Identification of gene networks associated with acute myeloid leukemia by comparative molecular methylation and expression profiling

Around 80% of acute myeloid leukemia (AML) patients achieve a complete remission, however many will relapse and ultimately die of their disease. The association between karyotype and prognosis has been studied extensively and identified patient cohorts as having favourable [e.g. t(8; 21), inv (16)/t(16; 16), t(15; 17)], intermediate [e.g. cytogenetically normal (NK-AML)] or adverse risk [e.g. complex karyotypes]. Previous studies have shown that gene expression profiling signatures can classify the sub-types of AML, although few reports have shown a similar feature by using methylation markers. The global methylation patterns in 19 diagnostic AML samples were investigated using the Methylated CpG Island Amplification Microarray (MCAM) method and CpG island microarrays containing 12,000 CpG sites. The first analysis, comparing favourable and intermediate cytogenetic risk groups, revealed significantly differentially methylated CpG sites (594 CpG islands) between the two subgroups. Mutations in the NPM1 gene occur at a high frequency (40%) within the NK-AML subgroup and are associated with a more favourable prognosis in these patients. A second analysis comparing the NPM1 mutant and wild-type research study subjects again identified distinct methylation profiles between these two subgroups. Network and pathway analysis revealed possible molecular mechanisms associated with the different risk and/or mutation sub-groups. This may result in a better classification of the risk groups, improved monitoring targets, or the identification of novel molecular therapies.

Analysis of HSC activity and compensatory Hox gene expression profile in Hoxb cluster mutant fetal liver cells

Overexpression of Hoxb4 in bone marrow cells promotes expansion of hematopoietic stem cell (HSC) populations in vivo and in vitro, indicating that this homeoprotein can activate the genetic program that determines self-renewal. However, this function cannot be solely attributed to Hoxb4 because Hoxb4(-/-) mice are viable and have an apparently normal HSC number. Quantitative polymerase chain reaction analysis showed that Hoxb4(-/-) c-Kit(+) fetal liver cells expressed moderately higher levels of several Hoxb cluster genes than control cells, raising the possibility that normal HSC activity in Hoxb4(-/-) mice is due to a compensatory up-regulation of other Hoxb genes. In this study, we investigated the competitive repopulation potential of HSCs lacking Hoxb4 alone, or in conjunction with 8 other Hoxb genes. Our results show that Hoxb4(-/-) and Hoxb1-b9(-/-) fetal liver cells retain full competitive repopulation potential and the ability to regenerate all myeloid and lymphoid lineages. Quantitative Hox gene expression profiling in purified c-KIt(+) Hoxb1-bg(-/-) fetal liver cells revealed an interaction between the Hoxa, b, and c clusters with variation in expression levels of Hoxa4, -a11, and -c4. Together, these studies show a complex network of genetic interactions between several Hox genes in primitive hematopoietic cells and demonstrate that HSCs lacking up to 30% of the active Hox genes remain fully competent.

Global down-regulation of HOX gene expression in PML-RAR alpha(+) acute promyelocytic leukemia identified by small-array real-time PCR

Acute promyelocytic leukemia (APL) is associated with a reciprocal and balanced translocation involving the retinoic acid receptor-alpha (RARalpha). All-trans retinoic acid (ATRA) is used to treat APL and is a potent morphogen that regulates HOX gene expression in embryogenesis and organogenesis. HOX genes are also involved in hematopoiesis and leukemogenesis. Thirty-nine mammalian HOX genes have been identified and classified into 13 paralogous groups clustered on 4 chromosomes. They encode a complex net-Work of transcription regulatory proteins whose precise targets remain poorly understood. The overall function of the network appears to be dictated by gene dosage. To investigate the mechanisms involved in HOX gene regulation in hematopoiesis and leukemogenesis by precise measurement of individual HOX genes, a small-array real-time HOX (SMART-HOX) quantitative polymerase chain reaction (PCR) platform was designed and validated. Application of SMART-HOX to 16 APL bone marrow samples revealed a global down-regulation of 26 HOX genes compared with normal controls. HOX gene expression was also altered during differentiation induced by ATRA in the PML-RARalpha(+) NB4 cell line. PML-RARalpha, fusion proteins have been reported to act as part of a repressor complex during myelold cell differentiation, and a model linking HOX gene expression to this PML-RARalpha repressor complex is now proposed.

Differential Hox Expression in Murine Embryonic Stem Cell Models of Normal and Malignant Hematopoiesis.

The Hox family are master transcriptional regulators of developmental processes, including hematopoiesis. The Hox regulators, caudal homeobox factors (Cdx1-4), and Meis1, along with several individual Hox proteins, are implicated in stem cell expansion during embryonic development, with gene dosage playing a significant role in the overall function of the integrated Hox network. To investigate the role of this network in normal and aberrant, early hematopoiesis, we employed an in vitro embryonic stem cell differentiation system, which recapitulates mouse developmental hematopoiesis. Expression profiles of Hox, Pbx1, and Meis1 genes were quantified at distinct stages during the hematopoietic differentiation process and compared with the effects of expressing the leukemic oncogene Tel/PDGFRß. During normal differentiation the Hoxa cluster, Pbx1 and Meis1 predominated, with a marked reduction in the majority of Hox genes (27/39) and Meis1 occurring during hematopoietic commitment. Only the posterior Hoxa cluster genes (a9, a10, a11, and a13) maintained or increased expression at the hematopoietic colony stage. Cdx4, Meis1, and a subset of Hox genes, including a7 and a9, were differentially expressed after short-term oncogenic (Tel/PDGFRß) induction. Whereas Hoxa4-10, b1, b2, b4, and b9 were upregulated during oncogenic driven myelomonocytic differentiation. Heterodimers between Hoxa7/Hoxa9, Meis1, and Pbx have previously been implicated in regulating target genes involved in hematopoietic stem cell (HSC) expansion and leukemic progression. These results provide direct evidence that transcriptional flux through the Hox network occurs at very early stages during hematopoietic differentiation and validates embryonic stem cell models for gaining insights into the genetic regulation of normal and malignant hematopoiesis.

Expression of osteopontin coregulators in primary colorectal cancer and associated liver metastases

Background: A transcription regulatory complex (TRC) that includes Ets1, Ets2, PEA3 and ß-catenin/T-cell factors regulates osteopontin (OPN) that is implicated in colorectal cancer (CRC) dissemination. The consistency of OPN transcriptional control between primary CRC and metastases is unclear. This study investigates expression and prognostic significance of the OPN–TRC in primary human CRC and associated colorectal liver metastases (CRLM).

Methods: Osteopontin–TRC factors were assayed by digital microscopy in 38 primary CRCs and matched CRLM specimens and assessed against clinical prognosis.

Results: In primary CRC, OPN expression intensity correlated with that of its co-activators, PEA3 (r=0.600; P<0.01), Ets1 (r=0.552; P<0.01), Ets2 (r=0.521; P<0.01) and had prognostic significance. Osteopontin intensity in primary CRC inversely correlated with the interval between diagnosis and resection of CRLM. Overall OPN intensity was lower in CRLM than primary CRC and correlations with co-activators were weaker, for example, Ets1 (P=0.047), PEA3 (P=0.022) or nonsignificant (Ets2). The ratio of OPN expression in CRLM vs primary CRC had prognostic significance.

Conclusion: This study supports transcriptional control of OPN by known coregulators in both primary and secondary CRC. Weaker associations in CRLM suggest involvement of other unknown factors possibly from the liver microenvironment or resulting from additional genetic or epigenetic changes that drive tumour metastatic capability in OPN transcriptional control.

Low cytosine triphosphate synthase 2 expression renders resistance to 5-fluorouracil in colorectal cancer

Understanding the determinants of resistance of 5-fluorouracil (5FU) is of significant value to optimizing administration of the drug, and introducing novel agents and treatment strategies. Here, the expression of 92 genes involved in 5FU transport, metabolism, co-factor (folate) metabolism and downstream effects was measured by real-time PCR low density arrays in 14 patient-derived colorectal cancer xenografts characterized for 5FU resistance. Candidate gene function was tested by siRNA and uridine modulation, and immunoblotting, apoptosis and cell cycle analysis. Predictive significance was tested by immunohistochemistry of tumors from 125 stage III colorectal cancer patients treated with and without 5FU. Of 8 genes significantly differentially expressed between 5FU sensitive and resistant xenograft tumors, CTPS2 was the gene with the highest probability of differential expression (p = 0.008). Reduction of CTPS2 expression by siRNA increased the resistance of colorectal cancer cell lines DLD1 and LS174T to 5FU and its analog, FUDR. CTPS2 siRNA significantly reduced cell S-phase accumulation and apoptosis following 5FU treatment. Exposure of cells to uridine, a precursor to the CTPS2 substrate uridine triphosphate, also increased 5FU resistance. Patients with low CTPS2 did not gain a survival benefit from 5FU treatment (p = 0.072), while those with high expression did (p = 0.003). Low CTPS2 expression may be a rationally-based determinant of 5FU resistance.

Activated oncogenic pathways and therapeutic targets in extranodal nasal-type NK/T cell lymphoma revealed by gene expression profiling

We performed comprehensive genome-wide gene expression profiling (GEP) of extranodal nasal-type natural killer/T-cell lymphoma (NKTL) using formalin-fixed, paraffin-embedded tissue (n = 9) and NK cell lines (n = 5) in comparison with normal NK cells, with the objective of understanding the oncogenic pathways involved in the pathogenesis of NKTL and to identify potential therapeutic targets. Pathway and network analysis of genes differentially expressed between NKTL and normal NK cells revealed significant enrichment for cell cycle-related genes and pathways, such as PLK1, CDK1, and Aurora-A. Furthermore, our results demonstrated a pro-proliferative and anti-apoptotic phenotype in NKTL characterized by activation of Myc and nuclear factor kappa B (NF-kappa B), and deregulation of p53. In corroboration with GEP findings, a significant percentage of NKTLs (n = 33) overexpressed c-Myc (45.4%), p53 (87.9%), and NF-kappa B p50 (67.7%) on immunohistochemistry using a tissue microarray containing 33 NKTL samples. Notably, overexpression of survivin was observed in 97% of cases. Based on our findings, we propose a model of NKTL pathogenesis where deregulation of p53 together with activation of Myc and NF-kappa B, possibly driven by EBV LMP-1, results in the cumulative up-regulation of survivin. Down-regulation of survivin with Terameprocol (EM-1421, a survivin inhibitor) results in reduced cell viability and increased apoptosis in tumour cells, suggesting that targeting survivin may be a potential novel therapeutic strategy in NKTL. Copyright (C) 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Measuring inconsistency in network intrusion rules

In this preliminary case study, we investigate how inconsistency in a network intrusion detection rule set can be measured. To achieve this, we first examine the structure of these rules which incorporate regular expression (Regex) pattern matching. We then identify primitive elements in these rules in order to translate the rules into their (equivalent) logical forms and to establish connections between them. Additional rules from background knowledge are also introduced to make the correlations among rules more explicit. Finally, we measure the degree of inconsistency in formulae of such a rule set (using the Scoring function, Shapley inconsistency values and Blame measure for prioritized knowledge) and compare the informativeness of these measures. We conclude that such measures are useful for the network intrusion domain assuming that incorporating domain knowledge for correlation of rules is feasible.

Measuring Inconsistency in a Network Intrusion Detection Rule Set Based on Snort

In this preliminary study, we investigate how inconsistency in a network intrusion detection rule set can be measured. To achieve this, we first examine the structure of these rules which are based on Snort and incorporate regular expression (Regex) pattern matching. We then identify primitive elements in these rules in order to translate the rules into their (equivalent) logical forms and to establish connections between them. Additional rules from background knowledge are also introduced to make the correlations among rules more explicit. We measure the degree of inconsistency in formulae of such a rule set (using the Scoring function, Shapley inconsistency values and Blame measure for prioritized knowledge) and compare the informativeness of these measures. Finally, we propose a new measure of inconsistency for prioritized knowledge which incorporates the normalized number of atoms in a language involved in inconsistency to provide a deeper inspection of inconsistent formulae. We conclude that such measures are useful for the network intrusion domain assuming that introducing expert knowledge for correlation of rules is feasible.

Received signal strength in large-scale wireless relay sensor network: a stochastic ray approach

The authors consider a point percolation lattice representation of a large-scale wireless relay sensor network (WRSN) deployed in a cluttered environment. Each relay sensor corresponds to a grid point in the random lattice and the signal sent by the source is modelled as an ensemble of photons that spread in the space, which may 'hit' other sensors and are 'scattered' around. At each hit, the relay node forwards the received signal to its nearest neighbour through direction-selective relaying. The authors first derive the distribution that a relay path reaches a prescribed location after undergoing certain number of hops. Subsequently, a closed-form expression of the average received signal strength (RSS) at the destination can be computed as the summation of all signal echoes' energy. Finally, the effect of the anomalous diffusion exponent ß on the mean RSS in a WRSN is studied, for which it is found that the RSS scaling exponent e is given by (3ß-1)/ß. The results would provide useful insight into the design and deployment of large-scale WRSNs in future. © 2011 The Institution of Engineering and Technology.

Rates for repair of pBR 322 DNA radicals by thiols as measured by the gas explosion technique: evidence that counter-ion condensation and co-ion depletion are significant at physiological ionic strength.

Rates of rapair of pBR 322 plasmid DNA radicals by thiols of varying net charge (Z) at pH 7 and physiological ionic strength were measured using the oxygen explosion technique. The extent of conversion of supercoiled to relaxed circular plasmid was measured by HPLC as a function of the time of oxygen exposure before or after irradiation, the time-courses being fitted by a pseudo-first-order kinetic expression with k1 = k2[RSH]. Values of k2 (M-1 S-1) were: 2.1 x 10(5) (GSH, Z = -1), 1.4 x 10(6) (2-mercaptoethanol, Z = 0), 1.2 x 10(7) (cysteamine, Z = +1), 6.6 x 10(7) (WR-1065 or N-(2-mercaptoethyl)-1,3-diamino?? propane, Z = +2). The approximately 6-fold increase in rate with each unit increase in Z is attributed to concentration of cationic thiols near DNA as a consequence of counter-ion condensation and reduced levels of anionic thiols near DNA owing to co-ion depletion. The results are quantitatively consistent with chemical repair as a significant mechanism for radioprotection of cells by neutral and cationic thiols under aerobic conditions, but indicate that repair by GSH will compete effectively with oxygen only at low oxygen tension.

Organizational structure and the periphery of the gene regulatory network in B-cell lymphoma

Background:
The physical periphery of a biological cell is mainly described by signaling pathways which are triggered by transmembrane proteins and receptors that are sentinels to control the whole gene regulatory network of a cell. However, our current knowledge about the gene regulatory mechanisms that are governed by extracellular signals is severely limited.Results: The purpose of this paper is three fold. First, we infer a gene regulatory network from a large-scale B-cell lymphoma expression data set using the C3NET algorithm. Second, we provide a functional and structural analysis of the largest connected component of this network, revealing that this network component corresponds to the peripheral region of a cell. Third, we analyze the hierarchical organization of network components of the whole inferred B-cell gene regulatory network by introducing a new approach which exploits the variability within the data as well as the inferential characteristics of C3NET. As a result, we find a functional bisection of the network corresponding to different cellular components.

Conclusions:
Overall, our study allows to highlight the peripheral gene regulatory network of B-cells and shows that it is centered around hub transmembrane proteins located at the physical periphery of the cell. In addition, we identify a variety of novel pathological transmembrane proteins such as ion channel complexes and signaling receptors in B-cell lymphoma. © 2012 Simoes et al.; licensee BioMed Central Ltd.

Genome-wide profiling of methylation identifies novel targets with aberrant hyper-methylation and reduced expression in low-risk myelodysplastic syndromes

Gene expression profiling signatures may be used to classify the subtypes of Myelodysplastic syndrome (MDS) patients. However, there are few reports on the global methylation status in MDS. The integration of genome-wide epigenetic regulatory marks with gene expression levels would provide additional information regarding the biological differences between MDS and healthy controls. Gene expression and methylation status were measured using high-density microarrays. A total of 552 differentially methylated CpG loci were identified as being present in low-risk MDS; hypermethylated genes were more frequent than hypomethylated genes. In addition, mRNA expression profiling identified 1005 genes that significantly differed between low-risk MDS and the control group. Integrative analysis of the epigenetic and expression profiles revealed that 66.7% of the hypermethylated genes were underexpressed in low-risk MDS cases. Gene network analysis revealed molecular mechanisms associated with the low-risk MDS group, including altered apoptosis pathways. The two key apoptotic genes BCL2 and ETS1 were identified as silenced genes. In addition, the immune response and micro RNA biogenesis were affected by the hypermethylation and underexpression of IL27RA and DICER1. Our integrative analysis revealed that aberrant epigenetic regulation is a hallmark of low-risk MDS patients and could have a central role in these diseases.

Rod photoreceptor loss in Rho(-/-) mice reduces retinal hypoxia and hypoxia-regulated gene expression

PURPOSE. This study was conducted to evaluate whether regions of the retinal neuropile become hypoxic during periods of high oxygen consumption and whether depletion of the outer retina reduces hypoxia and related changes in gene expression.

METHODS. Retinas from rhodopsin knockout (Rho(-/-)) mice were evaluated along with those of wild-type (WT) control animals. Retinas were also examined at the end of 12-hour dark or light periods, and a separate group was treated with L-cis-diltiazem at the beginning of a 12-hour dark period. Hypoxia was assessed by deposition of hypoxyprobe (HP) and HP-protein adducts were localized by immunohistochemistry and quantified using ELISA. Also, hypoxia-regulated gene expression and transcriptional activity were assessed alongside vascular density.

RESULTS. Hypoxia was observed in the inner nuclear and ganglion cell layers in WT retina and was significantly reduced in Rho (-/-) mice (P < 0.05). Retinal hypoxia was significantly increased during dark adaptation in WT mice (P < 0.05), whereas no change was observed in Rho(-/-) or with L-cis-diltiazem-treated WT mice. Hypoxia-inducible factor (HIF)-1 alpha DNA-binding and VEGF mRNA expression in Rho(-/-) retina was significantly reduced in unison with outer retinal depletion (P < 0.05). Retina from the Rho(-/-) mice displayed an extensive intraretinal vascular network after 6 months, although there was evidence that capillary density was depleted in comparison with that in WT retinas.

CONCLUSIONS. Relative hypoxia occurs in the inner retina especially during dark adaptation. Photoreceptor loss reduces retinal oxygen usage and hypoxia which corresponds with attenuation of the retinal microvasculature. These studies suggest that in normal physiological conditions and diurnal cycles the adult retina exists in a state of borderline hypoxia, making this tissue particularly susceptible to even subtle reductions in perfusion.

Progressive lung cancer determined by expression profiling and transcriptional regulation

Clinically, our ability to predict disease outcome for patients with early stage lung cancer is currently poor. To address this issue, tumour specimens were collected at surgery from non-small cell lung cancer (NSCLC) patients as part of the European Early Lung Cancer (EUELC) consortium. The patients were followed-up for three years post-surgery and patients who suffered progressive disease (PD, tumour recurrence, metastasis or a second primary) or remained disease-free (DF) during follow-up were identified. RNA from both tumour and adjacent-normal lung tissue was extracted from patients and subjected to microarray expression profiling. These samples included 36 adenocarcinomas and 23 squamous cell carcinomas from both PD and DF patients. The microarray data was subject to a series of systematic bioinformatics analyses at gene, network and transcription factor levels. The focus of these analyses was 2-fold: firstly to determine whether there were specific biomarkers capable of differentiating between PD and DF patients, and secondly, to identify molecular networks which may contribute to the progressive tumour phenotype. The experimental design and analyses performed permitted the clear differentiation between PD and DF patients using a set of biomarkers implicated in neuroendocrine signalling and allowed the inference of a set of transcription factors whose activity may differ according to disease outcome. Potential links between the biomarkers, the transcription factors and the genes p21/CDKN1A and Myc, which have previously been implicated in NSCLC development, were revealed by a combination of pathway analysis and microarray meta-analysis. These findings suggest that neuroendocrine-related genes, potentially driven through p21/CDKN1A and Myc, are closely linked to whether or not a NSCLC patient will have poor clinical outcome.