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N Huh

Publications and source records attributed to N Huh.

At least 37 records · Page 2Linked to original sources
PubMed1994

Isolation and characterization of a novel hair follicle-specific gene, Hacl-1.

We have isolated a hair-follicle-specific gene, termed hacl-1, from a cDNA library of ICR mouse skin. Hacl-1 is expressed specifically in skin, and its mRNA level is correlated with the active state of hair follicles in developmental and regenerative processes of hair. Its mRNA is approximately 1 kilobase pairs (kb). Its cDNA was completely co-linear with the genomic clone, indicating that the hacl-1 gene is composed of one exon. The hacl-1 gene has an open reading frame of 579 base pairs (bp). The deduced amino acid sequence showed six direct repeats of a decapeptide on the C-terminal side. The repetitive unit contains a CQP motif, which is present in repetitive peptide sequences of some hair- and epidermal-cell-specific proteins. In situ hybridization with a 3' untranslated region of the hacl-1 cDNA as a probe showed that hacl-1 was expressed specifically in the keratogenous zone of the cortical cells of the hair shaft. No other components of hair follicles or epidermis showed a positive signal. Thus, hacl-1 is a novel, hair-follicle-specific gene.

Amino Acid Sequence↗
PubMed1993

A highly sensitive and specific method for quantitation of O-alkylated DNA adducts and its application to the analysis of human tissue DNA.

Formation and accumulation of O6-alkylguanine and O4-alkylthymine in human tissues is possibly the most relevant marker for cancer risk. Because humans are chronically exposed to diverse kinds of chemicals and eventual DNA structural modifications are supposed to be a complex mixture of adducts at very low levels, it is essential to use an assay with extremely high sensitivity and specificity. We have established a quantitation method, called PREPI, for O6-methylguanine, O4-methylthymine, and O4-ethylthymine by the combination of prefractionation by HPLC, 32P-postlabeling, and immunoprecipitation. The detection limit was about 1 fmole for all three adducts, enabling us to analyze about 1 x 10(-8) levels as a molar ratio to normal counterpart using 100 micrograms of DNA. In a pilot experiment, we analyzed 11 peripheral blood samples from healthy volunteers. O6-Methylguanine was detected in all the cases with a mean value of 2.0 +/- 1.3 x 10(-8) (range, 0.78-4.6 x 10(-8)). Neither O4-methylthymine nor O4-ethylthymine was above the detection limit of 0.8 x 10(-8) as a ratio to thymine.

Alkylating Agents↗
PubMed1988

Enzymatic removal of O6-ethylguanine from mitochondrial DNA in rat tissues exposed to N-ethyl-N-nitrosourea in vivo.

DNA repair is essential for maintaining the integrity of the genetic material, and a number of DNA repair mechanisms have been fairly well characterized for the nuclear DNA of eukaryotic cells as well as prokaryotes. However, little is known about DNA repair in mitochondria. Using highly sensitive immunoanalytical methods to detect specific DNA alkylation products, we found active removal of O6-ethyl-2'-deoxyguanosine (O6-EtdGuo) from rat liver mitochondrial DNA after pulse-exposure to N-ethyl-N-nitrosourea in vivo. In the kidney, O6-EtdGuo was removed from mitochondrial DNA with moderate efficiency, but nearly no removal was observed from the DNA of brain mitochondria. Among the rat tissues examined, the kinetics of O6-EtdGuo elimination from mitochondrial DNA was very similar to the kinetics of removal from nuclear DNA. O4-Ethyl-2'-deoxythymidine, another premutagenic DNA ethylation product, was stable in both mitochondrial and nuclear DNA of rat liver.

Animals↗
PubMed1988

Detection of O4-ethylthymine in human liver DNA.

O4-Ethyl-2'-deoxythymidine (O4-EtdThy) in human liver DNA was quantified, in order to monitor possible human exposure to ethylating agents, using a highly sensitive immunological detection method. In 30 of 33 cases analysed, O4-EtdThy was detected at above the detection limit (i.e., 3 x 10(-8) O4-EtdThy/2'-deoxy-thymidine[dThy]), indicating actual chronic exposure to ethylating agents. The mean content of O4-EtdThy in 19 cases of malignant tumours was significantly higher than that in 11 nonmalignant cases (p less than 0.05).

DNA↗
PubMed1987

1 alpha,25-Dihydroxyvitamin D3 induces anchorage-independent growth and c-Ki-ras expression of BALB/3T3 and NIH/3T3 cells.

1 alpha,25-Dihydroxyvitamin D3, a hormonally active form of vitamin D, induces anchorage-independent growth of BALB/3T3 A31-1-1 and NIH/3T3 cells with concomitant increase of their mRNA level of c-Ki-ras but not of c-Ha-ras or c-myc, through a receptor-mediated mechanism. Under the same conditions, 12-O-tetradecanoylphorbol-13-acetate did not induce anchorage-independent growth in these cell lines.

Calcitriol↗
PubMed1987

[Oncogenes, their implications in cell growth, differentiation and carcinogenesis].

Cancer Research has changed substantially over the past several years since oncogenes were isolated from cancer cells. More than 40 oncogenes have been identified to date from tumor viruses and cancer cells. Many of the gene products seem related to signalling pathways that determine growth and differentiation of cells. This review attempts to summarize much of the currently available data and to gain future perspectives.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1986

Enzymatic elimination of O6-ethylguanine and stability of O4-ethylthymine in the DNA of malignant neural cell lines exposed to N-ethyl-N-nitrosourea in culture.

The capacity of cells for enzymatic repair of DNA structurally altered by DNA-reactive agents is of particular interest in relation to carcinogenesis and with regard to the sensitivity/resistance of cancer cells towards chemotherapeutic drugs. The developing rat brain is characterized both by a pronounced susceptibility to the tumorigenic effect of N-ethyl-N-nitrosourea (EtNU), and by its incapacity, relative to other rat tissues, for enzymatic removal of O6-ethylguanine from DNA. We have investigated whether the latter property is preserved or altered in a panel of nine tumorigenic neural cell lines derived from pre-natal BDIX-rat brain exposed to EtNU in vivo. Using a competitive radioimmunoassay (RIA) in conjunction with a monoclonal antibody (Mab ER-6) specific for O6-ethyl-2'-deoxyguanosine (O6-EtdGuo), the kinetics of O6-EtdGuo elimination from cellular DNA were determined after a 20-min exposure to EtNU in vitro (100 micrograms/ml; resulting average 'input' O6-EtdGuo/2'-deoxyguanosine molar ratio in DNA, 0.74 X 10(-5)). All of the cell lines showed rapid removal of O6-EtdGuo from DNA. During a 280-min period of culture at 37 degrees C following exposure to EtNU, 36-88% of the 'input' amount of O6-EtdGuo was eliminated. Several of the cell lines removed O6-EtdGuo at least as efficiently as fetal BDIX-rat liver (the most O6-EtdGuo elimination-proficient rat tissue thus far documented). In agreement with published data on postnatal rat tissues, O4-ethyl-2'-deoxythymidine (O4-EtdThd) was not eliminated from DNA to any significant degree in a cell line chosen on the basis of its high O6-EtdGuo removal capacity, as assayed by RIA using a Mab (Mab ER-01) specific for O4-EtdThd. Seven subclones derived from one of the cell lines exhibited considerable variation of O6-EtdGuo removal capacity (elimination of 43-93% of the 'input' O6-EtdGuo within 280 min). The relevance of the observed O6-alkylguanine repair-proficiency of malignant neural rat cell lines in relation to carcinogenesis by EtNU in rat brain is not yet clear; however, the apparent instability and intracell population heterogeneity of O6-alkylguanine repair capacity in malignant cells deserve attention with respect to chemotherapy with, e.g. chloroethylnitrosoureas.

Animals↗
PubMed1985

O(6)-methyldeoxyguanosine in oesophageal DNA among individuals at high risk of oesophageal cancer.

A radioimmunoassay using monoclonal antibodies (MAb) against 0(6)-medGuo and 0(6)-etdGuo enabled the presence of these DNA adducts to be detected in human tissues. With this methodology and with 1 mg of hydrolysed DNA it is possible to detect approximately 25 fmol 0(6)-medGuo or 12.5 fmol 0(6)-etdGuo per mg DNA. The analysis comprised a total of 37 human tissue specimens derived from patients who underwent surgery for cancer of the oesophagus in Linxian County (People's Republic of China); for comparison 12 tissue samples obtained from hospitals in Europe were similarly analysed. Seventeen samples from Linxian County showed a level of 0(6)-medGuo ranging from 15 to 50 fmol/mg DNA, 10 showed higher levels up to 160 fmol/mg DNA, and the remaining 10 samples were below the limit of detection. All the tissue samples from Europe showed levels below 45 fmol 0(6)-medGuo/mg DNA, 7 being below the limit of detection. No 0(6)-etdGuo was detected in any of the samples. 0(6)-alkylguanine DNA alkyltransferase activity observed in tissue extracts from Linxian County ranged from 190 to 326 fmol (mean values) of 0(6)-medGuo removed per mg protein and lower values were obtained for the removal of 0(6)-etdGuo. The findings obtained using this approach appear promising in assessing the role of alkylating agents, e.g. nitrosamines, in the aetiology of human cancer.

China↗
PubMed1985

Quantitation and visualization of alkyl deoxynucleosides in the DNA of mammalian cells by monoclonal antibodies.

Conventional radiochromatographic procedures for the quantitation of carcinogen/mutagen-induced structural DNA modifications have a number of limitations. Thus, these techniques for the most part require application of radioactively labeled carcinogens and the use of relatively large amounts of DNA for analysis at low levels of DNA modification. Radiochromatographic methods also preclude analyses at the level of single cells and DNA molecules. Recently developed immunoanalytical methods have improved this situation considerably. Monoclonal antibodies (Mab) characterized by a high substrate specificity and affinity, in combination with radio- and enzyme-immunoassays, or with "immuno-slot-blot" techniques, now permit the detection of femtomole to subfemtomole amounts of, e.g., alkyldeoxynucleosides in small samples of DNA isolated from tissues or cultured cells previously exposed to nonradioactive N-nitroso compounds. Furthermore, selected Mab can be used to quantitate by direct immunofluorescence (with the aid of computer-based image analysis of electronically intensified fluorescence signals), specific alkyldeoxynucleosides in the nuclear DNA of single cells. With this method, the detection limit for the alkylation product O6-ethyldeoxyguanosine (O6-EtdGuo) is presently of the order of 10(2) -10(3) O6-EtdGuo residues per diploid mammalian genome. Individual cells can thus be monitored for the presence of specific carcinogen-DNA adducts, and with respect to their capacity for enzymatic removal of such modified structures from DNA (as exemplified here by the kinetics of the enzymatic elimination of O6-EtdGuo from the DNA of malignant neurogenic rat cell lines). In combination with transmission electron microscopy, Mab also permit direct visualization (via Mab binding sites) of specific carcinogen-modified structures in individual DNA molecules.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkylating Agents↗
PubMed1984

O4-ethyldeoxythymidine, but not O6-ethyldeoxyguanosine, accumulates in hepatocyte DNA of rats exposed continuously to diethylnitrosamine.

In previous investigations into the mechanisms responsible for cell specificity in hepatocarcinogenesis, we have demonstrated that O6-methylguanine accumulates in the DNA of nonparenchymal cells (NPC) but is efficiently removed from hepatocellular DNA. O6-Alkylguanine may, therefore, be an important promutagenic lesion responsible for the induction of hepatic angiosarcomas after exposure to methylating agents, but other promutagenic DNA alkylation products--i.e., O4-alkylthymine--may be responsible for the initiation of hepatocellular carcinomas. F-344 male rats were provided drinking water containing diethylnitrosamine (DEN) at 40 ppm for 0, 2, 4, 8, 16, 28, 49, or 77 days, a regimen that selectively causes hepatocellular carcinomas. Hepatocytes and NPC were isolated by using low-speed differential centrifugation. DNA was purified by hydroxyapatite chromatography and hydrolyzed enzymatically, and O4-ethyldeoxythymidine (O4-EtdThd) and O6-ethyldeoxyguanosine (O6-EtdGuo) of hepatocyte and NPC DNA were quantitated by competitive radioimmunoassay using high-affinity monoclonal antibodies. O4-EtdThd accumulated in hepatocyte DNA during the first 28 days of DEN exposure, approximating a steady state at an O4-EtdThd-to-deoxythymidine molar ratio of approximately equal to 1 X 10(-5). This O4-EtdThd concentration was maintained from 28 to 77 days of DEN exposure. In contrast, O6-EtdGuo did not accumulate in hepatocyte DNA, its greatest concentration O6-EtdGuo-to-deoxyguanosine ratio (approximately equal to 3.7 X 10(-7) ) being detected after 2 days of exposure to DEN. O6-EtdGuo concentrations in hepatocyte DNA decreased with duration of exposure to DEN to an O6-EtdGuo-to-deoxyguanosine ratio of less than 2 X 10(-7) from 28 to 77 days. The data indicate that O4-EtdThd disappears from the DNA of hepatocytes less than 1/200th as fast as O6-EtdGuo. DNA from NPC contained approximately half as much O4-EtdThd as hepatocytes did, but greater than or equal to 2.5 times more O6-EtdGuo.

Alkylation↗
PubMed1984

High-affinity monoclonal antibodies for the specific recognition and quantification of deoxynucleosides structurally modified by N-nitroso compounds.

The applicability of conventional radiochromatographic procedures to the detection and quantification of specific, carcinogen-induced structural modifications in the DNA of mammalian cells is limited by the necessity of using radioactively labelled agents and by the relatively large amounts of DNA required for analysis of low levels of DNA modification. Recently developed immunoanalytical methods have improved this situation considerably. High-affinity monoclonal antibodies (MAB), in combination with radio- and enzyme-immunoassays, now permit the sensitive detection of alkyldeoxynucleosides in small samples of hydrolysed DNA from tissues and cultured cells exposed previously to non-radioactive (e.g., environmental) alkylating N-nitroso carcinogens. Furthermore, MAB can be used to quantify by direct immunofluorescence (and with the aid of computer-based image analysis of electronically intensified fluorescence signals) specific alkylation products in the DNA of individual cells. With this method, the present detection limit for, e.g. O6-ethyl-2'-deoxyguanosine (O6-EtdGuo) is of the order of 7 X 10(2) O6-EtdGuo molecules per diploid genome. Therefore, cells (e.g. from biopsy material) can now be monitored directly for the presence of specific carcinogen-DNA adducts, or with respect to their capacity to remove enzymatically such modified structures from DNA. In combination with transmission electron microscopy, MAB also permit the direct visualization of specific carcinogen-modified sites in DNA. Thus, O6-EtdGuo can be localized in double-stranded DNA molecules by the binding of a MAB specifically directed against this ethylation product.

Antibodies, Monoclonal↗
PubMed1982

Metabolic activation of benzo[a]pyrene, aflatoxin B1, and dimethylnitrosamine by a human hepatoma cell line.

The metabolism of chemical carcinogens by a human hepatoma cell line, huH-1, was studied. The huH-1 line has been derived from a hepatoma of a 57-year-old HBs-antigen carrier and cultivated for several years. The hepatoma cells metabolized about 90% of 5 microM benzo[a]pyrene into water-soluble products within 24 h. Aryl hydrocarbon hydroxylase activity in huH-1 cells was induced to 24 times higher than the basal level by treatment with 13 microM benz[a]anthracene for 24 h. Metabolic activation of benzo[a]pyrene, dimethylnitrosamine and aflatoxin B1 by huH-1 cells was observed by cell-mediated sister-chromatid exchange assay. Sister-chromatid exchanges in human diploid fibroblasts were observed in the cultures mixed with or without huH-1 cells. All 3 chemicals induced sister-chromatid exchanges in human fibroblasts far more efficiently in the cultures mixed with huH-1 cells than in those without huH-1 cells. Some characteristics of huH-1 cells as a human cell-mediated metabolic activation system for carcinogens are discussed.

Aflatoxin B1↗
PubMed1980

Double transformation of Indian muntjac cells by avian and murine sarcoma viruses.

Avian sarcoma virus-transformed Indian muntjac cells, SR-Mm-1, formed foci by murine sarcoma-xenotropic murine leukemia virus complex [MSV(X-MuLV)] superinfection. The response of SR-Mm-1 and parental normal Indian muntjac Mm-2K cells to MSV(X-MuLV) infection was compared. Focus formation by MSV(X-MuLV) followed two-hit kinetics in Mm-2K, but one-hit kinetics in SR-Mm-1 cells. MSV(X-MuLV)-infected SR-Mm-1 cells formed larger colonies than uninfected SR-Mm-1 cells in soft agar, while no colony was formed in the MSV(X-MuLV)-infected Mm-2K cells. After infection with MSV(X-MuLV), cell clones doubly transformed by avian and murine sarcoma viruses could be established in SR-Mm-1 cells, whereas no cell clone could be established in Mm-2K cells. The doubly transformed cells were more round and refractile than SR-Mm-1 cells. No specific chromosomal change could be detected among Mm-2K, SR-Mm-1, and the doubly transformed cells. By two-dimensional polyacrylamide gel electrophoresis of cellular proteins, several changes were seen between Mm-2K and SR-Mm-1 cells. In MSV(X-MuLV)-infected Mm-2K and SR-Mm-1 cells, several similar changes in polypeptide patterns were seen as compared with uninfected cells. These results indicate that Mm-2K cells were doubly transformed by avian and murine sarcoma viruses, and MSV transformation in SR-Mm-1 cells was different from that in Mm-2K cells.

Animals↗
PubMed1980

Establishment of tissue culture cell strains from normal fetal human liver and kidney.

Three cells strains of tissue culture were established from normal fetal human liver and kidney tissues. The rate of cell proliferation decreased with time after the primary culture. The cells were rarely subcultured, but the medium was renewed routinely twice a week. After 9 months of cultivation, the cells were found in both tissues to have abruptly begun to proliferate rapidly, but only in the group culture in the medium containing galactose and sodium pyruvate in place of glucose. No special treatment were given to these cultures, e.g. treatment with viruses, chemical carcinogens and others. The chromosome number was kept around diploid in the beginning but was shifted to hypotriploid after the establishment. The cell strain from liver consists of epithelial cells. The cell strains from kidney consist of mixed population of various kinds of cells. Doubling time of cells is about 33.4 hours in all of them, as determined by cinemicrography. This might be the first establishment of cell strains from untreated, normal human tissues.

Cell Division↗
PubMed1980

Neutralization of cytotoxicity of spermine on the proliferation of rat liver cells in tissue culture.

Cytotoxicity of spermine in tissue culture was found previously. To neutralize this toxicity, the addition of various high molecular weight substances and others was attempted in this paper, e.g. lysozyme, N-acetyl-D-glucosamine, chondroitin sulfate, poly-L-glutamic acid, bovine serum fractions V and VI, fetal calf serum, methyl cellulose, carboxymethyl cellulose, polyvinylpyrrolidone and others. Into the culture of rat liver cells, strain RLC-10(2), simultaneous addition of other substances with spermine did not neutralize the toxicity. However, by the pretreatment of spermine with fetal calf serum or bovine serum albumin (fraction V) at 37 degrees C for 24 hr, the toxicity of spermine was markedly reduced. This was probably due to the denaturation of spermine caused by the pretreatment.

Animals↗