Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 Dec 15;9(24):4893-9.
doi: 10.4161/cc.9.24.14157. Epub 2010 Dec 15.

MCPH1 patient cells exhibit delayed release from DNA damage-induced G2/M checkpoint arrest

Ioannis Gavvovidis  1 , Charlotte Pöhlmann, Juan Alberto Marchal, Markus Stumm, Daisuke Yamashita, Tatsuya Hirano, Detlev Schindler, Heidemarie Neitzel, Marc Trimborn
Affiliations

MCPH1 patient cells exhibit delayed release from DNA damage-induced G2/M checkpoint arrest

Ioannis Gavvovidis et al. Cell Cycle. .

Abstract

Mutations in the MCPH1 gene cause primary microcephaly associated with a unique cellular phenotype of misregulated chromosome condensation. The encoded protein contains three BRCT domains, and accumulating data show that MCPH1 is involved in the DNA damage response. However, most of this evidence has been generated by experiments using RNA interference (RNAi) and cells from non-human model organisms. Here, we demonstrate that patient-derived cell lines display a proficient G2/M checkpoint following ionizing irradiation (IR) despite homozygous truncating mutations in MCPH1. Moreover, chromosomal breakage rates and the relocation to DNA repair foci of several proteins functioning putatively in an MCPH1-dependent manner are normal in these cells. However, the MCPH1-deficient cells exhibit a slight delay in re-entering mitosis and delayed resolution of γH2AX foci following IR. Analysis of chromosome condensation behavior following IR suggests that these latter observations may be related to hypercondensation of the chromatin in cells with MCPH1 mutations. Our results indicate that the DNA damage response in human cells with truncating MCPH1 mutations differs significantly from the damage responses in cells of certain model organisms and in cells depleted of MCPH1 by RNAi. These subtle effects of human MCPH1 deficiency on the cellular DNA damage response may explain the absence of cancer predisposition in patients with biallelic MCPH1 mutations.

PubMed Disclaimer

Figures

Figure 1
Figure 1
DNA repair and G2/M checkpoint response of MCPH1 patient cells. (A) To analyze chromosomal breakage rates, lymphoblastoid cell lines (LCLs) derived from MCPH1 patients were exposed to different doses of IR. Metaphase spreads were prepared according to standard protocols at 0 h, 6 h and 24 h following irradiation. The number of chromosomal breaks per cell is shown to the left, and the percentage of aberrant metaphase cells is presented to the right. (B) G2/M checkpoint analysis. MCPH1 patients with the indicated homozygous mutations, LCLs derived from normal controls, and one ATM-mutated cell line (A-T) were irradiated with 1 Gy, and their mitotic indices were determined using flow cytometry by staining of mitotic cells with an antibody to phospho-histone H3. (C) G2/M checkpoint release. Cells were processed and analyzed as described above. Mitotic indices in patient and control LCLs were determined sequentially at the indicated time points after exposure to 1 Gy. (D) Immunofluorescence analysis of γH2AX IRIF in the cells analyzed in (C). The data represent the proportion of cells with >10 γH2AX foci. Error bars indicate the SD.
Figure 2
Figure 2
IRIF formation in MCPH1 patient cells. (A) Immunofluorescence micrographs showing 53BP1, NBS1 and RAD51 IRIF (red) in HeLa cells and transformed fibroblasts derived from a patient with a homozygous truncating MCPH1 mutation (c.427dupA/p.T143NfsX5) following irradiation with 10 Gy. The two righthand columns show fibroblasts from the same MCPH1 patient stably expressing EGFP or EGFP fused to wild-type MCPH1. Nuclei were counterstained with DAPI (blue). (B) Quantification of cells with >10 IRIF. Error bars indicate the SD of three different measurements, each numbering approximately 200 nuclei. Scale bar = 5 µm.
Figure 3
Figure 3
Chromosome condensation behavior and cell cycle distribution in response to IR. (A) LCLs derived from two MCPH1 patients with homozygous truncating mutations were irradiated with 0.5 Gy, 1 Gy or 2 Gy, and cytogenetic slides were prepared at varying times following IR as indicated. The fractions of mitotic and prophase-like cells were determined by counting 1,000 cells per time point. (B) Immunofluorescence micrographs showing 53BP1 IRIF (red) in fibroblasts from an MCPH1 patient with hypercondensed, prophase-like chromatin morphology. Nuclei were counterstained with DAPI (blue). Scale bar = 5 µm. (C) Cell cycle distribution of MCPH1 patient and control LCLs at varying times after 1 Gy IR as determined by flow cytometry. Error bars indicate SD. (D) Determination of the proportion of cells with a prophase-like appearance in the same experiment.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Neitzel H, Neumann LM, Schindler D, Wirges A, Tonnies H, Trimborn M, et al. Premature chromosome condensation in humans associated with microcephaly and mental retardation: a novel autosomal recessive condition. Am J Hum Genet. 2002;70:1015–1022. - PMC - PubMed
    1. Jackson AP, Eastwood H, Bell SM, Adu J, Toomes C, Carr IM, et al. Identification of microcephalin, a protein implicated in determining the size of the human brain. Am J Hum Genet. 2002;71:136–142. - PMC - PubMed
    1. Trimborn M, Bell SM, Felix C, Rashid Y, Jafri H, Griffiths PD, et al. Mutations in microcephalin cause aberrant regulation of chromosome condensation. Am J Hum Genet. 2004;75:261–266. - PMC - PubMed
    1. Trimborn M, Schindler D, Neitzel H, Hirano T. Misregulated chromosome condensation in MCPH1 primary microcephaly is mediated by condensin II. Cell Cycle. 2006;5:322–326. - PubMed
    1. Thornton GK, Woods CG. Primary microcephaly: Do all roads lead to Rome? Trends Genet. 2009;25:501–510. - PMC - PubMed

Publication types

LinkOut - more resources