Biomedical subjects

T F Orntoft

Publications and source records attributed to T F Orntoft.

At least 19 recordsLinked to original sources

PubMed2006

Stage-associated overexpression of the ubiquitin-like protein, ISG15, in bladder cancer.

Bladder cancer is among the most prevalent malignancies, and is characterised by frequent tumour recurrences and localised inflammation, which may promote tissue invasion and metastasis. Microarray analysis was used to compare gene expression in normal bladder urothelium with that in tumours at different stages of progression. The innate immune response gene, interferon-stimulated gene 15 kDa (ISG15, GIP2), was highly expressed at all stages of bladder cancer as compared to normal urothelium. Western blotting revealed a tumour-associated expression of ISG15 protein. ISG15 exhibited a stage-associated expression, with significantly (P<0.05) higher levels of ISG15 protein in muscle-invasive T2-T4 tumours, compared with normal urothelium. Although ISG15 is involved in the primary immune response, ISG15 expression did not correlate with bladder inflammation. However, immunohistochemical staining revealed expression of ISG15 protein in both cancer cells and stromal immune cells. Interestingly, a significant fraction of ISG15 protein was localised to the nuclei of tumour cells, whereas no nuclear ISG15 staining was observed in ISG15-positive stromal cells. Taken together, our findings identify ISG15 as a novel component of bladder cancer-associated gene expression.

Blotting, Western↗

PubMed2005

Gene expression profiling of noninvasive primary urothelial tumours using microarrays.

At present, the mechanism leading to bladder cancer is still poorly understood, and our knowledge about early events in tumorigenesis is limited. This study describes the changes in gene expression occurring during the neoplastic transition from normal bladder urothelium to primary Ta tumours. Using DNA microarrays, we identified novel differentially expressed genes in Ta tumours compared to normal bladder, and genes that were altered in high-grade tumours. Among the mostly changed genes between normal bladder and Ta tumours, we found genes related to the cytoskeleton (keratin 7 and syndecan 1), and transcription (high mobility group AT-hook 1). Altered genes in high-grade tumours were related to cell cycle (cyclin-dependent kinase 4) and transcription (jun d proto-oncogene). Furthermore, we showed the presence of high keratin 7 transcript expression in bladder cancer, and Western blotting analysis revealed three major molecular isoforms of keratin 7 in the tissues. These could be detected in urine sediments from bladder tumour patients.

Cluster Analysis↗

PubMed2005

UbcH10 overexpression may represent a marker of anaplastic thyroid carcinomas.

The hybridisation of an Affymetrix HG_U95Av2 oligonucleotide array with RNAs extracted from six human thyroid carcinoma cell lines and a normal human thyroid primary cell culture led us to the identification of the UbcH10 gene that was upregulated by 150-fold in all of the carcinoma cell lines in comparison to the primary culture cells of human normal thyroid origin. Immunohistochemical studies performed on paraffin-embedded tissue sections showed abundant UbcH10 levels in thyroid anaplastic carcinoma samples, whereas no detectable UbcH10 expression was observed in normal thyroid tissues, in adenomas and goiters. Papillary and follicular carcinomas were only weakly positive. These results were further confirmed by RT-PCR and Western blot analyses. The block of UbcH10 protein synthesis induced by RNA interference significantly reduced the growth rate of thyroid carcinoma cell lines. Taken together, these results would indicate that UbcH10 overexpression is involved in thyroid cell proliferation, and may represent a marker of thyroid anaplastic carcinomas.

Animals↗

PubMed2005

A genome-wide search for risk genes using homozygosity mapping and microarrays with 1,494 single-nucleotide polymorphisms in 22 eastern Cuban families with bipolar disorder.

Homozygosity mapping is a very powerful method for finding rare recessive disease genes in monogenic disorders and may also be useful for locating risk genes in complex disorders, late onset disorders where parents often are not available, and for rare phenotypic subgroups. In the present study, homozygosity mapping was applied to 24 persons with bipolar disorder from 22 inbred families. The families were selected irrespective of whether other affected family members were present or not. A genome wide screen using genotypes from only a single affected person in each family was performed using the AFFYMETRIX GeneChip HuSNP Mapping Assay, which contains 1,494 single nucleotide polymorphisms. At chromosome 17q24-q25 a parametric multipoint LOD score of 1.96 was found at WIAF-2407 and WIAF-2405. When analyzing 19 additional microsatellite markers on chromosome 17q the maximum parametric multipoint LOD score was 2.08, 1.5 cM proximal to D17S668. The present study replicates a recent significant linkage finding.

Alleles↗

PubMed2005

Differential gene expression in colon cancer of the caecum versus the sigmoid and rectosigmoid.

BACKGROUND AND AIMS: There are epidemiological, morphological, and molecular differences between normal mucosa as well as between adenocarcinomas of the right and left side of the large bowel. The aim of this study was to investigate differences in gene expression. METHODS: Oligonucleotide microarrays (GeneChip) were used to compare gene expression in 45 single samples from normal mucosa and sporadic colorectal carcinomas (Dukes' B and C) of the caecum compared with the sigmoid and rectosigmoid. Findings were validated by real time polymerase chain reaction. RESULTS: Fifty eight genes were found to be differentially expressed between the normal mucosa of the caecum and the sigmoid and rectosigmoid (p<0.01), including pS2, S100P, and a sialyltransferase, all being expressed at higher levels in the caecum. A total of 118 and 186 genes were differentially expressed between normal and right or left sided tumours of the colon, showing more pronounced differences in Dukes' C than B tumours. Thirty genes differentially expressed in tumour tissue were common to adenocarcinomas of both sides, including known tumour markers such as the matrix metalloproteinases. Keratins 8, 19, and 20 as well as carbonic anhydrases (II, IV, VII) showed side specific expression and were downregulated in left sided tumours whereas teratocarcinoma growth factor and cyclooxygenase 2 (COX-2) were upregulated in left sided adenocarcinomas. Immunohistochemical analysis confirmed differences in side specific expression for cytokeratin 20 and COX-2. CONCLUSIONS: Differences in gene expression between normal mucosa as well as between adenocarcinomas of the caecum and sigmoid or rectosigmoid exist and should be taken into account when examining new targeted therapeutic regimens.

Adenocarcinoma↗

PubMed2004

Suppressor of cytokine signalling (SOCS)-3 protects beta cells against IL-1beta-mediated toxicity through inhibition of multiple nuclear factor-kappaB-regulated proapoptotic pathways.

AIMS/HYPOTHESIS: The proinflammatory cytokine IL-1beta induces apoptosis in pancreatic beta cells via pathways dependent on nuclear factor-kappaB (NF-kappaB), mitogen-activated protein kinase, and protein kinase C. We recently showed suppressor of cytokine signalling (SOCS)-3 to be a natural negative feedback regulator of IL-1beta- and IFN-gamma-mediated signalling in rat islets and beta cell lines, preventing their deleterious effects. However, the mechanisms underlying SOCS-3 inhibition of IL-1beta signalling and prevention against apoptosis remain unknown. METHODS: The effect of SOCS-3 expression on the global gene-expression profile following IL-1beta exposure was microarray-analysed using a rat beta cell line (INS-1) with inducible SOCS-3 expression. Subsequently, functional analyses were performed. RESULTS: Eighty-two known genes and several expressed sequence tags (ESTs) changed expression level 2.5-fold or more in response to IL-1beta alone. Following 6 h of IL-1beta exposure, 23 transcripts were up-regulated. Of these, several, including all eight transcripts relating to immune/inflammatory response pathways, were suppressed by SOCS-3. Following 24 h of IL-1beta exposure, secondary response genes were detected, affecting metabolism, energy generation, protein synthesis and degradation, growth arrest, and apoptosis. The majority of these changes were prevented by SOCS-3 expression. Multiple IL-1beta-induced NF-kappaB-dependent proapoptotic early response genes were inhibited by SOCS-3 expression, suggesting that SOCS-3 inhibits NF-kappaB-mediated signalling. These observations were experimentally confirmed in functional analyses. CONCLUSIONS/INTERPRETATION: This study suggests that there is an unexpected cross-talk between the SOCS/IFN and the IL-1beta pathways of signalling in pancreatic beta cells, which could lead to a novel perspective of blocking two important proapoptotic pathways in pancreatic beta cells by influencing a single signalling molecule, namely SOCS-3.

Animals↗

PubMed2003

Study of the alteration of gene expression in adipose tissue of diet-induced obese mice by microarray and reverse transcription-polymerase chain reaction analyses.

In the present study we developed a model of diet-induced obesity (DIO) in male C57 BL/6J mice using an 8-wk high fat diet. This model should better reflect the physiology of the majority of the human obese patients than mouse genetic models of obesity with defects in leptin or leptin signaling. At the end of the diet, DIO mice displayed an increased weight (20%) and higher leptin, insulin, glucose, and corticosterone plasma levels compared with mice fed a standard diet during the same period. Moreover, they became resistant to the central effect of peripheral administration of leptin. Oligonucleotide microarray studies were conducted in adipose tissue. They showed that a great number of genes are differentially expressed. The majority of these genes (69%) are down-regulated in DIO mice. Among those are genes encoding enzymes of the lipid metabolism or markers of adipocyte differentiation, enzymes involved in detoxification processes, as well as structural components of the cytoskeleton. Some other groups of genes displayed increased expression, such as those encoding inflammatory markers. The results of the microarray analysis were confirmed by semiquantitative RT-PCR studies run on a selected number of genes that were differentially expressed or not modified.

Adipose Tissue↗

PubMed2001

Polyglutamine expansions cause decreased CRE-mediated transcription and early gene expression changes prior to cell death in an inducible cell model of Huntington's disease.

Huntington's disease (HD) is one of 10 known diseases caused by a (CAG)(n) trinucleotide repeat expansion that is translated into an abnormally long polyglutamine tract. We have developed stable inducible neuronal (PC12) cell lines that express huntingtin exon 1 with varying CAG repeat lengths under doxycycline (dox) control. The expression of expanded repeats is associated with aggregate formation, caspase-dependent cell death and decreased neurite outgrowth. Post-mitotic cells expressing mutant alleles were more prone to cell death compared with identical cycling cells. To determine early metabolic changes induced by this mutation in cell models, we studied changes in gene expression after 18 h dox induction, using Affymetrix arrays, cDNA filters and adapter-tagged competitive PCR (ATAC-PCR). At this time point there were low rates of inclusion formation, no evidence of mitochondrial compromise and no excess cell death in the lines expressing expanded compared with wild-type repeats. The expression profiles suggest novel targets for the HD mutation and were compatible with impaired cAMP response element (CRE)-mediated transcription, which we confirmed using CRE-luciferase reporter assays. Reduced CRE-mediated transcription may contribute to the loss of neurite outgrowth and cell death in polyglutamine diseases, as these phenotypes were partially rescued by treating cells with cAMP or forskolin.

Animals↗

PubMed2001

A subclass of HER1 ligands are prognostic markers for survival in bladder cancer patients.

Members of the epidermal growth factor (EGF) family have been suggested as prognostic markers in patients with bladder cancer. Thus far, there has been no consensus on their usefulness. We report an analysis of six ligands and two receptors of which a subset correlate to tumor stage and survival. Biopsies from bladder cancer tumors were obtained from 73 patients followed for a median of 28 months. The mRNA content for six ligands [EGF, transforming growth factor alpha (TGF-alpha), amphiregulin (AR), betacellulin (betaCL), heparin-binding EGF-like growth factor (HB-EGF), epiregulin (EPI)] and two receptors [EGF receptor I Human EGF Receptor (HER1) and 2 (HER2)] was examined by a newly developed quantitative reverse transcription-PCR method. Five ligands and two receptors (HER1 and HER2) were present in median concentrations of (10(-21) mol/microg RNA) 0.39 (AR), 11 (betaCL), 2.4 (EPI), 40 (HB-EGF), 1.4 (TGF-alpha), 75 (HER1), and 39,000 (HER2). EGF was barely detectable. A significantly higher expression of EPI (P < 0.001), HB-EGF (P < 0.001), and TGF-alpha (P < 0.05) were observed in T2-T4 tumors as compared with Ta tumors. Especially the expression of EPI mRNA correlated strongly to survival (P < 0.0005), but increased expression of TGF-alpha (P < 0.005), AR, and HB-EGF (P < 0.02) was also associated with a reduced life span. For the first time, mRNA expression of six ligands and two receptors of the EGF family have been examined in bladder cancer tumors. Our data emphasize that members of the EGF family, especially EPI, may be potential bladder tumor markers.

Adult↗

PubMed2001

Identification of gene expression patterns in superficial and invasive human bladder cancer.

Multiple transcriptional events take place when normal urothelium is transformed into tumor tissue. These can now be monitored simultaneously by the use of oligonucleotide arrays, and expression patterns of superficial and invasive tumors can be established. Single-cell suspensions were prepared from bladder biopsies (36 normal, 29 tumor). Pools of cells were made from normal urothelium and from pTa grade I and II and pT2 grade III and IV bladder tumors. From these suspensions, and from 10 single-tumor biopsies, labeled cRNA was hybridized to oligonucleotide arrays carrying probes for 6500 genes. The obtained expression data were sorted according to a weighting scheme and were subjected to hierarchical cluster analysis of tissues and genes. Northern blotting was used to verify the array data, and immunohistology was used to correlate between RNA and protein levels. Hierarchical clustering of samples correctly identified the stage using both 4076 genes and a subset of 400 genes covarying with the stages and grades of tumors. Hierarchical clustering of gene expression levels identified several stage-characteristic, functionally related clusters, encoding proteins that were related to cell proliferation, oncogenes and growth factors, cell adhesion, immunology, transcription, proteinases, and ribosomes. Northern blotting correlated well with array data. Immunohistology showed a good concordance between transcript level and protein staining. The study indicates that gene expression patterns may be identified in bladder cancer by combining oligonucleotide arrays and cluster analysis. These patterns give new biological insight and may form a basis for the construction of molecular classifiers and for developing new therapy for bladder cancer.

Algorithms↗

PubMed2001

Telomerase activity, MIB-1, PCNA, HPV 16 and p53 as diagnostic markers for cervical intraepithelial neoplasia.

The present investigation evaluated the relationship between dysplasia of the uterine cervix and telomerase activity, expression of p53, MIB-1 and PCNA. Telomerase activity was measured on cervical cytobrush material from 126 women suspected of having dysplasia and 61 controls using the telomeric repeat amplification protocol. Immunohistochemistry was used to detect the tumor suppressor protein p53 and cell proliferation, the latter by MIB-1 and PCNA expression. Infection with human papillomavirus 16 was detected by PCR amplification and Southern blot hybridization of DNA extracted from the same brush material. Positive telomerase activity was found in 5 of 43 (11.6%) normal samples, 12 of 57 (21.1%) samples with inflammation or koilocytosis, 7 of 17 (41.2%) CIN 1 (cervical intraepithelial neoplasia, grade 1), 8 of 20 (40.0%) CIN 2, and 25 of 42 (59.5%) CIN 3/ CIS. Telomerase activity was significantly related to the level of dysplasia (p<0.001) and proliferation measured by MIB-1 (p=0.019), but not to the level of PCNA (p=0.445), HPV 16 status (p=0.098) or staining for p53 (p=0.271). Dysplasia was also related to PCNA, MIB1, p53, and presence of HPV 16. A sequential increase in the examined parameters, paralleling the progression of abnormality, was observed. PCNA and telomerase showed an increase in CIN 1, MIB-1 and HPV16 in CIN 2, and finally p53 in CIN 3/CIS.

Adolescent↗

PubMed2000

Allelic deletions of cell growth regulators during progression of bladder cancer.

Cell growth regulators include proteins of the p53 pathway encoded by the genes CDKN2A (p16, p14arf), MDM2, TP53, and CDKN1A (p21) as well as proteins encoded by genes like RB1, E2F, and MYCL. In the present study we investigated allelic deletions of all these genes in each recurrent bladder tumor from well-defined clinical material with more than 3 years of follow-up. We followed three groups (22 or 23 patients/group) of patients with: (a) recurrent noninvasive tumors (Ta); (b) primary muscle-invasive tumors (T2-T4); and (c) progressing tumors (Ta/T1 --> T2/T4). We found a significant difference in the numbers of gene loci hit by deletions muscle-invasive versus noninvasive tumors (P = 0.0000002), with the genes most often hit by deletions in muscle-invasive tumors being TP53, RB1, and MYCL. A number of novel findings were made. Losses of MYCL and RB1 alleles were more pronounced in patients having concomitant field disease because 11 of 14 informative cases showed losses compared with 3 of 8 cases without field disease. A more pronounced deletion of TP53 (P = 0.002) and RB1 (P = 0.02) was found in the progressing tumor group compared with the recurrent noninvasive group, and, finally, the combined loss of TP53 and RB1 was present only in the progressing tumor or muscle-invasive groups. Deletion of two or more loci in TP53, MYCL, RB1, and CDKN2A was found in 10 patients in the progressing tumor group and in only 1 patient in the recurrent noninvasive group (P = 0.004). The data demonstrate that a characteristic difference between recurrent noninvasive and recurrent progressing bladder tumors is loss of cell cycle-regulatory genes in the latter group.

Adult↗

PubMed2000

Gene expression profiling: monitoring transcription and translation products using DNA microarrays and proteomics.

Novel and powerful technologies such as DNA microarrays and proteomics have made possible the analysis of the expression levels of multiple genes simultaneously both in health and disease. In combination, these technologies promise to revolutionize biology, in particular in the area of molecular medicine as they are expected to reveal gene regulation events involved in disease progression as well as to pinpoint potential targets for drug discovery and diagnostics. Here, we review the current status of these technologies and highlight some studies in which they have been applied in concert to the analysis of biopsy specimens.

Animals↗

PubMed2000

Microsatellite alterations in urinary sediments from patients with cystitis and bladder cancer.

Microsatellite instability (MIN) and loss of heterozygosity (LOH) in bladder cancer have been suggested for diagnosis and follow-up of bladder cancer based on urinary sediments, reflecting tumor alterations. We have examined 6 microsatellites in urine sediments from 11 patients with transitional-cell carcinomas (TCC) and in 31 patients with benign prostatic hyperplasia (BPH), 22 of whom had cystitis. In the TCC patients, tumor tissue was available for comparison with urine. Microsatellites were amplified by PCR and compared with leukocyte DNA from the same individual in silver-stained gels. Altered mobility of bands, new bands and loss of bands were scored. We found MIN and LOH at relatively high frequency in markers from chromosomes 8 and 14 in urine from patients with TCC, but also in BPH patients who had cystitis. Even control patients with BPH without cystitis showed some instability and some losses. Novel bands in urine occurred significantly more often among TCC patients than among BPH patients with or without cystitis (p < 0.001). Band shifts in urine appeared to be more associated with BPH plus cystitis than with TCC. The alterations we found in urine from patients with bladder cancer did not always reflect those found in their tumors, the occurrence of novel bands being significantly higher (p < 0.008) in tumor tissue than in corresponding urine. In conclusion, microsatellite alterations in urine are indicators not only of malignancy but also of inflammatory conditions.

Adult↗

PubMed2000

Efficient mutation detection in mismatch repair genes using a combination of single-strand conformational polymorphism and heteroduplex analysis at a controlled temperature.

Single-strand conformational polymorphism analysis (SSCP) and heteroduplex analysis (HD) were tested as methods for mutation screening with respect to experimental variation, sensitivity, and specificity. Thirty-nine fluorescently labeled PCR products covering the two mismatch repair genes, hMLH1 and hMSH2, were tested in 15 patients for pattern changes, using SSCP and HD at two temperatures, in a total of 2340 runs. SSCP was most efficient in detecting base changes, whereas HD was the method of choice when detecting deletions. SSCP and HD at 20 degrees C were most effective (sensitivity 97%, specificity 49%), and SSCP and HD at 10 degrees C gave no additional information, except in one case where an exon had two base changes. Several mutations only showed a small pattern change in one of the two strands, most explicit at 20 degrees C. No correlation between the type of base change and the size or direction of the pattern changes could be found.

Adaptor Proteins, Signal Transducing↗

PubMed2000

The C-terminal N-glycosylation sites of the human alpha1,3/4-fucosyltransferase III, -V, and -VI (hFucTIII, -V, adn -VI) are necessary for the expression of full enzyme activity.

The alpha1,3/4-fucosyltransferases are involved in the synthesis of fucosylated cell surface glycoconjugates. Human alpha1,3/4-fucosyltransferase III, -V, and -VI (hFucTIII, -V, and -VI) contain two conserved C-terminal N-glycosylation sites (hFucTIII: Asn154 and Asn185; hFucTV: Asn167 and Asn198; and hFucTVI: Asn153 and Asn184). In the present study, we have analyzed the functional role of these potential N-glycosylation sites, laying the main emphasis on the sites in hFucTIII. Tunicamycin treatment completely abolished hFucTIII enzyme activity while castanospermine treatment diminished hFucTIII enzyme activity to approximately 40% of the activity of the native enzyme. To further analyze the role of the conserved N-glycosylation sites in hFucTIII, -V, and -VI, we made a series of mutant genomic DNAs in which the asparagine residues in the potential C-terminal N-glycosylation sites were replaced by glutamine. Subsequently, the hFucTIII, -V, and -VI wild type and the mutants were expressed in COS-7 cells. All the mutants exhibited lower enzyme activity than the wild type and elimination of individual sites had different effects on the activity. The mutations did not affect the protein level of the mutants in the cells, but reduced the molecular mass as predicted. Kinetic analysis of hFucTIII revealed that lack of glycosylation at Asn185 did not change the Km values for the oligosaccharide acceptor and the nucleotide sugar donor. The present study demonstrates that hFucTIII, -V, and -VI require N-glycosylation at the two conserved C-terminal N-glycosylation sites for expression of full enzyme activity.

Amino Acid Sequence↗

PubMed2000

High-resolution genotyping of Chlamydia trachomatis from recurrent urogenital infections.

A method for Chlamydia trachomatis restriction fragment length polymorphism (RFLP) analysis and complete sequencing of omp1 was developed for use on samples collected at home, and results were compared. Genotyping by sequencing was superior to RFLP analysis. The omp1 gene in 31 clinical strains harbored few mutations compared to the same gene in ATCC reference strains. Follow-up samples obtained during a 24-week period from 31 patients showed recurrence with the same genotype in five cases and a new genotype in one case.

Bacterial Outer Membrane Proteins↗

PubMed2000

Evaluation of the performance of a p53 sequencing microarray chip using 140 previously sequenced bladder tumor samples.

BACKGROUND: Testing for mutations of the TP53 gene in tumors is a valuable predictor for disease outcome in certain cancers, but the time and cost of conventional sequencing limit its use. The present study compares traditional sequencing with the much faster microarray sequencing on a commercially available chip and describes a method to increase the specificity of the chip. METHODS: DNA from 140 human bladder tumors was extracted and subjected to a multiplex-PCR before loading onto the p53 GeneChip from Affymetrix. The same samples were previously sequenced by manual dideoxy sequencing. In addition, two cell lines with two different homozygous mutations at the TP53 gene locus were analyzed. RESULTS: Of 1464 gene chip positions, each of which corresponded to an analyzed nucleotide in the sequence, 251 had background signals that were not attributable to mutations, causing the specificity of mutation calling without mathematical correction to be low. This problem was solved by regarding each chip position as a separate entity with its own noise and threshold characteristics. The use of background plus 2 SD as the cutoff improved the specificity from 0.34 to 0.86 at the cost of a reduced sensitivity, from 0.92 to 0.84, leading to a much better concordance (92%) with results obtained by traditional sequencing. The chip method detected as little as 1% mutated DNA. CONCLUSIONS: Microarray-based sequencing is a novel option to assess TP53 mutations, representing a fast and inexpensive method compared with conventional sequencing.

Humans↗