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Increased Artemis levels confer radioresistance to both high and low LET radiation exposures.

Sridharan DM, Whalen MK, Almendrala D, Cucinotta FA, Kawahara M, Yannone SM, Pluth JM - Radiat Oncol (2012)

Bottom Line: However the exact function(s) of Artemis in DNA repair and its preferred substrate(s) in vivo remain undefined.Inhibitor studies reveal that the radioprotection imparted by Artemis is primarily dependent on DNA-PK activity, and to a lesser extent on ATM kinase activity.These findings indicate that Artemis levels significantly influence radiation toxicity in human cells and suggest that Artemis inhibition could be a practical target for adjuvant cancer therapies.

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Affiliation: Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.

ABSTRACT

Background: Artemis has a defined role in V(D)J recombination and has been implicated in the repair of radiation induced double-strand breaks. However the exact function(s) of Artemis in DNA repair and its preferred substrate(s) in vivo remain undefined. Our previous work suggests that Artemis is important for the repair of complex DNA damage like that inflicted by high Linear Energy Transfer (LET) radiation. To establish the contribution of Artemis in repairing DNA damage caused by various radiation qualities, we evaluated the effect of over-expressing Artemis on cell survival, DNA repair, and cell cycle arrest after exposure to high and low LET radiation.

Results: Our data reveal that Artemis over-expression confers marked radioprotection against both types of radiation, although the radioprotective effect was greater following high LET radiation. Inhibitor studies reveal that the radioprotection imparted by Artemis is primarily dependent on DNA-PK activity, and to a lesser extent on ATM kinase activity. Together, these data suggest a DNA-PK dependent role for Artemis in the repair of complex DNA damage.

Conclusions: These findings indicate that Artemis levels significantly influence radiation toxicity in human cells and suggest that Artemis inhibition could be a practical target for adjuvant cancer therapies.

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Artemis phosphorylation is delayed following high LET damage. Western blots were probed for total Artemis protein and mobility shifts of the protein indicate phosphorylation has occurred. Blots are shown 2 h post X-ray or iron nuclei exposure (A, left and right panel respectively) and 48 h post X-ray or iron nuclei exposure (B, left and right panel respectively). Arrows indicate location of basally-phosphorylated Artemis (lower arrow) and hyper-phosphorylated Artemis (upper arrow).
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Figure 3: Artemis phosphorylation is delayed following high LET damage. Western blots were probed for total Artemis protein and mobility shifts of the protein indicate phosphorylation has occurred. Blots are shown 2 h post X-ray or iron nuclei exposure (A, left and right panel respectively) and 48 h post X-ray or iron nuclei exposure (B, left and right panel respectively). Arrows indicate location of basally-phosphorylated Artemis (lower arrow) and hyper-phosphorylated Artemis (upper arrow).

Mentions: Artemis phosphorylation status was evaluated at 2 and 48 h following low and high LET exposures to determine if phosphorylation was temporally regulated with respect to radiation quality. We find that Artemis phosphorylation, which results in a mobility shift of the protein on SDS-PAGE, is delayed following high LET exposure relative to low LET (Figure3). More specifically, Artemis protein mobility is markedly altered at early time points (2 h) after X-rays while a parallel experiment with high LET radiation shows no change in Artemis protein mobility at this same timepoint (Figure3A). However, at 48 h after high LET exposure a mobility shift in Artemis protein is observed, indicative of phosphorylation (compare Figure3A and B). These data point towards a delay in Artemis phosphorylation following high LET as compared to low LET exposure, consistent with our previous studies showing ATM mediated signaling is delayed following high LET relative to low LET exposures in wild type fibroblast cells[23].


Increased Artemis levels confer radioresistance to both high and low LET radiation exposures.

Sridharan DM, Whalen MK, Almendrala D, Cucinotta FA, Kawahara M, Yannone SM, Pluth JM - Radiat Oncol (2012)

Artemis phosphorylation is delayed following high LET damage. Western blots were probed for total Artemis protein and mobility shifts of the protein indicate phosphorylation has occurred. Blots are shown 2 h post X-ray or iron nuclei exposure (A, left and right panel respectively) and 48 h post X-ray or iron nuclei exposure (B, left and right panel respectively). Arrows indicate location of basally-phosphorylated Artemis (lower arrow) and hyper-phosphorylated Artemis (upper arrow).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3585927&req=5

Figure 3: Artemis phosphorylation is delayed following high LET damage. Western blots were probed for total Artemis protein and mobility shifts of the protein indicate phosphorylation has occurred. Blots are shown 2 h post X-ray or iron nuclei exposure (A, left and right panel respectively) and 48 h post X-ray or iron nuclei exposure (B, left and right panel respectively). Arrows indicate location of basally-phosphorylated Artemis (lower arrow) and hyper-phosphorylated Artemis (upper arrow).
Mentions: Artemis phosphorylation status was evaluated at 2 and 48 h following low and high LET exposures to determine if phosphorylation was temporally regulated with respect to radiation quality. We find that Artemis phosphorylation, which results in a mobility shift of the protein on SDS-PAGE, is delayed following high LET exposure relative to low LET (Figure3). More specifically, Artemis protein mobility is markedly altered at early time points (2 h) after X-rays while a parallel experiment with high LET radiation shows no change in Artemis protein mobility at this same timepoint (Figure3A). However, at 48 h after high LET exposure a mobility shift in Artemis protein is observed, indicative of phosphorylation (compare Figure3A and B). These data point towards a delay in Artemis phosphorylation following high LET as compared to low LET exposure, consistent with our previous studies showing ATM mediated signaling is delayed following high LET relative to low LET exposures in wild type fibroblast cells[23].

Bottom Line: However the exact function(s) of Artemis in DNA repair and its preferred substrate(s) in vivo remain undefined.Inhibitor studies reveal that the radioprotection imparted by Artemis is primarily dependent on DNA-PK activity, and to a lesser extent on ATM kinase activity.These findings indicate that Artemis levels significantly influence radiation toxicity in human cells and suggest that Artemis inhibition could be a practical target for adjuvant cancer therapies.

View Article: PubMed Central - HTML - PubMed

Affiliation: Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.

ABSTRACT

Background: Artemis has a defined role in V(D)J recombination and has been implicated in the repair of radiation induced double-strand breaks. However the exact function(s) of Artemis in DNA repair and its preferred substrate(s) in vivo remain undefined. Our previous work suggests that Artemis is important for the repair of complex DNA damage like that inflicted by high Linear Energy Transfer (LET) radiation. To establish the contribution of Artemis in repairing DNA damage caused by various radiation qualities, we evaluated the effect of over-expressing Artemis on cell survival, DNA repair, and cell cycle arrest after exposure to high and low LET radiation.

Results: Our data reveal that Artemis over-expression confers marked radioprotection against both types of radiation, although the radioprotective effect was greater following high LET radiation. Inhibitor studies reveal that the radioprotection imparted by Artemis is primarily dependent on DNA-PK activity, and to a lesser extent on ATM kinase activity. Together, these data suggest a DNA-PK dependent role for Artemis in the repair of complex DNA damage.

Conclusions: These findings indicate that Artemis levels significantly influence radiation toxicity in human cells and suggest that Artemis inhibition could be a practical target for adjuvant cancer therapies.

Show MeSH
Related in: MedlinePlus