Home About us Contact
|
Home About us Contact | ||
|
|
||
Competitive Reverse Transcription (competitive + reverse_transcription)
Selected AbstractsSignificance of Fas expression alteration during tumor progression of renal cell carcinomaINTERNATIONAL JOURNAL OF UROLOGY, Issue 3 2006TAKEHIRO SEJIMA Background:, In order to characterize the alteration of apoptotic regulatory molecule expression during tumor progression of renal cell carcinoma (RCC), we compared the expression between tumor and normal tissues, and evaluated the relationship of the expression in tumors with pathological and clinical characteristics. Methods:, Competitive reverse transcription,polymerase chain reaction (RT,PCR) and immunohistochemistry (IHC) allowed the determination of Fas and bcl-2 mRNA and protein expression in surgically resected tumor and normal tissue of 50 RCC. Results:, The mRNA expression of Fas and bcl-2 in RCC was significantly reduced compared to that in normal tissues. An IHC analysis was supportive of the RT,PCR results. In terms of relationships with pathological and clinical characteristics, the mRNA and protein expression of Fas in high-stage or high-grade tumors was significantly higher than that in low-stage or low-grade tumors. Moreover, a statistically poor prognosis was observed in tumor cases expressing a high amount of mRNA. In bcl-2 analysis, the mRNA and protein expression was significantly reduced in clear cell tumors compared to chromophobe cell tumors. Conclusion:, It is suggested that the reduced expression of Fas and bcl-2 in RCC compared with the expression in normal kidney is a prominent alteration of apoptotic regulatory molecules. The alteration of the up-regulated Fas expression might be characterized during the tumor progression stage. It is also suggested that the effect of alteration of bcl-2 expression might be minimal during the tumor progression stage because of the reduced expression in tumors of the clear cell type, which is the most dominant cell type in RCC. [source] Nkx2.1 transcription factor in lung cells and a transforming growth factor-,1 heterozygous mouse model of lung carcinogenesis,MOLECULAR CARCINOGENESIS, Issue 4 2004Yang Kang Abstract The Nkx2.1 homeobox gene and transforming growth factor-,1 (TGF-,1) are essential for organogenesis and differentiation of the mouse lung. NKX2.1 is a marker of human lung carcinomas, but it is not known whether this gene participates in early tumorigenesis. Addition of TGF-,1 to TGF-,1-responsive nontumorigenic mouse lung cells cotransfected with a NKX2.1Luc luciferase reporter and either a Sp1 or Sp3 plasmid showed a significant increase or decrease, respectively, in NKX2.1Luc transcription. Cotransfection of Sp3 and dominant-negative TGF-, type II receptor plasmids negated the effect of Sp1. Cotransfected Sp1 plasmid with either dominant-negative Smad2 or Smad3 or Smad4 plasmids significantly decreased NKX2.1Luc transcription. Electrophoretic mobility shift assays revealed binding of Sp1 and Smad4 to the NKX2.1 promoter. With a TGF-,1 heterozygous mouse model, Nkx2.1 mRNA and protein in lungs of TGF-,1 heterozygous mice were significantly lower compared to wildtype (WT) littermates. Competitive reverse transcription (RT)-polymerase chain reaction (PCR) and immunostaining showed that Nkx2.1 mRNA and protein decreased significantly in adenomas and adenocarcinomas compared to normal lung tissue. Our in vitro data showed that regulation of Nkx2.1 by TGF-,1 occurs through TGF-, type II receptor and Smad signaling, with Sp1 and Sp3 in lung cells. Our in vivo data showed reduced Nkx2.1 in lungs of TGF-,1 heterozygous mice compared to WT mice, that is detectable in adenomas, and that is further reduced in carcinogenesis, and that correlates with reduction of Sp1, Sp3, and Smads in lung adenocarcinomas. Our findings suggest that reduced Nkx2.1 and TGF-,1 signaling components may contribute to tumorigenesis in the lungs of TGF-,1 heterozygous mice. Published 2004 Wiley-Liss, Inc. [source] Growth factors and proliferation of cultured rat gingival cells in response to cyclosporin AJOURNAL OF PERIODONTAL RESEARCH, Issue 1 2005Takumasa Yoshida Objective:, The prominent side-effect of cyclosporin A, an immunosuppressive drug, in oral tissues is gingival outgrowth, although the exact mechanism underlying this side-effect is unclear. The main purposes of the present study were to determine whether cyclosporin A induced the gingival outgrowth by promoting proliferation of gingival cells and whether growth factors such as transforming growth factor-,s (TGF-,s), fibroblast growth factor-2 (FGF-2), platelet-derived growth factors (PDGFs), and insulin-like growth factors (IGFs) are involved in the possible changes in the proliferation of gingival cells induced by cyclosporin A. Methods:, Cells isolated from rat gingival tissues were cultured with cyclosporin A or IGF-I for 3 days. The effects of cyclosporin A or IGF-I on the proliferation of cultured rat gingival cells were analyzed with a CellTiter 96 proliferation assay kit. The mRNA expression levels for TGF-,s, FGF-2, PDGFs, IGFs, insulin-like growth factor receptors (IGFRs), and insulin-like growth factor binding proteins (IGFBPs) in the rat gingival cells treated with cyclosporin A were measured using competitive reverse transcription,polymerase chain reaction (RT,PCR). Results:, Cyclosporin A induced 23,25% (p < 0.001) increases in the proliferation of rat gingival cells and approximately 130% (p < 0.05) and 60% (p < 0.05) elevations in the mRNA expression levels for TGF-,1 and FGF-2, respectively. On the other hand, exogenous IGF-I induced 8,11% (p < 0.05) increases in the proliferation, but cyclosporin A induced 30,80% (p < 0.05,0.01) reductions in the mRNA expression levels for endogenous IGF-I, IGFR1, IGFBP2, IGFBP3, IGFBP5, and IGFBP6. Conclusions:, Cyclosporin A stimulates the proliferation of rat gingival cells. TGF-,1 and FGF-2 could be involved, but IGFs, IGFRs and IGFBPs could not be directly involved in this cyclosporin A induced-stimulation of the gingival cell proliferation. [source] Response of anti-oxidant enzymes mRNA in the neonatal rat liver exposed to 1,2,3,4-tetrachlorodibenzo- p -dioxin via lactationPEDIATRICS INTERNATIONAL, Issue 5 2002Yumi Kono Abstract Background: The aim of this study was to assess the response to dioxin-induced oxidative stress in neonates via lactation in the model we have described previously. Methods: Maternal rats were treated with a single dose of 50 or 100 µmol/kg 1,2,3,4-tetrachlorodibenzo- p -dioxin (TCDD) on the first day postpartum (day 1). Messenger RNA levels of the key anti-oxidant enzymes (AOE), phospholipid hydroperoxide-glutathione peroxidase (PH-GPx), cellular-glutathione peroxidase (cell-GPx), copper-zinc superoxide dismutase (CuZn SOD), manganese superoxide dismutase (Mn SOD) and catalase (CAT) in the neonatal and maternal livers were determined by a competitive reverse transcription, polymerase chain reaction method. Results: Lactational transfer of 1,2,3,4-TCDD induced an inhibition of PH-GPx and cell-GPx mRNA in the neonatal liver on day 2 to 68 (P < 0.01) and 62% (P < 0.05) of the control at 100 µmol/kg, respectively. Both GPx mRNA returned to control levels on day 6 and thereafter increased to levels higher than the controls on day 10. In the dam rat, 10 days after the treatment, no remarkable change of PH-GPx or cell-GPx mRNA was observed. Copper-zinc superoxide dismutase and CAT mRNA of neonates on day 2 were also suppressed at 100 µmol/kg and then slightly increased on day 10. However, Mn SOD mRNA was not suppressed, but increased to a 2.1-fold level of the control (P < 0.05) on day 10 with 100 µmol/kg 1,2,3,4-TCDD. Conclusion: Quantitative analysis of AOE mRNA showed that PH-GPx and cell-GPx mRNA, as well as CuZn SOD and CAT mRNA in the neonatal liver were suppressed for a short period of time by 1,2,3,4-TCDD exposure via lactation. Dioxin induced oxidative stress by lactational transfer may alter pretranslation regulation of protective AOE in neonates. [source] Effect of lactational exposure to 1,2,3,4- tetrachlorodibenzo-p-dioxin on cytochrome P-450 1A1 mRNA in the neonatal rat liver: Quantitative analysis by the competitive RT-PCR methodPEDIATRICS INTERNATIONAL, Issue 5 2001Yumi Kono AbstractBackground and Methods: The aim of this study was to assess the effect of lactational exposure to dioxins in neonates on the cytochrome P450 1A1 (CYP1A1) induction in the level of gene expression. Maternal rats were treated with a single dose of 50 or 100 ,mol/kg 1,2,3,4-tetrachlorodibenzo-p-dioxin (1,2,3,4-TCDD), a low potent congener of dioxins, on the first day post-partum (day 1). Induction of CYP1A1 mRNA expression was quantitatively analyzed by the competitive reverse transcription,polymerase chain reaction (RT,PCR) method. Results: The CYP1A1 mRNA was detectable at extremely low amounts in the liver of control neonates and mothers. The mRNA ratios of CYP1A1 to ,-actin in neonates were dose-dependently increased by the treatment of 1,2,3,4-TCDD of their mothers. Its peak occurred on day 6 and was sustained at the same level on day 10. Increases of the ratio with 100 ,mol/kg 1,2,3,4-TCDD on day 2, 6 and 10 were 26-, 40- and 40-fold of the appropriate controls, respectively. These levels paralleled the activity of ethoxyresorufin-o-deethylase, representing CYP1A mediated monooxygenase. In the mother, the mRNA ratio was increased only to threefold of the control, 10 days after treatment. Conclusion: Current RT-PCR procedure enabled to assess both constitutive and induced levels of CYP1A1 mRNA in the neonatal rat livers. Although the dose of 1,2,3,4-TCDD selected in this study was about 5000 times higher than the daily intake of dioxins in breast-fed infants, CYP1A1 mRNA was highly induced for a longer period of time in neonatal rats receiving 1,2,3,4-TCDD via lactation than the treated maternal rats. [source] | ||||||||||