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. 2004 Aug 4;23(15):3164-74.
doi: 10.1038/sj.emboj.7600315. Epub 2004 Jul 29.

ATR functions as a gene dosage-dependent tumor suppressor on a mismatch repair-deficient background

Yanan Fang  1 , Cheng-Chung Tsao, Barbara K Goodman, Ryohei Furumai, Carlos A Tirado, Robert T Abraham, Xiao-Fan Wang
Affiliations

"V体育平台登录" ATR functions as a gene dosage-dependent tumor suppressor on a mismatch repair-deficient background

VSports注册入口 - Yanan Fang et al. EMBO J. .

"V体育官网入口" Abstract

The ataxia-telangiectasia mutated and rad3-related (ATR) kinase orchestrates cellular responses to DNA damage and replication stress. Complete loss of ATR function leads to chromosomal instability and cell death. However, heterozygous ATR mutations are found in human cancers with microsatellite instability, suggesting that ATR haploinsufficiency contributes to tumorigenesis. To test this possibility, we generated human cell line and mouse model systems in which a single ATR allele was inactivated on a mismatch repair (MMR)-deficient background. Monoallelic ATR gene targeting in MLH1-deficient HCT 116 colon carcinoma cells resulted in hypersensitivity to genotoxic stress accompanied by dramatic increases in fragile site instability, and chromosomal amplifications and rearrangements. The ATR(+/-) HCT 116 cells also displayed compromised activation of Chk1, an important downstream target for ATR. In complementary studies, we demonstrated that mice bearing the same Atr(+/-)/Mlh1(-/-) genotype were highly prone to both embryonic lethality and early tumor development VSports手机版. These results demonstrate that MMR proteins and ATR functionally interact during the cellular response to genotoxic stress, and that ATR serves as a haploinsufficient tumor suppressor in MMR-deficient cells. .

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Figures

Figure 1
Figure 1
Impaired survival of ATR+/− cells after genotoxic stress. (A) Schematic diagram of ATR-targeting construct. The neomycin resistance (Neo) gene-targeting construct is flanked by two loxP sites to allow Cre recombinase-mediated excision of the drug resistance cassette for targeting of the second ATR allele (not performed in the present study). Primers A, B, and C were used in the PCR screen to identify targeted clones. ‘A' primers (A1 and A2) recognize regions of the ATR gene located outside of the 5′-flanking region used in the targeting cassette. ‘C' primers (C1 and C2) hybridize to different sequences in the neomycin resistance marker (Neo). The dark line labeled ‘Probe' shows the source of ATR genomic probe used or the Southern blot analysis in Figure 6. (B) Identification of targeted clones by PCR analysis with primer pairs indicated in panel A. (C) Expression of ATR protein in HCT 116-derived clones. Sample lanes are identified in panel B legend. The relative ratio indicates the level of ATR protein in each sample lane after normalization of each sample to the tubulin loading control. The resulting ratios were then normalized to that obtained with the HCT 116 cell extract.
Figure 2
Figure 2
Reconstitution of ATR function in ATR+/− clones. (A) ATR+/− B24 cells were stably transfected with an AU1-tagged ATR expression plasmid. Transfected clones A29 and A30 were selected for clonogenic survival assays. (B) Clonogenic survival of HCT 116, ATR+/− B24, and clone A29 and A30 cells was determined after exposure to the indicated doses of UV.
Figure 3
Figure 3
Spontaneous chromosomal instability in ATR+/− cells. (A) Karyotypic analysis. The plot shows the percentage of metaphases with abnormalities for each cell line. The total number of metaphase spreads examined for each cell line were as follows: HCT 116, 11; clone B24, 16; clone 12, 15; clone B74, 17; HCT116.chr3: 11. (B) Representative metaphase spread from HCT 116 cells. Karyotype was stably maintained as 45, X, −Y, add 10q, add 16p, add 18p. The four signature alterations in these chromosomes are marked with open arrows. (C, D) Karyotype of two representative cells from the same ATR+/− clone B74 population. In addition to the four signature chromosomal alterations detected in the parental HCT 116 cells (open arrows), random gross chromosomal aberrations (solid arrows) and an HSR (curved arrow) located between the centromere and the dark band were observed in this metaphase spread.
Figure 4
Figure 4
S and G2 checkpoint defects in ATR+/− cells. (A) Parental HCT 116 cells and ATR+/− clone B74 and 12 cells were treated with nocodazole at 30 min after IR exposure. Approximately 200 cells were counted to determine mitotic index. Results shown are representative of two to three experiments for each sample. (B) The same cells were treated with nocodazole only to monitor normal G2-to-M-phase progression. (C) Cells were treated for 1 h with the indicated concentrations of APH, and detergent-soluble proteins were separated by SDS–PAGE and immunoblotted with the indicated antibodies.
Figure 5
Figure 5
Fragile site expression and SCE in ATR+/− cells. (A) Average numbers of chromosome breaks and gaps per metaphase observed in 25 metaphase spreads from each cell line. Fragile sites were induced by cellular exposure to 0.1 μM APH. *P<0.05. (B) Representative metaphase spread from APH-treated ATR+/− cells. The solid arrows point to fragile sites. (C) Enhanced phosphorylation of histone H2AX in ATR+/− cells after APH treatment. Cells from the parental line and two ATR+/− clones were treated as indicated, and cell extracts were analyzed by immunoblotting with anti-γH2AX antibody. (D) Representative metaphase from ATR+/− cells showing SCE. Sister chromatids were stained differentially and imaged as light or dark strands. The solid arrows point to SCE sites. (E) Average SCE frequencies in HCT 116 cells (n=20 metaphases) and two ATR+/− clones (n=25 metaphases per clone). *P<0.005.
Figure 6
Figure 6
Gene amplification and rearrangement events in ATR+/− cells. (A) Southern blot analysis with the DNA probe shown in Figure 1A. The ATR+/− clones used in this experiment were used at passages 3–5 after recovery from primary frozen stocks, and approximately 35 to 45 population doublings had occurred before the cells were harvested for these experiments. Lane 1, HCT 116; lane 2, a nonhomologously targeted clone; lane 4, clone 12; lane 8, clone B24; lane 9, clone B74. Lanes 3 and 5–7 show results from additional ATR+/− clones coisolated with the three named clones selected for further analyses. (B) Amplification of a chromosomal region containing the ATR locus (marked with red fluorophore) in metaphase and interphase nuclei from ATR+/− cells. (C) Appearance of amplified ATR locus in other chromosomes. The green fluorophore marks chromosome 3 alpha satellite (centromeric) DNA. Regions stained with red and green fluorophores are marked by arrows. (D) Amplification and rearrangement of p21/WAF1 locus during gene targeting. Lane 1, HCT 116 cells; lanes 2–5, hygromycin-resistant clones derived from HCT 116 cells transfected with the p21/WAF1-targeting vector; lane 6, ATR+/− cells (clone 12); lanes 7–11, hygromycin-resistant clones derived from ATR+/− cells transfected with the p21/WAF1-targeting vector. Southern blot was performed as described (Waldman et al, 1995). Restriction fragment from the wild-type p21/WAF1 allele is indicated with an arrow and the additional p21/WAF1-derived fragment found in ATR+/− cells (lane 6) is indicated with a cross. (E) PALA-induced CAD gene amplification. PALA-resistant cell colonies were scored as described in the text. Results shown are representative of two independent trials.
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"V体育ios版" References

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    1. Brown EJ, Baltimore D (2003) Essential and dispensable roles of ATR in cell cycle arrest and genome maintenance. Genes Dev 17: 615–628 - "VSports手机版" PMC - PubMed

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