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Establishment and characterisation of six human biliary tract cancer cell lines.

Ku JL, Yoon KA, Kim IJ, Kim WH, Jang JY, Suh KS, Kim SW, Park YH, Hwang JH, Yoon YB, Park JG - Br. J. Cancer (2002)

Bottom Line: In addition, we compared the genetic alterations in tumour cell lines and their corresponding tumour tissues.The culture success rate was 20% (six out of 30 attempts).Among the lines, three lines had p53 mutations; and homozygous deletions in both p16 and p15 genes were found three and three lines, respectively; one line had a heterozygous missense mutation in hMLH1; E-cadherin gene was hypermethylated in two lines.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biology, Korean Cell Line Bank, Cancer Research Center and Cancer Research Institute, Seoul National University College of Medicine, Seoul 110-744, Korea.

ABSTRACT
Human cell lines established from biliary tract cancers are rare, and only five have been reported previously. We report the characterisation of six new six biliary tract cancer cell lines (designated SNU-245, SNU-308, SNU-478, SNU-869, SNU-1079 and SNU-1196) established from primary tumour samples of Korean patients. The cell lines were isolated from two extrahepatic bile duct cancers (one adenocarcinoma of common bile duct, one hilar bile duct cancer), two adenocarcinomas of ampulla of Vater, one intrahepatic bile duct cancer (cholangiocarcinoma), and one adenocarcinoma of the gall bladder. The cell phenotypes, including the histopathology of the primary tumours and in vitro growth characteristics, were determined. We also performed molecular characterisation, including DNA fingerprinting analysis and abnormalities of K-ras, p15, p16, p53, hMLH1, hMSH2, DPC4, beta-catenin, E-cadherin, hOGG1, STK11, and TGF-betaRII genes by PCR-SSCP and sequencing analysis. In addition, we compared the genetic alterations in tumour cell lines and their corresponding tumour tissues. All lines grew as adherent cells. Population doubling times varied from 48-72 h. The culture success rate was 20% (six out of 30 attempts). All cell lines showed (i) relatively high viability; (ii) absence of mycoplasma or bacteria contamination; and (iii) genetic heterogeneity by DNA fingerprinting analysis. Among the lines, three lines had p53 mutations; and homozygous deletions in both p16 and p15 genes were found three and three lines, respectively; one line had a heterozygous missense mutation in hMLH1; E-cadherin gene was hypermethylated in two lines. Since the establishment of biliary tract cancer cell lines has been rarely reported in the literature, these newly established and well characterised biliary tract cancer cell lines would be very useful for studying the biology of biliary tract cancers, particularly those related to hypermethylation of E-cadherin gene in biliary tract cancer.

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Hypermethylation of E-cadherin gene in SNU-biliary tract cancer cell lines. (A) RT–PCR analysis of E-cadherin gene. Lane numbers (1–9) indicate cell lines: SNU-245, SNU-308, SNU-478, SNU-869, SNU-1079, SNU-1196, SNU-1 (gastric carcinoma cell line, positive control for methylation of E-cadherin gene, A-431(negative control), and water only. β-actin is RT–PCR control for mRNA expression. (B) RT–PCR analysis after 5-aza-2′-deoxycytidine treatment. It is note that E-cadherin gene is re-expressed after 5-aza-2′-deoxycytidine treatment. β-actin is RT–PCR control for mRNA expression. (C) Methylation specific PCR analysis after sodium bisulphite modification. It is evident that SNU-478 and SNU-1079 lines are methylated in CpG island of promoter region in the E-cadherin gene.
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fig5: Hypermethylation of E-cadherin gene in SNU-biliary tract cancer cell lines. (A) RT–PCR analysis of E-cadherin gene. Lane numbers (1–9) indicate cell lines: SNU-245, SNU-308, SNU-478, SNU-869, SNU-1079, SNU-1196, SNU-1 (gastric carcinoma cell line, positive control for methylation of E-cadherin gene, A-431(negative control), and water only. β-actin is RT–PCR control for mRNA expression. (B) RT–PCR analysis after 5-aza-2′-deoxycytidine treatment. It is note that E-cadherin gene is re-expressed after 5-aza-2′-deoxycytidine treatment. β-actin is RT–PCR control for mRNA expression. (C) Methylation specific PCR analysis after sodium bisulphite modification. It is evident that SNU-478 and SNU-1079 lines are methylated in CpG island of promoter region in the E-cadherin gene.

Mentions: By PCR-SSCP for all 16 exons, abnormal band shifts were not found in all cases. To determine the expression of E-cadherin gene in six biliary tract cancer cell lines, we used RT–PCR analysis. SNU-1 and A-431 cell lines were used for negative and positive controls for the expression of E-cadherin mRNA. As shown in Figure 5, SNU-245, SNU-308, SNU-869, SNU-1196 and control (A-431) cell lines expressed E-cadherin mRNA, whereas SNU-478, SNU-1079 and control (SNU-1) cell lines showed absence of expression (Figure 5AFigure 5


Establishment and characterisation of six human biliary tract cancer cell lines.

Ku JL, Yoon KA, Kim IJ, Kim WH, Jang JY, Suh KS, Kim SW, Park YH, Hwang JH, Yoon YB, Park JG - Br. J. Cancer (2002)

Hypermethylation of E-cadherin gene in SNU-biliary tract cancer cell lines. (A) RT–PCR analysis of E-cadherin gene. Lane numbers (1–9) indicate cell lines: SNU-245, SNU-308, SNU-478, SNU-869, SNU-1079, SNU-1196, SNU-1 (gastric carcinoma cell line, positive control for methylation of E-cadherin gene, A-431(negative control), and water only. β-actin is RT–PCR control for mRNA expression. (B) RT–PCR analysis after 5-aza-2′-deoxycytidine treatment. It is note that E-cadherin gene is re-expressed after 5-aza-2′-deoxycytidine treatment. β-actin is RT–PCR control for mRNA expression. (C) Methylation specific PCR analysis after sodium bisulphite modification. It is evident that SNU-478 and SNU-1079 lines are methylated in CpG island of promoter region in the E-cadherin gene.
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fig5: Hypermethylation of E-cadherin gene in SNU-biliary tract cancer cell lines. (A) RT–PCR analysis of E-cadherin gene. Lane numbers (1–9) indicate cell lines: SNU-245, SNU-308, SNU-478, SNU-869, SNU-1079, SNU-1196, SNU-1 (gastric carcinoma cell line, positive control for methylation of E-cadherin gene, A-431(negative control), and water only. β-actin is RT–PCR control for mRNA expression. (B) RT–PCR analysis after 5-aza-2′-deoxycytidine treatment. It is note that E-cadherin gene is re-expressed after 5-aza-2′-deoxycytidine treatment. β-actin is RT–PCR control for mRNA expression. (C) Methylation specific PCR analysis after sodium bisulphite modification. It is evident that SNU-478 and SNU-1079 lines are methylated in CpG island of promoter region in the E-cadherin gene.
Mentions: By PCR-SSCP for all 16 exons, abnormal band shifts were not found in all cases. To determine the expression of E-cadherin gene in six biliary tract cancer cell lines, we used RT–PCR analysis. SNU-1 and A-431 cell lines were used for negative and positive controls for the expression of E-cadherin mRNA. As shown in Figure 5, SNU-245, SNU-308, SNU-869, SNU-1196 and control (A-431) cell lines expressed E-cadherin mRNA, whereas SNU-478, SNU-1079 and control (SNU-1) cell lines showed absence of expression (Figure 5AFigure 5

Bottom Line: In addition, we compared the genetic alterations in tumour cell lines and their corresponding tumour tissues.The culture success rate was 20% (six out of 30 attempts).Among the lines, three lines had p53 mutations; and homozygous deletions in both p16 and p15 genes were found three and three lines, respectively; one line had a heterozygous missense mutation in hMLH1; E-cadherin gene was hypermethylated in two lines.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biology, Korean Cell Line Bank, Cancer Research Center and Cancer Research Institute, Seoul National University College of Medicine, Seoul 110-744, Korea.

ABSTRACT
Human cell lines established from biliary tract cancers are rare, and only five have been reported previously. We report the characterisation of six new six biliary tract cancer cell lines (designated SNU-245, SNU-308, SNU-478, SNU-869, SNU-1079 and SNU-1196) established from primary tumour samples of Korean patients. The cell lines were isolated from two extrahepatic bile duct cancers (one adenocarcinoma of common bile duct, one hilar bile duct cancer), two adenocarcinomas of ampulla of Vater, one intrahepatic bile duct cancer (cholangiocarcinoma), and one adenocarcinoma of the gall bladder. The cell phenotypes, including the histopathology of the primary tumours and in vitro growth characteristics, were determined. We also performed molecular characterisation, including DNA fingerprinting analysis and abnormalities of K-ras, p15, p16, p53, hMLH1, hMSH2, DPC4, beta-catenin, E-cadherin, hOGG1, STK11, and TGF-betaRII genes by PCR-SSCP and sequencing analysis. In addition, we compared the genetic alterations in tumour cell lines and their corresponding tumour tissues. All lines grew as adherent cells. Population doubling times varied from 48-72 h. The culture success rate was 20% (six out of 30 attempts). All cell lines showed (i) relatively high viability; (ii) absence of mycoplasma or bacteria contamination; and (iii) genetic heterogeneity by DNA fingerprinting analysis. Among the lines, three lines had p53 mutations; and homozygous deletions in both p16 and p15 genes were found three and three lines, respectively; one line had a heterozygous missense mutation in hMLH1; E-cadherin gene was hypermethylated in two lines. Since the establishment of biliary tract cancer cell lines has been rarely reported in the literature, these newly established and well characterised biliary tract cancer cell lines would be very useful for studying the biology of biliary tract cancers, particularly those related to hypermethylation of E-cadherin gene in biliary tract cancer.

Show MeSH
Related in: MedlinePlus