Search

In the setting of inactivated DNA damage-sensitive checkpoints, critically shortened telomeres promote chromosomal instability and the types of widespread cytogenetic alterations that characterize most human carcinomas. Using a direct telomere fluorescence in situ hybridization technique, we analyzed 114 invasive breast carcinomas, 29 carcinoma in situ lesions, 10 benign proliferative lesions, and different normal epithelial components of the male and female breast. We found marked telomere shortening in the majority (52.5%) of invasive carcinomas; smaller subsets of invasive carcinoma demonstrated moderate telomere shortening (17.5%) or normal telomere lengths (21%), while a small subgroup (5%) contained elongated telomeres. Strikingly, the majority (78%) of ductal carcinoma in situ demonstrated markedly or moderately shortened telomeres. Surprisingly, unlike all other normal epithelia studied to date, moderate telomere shortening was observed in benign secretory cells in approximately 50% of histologically-normal terminal duct lobular units (from which most breast cancer is thought to arise), while such shortening was not seen in myoepithelial cells or normal large lactiferous ducts of the female breast or male breast ducts (from which breast cancer infrequently arises). We postulate that such shortening is the result of hormonally driven, physiological proliferation, and may delineate a population of epithelial cells at risk for subsequent malignant transformation.

Most conditional expression vectors designed for mammalian cells have been valuable systems for studying genes of interest by regulating their expressions. The available vectors, however, are reliable for the short-length cDNA clones and not optimal for relatively long fragments of genomic DNA or long cDNAs. Here, we report the construction of two bacterial artificial chromosome (BAC) vectors, capable of harboring large inserts and shuttling among Escherichia coli, yeast, and mammalian cells. These two vectors, pEYMT and pEYMI, contain conditional expression systems which are designed to be regulated by tetracycline and mouse interferons, respectively. To test the properties of the vectors, we cloned in both vectors the green fluorescence protein (GFP) through an in vitro ligation reaction and the 17.8-kb-long X-inactive-specific transcript (Xist) cDNA through homologous recombination in yeast. Subsequently, we characterized their regulated expression properties using real-time quantitative RT-PCR (TaqMan) and RNA-fluorescent in situ hybridization (FISH). We demonstrate that these two BAC vectors are good systems for recombination-based cloning and regulated expression of large genes in mammalian cells., (Copyright 2001 Academic Press.)