MULTIPLE MECHANISMS OF P16(INK4A) INACTIVATION IN NON-SMALL-CELL LUNG-CANCER CELL-LINES

(1)p16(INK4A) specific inhibitor of cyclin-dependent kinase (cdk)4 and cdk6, is a candidate tumor suppressor in malignancies with wild-type retinoblastoma (Rb), Loss of p16(INK4A) frees these cdks from inhibiti on, permitting constitutive phosphorylation of Rb and inactivation of its growth suppressive properties. Consistent with this model, Rb-posi tive non-small cell lung cancers (NSCLCs) have little or no detectable p16(INK4A) protein, whereas Rb-negative lung cancers have abundant p1 6(INK4A). However, only some NSCLCs have homozygous deletions or nonse nse mutations in a remaining p16(INK4A) allele, suggesting that other mechanisms must account for absent or low levels of p16(INK4A) protein , Here, we analyzed 9 Rb-positive NSCLC cell lines for the controls go verning p16(INK4A) activity. Four lines had homozygous deletions of p1 6(INK4A) (SK-LU-1, SK-MES-1, A-427, and SW900), and three had a point mutation in a single allele, First, in H520 cells, the previously repo rted deletion at codon 45 results in a frameshift that produces no det ectable protein, Second, in Calu-3 cells, a His to Tyr substitution at codon 83 produced a variant with a shortened half-life that was unabl e to form complexes with cdk4 or cdk6. Third, in H661 cells, the previ ously reported point mutation in the second intron splice donor site r esulted in a smaller p16(INK4A) protein, Although this variant formed complexes with cdk4 and cdk6, it had a profoundly reduced half-life, p roducing low steady-state levels of p16(INK4A) and abundant levels of free cdks, Finally, Calu-1 and Calu-6 cells transcribed no detectable mRNA encoding authentic p16(INK4A). These cell lines displayed methyla tion of the CpG island surrounding the first exon of p16(INK4A) and ex pressed abundant levels of a nontranslated mRNA containing an alternat ive first exon (E1 beta), as did all other cell lines in which the p16 (INK4A) locus was not deleted. These data indicate that Rb-positive NS CLC cells have evolved a variety of pathways to suppress p16(INK4A) ex pression, Reintroduction of p16(INK4A) into these cell lines by retrov iral transfer resulted in a reduced growth rate, increased abundance o f hypophosphorylated Rb, accumulation of cells in G(1), and a less tra nsformed morphology in Rb-positive, but not Rb-negative cells, suggest ing that loss of p16(INK4A) is essential for maintenance of the transf ormed phenotype.