Expression of exon 13 from the Ki-67 gene in human cells and tissues by digoxigenin-labelled mRNA in situ hybridization

Objective To get insight on the regulatory mechanism of Ki-67 gene expressi on in malignant cell cycle. Methods Non-radioactive in situ hybridization (ISH) was undertaken, combine d with immunohistochemistry to study the Ki-67 gene transcription and trans lation in various human cells and tissues. Hela cells and fresh colon cance r cells, tonsil, normal pancreas and pancreatic cancer tissues were used in this study. A 435 bp cDNA fragment located in exon 13 of the Ki-67 antigen gene was amplified by polymerase chain reaction (PCR). Digoxigenin-labelle d antisense and sense RNA probes were prepared for detecting Ki-67 mRNA, co mbined with MIB-1 immunohistochemistry. Results Successful localization of Ki-67 mRNA in human Hela cells, colon ca ncer cells, tissues specimen of the tonsil and pancreatic cancer tissue sec tions was accomplished by digoxigenin-labelling in situ hybridization techn ique. ISH to colon cancer cells and pancreatic cancer tissue slides showed that much stronger cytoplasm and perinuclear mRNA signals of the Ki-67 gene were present in malignant cells than in normal cells, which was in accorda nce with MIB-1 nuclear protein signals. Conclusions A sensitive and practical in situ hybridization method for the analysis of Ki-67 antigen mRNA in human cell and tissue was developed. Abno rmal transcription of exon 13 of Ki-67 gene might be responsible for malign ant cell proliferation in colon and pancreatic cancer.