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Dection of mutations in cancer-related genes using Muts mismatch binging protein and its clinical application to diagnosis of digestive cancers.

Research Project

Project/Area Number 11670486
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Gastroenterology
Research InstitutionKanazawa University

Principal Investigator

WATANABE Hiroyuki  Cancer Research Institute, Kanazawa University Lecturer, がん研究所, 助手 (30242564)

Project Period (FY) 1999 – 2000
Project Status Completed (Fiscal Year 2000)
Budget Amount *help
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2000: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1999: ¥2,100,000 (Direct Cost: ¥2,100,000)
KeywordsMutS protein / homoduplex / heteroduplex / Cancer cell lines / pancreatic juice / K-ras mutation / p53 mutation / MutS protein / pancreatic cancer cell lines / K-ras / p53
Research Abstract

Recently, highly sensitive method of detecting genetic abnormalities including all single base mutations, as well as small deletions or insertions at any locations using Mut S mismatch binding protein was demonstrated in the experimental reports of Lishanski et al. and Wagner et al. I tried to apply this new method to detection of changes of cancer-related genes. First, DNA was extracted from 4 pancreatic cancer cell lines and pancreatic juice (PJ) from 5 patients with pancreatic carcinoma (PCa) and 10 with chronic pancreatitis (CP). Biotin-labeled sense primers for K-ras codon 12, exon 5 to 8 of p53, and exon 1 to 3 of p16 were synthesized. After the process of touchdown PCR amplification, heat denaturation and re-annealing, heteroduplex PCR products form in the case of presence of mispaired and unpaired bases and otherwise homoduplex PCR products alone form. Because of the specific binding of the heteroduplex not homoduplex to immobilized MutS protein on the nitrocellulose membrane, … More chemiluminescence method using streptavidin-HRP was performed for the detection of heteroduplex which is specific binding to MutS protein.
Among 4 pancreatic cancer cell lines, that is, PANC-1, PaCa-2, BxPC-3, and HPAF, PaCa-2 (homozygous mutation of K-ras codont 12) and BxPC-3 (no mutation of K-ras codon 12) would theoretically form homoduplex alone. PANC-1 and HPAF would also form heteroduplex due to heterozygous mutation of K-ras codon 12. On the other hand, p53 mutations were already detected in PANC-1 (exon 8), PaCa-2 (exon 7), BxPC-3 (exon 6), HPAF (exon 5) by direct sequencing. Therefore, as the same manner, these p53 mutations theoretically form heteroduplex. However, false positive was recognized in PaCa-2 for K-ras exon 1 and in PANC-1 for p53 exon 5, PaCa-2 for p53 exon 6, and BxPC-3 for p53 exon 7.Moreover, although the incidence of this heteroduplex using MutS assay was 80% (4/5) for K-ras exon 1 and 60% (3/5) for p53 in PJ with PCa, it was also 60% (6/10) for K-ras exon 1 and 50% (5/10) for p53 in PJ with CP.This detecting method has high sensitivity, but the problem of frequent false positivity.
The cause of the false positivity was regarded as the misincorporation during PCR and imperfect removal of non-specific biotion from the nitrocellulose membrane after the reaction with immobilized MutS binding protein. Further improvement will be needed for this detecting method. Less

Report

(3 results)
  • 2000 Annual Research Report   Final Research Report Summary
  • 1999 Annual Research Report
  • Research Products

    (27 results)

All Other

All Publications (27 results)

  • [Publications] Ha,A.,Watanabe,H., et al.: "Usefulness of supernatant of pancreatic juice for genetic analysis of K-ras in diagnosis of pancreatic carcinoms."Pancreas. (in press). (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Hu,YX.,Watanabe,H., et al.: "An immunohistochemical analysis of p27 expression in human pancreatic carcinomas."Pancreas. 21巻3号. 226-230 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Watanabe,H.,Ha,A., et al.: "K-ras mutations in duodenal aspirate without secretin stimulation for screening of pancreatic and biliary tract carcinoma"Cancer. 86巻8号. 1441-1448 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Yamaguchi,Y.,Watanabe,H., et al.: "Detection of mutations of p53 tumonsuppressior gene in panereatic juice and its application to diagnosis of patients with pancreatic canorcer comparing with K-ras mutations."Clin.Cancer Res.. 5巻5号. 1147-1153 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Wakabayashi,T.,Watanabe,H., et al.: "Clinical management of intraductal papillary mucinous tumors based on imaging findings."Pancreas. (in press). (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Su,S.B.,Watanabe,H., et al.: "Expression of p8 in human pancreatic cancer"Clin.Cancer Res.. 7巻2号. 309-313 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Yamaguchi, Y., Watanabe, H., Songur, Y., Ohtsubo, K., Motoo, Y., Okai, T.and Sawabu, N.: "Detection of mutations of p53 tumor suppressor gene in pancreatic juice and its application to diagnosis of patients with pancreatic cancer : comparing with K-ras mutation."Clin.Cancer Res.. 5. 1147-1153 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Hu Y.-X., Watanabe, H., Ohtsubo, K., Yamaguchi, Y., Ha, A., Motoo, Y., Okai, T.and Sawabu, N.: "Bcl-2 expression related to altered p53 protein and its impact on the progression of human pancreatic carcinoma."Br.J.Cancer. 80. 1075-1079 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Motoo, Y., Watanabe, H., Okai, T.and Sawabu, N.: "Urinary gonadotropin peptide as acute phase reactant : transient elevation after operation for digestive diseases."Eur.J.Endocrinol.. 140. 555-560 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Motoo, Y., Satomura, Y., Mouri, I., Mouri, H., Ohtsubo, K., Sakai, J., Fujii, T., Taga, H., Yamaguchi, Y., Watanabe, H., Okai T.and Sawabu.N.: "Serum levels of pancreatitis-associated protein in digestive diseases with special reference to gastrointestinal cancers."Dis.Dis.Sci.. 44. 1142-1147 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Okai, T., Watanabe, H., Yamaguchi, Y., Mouri, I., Motoo Y.and Sawabu N.: "Endoscopic ultrasonography with K-ras analysis of pure pancreatic juice for the diagnosis of pancreatic mass lesion : a prospective study."Gastrointest.Endosc.. 50. 797-803 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Watanabe, H., Ha, A., Hu, Y-X., Ohtsubo, K., Yamaguchi, Y., Motoo, Y., Okai, T., Toya, D., Tanaka, N.and Sawabu.N.: "K-ras mutations in duodenal aspirate without secretin stimulation for screening of pancreatic and biliary tract carcinoma."Cancer. 86. 1441-1448 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Okai, T., Kawashima, A., Watanabe, H., Takahashi, Y., Sakai, J., Ohtsubo, K., Motoo, Y., Matsui, O., Murakami, S., Nakabayashi, H.and Sawabu, N.: "Nonfunctioning islet cell carcinoma of the pancreas with high serum CEA & CA19-9, K-ras codon 12 mutation, and microsatellite instability."J.Clin.Gastroenterol.. 30. 307-310 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Hu, Y-X., Watanabe, H., Wang, Y., Li, P., Ohtsubo, K., Yamaguch, Y.and Sawabu, N.: "An immuohistochemical analysis of p27 expression in human pancreatic carcinomas."Pancreas. 21. 226-230 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Su, S.B., Motoo, Y., Iovanna, J.L., Xie, M.J., Ohtsubo, K., Mouri, H., Yamaguchi, Y., Watanabe, H., Okai, T., Matsubara, F.and Sawabu, N.: "Expression of p8 in human pancreatic cancer."Clin.Cancer Res.. 7. 309-313 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Ha, A., Watanabe, H., Yamaguchi, Y., Ohtsubo, K., Wang.Y., Motoo, Y., Okai, T.and Sawabu, N.: "Usefulness of supernatant of pancreatic juice for genetic analysis of K-rasin diagnosis of pancreatic carcinoma."Pancreas. (in press). (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Ha,A.,Watanabe,H., et al.: "Usefulness of supernatant of pancreatic juice for genetic analysis of K-ras in diagnosis of pancreatic carcinoma."Pancreas. (in press). (2001)

    • Related Report
      2000 Annual Research Report
  • [Publications] Hu,YX.,Watanabe,H., et al.: "An immunohistochemical analysis of p27 expression in human pancreatic carcinomas."Pancreas. 21巻3号. 226-230 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Watanabe,H.,Ha,A., et al.: "K-ras mutations in duodenal aspirate without secretin stimulation for screening of pancreastic and biliary tract carcinoma"Cancer. 86巻8号. 1441-1448 (1999)

    • Related Report
      2000 Annual Research Report
  • [Publications] Yamaguchi,Y.,Watanabe,H., et al.: "Detection of mutations of p53 tumor suppressor gene in pancreatic juice and its application to diagnosis of patients with pancreatic cancer comparing with K-ras mutations"Clin.Cancer Res.. 5巻5号. 1147-1153 (1999)

    • Related Report
      2000 Annual Research Report
  • [Publications] Wakabayashi,T.,Watanabe,H., et al.: "Clinical management of intraductal papillary mucinous tumors of the pancreas based on imaging findings"Pancreas. (in press). (2001)

    • Related Report
      2000 Annual Research Report
  • [Publications] Su,S.B.,Watanabe,H., et al.: "Expression of p8 in human pancraeatic cancer"Clin.Cancer Res.. 7巻2号. 309-313 (2001)

    • Related Report
      2000 Annual Research Report
  • [Publications] Y.Yamaguchi,et al.: "Detection of mutations of p53 tumor suppossor gene in pancreatin juice and its application to diagnosis of patients with pancreatin"Clin.Cancer Res.. 5(5). 1147-1153 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Y.-X.Hu et al.: "Bcl-2 expression related to altered p53 protein and its impact on the progression of human pancreatic carcinoma"Br.J.Cancer. 80. 1075-1079 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] H.Taga et al.: "Large duodenal tumor with positive k-ras mutations mimicking lateral spreading tumor of the colon: a case of report"Dig.Endosc.. 11(1). 70-75 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] T.Okai,et al.: "EUS and k-ras analysis of pure pancreatic juice collected us a duodeno scope after secretin stimulation for diagnosis of pancreatic mass lesion a prospect study."Gastrointest. Endosc.. 50(6). 797-803 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] H.Watanabe,et al.: "k-ras mutations in duodenal aspirate without secretin stimulation for screening of pancreatic and biliary tract carcinoma."Cancer. 86(8). 1441-1448 (1999)

    • Related Report
      1999 Annual Research Report

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Published: 1999-04-01   Modified: 2016-04-21  

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