BACKGROUND: Protein kinase C (PKC)s consist of three groups of isoenzyme; conventional, novel and atypical PKCs. Diacylglycerol (DAG) activates both conventional and novel PKCs, but not atypical PKCs. High glucose-induced fibronection production was shown to be mediated by activation of DAG-sensitive PKCs. In this study, we investigated whether PKC mediates IL-1beta-induced fibronectin mRNA expression, and the subtypes of PKC involved in the process. METHODS: Fibronectin mRNA level and phosphorylated PKC zeta/iota in total cell lysate were measured by Northern blot and Western blot, respectively. RESULTS: Pretreatment of HPMCs with calphostin C, a pan-PKC inhibitor, at doses of 500, 750 and 1, 000 nM caused dose-dependent inhibition of IL- 1beta (1 ng/mL)-induced fibronectin mRNA level. GF109203X, another pan-PKC inhibitor, at doses of 1, 5 and 10 microM also downregulated IL-1beta (1 ng/ mL)-induced fibronectin mRNA level in a dose-dependent manner. Phorbol 12-myristate 13-acetate (PMA), an activator of conventional and novel PKCs, stimulated fibronectin mRNA level at doses of 1, 10 and 100 nM. After prolonged treatment of the cells for 72 hr with PMA, another dose of PMA did not increase fibronectin mRNA level, while IL-1beta (1 ng/mL) still stimulated it. Pretreatment of the cells with 5, 10, 15 and 20 microM of myristoylated PKC zeta/iota pseudosubstrate inhibited IL-1beta (1 ng/mL)-induced fibronectin mRNA level in a dose-dependent manner, while 20 microM of myristoylated PKC [19-27] pseudosubstrate, given as a control, had no effect. Stimulation of fibronectin mRNA level by IL-1beta (1 ng/mL) was completely prevented by 20 microM of my ristoylated PKC zeta/iotapseudosubstrate. IL-1beta (1 ng/ mL) increased phosphorylated PKC zeta/iota, an active form of the enzyme. CONCLUSION: IL-1beta-induced fibronectin production in HPMCs occurs by way of activation of atypical PKCs (PKC zeta/iota).
Blotting, Northern ; Blotting, Western ; Fibronectins* ; Humans* ; Interleukin-1beta* ; Protein Kinase C* ; Protein Kinases* ; RNA, Messenger

PURPOSE: MMP-2, 72 kDa-type IV collagenase, plays a major role in the migration and growth of tumor cells, a process that requires the disintegration of basement membrane. Activation of MMP-2 is correlated with the invasiveness of various tumors. The aim of this study was to determine the sequence-specific phosphorothioated oligodeoxynucleotides (ODNs) inhibiting the translation of MMP-2 mRNA and the subsequent invasiveness of tumor cells. MATERIALS AND METHODS: Eight types of antisense ODNs were designed and each (8micro gram/ml) were transfected into HT1080 cells. The effects of these antisense ODNs on MMP expression were examined by gelatin zymography, Western blot, Northern blot and matrigel assay. RESULTS: Antisense-5 (+904~923), antisense-6 (+1274~+1293) and antisense-7 (+1646~+1665) reduced the MMP-2 activity of the culture supernatant in HT1080 fibrosarcoma cells. Treatment with antisense-6 showed inhibition of MMP-2 mRNA and protein, and in vitro invasion in a dose-dependent manner. CONCLUSION: Antisense-6 might be one of the therapeutic candidates for tumor invasion and metastasis.
Basement Membrane ; Blotting, Northern ; Blotting, Western ; Collagenases ; Fibrosarcoma* ; Gelatin ; Humans* ; Matrix Metalloproteinase 2* ; Neoplasm Metastasis ; Oligodeoxyribonucleotides ; RNA, Messenger

BACKGROUND: Peroxiredoxin I (Prx I) is part of an oxidative stress defense system with thioredoxin peroxidase activity to eliminate hydrogen peroxide (H2O2). UV irradiation is one of the major sources to produce H2O2, which should then be scavenged by antioxidant systems to maintain functional integrity of the skin. OBJECTIVE: This study aimed to evaluate the modulation of Prx I by ultraviolet B (UVB) irradiation in human epidermal keratinocytes. The modulation of Prx I expression by H2O2 was also evaluated. METHOD: Primary culture of epidermal keratinocytes was performed, and sub-confluent cells were irradiated with UVB irradiation (20mJ/cm(2)). Western blot and Northern blot analysis were performed after the cells were harvested at different time-points after UVB irradiation. Prx I expression and intracellular levels of H2O2 were evaluated in the cells which had been irradiated with different doses of UVB. The localization of Prx I expression was identified by immunocytochemical staining. RESULTS: UVB irradiation induced Prx I mRNA and protein expressions from 3 h and 6 h after irradiation, respectively, indicating that UVB induced Prx I expression at a transcription level. Intracellular H2O2 levels were steadily increased as keratinocytes were irradiated with increasing doses of UVB. Next, when keratinocytes were treated with 0.1-10.0mM of H2O2, the marked induction of Prx I protein expression was observed above 1 mM H2O2 at a time-dependent manner (after 6 h). The H2O2-induced Prx I expression was abolished by N-acetyl-L-cysteine, a H2O2 scavenger, pre-treatment. In 2D-gel electrophoresis, the active reduced form of Prx I was rapidly transformed into the oxidized, inactive form, and then it restored to the reduced form by H2O2 treatment, suggesting that Prx I was active in responding to the H2O2-induced oxidative stress. CONCLUSION: UVB irradiation up-regulates Prx I by the mediation of H2O2 in the keratinocytes.
Acetylcysteine ; Blotting, Northern ; Blotting, Western ; Electrophoresis ; Humans* ; Hydrogen Peroxide ; Keratinocytes* ; Negotiating ; Oxidative Stress ; Peroxiredoxins* ; RNA, Messenger ; Skin

PURPOSE: Membrane type-1-matrix metalloproteinase (MT1-MMP) plays a key role in endothelial cell (EC) migration, matrix remodeling, and angiogenesis. Previous studies demonstrated that a cyclic strain (CS) increases MT1-MMP expression by displacing specific protein 1(Sp1) with increased early growth response-1 (Egr-1) expression; and shear stress (SS) decreases MT1-MMP expression by Sp1 phosphorylation. However, the difference in MT1-MMP expression according to the change of SS is poorly understood. The aim of this study is to determine the effects of low or high SS on Egr-1 and MT1-MMP transcription and translation. METHOD: Bovine aortic ECs were exposed to oscillatory SS (low=0.1 dyne/cm2 or high=14 dyne/cm2) with orbital shaker for 0, 1, 4, or 8 hours. Activation of Egr-1 and MT1-MMP was assessed by the Northern blot and Western blot. RESULT: Although Egr-1 mRNA transcription and protein translation were induced (7.3-, 5.8-fold and 4.0-, 4.9-fold, respectively) in response to low SS (n=5, 0, 1, and 4 hr; P<0.05), MT1-MMP mRNA transcription and protein levels did not change remarkably. Egr-1 mRNA transcription and translation were induced (7.6-fold at 1 hr; 3.7- and 5.2-fold at 1 and 4 hr, respectively) in response to high SS (n=5; P<0.05). We observed that high SS decreased MT1-MMP mRNA transcription and translation in a time-dependent fashion (10%, 50%, and 90% reduction at 1, 4, and 8 hr, respectively; n=5, P<0.05). CONCLUSION: High SS induces Egr-1 up-regulation and inhibits MT1-MMP expression. But low SS has no effect on MT1-MMP expression in spite of Egr-1 up-regulation. These observations illustrate that the expression of MT1-MMP is dependent on, not only the type of hemodynamic forces, but also the strength of force (SS) in vascular endothelial cells.
Blotting, Northern ; Blotting, Western ; Endothelial Cells* ; Hemodynamics ; Matrix Metalloproteinase 14* ; Membranes ; Orbit ; Phosphorylation ; Protein Biosynthesis ; RNA, Messenger ; Up-Regulation

BACKGROUND: Nerve growth factor(NGF) is a neurotrophic polypeptide necessary for the survival and growth of some central neurons, as well as sensory afferent and sympathetic neurons. In addition to its actions on the nervous system, it also has a significant biologic effects on cells of the immune-inflammatory compartment. Recent studies suggest that NGF is an important autocrine growth factor and survival factor for keratinocytes which express both high- and low-affinity receptors for NGF. OBJECTIVE: The purpose of this study is to detect NFG receptors on cultured human keratinocytes and to evaluate the effect of NGF on proliferation of cultured human keratinocytes. METHODS: Cultured human keratinocytes were examined for the expression of high affinity receptor TrkA and low affinity receptor p75 by Northern blot, Western blot and immunocytochemistry. The effects of NGF on proliferation of cultured human keratinocytes were also evaluated. To specify the NGF effect on proliferation of human keratinocytes, excess of anti-NGF neutralizing polyclonal antibody was added. RESULTS: 1) NGF significantly stimulated the proliferation of keratinocytes in both 1% of keratinocyte growth supplement(KGS)-added medium(100ng/ml) and 0.2% KGS-added media(50, 100, 500ng/ml), (p<0.05). The cell number was dose-dependently increased in 0.2% KGS-added media. 2) Whenever we added 500 ng/ml of anti-NGF polyclonal antibody to the growth media, the cell number was statistically higher in 100ng/ml NGF-added group of 1% KGS-added medium, but there was not any statistical significance in 0.2% KGS-added media group. 3) Immunocytochemical staining with specific antibodies to TrkA and p75 revealed positive findings for these receptors, but TrkB and TrkC were not detected. 4) We could not detect both the mRNA and protein of TrkA and p75 by Northern and Western blot methods. CONCLUSION: These results suggest that both high affinity- and low affinity receptors for NGF are expressed in cultured human keratinocytes and NGF can induce keratinocyte proliferation.
Antibodies ; Blotting, Northern ; Blotting, Western ; Cell Count ; Cell Proliferation* ; Humans* ; Immunohistochemistry ; Keratinocytes* ; Nerve Growth Factor* ; Nervous System ; Neurons ; RNA, Messenger

OBJECTIVETo evaluate the expression of mitogen activated protein kinase phosphatase-1(MKP-1)in pancreatic cancer.

METHODSTotally 60 cases of normal pancreas, chronic pancreatitis(CP), and pancreatic cancer tissues were collected by operation in our hospital. Pancreatic tissues were analyzed by Northern blot analysis and Western blot analysis. Meanwhile, MKP-1 expression was detected in 6 pancreatic cancer cell lines by Western blot analysis.

RESULTSNorthern blot analysis of total RNA revealed relatively low MKP-1 mRNA expression in 7 of 20(35%)normal pancreatic samples. In the remaining 13 samples, the MKP-1 mRNA was absent to faint detectable. In 7 of the 20 CP samples, MKP-1 was demonstrated moderate to high expression. In contrast, 12 of 20(60%)pancreatic cancer samples MKP-1 mRNA was expressed at high levels, whereas in the remaining 8 cancer tissues this mRNA moiety was present at low to moderate levels. Densitometric analysis with normalization to 7S revealed that the median level of MKP-1 mRNA in CP and cancerous tissues was increased by 6.2 folds(P=0.035)and 8.1 folds(P=0.016)in comparison with the median level in the normal pancreatic samples, respectively. Overexpression of MKP-1 was also found in 6 pancreatic cancer cell lines, in which the expression of MKP-1 was slightly lower in one pancreatic cancer cell line but high in the remaining 5 cell lines.

CONCLUSIONSMKP-1 is over-expressed in pancreatic cancer, CP tissues, and pancreatic cell lines. It is speculated that MKP-1 may play an important role in tumorigenesis of pancreatic cancer.

Blotting, Northern ; Blotting, Western ; Dual Specificity Phosphatase 1 ; metabolism ; Humans ; Immunohistochemistry ; Pancreas ; Pancreatic Neoplasms ; metabolism ; RNA, Messenger

The present study was aimed at investigating the molecular regulation of the renin- angiotensin system (RAS) in two-kidney, one clip (2K1C) hypertension. The expression of renin, angiotensinogen and angiotensin II receptor genes was determined by Northern blot analysis in rats made 2K1C hypertensive for 2 or 4 weeks. The expression of renin gene was increased in the clipped kidney and decreased in the contralateral non-clipped kidney at weeks 2 and 4. The expression of angiotensinogen gene was not significantly altered at week 2, but increased at week 4 in the clipped kidney. However, it was not significantly altered in the contralateral kidney either at week 2 or 4. Nor was the expression of angiotensinogen gene significantly altered in the liver either at week 2 or 4. On the other hand, the expression of angiotensin II receptor gene was decreased at week 2, and increased at week 4 in the clipped kidney, whereas it was not significantly changed in the contralateral kidney either at week 2 or 4. In the liver, the expression of angiotensin II receptor gene was not significantly altered at week 2, but decreased at week 4. These results suggest that the components of RAS are transcriptionally regulated in 2K1C hypertension in a manner dependent on tissues and duration of hypertension.
Angiotensin II* ; Angiotensinogen* ; Angiotensins* ; Animals ; Blotting, Northern ; Hand ; Hypertension ; Kidney ; Liver ; Rats* ; Receptors, Angiotensin* ; Renin

Glutathione(GSH) has a very important role in detoxification of cells and is closely related to antitumor drug-resistance of cancer cells. In order to evaluate the importance of glutathione metabolism in the drug-resistant cancer cells, the concentration of celluar GSH and activities of y-glutamylcysteine synthetase(GCS), y-glutamyl transpeptidase (GGT) and glutathione S-transferases(GST) in the adriamycin, vincristine, or cisplatin resistant L1210 (L1210AdR; L1210VcR, or L12100s) sublines were measured. Expression and amplification of GCS, GGT, and GST-i7 genes were also observed in the parent L1210 and the drug-resistant L1210 sublines. The concentration of GSH was increased 5.34 fold in L12100s, 2.83 fold in L1210VcR, and 1.78 fo-d in L1210AdR, compared to L1210. The activities of GCS and GGT were -increased in drug-resistant L1210 sublines. The GST activity was increased in L1210VcR and L1210Cis but decreased in L1210AdR compared to L1210. Expression of GCS, GGT, and GST-rr genes were increased in the resistant L1210 sublines compare to the parent L1210 in northern blot analyses. Overexpression of GCS, GGT, and GST-77 were observed in the resistant sublines, and the increases of the concentration of glutathione and the activities of GCS and GGT in the resistant sublines may be involved in a part of the drug-resistance in the resistant sublines.
Blotting, Northern ; Cisplatin ; Doxorubicin ; Drug Resistance ; Glutathione* ; Humans ; Metabolism* ; Parents ; Vincristine

PURPOSE: The expression pattern of c-jun by ionizing radiation according to cell growth state (exponential growth phase vs. stationary phase) and its relationship with cell cycle redistribution were investigated. MATERIALS AND METHODS: The exponential growth phase (day 4) and stationary phase (day 9) cells were determined from cell growth curve according to the elapse of days in CaSki. The cells were irradiated using 6 MV X-ray with a dose of 2 Gy at a fixed dose rate of 3 Gy/min. Northern blot analysis was performed with total cellular RNA and cell cycle distribution was analyzed using flow cytometry according to time-course after irradiation. RESULTS: The maximum expression of c-jun occurred 1 hour after irradiation in both exponential growth and stationary phase cells. After then c-jun expression was elevated upto 6 hours in exponential growth phase cells, but the level decreased in stationary phase cells. Movements of cells from G0-G1 to S, G2-M phase after irradiation were higher in exponential growth phase than stationary phase. CONCLUSION: c-jun may be involved in the regulation of cellular proliferation according to the growth states after irradiation.
Blotting, Northern ; Cell Cycle ; Cell Line* ; Cell Proliferation ; Flow Cytometry ; Radiation, Ionizing ; RNA

OBJECTIVE: To investigated whether lowering oxygen tension affects invasion of cultured trophoblast. METHODS: Trophoblasts were isolated from the normal placenta in early pregnancy(6-10 weeks in gestation). Isolated trophoblasts were cultured under normoxic(5% CO2, 95% humid air in incubator) and hypoxic(MERCK, 1% O2, 99% CO2) conditions for 24, 48 and 72 hours, respectively. The proliferation ability was measured using [H3] thymidine assay. Total RNA was extracted from the cultured trophoblasts. The expressions of matrix metalloproteinase(MMP-2) and tissue inhibitor of metallo- proteinase(TIMP-2) were determined by reverse transcription- polymerase chain reaction(RT-PCR) and Northern blot analysis. The invasiveness of cultured trophoblast was observed using in vitro invasion assay. RESULTS: [H] thymidine assay indicated that cellular DNA synthesis was not affected by the culture condition. The expression of MMP-2 mRNA was decreased at 24 hours and then progressively increased in the time-dependent manner in each culture condition. The expression of TIMP-2 was decreased in the time-dependent manner under hypoxic condition. In vitro invasion assay revealed that the cultured trophoblasts under hypoxic condition has more invasive ability than them under normoxic condition. CONCLUSION: These data suggests that hypoxic condition may stimulates the invasion of trophoblast in the human placentation. And MMP-2 and TIMP-2 may be related to control their invasiveness under hypoxic condition.
Blotting, Northern ; DNA ; Humans ; Oxygen ; Placenta ; Placentation ; RNA ; RNA, Messenger ; Thymidine ; Tissue Inhibitor of Metalloproteinase-2 ; Trophoblasts*