Molecular analysis of the INK4A and INK4B gene loci in human breast cancer cell lines and primary carcinomas

The INK4A and INK4B loci are located at 9p21 and have been implicated in the tumorigenesis of various human malignancies. The INK4A gene encodes two cell cycle regulators, p16INK4A and ARF, while INK4B encodes p15INK4B. Previously, we have shown that the p16INK4 tumor suppressor was not mutated or deleted in primary breast carcinomas. However, primary and metastatic breast carcinomas exhibited a relative hypomethylation of p16INK4A, which is associated with expression, compared to normal breast tissue. The present study was conducted to determine if inactivation of p15INK4B and INK4A exon 1β (ARF) are common events in breast carcinoma. Mutational analysis was performed by PCR-SSCP, and mRNA expression was evaluated by RT-PCR. Methylation-specific PCR was used to determine the methylation status of the p15INK4B promoter. Our results demonstrate that the p15INK4B gene was altered in 3 (21%) of the 14 breast cell lines; one had a silent mutation and two had homozygous deletion of the gene. None of the cell lines showed methylation of p15INK4B. Two (14%) cell lines had homozygous deletion of INK4A exon 1β. All normal and malignant breast tissue samples were wild-type and non-methylated for p15INK4B and wild-type for exon 1β. Our results show that these structurally and functionally related genes are not invariably affected together, and the most frequently observed alteration at the INK4A and INK4B loci in breast carcinoma appears to be p16INK4A hypomethylation.