A Phage Display-Identified Peptide Selectively Binds to Kidney Injury Molecule-1 (KIM-1) and Detects KIM-1–Overexpressing Tumors In Vivo / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: This study was carried out to identify a peptide that selectively binds to kidney injury molecule-1 (KIM-1) by screening a phage-displayed peptide library and to use the peptide for the detection of KIM-1overexpressing tumors in vivo. MATERIALS AND METHODS: Biopanning of a phage-displayed peptide library was performed on KIM-1–coated plates. The binding of phage clones, peptides, and a peptide multimer to the KIM-1 protein and KIM-1–overexpressing and KIM-1–low expressing cells was examined by enzyme-linked immunosorbent assay, fluorometry, and flow cytometry. A biotin-peptide multimer was generated using NeutrAvidin. In vivo homing of the peptide to KIM-1–overexpressing and KIM1–low expressing tumors in mice was examined by whole-body fluorescence imaging. RESULTS: A phage clone displaying the CNWMINKEC peptide showed higher binding affinity to KIM-1 and KIM-1–overexpressing 769-P renal tumor cells compared to other phage clones selected after biopanning. The CNWMINKEC peptide and a NeutrAvidin/biotin-CNWMINKEC multimer selectively bound to KIM-1 over albumin and to KIM-1–overexpressing 769-P cells and A549 lung tumor cells compared to KIM-1–low expressing HEK293 normal cells. Co-localization and competition assays using an anti–KIM-1 antibody demonstrated that the binding of the CNWMINKEC peptide to 769-P cells was specifically mediated by KIM-1. The CNWMINKEC peptide was not cytotoxic to cells and was stable for up to 24 hours in the presence of serum. Whole-body fluorescence imaging demonstrated selective homing of the CNWM-INKEC peptide to KIM-1–overexpressing A498 renal tumor compared to KIM1–low expressing HepG2 liver tumor in mice. CONCLUSION: The CNWMINKEC peptide is a promising probe for in vivo imaging and detection of KIM-1‒overexpressing tumors.

T-CAM, a fastatin-FIII 9-10 fusion protein, potently enhances anti-angiogenic and anti-tumor activity via alphavbeta3 and alpha5beta1 integrins

We made fusion protein of fastatin and FIII 9-10, termed tetra-cell adhesion molecule (T-CAM) that can interact simultaneously with alphavbeta3 and alpha5beta1 integrins, both playing important roles in tumor angiogenesis. T-CAM can serve as a cell adhesion substrate mediating adhesion and migration of endothelial cells in alphavbeta3 and alpha5beta1 integrin-dependent manner. T-CAM showed pronounced anti-angiogenic activities such as inhibition of endothelial cell tube formation, endothelial cell proliferation, and induction of endothelial cell apoptosis. T-CAM also inhibited angiogenesis and tumor growth in mouse xenograft model. The anti-angiogenic and anti-tumoral activity of molecule like fastatin could be improved by fusing it with integrin-recognizing cell adhesion domain from other distinct proteins. The strategy of combining two distinct anti-angiogenic molecules or cell adhesion domains could facilitate designing improved anticancer agent of therapeutic value.

betaig-h3 triggers signaling pathways mediating adhesion and migration of vascular smooth muscle cells through alphavbeta5 integrin

Adhesion and migration of vascular smooth muscle cells (VSMCs) play an important role in the pathogenesis of atherosclerosis. These processes involve the interaction of VSMCs with extracellular matrix proteins. Here, we investigated integrin isoforms and signaling pathways mediating the adhesion and migration of VSMCs on betaig-h3, a transforming growth factor (TGF)-beta-inducible extracellular matrix protein that is elevated in atherosclerotic plaques. Adhesion assays showed that the alphavbeta5 integrin is a functional receptor for the adhesion of aortic VSMCs to betaig-h3. An YH18 motif containing amino acids between 563 and 580 of betaig-h3 was an essential motif for the adhesion and growth of VSMCs. Interaction between the YH18 motif and the alphavbeta5 integrin was responsible for the migration of VSMCs on betaig-h3. Inhibitors of phosphatidylinositide 3-kinase, extracellular signal-regulated kinase (ERK), and Src kinase reduced the adhesion and migration of VSMCs on betaig-h3. betaig-h3 triggered phosphorylation and activation of AKT, ERK, focal adhesion kinase, and paxillin mediating the adhesion and migration of VSMCs. Taken together, these results suggest that betaig-h3 and alphavbeta5 integrin play a role in the adhesion and migration of VSMCs during the pathogenesis of atherosclerosis.

Beta ig-h3 promotes renal proximal tubular epithelial cell adhesion, migration and proliferation through the interaction with alpha3beta1 integrin

Betaig-h3 (betaig-h3) is a secretory protein composed of fasciclin I-like repeats containing sequences that allows binding of integrins and glycosaminoglycans in vivo. Expression of betaig-h3 is responsive to TGF-beta and the protein is found to be associated with extracellular matrix (ECM) molecules, implicating betaig-h3 as an ECM adhesive protein of developmental processes. We previously observed predominant expression of betaig-h3 expression in the basement membrane of proximal tubules of kidney. In this study, the physiological relevance of such localized expression of betaig-h3 was examined in the renal proximal tubular epithelial cells (RPTEC). RPTEC constitutively expressed betaig-h3 and the expression was dramatically induced by exogenous TGF-beta1 treatment. betaig-h3 and its second and fourth FAS1 domain were able to mediate RPTEC adhesion, spreading and migration. Two known alpha3beta1 integrin-interaction motifs including aspartatic acid and isoleucine residues, NKDIL and EPDIM in betaig-h3 were responsible to mediate RPTEC adhesion, spreading, and migration. By using specific antibodies against integrins, we confirmed that alpha3beta1 integrin mediates the adhesion and migration of RPTECs on betaig-h3. In addition, it also enhanced proliferation of RPTECs through NKDIL and EPDIM. These results indicate that betaig-h3 mediates adhesion, spreading, migration and proliferation of RPTECs through the interaction with alpha3beta1 integrin and is intimately involved in the maintenance and the regeneration of renal proximal tubular epithelium.

The GSTT1 Genotype as A Marker for Susceptibility to Lung Cancer in Korean Female Never-Smokers / 결핵

BACKGROUND: Most previous studies regarding the role of GSTMl and GSTT1 on lung cancer risk have been focused mainly on male smokers. However, epidemiological characteristics, histologic types and risk factors are different in female and male lung cancers, we investigated the association between these genotypes and lung cancer risk in males and females separately. MATERIALS AND METHODS: The study population consisted of 253 lung cancer (153 males and 100 females) and 243 controls (140 males and 103 females). GSTM1 and GSTT1 genotypes were determined by a multiplex PCR. RESULTS: In the male population, neither GSTM1 nor GSTT1 null genotype showed significant difference between cases and controls. In the female population, the frequencies of GSTM1 null genotype showed no significant difference between cases and controls. However, the frequencies of GSTT1 null genotype was significantly higher in cases (70.3%) than controls (55.3%, odds ratio (OR)=2.18; 95% confidence interval (CI=l.21-3.93). When the female population was stratified by age and smoking status, the ORs for GSTT1 null genotype were significantly higher in subgroups of ≤60 years (OR=4.82; 95% CI=l.61-14.4) and never-smokers (OR=4.29; 95% CI=1.94-9.48) but not in subgroups of >60 years or smokers. When stratifying the female never-smokers by age, the ORs for GSTT1 null genotype were significantly higher in both age groups of ≤60 years (OR=7.64; 95% CI=2.00-29.2) and >60 years (OR=2.89; 95% CI=1.05-7.94). CONCLUSION: We found that GSTT1 null genotype was associated with an increased risk of lung cancer in Korean female never-smokers. This result suggests that GSTT1 null genotype could be used as a biomarker for genetic susceptibility to lung cancer in Korean female never-smokers.

Transcriptional repression of type I procollagen genes during adipocyte differentiation

Adipocyte differentiation is a very complex process in which whole-cell changes are accompanied. Among them, type I procollagen gene has been shown to specifically decrease during adipocyte differentiation; however, little is known about the molecular mechanism. To examine how type I procollagen gene expression is regulated at the level of transcription during adipocyte differentiation, 3T3-L1 preadipocyte cell line was used as an in vitro model. Northern blot analysis demonstrated that mRNA expression of type I procollagen gene was dramatically reduced during adipocyte differentiation. Time-course analysis indicated that decrease in mRNA expression occurred at early stage of differentiation. Studies on several stable cell lines showed that transcriptional activities of both alpha1 and alpha2 promoters decreased significantly during adipocyte differentiation. Despite extensive deletion-promoter analyses, however, we could not identify the cis-element responsible for the switch-off of type I procollagen gene during adipocyte differentiation, suggesting that the transcriptional repression of this gene occur through general transcription machinery rather than a specific cis-element. In conclusion, down-regulation of type I procollagen mRNA expression during adipocyte differentiation is due to repression of its promoter activity through general transcription machinery.

Transcriptional repression of type I procollagen genes during adipocyte differentiation

Adipocyte differentiation is a very complex process in which whole-cell changes are accompanied. Among them, type I procollagen gene has been shown to specifically decrease during adipocyte differentiation; however, little is known about the molecular mechanism. To examine how type I procollagen gene expression is regulated at the level of transcription during adipocyte differentiation, 3T3-L1 preadipocyte cell line was used as an in vitro model. Northern blot analysis demonstrated that mRNA expression of type I procollagen gene was dramatically reduced during adipocyte differentiation. Time-course analysis indicated that decrease in mRNA expression occurred at early stage of differentiation. Studies on several stable cell lines showed that transcriptional activities of both alpha1 and alpha2 promoters decreased significantly during adipocyte differentiation. Despite extensive deletion-promoter analyses, however, we could not identify the cis-element responsible for the switch-off of type I procollagen gene during adipocyte differentiation, suggesting that the transcriptional repression of this gene occur through general transcription machinery rather than a specific cis-element. In conclusion, down-regulation of type I procollagen mRNA expression during adipocyte differentiation is due to repression of its promoter activity through general transcription machinery.

Induction of fibronectin gene expression by inhibitors of protein phosphatase type 2B in normal and transformed fibroblasts

Two intracellular signal pathways mediated by cAMP and protein kinase C (PKC) were involved in the regulation of FN gene expression (Lee et al., Exp. Mol. Med. 30: 240, 1998). In this study, a possible involvement of protein phosphatase-dependent pathways in the regulation of FN gene expression was investigated by using protein phosphatase type 2B (PP2B) inhibitors, cyclosporin A and ascomycin. Both cyclosporin A and ascomycin increased the levels of FN mRNA in WI-38 human lung fibroblasts and the SV40-transformed WI-38 cells but not in MC3T3-E1 osteoblasts. The expression of FN appears to increase from six hours up to 48 hours after treatment suggesting that it is not an immediate effect. In addition, this effect required a new protein synthesis. Neither cyclosporin A nor ascomycin affects the phorbol myristate acetate (PMA)-induced stimulation of FN gene expression and the same result occurred in vice versa suggesting the mechanism of PMA and cyclosporin A/ascomycin in the regulation of FN gene expression may share a common downstream pathway. Taken together, this study suggests that PP2B is involved in the regulation of FN gene expression in normal and transformed fibroblasts but not in osteoblasts.

Regulation of fibronectin gene expression by cyclic AMP and phorbol myristate acetate in HT-1080 human fibrosarcoma cells

We studied the regulation of fibronectin (FN) gene expression by cAMP and phorbol-12-myristate-13-acetate (PMA) in HT-1080 human fibrosarcoma cells. Dibutyryl cAMP increased FN synthesis and mRNA levels, while PMA inhibited the cAMP-induced FN synthesis. In transient transfection assays, cAMP increased FN promoter activity, while PMA paradoxically enhanced the cAMP-induced promoter activity. Stable transfection experiments, however, showed that neither cAMP or PMA alone nor together affected FN promoter activity. These results suggest that PMA antagonizes the cAMP-induced FN gene expression and that both the action of cAMP and the inhibition of its action by PMA may occur at the posttranscriptional level in HT-1080 cells.

Plasma type IV collagen and fibronectin concentrations in diabetic patients with microangiopathy

In diabetes mellitus, thickening of basement membrane in capillaries and small vessels is a well-known finding and important in the progression of diabetic microangiopathy. We evaluated whether the plasma levels of type IV collagen and fibronectin, which are important factors of basement membrane, are related with the presence of diabetic microangiopathy. Plasma type IV collagen and fibronectin levels were measured in 40 healthy controls (Mean +/- SD, age; 50.3 +/- 5.5 yr) and 94 diabetic patients (age; 52.4 +/- 13.5 yr) with and without microvascular complications. The mean plasma levels of type IV collagen (5.3 +/- 2.9 ng/ml) and fibronectin (474.4 +/- 119.4 ug/ml) in diabetic patients were significantly higher (p < 0.01) than in healthy controls (3.7 +/- 1.3 ng/ml and 319 +/- 50.9 ug/ml). The mean plasma level of type IV collagen in diabetic patients with complications (6.6 +/- 3.7 ng/ml) was significantly higher (p < 0.01) than in those without complications (4.3 +/- 1.7 ng/ml) and became higher in more complicated patients. Furthermore, the severity of retinopathy and several indicators of nephropathy such as serum BUN, creatinine and proteinuria were closely associated with plasma type IV collagen level and a significant correlation was found between plasma type IV collagen and creatinine clearance (r = -0.31, p < 0.001). There was no significant difference in plasma fibronectin concentrations, however, between the diabetic patients with complications and those without complications.(ABSTRACT TRUNCATED AT 250 WORDS)

Topical Autologous Fibronectin in the Treatment of Persistent Corneal Epithelial defects

Fibronectin, a glycoprotein, is present in plasma and extracellular matrix and is responsible for cellular adhesion. Fibronectin was purified from autologous plasma of two persistent corneal epithelial defect patients by affinity chromatogaphy and administered topically 500 micro ml, 2 dropsper time, five times a day for 3 weeks. Reepithelization began three to seven days after initiation of treatment with autologous purified fibronectin eyedrops, and epithelial defects healed nearly completely and subjective symptoms were relieved. No side effects were observed. Over a follow-up period of 4 to 23 wks, no recurrences were noted.