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

The INK4A and INK4B loci are located at 9p21 and have been implicated in th e tumorigenesis of various human malignancies. The INK4A gene encodes two c ell cycle regulators, p16(INK4A) and ARF, while INK4B encodes p15(INK4B). P reviously, we have shown that the p16(INK4) tumor suppressor was not mutate d or deleted in primary breast carcinomas. However, primary and metastatic breast carcinomas exhibited a relative hypomethylation of p16(INK4A), which is associated with expression, compared to normal breast tissue. The prese nt study was conducted to determine if inactivation of p15(INK4B) and INK4A exon 1 beta (ARF) are common events in breast carcinoma. Mutational analys is was performed by PCR-SSCP, and mRNA expression was evaluated by RT-PCR. Methylation-specific PCR was used to determine the methylation status of th e p15(INK4B) promoter. Our results demonstrate that the p15(INK4B) 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 p15(INK4B). TWO (14%) cell Lines had homozygous deletion of INK4A exon Ip. All normal and malignant breast tissue samples were wild-typ e and non-methylated for p15(INK4B) and wild-type for exon 1 beta. Our resu lts show that these structurally and functionally related genes are not inv ariably affected together, and the most frequently observed alteration at t he INK4A and INK4B loci in breast carcinoma appears to be p16INK4A hypometh ylation. (C) 2001 Elsevier Science Inc. All rights reserved.