subjects with type 2 diabetes had lower glucose disposal (4.9 plusminus 1.9 vs. 7.5 plusminus 2.8 mg/min per kilogram fat-free mass; p < 0.001) and larger fat cells (0.90 plusminus 0.26 vs. 0.78 plusminus 0.17 mum; p = 0.04) as compared with obese control subjects. Three genes (SREBP1c, p < 0.01; STAT5A, p = 0.02; and PPARitalic gamma2, p = 0.02) had significantly lower expression in obese type 2 diabetics, whereas C/EBPbeta only tended to be lower (p = 0.07).

Discussion: This cross-sectional study supports the hypothesis that impaired expression of adipogenic genes may result in impaired adipogenesis, potentially leading to larger fat cells in subcutaneous adipose tissue and insulin resistance.

Identification of genes differentially regulated by the P210 BCR/ABL1 fusion oncogene using cDNA microarrays

Objective: The t(9;22) translocation is associated with more than 95% of cases of chronic myeloid leukemia. The resulting fusion of the BCR and ABL1 loci produces the constitutively active BCR/ABL1 tyrosine kinase. A wide range of signal transduction molecules are activated by BCR/ABL1, including MYC, PI-3 kinase, and different STAT molecules. In contrast, relatively few genes are known to be regulated by BCR/ABL1 at the level of transcription.

Materials and Methods: In an effort to better understand the transcriptional program activated by BCR/ABL1, we used cDNA microarrays to evaluate the relative expression of approximately 6450 human genes in U937 myelomonocytic cells expressing P210 BCR/ABL1 via a tetracycline-inducible promoter.

Results: We confirmed the previously reported up-regulation of the PIM1 and JUN oncogenes by BCR/ABL1. In addition, we identified 59 more genes up-regulated by BCR/ABL1. Interestingly, roughly one third of these were genes previously reported to be interferon (IFN)-responsive, including the OAS1, IFIT1, IFI16, ISGF3G, and STAT1 genes. An additional seven BCR/ABL1-regulated genes were found to be IFN-responsive in U937 cells. The expression profile also included genes encoding transcription factors, kinases, and signal transduction molecules, as well as genes regulating cell growth, differentiation, apoptosis, and cell adhesion, features previously suggested to be affected by BCR/ABL1.

Conclusion: These observations shed novel insight into the mechanism of BCR/ABL1 action and provide a range of targets for further investigation.

An effective non-parametric method for globally clustering genes from expression profiles

Clustering is widely used in bioinformatics to find gene correlation patterns. Although many algorithms have been proposed, these are usually confronted with difficulties in meeting the requirements of both automation and high quality. In this paper, we propose a novel algorithm for clustering genes from their expression profiles. The unique features of the proposed algorithm are twofold: it takes into consideration global, rather than local, gene correlation information in clustering processes; and it incorporates clustering quality measurement into the clustering processes to implement non-parametric, automatic and global optimal gene clustering. The evaluation on simulated and real gene data sets demonstrates the effectiveness of the algorithm.

Impaired expression of notch signaling genes in aged human skeletal muscle

Notch signaling is essential for myogenesis and the regenerative potential of skeletal muscle: however, its regulation in human muscle is yet to be fully characterized. Increased expression of Notch3, Jagged1. Hes1, and Hes6 gene transcripts were observed during differentiation of cultured human skeletal muscle cells. Furthermore, significantly lower expressions of Notch1, Jagged1, Numb, and Delta-like 1 were evident in muscle biopsies from older men (60-75 years old) compared to muscle from younger men (18-25 years old). Importantly, with supervised resistance exercise training, expression of Notch1 and Hes6 genes were increased and Delta-like 1 and Numb expression were decreased. The differences in Notch expression between the age groups were no longer evident following training. These results provide further evidence to support the role of Notch in the impaired regulation of muscle mass with age and suggest that some of the benefits provided by resistance training may be mediated through the Notch signaling pathway.