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Biomedical subjects

N Tsuchida

Publications and source records attributed to N Tsuchida.

At least 19 recordsLinked to original sources

The p53 tumor-suppressor gene and ras oncogene mutations in oral squamous-cell carcinoma.

The frequencies of mutations in the p53 tumor-suppressor gene and ras proto-oncogenes were investigated systematically in surgically resected oral squamous-cell carcinomas (SCCs) using single-strand conformation polymorphism (SSCP) and/or dot-blot hybridization analysis of DNA fragments which had been amplified by the polymerase chain reaction (PCR). p53 gene mutations, within the region of exons 5 to 8, were detected in 17 out of 27 (63%) tumor specimens. The role of p53 mutations in cell-line establishment was investigated. p53 gene mutations were detected in 5 out of 6 tissue samples from which cell lines were established and in 4 out of 5 specimens from which cell lines could not be established, suggesting that the presence of p53 gene mutations is not by itself sufficient for cell-line establishment. Tumor samples were also analyzed for point mutational activation of the ras proto-oncogenes. One out of 30 (3%) tumors showed an activating point mutation in codon 12 of H-ras, this being consistent with reports from Europe and USA but not with any from India. Compared to frequencies of the other genetic changes so far reported for oral SCC, the p53 mutations have been observed most often to undergo genetic change. p53 gene mutation is thus intimately involved in the genesis of oral SCC and consequently should be useful as a marker for the diagnosis of this neoplasm.

Adult

In vitro differentiation of the human osteosarcoma cell lines, HOS and KHOS.

The osteogenic potential of the two human osteosarcoma cell lines HOS and KHOS; a cell line produced by the transformation of the HOS cells by the Kirsten murine sarcoma virus, was studied in vitro. HOS cells cultured more than 2 weeks formed nodules composed of two morphologically distinct layers, an epithelial-like surface cell layer and a collagen-rich inner cell layer. Alkaline phosphatase (ALPase) activity occurred in the plasma membrane of the surface cell layer, and calcified substances developing along collagen fibers were detected in the collagen-rich inner cell layer. The calcified substances were further examined by analytical electron microscopy and were shown to be hydroxyapatite crystals. In contrast, there was neither ALPase nor the deposition of a calcified substance in the KHOS cells.

Alkaline Phosphatase

Mechanism of induction of cellular DNA synthesis by the adenovirus E1A 12S cDNA product.

The mechanism of induction of DNA synthesis in quiescent rat 3Y1 cells by the adenovirus E1A gene was investigated using the 3Y1 derivative cell lines g12-21, gn12RB1, and gn12RB2. The g12-21 cells express the E1A 12S cDNA and the latter two cells express both the E1A 12S cDNA and the human retinoblastoma susceptibility (Rb) gene at different levels in response to dexamethasone (dex). The cDNA sequences of E1A-inducible cell cycle-dependent genes, clone 3 and clone 16, were isolated by differential screening of a cDNA library constructed from dex-treated g12-21 cells. The quiescent 3Y1 cells induced c-fos and c-myc expression within 2 h after serum stimulation and expressed clone 16 and clone 3 transiently at around 8 h before the onset of DNA synthesis (10 h). In contrast, the quiescent g12-21 cells treated with dex expressed a high level of E1A at 6 to 8 h after treatment and expressed clone 16 and clone 3 at around 8 h without stimulation of c-fos and c-myc expression, suggesting that E1A bypasses the cell cycle early in G1. The half-maximal rate of DNA synthesis was reached in a much shorter time in dex-treated g12-21 cells (12 h) than in serum-treated 3Y1 cells (18 h), suggesting that E1A also bypasses the cell cycle at the G1/S boundary. The gn12RB1 and gn12RB2 cells were unable to induce DNA synthesis in response to dex presumably due to lower levels of E1A expression, although gn12RB2 but not gn12RB1 cells could express clone 16 and clone 3. These results suggest that the level of E1A required for bypass at the G1/S boundary is higher than that required early in G1.

Adenovirus Early Proteins

Detection of p53 gene mutations in human ovarian and endometrial cancers by polymerase chain reaction-single strand conformation polymorphism analysis.

The presence of mutations in the p53 gene was examined in ovarian cancers by a polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. The primers were designed to amplify exons 5 through 9 that contain phylogenetically conserved domains of the p53 gene. Mutations were detected in 5 out of 10 cases, one of which contained a deletion in the second allele. A single base substitution was detected in 4 cases at codons 162, 175, 205 and 273 and a single base insertion in one case within codon 315. A high frequency of p53 mutations in ovarian cancers and lack of mutation in 6 benign ovarian tumors and 2 normal ovaries suggested that the mutation of the p53 gene was associated with the genesis and/or progression of ovarian cancer. In 1 of 7 endometrial cancers, two mutations at codons 239 and 254 were detected.

Alleles

Most human squamous cell carcinomas in the oral cavity contain mutated p53 tumor-suppressor genes.

Oral squamous cell carcinoma cell lines and tumor tissues used for cell line establishment were examined for p53 tumor-suppressor gene mutation using cellular DNAs and RNAs. For sensitive and rapid detection, a newly designed two-stage filtration strategy was used. Full advantage was taken of the single-strand conformation polymorphism (SSCP) of cDNA and cellular DNA fragments amplified by the polymerase chain reaction (PCR), followed by DNA sequencing of mutated fragments. Fourteen out of 15 cells lines and the corresponding five tumor tissues had p53 mutations within the region of exons 5-8. Loss of normal alleles was noted in 14 lines, but not in one in which only mutated transcripts were detected. DNA sequencing indicated six out of 14 mutations to be in positions that have so far not been reported. In two special cases, novel mutations were found in the splicing donor sequence of exon 6, and consequently the cryptic splice site had to be used. Extremely frequent p53 gene mutations indicated that the mutations are likely be intimately involved in the carcinogenesis of oral squamous cell carcinoma.

Amino Acid Sequence

cDNA and deduced amino acid sequence of mouse matrix gla protein: one of five glutamic acid residues potentially modified to gla is not conserved in the mouse sequence.

A cDNA library was constructed using the mouse osteoblastic cell line MC3T3-E1 treated with 1 alpha,25-dihydroxyvitamin D3, based on the finding that the treatment increased ninefold the expression of 0.7 kb matrix gla protein (MGP) mRNA. cDNA clones encoding mouse MGP were isolated from the library. The nucleotide sequence showed an open reading frame of 312 nucleotides encoding 104 amino acids. Murine MGP shared 84-89% amino acid sequence homology with bovine, rat, and human MGP. However, there are five glutamic acid residues potentially modified to gamma-carboxyglutamic acid (gla) in those species; in murine MGP, lysine replaced glutamic acid 37. Also, an extra tyrosine was added at the carboxyl terminus. The significance of the substitution is discussed in relation to the gamma-carboxylation sites in MGP protein.

Amino Acid Sequence

Gene cloning of an Actinobacillus actinomycetemcomitans Y4 antigen which reacts with peripheral blood sera in patients with advanced destructive periodontitis.

Actinobacillus actinomycetemcomitans has been implicated in the aetiology of juvenile periodontitis and advanced destructive periodontitis. Levels of IgG antibody against A. actinomycetemcomitans in peripheral blood sera of patients with advanced destructive periodontitis are high, as are those against Bacteroides gingivalis. To clone the genes of antigens reactive with sera of such patients, a library of the A. actinomycetemcomitans strain Y4 DNA in lambda L47 was constructed and then screened, using an immunochemical detection method, with serum from a patient with the advanced disease. Six clones from among nearly 1000 reacted with the serum and also with that of another patient. They were designated 3, 4, 6, 7, 8 and 9. Restriction enzyme and Southern blot analyses indicated that clones 8 and 9 were identical and that all the clones were overlapping because they shared in common the 4 and 5 kbp HincII DNA fragments of A. actinomycetemcomitans. The cloned DNA fragment hybridized to the DNA of two other strains of A. actinomycetemcomitans but not to those of six periodontopathic bacteria examined. These findings suggest that a DNA sequence encoding an A. actinomycetemcomitans strain Y4 antigen strongly reactive with sera of patients with advanced destructive periodontitis was cloned. This sequence is present specifically in A. actinomycetemcomitans but not in other bacteria isolated from patients with periodontal diseases. Thus, the cloned DNA could serve as a probe for the diagnosis of periodontitis.

Actinobacillus

Nonlethal G0-ts mutant tsJT60 becomes lethal at the nonpermissive temperature after transformation: a hint for new cancer chemotherapeutics.

tsJT60 is a nonlethal temperature-sensitive (ts) mutant of a Fischer rat cell line (3Y1) classified as a G0 mutant; i.e., the ts defect is not expressed within the cell growth cycle but is expressed only between the G0 and S phase. tsJT60 clones transformed with oncogenes such as adenovirus E1A, polyoma large T, polyoma middle T, v-Ki-ras, and LTR activated c-myc, or with a chemical carcinogen N-methyl-N'-nitro-N-nitrosoguanidine, grew well at 34 degrees C. However, most of these clones grew slowly at 40 degrees C, producing many floating dead cells, and some clones were killed at 40 degrees C. When they were cultured under conditions inadequate for growth of untransformed cells, such as high cell density or serum restriction, they were killed at 40 degrees C. These and previous results from SV40- and adenovirus-transformed tsJT60 clones favour the idea that transformed tsJT60 cells occasionally enter the G0 phase and are metabolically imbalanced at 40 degrees C during self-stimulation from the G0 to S phase. We propose that a drug which exclusively block, G0-G1 transition would be cytocidal to transformed cells but cytostatic to normal cells.

Adenovirus Early Proteins

Altered ganglioside expression in ras-oncogene-transformed cells.

Alteration of ganglioside composition in mouse BALB/3T3 cells transformed either by DNA transfection with viral K-, H-, or cellular H-ras oncogene, or by infection with the K-ras oncogene-carrying murine sarcoma virus (Ki-KSV) was studied using a highly sensitive thin-layer chromatography/enzyme immunostaining method. Marked common decreases in the content of GD3 ganglioside and the increase of its metabolic precursor GM3 were bound in BALB/3T3 cell lines transformed by either K- or H-ras oncogenes. Moreover, a common decrease or loss in the contents of "A" series ganglio-tetraose gangliosides such as GM1a and GD1a was also found in all transformed cell lines, indicating that the alteration of cellular glycosphingolipids by ras oncogenes apparently does not depend on the type of ras-concogenes (K- and H-ras).

Animals

Activation of oncogenes in human oral cancer cells: a novel codon 13 mutation of c-H-ras-1 and concurrent amplifications of c-erbB-1 and c-myc.

By NIH3T3 transfection assay in conjunction with in vitro transient neomycin selection, activated c-H-ras-1 oncogenes were detected in two squamous cell carcinoma cell lines, ZA and HOC-313, newly established from human oral cancer patients. ZA had a point mutational activation at the 13th codon, this activation of c-H-ras-1 being novel in human cancer cells, while HOC-313 appeared to have an activation at the 12th codon. In ZA, 16- to 32-fold amplification of the EGF receptor gene, c-erbB-1 and a few-fold amplification of c-myc were detected. The significance of these findings is discussed in relation to multistep carcinogenesis in human cells.

Aged

Inactivation of the N-myc gene product by single amino acid substitution of leucine residues located in the leucine-zipper region.

To verify the importance of the hypothetical leucine-zipper structure in the N-myc protein, a series of mutants of the mouse N-myc gene were constructed, in which codons for the first and second leucine residues within this structure were systematically replaced by other amino acids. The expression plasmids which contained the mutated and wild type N-myc genes were cotransfected into rat embryo cells with activated c-Ha-ras gene and their transforming abilities were compared. It was shown that single amino acid substitutions in the leucine-zipper region inactivate the transforming ability of the N-myc gene product. In particular, proline, which is known to disrupt an alpha-helical structure, completely inactivated the transforming activity even when it was substituted for another amino acid located between these two leucine residues. Among several amino acid species used for substitution of the leucine residues, only methionine was able to retain the transforming activities in both the first and second leucine positions, although the activity was reduced as compared with wild-type N-myc gene product. It also appeared that the integrity of the first leucine is more important than the second leucine. Our results provide experimental evidence for the physiological importance of the hypothetical leucine-zipper structure.

Animals

Increased formation of unintegrated viral DNA in skin fibroblasts from patients with familial polyposis coli shortly after infection with Kirsten murine sarcoma virus.

Skin fibroblasts from patients with familial polyposis coli (FPC) exhibit increased frequency of morphological transformation, anchorage-independent growth, and enhanced production of viral RNA and viral particles after infection with murine sarcoma virus(murine leukemia virus), MSV(MLV). These FPC fibroblasts form greater amounts of unintegrated viral DNA as compared to normal fibroblasts during the first 4 to 24 hr after infection with Kirsten MSV(KiMLV), suggesting that increased susceptibility of FPC cells to MSV is determined at an early stage of viral transformation. Several MSV-transformed clones of both FPC and normal fibroblasts, isolated from agarose gel, were non-producers having integrated viral genome which was expressed into viral RNA and rescued by infection with MLV.

Adenomatous Polyposis Coli

Myc genes linked to viral promoters can collaborate with a transformation-deficient simian virus 40 early gene mutant to transform established rat cells.

Cotransfection of a transformation-deficient and immortalization-positive SV40 early gene mutant with various constructs of viral promoter-linked myc genes induced dense foci in 3Y1 cells, an established cell line derived from rat embryo fibroblasts, whereas transfections with either species alone did not. Four cell clones were isolated from colonies grown in soft agar medium, and found to contain at least a single copy of both DNA species and to express both sequences at relatively high levels. These results indicate that the two immortalizing genes used in the present study collaborate to transform 3Y1 cells.

Animals

Structure of the c-Ki-ras gene in a rat fibrosarcoma induced by 1,8-dinitropyrene.

Restriction enzyme maps were made of the region around exons 1 and 2 of activated c-Ki-ras of a fibrosarcoma (1,8-DNP2) induced in a rat by 1,8-dinitropyrene. Nucleotide sequence analysis revealed that activated c-Ki-ras shows a G----T transversion in codon 12 and consequently encodes cysteine instead of glycine in normal rat c-Ki-ras.

Amino Acid Sequence

Nucleotide sequence of exon I of the rat c-K-ras gene.

We have isolated a DNA fragment containing exon I of rat cellular proto-oncogene of K-ras and sequenced the exon I. The obtained sequence was compared with that of the corresponding region in viral oncogene of Kirsten strain of murine sarcoma virus (Ki-MSV). The results showed that the exon I sequences of cellular K-ras genes in rat and human cells could encode the same amino acid sequence, while the viral K-ras gene could code for two different amino acids corresponding to the 12th and 37th positions from the N-terminus of K-ras gene product. The amino acid substitutions found between viral and cellular K-ras genes are discussed in relation to the transforming ability of Ki-MSV.

Animals

Amplification of c-Ki-ras gene and aberrant expression of c-myc in WI-38 cells transformed in vitro by gamma-irradiation.

The expression of c-oncogenes was examined with normal human fibroblasts (WI-38) and the cells transformed in vitro by gamma-irradiation (CT-1). The amount of Ki-ras-specific mRNA in CT-1 cells was found to be approximately twice that in WI-38 cells. By Southern blot hybridization, the c-Ki-ras 2 gene of CT-1 cells was found to be amplified about two-fold. CT-1 cells have higher levels of c-myc mRNA than normal cells, although the gene dosage and the restriction nuclease pattern of the c-myc gene was the same. The content of c-myc mRNA in CT-1 cells was found to be constitutively high, whereas that in normal cells was increased by serum addition.

Cell Line

ISL1: a new transposable element in Lactobacillus casei.

The genome structures of a temperate Lactobacillus phage, phi FSW, and its virulent mutants, phi FSVs, were examined by restriction, heteroduplex and nucleotide-sequence analyses. The results showed that two out of three phi FSVs had the same 1.3 kbp insertion (designated as ISL1) at different positions in the phi FSW sequence. ISL1 was 1,256 bp long and contained at least two long open reading frames of 279 and 822 bases on one strand. Inverted repeats were found at the termini of the ISL1 which was bracketed by 3 bp direct repeats of the phi FSW sequence. From this evidence, we concluded that ISL1 was a transposable element in Lactobacillus casei.

Bacteriophages