ABSTRACT
Metastasis is the most frequent cause of death among patients with osteosarcoma. We have previously demonstrated in independent experiments that the forced expression of L/B/K ALP and CD99 in U-2 OS osteosarcoma cell lines markedly reduces the metastatic ability of these cancer cells. This behavior makes these cell lines a useful model to assess the intersection of multiple and independent gene expression signatures concerning the biological problem of dissemination. With the aim to characterize a common transcriptional profile reflecting the essential features of metastatic behavior, we employed cDNA microarrays to compare the gene expression profiles of L/B/K ALP- and CD99-transfected osteosarcoma clones showing low metastatic ability with those of osteosarcoma cell lines showing contrasting behavior. Changes in gene expression were validated by real-time PCR and immunohistochemistry in independent samples. In our study we identified several differentially expressed genes (GADD45alpha, VCP, DHX9, survivin, alpha-catulin, ARPC1B) related to growth arrest and apoptosis. Most of these genes are functionally related with the nuclear factor (NF)-kappaB cell survival pathway that appeared to be inhibited in the less malignant osteosarcoma cells. Hence, we propose the inhibition of the NF-kappaB pathway as a rational strategy for effective management of human osteosarcoma.
Subject(s)
Apoptosis , Bone Neoplasms/secondary , Osteosarcoma/secondary , Biomarkers, Tumor , Bone Neoplasms/genetics , Bone Neoplasms/pathology , Cell Cycle Proteins/genetics , Cell Line, Tumor , Cyclin D1/genetics , Gene Expression Profiling , Humans , NF-kappa B/genetics , NF-kappa B/physiology , Nuclear Proteins/genetics , Osteosarcoma/genetics , Osteosarcoma/pathology , Phenotype , Signal TransductionABSTRACT
BACKGROUND: All standard methods for cDNA cloning are affected by a potential inability to effectively clone the 5' region of mRNA. The aim of this work was to estimate mRNA open reading frame (ORF) 5' region sequence completeness in the model organism Danio rerio (zebrafish). RESULTS: We implemented a novel automated approach (5'_ORF_Extender) that systematically compares available expressed sequence tags (ESTs) with all the zebrafish experimentally determined mRNA sequences, identifies additional sequence stretches at 5' region and scans for the presence of all conditions needed to define a new, extended putative ORF. Our software was able to identify 285 (3.3%) mRNAs with putatively incomplete ORFs at 5' region and, in three example cases selected (selt1a, unc119.2, nppa), the extended coding region at 5' end was cloned by reverse transcription-polymerase chain reaction (RT-PCR). CONCLUSION: The implemented method, which could also be useful for the analysis of other genomes, allowed us to describe the relevance of the "5' end mRNA artifact" problem for genomic annotation and functional genomic experiment design in zebrafish.
Subject(s)
Expressed Sequence Tags , Open Reading Frames/genetics , RNA, Messenger/genetics , Zebrafish/genetics , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , Cluster Analysis , Computational Biology , DNA, Complementary/genetics , Databases, Factual , Molecular Sequence Data , Phylogeny , Reverse Transcriptase Polymerase Chain Reaction , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Software , Zebrafish Proteins/geneticsABSTRACT
AIM: A growing number of mutations mapped in the receptor gene for fibroblast growth factor have been implicated in several cranial development disorders including the Apert and Crouzon syndromes. The present paper investigated cellular mechanisms underlying Apert phenotype, by analyzing the effects of FGF2 in primary cultures of Apert periosteal fibroblasts carrying the FGFR2 Pro253Arg mutation. RESULTS: FGF2 administration significantly decreased extracellular matrix production in mutant cells by stimulating degradative enzymatic activities. Gene expression analysis revealed that decorin and biglycan, two proteoglycans involved in collagen fibrillogenesis, were more expressed in mutant cells and down-regulated by FGF2. FGF2 receptor binding showed little differences in high affinity receptor counts between mutant and wild-type cells, while we showed for the first time that low affinity receptors are significantly fewer in mutant cells. Differences were found in Crouzon syndrome, where both high and low affinity receptor counts were up-regulated. CONCLUSIONS: The different mutation and low affinity receptor regulation in mutant receptors support the hypothesis that the impact on the activity of the ligand-receptor complex could allow distinct modes of FGF2 activation in Apert and Crouzon syndromes, which interfere with the FGFR2 signalling cascade.
Subject(s)
Acrocephalosyndactylia/genetics , Craniofacial Dysostosis/genetics , Extracellular Matrix/metabolism , Fibroblast Growth Factor 2/metabolism , Periosteum/metabolism , Receptor, Fibroblast Growth Factor, Type 2/metabolism , Acrocephalosyndactylia/metabolism , Adolescent , Arginine/chemistry , Arginine/genetics , Cell Count , Collagen Type I/metabolism , Craniofacial Dysostosis/metabolism , DNA Mutational Analysis , Fibroblast Growth Factor 2/pharmacology , Fibroblasts/drug effects , Fibroblasts/metabolism , Fibronectins/metabolism , Glycoside Hydrolases/metabolism , Humans , Matrix Metalloproteinase 1/genetics , Matrix Metalloproteinase 1/metabolism , Mutation , Peptide Hydrolases/metabolism , Periosteum/cytology , Periosteum/drug effects , Phenotype , Proline/chemistry , Proline/genetics , Proteoglycans/genetics , Proteoglycans/metabolism , RNA, Messenger/analysis , RNA, Messenger/metabolism , Receptor, Fibroblast Growth Factor, Type 2/agonists , Receptor, Fibroblast Growth Factor, Type 2/geneticsSubject(s)
Calcineurin Inhibitors , Intracellular Signaling Peptides and Proteins/metabolism , Isoenzymes/metabolism , Multigene Family , Muscle Proteins/metabolism , Animals , Calcineurin/metabolism , DNA-Binding Proteins , Down Syndrome , Humans , Intracellular Signaling Peptides and Proteins/genetics , Isoenzymes/genetics , Muscle Proteins/geneticsABSTRACT
Clefts of the lip with or without cleft palate (CL/P) are one of the most common birth defects, occurring in 1/700-1/1,000 infants born alive. The nature of the genetic contribution is still to be clarified; however, some chromosome regions and candidate genes have been proposed for this malformation. Recently, a couple of genes, PVR and PVRL2, mapping in the candidate region OFC3 on chromosome 19q13.31, have been investigated because of their homology to PVRL1, a gene previously shown to cause the Margarita Island CL/P-ectodermal dysplasia syndrome. In the present work, we investigated PVR and PVRL2 genes by family-based linkage disequilibrium analysis using a sample collected from the Italian population. In contrast to previous analyses on other populations, we could not find any statistically significant association between the markers alleles and non-syndromic clefting.
Subject(s)
Cell Adhesion Molecules/genetics , Cleft Lip/genetics , Cleft Palate/genetics , Linkage Disequilibrium , Membrane Proteins/genetics , Receptors, Virus/genetics , Adult , Alleles , Child , Chromosomes, Human, Pair 19 , Female , Genetic Markers , Humans , Italy , Male , Nectins , Polymorphism, Single NucleotideABSTRACT
BACKGROUND: CYYR1 is a recently identified gene located on human chromosome 21 whose product has no similarity to any known protein and is of unknown function. Analysis of expressed sequence tags (ESTs) have revealed high human CYYR1 expression in cells belonging to the diffuse neuroendocrine system (DNES). These cells may be the origin of neuroendocrine (NE) tumors. The aim of this study was to conduct an initial analysis of sequence, splicing and expression of the CYYR1 mRNA in human NE tumors. METHODS: The CYYR1 mRNA coding sequence (CDS) was studied in 32 NE tumors by RT-PCR and sequence analysis. A subtle alternative splicing was identified generating two isoforms of CYYR1 mRNA differing in terms of the absence (CAG- isoform, the first described mRNA for CYYR1 locus) or the presence (CAG+ isoform) of a CAG codon. When present, this specific codon determines the presence of an alanine residue, at the exon 3/exon 4 junction of the CYYR1 mRNA. The two mRNA isoform amounts were determined by quantitative relative RT-PCR in 29 NE tumors, 2 non-neuroendocrine tumors and 10 normal tissues. A bioinformatic analysis was performed to search for the existence of the two CYYR1 isoforms in other species. RESULTS: The CYYR1 CDS did not show differences compared to the reference sequence in any of the samples, with the exception of an NE tumor arising in the neck region. Sequence analysis of this tumor identified a change in the CDS 333 position (T instead of C), leading to the amino acid mutation P111S. NE tumor samples showed no significant difference in either CYYR1 CAG- or CAG+ isoform expression compared to control tissues. CYYR1 CAG- isoform was significantly more expressed than CAG+ isoform in NE tumors as well as in control samples investigated. Bioinformatic analysis revealed that only the genomic sequence of Pan troglodytes CYYR1 is consistent with the possible existence of the two described mRNA isoforms. CONCLUSION: A new "subtle" splicing isoform (CAG+) of CYYR1 mRNA, the sequence and the expression of this gene were defined in a large series of NE tumors.
Subject(s)
Alternative Splicing , Gene Expression Regulation, Neoplastic , Membrane Proteins/genetics , Neuroendocrine Tumors/genetics , RNA, Messenger/genetics , RNA, Neoplasm/genetics , Adult , Aged , Alanine/analysis , Amino Acid Sequence , Animals , Base Sequence , Codon/genetics , Digestive System Neoplasms/genetics , Evolution, Molecular , Female , Humans , Male , Membrane Proteins/biosynthesis , Membrane Proteins/chemistry , Middle Aged , Molecular Sequence Data , Neoplasms/genetics , Pan troglodytes/genetics , Point Mutation , Polymorphism, Single Nucleotide , Protein Isoforms/genetics , Reverse Transcriptase Polymerase Chain Reaction , Sequence Alignment , Sequence Homology , Species SpecificityABSTRACT
Non-syndromic cleft lip with or without palate (CL/P) is one of the most common malformations among live births, but most of the genetic components and environmental factors involved remain to be identified. Among the different causes, MYH9, the gene encoding for the heavy chain of non-muscle myosin IIA, was considered a potential candidate, because it was found to be abundantly and specifically expressed in epithelial cells of palatal shelves before fusion. After fusion, its expression level was shown to decrease and to become limited to epithelial triangles before disappearing, as fusion is completed. To determine whether MYH9 plays a role in CL/P aetiology, a family-based association analysis was performed in 218 case/parent triads using single-nucleotide polymorphism (SNP) markers. Pairwise and multilocus haplotype analyses identified linkage disequilibrium between polymorphism alleles at the MYH9 locus and the disease. The strongest deviation from a null hypothesis of random sharing was obtained with two adjacent SNPs, rs3752462 and rs2009930 (global p value = 0.001), indicating that MYH9 might be a predisposing factor for CL/P, although its pathogenetic role needs to be investigated more accurately.
Subject(s)
Cleft Lip/genetics , Cleft Palate/genetics , Myosin Heavy Chains/genetics , Nonmuscle Myosin Type IIA/genetics , Alleles , Animals , Female , Gene Expression Regulation , Haplotypes , Humans , Linkage Disequilibrium/genetics , Mice , Myosin Heavy Chains/metabolism , Palate/embryology , Palate/pathology , Polymorphism, Single Nucleotide/genetics , Pregnancy , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
To improve current knowledge of the molecular mechanisms underlying exercise-induced cardioprotection in a rat model of mild exercise training, Sprague-Dawley rats were trained to run on a treadmill up to 55% of their maximal oxygen uptake for 1 h/day, 3 days/week, 14 weeks, with age-matched sedentary controls (n = 20/group). Rats were sacrificed 48 h after the last training session. Despite lack of cardiac hypertrophy, training decreased blood hemoglobin (7.94 +/- 0.21 mM vs. 8.78 +/- 0.23 mM, mean +/- SE, P = 0.01) and increased both plasma malondialdehyde (0.139 +/- 0.005 mM vs. 0.085 +/- 0.009 mM, P = 0.05) and the activity of Mn-superoxide dismutase (11.6 +/- 0.6 vs. 16.5 +/- 1.6 mU/microg, P = 0.01), whereas total superoxide dismutase activity was unaffected. When subjected to 30-min ischemia followed by 90-min reperfusion, hearts from trained rats (n = 5) displayed reduced infarct size as compared to controls (37.26 +/- 0.92% vs. 49.09 +/- 2.11% of risk area, P = 0.04). The biochemical analyses in the myocardium, which included gene expression profiles, real-time PCR, Western blot and determination of enzymatic activity, showed training-induced upregulation of the following mRNAs and/or proteins: growth-arrest and DNA-damage induced 153 (GADD153/CHOP), heme-oxygenase-1 (HO-1), cyclooxygenase-2 (Cox-2), heat-shock protein 70/72 (HSP70/72), whereas heat-shock protein 60 (HSP60) and glucose-regulated protein 75 (GRP75) were decreased. As a whole, these data indicate that mild exercise training activates a second window of myocardial protection against ischemia/reperfusion by upregulating a number of protective genes, thereby warranting further investigation in man.
Subject(s)
Cardiovascular Diseases/prevention & control , Gene Expression Profiling , Gene Expression , Oxidative Stress/genetics , Physical Conditioning, Animal , Animals , Blotting, Western , Cardiovascular Diseases/genetics , Cardiovascular Diseases/metabolism , Cyclooxygenase 2/genetics , Cyclooxygenase 2/metabolism , HSP70 Heat-Shock Proteins/genetics , HSP70 Heat-Shock Proteins/metabolism , Heme Oxygenase (Decyclizing)/genetics , Heme Oxygenase (Decyclizing)/metabolism , Hemoglobins/metabolism , Male , Malondialdehyde/blood , Membrane Proteins/genetics , Membrane Proteins/metabolism , Models, Animal , Myocardial Infarction/genetics , Myocardial Infarction/prevention & control , Myocardial Reperfusion Injury/genetics , Myocardial Reperfusion Injury/prevention & control , Myocardium/metabolism , Oligonucleotide Array Sequence Analysis , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction , Superoxide Dismutase/metabolism , Time Factors , Transcription Factor CHOP/genetics , Transcription Factor CHOP/metabolismABSTRACT
OBJECTIVE: This study was conducted to determine whether, in low risk women having labor induced using prostaglandin gel (dinoprostone gel), there is a relationship between the concentration of mRNA for the PLAC1 gene (a trophoblast-specific gene) in maternal blood and the time elapsed between the first gel administration and spontaneous delivery. STUDY DESIGN: Blood was collected from 49 selected women at 40.2-41.4 weeks' gestation. Total RNA was extracted by means of an ABI Prism 6100 nucleic acid Prep Station and quantitative real-time PCR analysis was performed by use of a PE Applied Biosystems 5700 Sequence Detection System. Sequence data were obtained from the Genebank Sequence Database. To determine the amount of cDNA, the PLAC1 locus was used. RESULTS: Thirty women (61.2%) had a spontaneous delivery. A caesarean section, either for fetal dystocia or fetal distress, was performed in 19 (38.8%). The crude delivery rates of the women who ended up with a spontaneous delivery were 30% at 24 h and 43% at 48 h. Women (n=19) with a blood concentration of logPLAC1 mRNA>or=2.00 displayed a median time to delivery of 23.50h, (95% CI: 13.13-33.87) while those with a logPLAC1 mRNA<2.00 (n=30) had a median time of 54 h. (95% CI: 37.86-70.14; p=0.0043, log-rank test). By means of multivariate analysis, quantitative Bishop score (from 2 to 7) at the time of the first gel administration and logPLAC1 mRNA>or=2.00 were associated with a higher rate of delivery per unit of time with an odds ratio of 1.35 (95% CI: 1.07-1.71) and 3.48 (95% CI: 1.55-7.80), respectively. CONCLUSIONS: In induced term pregnancies, PLAC1 mRNA in maternal blood at the beginning of the treatment correlates with the time elapsed before delivery. This evidence demonstrates that the fetomaternal trafficking of nucleic acids is more consistent when the labor is about to begin.
Subject(s)
Cesarean Section , Labor, Induced , Maternal-Fetal Exchange , Pregnancy Proteins/blood , RNA, Messenger/blood , Trophoblasts/physiology , Adult , Cross-Sectional Studies , Dinoprostone/therapeutic use , Female , Health Status Indicators , Humans , Oxytocics/therapeutic use , Pregnancy , Pregnancy Outcome , Pregnancy Proteins/geneticsABSTRACT
Doppler analysis of the uterine arteries is currently used for pre-eclampsia (PE) screening. PLAC1 is a trophoblast-specific gene, and it is known that in normal pregnancies, trophoblastic cells are released into the maternal circulation, where specific trophoblastic mRNA can be detected. In PE, as in women who eventually develop PE, an abnormal passage of fetal and placental cells is also present. In this study, we aimed to verify whether, in normal pregnancies, Doppler waveform of the uterine arteries correlates with PLAC1 mRNA concentrations. Thirteen cases of normal pregnancies at 37 weeks' gestation (23-41) were enrolled in the study. PLAC1 mRNA was extracted from 2 mL of blood by ABI PRISM 6100 nucleic acid Prep Station (Applied Biosystems, Foster City, CA) and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was performed by a PE 5700 Sequence detection system. Bulk RNA from normal placental tissue was used as the reference curve, and the amount of PLAC1 mRNA in the study samples was then expressed as the "relative amount" of weight of placental tissue (ng/mL). The uterine arterial mean resistance index (RI) and presence/absence of a dicrote waveform were calculated by using a 5 MHz transabdominal probe (Tecnos, ESAOTE) at the uterine cervico-corporal junction. Doppler measurement was performed on the same day as blood collection. The median of the means of uterine arterial RI was 0.52 (0.39-0.68). RI of uterine arteries and PLAC1 mRNA were significantly correlated in a log-linear regression (R(2) = 0.483, P = 0.024). Our data support that in normal pregnancy, the passage of trophoblast material into the maternal circulation is correlated with the quantitative measurement of uterine hemodynamics.
Subject(s)
Arteries/metabolism , Pregnancy Proteins , Pregnancy Trimester, Second , Pregnancy Trimester, Third , RNA, Messenger/blood , Uterus/blood supply , Female , Humans , Laser-Doppler Flowmetry , Maternal-Fetal Exchange , Pregnancy , Pregnancy Proteins/blood , Pregnancy Proteins/genetics , Regression AnalysisABSTRACT
BACKGROUND: The efficacy of screening for colorectal cancer using a simple blood-based assay for the detection of tumor cells disseminated in the circulation at an early stage of the disease is gaining positive feedback from several lines of research. This method seems able to reduce colorectal cancer mortality and may replace colonoscopy as the most effective means of detecting colonic lesions. METHODS: In this work, we present a new microarray-based high-throughput screening method to identifying candidate marker mRNAs for the early detection of epithelial cells diluted in peripheral blood cells. This method includes 1. direct comparison of different samples of colonic mucosa and of blood cells to identify consistent epithelial-specific mRNAs from among 20,000 cDNA assayed by microarray slides; 2. identification of candidate marker mRNAs by data analysis, which allowed selection of only 10 putative differentially expressed genes; 3. Selection of some of the most suitable mRNAs (TMEM69, RANBP3 and PRSS22) that were assayed in blood samples from normal subjects and patients with colon cancer as possible markers for the presence of epithelial cells in the blood, using reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Our present results seem to provide an indication, for the first time obtained by genome-scale screening, that a suitable and consistent colon epithelium mRNA marker may be difficult to identify. CONCLUSION: The design of new approaches to identify such markers is warranted.
Subject(s)
Colonic Neoplasms/blood , Epithelial Cells/chemistry , Neoplastic Cells, Circulating/chemistry , Oligonucleotide Array Sequence Analysis/methods , RNA, Messenger/blood , RNA, Neoplasm/blood , Reverse Transcriptase Polymerase Chain Reaction/methods , Adult , Aged , Automation , Female , Gene Expression Profiling/methods , Humans , Keratin-20 , Keratins/blood , Male , Membrane Proteins/genetics , Middle Aged , Nuclear Proteins/genetics , Nucleocytoplasmic Transport Proteins/genetics , Serine Endopeptidases/geneticsABSTRACT
OBJECTIVE: The aim of this study was to evaluate whether the quantitative distribution of a panel of circulating mRNAs from maternal whole blood of normal pregnancies is statistically different from those complicated with preeclampsia (PE) with or without intrauterine growth restriction (IUGR). METHODS: Maternal whole blood of six subjects with mild or severe PE with or without IUGR and 30 matched controls (1:5 match for gestational age) were retrospectively examined for circulating mRNA markers. Seven specific mRNA markers were identified and chosen based on previous microarray mRNA expressions performed on placental tissue from normal and PE patients. They were human placental lactogen (hPL), inhibin A, KISS-1, pregnancy-associated plasma protein-A (PAPP-A), plasminogen activator inhibitor type 1 (PAI-1), selectin-P and vascular endothelial growth factor receptor (VEGFR), which were therefore quantified for statistical purposes. RESULTS: Median gestational age was 229 (178-283) and 232 (194-262) days for controls and cases respectively. All mRNA markers but PAPP-A, showed statistically different median values. They were hPL, inhibin A, KISS-1, PAI-1, Selectin-P, and VEGFR. Inhibin A, Selectin-P and VEGFR showed higher values than expected for controls. Instead, hPL, KISS-1 and PAI-1 values of PE patients were lower than those of controls. Selectin-P was the marker with the most aberrant difference, followed by VEGFR and KISS-1. CONCLUSION: This preliminary analysis revealed that the median values of a panel of mRNAs from the maternal blood of PE patients were different from those of the same gestational age control group at the third trimester. If prospective studies at the second trimester could detect a related marker sufficiently able to discriminate between affected and unaffected patients and thus detect the disease before its clinical onset, then a screening project using a panel of mRNAs would be feasible.
Subject(s)
Biomarkers/blood , Pre-Eclampsia/genetics , RNA, Messenger/blood , Case-Control Studies , Female , Fetal Growth Retardation/genetics , Gestational Age , Humans , Inhibins/genetics , Kisspeptins , P-Selectin/genetics , Placental Lactogen/genetics , Plasminogen Activator Inhibitor 1/genetics , Pregnancy , Pregnancy Trimester, Third , Pregnancy-Associated Plasma Protein-A/genetics , Receptors, Vascular Endothelial Growth Factor/genetics , Reference Values , Tumor Suppressor Proteins/geneticsABSTRACT
UNLABELLED: UniGene Tabulator 1.0 provides a solution for full parsing of UniGene flat file format; it implements a structured graphical representation of each data field present in UniGene following import into a common database managing system usable in a personal computer. This database includes related tables for sequence, protein similarity, sequence-tagged site (STS) and transcript map interval (TXMAP) data, plus a summary table where each record represents a UniGene cluster. UniGene Tabulator enables full local management of UniGene data, allowing parsing, querying, indexing, retrieving, exporting and analysis of UniGene data in a relational database form, usable on Macintosh (OS X 10.3.9 or later) and Windows (2000, with service pack 4, XP, with service pack 2 or later) operating systems-based computers. AVAILABILITY: The current release, including both the FileMaker runtime applications, is freely available at http://apollo11.isto.unibo.it/software/
Subject(s)
Database Management Systems , Databases, Genetic , Information Storage and Retrieval/methods , Proteins/genetics , Software , User-Computer Interface , Base Sequence , Molecular Sequence DataABSTRACT
The causative molecular pathways underlying the pathogenesis of colorectal cancer (CRC) need to be better characterized. The purpose of our study was to better understand the genetic mechanism of oncogenesis for human colorectal cancer and to identify new potential tumor markers of use in clinical practice. We used cDNA microarrays to compare gene expression profiles of colorectal biopsies from 25 CRC patients and 13 normal mucosa from adjacent non-cancerous tissues. Findings were validated by real-time PCR; in addition, western blotting and immunochemistry analysis were carried out as further confirmation of differential expression at a protein level. Comparing cancerous tissues with normal colonic mucosa we identified 584 known genes differentially expressed to a significant degree (p<0.001). Many of the transcripts that were more abundant in tumors than in non-neoplastic tissues appear to reflect important events for colon carcinogenesis. For example, a significant number of these genes serve as apoptotic inhibitors (e.g. BFAR, BIRC1, BIRC6). Furthermore, we observed the simultaneous up-regulation of HLA-E and the down-regulation of beta2-microglobulin; these genes strongly support a potential tumor escape strategy from immune surveillance in colon cancer tissues. Our study provides new gene candidates in the pathogenesis of human CRC disease. From our results we hypothesize that CRC cells escape immune surveillance through a specific gene expression alteration; moreover, over-expression of several survival genes seems to confer a more anti-apoptotic phenotype. These genes are involved in pathways not previously implicated in CRC pathogenesis and they may provide new targets for therapy.
Subject(s)
Colorectal Neoplasms/genetics , Gene Expression Profiling/methods , Gene Expression Regulation, Neoplastic , Genes, Neoplasm , Intestinal Mucosa/metabolism , Oligonucleotide Array Sequence Analysis/methods , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Adult , Aged , Aged, 80 and over , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Female , HLA Antigens/genetics , HLA Antigens/metabolism , Histocompatibility Antigens Class I/genetics , Histocompatibility Antigens Class I/metabolism , Humans , Male , Membrane Proteins/genetics , Membrane Proteins/metabolism , Middle Aged , Neuronal Apoptosis-Inhibitory Protein/genetics , Neuronal Apoptosis-Inhibitory Protein/metabolism , RNA, Messenger/analysis , RNA, Messenger/metabolism , Reproducibility of Results , beta 2-Microglobulin/genetics , beta 2-Microglobulin/metabolism , HLA-E AntigensABSTRACT
The activation of the insulin-like growth factor 1/IGF1 receptor system (IGF1/IGF1R) is a critical event in the transformation and tumorigenicity processes in a wide variety of human tumors. The IGF1/IGF1R system has been recently studied in carcinoid tumors that often arise in the gastrointestinal tract; these tumors are characterized by hypersecretion of bioamines and neuropeptides, leading to functional tumor disease. Two alternatively spliced IGF1R mRNA transcripts have been described to differ by only three nucleotides (CAG) in the coding sequence, resulting in an amino-acid change from the originally described Thr-Gly to an Arg in the extracellular portion of the receptor beta subunit. In transfected Chinese hamster ovary cells, the form without CAG (CAG-) exhibited an approximate 2-fold increase in IGF1 stimulation of activities required for its mitogenic properties. In this study, we examine the relative expression of the two IGF1R mRNA isoforms by a semiquantitative RT-PCR approach using highly standardized conditions, beta-2 microglobulin (B2M) as a reference gene and gel imaging analysis. We analyzed a large series of human neuroendocrine tumors (32 samples) and 9 normal tissues. A significant higher expression of both isoforms in the tumor samples (approximately 2-fold increase) was found, while a constant CAG+/CAG- IGF1R mRNA isoforms of an approximate 3:1 ratio was observed in all tumoral and normal cell types studied. The phylogenetic study of the IGF1R locus in several species suggests that human IGF1R CAG- mRNA isoform is evolutionarily more recent compared to the IGF1R CAG+ mRNA isoform and it could be used by the splicing apparatus at this intron/exon junction with a lower efficiency. This study highlights the relevance of IGF1R mRNA expression in neuroendocrine tumor cells, and the constant presence of 'subtle' alternative splicing for the IGF1R locus.
Subject(s)
Alternative Splicing , Neuroendocrine Tumors/genetics , RNA, Messenger/metabolism , Receptor, IGF Type 1/genetics , Adult , Aged , Amino Acid Sequence , Carcinoma, Islet Cell/genetics , Carcinoma, Islet Cell/metabolism , Carcinoma, Islet Cell/pathology , Cell Differentiation , DNA Primers/chemistry , Female , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/metabolism , Head and Neck Neoplasms/pathology , Humans , Male , Middle Aged , Molecular Sequence Data , Neuroendocrine Tumors/metabolism , Neuroendocrine Tumors/pathology , Protein Isoforms , RNA, Neoplasm/genetics , RNA, Neoplasm/metabolism , Receptor, IGF Type 1/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino AcidABSTRACT
Down syndrome critical region gene 1-like 2 (DSCR1L2) belongs to the human DSCR1-like gene family, which also includes DSCR1 and DSCR1L1. Both DSCR1 and DSCR1L1 proteins interact with calcineurin, a calcium/calmodulin-dependent phosphatase. To date, no interactor has been described for DSCR1L2. The aim of this work was to perform a first functional study of DSCR1L2 using yeast two-hybrid analysis conducted on a human heart cDNA library. Here, we report the interaction between DSCR1L2 and the human cardiac troponin I (TNNI3), the heart-specific inhibitory subunit of the troponin complex, a central component of the contractile apparatus. This interaction was confirmed by both yeast cotransformation and GST (glutathione-sepharose transferase) fusion protein assay. Moreover, a new DSCR1L2 mRNA isoform, generated by alternative splicing, was identified and cloned in different tissues: it lacks two central exons, encoding the most conserved domains among the DSCR1-like protein family. A quantitative relative reverse transcription-polymerase chain reaction (RT-PCR) assay showed that in heart tissue the normalized expression level ratio for DSCR1L2 and DSCR1L2-E2E5 mRNA isoforms is 3.5:1, respectively. The yeast cotransformation and GST fusion protein assay demonstrated the interaction between this new DSCR1L2 variant and the human cardiac troponin I and the prominent role of DSCR1L2 exon 2 in determining binding between both DSCR1L2 isoforms and TNNI3. These data indicate an entirely new role for a DSCR1-like family gene, suggesting a possible involvement of DSCR1L2 in cardiac contraction.
Subject(s)
Proteins/genetics , Proteins/metabolism , RNA, Messenger/genetics , Troponin I/metabolism , Adaptor Proteins, Signal Transducing , Adult , Amino Acid Sequence , Cloning, Molecular , DNA, Complementary/genetics , DNA, Complementary/isolation & purification , DNA, Complementary/metabolism , Glutathione/metabolism , Humans , Molecular Sequence Data , Open Reading Frames/genetics , Protein Binding , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , Proteins/chemistry , RNA, Messenger/metabolism , Recombinant Fusion Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sequence Alignment , Two-Hybrid System Techniques , Yeasts/cytologyABSTRACT
OBJECTIVE: To retrospectively investigate whether the genetic profile from chorionic villous sampling (CVS) found in euploid fetuses with increased NT differs from matched controls. STUDY DESIGN: We employed cDNA microarray technology to characterize and compare the gene expression profile of chorionic villous tissues (which encompass the trophoblast and inner mesenchymal core) belonging to four singleton male fetuses with increased NT at 10-11 weeks' gestation. A pool of four normal chorionic villous tissues belonging to four respective fetuses, matched for gestational age and gender, was used as controls. RESULTS: In euploid fetuses, we found several underexpressed genes, possibly involved in mechanisms associated with the abnormal NT thickness. All these genes are likely to belong to the mesenchymal core of the villus that originates from the extraembryonic mesoderm, and thus might be closely representative of the embryonic genetic profile. They include: (1) genes of embryonic development and differentiation such as Endothelin 3 (EDN3) and secreted frizzled-related protein 4 (SFRP4); (2) genes of the extracellular matrix (ECM) metabolism such as tissue inhibitor of metalloproteinase1 (TIMP1), and disintegrin-like and matrix metalloproteinase (MMP) (reprolysin type) with thrombospondin type 1 Motif or ADAMTS2, exostoses (multiple)-like 1 (EXTL1), heparan sulfate (HS) 6-O-sulfotransferase 1 or HS6ST1, fibronectin 1 (FN1) and Integrin Alpha 10 (ITGA10) involved in HS and proteoglycan bio-synthesis, ECM synthesis and cell-matrix adhesion; (3) genes involved in vessel formation and differentiation such as angiogenic factor (VG5Q), and in blood pressure control and muscle contraction, like Endothelin 3 or EDN3 and sarcolemma associated protein (SLMAP). Such lower expressions of the villous tissues might be related to an immature genetic profile of the embryo development as well as abnormal regulation of ECM bio-synthesis and/or improper vessel growth and blood pressure control. Also, the results partially support the theories proposed for NT enlargement such as altered composition of ECM and abnormal/delayed development of the circulatory system. CONCLUSIONS: Abnormal extraembryonic genetic expression is found at 10-11 weeks' gestation in euploid fetuses with increased NT. If both extra- and intraembryonic mesoderms express the same genetic alterations, then microarray analyses on CVS could be used to screen several mesoderm-derivate anomalies.
Subject(s)
Chorionic Villi Sampling/methods , Chromosome Disorders/diagnosis , Fetal Diseases/diagnosis , Gene Expression Regulation, Developmental/genetics , Nuchal Translucency Measurement/methods , Case-Control Studies , Chromosome Disorders/genetics , Female , Fetal Diseases/genetics , Gene Expression Profiling , Gestational Age , Humans , Karyotyping , Maternal Age , Oligonucleotide Array Sequence Analysis , Pregnancy , Pregnancy Trimester, First , Retrospective StudiesABSTRACT
Cleft lip with or without cleft palate (CL/P) is the most common inborn craniofacial anomaly. Affected individuals require extensive medical and psychosocial support. Although CL/P has a complex and poorly understood etiology, increasing evidence of folate pathway involvement has been collected. So far, only the MTHFR gene has been extensively investigated as a risk factor for CL/P, while little has been done to test genetic variations in the folate biosynthetic pathways that may influence the infant's susceptibility to these birth defects. To date, this paper presents the first attempt to verify the involvement of four genes belonging to the folate pathway in nonsyndromic cleft onset. We used a case-parent triad design to test for linkage disequilibrium in the case of seven SNPs mapping on four different genes: transcobalamin 1 and 2 (TCN1 and TCN2), methionine synthase (MTR), and MTR reductase (MTRR). Our finding suggests that TCN2 is involved in causing CL/P. Indeed, significant overtransmission of the C allele was observed at the polymorphism c.776C>G (p.Pro259Arg) to the affected offspring (P=0.01). Results obtained with additional TCN2 polymorphisms suggest that c.776C>G may be functionally related to CL/P. However, because conflicting data exist with regard to the effect of the polymorphism in transcobalamin 2 function or in perturbing plasma levels of key molecules in the folate pathway, further investigation is warranted to confirm our data.
Subject(s)
Cleft Lip/genetics , Cleft Palate/genetics , Folic Acid/metabolism , Gene Expression Regulation , Genetic Predisposition to Disease , Transcobalamins/genetics , 5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase/genetics , Alleles , Ferredoxin-NADP Reductase/genetics , Genetic Markers , Humans , Polymorphism, Genetic , Polymorphism, Single NucleotideABSTRACT
During embryogenesis, a complex interplay between extracellular matrix (ECM) molecules, regulatory molecules, and growth factors mediates morphogenetic processes involved in palatogenesis. Transforming growth factor-beta (TGF-beta), retinoic acid (RA), and gamma-aminobutyric acid (GABA)ergic signaling systems are also potentially involved. Using [3H]glucosamine and [35S]methionine incorporation, anion exchange chromatography, semiquantitative radioactive RT-PCR, and a TGF-beta binding assay, we aimed to verify the presence of phenotypic differences between primary cultures of secondary palate (SP) fibroblasts from 2-year-old subjects with familial nonsyndromic cleft lip and/or palate (CLP-SP fibroblasts) and age-matched normal SP (N-SP) fibroblasts. The effects of RA--which, at pharmacologic doses, induces cleft palate in newborns of many species--were also studied. We found an altered ECM production in CLP-SP fibroblasts that synthesized and secreted more glycosaminoglycans (GAGs) and fibronectin (FN) compared with N-SP cells. In CLP-SP cells, TGF-beta3 mRNA expression and TGF-beta receptor number were higher and RA receptor-alpha (RARA) gene expression was increased. Moreover, we demonstrated for the first time that GABA receptor (GABRB3) mRNA expression was upregulated in human CLP-SP fibroblasts. In N-SP and CLP-SP fibroblasts, RA decreased GAG and FN secretion and increased TGF-beta3 mRNA expression but reduced the number of TGF-beta receptors. TGF-beta receptor type I mRNA expression was decreased, TGF-beta receptor type II was increased, and TGF-beta receptor type III was not affected. RA treatment increased RARA gene expression in both cell populations but upregulated GABRB3 mRNA expression only in N-SP cells. These results show that CLP-SP fibroblasts compared with N-SP fibroblasts exhibit an abnormal phenotype in vitro and respond differently to RA treatment, and suggest that altered crosstalk between RA, GABAergic, and TGF-beta signaling systems could be involved in human cleft palate fibroblast phenotype.
Subject(s)
Cleft Palate/pathology , Fibroblasts/pathology , Receptors, GABA-A/metabolism , Signal Transduction/drug effects , Transforming Growth Factor beta3/metabolism , Tretinoin/pharmacology , Cell Count , Cell Growth Processes/drug effects , Cell Survival/drug effects , Cells, Cultured , Child, Preschool , Female , Fibroblasts/drug effects , Fibronectins/metabolism , Gene Expression Regulation/drug effects , Glucosamine/metabolism , Glycosaminoglycans/metabolism , Humans , Male , Phenotype , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Transforming Growth Factor beta/genetics , Transforming Growth Factor beta3/geneticsABSTRACT
BACKGROUND: Formal description of a cell's genetic information should provide the number of DNA molecules in that cell and their complete nucleotide sequences. We pose the formal problem: can the genome sequence forming the genotype of a given living cell be known with absolute certainty so that the cell's behaviour (phenotype) can be correlated to that genetic information? To answer this question, we propose a series of thought experiments. RESULTS: We show that the genome sequence of any actual living cell cannot physically be known with absolute certainty, independently of the method used. There is an associated uncertainty, in terms of base pairs, equal to or greater than micros (where micro is the mutation rate of the cell type and s is the cell's genome size). CONCLUSION: This finding establishes an "uncertainty principle" in genetics for the first time, and its analogy with the Heisenberg uncertainty principle in physics is discussed. The genetic information that makes living cells work is thus better represented by a probabilistic model rather than as a completely defined object.