Regulation of telomere length and suppression of genomic instability in human somatic cells by Ku86 - PubMed (original) (raw)

Regulation of telomere length and suppression of genomic instability in human somatic cells by Ku86

Kyungjae Myung et al. Mol Cell Biol. 2004 Jun.

Abstract

Ku86 plays a key role in nonhomologous end joining in organisms as evolutionarily disparate as bacteria and humans. In eukaryotic cells, Ku86 has also been implicated in the regulation of telomere length although the effect of Ku86 mutations varies considerably between species. Indeed, telomeres either shorten significantly, shorten slightly, remain unchanged, or lengthen significantly in budding yeast, fission yeast, chicken cells, or plants, respectively, that are null for Ku86 expression. Thus, it has been unclear which model system is most relevant for humans. We demonstrate here that the functional inactivation of even a single allele of Ku86 in human somatic cells results in profound telomere loss, which is accompanied by an increase in chromosomal fusions, translocations, and genomic instability. Together, these experiments demonstrate that Ku86, separate from its role in nonhomologous end joining, performs the additional function in human somatic cells of suppressing genomic instability through the regulation of telomere length.

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Figures

FIG. 1.

FIG. 1.

Human Ku86-deficient cell lines have shortened telomeres. Genomic DNA was purified from the indicated cell lines, digested to completion with MboI and AluI, and then subjected to terminal restriction fragment Southern blot analysis under denaturing conditions with a (C3TA2)3 5′-end-radiolabeled oligonucleotide probe. Lanes: 1, HCT116 parental cell line; 2 and 3, HCT116 Ku86-heterozygous cell lines #44 and #70, respectively; 4, HCT116 p53-null cell line; 5 and 6, HCT116 p53-null Ku86-heterozygous cell lines #13 and #20, respectively. Approximate molecular size markers are shown on the far left.

FIG. 2.

FIG. 2.

Introduction of a human Ku86 cDNA into Ku86-heterozygous cells results in partial restoration of telomere length. (A) Whole-cell extracts were prepared from the indicated cell lines and subjected to immunoblot analysis with commercial monoclonal antibodies directed against either Ku86 or β-actin. The signals on the autoradiogram were quantitated by densitometry, and the ratio of the level of Ku86 to β-actin (K/β) expression is shown below each lane. (B) Genomic DNA was purified from the indicated cell lines and subjected to TRF analysis by denaturing gel electrophoresis. Lanes: 1, HCT116 parental cell line; 2 and 4, clone A6 cells harvested after 30 or 60 population doublings, respectively; 3, HCT116 Ku86-heterozygous cell line #70; 5 and 6, clone A10 cells harvested after 30 or 60 population doublings, respectively; 7 and 8, clone C7 and C5 cell lines (HCT116 Ku86+/− cell lines complemented with an empty vector), respectively. Approximate molecular size markers are shown on the far right.

FIG. 3.

FIG. 3.

The G-strand overhang is elongated in Ku86-heterozygous cells. (A) Genomic DNA was purified from the indicated cell lines and either left untreated (−) or digested overnight (+) with ExoI. The DNA was subsequently purified, digested to completion with MboI and AluI, and then subjected to terminal restriction fragment Southern blot analysis under nondenaturing (Native) conditions with a (C3TA2)3 5′-end-radiolabeled oligonucleotide probe. (B) The gels shown in panel A were denatured and rehybridized with the identical probe (Denatured). Exp., experiment.

FIG. 4.

FIG. 4.

Human Ku86-heterozygous cells contain chromosomes with telomere abnormalities. FISH analysis with a telomere-specific Cy3-(C3TA2)3 protein-nucleic acid probe of either wild-type cells (A) or three independent Ku86-heterozygous cells (B to D). Telomeres are seen as red dots, and the metaphase chromosomes are stained blue. In panel A, every chromosome contains four discrete spots of hybridization (two at each end). In panel B, the arrows point to chromosomes where no discernible hybridization was detected. In panel C, an example of two chromosomes that have fused is shown and the position of the internal telomere signal is designated by the arrow. In panel D, an example of a ring chromosome lacking any telomeric DNA is shown by the arrow.

FIG. 5.

FIG. 5.

Human Ku86-heterozygous cells contain chromosomal abnormalities. SKY analysis of either wild-type cells (A) or two independent Ku86-heterozygous cell lines (B and C). Each chromosome is represented in sets of three corresponding to the actual image, the 4′,6′-diamidino-2-phenylindole (DAPI)-stained image, and the computer-generated, false-color image, respectively. In panel A, the two derivative chromosomes (16 and 18) common to the parental cell line are marked with an asterisk. Panel B shows an example of a Ku86-heterozygous cell that contains—in addition to the parental translocations—a translocation of chromosome 15 to chromosome 10, each of which is also marked with an asterisk. Panel C shows an example of a Ku86-heterozygous cell that has become aneuploid and has sustained an amplification of chromosome 10. Panel D shows individual examples of the parental translocations, as well as some of the abnormalities detected in Ku86-heterozygous cells (see also Table 3).

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