Somatic INK4a-ARF locus mutations: A significant mechanism of gene inactivation in squamous cell carcinomas of the head and neck

The INK4a-ARF locus is located on human chromosome 9p21 and is known to enc ode two functionally distinct tumor-suppressor genes. The p16(INK4a) (p16) tumor-suppressor gene product is a negative regulator of cyclin-dependent k inases 4 and 6, which in turn positively regulate progression of mammalian cells through the cell cycle. The p14(ARF) tumor-suppressor gene product sp ecifically interacts with human double minute 2, leading to the subsequent stabilization of p53 and G(1) arrest. Previous investigations analyzing the p16 gene in squamous cell carcinomas of the head and neck (SCCHNs) have su ggested the predominate inactivating events to be homozygous gene deletions and hypermethylation of the p16 promoter. Somatic mutational inactivation of p16 has been reported to be low (0-10%, with a combined incidence of 25 of 279, or 9%) and to play only a minor role in the development of SCCHN. T he present study examined whether this particular mechanism of INK4a/ARF in activation, specifically somatic mutation, has been underestimated in SCCHN by determining the mutational status of the p16 and p14(ARF) genes in 100 primary SCCHNs with the use of polymerase chain reaction technology and a h ighly sensitive, nonradioactive modification of single-stranded conformatio nal polymorphism (SSCP) analysis termed "cold" SSCP. Exons 1 alpha, 1 beta, and 2 of INK4a/ARF were amplified using intron-based primers or a combinat ion of intron- and exon-based primers. A total of 27 SCCHNs (27%) exhibited sequence alterations in this locus, 22 (22%) of which were somatic sequenc e alterations and five (5%) of which were a single polymorphism in codon 14 8. Of the 22 somatic alterations, 20 (91%) directly or indirectly involved exon 2, and two (9%) were located within exon Ice. No mutations were found in exon 1 beta. All 22 somatic mutations would be expected to yield altered p16 proteins, but only 15 of them should affect p24(ARF) proteins. Specifi c somatic alterations included microdeletions or insertions (nine of 22, 41 %), a microrearrangement tone of 22, 5%), and single nucleotide substitutio ns (12 of 22, 56%). in addition, we analyzed the functional characteristics of seven unique mutant p16 proteins identified in this study by assessing their ability to inhibit cyclin-dependent kinase 4 activity. Six of the sev en mutant proteins tested exhibited reduced function compared with wild-typ e p16, ranging from minor decreases of function (twofold to eightfold) in f our samples to total loss of function (29- to 38-fold decrease) in two othe r samples. Overall, somatic mutation of the INK4a/ARF tumor suppressor locu s, resulting in functionally deficient p16 and possibly p14(ARF) proteins, seems to be a prevalent event in the development of SCCHN. (C) 2001 Wiley-L iss, Inc.