Fig. 5. Conservation of global chromosomal DNA methylation patterns. MIRA-assisted microarrays were conducted for pairs of normal lung tissue (N, green) and corresponding SCCs (T, red). The profile of methylated DNA sequences is displayed as the ratio of MIRA-enriched DNA versus input DNA. Representative tiling array data are shown demonstrating the general conservation of chromosomal methylation profiles between different individuals and, at this level of resolution, also between normal and tumor tissue. A segment of the long arm of chromosome 6 is shown.

Fig. 6. Examples of hypomethylation of SINE elements on chromosome 8. The low resolution methylation profile of the short arm of chromosome 8 is shown at the top. Selected tumor-specifically hypomethylated sequences are shown in the middle at high resolution. The blue bars indicated SINE elements. At the bottom, intra-SINE element hypomethylation in the tumor was confirmed by bisulfite-based COBRA assays. After digestion with BstUI after sodium bisulfite-treatment and locus-specific PCR, only the methylated DNA will be cut. Reduced BstUI cleavage indicates hypomethylation in the tumor.

Fig. 7. Hypomethylation of the subtelomeric region of chromosome 8. This region is rich in repetitive DNA elements. The lower scan shows that the sequences between 0.20 and 0.35 Mb are substantially undermethylated in the tumor.

Fig. 8. Hypomethylation of a CpG-rich sequence in an exon of the C8orf72 gene. The methylation profiles are shown at different levels of resolution. Bisulfite sequencing was used to verify the methylation differences between normal tissue and tumor for segments A and B of the hypomethylated region. The nearest LINE or SINE element is >5 kb away from the hypomethylated target.

Fig. 9. Hypomethylation of LINE and HERV sequences in lung SCCs. Methylation of LINE elements was analyzed by bisulfite conversion of DNA followed by PCR with consensus primers for the LINE1 promoter and HERV sequences. The PCR products were cleaved with HinfI, which cleaves only methylated DNA after bisulfite conversion. The percentage of methylation was determined after scanning of the gels and quantitation of the uncut (unmethylated) fragment relative to the total signal. LINE1 sequences were substantially hypomethylated, whereas HERV sequences showed only a small degree of hypomethylation in SCC tumors.

Fig. 10. Cancer-associated hypomethylation of an area of chromosome 8p that is rich in segmental duplications. The region between positions 7,608,000 and 7,670,000 is substantially hypomethylated in the tumor. This sequence contains LTR and LINE elements and simple repeats. It is rich in segmental duplications. One of these duplications is a direct repeat of 30.5 kb (7,608,484-7,639,048 and 7,639,049-7,669,649). Additional duplicate copies of this region are present on chromosomes 8, 10, 11, 12, and 13. Although it is not possible to tell which particular chromosomal segment hybridizes to the probes on the array, it is clear that these duplicated sequences underwent extensive demethylation in the tumor sample. Information on repeats and duplications was taken from the UCSC Genome Browser, March 2006 assembly (http://genome.ucsc.edu/cgibin/hgGateway). Gray, 90-98% similarity; yellow, 98-99% similarity; orange, >99% similarity.

Fig. 11. Absence of methylation of SCC marker genes in normal blood and lung DNA. (A) (Upper) DNA was isolated from pooled leukocytes of normal healthy individuals. (Lower) DNA from noncancerous lung was pooled from two patients who underwent lung surgery for necrotizing granulomatous infection. PCR was performed on sodium bisulfite-treated DNA, and the methylation status of the individual CpG islands was analyzed by COBRA assay by using BstUI digestion. Digestion by BstUI indicates methylation of the sequence tested. The positive control (+Ctrl) is the PAX6 CpG island from an SCC sample. (B) Bisulfite sequence analysis of six frequently methylated CpG islands in DNA from a tumor (SCC, stage I), noncancerous lung, and leukocyte DNA.