ABSTRACT
BACKGROUND: Activation of the enzyme telomerase, which has been associated with cellular immortality, may constitute a key step in the development of human cancer. Telomerase is repressed in most normal human somatic cells. This study was conducted, using a genetic complementation approach, with the aim of identifying and mapping the genes responsible for repressing telomerase and, simultaneously, to establish the effect of experimentally induced telomerase repression on human tumor cell growth. METHODS: Individual human chromosomes isolated from normal diploid cells and tagged with bacterial antibiotic resistance genes (for later selection) were introduced into cells of the human breast carcinoma cell line 21NT by means of microcell transfer. Selected hybrid clones were screened for telomerase activity by use of the polymerase chain reaction-based telomere repeat amplification protocol (TRAP) assay, and the proliferative fate of the hybrid clones was determined. Regions of the introduced chromosomes associated with telomerase repression were mapped using segregant hybrids and a deletion analysis that employed microsatellite DNA markers. RESULTS: Strong repression of telomerase was observed following transfer of human chromosome 3 into 21NT cells but not after transfer of chromosomes 8, 12, or 20. The vast majority of hybrid clones with repressed telomerase entered permanent growth arrest after 10-18 population doublings. Deletion analysis of nonrepressed segregant monochromosome 3 hybrids indicated two regions on the short arm of chromosome 3 (3p21.3-p22 and 3p12-21.1) where telomerase regulator genes may be located. CONCLUSIONS: Telomerase in human breast cancer cells is efficiently repressed by a gene or genes on normal human chromosome 3p, and this repression is associated with permanent growth arrest of the tumor cells.
Subject(s)
Breast Neoplasms/pathology , Carcinoma, Intraductal, Noninfiltrating/pathology , Chromosomes, Human, Pair 3/genetics , Gene Expression Regulation, Enzymologic , Gene Transfer Techniques , Growth Inhibitors/genetics , Neoplasm Proteins/biosynthesis , Repressor Proteins/genetics , Telomerase/biosynthesis , Adult , Carcinoma, Intraductal, Noninfiltrating/enzymology , Cell Division/drug effects , Cell Fusion , Chromosomes/metabolism , Chromosomes/ultrastructure , Female , Growth Inhibitors/physiology , Humans , Hybrid Cells , In Situ Hybridization, Fluorescence , Neoplasm Proteins/genetics , Neoplasm Proteins/physiology , Polymerase Chain Reaction , Repressor Proteins/physiology , Telomerase/genetics , Telomerase/physiology , Telomere/metabolism , Tumor Cells, CulturedABSTRACT
We have used single-strand conformation polymorphism (SSCP) analysis to screen for mutations in the BCL10 gene in 81 primary prostate carcinomas, 20 squamous cell cancers of the head and neck, 15 small-cell lung cancer cell lines, 24 renal carcinoma cell lines and 13 sarcoma cell lines. We failed to find evidence of somatically acquired mutations of the BCL10 gene suggesting that BCL10 does not play a major role in the development of these malignancies.