Yoshiro Koda

A novel myc target gene, Mina53, that is involved in cell proliferation

Myc is a ubiquitous mediator of cell proliferation and can transactivate the expression of various genes through E-box sites. Here we report a novel gene, mina53(Myc-inducednuclear antigen with a molecular mass of 53 kDa). The mina53 gene encodes a protein with a molecular weight of 53 kDa, which is localized in the nucleus and with part of the protein concentrated in the nucleolus. When serum-starved cells were activated by serum, the level of c-myc mRNA was elevated, and an increase in mina53 mRNA followed the elevation of c-myc mRNA. When expression of c-myc was reduced in human promyelocytic leukemia HL60 cells by phorbol 12-myristate 13-acetate, the expression ofmina53 mRNA and protein was reduced. The expression ofmina53 mRNA and Mina53 protein was induced by ectopic introduction of wild type c-Myc but not by a mutant c-Myc lacking the transactivation domain. When c-Myc in the c-MycER chimeric protein was activated, mina53 mRNA was increased, even in the presence of an inhibitor for protein synthesis. E-box sites are present in a region proximal to the transcription initiation sites of themina53 gene. The gene expression from themina53 promoter was elevated by c-Myc through E-box sites. c-Myc protein bound to the mina53 promoter region in vivo in HL60 cells in the proliferating phase but not after treatment of cells with phorbol 12-myristate 13-acetate. Specific inhibition of mina53 expression by an RNA interference method severely suppressed cell proliferation. Taken together, these results indicate that mina53 is a direct target gene of Myc, suggesting that mina53 is involved in mammalian cell proliferation.

Population differences of two coding SNPs in pigmentation-related genes SLC24A5 and SLC45A2

The two genes SLC24A5 and SLC45A2 were recently identified as major determinants of pigmentation in humans and in other vertebrates. The allele p.A111T in the former gene and the allele p.L374F in the latter gene are both nearly fixed in light-skinned Europeans, and can therefore be considered ancestry informative marker (AIMs). AIMs are becoming useful for forensic identification of the phenotype from a DNA profile sampled, for example, from a crime scene. Here, we generate new allelic data for these two genes from samples of Chinese, Uygurs, Ghanaians, South African Xhosa, South African Europeans, and Sri Lankans (Tamils and Sinhalese). Our data confirm the earlier results and furthermore demonstrate that the SLC45A2 allele is a more specific AIM than the SLC24A5 allele because the former clearly distinguishes the Sri Lankans from the Europeans.

The polymorphisms of fucosyltransferases

The alpha(1,2)fucosyltransferase Se enzyme regulates the expression of the ABH antigens in secretion. Secretors, who have ABH antigens in their saliva, have at least one functional Se allele in the FUT2 locus, while non-secretors, who fail to express ABH antigens in saliva, are homozygous for the non-functional se allele. Molecular analyses of the FUT2 polymorphism of various populations have indicated the ethnic specificity of null alleles: the null allele se(428) is a common Se enzyme-deficient allele in Africans and Caucasians but does not occur in Asians, whereas the null allele se(357,385) is specific to Asians. The gene frequency of se(428) or se(357,385) is about 0.5 in each respective population. Why the se(428) is absent in Asians is of interest. Also here, we describe the polymorphisms of the fucosyltransferase genes (FUT1, FUT3 and FUT6).

Extensive polymorphism of the FUT2 gene in an African (Xhosa) population of South Africa

The human secretor type α(1,2)fucosyltrans-ferase gene (FUT2) polymorphism was investigated in Xhosa and Caucasian populations of South Africa by polymerase chain reaction–restriction fragment length polymorphism and DNA sequencing. Six new base substitutions were found in the coding region of FUT2. A single base (C) deletion at nucleotide 778, which led to a frame shift and produced a stop codon at codon 275, was responsible for the enzyme inactivation. Three nonsynonymous base substitutions, A40G (Ile14Val), C379T (Arg127Cys), and G481A (Asp161Asn), and two synonymous base substitutions, A375G (Glu125) and C480T (His160), were also identified in functional alleles. As a result, seven new alleles, Se40, Se481, Se40,481, Se357,480, Se357,379,480, Se375, and se357,480,778 were identified. Population studies revealed that an allele containing a nonsense mutation G428A (Trp143stop) (se428) was the common null allele in both Xhosa and Caucasian populations, whereas an allele containing a missense A385T (Ile129Phe) mutation (se357,385), which is the common null allele in Orientals, was found to be absent from both populations. The heterozygosity rates of FUT2 genotypes were as high as 0.75 in the Xhosa population and 0.65 in the Caucasian population. Therefore, the extensive polymorphism and race specificity of the FUT2 gene make it suitable for application as a new tool in genetic studies of modern human evolutionary history.

Distinctive distribution of AIM1 polymorphism among major human populations with different skin color

AbstractThe genetic background for human skin color has been a major topic in human genetics; however, its molecular basis is still unclear. The gene for the AIM-1 protein (AIM1) was recently found to be responsible for the body color of medaka fish. In the search for the genes controlling human skin color variations, we have investigated genetic polymorphisms of this gene, and we have found a single-nucleotide polymorphism that has clear association with major human populations in terms of skin color.

Increased Expression of a Myc Target Gene Mina53 in Human Colon Cancer

Mina53 is a novel Myc target gene that we previously demonstrated to be involved in cell proliferation. We studied, here, the expression of Mina53 in colon cancer to examine its possible role in carcinogenesis. We generated a specific monoclonal anti-human Mina53 antibody and found that colon tumor cell lines expressed Mina53 highly. We also found that expression of Mina53 was elevated in colon tumor tissues by immunoblotting analysis. Tissue sections of 23 surgical cases of adenocarcinoma and 1 case of adenoma were stained immunohistochemically, and the expression of Mina53 was found to be elevated in all of the adenocarcinomas compared to adjacent nonneoplastic tissues, which showed little staining. Deeply invading tumors as well as tumors that have invaded lymphatic vessels showed strong immunoreactivity against anti-Mina53 antibody. Mina53 was expressed in all pathological grades of cancer as well as in the adenoma. Staining patterns of Ki-67, a biomarker for cell proliferation, were similar to those of Mina53 in most cases, but the percentage of tumor cells stained by anti-Mina53 was higher. Although anti-Ki-67 antibody strongly stained some well-proliferating nonneoplastic cells including cells in the deeper part of the crypts and in lymphoid germinal centers, antibody to Mina53 rarely stained those cells. Suppression of mina53 expression severely suppressed proliferation of colon tumor cells in vitro. Together, our results indicate that the elevated expression of Mina53 is a characteristic feature in colon cancer, one that may have therapeutic applications.

Mina53 as a Potential Prognostic Factor for Esophageal Squamous Cell Carcinoma

Purpose: We previously identified mina53, a novel Myc target gene. Here we investigated whether mina53 is related to esophageal squamous cell carcinoma (ESCC), a disease with poor prognosis. Experimental Design: Mina53 expression was suppressed in ESCC cell lines by a RNA interference method to investigate whether Mina53 is involved in cell proliferation. Expression of Mina53 was investigated by Western blotting in tissue sections from patients with ESCC. Immunohistochemical analysis of Mina53 was carried out and compared with that using anti–Ki-67 antibody. Finally, the level of Mina53 expression was compared with the length of survival of patients with ESCC. Results: Reduction of mina53 expression by RNA interference suppressed cell proliferation in ESCC cell lines. Western blot analysis of surgically resected ESCC specimens indicated that the expression of Mina53 in tumors was increased compared with that in adjacent nonneoplastic tissues in all four specimens examined. When formalin-fixed specimens from 52 patients with ESCC were stained immunohistochemically, it was found that Mina53 was highly expressed in 83% of specimens. Anti-Mina53 antibody stained tumors more efficiently than antibody against Ki-67, a cell proliferation biomarker, in some cancer specimens. Patients with high expression of Mina53 had shorter survival periods, whereas the expression level of Ki-67 in ESCC showed no relationship to patient outcome. Conclusions: Taken together, our results indicate that expression of Mina53 is a characteristic feature of ESCC and suggest that immunostaining by anti-Mina53 antibody may be useful as a potential prognostic indicator.

The fusion gene at the ABO-secretor locus (FUT2): absence in Chinese populations.

AbstractThe fusion gene (sefus) is a null allele of the secretor type α (1, 2) fucosyltransferase gene (FUT2) and was first found in a Japanese population. It has not yet been reported in any other ethnic population. In the present study, we investigated the distribution of the fusion gene of the FUT2 locus in five populations from three ethnic groups in East Asia. The fusion gene was found in two additional Japanese populations with a high frequency (0.0551 in Okinawa and 0.0792 in Akita) and, for the first time outside Japan, in a Korean population, at a very low frequency (0.0063 in Seoul). In contrast, we found no fusion gene in two Chinese populations. These findings showed that the FUT2 fusion gene was ubiquitous in Japanese, but was rare in neighboring populations, suggesting that the FUT2 fusion gene had emerged from within the Japanese. Additionally, a new null allele with a C-to-T substitution at nucleotide 658 was found in one individual native of southern China.

A-61C and C-101G Hp gene promoter polymorphisms are, respectively, associated with ahaptoglobinaemia and hypohaptoglobinaemia in Ghana.

We have investigated the genetic basis for the Hp0 phenotype amongst 123 randomly selected Ghanaians. A total of 17 individuals were determined to be Hp0 phenotype, based on the classical method for Hp phenotyping of Hb‐supplemented plasma. Out of the 17 Hp0 individuals, nine subjects were further classified as ahaptoglobinaemic and eight as hypohaptoglobinaemic by Western blots and double immunodiffusion. We identified three previously known base substitutions (A−55G, A−61C and T−104A) and three new ones (C−101G, T−191G and C−242T) within the 5′ flanking region of the Hp gene. The A−61C base substitution significantly decreased transcriptional activity and was associated strongly with Hp2 allele and ahaptoglobinaemia. The C−101G substitution was similar in transcriptional activity to the wild‐type and was associated with Hp1S allele and hypohaptoglobinaemia. The Hpdel allele seen in Asian populations was absent. We conclude that the Hp0 phenotype in Ghana has a genetic basis that differs significantly from that seen in Asia.

Structure and Expression of H-type GDP-L-Fucose:β-D-Galactoside 2-α-L-Fucosyltransferase Gene (FUT1) TWO TRANSCRIPTION START SITES AND ALTERNATIVE SPLICING GENERATE SEVERAL FORMS OF FUT1 mRNA

The expression of the ABO antigens on erythrocyte membranes is regulated by H gene (FUT1)-encoded α(1,2)fucosyltransferase activity. We have examined the expression of the FUT1 in several tumor cell lines, including erythroid lineage and normal bone marrow cells, by Northern blot and/or reverse transcription-polymerase chain reaction (RT-PCR) analyses. RT-PCR indicated that bone marrow cells, erythroleukemic cells (HEL), and highly undifferentiated leukemic cells (K562) that have erythroid characteristics expressed the FUT1 mRNA while four leukemic cell lines did not. The FUT1 mRNA was also demonstrated in gastric, colonic, and ovarian (MCAS) cancer cell lines by RT-PCR. Northern blot analysis indicated that a 4.0-kilobase FUT1 transcript was expressed in some of these tumor cell lines. Rapid amplification of 5′ cDNA end (RACE) analysis suggested that the FUT1 transcript had several forms generated by two distinct transcription start sites and alternative splicing. The results of RT-PCR using specific primers for each starting exon suggested that two transcription initiation sites (exon 1A and exon 2A) of the FUT1 were identified in gastric cancer cells and in ovarian cancer cells. Only exon 1A was identified as a transcription start site in another gastric cancer cell line, two colonic cancer cell lines, and in K562 cells, whereas only exon 2A was identified in HEL cells and in bone marrow cells. These two transcription start sites were located 1.8 kilobases apart. Therefore, two distinct promoters appeared to be present in the FUT1 The distinct promoters of the FUT1 and alternative splicing of the FUT1 mRNA may be associated with time- and tissue-specific expression of the FUT1