Differences In Transcriptional Profiles Research Articles

Reversine: A Chemical Probe Providing Insight into Myeloma Biology and Potential Therapeutic Applications.

Multiple myeloma (MM) is proposed to be driven by a CD138− CD20+ population of “MM stem cells” (MMSCs) which give rise to the more “differentiated” CD138+ malignant plasma cells (PCs), which have historically been the main target of anti-MM therapeutics. We hypothesized that inhibition of “differentiation” of these MMSCs to CD138+ derivative MM PCs could allow for expansion of the MMSC compartment and facilitate its cellular and molecular characterization. To test this hypothesis, we performed in vitro studies of MM cells (cell lines and primary MM cells) with reversine (2-(4-Morpholinoanilino)-6-cyclohexylaminopurine), a small molecule known to suppress differentiation (e.g. of myoblasts to myocytes) and/or cause de-differentiation of lineage-committed cells. We observed no reversine-induced expansion in CD138− populations deemed to represent MMSCs and no de-differentiation of CD138+ CD20− MM PCs to more “immature” CD138− CD20+ phenotype. Instead, sub-uM doses of reversine (as low as 0.1 uM, and significantly lower than the >5 uM required for its de-differentiation effects in other tissue type models) triggered major increases in MM cell death, including cells resistant to conventional (e.g. Dex, alkylating agents, anthracyclines, thalidomide, bortezomib, immunomodulatory thalidomide derivatives) and/or investigational agents (e.g. Apo2L/TRAIL) anti-MM agents. PBMCs, stromal cells, and immortalized hepatocytes were less sensitive to reversine than MM cells. Reversine was also active against a broad panel of neoplastic cells, e.g. various leukemia and lymphoma cell lines, suggesting a potentially broader spectrum of anti-neoplastic properties. Reversine was active against MM cells which are independent from or unresponsive to bone marrow stromal cell (BMSC) and was also able to overcome the effect of cytokine- (e.g.IL-6, IGF-1) or stroma-derived protection to BMSCs-responsive MM cells. The anti-MM effect of reversine involved G2 cell cycle arrest and commitment to cell death by drug exposure as short as 8hrs. Cleavage of PARP and caspase 3 were observed, and protein levels for diverse signaling effectors in the B-Raf and Akt cascades were decreased. Reversine enhanced responses of primary MM cells to other anti-MM agents, e.g. Dex, and had additive effects with cytotoxic chemotherapeutics or bortezomib. Reversine affected the molecular profiles of MM cells in a manner distinct from other anti-MM agents studied to date. Unlike bortezomib, reversine did not decrease NF-κB transcriptional activity, or affect the levels of several Bcl-2 family members, inhibitors of apoptosis, or heat shock proteins. These data suggest that reversine kills MM cells in an NF-κB independent manner, and also supports a broader conclusion that reversine has distinct profiles of molecular effects and anti-MM activity. Differential transcriptional profiling of MM cells with >1 vs. <1 log differential sensitivity to the normal tissues tested identified candidate markers correlating with increased reversine sensitivity, including molecules with known roles in malignant transformation, e.g. PKCiota, polo-like kinase-1 and c-myb. These results, coupled with ongoing in vivo studies, suggest that reversine may represent a distinct class of agents with therapeutic potential for specific subset(s) of MM and perhaps other hematologic neoplasias.

Differential transcription profiles in Trypanosoma cruzi associated with clinical forms of Chagas disease: Maxicircle NADH dehydrogenase subunit 7 gene truncation in asymptomatic patient isolates

The majority of individuals in the chronic phase of Chagas disease are asymptomatic (indeterminate form). Every year 2–3% of these individuals develop severe clinical manifestations (cardiac and digestive forms). In this study a Trypanosoma cruzi DNA microarray was used to compare the transcript profiles of six human isolates: three from asymptomatic and three from cardiac patients. Seven signals were expressed differentially between the two classes of isolates, including tryparedoxin, surface protease GP63, cyclophilin, some hypothetical proteins and the pre-edited maxicircle gene NADH dehydrogenase subunit 7 (ND7). The approximately 30-fold greater signal in cardiac strains for ND7 was the most pronounced of the group, and differential levels of pre-edited ND7 transcript confirmed the microarray analysis. The ND7 gene from asymptomatic isolates showed a deletion of 455bp from nt 222 to nt 677 relative to ND7 of the CL Brener reference strain. The ND7 gene structure correlated with disease manifestation for 20 isolates from clinically characterised, chronic phase patients. The ND7 lesion produces a truncated product that could impair the function of mitochondrial complex I. Possible links between the integrity of the electron transport chain and symptom presentation are discussed. We propose that ND7 and other genes of the pathway constitute valuable targets for PCR assays in the differential diagnosis of the infective T. cruzi strain. While this hypothesis requires validation by the examination of additional recent parasite isolates from patients with defined pathologies, the identification of specific molecular markers represents a promising advance in the association between parasite genetics and disease pathology.

DNA micro-array-based identification of bile-responsive genes in Lactobacillus plantarum

The purpose of this study was to determine the global transcriptional response in a food-associated lactic acid bacterium during bile stress. Clone-based DNA micro-arrays were employed to describe the global transcriptional response of Lactobacillus plantarum WCFS1 towards 0.1% porcine bile. Comparison of differential transcript profiles obtained during growth of Lact. plantarum on plates with and without bile revealed 28 and 62 putative genes, of which the expression was at least 2.5-fold up- or down-regulated by bile, respectively. Approximately, 50% of these genes appeared genetically linked, and 12 bile-responsive gene clusters were identified. Seven of the identified bile-responsive genes and gene clusters encode typical stress-related functions, including glutathione reductase and glutamate decarboxylase, involved in oxidative and acid stress, respectively. Moreover, 14 bile-responsive genes and gene clusters were identified that encode proteins that are located in the cell envelope, including the dlt operon and the F1F0 ATPase. The identification of a relatively high number of genes encoding cell envelope functions indicates a major impact of bile acids on the integrity and/or functionality of the cytoplasmic membrane and cell wall. The data presented here provide valuable clues towards the defence mechanisms that play a role during bile stress in Lact. plantarum.

Physiological genomics in PG and beyond: October to December 2005

physiological genomics ( PG ) publishes, on a quarterly basis, brief editorial perspectives entitled “Physiological genomics in PG and beyond.” These editorial perspectives provide brief, accessible highlights of the studies published in PG in the most recent quarter, and relate those studies to

Acclimatization to chronic hypobaric hypoxia is associated with a differential transcriptional profile between the right and left ventricle

Acute hypobaric hypoxia induces a transient reactivation of the fetal-metabolic gene program in the rat heart. Although chronic hypobaric hypoxia causes alterations in metabolism and cardiac function, little is known about the transcriptional profile associated with acclimatization to chronic hypoxia. Because in chronic hypoxia only the right ventricle is exposed to pressure overload (pulmonary hypertension), we hypothesized that chronic hypobaric hypoxia induces a differential transcriptional profile in the right and left ventricle. Male Wistar rats were exposed to a hypobaric environment (11% O2) for 4, 10, and 12 weeks. Right and left ventricular tissue was isolated for histology and candidate gene expression. Chronic hypobaric hypoxia induced right ventricular hypertrophy without fibrosis. In the right ventricle, changes in metabolic gene expression suggested a downregulation of fatty acid metabolism and an increase in glucose metabolism, while left ventricular metabolic gene expression suggested restoration of fatty acid metabolism. While myosin heavy chain isoform transcript levels in the right ventricle indicated a progressive reactivation of the fetal iso-gene pattern, there was normalization of myosin iso-gene expression in the left ventricle. Similarly, sarcoendoplasmic reticulum ATPase 2a (SERCA2a) transcript levels in the right ventricle decreased by 12 weeks of chronic hypoxia exposure, whereas, left ventricular SERCA2a expression was unchanged. In conclusion, acclimatization to chronic hypobaric hypoxia induced a differential transcriptional response between the right and left ventricle. We speculate that reactivation of the fetal-metabolic program in the right ventricle is adaptive to pressure overload.

Differential Transcriptional Expression Profiles of Juvenile and Adult Calvarial Bone

It has widely been observed that young children are capable of reossifying large calvarial defects, while adults lack this endogenous tissue-engineering capacity. The ability of juvenile animals to regenerate calvarial defects has been investigated in multiple animal models, including mice. In this study, the authors used cDNA microarrays to investigate the expression of osteogenesis-associated genes upstream and downstream of Runx2 in juvenile and adult mouse calvaria. Nonsuture-associated parietal bone discs were harvested from 6-day-old (n = 50) and 60-day-old (n = 35) male CD-1 mice. After separation of the underlying dura mater and overlying pericranium, the calvarial discs were snap-frozen and RNA was extracted from pooled samples of calvaria for microarray analysis. Genes analyzed included cytokines, receptors, and cell-surface and matrix proteins both upstream and downstream of Runx2. Genes associated with the Runx2 pathway had notably higher levels in the juvenile versus adult calvaria. All genes except for osteocalcin were expressed at least twofold higher in the juvenile calvaria. This pattern was validated with quantitative real-time polymerase chain reaction. In addition, mRNA for potent osteoinductive growth factors was present at higher levels in the juvenile compared with the adult calvaria. These findings reflect a genomic environment of active osteoblast differentiation and ossification in the juvenile calvaria compared with the adult "quiescent" calvarial tissue. These data suggest that a decreased osteogenic potential of adult calvarial osteoblasts may, in part, explain the inability of adult animals to heal calvarial defects.

Molecular Characterization of Thyroid Toxicity: Anchoring Gene Expression Profiles to Biochemical and Pathologic End Points

Organic iodides have been shown to induce thyroid hypertrophy and increase alterations in colloid in rats, although the mechanism involved in this toxicity is unclear. To evaluate the effect that free iodide has on thyroid toxicity, we exposed rats for 2 weeks by daily gavage to sodium iodide (NaI). To compare the effects of compounds with alternative mechanisms (increased thyroid hormone metabolism and decreased thyroid hormone synthesis, respectively), we also examined phenobarbital (PB) and propylthiouracil (PTU) as model thyroid toxicants. Follicular cell hypertrophy and pale-staining colloid were present in thyroid glands from PB-treated rats, and more severe hypertrophy/colloid changes along with diffuse hyperplasia were present in thyroid glands from PTU-treated rats. In PB-and PTU-treated rats, thyroid-stimulating hormone (TSH) levels were significantly elevated, and both thyroxine and triiodothyronine hormone levels were significantly decreased. PB induced hepatic uridine diphosphate-glucuronyltransferase (UDPGT) activity almost 2-fold, whereas PTU reduced hepatic 5′-deiodinase I (5′-DI) activity to < 10% of control in support of previous reports regarding the mechanism of action of each chemical. NaI also significantly altered liver weights and UDPGT activity but did not affect thyroid hormone levels or thyroid pathology. Thyroid gene expression analyses using Affymetrix U34A GeneChips, a regularized t-test, and Gene Map Annotator and Pathway Profiler demonstrated significant changes in rhodopsin-like G-protein–coupled receptor transcripts from all chemicals tested. NaI demonstrated dose-dependent changes in multiple oxidative stress–related genes, as also determined by principal component and linear regression analyses. Differential transcript profiles, possibly relevant to rodent follicular cell tumor outcomes, were observed in rats exposed to PB and PTU, including genes involved in Wnt signaling and ribosomal protein expression.

A comprehensive analysis of gene expression alterations in a newly synthesized Paspalum notatum autotetraploid

Although the causes of novel variation in polyploids are not well understood, they could involve changes in gene expression through dosage-regulation, altered regulatory interactions and rapid genetic/epigenetic changes. The objective of this work was to contribute to the elucidation of the patterns and mechanisms of gene regulation during the early stages of polyploid formation. The genome-wide expression of ∼10,000 genes was monitored by using differential display transcript profiling on flowers of a diploid genotype and a newly-formed autotetraploid derivative. Differential expression of 64 clones was validated by reverse-Northern blot. Sequence analysis showed that 42 were homologous to 26 distinct genes of known function while the remaining 22 were classified as novel sequences. Genes detected were involved in processes of DNA repair, chromatin structure modification, regulation of transcription, proteolysis, protein folding, carbohydrate and lipid metabolism and signal transduction. Our results show evidence for gene expression alterations occurring immediately after polyploidization in Paspalum.

Spatial Heterogeneity of Endothelial Phenotypes Correlates With Side-Specific Vulnerability to Calcification in Normal Porcine Aortic Valves

Calcific aortic valve sclerosis involves inflammatory processes and occurs preferentially on the aortic side of endothelialized valve leaflets. Although the endothelium is recognized to play critical roles in focal vascular sclerosis, the contributions of valvular endothelial phenotypes to aortic valve sclerosis and side-specific susceptibility to calcification are poorly understood. Using RNA amplification and cDNA microarrays, we identified 584 genes as differentially expressed in situ by the endothelium on the aortic side versus ventricular side of normal adult pig aortic valves. These differential transcriptional profiles, representative of the steady state in vivo, identify globally distinct endothelial phenotypes on opposite sides of the aortic valve. Several over-represented biological classifications with putative relevance to endothelial regulation of valvular homeostasis and aortic-side vulnerability to calcification were identified among the differentially expressed genes. Of note, multiple inhibitors of cardiovascular calcification were significantly less expressed by endothelium on the disease-prone aortic side of the valve, suggesting side-specific permissiveness to calcification. However, coexisting putative protective mechanisms were also expressed. Specifically, enhanced antioxidative gene expression and the lack of differential expression of proinflammatory molecules on the aortic side may protect against inflammation and lesion initiation in the normal valve. These data implicate the endothelium in regulating valvular calcification and suggest that spatial heterogeneity of valvular endothelial phenotypes may contribute to the focal susceptibility for lesion development.

Transcriptome analysis of acetate metabolism in Corynebacterium glutamicum using a newly developed metabolic array.

Following the determination of the whole-genome sequence of Corynebacterium glutamicum, we have developed a DNA array to extensively investigate gene expression and regulation relevant to carbon metabolism. For this purpose, a total of 120 C. glutamicum genes, including those in central metabolism and amino acid biosyntheses, were amplified by PCR and printed onto glass slides. The resulting array, designated a "metabolic array", was used for hybridization with fluorescently labeled cDNA probes generated by reverse transcription from total RNA samples. As the first demonstration of transcriptome analysis in this industrially important microorganism, we applied the metabolic array to study differential transcription profiles between cells grown on glucose and on acetate as the sole carbon source. The changes in gene expression observed for the known acetate-regulated genes (aceA, aceB, pta, and ack) were well consistent with the literature data of northern analyses and enzyme assays, indicating the utility of the metabolic array in transcriptome analysis of C. glutamicum. In addition to the known responses, many previously unrecognized co-regulated genes were identified. For example, several TCA cycle genes, such as gltA, sdhA, sdhB, fumH, and mdh, and the gluconeogenic gene pck were up-regulated in the acetate medium. On the other hand, a few genes involved in glycolysis and the pentose phosphate pathway, as well as many amino acid biosynthetic genes, were down-regulated in acetate. Furthermore, two gap genes, gapA and gapB, were found to be inversely regulated, suggesting the presence of a new regulatory step for carbon metabolism between glycolysis and gluconeogenesis.