IN-SITU ANALYSIS OF CHANGES IN TELOMERE SIZE DURING REPLICATIVE AGINGAND CELL-TRANSFORMATION

Telomeres have been shown to gradually shorten during replicative agin g in human somatic cells by Southern analysis. This study examines tel omere shortening at the single cell level by fluorescence in situ hybr idization (FISH). FISH and confocal microscopy of interphase human dip loid fibroblasts (HDFs) demonstrate that telomeres are distributed thr oughout the nucleus with an interchromosomal heterogeneity in size. An alysis of HDFs at increasing population doubling levels shows a gradua l decrease in spot size, intensity, and detectability of telomeric sig nal. FISH of metaphase chromosomes prepared from young and old HDFs sh ows a heterogeneity in detection frequency for telomeres on chromosome s 1, 9, 15, and Y. The interchromosomal distribution of detection freq uencies was similar for cells at early and late passage. The telomeric detection frequency for metaphase chromosomes also decreased with age . These observations suggest that telomeres shorten at similar rates i n normal human somatic cells. T-antigen transformed HDFs near crisis c ontained telomere signals that were low compared to nontransformed HDF s. A large intracellular heterogeneity in telomere lengths was detecte d in two telomerase-negative eel lines compared to normal somatic cell s and the telomerase-positive 293 cell line. Many telomerase-negative immortal cells had telomeric signals stronger than those in young HDFs , suggesting a different mechanism for telomere length regulation in t elomerase-negative immortal cells. These studies provide an in situ de monstration of interchromosomal heterogeneity in telomere lengths. Fur thermore, FISH is a reliable and sensitive method for detecting change s in telomere size at the single cell level.