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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.

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.

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Related in: MedlinePlus

Quantitation of cell cycle distribution of WT or Artemis over-expressing cells following exposure to varied doses of different radiation qualities. Normal wild-type (HEK293) or these same cells over-expressing Artemis (293-Art) were exposed to various doses and qualities of radiation. Following radiation cells were incubated for 24 hours, then labeled for 24 hours with BrdU, and fixed at 48 hours post exposure. Flow cytometry was used to quantify the percentage of cells in G1 or G2.
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Figure 5: Quantitation of cell cycle distribution of WT or Artemis over-expressing cells following exposure to varied doses of different radiation qualities. Normal wild-type (HEK293) or these same cells over-expressing Artemis (293-Art) were exposed to various doses and qualities of radiation. Following radiation cells were incubated for 24 hours, then labeled for 24 hours with BrdU, and fixed at 48 hours post exposure. Flow cytometry was used to quantify the percentage of cells in G1 or G2.

Mentions: High Artemis levels had no discernable effect on cell cycle distributions in the absence of DNA damage. As expected, both 293Art and HEK293 cells showed a marked accumulation of cells at the G2/M boundary following radiation exposure and this accumulation increased at higher doses, and was greater with high LET as compared to low LET exposure (Figure4). In previous studies with Artemis defective cells we observed persistent cell cycle arrest and an absence of cycling 48 h post radiation exposure[16], in contrast, cells over-expressing Artemis traverse the G2/M checkpoint more readily and move into G1 to a greater extent than control wild type HEK293 (Figure4). Similar results are also observed at 72 h post radiation exposure suggesting that the proliferation defect in wild type cells is due to a damage-induced cell cycle block, which is not rectified even at 72 h after irradiation (not shown). Importantly, Artemis over-expression does not eliminate or bypass the G2/M cell cycle checkpoint, but rather cells with elevated Artemis levels have an attenuated biological response to irradiation with cell cycle kinetics similar to the parental line albeit at a lower dose (i.e. cell cycle profile of HEK293 at 2 Gy is comparable to 293Art at 3 Gy, Figure4). This effect was observed at different doses and with different radiation qualities (Fe and Ti), rendering the cell cycle distribution of 293Art cells at higher doses nearly identical to that of the parental HEK293 at lower doses (Figure5, note patterns highlighted by dotted lines).


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)

Quantitation of cell cycle distribution of WT or Artemis over-expressing cells following exposure to varied doses of different radiation qualities. Normal wild-type (HEK293) or these same cells over-expressing Artemis (293-Art) were exposed to various doses and qualities of radiation. Following radiation cells were incubated for 24 hours, then labeled for 24 hours with BrdU, and fixed at 48 hours post exposure. Flow cytometry was used to quantify the percentage of cells in G1 or G2.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Quantitation of cell cycle distribution of WT or Artemis over-expressing cells following exposure to varied doses of different radiation qualities. Normal wild-type (HEK293) or these same cells over-expressing Artemis (293-Art) were exposed to various doses and qualities of radiation. Following radiation cells were incubated for 24 hours, then labeled for 24 hours with BrdU, and fixed at 48 hours post exposure. Flow cytometry was used to quantify the percentage of cells in G1 or G2.
Mentions: High Artemis levels had no discernable effect on cell cycle distributions in the absence of DNA damage. As expected, both 293Art and HEK293 cells showed a marked accumulation of cells at the G2/M boundary following radiation exposure and this accumulation increased at higher doses, and was greater with high LET as compared to low LET exposure (Figure4). In previous studies with Artemis defective cells we observed persistent cell cycle arrest and an absence of cycling 48 h post radiation exposure[16], in contrast, cells over-expressing Artemis traverse the G2/M checkpoint more readily and move into G1 to a greater extent than control wild type HEK293 (Figure4). Similar results are also observed at 72 h post radiation exposure suggesting that the proliferation defect in wild type cells is due to a damage-induced cell cycle block, which is not rectified even at 72 h after irradiation (not shown). Importantly, Artemis over-expression does not eliminate or bypass the G2/M cell cycle checkpoint, but rather cells with elevated Artemis levels have an attenuated biological response to irradiation with cell cycle kinetics similar to the parental line albeit at a lower dose (i.e. cell cycle profile of HEK293 at 2 Gy is comparable to 293Art at 3 Gy, Figure4). This effect was observed at different doses and with different radiation qualities (Fe and Ti), rendering the cell cycle distribution of 293Art cells at higher doses nearly identical to that of the parental HEK293 at lower doses (Figure5, note patterns highlighted by dotted lines).

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