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J Welsh

Publications and source records attributed to J Welsh.

At least 37 records · Page 2Linked to original sources
PubMed1999

Kinesin-like protein CENP-E is upregulated in rheumatoid synovial fibroblasts.

UNLABELLED: Articular destruction by invading synovial fibroblasts is a typical feature in rheumatoid arthritis (RA),. Recent data support the hypothesis that key players in this scenario are transformed-appearing synovial fibroblasts at the site of invasion into articular cartilage and bone. They maintain their aggressive phenotype toward cartilage, even when first cultured and thereafter coimplanted together with normal human cartilage into severe combined immunodeficient mice for and extended period of time. However, little is known about the upregulation of genes that leads to this aggressive fibroblast phenotype. To inhibit this progressive growth without interfering with pathways of physiological matrix remodelling, identification of pathways that operate specifically in RA synovial fibroblasts is required. In order to achieve this goal, identification of genes showing upregulation restricted to RA synovial fibroblasts is essential. AIMS: To identify specifically expressed genes using RNA arbitrarily primed (RAP)-polymerase chain reaction (PCR) for differential display in patients with RA. METHODS: RNA was extracted from cultured synovial fibroblasts from 10 patients with RA, four patients with osteoarthritis (OA), and one patient with psoriatic arthritis. RAP-PCR was performed using different arbitrary primers for first-strand and second-strand synthesis. First-strand and second-strand synthesis were performed using arbitrary primers: US6 (5'-GTGGTGACAG-3') for first strand, and Nuclear 1+ (5'ACGAAGAAGAG-3'), OPN28 (5'GCACCAGGGGG-3'), Kinase A2+(5'-GGTGCCTTTGG-3') and OPN24 (5'AGGGGCACCA-3') for second strand synthesis. PCR reactions were loaded onto 8 mol/l urea/6% polyacrylamide-sequencing gels and electrophoressed. Gel slices carrying the target fragment were then excised with a razor blade, eluated and reamplified. After verifying their correct size and purity on 4% agarose gels, the reamplified products derived from the single-strand confirmation polymorphism gel were cloned, and five clones per transcript were sequenced. Thereafter, a genbank analysis was performed. Quantitative reverse transcription PCRj of the segments was performed using the PCR MIMIC technique. In-situ expression of centromere kinesin-like protein-E (CENP-E) messenger (m)RNA in RA synovium was assessed using digoxigenin-labelled riboprobes, and CENP-E protein expression in fibroblasts and synovium was performed by immunogold-silver immunohistochemistry and cytochemistry. Functional analysis of CENP-E was done using different approaches (eg glucocorticoid stimulation, serum starvation and growth rate analysis of synovial fibroblasts that expressed CENP-E). RESULTS: In RA, amplification of a distinct PCR product suitable for sequencing could be observed. The indicated complementary DNA fragment of 434 base pairs from RA mRNA corresponded to nucleotides 6615-7048 in the human centromere kinesin-like protein CENP-E mRNA (GenBank accession No. emb/Z15005). The isolated sequence shared greater than 99% nucleic acid (P=2.9e(-169)) identify with the human centromere kinesin-like protein CENP-E. Two base changes at positions 6624 (A to C) and 6739 (A to G) did not result in alteration in the amino acid sequence, and therefore 100% amino acid identity could be confirmed. The amplification of 10 clones of the cloned RAP product revealed the presence of CENP-E mRNA in every fibroblast culture examined, showing from 50% (271.000 +/- 54.000 phosphor imager arbitrary units) up to fivefold (961.000 +/- 145.000 phosphor image arbitrary units) upregulation when compared with OA fibroblasts. Neither therapy with disease-modifying antirheumatic drugs such as methotrexate, gold, resochine or cyclosporine A, nor therapy with oral steroids influenced CENP-E expression in the RA fibroblasts. Of the eight RA fibroblast populations from RA patients who were receiving disease-modifying antirheumatic drugs, five showed CENP-E upregulation; and of the eight fibroblast populations from RA patients receiving steroids, four showed CENP-E upregulation. Numerous synovial cells of the patients with RA showed a positive in situ signal for the isolated CENP-E gene segment confirming CENP-E mRNA production in rheumatoid synovium, whereas in OA synovial tissue CENP-E mRNA could not be detected. In addition, CENP-E expression was independent from medication. This was further confirmed by analysis of the effect of prednisolone on CENP-E expression, which revealed no alteration in CENP-E mRNA after exposure to different (physiological) concentrations of prednisolone. Serum starvation also could not suppress CENP-E mRNA completely. DISCUSSION: Since its introduction in 1992, numerous variants of the differential display method and continuous improvements including RAP-PCR have proved to have both efficiency and reliability in examination of differentially regulated genes. The results of the present study reveal that RAP-PCR is a suitable method to identify differentially expressed genes in rheumatoid synovial fibroblasts. The mRNA, which has been found to be upregulated in rheumatoid synovial fibroblasts, codes for a kinesin-like motor protein named CENP-E, which was first characterized in 1991. It is a member of a family of centromere-associated proteins, of which six (CENP-A to CENP-F) are currently known. CENP-E itself is a kinetochore motor, which accumulates transiently at kinetochores in the G2 phase of the cell cycle before mitosis takes place, appears to modulate chromosome movement and spindle elongation,and is degraded at the end of mitosis. The presence or upregulation of CENP-E has never been associated with RA.The three-dimensional structure of CENP-E includes a coiled-coil domain. This has important functions and shows links to known pathways in RA pathophysiology. Coiled-coil domains can also be found in jun and fos oncogene products, which are frequently upregulated in RA synovial fibroblasts. They are also involved in DNA binding and transactivation processes resembling the situation in AP-1 (Jun/Fos)-dependent DNA-binding in rheumatoid synovium. Most interestingly, these coiled-coil motifs are crucial for the assembly of viral proteins, and the upregulation of CENP-E might reflect the influence of infectious agents in RA synovium. We also performed experiments showing that serum starvation decreased, but did not completely inhibit CENP-E mRNA expression. This shows that CENP-E is related to, but does not completely depend on proliferation of these cells. In addition, we determined the growth rate of CENP-E high and low expressors, showing that it was independent from the amount of CENP-E expression. supporting the statement that upregulation of CENP-E reflects an activated RA fibroblast phenotype. In summary, the results of the present study support the hypothesis that CENP-E, presumably independently from medication, may not only be upregulated, but may also be involved in RA pathophysiology.

Adult↗
PubMed1999

[Analysis of gene expression patterns in rheumatoid synovial fibroblasts using RAP-PCR for differential display].

OBJECTIVE: Destruction of articular cartilage and bone by invading synovial fibroblasts is a typical histopathologic feature in rheumatoid arthritis (RA). However, little is known about specific up- or downregulation of genes leading to this aggressive phenotype. Thus, our aim was to identify genes, which are differentially expressed in RA synovial fibroblasts as compared to synovial fibroblasts derived from patients with osteoarthritis (OA) using RAP-PCR for differential display. METHODS: After extraction of total RNA, the first step of RAP-PCR was performed using various different arbitrary 10-12-base primers for first-strand cDNA synthesis. Second-strand synthesis was achieved by cycling at low stringency conditions for 35 cycles using different arbitrary 10-base primers, followed by electrophoretic separation and sequence analysis of the amplified fingerprint products. RESULTS: On average, approximately 70 different RNAs were obtained per primer, of which most were expressed both by RA and OA synovial fibroblasts. Using 26 different primer combinations, in total 12 cDNAs were differentially expressed between RA and OA synovial fibroblasts. In the RA group strong amplification of distinct PCR products suitable for sequencing could be observed. Sequence analysis identified these PCR products as highly homologous to various genes involved in regulation of cell cycle and metabolism. CONCLUSION: The data indicate that RAP-PCR is a suitable method to identify differentially expressed genes in rheumatoid synovial fibroblasts potentially involved in the specific pathophysiology of RA.

Arthritis, Rheumatoid↗
PubMed1999

SUM-159PT cells: a novel estrogen independent human breast cancer model system.

Breast cancer remains one of the most common malignant diseases in women in North America and Western Europe, yet therapies for the more aggressive estrogen independent tumors are limited and few model systems are available for the study of this type of breast cancer. In these studies, we characterized a novel estrogen independent breast cancer cell line, SUM-159PT. SUM-159PT cells are epithelial in origin, demonstrated by expression of cytokeratin 18. SUM-159PT cells are estrogen independent, demonstrated by lack of estrogen receptor (ER) protein and ER ligand binding studies. Furthermore, SUM-159PT cells injected subcutaneously or orthotopically are tumorigenic in ovariectomized athymic nude mice in the absence of estradiol supplementation. SUM-159PT cells are capable of invading through an 8 microm Matrigel membrane and display a stellate morphology in Matrigel, indicative of a metastatic phenotype. Correlating with this phenotype, we have detected secondary tumors upon inoculation of SUM-159PT cells into the mammary fat pad. To further investigate the metastatic potential of the SUM-159PT cells, we examined the expression of two proteins, vimentin and E-cadherin, implicated in the transition of carcinoma cells to a metastatic phenotype. Western blot and immunohistochemical analysis demonstrated that both SUM-159PT cells and xenografts express vimentin. No expression of E-cadherin was detected in SUM-159PT cells. Our data indicate that despite estrogen independence, SUM-159PT cells are growth inhibited in vitro by compounds such as 1,25(OH)2D3, transforming growth factor beta (TGF-beta), and the phorbol ester TPA. These studies indicate that SUM-159PT cells represent a good model system for the study of late stage estrogen independent, invasive breast cancer.

Animals↗
PubMed1999

Clinical and pathologic predictors of survival in patients with thymoma.

OBJECTIVE: To evaluate the Johns Hopkins Hospital experience with 136 thymomas over the past 40 years. This number of patients allowed quantitative estimation of the independent influence of common clinicopathologic risk factors using multivariate analysis. SUMMARY BACKGROUND DATA: Thymomas vary widely in terms of recurrence and influence on overall survival. Several series have indicated the importance of initial tumor invasion, as well as the extent of surgical resection, as predictors of recurrence and survival after thymoma resection. However, findings have been equivocal when other predictors of prognosis were examined. METHODS: The authors evaluated 136 patients seen at the Johns Hopkins Hospital between 1957 and 1997 with a pathologic diagnosis of thymoma. Demographic information, clinical staging data, surgical and adjuvant treatment details, and patient follow-up data were obtained from the patient record and from detailed patient or family interviews. Microscopic sections of all 136 patients were reviewed by two pathologists blinded to the clinical data. All data were analyzed by multivariate Cox regression analysis, which allowed the quantification of the independent predictive value of 12 putative clinicopathologic prognostic indicators. RESULTS: Completeness of follow-up was 99%, 99%, and 98% of eligible patients at 5, 10, and 15 years, respectively. Forty percent of the patients had associated myasthenia gravis and 27% had a secondary primary malignancy. Overall patient survival rates were 71%, 56%, 44%, 38%, and 33% at 5, 10, 15, 20, and 25 years, respectively. Overall, the thymoma-related mortality rate was 14%; the nonthymoma-related mortality rate was 26%. Incomplete resection, preoperative absence of myasthenia gravis, and advanced Lattes/Bernatz pathologic class were found to be independent predictors of poorer overall survival. CONCLUSIONS: These findings support a policy of aggressive, complete surgical resection of all thymomas when feasible. Thymoma behaves as a rather indolent tumor, with most deaths from causes unrelated to thymoma or its direct treatment. Clinicians should have an increased awareness of the possibility of second primary malignancies in patients with thymoma.

Adolescent↗
PubMed1999

Differential display probes for cDNA arrays.

PCR with a combination of one arbitrary and one oligo(dT) anchor primer can be used to generate an effective probe for cDNA arrays. The method uses less than 1/200 of the amount of RNA used in some other array hybridization methods. Each fingerprint detects approximately 5% of the transcribed mRNAs, sampled almost independent of abundance, using inexpensive E. coli colony arrays of expressed sequence tag (EST) clones. It proved necessary to alter the differential display (DD) protocol to generate a sufficient mass of PCR products for use as a probe. The use of different oligo(dT) anchor primers with the same arbitrary primer resulted in considerable overlap among the genes sampled by each probe. This can be avoided by using different arbitrary primers with each oligo(dT) anchor primer. Four genes not previously known to be regulated by epidermal growth factor (EGF) and three genes known to be regulated by EGF in other cell types were characterized using DD fingerprints as probes for arrays. It should be possible to convert archived DD fingerprints into effective probes for arrays, allowing thousands of experiments that have already been performed to yield further information. The use of DD fingerprints as probes should increase the rate of identification of differentially regulated genes several fold while obviating the need for cloning and sequencing.

Cell Line↗
PubMed1998

Non-stoichiometric reduced complexity probes for cDNA arrays.

A method is presented in which the reduced complexity and non-stoichiometric amplification intrinsic to RNA arbitrarily primed PCR fingerprinting (RAP-PCR) is used to advantage to generate probes for differential screening of cDNA arrays. RAP-PCR fingerprints were converted to probes for human cDNA clones arrayed as Escherichia coli colonies on nylon membranes. Each array contained 18 432 cDNA clones from the IMAGE consortium. Hybridization to approximately 1000 cDNA clones was detected using each RAP-PCR probe. Different RAP-PCR fingerprints gave hybridization patterns having very little overlap (<3%) with each other or with hybridization patterns from total cDNA probes. Consequently, repeated application of RAP-PCR probes allows a greater fraction of the message population to be screened on this type of array than can be achieved with a radiolabeled total cDNA probe. This method was applied to RNA from HaCaT keratinocytes treated with epidermal growth factor. Two RAP-PCR probes detected hybridization to 2000 clones, from which 22 candidate differentially expressed genes were observed. Differential expression was tested for 15 of these clones using RT-PCR and 13 were confirmed. The use of this cDNA array to analyze RAP-PCR fingerprints allowed for an increase in detection of 10-20-fold over the conventional denaturing polyacrylamide gel approach to RAP-PCR or differential display. Throughput is vastly improved by the reduction in cloning and sequencing afforded by the use of arrays. Also, repeated cloning and sequencing of the same gene or of genes already known to be regulated in the system of interest is minimized. The procedure we describe uses inexpensive arrays of plasmid clones spotted as E.coli colonies to detect differential expression, but these reduced complexity probes should also prove useful on arrays of PCR-amplified fragments and on oligonucleotide chips. Genesobserved in this manuscript: H11520, U35048, R48633, H28735, M13918, H12999, H05639, X79781, M31627, H23972, AB000712, R75916, U66894, AF067817.

DNA, Complementary↗
PubMed1998

Differentially expressed genes in the Trypanosoma brucei life cycle identified by RNA fingerprinting.

RNA fingerprinting by arbitrarily primed polymerase chain reaction (RAP-PCR) was used to identify genes that were differentially expressed during the life cycle of Trypanosoma brucei, as well as in response to heat shock. The standard RAP-PCR protocol was varied in two ways. First, the PCR reactions sometimes included a primer derived from the 5' mini-exon sequence, to ensure that most of the products contained the 5' end of mRNAs. Second, differentially amplified products were reamplified, isolated on single strand conformation polymorphism (SSCP) gels, cloned, and sequenced. Clones representing 32 different expressed sequence tags (ESTs) were obtained. Twenty-four ESTs were confirmed as differentially expressed by RT-PCR between different stages of the parasite cycle, or in response to temperature elevation. Nine clones had significant similarities to sequences already in the database. These transcripts included genes encoding cell surface proteins, metabolic enzymes, and heat shock proteins, either from trypanosomes or other organisms. Of particular interest, ESAG1 was shown to be heat-inducible in the procyclic stage. Most of the transcripts were unrelated to any other sequences in the database, and were deposited as new ESTs. The identification of stage-specific and heat shock-regulated transcripts will complement the growing T. brucei database. In addition, this experimental approach allows previous entries in the sequence database to be annotated with regulatory information.

Animals↗
PubMed1998

Solitary plasmacytoma of the epiglottis: a case report and review of the literature.

A 59-year-old white male presented with a two-month history of dry cough and shortness of breath. At bronchoscopy, a 1 cm mass on the laryngeal surface of the epiglottis was found. Immunohistochemical stain of the biopsy specimen revealed a monoclonal proliferation of plasma cells. Serological tests revealed normal serum immunoglobulin levels, and a bone marrow aspirate and biopsy was also normal; no abnormalities were found on serum or urine electrophoresis. He received radiotherapy (50 Gy), and was doing well three years following therapy without evidence of disease.

Epiglottis↗
PubMed1998

Estrogen-responsive RING finger mRNA induction in gastrointestinal carcinoma cells following bile acid treatment.

The underlying molecular mechanisms of the tumor-promoting activity of bile acids such as chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA) and the protective effect of ursodeoxycholic acid (UDCA) remain largely unclear. Using RNA arbitrarily primed PCR (RAP-PCR) for differential display, we identified, cloned and sequenced differentially expressed transcripts after treating gastric carcinoma cells (St 23132) with the bile acids CDCA, DCA and UDCA. One of these transcripts was identified to be an estrogen-responsive RING finger protein (efp) mRNA. The differential expression of efp in gastric cancer cells was confirmed by low stringency RT-PCR. efp mRNA levels were induced 3-fold in gastric carcinoma cells after CDCA and DCA treatment, whereas no change in expression was detected after UDCA treatment. Finally, treatment of the colon carcinoma cell line HT 29 with DCA resulted in a 2- to 5-fold induction of efp mRNA levels whereas UDCA did not induce efp. As expected, efp expression was also increased after 24 h of estrogen treatment. In summary, a synergy or a common pathway of tumor enhancement of bile acids and estrogen via efp in gastrointestinal carcinogenesis can be envisioned.

Bile Acids and Salts↗