In the article on DNA methylation changes in TRAMP in the December 15, 2006 issue of Cancer Research (1), the authors' description of the methylation status of the promoter region of p16INK4a in Fig. 6A (left-hand region) was in error. The methylation pattern shown, while correct, actually corresponds to the promoter of p19ARF, which, as indicated in the figure, lies ∼20 kB upstream of the RLGS-identified Not I site (1). In contrast, the p16INK4a start site lies ∼8 kB upstream of the Not I site (Fig. 1A). To define the methylation pattern of the region encompassing the p16INK4a transcriptional start site in normal prostates and TRAMP, the authors utilized sodium bisulfite sequencing (Fig. 1A-B). This analysis revealed a heterogeneous methylation pattern in approximately one-third of TRAMP tumor samples, and very little methylation in the remainder of TRAMP tumor samples, as well as in all normal prostates examined (Fig. 1B and data not shown). Interestingly, the authors reported that both the p16INK4a mRNA and protein are overexpressed in TRAMP tumors relative to normal prostate (1). To validate these data, the authors specifically reanalyzed both p16INK4a and p19ARF mRNA expression in TRAMP using quantitative PCR assays specific for either gene (Fig. 1C-D). The authors find that both genes are overexpressed in TRAMP tumors relative to normal prostates (Fig. 1C-D). The mechanism of p16INK4a and p19ARF overexpression in TRAMP, as well as its functional consequences, remains an important area for future investigation.

Figure 1.

DNA methylation and mRNA expression of p16INK4a and p19ARF in TRAMP. A, diagram of the CDKN2A locus in mice, illustrating the exons of p19ARF (open bars) p16INK4a (hatched bars), and the intervening introns. Bent arrows, transcriptional start sites; vertical arrow, the approximate position of the Not I site identified by RLGS (see ref. 1 for detailed explanation of RLGS); black bars, CpG islands; Regions A-C, the three regions in the CDKN2A locus that were analyzed by sodium bisulfite sequencing. Nucleotide sequence positions given are relative to the transcriptional start site of p19ARF. B, sodium bisulfite DNA sequencing methlyation analysis of the three regions indicated in (A). Data from two normal prostates of strain-matched animals (N1 and N2), and two primary tumors (P5 and P9) indentified as methlyated by RLGS are shown. Rows, individually sequenced alleles; open and filled circles, unmethylated and methylated CpG sites, respectively. C and D, mRNA expression of p16INK4a (C) and p16ARF (D) in different normal prostates (N #) and TRAMP primary tumor samples (P #). mRNA expression was measured by quantitatve real-time PCR using the SYBR green method and mRNA copy number was normalized relative to 18s rRNA copy number. Experimental conditions and primer sequences are available on request. The numbering scheme for the samples is the same as used in ref. 1.

Figure 1.

DNA methylation and mRNA expression of p16INK4a and p19ARF in TRAMP. A, diagram of the CDKN2A locus in mice, illustrating the exons of p19ARF (open bars) p16INK4a (hatched bars), and the intervening introns. Bent arrows, transcriptional start sites; vertical arrow, the approximate position of the Not I site identified by RLGS (see ref. 1 for detailed explanation of RLGS); black bars, CpG islands; Regions A-C, the three regions in the CDKN2A locus that were analyzed by sodium bisulfite sequencing. Nucleotide sequence positions given are relative to the transcriptional start site of p19ARF. B, sodium bisulfite DNA sequencing methlyation analysis of the three regions indicated in (A). Data from two normal prostates of strain-matched animals (N1 and N2), and two primary tumors (P5 and P9) indentified as methlyated by RLGS are shown. Rows, individually sequenced alleles; open and filled circles, unmethylated and methylated CpG sites, respectively. C and D, mRNA expression of p16INK4a (C) and p16ARF (D) in different normal prostates (N #) and TRAMP primary tumor samples (P #). mRNA expression was measured by quantitatve real-time PCR using the SYBR green method and mRNA copy number was normalized relative to 18s rRNA copy number. Experimental conditions and primer sequences are available on request. The numbering scheme for the samples is the same as used in ref. 1.

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1
Morey SR, Smiraglia DJ, James SR, Yu J, Moser MT, Foster BA, Karpf AR. DNA methylation pathway alterations in an autochthonous murine model of prostate cancer.
Cancer Res
2006
;
66
:
11659
–67.