MOLECULAR ANALYSIS OF INK4 GENES IN BREAST CARCINOMAS

Cell cycle regulators have recently been implicated in oncogenic trans formation of cells, including the cyclins active in the G1 phase of th e cell cycle and their respective cyclin-dependent kinases (CDK) whose activities are regulated by a set of inhibitors of CDK (CDKI). Since CDKIs can inhibit cell proliferation, they may have a role as tumor su ppressor genes. To determine if alterations of CDKI genes may be invol ved in tumorigenesis of breast cancer, we examined the mutational stat us of p16(INK4A), p15(INK4B), p18(INK4C), p19(INK4D) CDKI genes in 36 primary breast carcinomas and 9 breast cancer cell lines using polymer ase chain reaction-single strand conformational polymorphism (PCR-SSCP ), direct DNA sequencing, and Southern blot analysis. Furthermore, amp lification of cyclin D1, D2, D3 genes were also examined in these samp les. One mutation of p15(INK4B) gene occurred, resulting in change of aspartic acid to asparagine at codon 85. Since aspartic acid at this p osition is conserved between all four human and murine INK4 proteins, this missense mutation may have functional significance. The sample wi th a p15(INK4B) point mutation was accompanied by amplification of the cyclin D1 gene. A deletion of the p18(INK4C) gene was found in a prim ary tumor. Three deletions of the p16(INK4A) gene and two deletions of the p15(INK4B) gene were found in the cell lines. Also, we found ampl ification of the p15(INK4B) and p16(INK4A) loci in a clinical sample a s well as amplification of the p19(INK4D) in another sample, and ampli fication of the myeloperoxidase (MPO) gene in one cell line and two pr imary tumors. We suspect that a critical gene for breast cancer is amp lified near the MPO gene. These data indicate that CDKI mutations are moderately rare in breast cancer and are often associated with the sim ultaneous alteration of more than one cell-cycle regulatory gene.