Methylation of p16 in sputum from
smokers. Bisulfite modified DNA was amplified by primers specific to
methylated or unmethylated alleles of the p16 gene as
described. Unmethylated p16 alleles were detected in all
16 sputum samples and in DNA from normal human bronchial epithelial
(NHBE) cells obtained from a never-smoker. The Calu1 cell line
(positive control for p16 methylation) is completely
methylated. Methylated alleles of p16 were observed in
DNA from 4 cancer-free persons (lanes 3, 10, 13, and 14), 1 person
diagnosed at the time of sputum induction with a SCLC (lane 2) who
developed a NSCLC 2 years later, 1 smoker (lane 15) who was positive
for lung cancer at the time of sputum induction, and in the positive
control (Calu1). No methylation was observed in the negative control
normal human bronchial epithelial cells. Samples negative for
p16 methylation are shown in lanes 1, 4–9, 11–12, and
16.