Preparation of monoclonal antibody against human KIAA0100 protein and Northern blot analysis of human KIAA0100 gene / 药物分析学报

Monoclonal antibodies (MAbs) are important tools for the study of proteins′ function and structure. But there has been no report on the preparation of MAbs against human KIAA0100 protein up to date. Here, first, we generated the mouse MAb against human KIAA0100 protein using purified recombinant 6×Histidinc (6×His)- tagged human KIAA0100 protein segment (1557–2234) as an antigen; then, the mRNA expression of human KIAA0100 gene was detected in U937 cells using Northern blot analysis. The results showed that the mouse MAb against human KIAA0100 protein could sensitively recognize the human KIAA0100 protein using Western blot analysis and immunocytochemistry analysis. Besides, Western blot analysis revealed that human KIAA0100 gene possibly encoded two different protein products (254 kDa and < 250 kDa) in U937 cells. Moreover, Northern blot analysis confirmed that human KIAA0100 gene might produced two different mRNA products (6000–10000 bp and 5000–6000 bp) in U937 cells. The results provide a basis for large-scale production of the MAb against human KIAA0100 protein, which will be useful for the study of human KIAA0100 protein′s function/structure and MAb-targeted drugs in the future.

Estandarización de un protocolo para Northern blot no radioactivo usado en la detección de pequeños RNA en células Vero / Standardization of Northern blot non-radioactive protocol used in the detection of small RNAs in Vero line cells

El interés en la detección, identificación, y caracterización funcional de los pequeños RNAs no codificantes (sRNAs), ha generado la necesidad de optimizar las metodologías comúnmente usadas en su detección, la reacción en cadena de la polimerasa cuantitativa (RT-qPCR) y Northern blot, con el fin de que sean más sensibles y específicas. A pesar de la baja sensibilidad del Northern blot, esta metodología continúa siendo de uso común en la detección de sRNAs porque permite detectar el RNA pequeño así como a sus precursores, razón por la cual se usa como una metodología complementaria en este tipo de investigaciones. En este trabajo se describe la implementación de un nuevo protocolo para Northern blot no radioactivo, con modificaciones dirigidas a mejorar su sensibilidad y especificidad. El diseños de la sonda con la tecnología LNA, el marcaje de esta con Digoxigenina y por último la fijación del RNA a la membrana mediante 1-Ethyl-3-(-3-dimethylaminopropyl) carboniimide (EDC) y finalmente se discuten los fundamentos teóricos de estos cambios.

The interest in detection, identification and functional characterization of small non-coding RNAs (snRNAs), has generated the need to optimize the methodologies commonly used in its detection in specificity and sensitivity, The Quantitative reverse transcription PCR (RT-qPCR) and Northern blot. Even though the low sensitivity of Northern blot, this method continues to be commonly used in the sRNAs, because its capacity to detect the sRNA and its precursor, which is the reason why Northern blot is used as complementary method in this sort of Research. This work describes the implementation of an innovative non-radioactive Northern blot protocol, with modifications that improving the sensibility and specificity, with the discussion of the theoretical foundations of such modifications.

Suppression of RNA Interference Pathway in vitro by Grass Carp Reovirus / 中国病毒学·英文版

The means of survival of genomic dsRNA of reoviruses from dsRNA-triggered and Dicer-initiated RNAi pathway remains to be defined.The present study aimed to investigate the effect of Grass carp reovirus (GCRV) replication on the RNAi pathway of grass carp kidney cells (CIK).The dsRNA-triggered RNAi pathway was demonstrated unimpaired in CIK cells through RNAi assay.GCRV-specific siRNA was generated in CIK cells transfected with purified GCRV genomic dsRNA in Northern blot analysis; while in GCRV-infected CIK cells,no GCRV-specific siRNA could be detected.Infection and transfection experiments further indicated that replication of GCRV correlated with the increased transcription level of the Dicer gene and functional inhibition of in vitro synthesized egfp-siRNA in silencing the EGFP reporter gene.These data demonstrated that although only the genomic dsRNA of GCRV was sensitive to the cellular RNAi pathway,unidentified RNAi suppressor protein(s) might contribute to the survival of the viral genome and efficient viral replication.

The Effect of HSVⅠ Infection on the Expression of hnRNP H2 in Human Fetal Liver Cell / 中国生物工程杂志

Herpes simplex virusⅠ(HSVⅠ) regulating the pathway of transcription and translation modify in host cell is a very systematic and complicate system. A clear understanding of the concrete mechanisms of infection will greatly help to comprehend the virus replication and the interaction with the host cell. By the analysis of 2-DE, the heterogeneous nuclear ribonucleoprotein H2 in human fetal liver cell represent distinction after the HSVⅠinfection.Utilization of Northern blot and Western blot technologies verified the expression of hnRNP H2 in different stage of virus infection is varied.

OsCOI1, a Putative COI1 in Rice, Show MeJA and ABA Dependent Expression / 生物化学与生物物理进展

A novel gene, which was a homologue of Arabidopsis COI1 was isolated from rice (Oryza sativa L.) by RT-PCR and designated as OsCOI1. It encoded a protein of 595 amino acids. The similar F-box motif and 16 leucine-rich repeats were found in the deduced protein OsCOI1. OsCOI1 and COI1 showed high homology (74%) at amino acid level. Semi-quantitative RT-PCR and Northern blot analysis demonstrated that the expression of OsCOI1 in rice varied obviously after treatment with MeJA and ABA but was not affected by SA and ET, suggesting that the specific function of OsCOI1 in JA signal pathway and related ABA pathway.

Changes of beta-Adrenergic Receptor mRNA in the Visual Cortex and Superior Colliculus of Monocular Deprivated Rat

PURPOSE: To investigate the change of the Beta adrenergic system in the rat visual cortex and superior colliculus after visual deprivation during a critical period of postnatal development. METHODS: The changes of beta 1 and beta 2 adrenergic receptor mRNA were investigated by using northern blot analysis in the rat visual cortex and superior colliculus. The right eyelid of visually deprived rat was sutured at the 10th postnatal days. After visual deprivation for 4 weeks, the rat were sacrificed and the visual cortex and superior colliculus tissues were removed for analysis. RESULTS: Beta 1 and beta 2 adrenergic receptor mRNA expression was decreased in the contralateral visual cortex to the deprived eye. In the superior colliculus, beta 2 adrenergic receptor mRNA expression increased in both sides, but a much greater increase was shown in the ipsilateral superior colliculus than the contralateral side. CONCLUSIONS: These results suggests that visual deprivation during a critical period of postnatal development influences the beta adrenergic system in the rat visual cortex and superior colliculus.

Hypoxia Induced Expression of Vascular Endothelial Growth Factor in Rat Pulmonary Artery Smooth Muscle Cells

PURPOSE: Pulmonary vascular hypertension is a common problem in congenital heart disease, the most common cardiac condition in childhood. However, the mechanisms responsible for this pathologic change, treatment, and prevention are poorly understood. Therefore, we studied the gene expression of vascular endothelial growth factor(VEGF) by using a hypoxic model of the pulmonary artery smooth muscle cells. METHODS: The main pulmonary artery and its proximal branches of a 6 wk old Fischer rat were excised. They were cut into multiple small pieces and suspended in DMEM medium supplemented with 20% fetal bovine serum and incubated in 5% CO2-95% air atmosphere. The smooth muscle cells were confirmed by immunostaining with smooth muscle myosin and alpha-smooth muscle actin antibodies. The VEGF gene expression in the hypoxic group was compared with the one in control the group as well as the one in the starved group by RT-PCR and Northern blot hybridization. RESULTS: There was no statistically significant difference among the control, hypoxic and starved groups. CONCLUSION: There are few studies of pulmonary vascular hypertension at the molecular level in Korea. Therefore, we studied the expression of VEGF gene in hypoxic pulmonary vascular smooth muscle cells. Further studies will be needed to find the difference between newly born and adult rats, or human and rat pulmonary vascular smooth muscle cells in gene expression. We hope that the study will lead to a better understanding of pulmonary vascular hypertension.

Expression of hypoxia-inducible factor-1? in the lungs of rats with hypoxic pulmonary hypertension / 中国呼吸与危重监护杂志

Objective To study expression of hypoxia inducible factor 1? (HIF 1?) in the lungs of hypoxic rats and to explore the role of HIF 1? in the pathogenesis of hypoxic pulmonary hypertension Methods Twenty Wistar rats were randomly divided into control group and hypoxia group The models of hypoxic pulmonary hypertension were established by exposure to hypoxia for 4 weeks according to our laboratory protocol Digoxin labelled cRNA probe for HIF 1? was prepared by in vitro transcription Northern blot was performed by using the HIF 1? cRNA probe and In situ hybridization was also conducted with rat lung tissue sections Results Northern blot hybridization showed minimally positive in the lung tissue of control group ,but strongly positive in hypoxia group In situ hybridization analysis with hypoxic rat lung tissue revealed that HIF 1? mRNA was expressed in bronchial epithelial cells (strongly positive reaction) and peribronchial proliferative lymphomatic tissue (weakly positive reaction),casually in alveolar wall cells (weakly positive reaction),but not in pulmonary arterial endothelium and smooth muscle cells Conclusions Our data suggested that chronic hypoxia could induce pulmonary hypertension characterized by pulmonary vascular remodeling HIF 1? mRNA expression was elevated in the lungs of rat under hypoxic condition HIF 1 may be involved in the pathophysiological changes in response to hypoxia and play an important role in the development of hypoxic pulmonary hypertension

Molecular Biological Responses of TCDD Transformed Human Keratinocytes Induced by UVB Irradiation / 대한해부학회지

The expression of nm23-H1, nm23-H2, p53, c-myc, cyclin D1, and k-ras genes were investigated in TCDD transformed human keratinocyte RHEK1 cells by exposure to UVB 200 J/m2. For 3 days after irradiation the transcriptional kinetics of these genes were evaluated. The expression of nm23-H1 and H2 were highly increased at 6 hours post-irradiation and recovered in about 3 days to level of pre-irradiation. In cyclin D1 there was a temporary increased expression at 3 hours post-irradiation, but after that time its expression increased minimally. The kinetic of p53 expression was not constant, but showed a decreased tendency. K-ras expression level was decreased by degrees. The decrease in c-myc expression might be related to wavelength-specific induction. In this experiment, S-phase prolongation was a typical alternation in early phase after UVB irradiation. The decreased p53 and k-ras expression and the increased expression of cyclin D1 pushed G1-epsilonS phase transition, and during prolonged S-phase the increased expression of nm23-H1 and H2 might be involved in cell cycle progression, suggesting a function concomitant with prolongation of S-phase after UVB irradiation. TCDD transformed human keratinocyte RHEK1 cell line had 2 heterogeneous cell clones, near diploid and hypotetraploid, and were more resistant against UVB irradiation than RHEK1 cell line. And it had S-phase prolongation instead of G1 arrest in response to UVB irradiation, which may have negative effect to be accumulated DNA mutation without DNA repair.

Expression of Osteopontin in the Adult and Developing Rat Kidney / 대한해부학회지

Osteopontin (OPN), originally considered to be a bone protein, is now reported to be expressed in other tissues, notably in kidney. OPN has been demonstrated in the kidney by Northern and Western analyses, immunohistochemistry and in situ hybridization. However, studies of the cellular distribution of OPN in the kidney, especially in normal condition, have given highly conflicting results. This study is designed to establish the expression of OPN in kidney from the fetus to adult. Kidneys from 16-(F16), 18-(F18), and 20 day-old (F20) fetuses and 1-(P1), 3-(P3), 7-(P7), 14-(P14), 21 day-old pups, and adult were studied by in situ hybridization and immunohistochemistry. OPN mRNA and protein were expressed in the thick ascending limb (TAL) at F18 and F20, but disappeared gradually after birth. In the collection duct (CD), weak labeling appeared in a few cells at F20. After birth cells with strong labeling were scattered throughout the CD in the medulla and inner cortex at P1-P7 and in the outer cortex at P14. There was little OPN expression in the CD at P21. OPN mRNA and immunoreactivity appeared in the papillary surface epithelium (PSE) at F20 and in the descending thin limb (DTL) at P1. After birth OPN expression gradually increased to adult levels in the PSE. In the DTL, adult levels of expression were reached at P21. Proximal tubules exhibited a punctate subapical OPN immunos-taining from F18, but no hybridization signal. During renal development, the transient expression of OPN in the TAL and CD suggests that OPN may have a role in the developing kidney, and from P21 OPN expression was localized at DTL and PSE exclusively, which was similar to adult OPN expression pattern.

Tissue-specific regulation of angiotensinogen and angiotensin II receptor gene expression in deoxycorticosterone acetate-salt hypertensive rats

Molecular regulation of the renin-angiotensin system (RAS) was investigated in deoxycorticosterone acetate (DOCA)-salt hypertension. The expression of renin, angiotensinogen and angiotensin II receptor genes in the kidney and liver was determined by Northern blot analysis in rats which were made DOCA-salt hypertensive over the period of 2 or 4 weeks. Along with the hypertension, renin mRNA was decreased in the remnant kidney. The expression of angiotensinogen gene was not significantly altered in the kidney, but was significantly decreased in the liver. The expression of angiotensin II receptor gene was increased in the kidney, while it remained unaltered in the liver. The duration of hypertension did not affect the altered gene expression. It is suggested that the components of RAS are transcriptionally regulated in DOCA-salt hypertension in a tissue-specific manner.

Tissue-Specific Regulation of Angiotensinogen and Angiotensin II Receptor Genes in Two-Kidney, One Clip Hypertensive Rats / 대한신장학회잡지

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.

Apoptosis and Expression of bcl-2, bax in the Cyclic Human Endometrium / 대한산부인과학회잡지

OBJECTIVE: To understand the regulatory mechanism of apoptosis by pro- and anti-apoptotic genes in the cyclic human endometrium. METHODS: Each case of endometrial status was classified by Noyes criteria, and grouped into early proliferative(n=13), late proliferative(n=14), early secretory(n=15), and late secretory phases(n=15). Expression of bcl-2, and bax were assessed by Northern blot and immunohistochemistry in relation to apoptotic index by TUNEL. Results: Apoptotic index showed increasing tendency as progressing to the late secretory phase, which phase showed significantly higher apoptotic index compared to the other phases(p<0.05). The intensity of bcl-2 8.5kb transcript by Northern blot was highest in the late proliferative phase significantly(p<0.05), decreasing to nadir in the late secretory phase. In contrast to bcl-2 expression, bax mRNA expression was highest in the late secretory phase significantly(p<0.05). Both the relative ratio of bcl-2 8.5kb transcript and bcl-2 5.5kb trascript to bax showed that the ratio was higher in the early, and late proliferative phase, but, reversed in the late secretory phase. Both the immunoreactivity of bcl-2 and bax proteins could be detected in the basal, functional, and stromal layers of endometrium. The immunoreactivity of bcl-2 protein was more prominent in the proliferative phase, however, bax protein was more prominent in the secretory phase. CONCLUSION: This study suggests that apoptosis could be regulated by the relative dominance of bcl-2 or bax expression in the human endometrium. Thus, bcl-2 and bax expressions might be one of the possible mechanism in the regulation of normal menstruation.

Effects of angiotensin converting enzyme inhibition on gene expression of the renin-angiotensin system in rats

To investigate interaction of angiotensin converting enzyme (ACE) inhibitor with local tissue renin-angiotensin system (RAS), changes in gene expression of the RAS components in various tissues in response to chronic administration of an ACE inhibitor, enalapril, were examined in Sprague-Dawley male rats. Enalapril was administered in their drinking water (3 ~ 4 mg/day) over 8 wk. Plasma and renal ACE activity increased significantly after 4 and 8 wk of enalapril treatment. Renin levels of the plasma and kidney of the enalapril-treated rats markedly increased after 4 wk and decreased thereafter, but still remained significantly higher than those of control rats. Kidney mRNA levels of renin markedly increased after 4 and 8 wk of enalapril treatment, but those of angiotensinogen and ANG II-receptor subtypes, AT1A and AT1B, did not change significantly. The liver expressed genes for renin, angiotensinogen and AT1A receptor subtype, but AT1B receptor subtype mRNA was not detectable by RT-PCR. None of mRNA for these RAS components in the liver changed significantly by enalapril treatment. The hypothalamus showed mRNA expressions of renin, angiotensinogen, AT1A and AT1B receptor subtypes. AT1A receptor subtype mRNA was more abundant than AT1B receptor subtype in the hypothalamus as shown in the kidney. However, gene expression of the RAS components remained unchanged during 8-wk treatment of enalapril. In the present study, chronic ACE inhibition increased plasma and renal levels of ACE and renin, but did not affect mRNA levels of other RAS components such as angiotensinogen, ANG II receptor subtypes in the kidney. Gene levels of the RAS components in the liver and hypothalamus were not altered by chronic treatment of enalapril. These results suggest the differential expression of the RAS components in response to enalapril, and localized action and some degree of tissue specificity of enalapril.

Change of TypeI and TypeII Collagen Biosynthesis by Growth Factors in Cultured Cells Isolated from Rabbit Intervertebral Disc / 대한정형외과학회잡지

Growth factors influencing the function of chondrocytes are insulin-like growth factor I(IGF-I), basic fibroblast growth factor(bFGF), transforming growth factor-beta1(TGF-beta1), and epidermal growth factor(EGF). To find out the role of four kinds of growth factors in the biosynthesis of type I and II collagen represented as the phenotype of the disc cells, we cultured the disc cells isolated from rabbit intervertebral discs primarily and then checked cell proliferation, the expression of type I and II procollagen mRNA, and the immunohistochemical stains with type I and II collagen antibodies during in vitro culture in the maintenance medium containing low serum concentration with adding four kinds of growth factors. The results are as follows. FBS(10% Fetal bovine serum) group showed the highest cell proliferation potential. EGF and TGF groups showed remarkable cell proliferation, but there was no significant difference in IGF and FGF groups comparing to control group. A partial clone that encodes the rabbit type II procollagen C-propeptide region(RbCo12A1) was successfully isolated by reverse transcription-polymerase chain reaction using total RNA extracted from articular chondrocytes of rabbits. The identity of the cDNA clone was confirmed by DNA sequencing of the polymerase chain reaction products. A comparison of human al(II) cDNA sequence showed high sequence homology(83.6%). Type I procollagen mRNA expressed highly in EGF group. FGF, IGF, and TGF groups showed no significant expression comparing to control group. FBS group showed lower expression than control group. Type II procollagen expression was increased with passage of time, so at Day 10 it was the highest in all groups. Control group showed the highest expression among 6 experimental groups. The expression of type II procollagen in FGF and TGF groups was slightly lower than that of control. EGF and IGF groups showed markedly decreased expression comparing to control group. That in FBS group was the lowest, so it was three times lower than control group. In immunohistochemical stains with type I collagen, there was no difference among control, FBS, and EGF groups. FGF, IGF, and TGF groups showed increased positivity on stain comparing to control group, but the positivity didnt exceed 10%. For type II collagen, EGF and FGF groups showed decreased positivity, but there was no significant difference in FBS, IGF, and TGF groups comparing to control group. On the basis of this study, it may be concluded that TGF-pl showed the possibility of regeneration or delay the degeneration process of the intervertebral disc through the contribution to the stimulatory effects of cell proliferation and the synthesis of type II collagen. For the clinical use of this, more studies about the combination effects with FBS or other kinds of growth factors and finding out the ideal concentration about TGF-pl will be needed.

The Establishment of IL - 2 Dependent Cell Line from IL - 3 Dependent Mouse Hemopoietic Progenitor Cell Line and Its Analysis / 대한면역학회지

No abstract available.

Effect of hemorrhage on mRNA expressions of renin, angiotensinogen and AT-1 receptors in rat central and peripheral tissues

In an attempt to investigate whether hemorrhage affects the gene expression of the renin-angiotensin system (RAS) components in the brain and peripheral angiotensin-generating tissues, changes in mRNA levels of the RAS components in response to hemorrhage were measured in conscious unrestrained rats. Wistar rats were bled at a rate of 3 ml/kg/min for 5 min, and then decapitated 7 h after hemorrhage. Levels of mRNA for renin, angiotensinogen and angiotensin II-AT-1 receptor subtypes (AT-1A and AT-1B) were determined with the methods of northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR). Hemorrhage produced a profound hypotension with tachycardia, but blood pressure and heart rate recovered close to the basal level at 7 h. Plasma and renal renin levels were significantly increased at 7 h. Hemorrhage induced rapid upregulation of gene expression of both AT-1A and AT-1B receptor subtypes in the brainstem and hypothalamus, downregulation of them in the adrenal gland and liver. However, renin mRNA level increased in the brainstem, decreased in the liver, but was not changed in the hypothalamus, kidney and adrenals after hemorrhage. Angiotensinogen mRNA level was not significantly changed in any of the tissue except a slight increase in the liver. The kidney and liver did not show any significant change in gene expression of the RAS components. These results suggest that gene expression of the RAS in central and peripheral tissues are, at least in part, under independent control and the local RAS in each organ plays specific physiologic role.

Gene expression of intrarenal renin-angiotensin system in streptozotocin-induced diabetic rats

In humans and many animal models with chronic progressive renal diseases, angiotensin-converting enzyme (ACE) inhibitor markedly attenuates the progression of nephropathy. Several studies have reported augmented gene expression and redistribution of renal renin in partial nephrectomized rats. Although precise mechanism(s) is not known, the renin-angiotensin system (RAS) may play an important role in the progression of renal diseases. Thus, this study was undertaken to examine the gene expression of renal renin, angiotensinogen, and AT-1 subtypes (AT-1A and AT-1B) in rats with diabetic nephropathy, and the influences of lipopolysaccharide (LPS)-induced septicemia on the gene expression. Four weeks after streptozotocin (STZ) treatment (55 mg/kg, i.p.), rats were randomly divided into LPS-treated (1.6 mg/kg, i.p.) and control rats. At 6 hours after LPS treatment, the rats were killed and the kidney was removed from each rat. Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) techniques were used to detect mRNA expression. STZ treatment markedly attenuated body weight gain and significantly increased blood glucose level. Renal renin content (RRC) was significantly decreased in the STZ-treated rats compared to that in control rats. The renal ACE activity between STZ-treated and control rats was not significantly different. Renal renin mRNA level was prominently increased, while angiotensinogen and AT-1A mRNA levels were slightly decreased in STZ-treated rats compared to those in controls. AT-1B mRNA level did not differ in both groups. Acute LPS treatment did not show any significant changes of mRNA levels of intrarenal RAS components in both groups. These results suggest that intrarenal RAS components were differentially regulated in STZ-treated diabetic rats. Further studies are required to evaluate the relationship between intrarenal RAS and other vasomodulatory systems.

Overexpression of tissue inhibitors of metalloproteinase-1 and -2 in the stroma of gastric cancer

The fundamental event of cancer invasion and metastasis is the complicated interaction of cancer cells with host cells, in which event, a number of proteases and their inhibitors are involved. Matrix metalloproteinases are the potent proteases in degrading the basement membrane and extra cellular matrix and are inhibited by specific endogeneous inhibitors, tissue inhibitors of metalloproteinases-1(TIMP-1) and TIMP-2. The expression of mRNA for TIMP-1 and -2 was investigated by Northern blot analysis in specimens taken from 27 patients with primary gastric adenocarcinoma; 25 samples from the primary site, six from the metastatic lymph nodes and two from the peritoneal fluids. The expression for TIMP-1 and -2 was compared in primary gastric cancer tissues, metastatic lymph nodes and normal gastric mucosae. TIMP-1 mRNA was overexpressed in 24 (96%) out of 25 primary cancer tissues compared with the paired normal mucosae, while TIMP-2 was in 10 (40%). In six specimens of metastatic lymph nodes, TIMP-1 and -2 were overexpressed in 6 (100%) and 4 (67%) specimens, respectively. Of two specimens prepared from the peritoneal fluids, all specimens overexpressed TIMP-1 compared with the those of primary cancer tissues, while one (50%) specimen overexpressed TIMP-2. Immunohistochemical staining was done to investigate the localization of TIMP-1 and -2, demonstrating that the immunoreactivity for TIMP-1 and -2 was clearly detected in the cytoplasm of the stromal cells. These results suggest that both TIMP-1 and -2 are overexpressed by stromal cells in most of primary and some metastatic gastric cancer tissues and that TIMP-1 and TIMP-2, produced by stromal cells, may play an important role in inhibiting the proteolytic activity of matrix metalloproteinases originated from cancer cells, in gastric cancer.

Overexpression of tissue inhibitors of metalloproteinase-1 and -2 in the stroma of gastric cancer

The fundamental event of cancer invasion and metastasis is the complicated interaction of cancer cells with host cells, in which event, a number of proteases and their inhibitors are involved. Matrix metalloproteinases are the potent proteases in degrading the basement membrane and extra cellular matrix and are inhibited by specific endogeneous inhibitors, tissue inhibitors of metalloproteinases-1(TIMP-1) and TIMP-2. The expression of mRNA for TIMP-1 and -2 was investigated by Northern blot analysis in specimens taken from 27 patients with primary gastric adenocarcinoma; 25 samples from the primary site, six from the metastatic lymph nodes and two from the peritoneal fluids. The expression for TIMP-1 and -2 was compared in primary gastric cancer tissues, metastatic lymph nodes and normal gastric mucosae. TIMP-1 mRNA was overexpressed in 24 (96%) out of 25 primary cancer tissues compared with the paired normal mucosae, while TIMP-2 was in 10 (40%). In six specimens of metastatic lymph nodes, TIMP-1 and -2 were overexpressed in 6 (100%) and 4 (67%) specimens, respectively. Of two specimens prepared from the peritoneal fluids, all specimens overexpressed TIMP-1 compared with the those of primary cancer tissues, while one (50%) specimen overexpressed TIMP-2. Immunohistochemical staining was done to investigate the localization of TIMP-1 and -2, demonstrating that the immunoreactivity for TIMP-1 and -2 was clearly detected in the cytoplasm of the stromal cells. These results suggest that both TIMP-1 and -2 are overexpressed by stromal cells in most of primary and some metastatic gastric cancer tissues and that TIMP-1 and TIMP-2, produced by stromal cells, may play an important role in inhibiting the proteolytic activity of matrix metalloproteinases originated from cancer cells, in gastric cancer.