Biomedical subjects

Masahiro Seike

Publications and source records attributed to Masahiro Seike.

29 records · Page 2Linked to original sources

PubMed2004

Mutational analysis of the macrophage scavenger receptor 1 (MSR1) gene in primary lung cancer.

Allelic deletion at chromosome 8p21-25 is an early and frequent event in the carcinogenesis and development of various cancers. To facilitate investigation of alterations of the macrophage scavenger receptor 1 (MSR1), which is located on 8p22, and to determine the role of this gene in human carcinogenesis and tumor progression, we determined intronic primers designed to amplify the coding region. Since frequent deletion of 8p21-23 has been previously reported in lung cancer, we searched for mutations throughout the coding sequence of the MSR1 gene within a panel of genomic DNA samples obtained from 30 primary lung cancers. Our approach, which involved polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis and direct DNA sequencing, revealed nucleotide variants of the MSR1 gene in only one of the 30 cases examined, with this sample displaying both a 6 bp deletion and a thymine-to-cytosine substitution, the latter occurring within intron 7. The 6 bp deletion was located at a DNA microsatellite region and the thymine-to-cytosine substitution seemed to be a polymorphism. These results suggest that the MSR1 gene is not commonly mutated in lung cancer and not important in susceptibility to lung cancer. Further studies may focus on alternative mechanisms through which the MSR1 gene might be inactivated, such as aberrant DNA methylation, and/or pursue analyses of other genes on 8p21-23 for mutational events. Nevertheless, the panel of intronic PCR primer pair sequences presented here will facilitate future studies to determine the full spectrum and frequency of genetic events that may affect expression/activity of the MSR1 gene in human tumors.

Chromosomes, Human, Pair 8↗

PubMed2003

Proteomic analysis of intestinal epithelial cells expressing stabilized beta-catenin.

Aberrant accumulation of beta-catenin protein because of mutation of either the beta-catenin or adenomatous polyposis coli gene plays an essential role in the development of colorectal carcinoma. We established previously a stable clone of the rat small intestinal epithelial cell line IEC6, which is capable of inducing stabilized beta-catenin protein lacking NH(2)-terminal glycogen synthase kinase-3beta phosphorylation site under a strict control of the tetracycline-regulatory system. This clone, IEC6-TetOFF-beta-catenin DeltaN89, shows in vitro polypoid growth on the removal of doxycycline and seems to be an appropriate model for analyzing the molecular mechanisms of early intestinal carcinogenesis. Of >2000 protein spots displayed by newly developed two-dimensional difference gel electrophoresis, 22 were found to be up- or down-regulated on the induction of stabilized beta-catenin. The majority of these proteins fell into two categories: (a) redox-status regulatory proteins and (b) cytoskeleton-associated proteins. Representatively, a key redox-status regulatory protein, manganese superoxide dismutase, up-regulated in IEC6 cells expressing stabilized beta-catenin protein, was overexpressed in adenoma and adenocarcinoma cells of familial adenomatous polyposis patients in parallel with the accumulation of beta-catenin. These results suggest that aberrant accumulation of beta-catenin might contribute to colorectal carcinogenesis by affecting redox status in the mitochondria of intestinal epithelial cells.

Adenocarcinoma↗

PubMed2003

Increased expression of the LGALS3 (galectin 3) gene in human non-small-cell lung cancer.

Patients with lung cancer have a poor prognosis because of the high metastatic potential of the neoplasm. Therefore, identifying new molecular targets for anti-metastatic therapy is very important. To identify novel key factors of tumor metastasis in lung cancer, we established the gene expression profiles of two adenocarcinoma cell line variants, PC9/f9 and PC9/f14, by use of genome-wide human cDNA microarray analysis and comparing these profiles with that of the parental cell line, PC9. The PC9/f9 and PC9/f14 cell lines were selected for analysis because of their high metastatic potential. We identified five genes in the highly metastatic cell lines that showed a significantly enhanced or reduced expression and that had not been reported to be involved in metastasis of lung cancer. One of the overexpressed genes that was identified encoded the beta-galactoside-binding protein LGALS3 (Galectin 3). LGALS3 has been reported to be overexpressed in a variety of human cancers, but not in lung cancer, and to be involved in tumor metastasis. We examined the expression of LGALS3 by use of real-time quantitative reverse transcription-polymerase chain reaction in 38 lung cancer cell lines and in tumor tissue obtained by thoracoscopic biopsy. A population (10/30) of the non-small-cell lung cancers examined was found to overexpress the LGALS3 gene at levels three times higher than those of normal epithelial cells. In contrast, all small-cell lung cancers either failed to express the gene or expressed it at a very low level. The mean of the relative expression of the LGALS3 gene in non-small-cell lung cancer (3.065 +/- 3.976) was significantly higher than those of small-cell lung cancer (0.02 +/- 0.03) (P < 0.025). This is the first report of alterations of LGALS3 gene expression in lung cancer. These results, together with the previous reports on Galectin 3 function, suggest that Galectin 3 may play a role in the process of metastasis in non-small-cell lung cancer that overexpresses Galectin 3, but not in small-cell cancer. Accordingly, LGALS3 may be a phenotypic marker that excludes small-cell lung cancer and may represent a novel target molecule in non-small-cell lung cancer therapy.

Adenocarcinoma↗

PubMed2003

Reduced transcription of the RB2/p130 gene in human lung cancer.

Reduced expression of the retinoblastoma gene (RB)2/p130 protein, as well as mutation of exons 19, 20, 21, and 22 of the same gene, has been reported in primary lung cancer. However, it has been suggested by other investigators that mutational inactivation and loss of the RB2/p130 gene and protein, respectively, are rare events in lung cancer. In order to determine the contribution and mechanisms of RB2/p130 gene inactivation to lung cancer development and progression, we quantified RB2/p130 mRNA expression levels in a range of human lung cancer cell lines (n = 13) by real-time reverse transcription (RT)-polymerase chain reaction (PCR) analysis. In comparison to normal lung tissue, reduced transcription of the RB2/p130 gene was found in all small cell lung cancer cell lines examined, along with six out of the eight nonsmall cell lung cancers tested, most of which had inactivation of RB/p16 pathway. On the basis of Western blot analysis, the expression of RB2/p130 protein was consistent with RNA expression levels in all lung cancer cell lines examined. In addition, the mutational status of the RB2/p130 gene (specifically, exons 19, 20, 21, and 22) was determined in 30 primary lung cancers (from patients with distant metastasis) and 30 lung cancer cell lines by PCR-single strand conformation polymorphism (SSCP) analysis and direct DNA sequencing. There was no evidence of somatic mutations within the RB2/p130 gene in the 60 lung cancer samples (both cell lines and tumors) assessed, including the 11 lung cancer cell lines that displayed reduced expression of the gene. Furthermore, hypermethylation of the RB2/p130 promoter was not found in any of the above-mentioned 11 cell lines, as determined by a DNA methylation assay, combined bisulfite restriction analysis (COBRA). The results of the present study suggest that the reduced RB2/p130 expression seen in lung cancer may be in part transcriptionally mediated, albeit not likely via a mechanism involving hypermethylation of the RB2/p130 promoter. The observed reduction in RB2/p130 gene expression may be due to histone deacetylation, altered mRNA stability, and/or other forms of transcriptional regulation.

Adenocarcinoma↗

PubMed2003

Application of sensitive fluorescent dyes in linkage of laser microdissection and two-dimensional gel electrophoresis as a cancer proteomic study tool.

The combination of laser microdissection and two-dimensional gel electrophoresis (2-D PAGE) has been developed to perform proteomic analysis on specific populations of cells in cancer tissues. However, as conventional low sensitivity silver staining was used for spot detection, the microdissection required to obtain an adequate amount of protein for 2-D PAGE is laborious and only a restricted number of protein spots could be visualized. As a consequence, this technology was impractical for direct clinical applications and had a limited impact on cancer studies. To solve these problems, we developed an application in which fluorescent dyes label the proteins extracted from microdissected tissues prior to 2-D PAGE separation. In this application, a small amount of protein, less than 6.6 microg, was enough to generate a 2-D profile with approximately 1500 protein spots. This technique was applied to compare the proteome of normal intestinal epithelium with that of adenoma in Min mice. Thirty-seven protein spots reproducibly showed significant differences in intensities. Mass spectrometric analysis and Western blotting identified eight of them, including prohibitin, 14-3-3zeta, tropomyosin 3 and Hsp84. These results indicate that fluorescence labeling of proteins from microdissected tissues prior to 2-D PAGE is a powerful cancer proteomic study tool.

Adenoma↗

PubMed2003

Different mechanisms of adhesion molecule expression in human dermal microvascular endothelial cells by xanthoma tissue-mediated and copper-mediated oxidized low density lipoproteins.

BACKGROUND: Oxidation of low density lipoprotein (LDL) has been implicated in infiltration of foam cells derived from circulating monocytes. Monocyte adhesion to endothelial cells and migration into dermis are essential steps for infiltration of foam cells. OBJECTIVE: We investigated the role of adhesion molecules contributing to the process of monocyte adhesion to human dermal microvascular endothelial cells (HDMEC). Special attention was paid to the signal transduction for adhesion molecule expression induced by two distinct types of oxidized LDL. METHODS: HDMEC were incubated with xanthoma tissue-modified LDL (x-LDL), a model of extravasated LDL oxidized in xanthoma lesions, or Cu(2+)-treated LDL (Cu-LDL), a model of oxidized LDL. Adhesion of U937 cells, a human monocytic leukemia cell line, to HDMEC and expression of endothelial cell adhesion molecules on HDMEC were examined. Signal transduction pathways for the adhesion molecule expression were evaluated by employing specific inhibitors. RESULTS: x-LDL induced adhesion of U937 cells to HDMEC through vascular cell adhesion molecule-1 (VCAM-1) and E-selectin by activating tyrosine kinase pathway. Cu-LDL up-regulated the adhesion through not only VCAM-1 and E-selectin but also intercellular cell adhesion molecule-1 (ICAM-1) by activating G(i) protein pathway. CONCLUSION: Extravasated and oxidized LDL in xanthoma lesions contributes to foam cell recruitment by activating tyrosine kinase pathway and inducing adhesion of monocytes to HDMEC through VCAM-1 and E-selectin. Cu-LDL, on the other hand, activates G(i) protein pathway and induces the adhesion through ICAM-1, VCAM-1 and E-selectin.

Cell Adhesion↗

PubMed2003

Coexistence of Riehl's melanosis and lichen planus.

A 61-year-old man presented with purple-red papules and plaques. These lesions on the nape and dorsal aspects of both hands displayed the histological findings characteristic of lichen planus. Riehl's melanosis, which is synonymous with pigmented contact dermatitis, was present on the forehead and bald scalp. Riehl's melanosis typically appears on the face and is induced by cosmetics. Causative factors in this case, however, remained undefined. The coexistence of lichen planus and Riehl's melanosis at separate sites in a single patient suggests that the two diseases represent manifestations of specific immune responses characteristic of lichenoid tissue reaction and situate in the same spectrum. The differences in the skin manifestations of lichen planus and Riehl's melanosis might depend on the severity of immune responses against intrinsic or extrinsic antigens. In addition, the skin manifestations of lichenoid tissue reaction might depend on the disposition of the lesion sites.

Humans↗

PubMed2002

The promoter region of the human BUBR1 gene and its expression analysis in lung cancer.

Mitotic checkpoint impairment is present in human lung cancers with chromosomal instability (CIN). Spindle-checkpoint genes have been reported to be mutated in several human cancers, but these mutations are infrequent. Recent reports suggest that the hBUBR1 gene may play an important role in mitotic checkpoint control and in mitotic checkpoint impairment in human cancers. We analyzed the expression of hBUBR1 in lung cancer cell lines using real time quantitative RT-PCR. The expression of BUBR1 was found to be up-regulated in all of these cell lines. In addition, we cloned and characterized the promotor region of hBUBR1 and determined its genomic structure, which includes 23 exons. The open reading frame (ORF) of the hBUBR1 gene comprises exons 1 through 23. There are GC-rich regions located at the flanking region and about 150 bp upstream from exon 1. The promoter region (424 bp upstream from exon 1) showed promoter activity and includes multiple transcription factor consensus binding motifs, including those for Sp1, Nkx-2, CdxA, SRY, MyoD, Ik-2, HNF-3b, Staf, Oct-1, Nkx-2, v-Myb, and AML 1a. Multiple pathways leading to activation of those binding factors may contribute to hBUBR1 gene transcription. Knowledge of the genomic structure and the promoter region of the hBUBR1 gene will facilitate investigation of its role in mitotic checkpoint control and tumor progression in human cancers.

Cell Cycle Proteins↗

PubMed2002

Increased synthesis of calcitonin gene-related peptide stimulates keratinocyte proliferation in murine UVB-irradiated skin.

Repeated ultraviolet (UV) irradiations have been shown to induce keratinocyte proliferation with acanthosis, stimulate the cutaneous nerve proliferation, and increase the synthesis of calcitonin gene-related peptide (CGRP). In the current study, we examined the role of CGRP in the UVB-induced proliferation of murine keratinocytes. UVB irradiation increased the number of bromodeoxyuridine (BrdU)-labeled basal keratinocytes and caused acanthosis. In addition, CGRP expression was up-regulated in the peripheral nerves of the upper dermis and lower epidermis. Repeated intradermal injections of CGRP increased the number of BrdU-labeled basal cells and caused acanthosis. Intradermal injections of capsaicin prior to UVB-irradiation inhibited the UVB-induced CGRP expression, BrdU labeling in basal keratinocytes and epidermal thickening. Intradermal injections of anti-CGRP antibody inhibited the UVB-induced BrdU labeling in basal keratinocytes, but epidermal thickening was not significantly inhibited. These results indicate that CGRP is one of the stimulators to UVB-induced keratinocyte proliferation. On the other hand, expression of substance P, another neuropeptide in the peripheral nerve, was not up-regulated by UVB irradiation.

Animals↗

PubMed2002

Low density lipoprotein oxidized in xanthoma tissue induces the formation and infiltration of foam cells.

Human low density lipoprotein (LDL) was incubated with rabbit xanthoma tissue or non-lesional dermis. The xanthoma tissue-modified LDL (x-LDL) was oxidized showing a 12-fold higher level of thiobarbituric acid-reactive substances (TBARSs) and a faster anodic electrophoretic mobility than native LDL (n-LDL). The LDL treated with non-lesional dermis (d-LDL) had a twofold higher TBARS level compared with n-LDL, but the electrophoretic mobility of d-LDL and n-LDL was similar. Cholesterol esterifying activity in mouse peritoneal macrophages, an indicator of LDL uptake, was up-regulated 5-fold and 1.8-fold by incubation with x-LDL and d-LDL, respectively, compared with n-LDL. Macrophages transformed into foam cells in incubation with x-LDL, and intradermal injections of x-LDL induced infiltration of great many foam cells in the normolipemic rabbit dermis. d-LDL had much less effects on the foam cell formation and foam cell infiltration than x-LDL. Cholesterol:protein ratio was higher in x-LDL than in n-LDL and d-LDL, suggesting that x-LDL-induced foam cells accumulated the lipids by incorporating the cholesterol-rich x-LDL. In conclusion, extravasated LDL receives oxidation and contributes to foam cell recruitment in xanthoma lesions. On the other hand, extravasated LDL in non-lesional dermis receives limited oxidation and additional promoting factors are necessary for initiation of xanthoma development.

Animals↗

PubMedDate not supplied

Phase I/II study of paclitaxel + carboplatin for refractory or recurrent non-small cell lung cancer.

A gene-drug correlation analysis was previously performed in lung cancer cell lines using the NC160 program. On the basis of this work, a phase I/II pilot study of weekly paclitaxel and carboplatin (CBDCA) was subsequently planned for refractory or recurrent non-small cell lung cancer (NSCLC). Safety and antitumor effects were evaluable in all 30 patients registered for this study. Seven patients were stage IIIB and 23 were stage IV. At level 5 (paclitaxel 100 mg/m2 and CBDCA AUCS), toxicities were not dose-limiting factors, but three out of the initial six cases had infusion skips. Our recommended dose was paclitaxel 100 mg/m2 and CBDCA AUC5. The response rate was 50% (9/18)(95% CI: 27-73%) in step 5. The median survival time was 12 months. This combination showed a promising clinical activity with mild toxicity and should be selected for the investigational arm of phase III trials to be compared with either docetaxel or pemetrexed.

Adult↗