ΔCt Values Research Articles

Tropism between hepatic and pulmonary metastases in colorectal cancers

In metastatic colorectal cancers, tumor cells are disseminated prior to surgical resection of the primary tumor but remain dormant until proper colonization mechanisms are activated. To identify the colonization mechanisms of the metastatic tumors, we conducted a pairwise comparison between primary colorectal cancers and metastatic tumors (n=12 pairs), including six hepatic pairs and six pulmonary pairs. The mRNA levels of 224 genes previously reported to be associated with metastasis, cytokines and angiogenesis were quantitatively determined by PCR arrays. Among them, 27 genes were duplicated or triplicated to show consistent expression. Unsupervised hierarchical clustering of the Ct values of metastasis-related genes revealed that liver metastases were indistinguishable from primary colorectal cancers (n=5/6), whereas lung metastases were highly diversified from one another and from the primary tumors (n=6/6). Cytokines and receptor gene expression array data also confirmed the divergence of pulmonary metastases from primary colorectal cancers (n=6/6). Heat map analyses of ΔCt values of the metastasis-related genes identified a 17-gene tropism signature that was sufficient not only to distinguish liver and the lung metastases, but also reconstituted the clustering of primary tumors with the hepatic metastases (n=17/18). In this pilot experiment, pulmonary metastases were significantly diverged from hepatic metastases that were indistinguishable from primary colorectal cancers. Further genomic and clinical studies are in progress to evaluate the potential of the tropism signature as a therapeutic target to inhibit the colonization of metastatic colorectal cancers.

TRPV1 in GABAergic Interneurons Mediates Neuropathic Mechanical Allodynia and Disinhibition of the Nociceptive Circuitry in the Spinal Cord

Neuropathic pain and allodynia may arise from sensitization of central circuits. We report a mechanism of disinhibition-based central sensitization resulting from long-term depression (LTD) of GABAergic interneurons as a consequence of TRPV1 activation in the spinal cord. Intrathecal administration of TRPV1 agonists led to mechanical allodynia that was not dependent on peripheral TRPV1 neurons. TRPV1 was functionally expressed in GABAergic spinal interneurons and activation of spinal TRPV1 resulted in LTD of excitatory inputs and a reduction of inhibitory signaling to spinothalamic tract (STT) projection neurons. Mechanical hypersensitivity after peripheral nerve injury was attenuated in TRPV1(-/-) mice but not in mice lacking TRPV1-expressing peripheral neurons. Mechanical pain was reversed by a spinally applied TRPV1 antagonist while avoiding the hyperthermic side effect of systemic treatment. Our results demonstrate that spinal TRPV1 plays a critical role as a synaptic regulator and suggest the utility of central nervous system-specific TRPV1 antagonists for treating neuropathic pain.

Role of charge transfer state and host matrix in Eu3+-doped alkali and earth alkali fluoro-aluminoborate glasses

Judd–Ofelt parameters of Eu3+-doped alkali and earth alkali fluoro-aluminoborate glasses are determined from the fluorescence spectra. The dependence of these parameters on the compositional changes of the materials is discussed. The glass containing sulphate component shows the larger values of Ω compared with the fluoro-aluminoborate glasses because of higher ΔCT (Eu3+–F−) values. The difference of ionic radius and electronegativity between Na and Ca lead to higher Ω2 parameter of CaF2–Al2O3–B2O3:Eu3+ glass.Persistent spectral hole burning has been observed in Eu3+ ions doped alkali-and earth alkali fluoro-aluminoborate glasses with the compositions of 16NaF·73B2O3·8Al2O3·3Eu2O3 and 16CaF2·73B2O3·8Al2O3·3Eu2O3, which have been prepared using conventional melting technique.

Evaluation of candidate diagnostic microRNAs in thyroid fine-needle aspiration biopsy samples.

Thyroid cancer diagnosis in the United States has increased by 2.3-folds in the last three decades. Up to 30% of thyroid fine-needle aspiration biopsy (FNAB) results are inconclusive. Several differentially expressed microRNAs (miRNAs) have been identified as candidate diagnostic markers for thyroid nodules. We hypothesized that these differentially expressed miRNAs may improve the accuracy of FNAB in difficult to diagnose thyroid nodules. Expression levels of four miRNAs (miR-7, -126, -374a, and let-7g) were analyzed using quantitative real-time reverse transcription-polymerase chain reaction in 95 FNAB samples as the training set. A predictor model was formulated based on the most differentially expressed miRNA (miR-7) ΔCt value and the model was applied on a separate cohort of 59 FNAB samples as the validation set. miR-7 was the best predictor to distinguish benign from malignant thyroid FNAB samples. The other three miRNAs were co-expressed and did not significantly contribute to the predictor model. miR-7 had a sensitivity of 100%, specificity of 29%, positive predictive value (PPV) of 36%, negative predictive value (NPV) of 100%, and overall accuracy of 76% when applied to the validation set. In subgroup analysis of preoperative nondiagnostic, indeterminate, or suspicious FNAB samples, the predictor model had an overall accuracy of 37% with sensitivity of 100%, specificity of 20%, PPV of 25%, and NPV of 100%. miR-7 may be a helpful adjunct marker to thyroid FNAB in tumor types which are inconclusive. Given the high NPV of miR-7, a patient with a benign result based on the predictor model may be followed as opposed to performing an immediate diagnostic thyroidectomy. Future prospective clinical trials evaluating its accuracy in a larger cohort are warranted to determine its clinical utility.

Expression of Ara-C Metabolizing Enzymes Mediates in Vitro Sensitivity to Ara-C in Primary AML Cells From Patients with Denovo AML,

Abstract 3481Wide inter-individual variation in terms of treatment outcome and toxic side effects of treatment exist among patients with AML receiving chemotherapy with cytarabine (ara-C) and daunorubicin. The pre-requisite for the cytotoxic action of pro-drug Ara-C is the enzymatic conversion to its active tri-phosphorylated form ara-CTP. Many drug activating (Deoxycytidine kinase (dCK) and human Equilibrative Nucleoside Transporter 1 (hENT1) and deactivating (Cytidine deaminase (CDA), 5'nucleotidase (NT5C2) genes and ribonucleoside reductase (RRM1), which are involved in transport and biotransformation of cytarabine contribute to the variation in ara-C sensitivity in AML patients. FLT3-ITD and NPM1 mutations act as major poor and good prognostic markers respectively in cytogenetically normal AML. The effect of these mutations in ara-C metabolism remains to be elucidated.The present study aims to determine independent as well as the combined effect of ara-C metabolizing genes mRNA expression on in-vitro ara-C cytotoxicity and the role of FLT3-ITD and NPM mutations on mRNA expression of these genes. Diagnostic bone marrow sample (median blasts 65%; range 21 – 98%) from 98 adult patients with de novo AML (other than AML-M3) were included in this study. mRNA expression levels for each target gene relative to housekeeping gene GAPDH was analyzed using Taqman based gene expression assays. In vitro cytotoxicity was assessed using MTT cell viability assay and IC-50 was calculated. In vitro sensitivity or resistance was classified on the basis of the IC-50 values <6uM and >6uM ara-C respectively. FLT3 ITD and NPM mutation status at diagnosis were determined through PCR followed by Genescan analysis using genomic DNA samples. Type of NPM mutation was identified by sequencing.When ara-C IC-50 values were compared with the mRNA expression levels of these candidate genes, Ara-C sensitive samples (n= 30; IC-50 < 6uM) showed significantly higher mRNA expression of dCK and hENT1 compared to those with Ara-C resistance (n=51) IC50 >6uM (median 314 (61.56 – 1232) vs. 180 (31.87 – 749.2); p = 0.0004 and median 172.1 (44.12 – 657.6) vs. 96.19 (37.49 – 432.4), p= 0.0008 respectively. RRM1 and NT5C2 did not show any association with in vitro Ara-C cytotoxicity, while CDA showed a trend towards association with lower CDA expression in ara-C sensitive samples. Based on these findings we put forward Ara-C resistance index (RI). RI is calculated by the formulaRI = ΔCT (dCK X ENT1)/ ΔCT CDA.(Smaller ΔCT value= higher mRNA expression). RI values were significantly higher in resistant (IC50 >6uM) compared to sensitive cells (median: 6.084; range 1.89–11.82) vs. 3.702 (1.89–9.80); p=<0.0001). This association should now be validated in an independent cohort.Effects of NPM and FLT3 mutation status on Ara-C metabolizing genes were then evaluated. No significant association was found between FLT3-ITD status and the mRNA expression of these candidate genes. Interestingly, dCK mRNA levels were significantly higher in samples with NPM mutation (n=39) compared to NPM wild type (n=59); median 272.3 (41.64–1232) vs. 188.6 (31.87–1030); p value= 0.01. When analysed separately, patients with NPM type A mutation (n=27) showed significantly higher dCK expression (median 347.4 (41.64–1232) vs. 188.6 (31.87–1030); p value= 0.003 compared to those with wild type NPM1. This first report showing an association between expression profiles of ara-C metabolizing genes and NPM mutation should form the basis for evaluating their clinical correlations. Disclosures:No relevant conflicts of interest to declare.

Evaluation of the presence of Leishmania spp. by real-time PCR in the lacrimal glands of dogs with leishmaniosis

Leishmania infantum infection is highly prevalent in endemic areas. Dogs with leishmaniosis may develop keratoconjunctivitis sicca (KCS). The goals of this study were (1) to quantify Leishmania amastigotes in the Meibomian glands (MG), main lacrimal gland (MLG) and nictitating membrane gland (NMG) from dogs with leishmaniosis; (2) to compare these results to immunohistochemistry (IHC), and (3) to explore the association between the Leishmania parasite load and the presence of ocular clinical signs. Twenty-five dogs diagnosed with leishmaniosis were included. MG, MLG and NMG from both eyes were collected. Histopathology, IHC and real-time PCR were performed. All specimens yielded positive real-time PCR results. For all three glands, samples from dogs with ocular clinical signs had mean ΔCt (cycle threshold) values significantly lower (higher parasite loads) than those from dogs without signs. Cut-off values of ΔCt<0, ΔCt<4 and ΔCt<4.9 for MG, MLG and NMG, resulted in a likelihood ratio of positives of 5.9, 6.38 and 6.38, respectively. Samples with ΔCt values below the reported cut-off were significantly more likely to display clinical signs related to KCS than those with results above the cut-off, for all three glands. Similarly, ΔCt values below the cut-off were significantly associated with positive IHC. In this study real-time PCR has been standardised for use in MG, MLG and NMG. A cut-off value established for each of these tissues may aid the clinician in the discrimination between ocular signs related to Leishmania from those associated with other causes of KCS.

DNA Microarray Analysis Identifies CKS2 and LEPR as Potential Markers of Meningioma Recurrence

Meningiomas are the most frequent intracranial tumors. Surgery can be curative, but recurrences are possible. We performed gene expression analyses and loss of heterozygosity (LOH) studies looking for new markers predicting the recurrence risk. We analyzed expression profiles of 23 meningiomas (10 grade I, 10 grade II, and 3 grade III) and validated the data using quantitative polymerase chain reaction (qPCR). We performed LOH analysis on 40 meningiomas, investigating chromosomal regions on 1p, 9p, 10q, 14q, and 22q. We found 233 and 268 probe sets to be significantly down- and upregulated, respectively, in grade II or III meningiomas. Genes downregulated in high-grade meningiomas were overrepresented on chromosomes 1, 6, 9, 10, and 14. Based on functional enrichment analysis, we selected LIM domain and actin binding 1 (LIMA1), tissue inhibitor of metalloproteinases 3 (TIMP3), cyclin-dependent kinases regulatory subunit 2 (CKS2), leptin receptor (LEPR), and baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5) for validation using qPCR and confirmed their differential expression in the two groups of tumors. We calculated ΔCt values of CKS2 and LEPR and found that their differential expression (C-L index) was significantly higher in grade I than in grade II or III meningiomas (p < .0001). Interestingly, the C-L index of nine grade I meningiomas from patients who relapsed in <5 years was significantly lower than in grade I meningiomas from patients who did not relapse. These findings indicate that the C-L index may be relevant to define the progression risk in meningioma patients, helping guide their clinical management. A prospective analysis on a larger number of cases is warranted.

Gene expression signature comparison for solid tumor chemotherapy.

e21045 Background: It is unclear which one of signatures of ΔCT or RQ values from gene expression respond better in chemotherapy evaluation. Improved understanding is required for the comparison analysis in FFPE and blood samples. Methods: 370 solid tumor consecutive FFPE samples were tested by quantitative PCR. Among all the samples, 251 cases of solid tumor (108 lung cancer, 72 colon cancer, 44 gastric cancer, 27 breast cancer) with self-blood control were enrolled and thresholds were set at 15-35 cycles, which involved in predictive chemotherapy biomarkers ERCC1, BRCA1, TSand RRM1. For source dataset, a P value was computed for genes ΔCT and genes RQ value between the specimens of solid tumor, tumor subgroups and blood. A summary statistic to solid tumor, subgroup tumors and target gene signatures including comparison algoriths was analyzed with t test, chitest X2 and Pearson’s correlation coefficients. Results: Overall, RNA was isolated for 324 cases (87.6%) among 370 FFPE tissue successfully and over 1.8ng/ul per reaction. Total 251 solid cancers and its subgroups were examined for genes average ΔCT of tumor is 4.698±2.066 (ERCC1), 6.918±2.533 (BRCA1), 5.364±2.452 (RRM1), 6.355±2.459 (TS). Average RQ value of tumor is 38.157±83.086 (ERCC1), 65.030±245.821 (BRCA1), 42.841±166.933 (RRM1), 111.820±545.89 (TS). The data extracted from tumor ΔCT and RQ value (ΔCT/ΔΔCT(X+SD) were categorized and compared, statistics analysis indicated no significant differences of ΔCT value between the solid tumor, 4 subgroups tumors and 4 genes (P>0.05), but relative RQ value were observed in odds repetition and dispersed CV value with more groups difference (p<0.05), without correlation coefficients between ΔCTand RQ value in subgroups of cancer (P>0.05, pearson correlation), analyzed 4 gene synergism expression values based on cutoffs (ERCC1:4.3, TS:8.5, RRM1:8.0, BRCA1:6.5) in cancer subgroup, only half situation of coordinate up or down-regulation occured (p<0.05). Conclusions: ΔCT value- based signature make a stable and accurate resources data from solid tumor FFPE mRNA, which could contribute to the personality chemotherapy evaluation and analysis is more reliable than blood sample.

Relationship of CRLF2 expression and outcome in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL): A report from the Children’s Oncology Group.

9505 Background: We previously reported a highly significant association between over-expression of CRLF2 (cytokine receptor-like factor 2) and poor clinical outcomes in 207 uniformly-treated children with high-risk B-ALL (Blood 115:5312, 2010). To validate and extend these studies to standard risk cases we studied 896 B-ALL patients from COG P9905. Eligible patients included 344 with NCI high-risk (HR) features and 552 with standard-risk (SR) features. Methods: Quantitative RT-PCR assays were used to determine CRLF2 expression in the diagnostic leukemic cells. Averages of duplicate assays were used to establish ΔCt values relative to a control gene (EEF2). Using the ΔCt threshold determined from our prior studies, 117/896 B-ALL samples had elevated CRLF2 expression. These 117 samples and other 35 samples below the threshold were tested for genomic rearrangements of CRLF2 to detect the P2RY8-CRLF2 fusion or CRLF2 F232C mutations. Studies to detect the IGH@-CRLF2 translocation, IKZF1 (IKAROS) deletions, and JAK mutations are ongoing. Outcomes were evaluated using relapse-free survival (RFS) and other known risk factors. Results: CRLF2 overexpression was associated with a poorer outcome in HR, but not in SR, B-ALL. Among the 246 HR cases lacking detectable end-induction minimal residual disease, CRLF2 expression levels could further distinguish those B-ALL patients who had a very good (>80%) vs. poor (<40%) RFS (Hazard Ratio = 2.59; P=0.002). While P2RY8/CRLF2 fusions were seen only in high CRLF2-expressors, 6/17 CRLF2 F232C mutations were among low CRLF2 expressors. Down syndrome ALL cases had a relatively good outcome regardless of CRLF2 expression levels or NIH risk status. Conclusions: Elevated CRLF2 expression predicts a significantly poorer outcome in HR, but not SR, B-ALL and can be used with other prognostic factors to improve ALL risk classification. Standard-risk RFS High-risk RFS N Hazard ratio P N Hazard ratio P All cases (n=896) 552 – – 344 – – High CRLF2 81 1.20 .510 36 1.84 .019 Down syndrome (DS) 35 1.25 .571 5 0.94 .952 Genomic abnormalities 99 53 P2RY8 fusion 37 1.00 .988 14 1.64 .034 F232C mutation 15 – – 2 – –

The expression of intestinal epithelial stem cell markers in mouse radiation enteritis

Objective To investigate the expression of intestinal epithelial stem cell markers Msil and Hesl in mouse acute radiation enteritis (RE) model. Methods Forty female BALB/c mice were undergone one-time whole abdominal irradiation by 300 cGy/min β-ray. Thirty mice were selected before irradiation (Day 0) and every six mice were euthanized at day 3,5,7 and 14 after irradiation. The small intestinal were detached and the expression of Msil and Hesl were detected in middle part of small intestinal by quantitative RT-PCR, Western blots, and immunohistochemistry. Subsequently,the difference of Msil and Hesl expression was compared. Results The mouse acute RE model was successfully established. At the 5th day after radiation, the results of quantitative RT-PCR indicated that the relative ΔCt value of Msil and Hesl expression were 15. 17 ± 0. 47 and 15. 83 ± 0. 24respectively, the results of Western blot showed that the integrated intensity were 0. 443±0. 055 and 0. 301 + 0. 047 for Msil and Hesl. The expressions were significantly higher than other time points at RNA and protein levels (P<0. 05). The results of immunohistochemistry revealed that Msil and Hesl were highly expressed in intestinal crypts at 5th day and 7th day. Conclusions The expression of Msil and Hesl, upregulated before wound repair in RE model, indicates that intestinal epithelial repair is related to intestinal stem cell proliferation. Key words: Enteritis; Radiation injuries,experimental; Intestines; Stem cell; RNA-binding proteins; Hesl protein,mouse; Gene expression

A novel method for detection of mutation in epidermal growth factor receptor

Background For the rapid and sensitive screening of epidermal growth factor receptor ( EGFR) hot-spot mutations, we developed a novel method combining mutant-enriched PCR with amplification refractory mutation system (ARMS) TaqMan real-time PCR in a one-step reaction tube. Methods and results We designed two pairs of primers to enrich and genotyping each mutation ( E746_A750del and L858R): nest primers and ARMS primers. Before the PCR assays were carried out, the restriction enzymes were used to cut wild alleles. The results showed that this method could detect mutant alleles mixed samples containing 0.1% with a cutoff ΔCt value of 12. We used this method in a survey of 73 non-small cell lung cancer (NSCLC) samples, detecting 14 mutant samples of E746_A750del and 12 mutant samples of L858R. The results well agreed with the results of DxS. All unmatched samples were identified by sequencing and the results showed that our method has high specificty. Conclusion The mutant-enriched ARMS TaqMan PCR could be useful in the detection of mutation in clinical samples containing only a small number of mutant alleles.

Altered Immune Response in Mice Deficient for the G Protein-coupled Receptor GPR34

The X-chromosomal GPR34 gene encodes an orphan G(i) protein-coupled receptor that is highly conserved among vertebrates. To evaluate the physiological relevance of GPR34, we generated a GPR34-deficient mouse line. GPR34-deficient mice were vital, reproduced normally, and showed no gross abnormalities in anatomical, histological, laboratory chemistry, or behavioral investigations under standard housing. Because GPR34 is highly expressed in mononuclear cells of the immune system, mice were specifically tested for altered functions of these cell types. Following immunization with methylated BSA, the number of granulocytes and macrophages in spleens was significantly lower in GPR34-deficient mice as in wild-type mice. GPR34-deficient mice showed significantly increased paw swelling in the delayed type hypersensitivity test and higher pathogen burden in extrapulmonary tissues after pulmonary infection with Cryptococcus neoformans compared with wild-type mice. The findings in delayed type hypersensitivity and infection tests were accompanied by significantly different basal and stimulated TNF-α, GM-CSF, and IFN-γ levels in GPR34-deficient animals. Our data point toward a functional role of GPR34 in the cellular response to immunological challenges.

Neurodegenerative Mutation in Cytoplasmic Dynein Alters Its Organization and Dynein-Dynactin and Dynein-Kinesin Interactions

A single amino acid change, F580Y (Legs at odd angles (Loa), Dync1h1Loa), in the highly conserved and overlapping homodimerization, intermediate chain, and light intermediate chain binding domain of the cytoplasmic dynein heavy chain can cause severe motor and sensory neuron loss in mice. The mechanism by which the Loa mutation impairs the neuron-specific functions of dynein is not understood. To elucidate the underlying molecular mechanisms of neurodegeneration arising from this mutation, we applied a cohort of biochemical methods combined with in vivo assays to systemically study the effects of the mutation on the assembly of dynein and its interaction with dynactin. We found that the Loa mutation in the heavy chain leads to increased affinity of this subunit of cytoplasmic dynein to light intermediate and a population of intermediate chains and a suppressed association of dynactin to dynein. These data suggest that the Loa mutation drives the assembly of cytoplasmic dynein toward a complex with lower affinity to dynactin and thus impairing transport of cargos that tether to the complex via dynactin. In addition, we detected up-regulation of kinesin light chain 1 (KLC1) and its increased association with dynein but reduced microtubule-associated KLC1 in the Loa samples. We provide a model describing how up-regulation of KLC1 and its interaction with cytoplasmic dynein in Loa could play a regulatory role in restoring the retrograde and anterograde transport in the Loa neurons.

Folic Acid Remodels Chromatin on Hes1 and Neurog2 Promoters during Caudal Neural Tube Development

The mechanism(s) behind folate rescue of neural tube closure are not well understood. In this study we show that maternal intake of folate prior to conception reverses the proliferation potential of neural crest stem cells in homozygous Splotch embryos (Sp(-/-)) via epigenetic mechanisms. It is also shown that the pattern of differentiation seen in these cells is similar to wild-type (WT). Cells from open caudal neural tubes of Sp(-/-) embryos exhibit increased H3K27 methylation and decreased expression of KDM6B possibly due to up-regulation of KDM6B targeting micro-RNAs such as miR-138, miR-148a, miR-185, and miR-339-5p. In our model, folate reversed these epigenetic marks in folate-rescued Sp(-/-) embryos. Using tissue from caudal neural tubes of murine embryos we also examined H3K27me2 and KDM6B association with Hes1 and Neurog2 promoters at embryonic day E10.5, the proliferative stage, and E12.5, when neural differentiation begins. In Sp(-/-) embryos compared with WT, levels of H3K27me2 associated with the Hes1 promoter were increased at E10.5, and levels associated with the Neurog2 promoter were increased at E12.5. KDM6B association with Hes1 and Neurog2 promoters was inversely related to H3K27me2 levels. These epigenetic changes were reversed in folate-rescued Sp(-/-) embryos. Thus, one of the mechanisms by which folate may rescue the Sp(-/-) phenotype is by increasing the expression of KDM6B, which in turn decreases H3K27 methylation marks on Hes1 and Neurog2 promoters thereby affecting gene transcription.

Enzymatic activity of the human 1-acylglycerol-3-phosphate-O-acyltransferase isoform 11: upregulated in breast and cervical cancers

The conversion of lysophosphatidic acid (LPA) to phosphatidic acid is carried out by the microsomal enzymes 1-acylglycerol-3-phosphate-O-acyltransferases (AGPATs). These enzymes are specific for acylating LPA at the sn-2 (carbon 2) position on the glycerol backbone and are important, because they provide substrates for the synthesis of phospholipids and triglycerides. At least, mutations in one isoform, AGPAT2, cause near complete loss of adipose tissue in humans. We cloned a cDNA predicted to be an AGPAT isoform, AGPAT11. This cDNA has been recently identified also as lysophosphatidylcholine acyltransferase 2 (LPCAT2) and lyso platelet-activating factor acetyltransferase. When AGPAT11/LPCAT2/lyso platelet-activating factor acetyltransferase cDNA was expressed in CHO and HeLa cells, the protein product localized to the endoplasmic reticulum. In vitro enzymatic activity using lysates of Human Embryonic Kidney-293 cells infected with recombinant AGPAT11/LPCAT2/lyso platelet-activating factor-acetyltransferase cDNA adenovirus show that the protein has an AGPAT activity but lacks glycerol-3-phosphate acyltransferase enzymatic activity. The AGPAT11 efficiently uses C18:1 LPA as acyl acceptor and C18:1 fatty acid as an acyl donor. Thus, it has similar substrate specificities for LPA and acyl-CoA as shown for AGPAT9 and 10. Expression of AGPAT11 mRNA was significantly upregulated in human breast, cervical, and colorectal cancer tissues, indicating its adjuvant role in the progression of these cancers. Our enzymatic assays strongly suggest that the cDNA previously identified as LPCAT2/lyso platelet-activating factor-acetyltransferase cDNA has AGPAT activity and thus we prefer to identify this clone as AGPAT11 as well.

Assessment of nociceptin/orphanin FQ and μ-opioid receptor mRNA in the human right atrium

BackgroundThe expression of µ (mu: MOP) and nociceptin/orphanin FQ (NOP) receptors in the human myocardium is controversial. In this polymerase chain reaction (PCR)-based study using human right atrial biopsies, we have (i) probed for mRNA encoding NOP receptor and its endogenous peptide precursor, ppN/OFQ, and mRNA encoding MOP and (ii) attempted to correlate expression with cardiac function. MethodsmRNA encoding MOP, NOP, and the precursor for NOP (ppN/OFQ) was assessed by quantitative real-time PCR (Q-PCR) using validated TaqMan® primers and compared with a housekeeper (glyceraldehyde-3-phosphate dehydrogenase, GAPDH). Q-PCR data are expressed as the difference in cycle threshold (ΔCt=CtGene of interest−CtGAPDH: high value, low expression) and patients were grouped according to left ventricular ejection fraction (LVEF). ResultsForty patients were recruited; NOP, MOP, and ppN/OFQ mRNA were measured in 38, 29, and 10 patients, respectively. ΔCt (median and range) values for NOP and MOP were 10.9 (7.8–13.7) and 16.0 (12.3–18.9), respectively, representing low expression of MOP and ∼34-fold more NOP. MOP mRNA was not detected in seven samples and with ΔCt values of ∼20, ppN/OFQ was considered absent. When patients were grouped into normal (>50%) and impaired (<50%) LVEF, there was no difference between the groups for either NOP or MOP. In some patients, intraoperative LVEF was estimated using transoesophageal echocardiography, and there was no correlation with either NOP or MOP. ConclusionsThe human right atrium of patients with coronary artery disease and heart failure expresses mRNA encoding NOP and possibly low levels of MOP. This does not correlate with degree of cardiac dysfunction. In addition, the atrium does not express ppN/OFQ mRNA.

Microarray-based Transcriptional and Epigenetic Profiling of Matrix Metalloproteinases, Collagens, and Related Genes in Cancer

Epigenetic parameters (DNA methylation, histone modifications, and miRNAs) play a significant role in cancer. To identify the common epigenetic signatures of both the individual matrix metalloproteinases (MMPs) and the additional genes, the function of which is also linked to proteolysis, migration, and tumorigenesis, we performed epigenetic profiling of 486 selected genes in unrelated non-migratory MCF-7 breast carcinoma and highly migratory U251 glioma cells. Genome-wide transcriptional profiling, quantitative reverse transcription-PCR, and microRNA analyses were used to support the results of our epigenetic studies. Transcriptional silencing in both glioma and breast carcinoma cells predominantly involved the repressive histone H3 Lys-27 trimethylation (H3K27me3) mark. In turn, epigenetic stimulation was primarily performed through a gain in the histone H3 Lys-4 dimethylation (H3K4me2) and H3 hyperacetylation and by a global reduction of H3K27me3. Inactive pro-invasive genes in MCF-7 cells but not in U251 cells frequently exhibited a stem cell-like bivalent mark (enrichment in both H3K27me3 and H3K4me2), a characteristic of developmental genes. In contrast with other MMPs, MMP-8 was epigenetically silenced in both cell types, thus providing evidence for the strict epigenetic control of this anti-tumorigenic proteinase in cancer. Epigenetic stimulation of multiple collagen genes observed in cultured glioma cells was then directly confirmed using orthotopic xenografts and tumor specimens. We suggest that the epigenetic mechanisms allow gliomas to deposit an invasion-promoting collagen-enriched matrix and then to use this matrix to accomplish their rapid migration through the brain tissue.

Abstract 3035: MicroRNA expression in early stage endometrial cancers

Abstract The molecular signatures of endometrial cancers from whole genome gene expression studies are now available setting a stage to explore the key regulators involved in these cancers. Non-coding micro-RNAs (miRNAs) are functionally important regulators of numerous cellular processes affecting growth and dissemination and possibly possess prognostic significance for several cancer types. In a first step toward understanding the miRNA biology of endometrial cancers, we performed an unbiased characterization of about 667 unique human miRNAs in stage-1 endometrial cancers including 51 endometrioid (16 grade-1, 23 grade-2 and 12 grade-3) and 7 serous (grade-3) cancer types and 17 endometrial epithelial samples from healthy individuals. In total, there were 8 stage IA, 32 stage IB and 18 stage IC cancers. RNA was extracted from samples prepared by laser capture micro-dissection which contained minimal non-epithelial cells. TaqMan Low Density Arrays (TLDA) were used to quantitatively determine the expression levels of miRNAs (TaqMan® MicroRNA Arrays A and B from Applied Biosystems). Isolated RNA was transcribed into cDNA and used to quantitatively assess the expression of 768 miRNAs arrayed within the TLDA. The Ct-values were initially normalized by subtracting a reference Ct-value derived from multiple positive controls on the array. These ΔCt values were filtered to exclude the miRNAs that were not expressed in any of the arrays. Statistical calculations were performed on -ΔCt values. In summary, about 158 miRNAs were up or down regulated in cancers at least by four-fold (p&amp;lt;0.001) including many novel candidates for this cancer type. Notably, miR-183* was on average the most differentially over-expressed in both endometrioid and serous cancers compared to normal endometrium. The numbers of miRNAs and their statistical significance to distinguish the histological type, grade, and stage among cancers were found to be in decreasing order respectively. The putative mRNA targets of miRNAs were predicted using Targetscan v. 5.1. These predicted targets were further examined using gene expression data (Affymetrix Human Genome Plus 2.0) of the same RNAs. In conclusion, we identified numerous miRNAs that are disregulated in endometrial cancer. The potential of these miRNAs to define the biology of endometrial cancer is further discussed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3035.

Detection of oocyte mRNA in starfish polar bodies

We report the first demonstration of oocyte mRNA detected in its sibling polar body. Oogenesis is a conserved process that requires gene transcription and storage of RNA for development until the embryonic genome is activated. Analysis of oocyte mRNA profiles detected in polar bodies may allow evaluation of gene expression in single oocytes without destroying the cell. We biopsied the polar bodies from eggs of the starfish, Asterina miniata, without disrupting either cell. The estimated volume of an A. miniata oocyte is 3.05×10−3pl, nearly 2,000 times greater than that of its polar body, which has an estimated volume of 1.77×10−6pl. Individual polar bodies and oocytes were isolated and transferred to a reaction buffer, heat lysed, DNAse treated, and reverse transcribed without isolation of the RNA (Protocol described in supplemental material). We first tested whether the polar body had detectable ribosomal RNA using 1/30th of the RT-reaction from each cell and we consistently amplified the appropriate product from each cell (supplemental figure 2). We then tested for specific mRNAs; these too were detectable with greater success for transcripts that were more abundant (had lower Ct values) in the sibling oocyte (Table I). Detection of a specific mRNA transcript in the polar body decreased 60% for every unit increase in corresponding Ct value for that transcript in its sibling oocyte (odds ratio=0.40; p=0.01). This result provides a baseline from which to predict transcripts that may be detectable in polar bodies if levels in the oocyte are known. Confocal imaging supports the hypothesis that representative ooplasm containing mitochondria is extruded with the first polar body (supplemental figure 3). TABLE 1 Relationship of Sibling Oocyte and Polar Body mRNA We compared the level of mRNA for six genes in seven different sibling pairs. The ΔCt value between oocytes and polar bodies for a given gene ranged from 4.6 for eukaryotic initiation factor 2 (eif2) transcripts to 14.0 for histone2A (h2a) transcripts. These values correspond to 25 to 16,000 fold relative differences in mRNA abundance between oocytes and polar bodies. Since the variance within sample replicates is less than 2%, variability between transcripts may reflect a difference in retention or localization between these sibling cells. Further research is needed to correlate variation of mRNA levels in a polar body with its sibling oocyte. It will be important to examine cell-to-cell variability among developmentally critical gene transcripts in polar bodies and to test whether variability between individuals may reflect biological differences between oocytes. Variation between different oocytes may reflect dynamic changes in mRNA abundance during oocyte development or the maturation process. Translating this work into humans is promising; although the human oocyte is smaller (120μm diameter compared to 180μm), the polar body is comparable or larger than the starfish polar body (Veeks, 1990). Technological advances including incorporation of non-biased amplification of mRNA may permit clinical analysis of human oocyte gene expression from a single polar body. Such a development may aid with the assessment of oocyte quality and developmental competence in patients using assisted reproductive technologies.

Adipocyte Apoptosis, a Link between Obesity, Insulin Resistance, and Hepatic Steatosis

Adipocyte death has been reported in both obese humans and rodents. However, its role in metabolic disorders, including insulin resistance, hepatic steatosis, and inflammation associated with obesity has not been studied. We now show using real-time reverse transcription-PCR arrays that adipose tissue of obese mice display a pro-apoptotic phenotype. Moreover, caspase activation and adipocyte apoptosis were markedly increased in adipose tissue from both mice with diet-induced obesity and obese humans. These changes were associated with activation of both the extrinsic, death receptor-mediated, and intrinsic, mitochondrial-mediated pathways of apoptosis. Genetic inactivation of Bid, a key pro-apoptotic molecule that serves as a link between these two cell death pathways, significantly reduced caspase activation, adipocyte apoptosis, prevented adipose tissue macrophage infiltration, and protected against the development of systemic insulin resistance and hepatic steatosis independent of body weight. These data strongly suggest that adipocyte apoptosis is a key initial event that contributes to macrophage infiltration into adipose tissue, insulin resistance, and hepatic steatosis associated with obesity in both mice and humans. Inhibition of adipocyte apoptosis may be a new therapeutic strategy for the treatment of obesity-associated metabolic complications.