Mutations to Ku reveal differences in human somatic cell lines

Abstract

NHEJ (non-homologous end joining) is the predominant mechanism for repairing DNA double-stranded breaks in human cells. One essential NHEJ factor is the Ku heterodimer, which is composed of Ku70 and Ku86. Here we have generated heterozygous loss-of-function mutations for each of these genes in two different human somatic cell lines, HCT116 and NALM-6, using gene targeting. Previous work had suggested that phenotypic differences might exist between the genes and/or between the cell lines. By providing a side-by-each comparison of the four cell lines, we demonstrate that there are indeed subtle differences between loss-of-function mutations for Ku70 versus Ku86, which is accentuated by whether the mutations were derived in the HCT116 or NALM-6 genetic background. Overall, however, the phenotypes of the four lines are quite similar and they provide a compelling argument for the hypothesis that Ku loss-of-function mutations in human somatic cells result in demonstrable haploinsufficiencies. Collectively, these studies demonstrate the importance of proper biallelic expression of these genes for NHEJ and telomere maintenance and they provide insights into why these genes are uniquely essential for primates.

Fig. 1

Scheme for functional inactivation of the human Ku70 and Ku86 loci. (A) Partial Ku70 and Ku86 genomic loci. Open rectangles with numbers represent exons. (B) The cartoon of the targeting vector. In the targeting vector open boxes designate the left and right inverted terminal repeats; triangles, loxP sites; PGK, phosphoglycerate kinase eukaryotic promoter; Neo, neomycin resistance gene; EM7, EM7 prokaryotic promoter; Zeo, zeomycin resistance gene; dotted rectangles, left and right homology arms to facilitate targeting by homologous recombination. (C) In the targeted allele of Ku70, exons 3 & 4 have been replaced by the targeting vector. a and b are external probes while c is an internal probe. HindIII (Hi) restriction enzyme sites and an unique FspI (Fs) restriction site in the targeting construct are shown. Also shown are the primers used for the diagnostic PCR screening. (D) In the targeted allele of Ku86, exon 1 has been replaced by the targeting vector. a’ and b’ are 5′ and 3′ external probes respectively while c’ is an internal probe. Ap is a ApaLI restriction site in the targeting construct. Also shown are the primers used for the diagnostic PCR screening.

Fig. 2

Identification of Ku70 and Ku86 heterozygous cell lines. (A) Diagnostic genomic PCR carried out with HCT116 Ku70^+/- cells. The 3′-PCR was carried out using the primer set RArmF and Ku703,4R1 (Fig. 1C) while the 5′-PCR was performed using the primers LArmR and Ku703,4F1 (Fig. 1C). An ethidium-bromide stained agarose gel picture is shown. Genomic DNA was isolated from two heterozygous clones (#53 and #106), a randomly targeted clone (#108) and from WT HCT116 cell line. (B) Diagnostic genomic PCR performed with the NALM-6 clones. The same primer sets were used as described above in (A). The gel shows the PCR performed with DNA from WT, two Ku70^+/- clones (N31 and N35) and a randomly targeted clones (N50). (C) Southern blot analysis of WT (HCT116 and NALM-6) and Ku70^+/- cell lines (#53, #106, N31 and N35). Genomic DNA was doubly digested with HindIII and FspI and hybridized with probe c (Fig. 1C). (D) Genomic PCR carried out on WT NALM-6, Ku86^+/- clones (N197 and N397) and two randomly targeted clones (N205 and N310). The upper panel shows the 3′-PCR using the primers RArmF and KU861R while the lower panel shows the 5′-PCR using the primers LArmR and KU861F. M indicates the DNA ladder lane. (E) Southern hybridization of Ku86^+/- NALM-6 clones using 5′- and 3′-flanking probes, a’ and b’, respectively. Approximate molecular markers are shown on the left. The 5′-probe and 3′ probe are shown in Fig. 1D. (F) Southern hybridization of Ku86^+/- NALM-6 clones using an internal probe, c’ (Fig. 1D).

Fig. 3

Growth defects of human somatic cells with reduced Ku expression. (A) Ku70^+/- HCT116 cells show defects in their proliferation rate. 3 × 10³ cells from the indicated clones were seeded on tissue culture plates and the increase in cell number was determined using trypan blue staining and a hemocytometer in daily intervals. The average of three experiments, each done in triplicate is shown. (B) Ku70^+/- NALM-6 clones. The proliferation rate was determined as described above. (C) Ku86^+/- NALM-6 cells. The proliferation rate was determined as in (A).

Fig. 4

The IR sensitivity profiles of human somatic cells expressing reduced levels of Ku. HCT116-derived, but not NALM-6-derived, cell lines are IR^s. For HCT116 (A), 300 cells of the indicated cell lines were seeded on tissue culture plates and X-irradiated at the indicated doses. Cells surviving to form colonies between 10-14 days later were scored. For NALM-6 Ku70^+/- clones (B), control plates (no irradiation) contained 10, 5 and 2.5 cells per well of a 96-well plate. Plates that were used for irradiation had 10, 100 or 1000 cells per well of a 96-well plate. For NALM-6 Ku86^+/- clones (C), control plates (no irradiation) contained 5, 10 and 20 cells per well while the plates used for irradiation had 100, 500 and 1000 cells per well of a 96-well tissue culture plate. Following irradiation, all the plates were allowed to recover for 20 days before wells containing colonies were scored.

Fig. 5

Telomere shortening in human Ku70 and Ku80-deficient cell lines. Genomic DNA was purified from the indicated cell lines, digested to completion with AluI and MboI, and then subjected to terminal restriction fragment Southern blot analysis under denaturing conditions with a (C₃TA₂)₃ 5′-end-radiolabeled oligonucleotide probe. (A) Telomere lengths of Ku70^+/- HCT116 clones. (B) Telomere lengths of Ku70^+/- NALM-6 clones. (C) Telomere lengths of Ku86^+/- NALM-6 clones. Approximate molecular size markers are shown.