Large Differences In Expression Research Articles

Survivin: A promising tumor biomarker

Survivin is a 16.5 kDa protein overexpressed in almost all malignancies but rarely detected in normal differentiated adult tissues. Functionally, survivin has been shown to inhibit apoptosis, promote cell proliferation and enhance angiogenesis. Consistent with its role in these processes, survivin has been shown to play a key role in cancer progression. Because of the large difference in expression between normal and malignant tissue and its causal role in cancer progression, survivin is currently undergoing intensive investigation as a potential tumor marker. Emerging data suggests that measurement of survivin can aid the early diagnosis of bladder cancer, determine prognosis in multiple cancer types and predict response to diverse anti-cancer therapies. These preliminary findings on the diagnostic, prognostic and predictive potential of survivin should now be confirmed in large prospective trials. Furthermore, assays for the measurement of survivin should be simplified, standardized and evaluated in external quality assurance schemes.

Proteome Analysis of Responses to Ascochlorin in a Human Osteosarcoma Cell Line by 2-D Gel Electrophoresis and MALDI-TOF MS

Ascochlorin is a prenyl-phenol compound that was isolated from the fungus Ascochyta viciae. Ascochlorin reduces serum cholesterol and triglyceride levels, suppresses hypertension and tumor development, and ameliorates type I and II diabetes. Here, to better understand the mechanisms by which ascochlorin regulates physiological or pathological events and induces responses in the pharmacological treatment of cancer, we performed differential analysis of the proteome of the human osteosarcoma cells U2OS in response to ascochlorin. In addition, we established the first two-dimensional map of the U2OS proteome. The U2OS cell proteomes with and without treatment with ascochlorin were compared using two-dimensional electrophoresis, matrix-assisted laser desorption/ionization mass spectrometry and bioinformatics. The largest differences in expression were observed for the epidermal growth factor receptor (4-fold decrease), ribulose-5-phosphate-epimerase (13-fold decrease), ATP-dependent RNA helicase (8-fold decrease), and kelch-like ECH-associated protein 1 (6-fold decrease). The abundance of heterogeneous nuclear ribonucleoprotein L and minichromosome maintenance protein 7 increased 12- and 8.2-fold, respectively. In addition, Erk 2 was increased 3-fold in U2OS cells treated with ascochlorin. The expression of some selected proteins was confirmed by western blotting, zymography and RT-PCR analysis.

Quantitative mapping of tryptophan hydroxylase‐2, 5‐HT1A, 5‐HT1B, and serotonin transporter expression across the anteroposterior axis of the rat dorsal and median raphe nuclei

Depression and anxiety disorders are among the leading causes of morbidity, mortality, and disability in the United States. Impaired serotonin neurotransmission appears to be a central mechanism inducing depressive and anxiety symptoms. Most serotonergic innervation of the forebrain arises from the median raphe nucleus (MRN) and dorsal raphe nucleus (DRN). The DRN displays a complex internal morphology, with distinct subregions varying across the anteroposterior (A-P) axis. However, many studies have considered the DRN as a whole or used easily confused terminology to describe position. Given the large differences in receptor expression, electrophysiological properties, and connectivity between various subregions of the DRN, it appears probable that they have distinct functional roles in the regulation of behavior. To foster uniform definitions of locations within these nuclei, we have quantitatively mapped gene expression in DRN and MRN for tryptophan hydroxylase-2 (Tph2), the serotonin transporter, as well as 5-HT1A and 5-HT1B receptors. These quantitative studies revealed differences in the density of expression of each gene in the ventromedial, dorsomedial, and dorsolateral subnuclei of the DRN, as well as distinct variation in expression across the A-P axis. These findings provide additional evidence that subregions of the DRN are heterogeneous and need to be considered independently. In addition, a fine scale map of Tph2 expression suggests definitions for categorical divisions of the DRN across the A-P axis. These are based on distinct morphological patterns of Tph2 expression and may be more reflective of physiology than the classic terminology dividing the DRN into equal thirds.

Search for an association between the human CYP1A2 genotype and CYP1A2 metabolic phenotype

The genotype responsible for more than 60-fold interindividual differences in human hepatic CYP1A2 constitutive expression is not understood. Resequencing the human CYP1A1_CYP1A2 locus (39.6 kb) in five major geographically isolated subgroups recently led to the identification of 85 single nucleotide polymorphisms (SNPs), 57 of which were double-hit SNPs. Here, we attempted to correlate the CYP1A2 genotype with a metabolic phenotype. We chose 16 SNPs (all having a minor allele frequency > or =0.05 in Caucasians) to genotype 32 DNA samples (26 Caucasians, six Ethiopians) in which CYP1A2 metabolism had previously been determined. From 280 subjects (five locations worldwide) that had been CYP1A2-phenotyped, we genotyped the 10 highest, 14 lowest and eight intermediate DNA samples. Although no SNP was significant (P<0.05), possibly due to the small sample size, we found a trend for several of the six SNPs across the CYP1A2 linkage disequilibrium block associated with the trait. Five CYP1A2 haplotypes were inferred, two of which had not previously been reported; haplotype 1A2H10 showed the greatest association with CYP1A2 activity. Regulatory sequences responsible for the large interindividual differences in hepatic CYP1A2 gene basal expression might reside, in part, with some of these CYP1A2 SNPS but, in large part, might be located either cis (in nearby sequences not yet haplotyped) or trans in that they are not linked to the gene. We conclude that no SNP or haplotype in the CYP1A2 gene has yet been identified that can unequivocally be used to predict the metabolic phenotype in any individual patient.

Proteome analysis of human monocytic THP‐1 cells primed with oxidized low‐density lipoproteins

Native low-density lipoprotein (LDL) and oxidized LDL (oxLDL) possess a wide variety of biological properties, and play a central role in atherogenesis. In this study, we used a proteomic analysis of human monocyte THP-1 cells induced with oxLDL or with LDL, to identify proteins potentially involved in atherosclerotic processes. Of the 2500 proteins detected, 93 were differentially expressed as a result of priming with LDL or oxLDL. The proteins were unambiguously identified by comparing the masses of their tryptic peptides with those of all known proteins using MALDI-TOF MS and the NCBI database. The largest differences in expression were observed for vimentin (94-fold increase), meningioma-expressed antigen 6 (48-fold increase), serine/threonine protein phosphatase 2A (40-fold increase), and beta-1,3-galactosyltransferase (15-fold increase). In contrast, the abundance of an unnamed protein product and phosphogluconate dehydrogenase decreased 30-fold and 25-fold, respectively. The expression of some selected proteins was confirmed by Western blot and RT-PCR analyses. The proteins identified in this study are attractive candidates for further biomarker research. This description of the altered protein profiles induced by oxLDL in human monocytes will support functional studies of the macrophage-derived foam cells involved in the pathogenesis of atherosclerosis.

Identification of PML-RARa Target Genes Using Microarray and ChIP-on-Chip Analysis.

PML-RARa is very toxic to early myeloid cell lines that express neutrophil elastase. As a consequence, myeloid cell lines that stably express PML-RARa must undergo significant adaptations to avoid toxicity (eg. loss of expression of neutrophil elastase (Lane and Ley, MCB, 2005). To identify the direct target genes of PML-RARa, we decided to bypass the issue of adaptation by profiling naive U937 cells that transiently expressed PML-RARa. To define “immediate-early” expression changes caused by PML-RARa, we harvested U937 cells electroporated with expression vectors containing eGFP-PML-RARa (known to possess all the measurable functional properties of PML-RARa) vs. eGFP alone, at the earliest time point when adequate numbers of eGFP positive cells could be collected via high speed cell sorting (6 hours). RNA obtained from these cells was subjected to linear amplification and hybridized with Affymetrix U133 Plus 2.0 arrays. Probesets that demonstrated the largest differences in expression between cells transfected with eGFP vs. eGFP-PML-RARa were PML and RARa themselves (15-fold increase for PML, 9-fold for RARa), providing an important validation for the experimental system. Differentially expressed genes were selected by three filters for further study: (i) ANOVA statistic (P < 0.05); (ii) “present” call and >150 average expression units in at least one sample; and (iii) Reproducible (n=2) >2-fold differences between eGFP-PML-RARa vs. eGFP vector transfected cells. Only 280 probesets (out of 54,613 total) met all of these criteria. Among them, 37 genes were involved in signal transduction, 15 in cell proliferation and differentiation, 7 in apoptosis, 14 in cell cycle, 15 in transport, 27 in protein metabolism and modification, 13 in oncogenesis, and 13 in immunity and defense (Panther classification system). 15/15 genes selected from a variety of categories were further validated with Q-PCR in duplicate using replicate samples, and all differences were statistically significant (p<0.05). Q-PCR validated genes that were up-regulated at 6 hours by eGFP-PML-RARa included CD36 (196% increase over eGFP vector control), LATS2 (204%), Sox4 (395%), COPEB (325%), HoxA5 (294%), IFITM3 (139%), AIF1 (197%), and PLD1 (315%). Q-PCR validated genes that were down-regulated by eGFP-PML-RARa at 6 hours included BTG2 (60% the level of eGFP vector control), KLF7(52%), CDKN1A (p21)(39%), Pim1 (58%), SLC23A2 (36%), HoxA13 (51%), and PDCD1LG1 (42%). Among these genes, Sox4, Pim1, BTG2, and PDCD1LG1 have previously been implicated in cancer pathogenesis by retroviral insertional mutagenesis (http://RTCGD.ncifcrf.gov ). To identify promoters that were directly in contact with the eGFP-PML-RARa fusion protein, we performed “ChIP-on-chip” analysis using chromatin immunoprecipitation coupled with a NimbleGen promoter tiling array containing 15 50-mer probes (~100 bp spacing) between positions −1500 and +500 of the transcription initiation sites of a curated subset of 24,275 known human genes (HG17, Build 35). By subtracting non-specific hybridization signals from the eGFP control chip, we identified and validated several promoter regions as direct binding targets of eGFP-PML-RARa, including HoxA13 and CDKN1A (p21). This experimental system provides a new approach for the identification of the direct transcriptional targets of PML-RARa, which may lead to new insights into APL pathogenesis and treatment.

Human CXCR2 (hCXCR2) takes over functionalities of its murine homolog in hCXCR2 knockin mice

Human CXCR2 (hCXCR2) has been implicated in diverse inflammatory diseases. When roles of this receptor studied in animal models are extrapolated into men, large species differences in expression of the receptor and its ligands must be considered. These differences seriously weaken conclusions toward the role of hCXCR2 in the development of human diseases. It furthermore hampers straightforward testing of CXCR2 antagonists, especially when compounds discriminate between human and other species' receptors. Using gene targeting in embryonic stem cells, a hCXCR2 knockin mouse strain was generated in which endogenous murine CXCR2 (mCXCR2) sequences are replaced by the hCXCR2 gene. Correct targeting and expression on neutrophils were confirmed by Southern blot and immunohistochemical analyses. A phenotypic analysis of the hCXCR2 knockin mice, in comparison to wild-type and CXCR2 knockout mice, confirmed proper function of the hCXCR2 gene. In vivo migratory responses of neutrophils were intact in hCXCR2 knockin mice. Finally, an experiment with a CXCR2 antagonist demonstrated that the knockin model is indeed useful for in vivo evaluation of low-molecular weight compounds. In conclusion, our data unequivocally show that hCXCR2 can functionally replace mCXCR2, making this an attractive model to test novel pharmaceuticals designed to antagonize human CXCR2 in vivo.

Transcriptional synergy and the regulation of Ucp1 during brown adipocyte induction in white fat depots.

Induction of brown adipocytes in white fat depots by adrenergic stimulation is a complex genetic trait in mice that affects the ability of the animal to regulate body weight. An 80-fold difference in expression of the mitochondrial uncoupling gene (Ucp1) at the mRNA and protein levels between A/J and C57BL/6J (B6) mice is controlled by allelic interactions among nine quantitative trait loci (QTLs) on eight chromosomes. Overlapping patterns of these QTLs also regulate expression levels of Pgc-1alpha, Pparalpha, and type 2 deiodinase. Independent validation that PPARalpha is associated with Ucp1 induction was obtained by treating mice with the PPARalpha agonist clofibrate, but not from the analysis of PPARalpha knockout mice. The most upstream sites of regulation for Ucp1 that differed between A/J and B6 were the phosphorylation of p38 mitogen-activated protein kinase and CREB and then followed by downstream changes in levels of mRNA for PPARgamma, PPARalpha, PGC-1alpha, and type 2 deiodinase. However, compared to Ucp1 expression, the two- to fourfold differences in the expression of these regulatory components are very modest. It is proposed that small variations in the levels of several transcriptional components of the Ucp1 enhanceosome interact synergistically to achieve large differences in Ucp1 expression.

Clinical Significance of Heparin-Binding Epidermal Growth Factor–Like Growth Factor and A Disintegrin and Metalloprotease 17 Expression in Human Ovarian Cancer

Lysophosphatidic acid, which is enriched in the peritoneal fluid of ovarian cancer patients, plays a key role in the progression of ovarian cancer. Lysophosphatidic acid can generate epidermal growth factor receptor (EGFR) signal transactivation involving processing of EGFR ligands by ADAM (a disintegrin and metalloprotease) family metalloproteases. We aimed to investigate the clinical significance of EGFR ligands and ADAM family in the lysophosphatidic acid-induced pathogenesis of ovarian cancer. We examined the expression of EGFR ligands and ADAM family members in 108 patients with normal ovaries or ovarian cancer, using real-time PCR, immunohistochemistry, and in situ hybridization, and analyzed the clinical roles of these molecules. Statistical analyses of these data were done using the Mann-Whitney test, Kaplan-Meier method, or Spearman's correlation analysis. Large differences in expression were found for heparin-binding EGF-like growth factor (HB-EGF) and other EGFR ligands and for ADAM 17 and other ADAM family members. HB-EGF expression was significantly increased in advanced ovarian cancer compared with that in normal ovaries (P < 0.01). HB-EGF expression was significantly associated with the clinical outcome (P < 0.01). ADAM 17 expression was significantly enhanced in both early and advanced ovarian cancer compared with that in normal ovaries (both P < 0.01), although it had no clinical significance in the progression-free survival. HB-EGF expression was significantly correlated with ADAM 17 expression (gamma = 0.437, P < 0.01). Our findings suggest that HB-EGF and ADAM 17 contribute to the progression of ovarian cancer and that HB-EGF plays a pivotal role in the aggressive behavior of a tumor in ovarian cancer.

Changes in the proteomic profile during differentiation and maturation of human monocyte-derived dendritic cells stimulated with granulocyte macrophage colony stimulating factor/interleukin-4 and lipopolysaccharide

Dendritic cells (DCs) are highly specialized antigen-presenting cells that play an essential role in the immune response. We used the proteomic approach based on two-dimensional gel electrophoresis and mass spectrometry to identify the protein changes that occur during differentiation of DCs from monocytes (Mo) stimulated with granulocyte macrophage colony stimulating factor/interleukin-4 (GM-CSF/IL-4) and during the maturation of immature DCs stimulated with lipopolysaccharide. Sixty-three differentially expressed proteins (+/- two-fold) were unambiguously identified with sequence coverage greater than 20%. They corresponded to only 36 different proteins, because 11 were present as 38 electrophoretic forms. Some proteins such as tropomyosin 4 and heat shock protein 71 presented differentially expressed electrophoretic forms, suggesting that many of the changes in protein expression that accompany differentiation and maturation of DCs occur in post-translationally modified proteins. The largest differences in expression were observed for actin (21-fold in Mo), Rho GDP-dissociation inhibitor 2 (20-fold in Mo), vimentin (eight-fold in immature DCs), lymphocyte-specific protein 1 (12-fold in mature DCs) and thioredoxin (14-fold in mature DCs). Several proteins are directly related to functional and morphological characteristics of DCs, such as cytoskeletal proteins (cytoskeleton rearrangement) and chaperones (antigen processing and presentation), but other proteins have not been assigned specific functions in DCs. Only a few proteins identified here were the same as those reported in proteomic studies of DCs, which used different stimuli to produce the cells (GM-CSF/IL-4 and tumor necrosis factor-alpha). These data suggest that the DC protein profile depends on the stimuli used for differentiation and especially for maturation.

Quantitation of H. pylori cytotoxin mRNA by real-time RT-PCR shows a wide expression range that does not correlate with promoter sequences

Up to 28-fold differences in vacA expression in Helicobacter pylori strains grown in vitro were demonstrated by real time quantitative RT-PCR. These large differences in expression were unrelated to putative −35 and −10 motifs or to other untranslated sequences upstream of the ATG start site. The lack of correlation between promoter sequences and the vacA expression levels suggest the potential existence of a bacterial strain-specific factor, as earlier proposed by others on the basis of reporter gene fusions.

A preoperative diagnostic test that distinguishes benign from malignant thyroid carcinoma based on gene expression

Accurate diagnosis of thyroid tumors is challenging. A particular problem is distinguishing between follicular thyroid carcinoma (FTC) and benign follicular thyroid adenoma (FTA), where histology of fine-needle aspirates is not conclusive. It is often necessary to remove healthy thyroid to rule out carcinoma. In order to find markers to improve diagnosis, we quantified gene transcript expression from FTC, FTA, and normal thyroid, revealing 73 differentially expressed transcripts (P ≤ 0.0001). Using an independent set of 23 FTCs, FTAs, and matched normal thyroids, 17 genes with large expression differences were tested by real-time RT-PCR. Four genes (DDIT3, ARG2, ITM1, and C1orf24) differed between the two classes FTC and FTA, and a linear combination of expression levels distinguished FTC from FTA with an estimated predictive accuracy of 0.83. Furthermore, immunohistochemistry for DDIT3 and ARG2 showed consistent staining for carcinoma in an independent set 59 follicular tumors (estimated concordance, 0.76; 95% confidence interval, [0.59, 0.93]). A simple test based on a combination of these markers might improve preoperative diagnosis of thyroid nodules, allowing better treatment decisions and reducing long-term health costs.

A preoperative diagnostic test that distinguishes benign from malignant thyroid carcinoma based on gene expression.

Accurate diagnosis of thyroid tumors is challenging. A particular problem is distinguishing between follicular thyroid carcinoma (FTC) and benign follicular thyroid adenoma (FTA), where histology of fine-needle aspirates is not conclusive. It is often necessary to remove healthy thyroid to rule out carcinoma. In order to find markers to improve diagnosis, we quantified gene transcript expression from FTC, FTA, and normal thyroid, revealing 73 differentially expressed transcripts (P < or = 0.0001). Using an independent set of 23 FTCs, FTAs, and matched normal thyroids, 17 genes with large expression differences were tested by real-time RT-PCR. Four genes (DDIT3, ARG2, ITM1, and C1orf24) differed between the two classes FTC and FTA, and a linear combination of expression levels distinguished FTC from FTA with an estimated predictive accuracy of 0.83. Furthermore, immunohistochemistry for DDIT3 and ARG2 showed consistent staining for carcinoma in an independent set 59 follicular tumors (estimated concordance, 0.76; 95% confidence interval, [0.59, 0.93]). A simple test based on a combination of these markers might improve preoperative diagnosis of thyroid nodules, allowing better treatment decisions and reducing long-term health costs.

Ceramic hydroxyapatite coating on titanium implants drives selective bone marrow stromal cell adhesion.

The aim of this study was to determine the cell characteristics that regulate implant osseointegration. The heterogeneity of bone marrow stromal cells obtained from 11 donors was assessed by measuring the expression of a large panel of adhesion molecules. Large differences in expression of adhesion molecules were detected depending on the culture conditions used. Cells cultured in fetal bovine serum induced the expression of different adhesion molecules from cells cultured in human serum. Donor-to-donor variation was determined by measuring the expression of adhesion molecules for stromal cells obtained from different donors that were processed identically. Fat adherent cells but also loose bone marrow cells showed large differences in expression of some but not all adhesion molecules. The flow cytometric data demonstrated large heterogeneity in expression of adhesion molecules, and this heterogeneity was influenced by culture conditions and varied from donor to donor. This demonstrates that the implant encounters different cell types, which could lead to different levels of integration. Surprisingly, in vitro only a subfraction of bone marrow stromal cells attached to titanium coated with ceramic hydroxyapatite. Adaptation of all cell types present in heterogeneous bone marrow to a coated surface is apparently not possible. Differential binding was not caused by aberrant staining of the stromal cells as the results were confirmed with bone marrow cells obtained from transgenic GFP mice. These results demonstrate that hydroxyapatite ceramics are selective in cell recruitment from the bone marrow, explaining the differences found in vivo for these coatings compared with titanium.

ICAM-1 and VCAM-1 expression by endothelial cells grown on fibronectin-coated TCPS and PS.

Small-diameter vascular grafts rapidly fail as a result of blood coagulation and platelet deposition. Endothelial cells lining the inner side of blood vessels can provide the graft lumen with an antithrombogenic surface. One of the remaining problems is cell detachment after restoration of blood flow, because of infiltration of leukocytes that respond to an inflammatory-like activation of the endothelial cells. This endothelial activation is possibly caused by the surface characteristics of the underlying polymer. To get more insight into the effects of the polymer surface on endothelial cell activation, we seeded human umbilical vein endothelial cells (HUVECs) in various densities and subsequently grew them on tissue culture polystyrene (TCPS; hydrophilic) and polystyrene (PS; hydrophobic) surfaces. To improve cell adhesion, surfaces were coated with purified fibronectin prior to cell seeding. During proliferation, the expressions of the leukocyte adhesion molecules ICAM-1 and VCAM-1 were determined. Results indicate that ICAM-1 expression is not influenced by the character of the polymer surface, and that VCAM-1 expression is slightly higher on the TCPS surface. Expressions of both adhesion molecules are influenced by the seeding density and time of proliferation. At low seeding densities (< or = 10,000 cells/cm(2)), a relatively low percentage of nonexogenously activated cells expressed ICAM-1 during the first 3 days of proliferation compared to higher seeding densities. Although less pronounced, this was also observed for the percentage of cells expressing VCAM-1. During proliferation, the amount of ICAM-1 per endothelial cell increased, whereas the expression of VCAM-1 remained low. The absence of large differences in leukocyte adhesion molecule expression by endothelial cells grown on TCPS or PS is possibly caused by coating of the surfaces with fibronectin. It is known that surface hydrophilicity influences protein adsorption. Although this had no or little effect on leukocyte adhesion molecule expression, endothelial cell growth was affected, because proliferation was slower on the hydrophobic PS.

Interferon-gamma enhances cytotoxic T lymphocyte recognition of endogenous peptide in keratinocytes without lowering the requirement for surface peptide.

Keratinocytes expressing the human papillomavirus (HPV) type 16 E7 protein, as a transgene driven by the K14 promoter, form a murine model of HPV-mediated epithelial cancers in humans. Our previous studies have shown that K14E7 transgenic skin grafts onto syngeneic mice are not susceptible to immune destruction despite the demonstrated presence of a strong, systemic CTL response directed against the E7 protein. Consistent with this finding, we now show that cultured, E7 transgenic keratinocytes (KC) express comparable endogenous levels of E7 protein to a range of CTL-sensitive E7-expressing cell lines but are not susceptible to CTL-mediated lysis in vitro. E7 transgenic and non-transgenic KC are susceptible to conventional mechanisms of CTL-mediated lysis, including perforin and Fas/FasL interaction when an excess of exogenous peptide is provided. The concentration of exogenous peptide required to render a cell susceptible to lysis was similar between KC and other conventional CTL targets (e.g. EL-4), despite large differences in H-2Db expression at the cell surface. Furthermore, exposure of KC to IFN-gamma increased H-2Db expression, but did not substantially alter the exogenous peptide concentration required to sensitize cells for half maximal lysis. In contrast, the lytic sensitivity of transgenic KC expressing endogenous E7 is modestly improved by exposure to IFN-gamma. Thus, failure of CTL to eliminate KC expressing endogenous E7, and by inference squamous tumours expressing E7, may reflect the need for a sustained, local inflammatory environment during the immune effector phase.

Effect of the storage period of paraffin sections on the detection of mRNAs by in situ hybridization.

In this study we evaluated whether storing non-deparaffinized sections can affect the detection of specific mRNAs by radioactive in situ hybridization (ISH). Using a standard ISH protocol, we hybridized serial sections of paraffin blocks stored for different periods of time with (33)P-labeled riboprobes specific for rat Type III collagen and matrix metalloproteinase-2 (MMP-2). Signal intensities were evaluated using a phosphorimager and by blinded microscopic examination. For slides hybridized with the Type III collagen riboprobe, signal intensities measured with the phosphorimager or evaluated by microscopic examination were negatively correlated with the storage period of the sections. For slides hybridized with the MMP-2 riboprobe, differences in signal intensity could be detected, albeit inconsistently, with the phosphorimager, although microscopic examination consistently indicated stronger signals in freshly sectioned slides compared to slides stored for 2 weeks or more. We concluded that it was preferable to use recently prepared sections for trying to locate mRNAs in paraffin-embedded tissues by ISH. In addition, our results suggest that quantifying signal intensity using a phosphorimager is feasible for abundant mRNAs or when large differences in expression are anticipated.(J Histochem Cytochem 49:927-928, 2001)

Mutation analysis ofNTRK2 andNTRK3, encoding 2 tyrosine kinase receptors, in sporadic human medullary thyroid carcinoma reveals novel sequence variants

Somatic mutations in the proto-oncogene RET are found in 25% to 80% of sporadic medullary thyroid carcinomas (MTCs). The significance of somatic RET mutation in MTC initiation and progression, however, remains unknown. Like RET, TRK is a neurotrophic receptor tyrosine kinase. Immunostaining has shown that only a subset of normal C cells expresses Trk family receptors, but in C-cell hyperplasia, they consistently express NTRK2, with variable expression of NTRK1 and NTRK3. In later stages of MTC, NTRK2 expression was reduced while NTRK3 expression was increased. In the context of these data, we sought to determine whether sequence variants in NTRK2 and NTRK3 are responsible for these differences in protein expression. We determined the genomic structure of NTRK2 and found that it consists of at least 17 exons varying in size from 36 to 306 bp. Mutation analysis of sporadic MTC did not reveal any sequence variants in NTRK2 but did reveal 3 variants in NTRK3, c.573C >T (N191N, exon 5), c.678T > C (N226N, exon 6) and c.1488C > G (A496A, exon 12) occurring among 19 chromosomes (31%), 1 chromosome (2%) and 24 chromosomes (39%), respectively. Corresponding germline also harbored these variants. There was a trend toward excess association of the NTRK3 variant c.1488C > G (A496A) in cases (24/62 chromosomes, 39%) compared to controls (18/62, 29%), but this difference did not reach significance (p > 0.05). The remaining 2 NTRK3 variants occurred with similar frequencies between MTC cases and population-matched controls (19 vs. 17 and 1 vs. 0, p > 0.05). We conclude that sequence variants in NTRK2 and NTRK3 are not likely to be responsible for large differences in expression at the protein level, but we cannot exclude very low penetrance effects.

Impact of endotoxin on UCP homolog mRNA abundance, thermoregulation, and mitochondrial proton leak kinetics.

Linking tissue uncoupling protein (UCP) homolog abundance with functional metabolic outcomes and with expression of putative genetic regulators promises to better clarify UCP homolog physiological function. A murine endotoxemia model characterized by marked alterations in thermoregulation was employed to examine the association between heat production, UCP homolog expression, and mitochondrial proton leak ("uncoupling"). After intraperitoneal lipopolysaccharide (LPS, approximately 6 mg/kg) injection, colonic temperature (T(c)) in adult female C57BL6/J mice dropped to a nadir of approximately 30 degrees C by 8 h, preceded by a four- to fivefold drop in liver UCP2 and UCP5/brain mitochondrial carrier protein 1 mRNA levels, with no change in their hindlimb skeletal muscle (SKM) expression. SKM UCP3 mRNA rose fivefold during development of hypothermia and was correlated with an LPS-induced increase in plasma free fatty acid concentration. UCP2 and UCP5 transcripts recovered about three- to sixfold in both tissues starting at 6-8 h, preceding a recovery of T(c) between 16 and 24 h. SKM UCP3 followed an opposite pattern. Such results are not consistent with an important influence of UCP3 in driving heat production but do not preclude a role for UCP2 or UCP5 in this process. The transcription coactivator PGC-1 displayed a transient LPS-evoked rise (threefold) or drop (two- to fivefold) in SKM and liver expression, respectively. No differences between control and LPS-treated mouse liver or SKM in vitro mitochondrial proton leak were evident at time points corresponding to large differences in UCP homolog expression.

Modulation of transforming growth factor beta1 gene expression in the mammary gland by insulin-like growth factor I and octreotide.

Transforming growth factor beta1 (TGF-beta1) has been shown to exhibit anti-proliferative activity for mammary gland epithelial cells and for human breast cancer cells. Insulin-like growth factor I (IGF-I), in contrast, is a well-characterized mitogenic and anti-apoptotic factor involved in mammary gland physiology. In order to examine the hypothesis that IGF-I suppresses TGF-beta1 expression in the mammary gland, we studied the effect of various manipulations of the growth hormone - IGF-I axis on TGF-beta1 mRNA abundance. Administration of IGF-I to intact animal suppressed TGF-beta1 mRNA levels in a dose-dependent manner to approximately 20% of control levels. Administration of the somatostatin analogue octreotide in a manner previously shown to acutely suppress the growth hormone - IGF-I axis increased mammary gland TGF-beta1 expression approximately 3-fold. Transgenic mice overexpressing growth hormone expressed TGF-beta1 in the mammary gland at only approximately 12% of the level of control animals, while mice IGF-I deficient due to the mutation expressed TGF-beta1 at slightly higher levels than control animals. The large differences in TGF-beta1 expression between control and GH-transgenic animals were correlated with major differences in architecture of the mammary gland, while the appearance of mammary glands of normal and animals was similar. These data document a previously unrecognized relationship between TGF-beta1 and IGF-I physiology in the mammary gland, and suggest a novel mechanism by which somatostatin analogues influence the proliferative behaviour of breast epithelial cells.