Expression analysis of mRNA and proteins related to angiogenesis following management with cytotoxic agents in gynecologic cancer cell lines / 대한산부인과학회지

OBJECTIVE: Angiogenesis is central role to both the proliferation and metastasis of malignant tumor. The intense interest in angiogenesis has also lead to a re-examination of the activity of established cytotoxic agents which are known to be an antiangiogenic effect anecdotally. In this study, anti-angiogenic effect of cisplatin, paclitaxel and thalidomide was evaluated in human ovarian cancer cell lines and cervical cancer cell line. METHODS: Human ovarian cancer cell line A2780, cisplatin resistant human ovarian cancer cell line A2780-CDDP, human breast cancer cell line MCF-7, and squamous cell uterine cervical carcinoma cell line SiHa were used to evaluate the level of mRNA and protein expression of VEGF, bFGF and TSP-1, 2 before and after the treatment with cisplatin, paclitaxel, and thalidomide using RT-PCR, protein extraction, and Western blot. The results were analyzed with Wilcoxon signed rank test in the SAS ver 8.1. RESULTS: Targeted mRNAs were synthesized as 212 bp VEGF, 238 bp bFGF, and 492 bp band sized except mRNA of TSP-2 via RT-PCR. The protein of VEGF and bFGF were appeared as 21KDa and 17 KDa size, however, the protein of TSP-1 was not appeared through western blot. No effect of cisplatin on protein expression was measured in these cell lines, but paclitaxel influenced the expression of bFGF in MCF-7 cell line and the expression of TSP-1 in MCF-7 and SiHa cell lines. TSP-1 expression was influenced by thalidomide in A2780 cell line. The protein expression of VEGF and bFGF were not influenced following treatment with cisplatin, paclitaxel, and thalidomide. CONCLUSION: These results were suggested that bFGF and TSP-1 will be used as a target gene for the assay of antiangiogenic effect of paclitaxel in breast and uterine cervical cancer tissue and TSP-1 will be used as that of thalidomide in ovarian cancer. Furthermore, thalidomide will be tried as an adjunctive agent for the improvement of the survival in the case of the patient with ovarian cancer.

Expression analysis of genes related to multidrug resistance (MDR) in ovarian cancer cell line A2780 and cisplatinum resistant cell line A2780cp / 대한산부인과학회잡지

OBJECTIVE: Expressions of P-glycoprotein, the multidrug resistance-associated protein (MRP) and the lung resistance protein (LRP) with the MDR phenotype widely divergent in human cancer cell lines. This study focused on the altered gene expression related drug transport. METHODS: To examine correlations between MDR-associated genes and PKC isozyme with cisplatin resistance on the level of the mRNA expression, we analyzed MDR-associated gene (LRP, MDR1/P-gp and MRP) expression and PKC isozyme, topoisomerase II alpha and beta in cisplatin-sensitive ovarian cancer cell line A2780 and cisplatin - resistant cell line A2780cp using cDNA-PCR approach. RESULTS: LRP mRNA levels were significantly increased in A2780cp compared to the drug sensitive variant. In contrast, MRP mRNA levels were not significantly correlated with drug sensitivity. A modest increase in PKC(eta) and MDR1/P-gp mRNA expression activity was also observed in ovarian cancer A2780cp cell lines that were resistant to CDDP. The level of topoisomerase II alpha and beta were not affected. CONCLUSION: These results showed that MDR1/P-gp expression may be an important determinant of the MDR phenotype in cell lines intrinsically resistant to cancer chemotherapeutic agents and a multifactorial emergence of MDR phenotype of tumor with a possible involvement of the PKC isozymes may be associated with CDDP resistant ovarian cancer cell line.

The Frequency of Serum IgA autoantibody in Patients with Pemphigus / 대한피부과학회지

BACKGROUND: Pemphigus is an autoimmune bullous disease with circulating desmosomal autoantibodies of IgG. In direct IF studies with perilesional tissue, IgA or IgM antibodies can be seen in addition to IgG. OBJECTIVE: We examined sera of patients with pemphigus for the presence/frequency of IgA autoantibodies as well as IgG by indirect IF and immunoblot assay. Patients: Twenty patients of pemphigus (PV 10, PF 10) who showed positive findings in indirect IF examinations. METHODS: Indirect IF study with normal human skin substrates and immunoblot analysis using A431 cell extracts (with multi-step immunostaining) were performed with patients sera. RESULTS: In indirect IF, IgA autoantibodies that bind to the epidermal keratinocyte antigens were detected in 4 cases among the 20 patients (PV 2 and PF 2). In immunoblot analysis IgA bands reacting to PV/PF antigens were observed in 7 cases from the 20 patients with pemphigus (PV 3, PF 4). The serum titers of IgA autoantibodies were lower than those of IgG in every single case. CONCLUSION: In patients with pemphigus (PV/PF), 35% of cases have serum IgA autoantibodies as well as IgG autoantibodies specific to the pemphigus antigens (Dsg 1/Dsg 3). However, pathogenic roles of the associated IgA autoantibody are not clear.

Induction of Apoptosis in Synovial Cells from Patients with Rheumatoid Arthritis / 대한면역학회지

Homeostasis of multicellular organism is controlled by proliferation and differentiation of cells as well as by cell death. The defects in programmed cell death contribute to the pathogenesis of rheumatoid arthritis (RA) and systemic lupus erythematous. RA is considered to be a proliferating disorder of synovial tissue which is accompanied by inflammatory cell infiltration and bone erosion. The aim of the study was to find whether potent inducers of apoptosis could be induced apoptosis in RA synovial cells. We examined the effects of drugs, such as dexamethasone, methotrexate, hydrogen peroxide, and ceramide on induction of apoptosis in cultured RA synovial cells. Used drugs did not induced apoptosis in RA synovial cells. Finally Fas antigen-mediated apoptosis of RA synovial cells was investigated by the addition of anti-Fas antibody. To examine the ICE (interleukin-1p-convertase; caspase-1) expression in synovial cells, RT-PCR of caspase-1 gene was performed. In synovial cells of RA, Fas induces that caspase-1 activation cause apoptosis.

Type 2 Procollagen Gene Mutation in Osteoarthritis / 대한류마티스학회지

OBJECTIVES: Human osteoarthritis is a heterogeneous and multifactorial disease characterized by the progressive deterioration of the cartilage of diarthrodial joints. In some instances, there is an identifiable cause, such as trauma or congenital malformation, but, mostly the etiology remains unknown. Since familial aggregation is seen, genetic factors may be important, particularly in osteoarthritis of the hand. METHODS: Blood samples from patients and controls were obtained for DNA analysis. Exon 31 of type II procollagen gene was amplified by polymerase chain reaction, and screening for the mutation was undertaken using a restriction enzyme digestion (Dsa I). RESULTS: The patients phenotype represented typical, but earlyonset and family history, osteoarthritis. No mutation in exon 31 of type II procollagen gene could be identified. CONCLUSION: Screening of the 31 exon did not, however, reveal any mutation. It needs further evaluation in other sites of type II procollagen genes.

Nitric Oxide(NO) in Inflammatory Arthritis / 대한내과학회지

OBJECTIVES:Nitric Oxide(NO) is a toxic, inorganic, gaseous free radical produced during the metabolism of L-Arginine by NO synthase(NOS). It has been implicated in a rapidly growing number of physiological and pathophysiological processes such as cytotoxic effects against microbes and tumor cells, blood vessel dilation and neurotransmitter. Recently there is growing evidence implicating NO in immune regulation, inflammation, autoimmunity, and arthritis. We performed this study to determine a role for nitric oxide in inflammatory arthritis especially rheumatoid arthritis(RA). METHODS: We measured (1) the concentrations of nitrite, a breakdown product of nitric oxide, in serum and synovial fluid from patients with RA and osteoarthritis(OA) and in the serum of controls (2) the concentrations of nitrite in the supernatant of cultured synovial tissue with RA and OA and (3) determined whether human chondrocytes and synoviocytes can synthesize nitric oxide and if so, how production is regulated by cytokines and antirheumatic drugs. RESULTS: 1) Serum nitrite concentrations in patients with RA and OA were higher than in controls. In both disease groups synovial fluid nitrite was higher than serum nitrite. Serum and synovial fluid nitrite concenrations in RA were higher than those in OA. However, those findings are not statistically significant. 2) Although these findings are not statistically significant, the concentration of nitrite in the supernatant of cultured synavial tissue with RA was higher than that in OA. 3) IL-1beta and TNF-alpah induced the biosynthesis of NO by chondrocytes and synoviocytes. IGF-1 and TGF-beta failed to provoke the production of NO. The biosynthesis of NO required an induction period of approximately 6 hours and was inhibited by L-NMMA and cycloheximide. Dexamethasone, indomethacin, gold sodium thiomalate and methotrexate had no effect on the induction of NO biosynthesis. CONCLUSION: These results suggest a role for nitric oxide as an inflommatory mediator in inflammatory arthritis.

Analysis of TCR Vfi Gene Repertoire in Patients with Rheumatoid Arthritis / 대한류마티스학회지

OBJECTIVES: Polymerase chain reaction (PCR) technology was eamine synovial fluid and peripheral T cells in patients with rheumatoid arthritis(RA) to determine the preferential usage of the T cell receptor(TCR) variable region(V) gene. METHODS: Oligonucleotide primers specific for individual TCR Vfi gene families were used to amplify the TCR gene products in a semiquantitative assay of their relative utilization in unselected T cell populations. RESULTS: The result of Vfi utilization was generally heterogenous, similar with previous reports. However, the mean expression of Vfi16 and Vfi18 in RA was more preferentially utilized compared to normal donors. The usage of Vfi in peripheral blood from 3 patients with RA demonstrated restrictions in Vfi16, Vfi 20 and Vfi18 genes, respectively. Analyses of synovial fluid resulted in restriction in Vfi12, Vfi20 and Vfi20, respectively. Although there was no significant pattern of skewed Vfi gene mean usage when comparing the synovial fluids with the peripheral blood T cells from RA patients, there were significant biased Vfi genes, Vfi12, V~I and Vfi20, each 3 patients. As the HLA type is a determining factor in shaping TCR repertoire of peripheral T cells, we compared the Vfi utilization in HLA-DR4 expressing groups that have susceptibility and gene dosage effect in disease progression. It was a little different that comparing the pattern of Vfi usage in peripheral blood and synovial fluid from RA patients between HLA-DR4 positive and negative group. CONCLUSION: The results were consistent with the conclusion that the increased Vfi family T cells infiltrate synovium and are dependent on each patient and may be involved in inducing and maintaining the synovitis that characterizes RA. The different outcome of each patient may be due to the difference in disease duration, genetic background and geographic region. A more important factor may be the stage of disease, because epitope 'induced immune reaction may change over time. Therefore, selecting patients early in the course of disease may be important and may facilitate the need for more in-depth TCR analysis in the future.

Characteristics of Insulin-Like Growth Factor I in Serum; Pattern of Insulin-Like Growth Factor Binding Protein(Large Complex) in Short Stature Patients with Complete Growth Hormone Deficiency

To elucidate the complex of insulin-like growth factor binding proteins (IGF-BPs) in short stature patients, we carried out a prospective study on three patients who were diagnosed as complete GH deficiency at the department of pediatrics from July 1992 to June 1993. The results were summarized as follows: 1) Two circulating IGFs complexed to specific binding protein existed in normal serum. Binding activity was found to be in the 150,000 molecular weight area (the large complex) and 50~60,000 molecular weight area (the small complex). 2) Binding activity for the large complex was seen to be dependent on advancing age, level of large IGF-BP3 complex peacked at the age of 15~16 years. 3) The binding activity for large complex diminished in three GH deficient patients and increased after hGH injection to near or above normal level. 4) Increased growth rate after GH treatment in GH deficient patient was closely related with increasing level of the large IGF-BP3 complex. Therefore we suggest that the large IGF-BP3 complex is regulated by GH. Estimating its serum level is useful for screening of GH deficiency and the monitoring of response to GH therapy.