Synthesis of a family of proteins called "heat shock" proteins is induced or enhanced in cells in response to various environmental stresses, suggesting that these proteins may perform functions essential to cell survival. Because a brief, nonlethal heat treatment can dramatically induce a transient resistance to a subsequent lethal heat treatment (thermotolerance), we examined the effect of heat treatment (41-460C) on protein synthesis and cell survival in.plateau-phase Chinese hamster fibroblast (HA-1) cells. After heat treatments that either drastically inhibited total protein synthesis (46C) or did not suppress it (41°C), the synthesis of'heat shock proteins was greatly enhanced over that in unheated cells, and cell survival was increased 102_ to 106-fold when cells were challenged by a subsequent lethal heat treatment. The synthesis of heat shock proteins correlated well with the development of thermotolerance, and the stability of these proteins correlated well with the persistence of thermotolerance up to 36 hr. Sodium arsenite, hypoxia, and ethanol also induced both the synthesis of heat shock proteins and transient thermotolerance. A qualitative analysis of individual proteins suggests that the synthesis and persistence ofpolypeptides of Mr 70,000 or 87,000 most closely conformed to the kinetics of thermotolerance.In the past few years, the induction or enhanced synthesis of a family of proteins in response to heat, other environmental stresses, and various chemical and mechanical treatments has been reported in cells from. yeast to mammals (1-6). The function of these "heat shock" proteins is not well understood, but they may be essential to cell survival after certain kinds of environmental stress (3, 7, 8 Heating. Thermotolerance can be induced in Chinese hamster ovary cells either by a short 450C heat treatment, followed by an incubation period at near-physiologic temperatures, or by a prolonged incubation at <430C (10). Heating of monolayers ofcells by the protocols described in Results was carried out in hot waterbaths in incubators (15). The pH of the medium overlaying the cells was maintained at 7.2-7.4 by a regulated gas flow of a mixture of air and CO2 and was monitored immediately before and after heating. The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

Recent findings intriguingly place DNA double-strand break repair proteins at chromosome ends in yeast, where they help maintain normal telomere length and structure. In the present study, an essential telomere function, the ability to cap and thereby protect chromosomes from end-to-end fusions, was assessed in repairdeficient mouse cell lines. By using fluorescence in situ hybridization with a probe to telomeric DNA, spontaneously occurring chromosome aberrations were examined for telomere signal at the points of fusion, a clear indication of impaired end-capping. Telomeric fusions were not observed in any of the repair-proficient controls and occurred only rarely in a p53 null mutant. In striking contrast, chromosomal end fusions that retained telomeric sequence were observed in nontransformed DNA-PKcs-deficient cells, where they were a major source of chromosomal instability. Metacentric chromosomes created by telomeric fusion became even more abundant in these cells after spontaneous immortalization. Restoration of repair proficiency through transfection with a functional cDNA copy of the human DNA-PKcs gene reduced the number of fusions compared with a negative transfection control. Virally transformed cells derived from Ku70 and Ku80 knockout mice also displayed end-to-end fusions. These studies demonstrate that DNA double-strand break repair genes play a dual role in maintaining chromosomal stability in mammalian cells, the known role in repairing incidental DNA damage, as well as a new protective role in telomeric end-capping.

Telomeres are specialized DNA/protein structures that act as protective caps to prevent end fusion events and to distinguish the chromosome ends from double-strand breaks. We report that TRF1 and Ku form a complex at the telomere. The Ku and TRF1 complex is a specific high-affinity interaction, as demonstrated by several in vitro methods, and exists in human cells as determined by coimmunoprecipitation experiments. Ku does not bind telomeric DNA directly but localizes to telomeric repeats via its interaction with TRF1. Primary mouse embryonic fibroblasts that are deficient for Ku80 accumulated a large percentage of telomere fusions, establishing that Ku plays a critical role in telomere capping in mammalian cells. We propose that Ku localizes to internal regions of the telomere via a high-affinity interaction with TRF1. Therefore, Ku acts in a unique way at the telomere to prevent end joining.