897

Introduction: Tumor suppressor gene silencing by DNA methylation is common in cancers. Methylation-sensitive arbitrarily primed PCR (Ms-APPCR) was used to identify a novel sequence hypermethylated in breast cancer. Methods: Breast tissue from patients undergoing reduction mammoplasty (n=5) or surgery for breast cancer (both normal, n=44, and cancer, n=43) was obtained fresh from the operating room and snap frozen. DCIS tissue was microdissected from parafinized blocks. Genomic DNA was extracted from the tissues. MCF7 and MDA231 cells were grown under standard conditions, harvested, and DNA and RNA isolated. Ms-APPCR was performed on genomic DNA from matched tumor/normal tissue. A 222 bp fragment hypermethylated in cancer was identified, gel extracted, cloned and sequenced. BLAST search revealed a single match with 100% homology. DNA from tissues and cell lines was bisulfite converted and used for methylation assays using the MethyLight technique targeting 2 different regions of the 5’ CpG island (HPP1A (bp197-289), and HPP1B (bp332-478)). Normal and malignant tissue cDNA panels (Clonetech) and cell line RNA were used for expression analyses by RT-PCR. Primers/probes for MethyLight and RT-PCR were designed using the full-length sequence obtained from GenBank. Results: A sequence hypermethylated in breast cancer was identified by Ms-APPCR. BLAST search identified the sequence as the 5’UTR of HPP1 (AF264150), a gene originally identified as a sequence hypermethylated in hyperplastic polyps of the colon. Results of the methylation assays are shown in the table. HPP1 was strongly expressed in normal prostate, testis, ovary, small and large intestine, esophagus, stomach, and liver, but weakly or not expressed in spleen, thymus, peripheral blood leukocyte. HPP1 was strongly expressed in 1 of 2 colon cancers and in an ovarian cancer, whereas weak or no HPP1 expression was seen in cancers of the breast, lung, prostate, and pancreas, or in 2 breast cancer cell lines. Conclusion: MS-AP-PCR is an effective method to identify sequences that are hypermethyalted in cancer. HPP1 is hypermethylated in breast cancers compared to normal tissue. Different regions of the 5’UTR display different methylation patterns. HPP1 does not appear to be hypermethylated in preinvasive breast cancer (DCIS), but more data is needed to confirm this. HPP1 is expressed in most normal tissues but not in the normal cells and tissues of the hematologic system. Expression is often lost or down-regulated in tumor tissue. HPP1 may be an important tumor suppressor gene in many tumors. Its function remains unclear.

[Proc Amer Assoc Cancer Res, Volume 45, 2004]