Biomedical subjects

T Sugimura

Publications and source records attributed to T Sugimura.

At least 217 records · Page 12Linked to original sources

PubMed1996

Aberrant DNA methylation on chromosome 16 is an early event in hepatocarcinogenesis.

In order to clarify the significance of DNA methylation in both earlier and later stages of hepatocarcinogenesis, the DNA methylation state on chromosome 16, on which loss of heterozygosity (LOH) has frequently been detected in human hepatocellular carcinomas (HCCs), was examined. DNA from primary HCCs and tissues showing chronic hepatitis and liver cirrhosis, which are considered to be precancerous conditions, was analyzed by digestion with methylation-sensitive and non-sensitive restriction enzymes. DNA hypermethylation at the D16S32, tyrosine aminotransferase (TAT) and D16S7 loci and hypomethylation at the D16S4 locus were detected in 18%, 58%, 20% and 48% of examined HCCs, respectively. Aberrant DNA methylation occurred more frequently in advanced HCCs than in early HCCs. Moreover, DNA hypermethylation at the D16S32, TAT and D16S7 loci was frequently observed in chronic hepatitis and liver cirrhosis. The incidence of DNA hypermethylation was higher than that of LOH (42% at the TAT locus). These data suggest that DNA hypermethylation might predispose the locus to allelic loss. Aberrant DNA methylation is a significant change which may participate in the early developmental stages of HCCs.

Carcinoma, Hepatocellular↗

PubMed1996

Presence in Pieris rapae of cytotoxic activity against human carcinoma cells.

Cytotoxic activity in extracts of pupae and adults of various kinds of butterflies and moths was tested in vitro against the human gastric carcinoma cell line, TMK-1, which was chosen as an example of human carcinoma cells. Among the species examined, cytotoxicity was limited to Pieris rapae, Pieris napi and Pieris brassicae. Activity was found down to a dilution of 1/10(4), while with the other butterflies and moths no activity was observed, even at 1/10(2). When the cytotoxicity of the three developmental stages, larvae, pupae and adults, of Pieris rapae was compared, the pupae showed the strongest activity, the IC50 against TMK-1 cells being at the 1/10(6) dilution. For larvae and adults, the respective IC50 values were at the 1/10(5) and 5/10(5) dilutions. The active principle in the pupae of Pieris rapae was found to be heat-labile and not extractable with organic solvents, but precipitated with ammonium sulfate and digested by proteases, suggesting that it is a protein. This cytotoxic factor was named pierisin.

Animals↗

PubMed1996

Portal-systemic shunting in a patient with normal portal vain pressures and histological evidence of idiopathic portal hypertension.

Although idiopathic portal hypertension (IPH) is clinically characterized by portal hypertension and marked splenomegaly, we have experienced a case of spontaneous portal-systemic shunt without splenomegaly in whom the liver histology resembled IPH but with normal portal pressure. We admitted a 64 year old man who had suffered from hepatic encephalopathy for 2 years. Laparoscopy revealed a dark grey liver with a sharp edge and a concave surface. Examination of a liver biopsy specimen revealed peri-portal fibrosis consistent with IPH. A single, large, portal-systemic shunt was identified by percutaneous transhepatic portography. The shunt arose from the left gastric vein and flowed through the left renal vein into the inferior vena cava. No varices were identified. There were no morphological changes in the hepatic or portal veins. Portal vain pressure was normal. There was a slight difference between the portal pressure and the wedged hepatic vein pressure, suggesting a presinusoidal block. This case raises important questions concerning the aetiology of IPH and the relationship between portal hypertension and the development of collateral venous circulation.

Collateral Circulation↗

PubMed1996

Carcinogenicity of food mutagens.

Cancer cells are produced by the accumulation of genetic alterations in somatic cells. Those genetic alterations are produced by xenobiotics, which enter the human body from the environment, and by autobiotics, which are produced in the human body. Food contains many different types of xenobiotic mutagens/carcinogens and tumor promoters. Food can influence the formation of autobiotic mutagens/carcinogens and give rise to tumor-promoting conditions. In spite of this, it can also contain many antimutagenic, anticarcinogenic, and antitumor-promoting substances. Carcinogenic risk and anticarcinogenic efficacy are hard to express quantitatively; however, holistic approaches that are designed to improve lifestyle are realistic for cancer prevention.

Amines↗

PubMed1996

Human exposure to carcinogenic heterocyclic amines and their mutational fingerprints in experimental animals.

Heterocyclic amines (HCAs) are mutagens/carcinogens to which humans are exposed on almost a daily basis. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhlP) is the most abundant of the various carcinogenic HCAs (present at a level of 0.56 to 69.2 ng/g of cooked meat or fish), with 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MelQx) following it at 0.64 to 6.44 ng/g. HCAs have been found in the urine of healthy people who consume ordinary diets, while patients receiving parenteral alimentation lack, for example, PhlP and MelQx in their urine. Based on the concentrations of PhlP and MelQx in urine samples from 10 healthy volunteers, daily intake of MelQx in Japanese was calculated to be 0.3 to 3.9 micrograms/person, while that of PhlP was 0.005 to 0 micrograms. The Japanese consume more MelQx than Americans, whereas Japanese intake of PhlP was about one-third that of Americans. MelQx-DNA adducts have also detected in Japanese Kidney, colon, and rectum samples using the 32P-postlabeling method followed by identification using high-performance liquid chromatography (HPLC) analysis; the levels were 0.18, 1.8, and 1.4 per 10(9) nucleotides, respectively. In addition, we elucidated the mutational fingerprints of Phlp by analyzing Apc mutations in rat colon cancers induced by this carcinogen. Four of eight tumors had a total of five mutations in the Apc gene, four of which featured a guanine deletion from 5'-GTGGGAT-3' sequences. This specific mutation spectrum may be used as a fingerprint of PhlP in evaluating its risk potential for human colon carcinogenesis. Mutations were not found in similar 2-amino-3-methylimidazo[4,5-f]quinoline-induced colon lesions. Microsatellite instability was detected in both colon and mammary tumors induced by PhlP. The mechanisms involved in this development of microsatellite instability in PhlP. The mechanisms involved in this development of microsatellite instability in PhlP-induced cancers remain to be elucidated.

Amines↗

PubMed1996

Preparation of virus-infection-associated (VIA) antigen of foot-and-mouth disease (FMD) virus from inactivated vaccine.

Virus-infection-associated (VIA) antigen of foot-and-mouth disease (FMD) virus was prepared from an inactivated FMD vaccine. The VIA antigen coupled with an adjuvant of aluminium hydroxide gel supplemented vaccine was efficiently eluted by suspending and stirring in high concentration of phosphate buffer solution (0.3M, pH 7.6). The final elute purified by DEAE-Sephadex A50 from the vaccine was concentrated in 1/500-1/1,000 of the original volume. VIA antigens prepared from two kinds of vaccine (Type O and Asia-1) were antigenically identical to one prepared from a cell culture infected with live virus. Two cattle were infected with FMD virus (Type O) and sera were obtained from each cattle every week. The VIA antigens from the inactivated vaccine were compared with the ones from the cell culture infected with live virus in agar gel diffusion tests using sera from cattle infected with live virus. The antibodies against the VIA antigen were detected between the second week and the 13th or 14th week after infection. The VIA antigen from an inactivated vaccine would be very useful in FMD free countries like Japan to avoid the risk of using live FMD viruses.

Animals↗

PubMed1996

Immunohistochemical detection of K-sam protein in stomach cancer.

The K-sam gene, originally isolated as an amplified gene from the stomach cancer cell line KATO-III, is characterized by its preferential amplification in the undifferentiated type (diffuse type) of stomach cancer and encodes one of the receptors for heparin-binding growth factors or fibroblast growth factors. The K-sam gene has been isolated by different methods and has been designated BEK, TK14, and Cek2. The receptor for keratinocyte growth factor was also found to be encoded by the same gene. To examine the expression of the K-sam protein in stomach cancer, polyclonal antibody pK1-2 was raised against the extracellular domain of the gene product. This antibody detected K-sam proteins by Western blot and flow cytometry analyses in stomach cancer cell lines KATO-III and HSC39, in which the K-sam gene is amplified and overexpressed. By immunohistochemical analysis, 20 of 38 cases of the undifferentiated type of advanced stomach cancer were K-sam positive, whereas none of 11 cases of the differentiated or intestinal type revealed K-sam staining. The K-sam product was observed predominantly in diffusely infiltrative lesions. In one autopsy case, the K-sam protein was detected only focally in the primary tumor, whereas markedly increased staining for the K-sam product was detected diffusely in the metastasized tumor in the lymph node and liver. These results suggest that K-sam overexpression is associated with the malignant phenotype of the undifferentiated type of stomach cancer, such as infiltrative growth and metastasis.

Amino Acid Sequence↗

PubMed1995

Tumorigenicity and gene amplification potentials of cyclin D1-overexpressing NIH3T3 cells.

Cyclin D1 is a key regulator of the G1-S transition in cell cycle, and its gene is amplified and overexpressed in many cancers. To address the gene amplification potential of the cells in which the cyclin D1 gene expression is deregulated, we have established NIH3T3 clones with various levels of cyclin D1 transgene message. Those transfectants showed anchorage independent growth and tumorigenicity without in vitro morphological transformation. The degree of the transformed phenotype apparently correlated with the cyclin D1 expression level. Upon selection by N-(phosphonoacetyl)-L-aspartate (PALA), the cyclin D1-transfected NIH3T3 cells showed a higher ability to develop PALA-resistant colonies by amplifying the CAD gene, as compared to the parental NIH3T3 cells.

3T3 Cells↗

PubMed1995

Increased risk of mammary carcinoma development following transplacental and trans-breast milk exposure to a food-derived carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), in Sprague-Dawley rats.

Effects of transplacental and trans-breast milk exposure to a food-derived mammary and colon carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), were investigated in rats. Female Sprague-Dawley rats were administered PhIP in the diet (100 ppm) for 4 weeks before mating with nontreated males and also during gestation and lactation. As controls, additional females were maintained on the basal diet without PhIP and mated as with the treated animals. The offspring of both groups were subdivided for each sex at weaning into three dietary groups receiving 100, 25, and 0 ppm and were killed at 47 weeks of age. Effects of the transplacental and neonatal exposure to PhIP on mammary carcinogenesis were most evident in females administered 25 ppm PhIP after weaning; the incidence and multiplicity of adenocarcinomas in offspring from the PhIP fed dams (42.9%, 0.62/rat) was significantly higher than the value for offspring from nontreated dams (4.8%, 0.05/rat). Furthermore, in the basal diet groups, the incidence of adenocarcinomas in females was higher, albeit not significantly, in offspring of the PhIP-treated than the nontreated dams (16.7%, 0.22/rat as compared with 3.3%, 0.07/rat). Although the highest incidence of mammary adenocarcinomas was found in the female progeny given 100 ppm PhIP from PhIP-treated dams (70.0%, 1.55/rat), this was only slightly higher than the 61.9% and 0.90/rat of the same dose group from the nontreated dams. In males, no apparent effects of transplacental and neonatal exposures were evident. In a separate experiment, excretion of PhIP into breast milk and transfer of PhIP to fetuses and neonates with resultant hepatic PhIP-DNA adduct formation were demonstrated. Thus, maternal exposure to this food-derived carcinogen may be a critical risk factor for generation of mammary carcinomas.

Animals↗

PubMed1995

Analysis by in vitro mutagenesis of PP2A alpha okadaic acid responsive sequences.

It has been shown that the protein serine/threonine phosphatase type 2A alpha catalytic subunit (PP2A alpha) has a mutation consisting of a glycine substitution for cysteine at 269 in the okadaic acid resistant variant of CHO cells, and that the mutant protein is resistant to okadaic acid (Shima, H. et al. Proc. Natl. Acad. Sci. USA 91, 9267-9271, 1994). In this study we analyzed okadaic acid responsive sequences in PP2A alpha by introducing a mutation at around codon 269 of the cDNA strand. The recombinant mutant PP2A alpha proteins Y265F, C266G, Y267G and C269Y were resistant to okadaic acid, with IC50s of 10, 24, 20 and 10 nM, respectively, as opposed to the recombinant wild PP2A alpha protein which had an IC50 of 0.24 nM. This observation suggests that not only cysteine at 269, but also other YCY amino acids, between 265-267, are necessary for the high sensitivity of PP2A to okadaic acid.

Amino Acid Sequence↗

PubMed1995

Rapid isolation of cell-type-specific protein tyrosine kinases by degenerate polymerase chain reaction combined with differential hybridization technique.

To identify protein tyrosine kinase (PTK) genes preferentially expressed in renal cell carcinoma cell line, we screened a PTK-cDNA-enriched library constructed from RNA of an renal cell carcinoma cell line with a PTK probe, each produced from renal cell carcinoma, gastric cancer or esophageal cancer cell lines by degenerate polymerase chain reaction. Two cDNA fragments of PTK genes, FRK and FLT-3, were isolated from the PTK-cDNA-enriched library of the renal cell carcinoma cell line by differential hybridization technique. The FRK cDNA clone represented 15.8% of the PTK-cDNA-enriched library from the renal cell carcinoma cell line, while the FLT-3 cDNA clone was 2.8% of the same library. Both of the two PTK genes were expressed preferentially in renal cell carcinoma cell lines. This method, described here, is useful for the rapid isolation of PTK cDNA fragments, including a low abundant cDNA, preferentially expressed in a specific cell line.

Base Sequence↗

PubMed1995

Liposome-mediated in vivo gene transfer of antisense K-ras construct inhibits pancreatic tumor dissemination in the murine peritoneal cavity.

K-ras point mutation occurs at a characteristically high incidence in human pancreatic cancer. Plasmids expressing antisense (AS), AS-K-ras-LNSX or sense K-ras gene fragment, were first transduced into three human pancreatic cancer cell lines (AsPC-1, MIAPaCa-2, and BxPC-3) by liposome-mediated transfection. A stable expression of antisense or sense K-ras RNA was detected by Northern blot analysis, and Western blot analysis confirmed a reduction of up to 20% of K-ras-specific p21 protein in AsPC-1 cells transduced with AS-K-ras-LNSX. The growth of pancreatic cancer cells with K-ras point mutations (AsPC-1 and MIAPaCa-2) was significantly suppressed after transduction of AS-K-ras-LNSX, although the effect of antisense construct was not found in cells with a wild-type K-ras gene (BxPC-3). Next, to test the efficacy in vivo, AsPC-1 cells were inoculated into the intraperitoneal cavity of nude mice, and 3 days later, the AS-K-ras-LNSX:liposome complex was injected i.p. 3 times. Twenty-eight days after tumor cell inoculation, 9 of 10 control mice developed peritoneal dissemination and/or solid tumors on the pancreas, whereas only 2 of 12 mice treated with AS-K-ras-LNSX showed any evidence of tumors. Although PCR analysis indicated that the injected DNA was delivered to various organs except for the brain, treatment-related toxicity was not observed. This study shows that the liposome-mediated in vivo gene transfer of antisense K-ras construct may be a useful therapeutic strategy for pancreatic cancer.

Animals↗

PubMed1995

Germline mutation of BRCA1 in Japanese breast cancer families.

We analyzed germline mutations of the BRCA1 gene in 18 Japanese breast cancer families and two Japanese breast-ovarian cancer families. In two site-specific breast cancer families, the same mutation was detected; a nonsense mutation at codon 63 encoding a truncated small protein. It was demonstrated that the mutant allele cosegregated with breast cancer patients within a family and was absent in healthy Japanese, suggesting a breast cancer-predisposing allele. The average age at diagnosis was 44 and 55 years in each family with BRCA1 mutation. No bilateral breast cancer patients were present in the BRCA1 mutation-positive families, although five were present in the BRCA1-negative families. No germline mutations of BRCA1 were detected in the two breast-ovarian cancer families examined in this study, although BRCA1 mutation plays a major role in breast-ovarian cancer families in Western countries. Thus, the proportion of families who inherit the mutated BRCA1 allele seems to be small among Japanese breast cancer families and Japanese breast-ovarian cancer families.

Adult↗

PubMed1995

Silencing of the E-cadherin invasion-suppressor gene by CpG methylation in human carcinomas.

E-Cadherin, a cell adhesion molecule, which plays a key role in maintaining the epithelial phenotype, is regarded as an invasion-suppressor gene in light of accumulating evidence from in vitro experiments and clinical observations. In an attempt to clarify the mechanism responsible for inactivation of this gene in carcinomas, we investigated the methylation state around the promoter region by digestion of DNA with the methylation-sensitive restriction enzyme Hpa II, as CpG methylation of the promoter has been postulated to be a mechanism of transcriptional inactivation of some genes. We found that E-cadherin expression-negative carcinoma cell lines were accompanied by the hypermethylation state, whereas E-cadherin-positive cell lines were not. Furthermore, treatment of E-cadherin-negative carcinoma cells with the demethylating agent 5-azacytidine resulted in reexpression of the gene and reversion of scattered spindle-shaped cells to cells with epithelial morphology. These results suggest that hypermethylation around the promoter may be a mechanism of E-cadherin inactivation in human carcinomas and that treatment of E-cadherin-inactivated cells with a demethylating agent may cause gene expression reversion leading to epithelial morphogenesis with acquisition of the homophilic cell-cell adhesive property.

Azacitidine↗

PubMed1995

Mutation frequency of the p16/CDKN2 gene in primary cancers in the upper digestive tract.

We report a highly frequent homozygous deletion of the p16/CDKN2 gene in the esophageal cancer cell line and a relatively high frequency of homozygous deletion in gastric cancer cell lines. In contrast, in primary esophageal carcinomas, mutation frequency of the p16/CDKN2 gene has been controversial (0, 21, and 52% previously reported), and no reports are available for the mutation frequency of this gene in surgical specimens of gastric carcinomas. Here we report that four (16%) of 25 primary esophageal squamous cell carcinomas were found to be mutated, one in exon 1 and three in exon 2, and that no mutations were observed in 19 surgical specimens of gastric adenocarcinomas. This is the first report showing the absence or quite low frequency of mutation in surgical specimens of gastric carcinomas.

Base Sequence↗

PubMed1995

cDNA cloning of BR gamma, a novel brain-specific isoform of the B regulatory subunit of type-2A protein phosphatase.

A cDNA clone for a novel isoform of the B regulatory subunit of type 2A protein phosphatase (BR) was isolated from a rat brain cDNA library by hybridization under low-stringency conditions with the BR beta cDNA fragment as a probe. The novel cDNA encodes a protein of 447 amino acids. The identities of amino acid sequences of the novel isoform with those of rat BR alpha and BR beta were 80% and 85%, respectively, with a unique sequence at the C-terminal. Since this isoform is thought to be encoded by a third gene for the B regulatory subunit, it was named BR gamma. The mRNA for BR gamma was 4.2 kb. The expression level of BR gamma was high in the brain and spinal cord of rats, and its expression was also detected in mouse and human brains, but in contrast to BR beta, it was not detected in the testes of rats or mice. Thus BR gamma is though to be a brain-specific isoform of the B regulatory subunit.

Amino Acid Sequence↗

PubMed1995

Cloning and characterization of MST, a novel (putative) serine/threonine kinase with SH3 domain.

Protein kinases play a key role in cell growth regulation. We have isolated a cDNA fragment of the MST gene from the MKN28 gastric cancer cell line cDNA pool by degenerate polymerase chain reaction. MST-cDNAs were cloned from the human brain cDNA library. Nucleotide sequence analysis indicated that the MST gene encodes a novel putative non-receptor type of serine/threonine kinase with Src homology 3 (SH3) domain, two leucine zipper domains and proline rich domain. The deduced amino acid sequence corresponding to a part of kinase domain and leucine zipper domains of MST (amino acid codons 244-461) is almost identical to the published partial amino acid sequence of MLK2. MST is the first non-receptor type of serine/threonine kinase containing SH3 domain, leucine zipper domain and proline rich domain other than PTK1/Sprk. The MST gene was moderately expressed in brain, skeletal muscle and testis as a 3.8 kb mRNA, and the MST gene has been mapped to human chromosome 19q13.1-q13.2.

Amino Acid Sequence↗

PubMed1995

Mutant K-ras in apparently normal mucosa of colorectal cancer patients. Its potential as a biomarker of colorectal tumorigenesis.

BACKGROUND: The best way to reduce the incidence of colorectal cancer mortality would be to prevent this cancer. However, none of the biomarkers proposed can accurately identify persons at increased risk of colorectal cancer or those at low risk. As a possible genetic biomarker, K-ras mutations, which are frequently found in colorectal cancers, were analyzed in apparently normal colorectal mucosa. METHODS: Nonneoplastic mucosa and tumor tissues were collected at surgery from 70 patients with colorectal cancer: one sample each from 50 patients (group A) and multiple samples from the other 20 patients (group B). Mutant K-ras codon 12 was analyzed by the enriched polymerase chain reaction (EPCR), by which one mutant can be detected among 10(3) to 10(4) normal alleles. RESULTS: Only with the aid of EPCR was mutant K-ras detected in nonneoplastic mucosa of nine patients (18%) in Group A and five patients (25%) in Group B. This increased incidence could be attributed to the multiple tissue sampling. The presence of mutant K-ras in nonneoplastic mucosae was not consistently correlated with that in the tumors. These findings suggest that the mutant K-ras identified in nonneoplastic mucosa actually represents de novo mutations, which may be initiated by different etiologic factors and at different times. CONCLUSION: Mutant K-ras detected in apparently normal mucosa should be a useful biomarker for identifying persons at higher risk of colorectal cancer. Our study also emphasizes the need for improving the method for sample collection to achieve true representation of the colorectal mucosa.

Adenocarcinoma↗