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DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis

Abstract

During the evolution of cancer, the incipient tumour experiences ‘oncogenic stress’, which evokes a counter-response to eliminate such hazardous cells. However, the nature of this stress remains elusive, as does the inducible anti-cancer barrier that elicits growth arrest or cell death. Here we show that in clinical specimens from different stages of human tumours of the urinary bladder, breast, lung and colon, the early precursor lesions (but not normal tissues) commonly express markers of an activated DNA damage response. These include phosphorylated kinases ATM and Chk2, and phosphorylated histone H2AX and p53. Similar checkpoint responses were induced in cultured cells upon expression of different oncogenes that deregulate DNA replication. Together with genetic analyses, including a genome-wide assessment of allelic imbalances, our data indicate that early in tumorigenesis (before genomic instability and malignant conversion), human cells activate an ATR/ATM-regulated DNA damage response network that delays or prevents cancer. Mutations compromising this checkpoint, including defects in the ATM–Chk2–p53 pathway, might allow cell proliferation, survival, increased genomic instability and tumour progression.

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Figure 1: Constitutive activation of the ATM–Chk2–p53 pathway in human urinary bladder cancer.
Figure 2: Chk2 activation precedes p53 mutations and genomic instability in bladder cancer.
Figure 3: Phosphorylation and activation of Chk2 in early breast and colon tumours.
Figure 4: Overexpressed cyclin E, Cdc25A and E2F1 induce a DNA damage response in U-2-OS cells.
Figure 5: DNA damage response to overexpressed oncogenes in U-2-OS cells.
Figure 6: Oncogenes and DNA damage response in early lesions.

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Acknowledgements

We thank Y. Shiloh, C. Bakkenist, M. Kastan, M. Welcker, B. Clurman, V. Gorgoulis and K. Helin for reagents; M.H. Lee, D. Lützhoft, A. Arnt Kjerulff and L.-L. Christensen for technical assistance; and C. Wiuf for data analysis. This work was supported by the Danish Cancer Society, the Alfred Benzon Foundation and the European Commission.

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Correspondence to Jirina Bartkova or Jiri Bartek.

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Supplementary information

Supplementary Figure Legends

Legends to accompany the below Supplementary Figures. (DOC 38 kb)

Supplementary Figure S1

Immunostaining of γH2AX in human Ta lesions of the urinary bladder, shown at high magnification by confocal laser microscope or immunoperoxidase staining to document the focal pattern of γH2AX in tumour cell nuclei. (PDF 513 kb)

Supplementary Figure S2

Summary data for Ser1981-phosphorylated ATM, Thr68-phosphorylated Chk2, and classification of bladder tumours according to their genomic instability deduced from the SNP array analysis of allelic imbalances. (JPG 727 kb)

Supplementary Figure S3

Examples of immunohistochemistry images to illustrate that mononuclear cell infiltrate is insufficient to activate the DNA damage response in human colon tissues. (JPG 663 kb)

Supplementary Figure S4

Cytological detection (confocal laser microscope and light microscope images) of activated DNA damage checkpoint markers in U-2-OS cells exposed to various hyperproliferative, oncogenic stimuli. (PDF 547 kb)

Supplementary Figure S5

Examples of immunoperoxidase and immunofluorescence images of cyclin E-activated DNA damage checkpoint markers in normal human fibroblasts. (PDF 920 kb)

Supplementary Figure S6

Evidence for altered dynamics of DNA replication and chromatin association of the RPA protein) on induced overexpression of cyclin E in human U-2-OS cells. (JPG 331 kb)

Supplementary Figure S7

Immunohsitochemical evidence for aberrantly enhanced cyclin E and γH2AX in colon adenomas and summary to document the correlation of γ-H2AX and elevated cyclin E. (JPG 358 kb)

Supplementary Figure S8

Examples of immunohistochemical patterns of Tyr15-phosphorylated Cdk1, Ki67 and γH2AX in colon adenomas, and summary of frequencies and correlation between the γH2AX and pTyr-Cdk1 markers in adenomas and carcinomas, respectively. (JPG 492 kb)

Supplementary Data

Data from the SNP array analysis of DNA isolated from the blood and microdissected lesions from samples of different stages of human bladder tumourigenesis, MIAME compliant. (DOC 70 kb)

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Bartkova, J., Hořejší, Z., Koed, K. et al. DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis. Nature 434, 864–870 (2005). https://doi.org/10.1038/nature03482

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