Effect of non-tumor cell contamination on detection of p53 gene mutations in human gastric cancer cells by polymerase chain reaction single-strand conformation polymorphism analysis.

Abstract

Background:We have previously studied p53 gene mutations in 25 primary gastric cancer tissues by polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis for exon 4–8 and immunohistochemical staining with anti-p53 antibody. In four cases, the discrepancy of the results was observed between the two methods. In one case, positive by PCR-SSCP but negative by immunohistochemical staining, the mutation was silent. In three cases, the p53 gene mutations were detected only by immunohistochemical staining. This discrepancy may be due to the contamination of the samples by cells without p53 gene mutation, such as non-tumor cells. This study was conducted to investigate the sensitivity of PCR-SSCP analysis to p53 gene mutations when the sample was contaminated with non-tumor cells.Methods:Genomic DNA was extracted by the digestion with proteinase K and phenol-chloroform-ethanol method from two human gastric adenocarcinoma cell lines, MKN-45 and KATO III. To investigate the sensitivity of PCR-SSCP, DNA extracted from cancer cells was mixed with DNA obtained from normal gastric mucosal cells at various ratios. PCR-SSCP analysis for exon 4–8 of the p53 gene was performed with the mixed DNA samples.Results:In KATO III, no PCR products were generated in exon 4–8 of the p53 gene by PCR, suggesting that both alleles from exon 4–8 of the p53 gene were deleted. In MKN-45, the mobility shift was observed in exon 4. Therefore, the effect of non-tumor cell contamination on the detection of p53 gene mutations was conducted using MKN-45 and normal gastric mucosal cells. In the mixed DNA samples of MKN-45 and normal gastric mucosal cells, an extra band with the migration similar to that of MKN-45 was found in the samples of 1:8 dilution or less, while no extra band was grossly detectable in DNA of normal gastric mucosal cells and in the samples of more than 1:16 dilution.Conclusions:These results suggest that the detection of p53 mutations by PCR-SSCP analysis may be underestimated in samples contaminated by a large number of non-tumor cells.