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ATM-independent, high-fidelity nonhomologous end joining predominates in human embryonic stem cells.

Adams BR, Hawkins AJ, Povirk LF, Valerie K - Aging (Albany NY) (2010)

Bottom Line: ATM and DNA-PKcs inhibitors were ineffective or partially effective, respectively, at inhibiting NHEJ in hESCs, whereas progressively more inhibition was seen in NPs and astrocytes.The lack of any major involvement of DNA-PKcs in NHEJ in hESCs was supported by siRNA-mediated DNA-PKcs knockdown.Furthermore, as hESCs differentiated a progressive decrease in the accuracy of NHEJ was observed.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, Virginia Commonwealth University, Richmond, Virginia 23298, USA.

ABSTRACT
We recently demonstrated that human embryonic stem cells (hESCs) utilize homologous recombination repair (HRR) as primary means of double-strand break (DSB) repair. We now show that hESCs also use nonhomologous end joining (NHEJ). NHEJ kinetics were several-fold slower in hESCs and neural progenitors (NPs) than in astrocytes derived from hESCs. ATM and DNA-PKcs inhibitors were ineffective or partially effective, respectively, at inhibiting NHEJ in hESCs, whereas progressively more inhibition was seen in NPs and astrocytes. The lack of any major involvement of DNA-PKcs in NHEJ in hESCs was supported by siRNA-mediated DNA-PKcs knockdown. Expression of a truncated XRCC4 decoy or XRCC4 knock-down reduced NHEJ by more than half suggesting that repair is primarily canonical NHEJ. Poly(ADP-ribose) polymerase (PARP) was dispensable for NHEJ suggesting that repair is largely independent of backup NHEJ. Furthermore, as hESCs differentiated a progressive decrease in the accuracy of NHEJ was observed. Altogether, we conclude that NHEJ in hESCs is largely independent of ATM, DNA-PKcs, and PARP but dependent on XRCC4 with repair fidelity several-fold greater than in astrocytes.

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Specific DNA-PKcs and ATM kinase inhibitors become more effective as hESCs differentiate.(A) DNA-PKi and ATMi are functioning in hESCs. hESCs were harvested 5, 10, and 15 min after exposure to 6 Gy with or without ATMi (10 μM) and DNA-PKi (2.5 μM) or both. Drugs were added 15 min prior to radiation. Fold change depicts phosphorylation of KAP1 (S824) and H2AX (S139) after normalization to CHK1 (and GAPDH) which served as loading controls. (B) BG01V/NHEJ-red (C) NP/NHEJ-red and (D) astrocyte/NHEJ-red cells were infected with Ad-SceI and then treated with either ATMi at 10 μM or DNA-PKi at 2.5 μM 1 h after infection. Cells were collected at 24 h post-infection. (Columns) Relative NHEJ levels were normalized to β-actin; (Error bars) SEM for data sets n = 3. Fold (x) indicates changes in the relative repair levels when compared to the hESC sample. Differences in the scale of the separate cell populations (B-D) are due to variation in the uninfected sample PCR amplification from 3 separate experiments. Statistical significance of differences in NHEJ with respect to cells expressing I SceI with no inhibitor, are indicated.
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Figure 4: Specific DNA-PKcs and ATM kinase inhibitors become more effective as hESCs differentiate.(A) DNA-PKi and ATMi are functioning in hESCs. hESCs were harvested 5, 10, and 15 min after exposure to 6 Gy with or without ATMi (10 μM) and DNA-PKi (2.5 μM) or both. Drugs were added 15 min prior to radiation. Fold change depicts phosphorylation of KAP1 (S824) and H2AX (S139) after normalization to CHK1 (and GAPDH) which served as loading controls. (B) BG01V/NHEJ-red (C) NP/NHEJ-red and (D) astrocyte/NHEJ-red cells were infected with Ad-SceI and then treated with either ATMi at 10 μM or DNA-PKi at 2.5 μM 1 h after infection. Cells were collected at 24 h post-infection. (Columns) Relative NHEJ levels were normalized to β-actin; (Error bars) SEM for data sets n = 3. Fold (x) indicates changes in the relative repair levels when compared to the hESC sample. Differences in the scale of the separate cell populations (B-D) are due to variation in the uninfected sample PCR amplification from 3 separate experiments. Statistical significance of differences in NHEJ with respect to cells expressing I SceI with no inhibitor, are indicated.

Mentions: We, and another group, showed recently that an ATMi was only partially effective at abrogating DSB repair and DNA damage checkpoint signaling in hESCs [5,26]. In order to first confirm that the KU-55933 (ATMi) and KU-57788 (DNA-PKi) small molecule inhibitors were entering the cells, the effect on radiation-induced H2AX (S139) and KAP1 (S824) phosphorylation was examined. KAP1 is involved in chromatin remodeling after DNA damage and its activation is dependent on ATM and DNA-PKcs at early time points [29]. Furthermore, we showed recently that H2AX phosphorylation is completely blocked at early times (≤ 15 min) after irradiation when both drugs are applied to glioma cells [30]. Here, we show that after irradiation p-KAP1 and γ-H2AX are reduced to near basal levels in a time-dependent manner when treated with a combination of ATMi and DNA-PKi (Figure 4A). Therefore, we conclude that both drugs enter hESCs and inhibit the DDR similar to what is seen with glioma cells [30].


ATM-independent, high-fidelity nonhomologous end joining predominates in human embryonic stem cells.

Adams BR, Hawkins AJ, Povirk LF, Valerie K - Aging (Albany NY) (2010)

Specific DNA-PKcs and ATM kinase inhibitors become more effective as hESCs differentiate.(A) DNA-PKi and ATMi are functioning in hESCs. hESCs were harvested 5, 10, and 15 min after exposure to 6 Gy with or without ATMi (10 μM) and DNA-PKi (2.5 μM) or both. Drugs were added 15 min prior to radiation. Fold change depicts phosphorylation of KAP1 (S824) and H2AX (S139) after normalization to CHK1 (and GAPDH) which served as loading controls. (B) BG01V/NHEJ-red (C) NP/NHEJ-red and (D) astrocyte/NHEJ-red cells were infected with Ad-SceI and then treated with either ATMi at 10 μM or DNA-PKi at 2.5 μM 1 h after infection. Cells were collected at 24 h post-infection. (Columns) Relative NHEJ levels were normalized to β-actin; (Error bars) SEM for data sets n = 3. Fold (x) indicates changes in the relative repair levels when compared to the hESC sample. Differences in the scale of the separate cell populations (B-D) are due to variation in the uninfected sample PCR amplification from 3 separate experiments. Statistical significance of differences in NHEJ with respect to cells expressing I SceI with no inhibitor, are indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 4: Specific DNA-PKcs and ATM kinase inhibitors become more effective as hESCs differentiate.(A) DNA-PKi and ATMi are functioning in hESCs. hESCs were harvested 5, 10, and 15 min after exposure to 6 Gy with or without ATMi (10 μM) and DNA-PKi (2.5 μM) or both. Drugs were added 15 min prior to radiation. Fold change depicts phosphorylation of KAP1 (S824) and H2AX (S139) after normalization to CHK1 (and GAPDH) which served as loading controls. (B) BG01V/NHEJ-red (C) NP/NHEJ-red and (D) astrocyte/NHEJ-red cells were infected with Ad-SceI and then treated with either ATMi at 10 μM or DNA-PKi at 2.5 μM 1 h after infection. Cells were collected at 24 h post-infection. (Columns) Relative NHEJ levels were normalized to β-actin; (Error bars) SEM for data sets n = 3. Fold (x) indicates changes in the relative repair levels when compared to the hESC sample. Differences in the scale of the separate cell populations (B-D) are due to variation in the uninfected sample PCR amplification from 3 separate experiments. Statistical significance of differences in NHEJ with respect to cells expressing I SceI with no inhibitor, are indicated.
Mentions: We, and another group, showed recently that an ATMi was only partially effective at abrogating DSB repair and DNA damage checkpoint signaling in hESCs [5,26]. In order to first confirm that the KU-55933 (ATMi) and KU-57788 (DNA-PKi) small molecule inhibitors were entering the cells, the effect on radiation-induced H2AX (S139) and KAP1 (S824) phosphorylation was examined. KAP1 is involved in chromatin remodeling after DNA damage and its activation is dependent on ATM and DNA-PKcs at early time points [29]. Furthermore, we showed recently that H2AX phosphorylation is completely blocked at early times (≤ 15 min) after irradiation when both drugs are applied to glioma cells [30]. Here, we show that after irradiation p-KAP1 and γ-H2AX are reduced to near basal levels in a time-dependent manner when treated with a combination of ATMi and DNA-PKi (Figure 4A). Therefore, we conclude that both drugs enter hESCs and inhibit the DDR similar to what is seen with glioma cells [30].

Bottom Line: ATM and DNA-PKcs inhibitors were ineffective or partially effective, respectively, at inhibiting NHEJ in hESCs, whereas progressively more inhibition was seen in NPs and astrocytes.The lack of any major involvement of DNA-PKcs in NHEJ in hESCs was supported by siRNA-mediated DNA-PKcs knockdown.Furthermore, as hESCs differentiated a progressive decrease in the accuracy of NHEJ was observed.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, Virginia Commonwealth University, Richmond, Virginia 23298, USA.

ABSTRACT
We recently demonstrated that human embryonic stem cells (hESCs) utilize homologous recombination repair (HRR) as primary means of double-strand break (DSB) repair. We now show that hESCs also use nonhomologous end joining (NHEJ). NHEJ kinetics were several-fold slower in hESCs and neural progenitors (NPs) than in astrocytes derived from hESCs. ATM and DNA-PKcs inhibitors were ineffective or partially effective, respectively, at inhibiting NHEJ in hESCs, whereas progressively more inhibition was seen in NPs and astrocytes. The lack of any major involvement of DNA-PKcs in NHEJ in hESCs was supported by siRNA-mediated DNA-PKcs knockdown. Expression of a truncated XRCC4 decoy or XRCC4 knock-down reduced NHEJ by more than half suggesting that repair is primarily canonical NHEJ. Poly(ADP-ribose) polymerase (PARP) was dispensable for NHEJ suggesting that repair is largely independent of backup NHEJ. Furthermore, as hESCs differentiated a progressive decrease in the accuracy of NHEJ was observed. Altogether, we conclude that NHEJ in hESCs is largely independent of ATM, DNA-PKcs, and PARP but dependent on XRCC4 with repair fidelity several-fold greater than in astrocytes.

Show MeSH
Related in: MedlinePlus