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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 expression in malignant cell cycle.


METHODS
Non-radioactive in situ hybridization (ISH) was undertaken, combined with immunohistochemistry to study the Ki-67 gene transcription and translation in various human cells and tissues. HeLa cells and fresh colon cancer 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-labelled antisense and sense RNA probes were prepared for detecting Ki-67 mRNA, combined with MIB-1 immunohistochemistry.


RESULTS
Successful localization of Ki-67 mRNA in human HeLa cells, colon cancer cells, tissues specimen of the tonsil and pancreatic cancer tissue sections was accomplished by digoxigenin-labelling in situ hybridization technique. 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 accordance 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. Abnormal transcription of exon 13 of Ki-67 gene might be responsible for malignant cell proliferation in colon and pancreatic cancer.