No abstract

Expression of 14-3-3 () is induced in response to DNA damage, and causes cells to arrest in G2. By SAGE (serial analysis of gene expression) analysis, we identified as a gene whose expression is 7-fold lower in breast carcinoma cells than in normal breast epithelium. We verified this finding by Northern blot analysis. Remarkably, mRNA was undetectable in 45 of 48 primary breast carcinomas. Genetic alterations at such as loss of heterozygosity were rare (1͞20 informative cases), and no mutations were detected (0͞34). On the other hand, hypermethylation of CpG islands in the gene was detected in 91% (75͞82) of breast tumors and was associated with lack of gene expression. Hypermethylation of is functionally important, because treatment of -non-expressing breast cancer cell lines with the drug 5-aza-2-deoxycytidine resulted in demethylation of the gene and synthesis of mRNA. Breast cancer cells lacking expression showed increased number of chromosomal breaks and gaps when exposed to ␥-irradiation. Therefore, it is possible that loss of expression contributes to malignant transformation by impairing the G 2 cell cycle checkpoint function, thus allowing an accumulation of genetic defects. Hypermethylation and loss of expression are the most consistent molecular alterations in breast cancer identified so far.A lthough many studies have identified critical genetic and epigenetic changes that mark the transformation of cells in tissues such as colon, pancreas, and lung, similar studies in breast cancer have met with limited success. In this paper we report the identification of a gene, 14-3-3 (), whose expression is undetectable in 94% (45͞48) of breast tumors.was originally identified as an epithelial-specific marker, HME1, which was down-regulated in a few breast cancer cell lines but not in cancer cell lines derived from other tissue types (1). Later studies showed that protein (also called stratifin) was abundant in differentiated squamous epithelial cells, but decreased by 95% in simian virus 40-transformed epithelial cells and in primary bladder tumors (2-4).We investigated the molecular mechanism underlying the low expression of in breast cancers. We find that genetic alterations such as loss of heterozygosity (LOH) and intragenic mutations are not major contributing mechanisms for lack of expression. Instead, we show that hypermethylation of the CpG-rich region in the gene is associated with its transcriptional silencing in the majority of breast tumors. Treatment of breast cancer cell lines that do not express with the DNA methyltransferase inhibitor, 5-aza-2Ј-deoxycytidine (5-aza-dC), leads to partial demethylation of this CpG island and synthesis of mRNA. Thus, hypermethylation appears to be responsible for silencing of gene expression.Recent studies have shed light on the function of . It was originally identified as a p53-inducible gene that is responsive to DNA damaging agents (5). apparently sequesters the mitotic initiation complex, cdc2-cyclin B1, in the cytoplasm after DNA damage (6). This prevents cdc...

We have identi®ed 14-3-3 s (s) as a gene whose expression is lost in breast carcinomas, primarily by methylation-mediated silencing. In this report, we investigated the timing of loss of s gene expression during breast tumorigenesis in vivo. We analysed the methylation status of s in breast cancer precursor lesions using microdissection for selective tissue sampling. We found hypermethylation of s in 24 of 25 carcinomas (96%), 15 of 18 (83%) of ductal carcinoma in situ, and three of eight (38%) of atypical hyperplasias. None of the ®ve hyperplasias without atypia showed s-hypermethylation. Unexpectedly, patients with breast cancer showed s hypermethylation in adjacent histologically normal breast epithelium, while this was never observed in individuals without evidence of breast cancer. Also, samples of periductal stromal breast tissue were consistently hypermethylated, underscoring the importance of selective tissue sampling for accurate assessment of 14-3-3-s methylation in breast epithelium. These results suggest that hypermethylation of 14-3-3-s occurs at an early stage in the progression to invasive breast cancer, and may occur in apparently normal epithelium adjacent to breast cancer. These results provide evidence that loss of expression of s is an early event in neoplastic transformation. Oncogene (2001) 20, 3348 ± 3353.