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Telomere-dependent and telomere-independent origins of endogenous DNA damage in tumor cells.

Nakamura AJ, Redon CE, Bonner WM, Sedelnikova OA - Aging (Albany NY) (2009)

Bottom Line: The distinct heterogeneity in the numbers of gamma-H2AX foci in these tumor cell lines was found to be due to foci associated with uncapped telomeres, and the amount of total telomeric damage also appeared to inversely correlate with the telomerase activity present in these cells.These results indicate that damaged telomeres are the major factor accounting for the variability in the amount of DNA DSB damage in tumor cells.This characterization of DNA damage in tumor cells helps clarify the contribution of non-telomeric DSBs and damaged telomeres to major genomic alterations.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
Human tumors and cultured cells contain elevated levels of endogenous DNA damage resulting from telomere dysfunction, replication and transcription errors, reactive oxygen species, and genome instability. However, the contribution of telomere-associated versus telomere-independent endogenous DNA lesions to this damage has never been examined. In this study, we characterized the relative amounts of these two types of DNA damage in five tumor cell lines by noting whether gamma-H2AX foci, generally considered to mark DNA double-strand breaks (DSBs), were on chromosome arms or at chromosome ends. We found that while the numbers of non-telomeric DSBs were remarkably similar in these cultures, considerable variation was detected in the level of telomeric damage. The distinct heterogeneity in the numbers of gamma-H2AX foci in these tumor cell lines was found to be due to foci associated with uncapped telomeres, and the amount of total telomeric damage also appeared to inversely correlate with the telomerase activity present in these cells. These results indicate that damaged telomeres are the major factor accounting for the variability in the amount of DNA DSB damage in tumor cells. This characterization of DNA damage in tumor cells helps clarify the contribution of non-telomeric DSBs and damaged telomeres to major genomic alterations.

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Distribution of γ-H2AX foci on metaphases of human tumor cells. (A) Metaphase spread of HCT116 cells stained for γ-H2AX (green) and telomeric DNA (red). (B)                                             Scoring of γ-H2AX foci as along chromatid                                             arms (Arms) or on chromatid ends (Ends). (C) The numbers of γ-H2AX foci in metaphases from five tumor cell                                             lines as noted. Foci are noted as non-telomeric (Arms, gray), telomeric                                             (Ends, blue), and total (black). (D) Telomeric γ-H2AX foci with (yellow) and without (green)                                             telomere FISH signal in the five tumor lines. At least 10 metaphases were                                             screened per data point in independent experiments. Error bars signify                                             standard errors. (E) Numbers of telomeric (open circles) and                                             non-telomeric (cross hatches) foci vs. the total numbers of γ-H2AX foci on the metaphase spreads of the five                                             tumor cell lines. The data from all five tumor cell lines was pooled for                                             this analysis. (F) Numbers of FISH negative (open squares) and FISH                                             positive telomeric (filled triangle) γ-H2AX                                             foci vs. total telomeric foci in all checked metaphases of the five tumor                                             cell lines. (G) Reverse correlation of the numbers of γ-H2AX foci and telomerase activity in the five                                             tumor cell lines. TPG is a total product generated corresponding to 600                                             molecules of telomerase substrate primers extended with at least four                                             telomere repeats [28].
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Figure 3: Distribution of γ-H2AX foci on metaphases of human tumor cells. (A) Metaphase spread of HCT116 cells stained for γ-H2AX (green) and telomeric DNA (red). (B) Scoring of γ-H2AX foci as along chromatid arms (Arms) or on chromatid ends (Ends). (C) The numbers of γ-H2AX foci in metaphases from five tumor cell lines as noted. Foci are noted as non-telomeric (Arms, gray), telomeric (Ends, blue), and total (black). (D) Telomeric γ-H2AX foci with (yellow) and without (green) telomere FISH signal in the five tumor lines. At least 10 metaphases were screened per data point in independent experiments. Error bars signify standard errors. (E) Numbers of telomeric (open circles) and non-telomeric (cross hatches) foci vs. the total numbers of γ-H2AX foci on the metaphase spreads of the five tumor cell lines. The data from all five tumor cell lines was pooled for this analysis. (F) Numbers of FISH negative (open squares) and FISH positive telomeric (filled triangle) γ-H2AX foci vs. total telomeric foci in all checked metaphases of the five tumor cell lines. (G) Reverse correlation of the numbers of γ-H2AX foci and telomerase activity in the five tumor cell lines. TPG is a total product generated corresponding to 600 molecules of telomerase substrate primers extended with at least four telomere repeats [28].

Mentions: Yu et al. reported that tumor cell cultures exhibited large numbers of endogenous γ-H2AX foci per cell, sometimes equivalent to several Gy of ionizing radiation. Strikingly however, they found no difference in tail moments when these cultures were identically irradiated and the cells were analyzed by the comet assay [12]. This discrepancy suggests the hypothesis that a substantial fraction of the endogenous γ-H2AX foci might be marking uncapped telomeres rather than DSBs. Since the damage is at the end of the DNA, the comet or any other DNA fragmentation assay would not detect it. To examine this notion, we analyzed metaphases of five tumor cell lines for γ-H2AX and telomeric DNA FISH signals to score the numbers of telomere-associated and telomere-independent γ-H2AX foci (Figure 3). This procedure permits the localization of γ-H2AX foci to either the chromatid arms, corresponding to DNA DSBs of non-telomeric origin, or to the ends of the chromatids, corresponding to either DSB-damaged telomeres (FISH-positive terminal foci), or double-strand ends at critically short telomeres lacking detectable telomere repeats (FISH-negative foci) (Figure 3A, B).


Telomere-dependent and telomere-independent origins of endogenous DNA damage in tumor cells.

Nakamura AJ, Redon CE, Bonner WM, Sedelnikova OA - Aging (Albany NY) (2009)

Distribution of γ-H2AX foci on metaphases of human tumor cells. (A) Metaphase spread of HCT116 cells stained for γ-H2AX (green) and telomeric DNA (red). (B)                                             Scoring of γ-H2AX foci as along chromatid                                             arms (Arms) or on chromatid ends (Ends). (C) The numbers of γ-H2AX foci in metaphases from five tumor cell                                             lines as noted. Foci are noted as non-telomeric (Arms, gray), telomeric                                             (Ends, blue), and total (black). (D) Telomeric γ-H2AX foci with (yellow) and without (green)                                             telomere FISH signal in the five tumor lines. At least 10 metaphases were                                             screened per data point in independent experiments. Error bars signify                                             standard errors. (E) Numbers of telomeric (open circles) and                                             non-telomeric (cross hatches) foci vs. the total numbers of γ-H2AX foci on the metaphase spreads of the five                                             tumor cell lines. The data from all five tumor cell lines was pooled for                                             this analysis. (F) Numbers of FISH negative (open squares) and FISH                                             positive telomeric (filled triangle) γ-H2AX                                             foci vs. total telomeric foci in all checked metaphases of the five tumor                                             cell lines. (G) Reverse correlation of the numbers of γ-H2AX foci and telomerase activity in the five                                             tumor cell lines. TPG is a total product generated corresponding to 600                                             molecules of telomerase substrate primers extended with at least four                                             telomere repeats [28].
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Figure 3: Distribution of γ-H2AX foci on metaphases of human tumor cells. (A) Metaphase spread of HCT116 cells stained for γ-H2AX (green) and telomeric DNA (red). (B) Scoring of γ-H2AX foci as along chromatid arms (Arms) or on chromatid ends (Ends). (C) The numbers of γ-H2AX foci in metaphases from five tumor cell lines as noted. Foci are noted as non-telomeric (Arms, gray), telomeric (Ends, blue), and total (black). (D) Telomeric γ-H2AX foci with (yellow) and without (green) telomere FISH signal in the five tumor lines. At least 10 metaphases were screened per data point in independent experiments. Error bars signify standard errors. (E) Numbers of telomeric (open circles) and non-telomeric (cross hatches) foci vs. the total numbers of γ-H2AX foci on the metaphase spreads of the five tumor cell lines. The data from all five tumor cell lines was pooled for this analysis. (F) Numbers of FISH negative (open squares) and FISH positive telomeric (filled triangle) γ-H2AX foci vs. total telomeric foci in all checked metaphases of the five tumor cell lines. (G) Reverse correlation of the numbers of γ-H2AX foci and telomerase activity in the five tumor cell lines. TPG is a total product generated corresponding to 600 molecules of telomerase substrate primers extended with at least four telomere repeats [28].
Mentions: Yu et al. reported that tumor cell cultures exhibited large numbers of endogenous γ-H2AX foci per cell, sometimes equivalent to several Gy of ionizing radiation. Strikingly however, they found no difference in tail moments when these cultures were identically irradiated and the cells were analyzed by the comet assay [12]. This discrepancy suggests the hypothesis that a substantial fraction of the endogenous γ-H2AX foci might be marking uncapped telomeres rather than DSBs. Since the damage is at the end of the DNA, the comet or any other DNA fragmentation assay would not detect it. To examine this notion, we analyzed metaphases of five tumor cell lines for γ-H2AX and telomeric DNA FISH signals to score the numbers of telomere-associated and telomere-independent γ-H2AX foci (Figure 3). This procedure permits the localization of γ-H2AX foci to either the chromatid arms, corresponding to DNA DSBs of non-telomeric origin, or to the ends of the chromatids, corresponding to either DSB-damaged telomeres (FISH-positive terminal foci), or double-strand ends at critically short telomeres lacking detectable telomere repeats (FISH-negative foci) (Figure 3A, B).

Bottom Line: The distinct heterogeneity in the numbers of gamma-H2AX foci in these tumor cell lines was found to be due to foci associated with uncapped telomeres, and the amount of total telomeric damage also appeared to inversely correlate with the telomerase activity present in these cells.These results indicate that damaged telomeres are the major factor accounting for the variability in the amount of DNA DSB damage in tumor cells.This characterization of DNA damage in tumor cells helps clarify the contribution of non-telomeric DSBs and damaged telomeres to major genomic alterations.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
Human tumors and cultured cells contain elevated levels of endogenous DNA damage resulting from telomere dysfunction, replication and transcription errors, reactive oxygen species, and genome instability. However, the contribution of telomere-associated versus telomere-independent endogenous DNA lesions to this damage has never been examined. In this study, we characterized the relative amounts of these two types of DNA damage in five tumor cell lines by noting whether gamma-H2AX foci, generally considered to mark DNA double-strand breaks (DSBs), were on chromosome arms or at chromosome ends. We found that while the numbers of non-telomeric DSBs were remarkably similar in these cultures, considerable variation was detected in the level of telomeric damage. The distinct heterogeneity in the numbers of gamma-H2AX foci in these tumor cell lines was found to be due to foci associated with uncapped telomeres, and the amount of total telomeric damage also appeared to inversely correlate with the telomerase activity present in these cells. These results indicate that damaged telomeres are the major factor accounting for the variability in the amount of DNA DSB damage in tumor cells. This characterization of DNA damage in tumor cells helps clarify the contribution of non-telomeric DSBs and damaged telomeres to major genomic alterations.

Show MeSH
Related in: MedlinePlus