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We have identified 14-3-3 sigma (sigma) 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 sigma gene expression during breast tumorigenesis in vivo. We analysed the methylation status of sigma in breast cancer precursor lesions using microdissection for selective tissue sampling. We found hypermethylation of sigma 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 five hyperplasias without atypia showed sigma-hypermethylation. Unexpectedly, patients with breast cancer showed sigma 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-sigma methylation in breast epithelium. These results suggest that hypermethylation of 14-3-3-sigma 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 sigma is an early event in neoplastic transformation.

The increasing number of breast carcinoma in situ detected by screening procedures makes it imperative to develop improved markers to stratify the risk of invasive cancer. Telomerase is detectable in invasive cancer, but not in normal tissues. We have microdissected frozen tissue blocks containing both DCIS and invasive cancer to assay the telomerase activity of these two lesions. The 46 available cases of concurrent DCIS and invasive breast cancer resulted in 43 DCIS samples and 38 invasive cancer samples adequate for analysis. Seventy per cent of the DCIS and all invasive cancer samples tested had detectable telomerase activity. In addition, we analysed telomerase activity in ten cases of DCIS that were not associated with invasive cancer, and detected telomerase activity in seven (70%). Mixing experiments showed no evidence of telomerase inhibitors in telomerase negative samples. Furthermore, periductal inflammatory infiltrates were shown to be a potential confounding source of telomerase activity. Since DCIS lesions appear to be heterogeneous with respect to telomerase activity, and telomerase activation appears to precede the development of invasive cancer, telomerase activity may be a useful adjunct in stratifying the risk of developing invasive breast cancer in patients with DCIS.

Chinese hamster cells frequently have altered karyotypes. To investigate the basis of recent observations that karyotypic alterations are related to telomeric fusions, we asked whether these alterations are due to lack of telomere repeat binding factor/s. Further, Chinese hamster chromosomes contain large blocks of interstitial telomeric repeats, which are preferentially involved in chromosome breakage and exchange, rendering it an interesting model for such studies. Here, we report on the cloning and the chromosomal localization of the Chinese hamster telomere repeat binding factor, chTRF1. The sequence analysis revealed, similar to human TRF1 (hTRF1), an N-terminal acidic domain, a TRF1 specific DNA binding motif and a C-terminal Myb type domain. Unlike mouse TRF1 (mTRF1), chTRF1 shows 97.5% identity to hTRF1. chTRF1 gene was localized on the long arm of chromosome 5. In vitro translation of chTRF1 resulted in protein product similar in molecular weight to hTRF1. Immunostaining of Chinese hamster ovary cells (CHO) with anti-TRF1 antibody revealed punctate nuclear staining. At metaphase, antibodies failed to detect TRF1 on most of the chromosome ends and the interstitial telomeric repeat bands. These studies suggest that chTRF1 does not bind the interstitial telomeric repeats, and its presence at the metaphase chromosome ends is limited. The later could be a factor contributing to frequent karyotypic alterations observed in Chinese hamster cells.