Yoji Fukuda

Enhanced expression of PAI-1 in visceral fat: possible contributor to vascular disease in obesity.

The presence of obesity increases the risk of thrombotic vascular diseases. The role of fat accumulation and its effect on plasminogen activator inhibitor–1 (PAI–1) levels was investigated in humans and animals. Plasma PAI–1 levels were closely correlated with visceral fat area but not with subcutaneous fat area in human subjects. PAI–1 mRNA was detected in both types of fat tissue in obese rats but increased only in visceral fat during the development of obesity. These data suggest that an enhanced expression of the PAI–1 gene in visceral fat may increase plasma levels and may have a role in the development of vascular disease in visceral obesity.

Comparative genomic hybridization of squamous cell carcinoma of the esophagus: The possible involvement of the DP1 gene in the 13q34 amplicon

We investigated copy number aberrations in 29 primary tumors and 12 cell lines of esophageal squamous cell carcinoma (ESC) using comparative genomic hybridization. In the primary tumors, the most common sites of copy number gains were 3q26.3–27 (45%), 8q24 (41%), 5p15 (38%), Xq27–28 (38%), 14q32 (31%), 11q13 (28%), and 20q13.3 (28%). High‐level gains (HLGs) indicative of gene amplifications were identified at 11q13 in two cases, and in one case each at 2q33–34, 3q25–29, 5p15.1–15.2, 7q21–22, 11p11.2, 12p11.2–12, and 13q34. Recurrent losses were observed only at 9p13 (17.2%). In the 12 ESC cell lines, the most common sites of HLGs were 5p15.1–15.3 (four cases), 11q13 (four cases), 8q24.1–24.2 (three cases), 20q13.2–13.3 (three cases), 3q26.3 (two cases), and 7p15–22 (two cases). Less frequent HLGs (one case each) were observed at 2p16–22, 3q25, 7p12–14, 7q21–22, 9q34, 10q21, 11p11.2, 14q13–14, 14q31–32, 15q22–26, and 17p11.2. Chromosomes and chromosome arms that showed frequent losses in the cultured lines were 18q (58%), 4 (50%), 9p (50%), and 3p (42%). These findings provide evidence for a number of previously unknown genomic aberrations in ESC, suggesting target regions for positional cloning of genes relevant to carcinogenesis in the esophagus. In particular, we identified a significant amplification of the DP1 gene (TFDP1), a transcription factor that forms heterodimers with E2F1, in the single primary tumor that exhibited HLG at 13q34. Genes Chromosomes Cancer 24:337–344, 1999.

A new mutation of the fukutin gene in a non‐Japanese patient

Fukuyama‐type congenital muscular dystrophy (FCMD), Walker–Warburg syndrome, and muscle‐eye‐brain disease are clinically similar autosomal recessive disorders characterized by congenital muscular dystrophy, cobblestone lissencephaly, and eye anomalies. FCMD is frequent in Japan, but no FCMD patient with confirmed fukutin gene mutations has been identified in a non‐Japanese population. Here, we describe a Turkish CMD patient with severe brain and eye anomalies. Sequence analysis of the patients DNA identified a homozygous 1bp insertion mutation in exon 5 of the fukutin gene. To our knowledge, this is the first case worldwide in which a fukutin mutation has been found outside the Japanese population. This report emphasizes the importance of considering fukutin mutations for diagnostic purposes outside of Japan. Ann Neurol 2003

Human arylhydrocarbon receptor repressor (AHRR) gene: genomic structure and analysis of polymorphism in endometriosis

AbstractThe diversity of biological effects resulting from exposure to dioxin may reflect the ability of this environmental pollutant to alter gene expression by binding to the arylhydrocarbon receptor (AHR) gene and related genes. AHR function may be regulated by structural variations in AHR itself, in the AHR repressor (AHRR), in the AHR nuclear translocator (ARNT), or in AHR target molecules such as cytochrome P-4501A1 (CYP1A1) and glutathione S-transferase. Analysis of the genomic organization of AHRR revealed an open reading frame consisting of a 2094-bp mRNA encoded by ten exons. We found one novel polymorphism, a substitution of Ala by Pro at codon 185 (GCC to CCC), in exon 5 of the AHRR gene; among 108 healthy unrelated Japanese women, genotypes Ala/Ala, Ala/Pro, and Pro/Pro were represented, respectively, by 20 (18.5%), 49 (45.4%), and 39 (36.1%) individuals. We did not detect previously published polymorphisms of ARNT (D511N) or the CYP1A1 promoter (G-469A and C-459T) in our subjects, suggesting that these polymorphisms are rare in the Japanese population. No association was found between uterine endometriosis and any polymorphisms in the AHRR, AHR, ARNT, or CYP1A1 genes analyzed in the present study.

IQGAP1, a negative regulator of cell-cell adhesion, is upregulated by gene amplification at 15q26 in gastric cancer cell lines HSC39 and 40A.

AbstractOur previous comparative genomic hybridization (CGH) study revealed a novel amplified region at 15q26 in two cell lines established from diffuse types of gastric cancer (GC). In this amplified region, FES and IGF1R, known targets on 15q26, were located telomeric to the amplicon in the two cell lines, HSC39 and 40A, suggesting that another tumor-associated gene exists in this region. While screening expressed sequence tags (ESTs) for novel genes in this region, we identified the IQGAP1 amplification. IQGAP1 has been reported to encode a ras GAP-related protein, and its interaction with cadherin and/or β-catenin induces a dissociation of β-catenin from the cadherin-catenin complex, one of the mechanisms for cell-cell adhesion. Northern and Western blot analyses revealed that amplification of this gene was accompanied by corresponding increases in mRNA and protein expression. Moreover, immunocytochemical staining showed that overexpressed IQGAP1 accumulated at the membrane, suggesting its colocalization with β-catenin. Taken together, these findings suggest that IQGAP1 may be one of the target genes in the 15q26 amplicon correlated with a malignant phenotype of gastric cancer cells, such as diffuse and invasive characteristics, through the disruption of E-cadherin-mediated cell-cell adhesion.

Amplification on double-minute chromosomes and partial-tandem duplication of the MILL gene in leukemic cells of a patient with acute myelogenous leukemia

Partial‐tandem duplication (PTD) of an internal portion of MLL occurs in some cases of acute myelogenous leukemia (AML) with trisomy 11 or a normal karyotype. This type of MLL rearrangement may be transcribed into an mRNA species that is capable of encoding a partially duplicated protein associated with leukemogenesis. However, although several kinds of oncogenes, especially MYC, are often amplified on double‐minute chromosomes (dmins) in hematological malignancies, no amplification of MLL has been reported in AML. Here, we report the first documented case of a patient with AML whose leukemic cells exhibited amplification of MLL on dmins. Furthermore, in this patient, MLL was rearranged in a PTD manner, with in‐frame fusion of exons 2 and 6. Genes Chromosomes Cancer 23:267–272, 1998.

CD44 is a potential target of amplification within the 11p13 amplicon detected in gastric cancer cell lines.

Classical cytogenetic approaches have revealed many of the chromosomal aberrations that may occur in gastric cancers (GC), although few alterations of specific genes have been identified so far. Genes that affect progression of this disease need to be identified if clinicians are to achieve optimal management of patients with GC. As the first step toward the cloning of gene(s) that may be involved in gastric carcinogenesis, we examined 25 GC cell lines for aberrations in DNA copy number to detect chromosomal gains and losses, as well as gene amplifications, by comparative genomic hybridization (CGH). Our CGH study revealed high‐level amplifications in chromosomal regions that had been well defined in GC but also in sites that had not, including 3p24, 5p15, 11p11.2–14, 13q34, 15q26, Xp24, and Xq26–28. The minimal common region at 11p13, within the 11p11.2–14 amplicon, harbors the CD44 gene. Northern and Western blot analyses showed that an alternatively spliced form of CD44, with variant exons 8–10 (CD44E), was overexpressed in all cell lines bearing the 11p13 amplicon. However, cell adhesion activity was no greater in these lines than in cell lines without amplifications at the CD44 locus, suggesting that the major property of upregulated CD44 in these cases might be to transduce signals critically associated with growth and proliferation of the tumor cells.

Structural organization, complete genomic sequences and mutational analyses of the Fukuyama-type congenital muscular dystrophy gene, fukutin.

Fukuyama‐type congenital muscular dystrophy (FCMD) is an autosomal recessive severe muscular dystrophy in combination with cerebral cortical dysplasia. Previously, we identified the gene responsible for FCMD, termed fukutin, through positional cloning. In this study, we have sequenced 131 892 bp of genomic DNA in the region of the fukutin gene on chromosome 9q31 and obtained its complete genomic structure. The fukutin genomic sequence spans approximately 100 kb and is organized into 10 exons (41–6067 bp) and nine introns (1841–21 460 bp). Using these sequence data, we have identified three novel fukutin mutations in FCMD patients. We have also located a putative TATA box in the flanking 5′ region and identified numerous alternatively spliced fukutin mRNA transcripts. Analysis of expressed sequence tag clusters within the region revealed two novel genes upstream of the fukutin gene. These data provide fundamental information to support detailed genetic and functional analyses of the fukutin gene.

Molecular cytogenetic analysis of 17 renal cancer cell lines: increased copy number at 5q31-33 in cell lines from nonpapillary carcinomas.

Comparative genomic hybridization (CGH) was used to screen for genomic imbalances in cell lines derived from 13 nonpapillary renal‐cell carcinomas (RCCs), two papillary RCCs, one renal squamous‐cell carcinoma, and one transitional‐cell carcinoma of the renal pelvis. Aberrations were found in all 17 lines. The most frequent changes in nonpapillary RCC cell lines were gains of 5q (85%), 7q (69%), 8q (69%) and 1q (54%) and losses of 3p (92%), 8p (77%), 4q (62%) and 14q (54%). High‐level gains (HLGs) were detected at 4q12, 5p, 5q23‐33, 7q22‐qter, 8q23‐24, 10q21‐qter, 12p and 12q13‐22. By means of fluorescence in situ hybridization (FISH) we narrowed the smallest common region involving 5q gains to the genomic segment between D5S642 and D5S673, and found that the HLG at 4q12 possibly involved amplifications of c‐kit and PDGFRA. Two papillary RCC cell lines showed gains of entire chromosomes 7, 12 and 17. The CGH data reported here should help to facilitate the choice of individual renal‐tumor cell lines for exploring target genes in regions of interest.

Identification of target genes within an amplicon at 14q12-q13 in esophageal squamous cell carcinoma.

Comparative genomic hybridization studies have revealed frequent amplification of the 14q12‐q13 region in esophageal squamous cell carcinoma (ESC) cell lines. To identify genes targeted for amplification, we first defined the minimal common region of amplification using fluorescence in situ hybridization in affected ESC cell lines. The amplicon covered about 6 Mb, between markers D14S1034 and L18528. Then we screened 32 ESC cell lines to discern amplifications and expression levels of 26 expressed sequence tags (ESTs) that had been localized to the amplified region. Five known genes (BAZ1A, SRP54, NFKBIA, MBIP, and HNF3A) and two uncharacterized ESTs (GenBank Accession numbers AA991861 and AA167732) within the amplicon showed amplification and consequent overexpression. Two of these transcripts were amplified in three of the primary ESCs we examined. Our findings suggest that these seven genes are candidate targets of the amplification mechanism and therefore may be associated, together or separately, with development and progression of ESC.