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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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Elevated Artemis levels confer a radioprotective effect against High and Low LET radiation. Western blots of lysates from human HEK293 cells carrying integrated vectors harboring tetracycline-inducible Artemis expression constructs (293Art), or control vector (HEK293), probed with antibodies recognizing Artemis (top) or tubulin for loading controls (bottom) (1A-inset). Results from a clonogenic survival experiment using HEK293 (open circles) and 293Art Artemis over expressing cells (solid triangles), stained and colonies scored 14 days after exposure to varied doses of X-rays (A) compared to a BrdU proliferation experiment set up in parallel and harvested 48 h post exposure (B). Results from BrdU proliferation experiments following high LET exposures in the form of iron nuclei (C) or titanium ions (D) carried out using the same dose range as X-ray experiments and processed at the same time points.
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Figure 1: Elevated Artemis levels confer a radioprotective effect against High and Low LET radiation. Western blots of lysates from human HEK293 cells carrying integrated vectors harboring tetracycline-inducible Artemis expression constructs (293Art), or control vector (HEK293), probed with antibodies recognizing Artemis (top) or tubulin for loading controls (bottom) (1A-inset). Results from a clonogenic survival experiment using HEK293 (open circles) and 293Art Artemis over expressing cells (solid triangles), stained and colonies scored 14 days after exposure to varied doses of X-rays (A) compared to a BrdU proliferation experiment set up in parallel and harvested 48 h post exposure (B). Results from BrdU proliferation experiments following high LET exposures in the form of iron nuclei (C) or titanium ions (D) carried out using the same dose range as X-ray experiments and processed at the same time points.

Mentions: To examine the influence of Artemis levels on cellular survival after exposure to high and low LET radiation, we used an extensively characterized human expression embryonic kidney cell line. The wild type line, T-Rex-293 (hereafter, HEK293), expresses native levels of Artemis and the derivative cell line carries a stably integrated doxycycline-inducible Artemis cDNA (hereafter, 293Art)[16] which can be induced to express high levels of Artemis. The T-Rex-293 cell line was chosen for this work due to its ease of transfection, however our studies have shown survival responses after radiation treatments comparable to primary fibroblasts when measured by a standard clonal survival assay and the BrdU proliferation assay (unpublished data). Artemis protein levels were evaluated twenty-four hours after doxycycline treatment by Western blots of whole cell extracts. Artemis protein levels in HEK293 were very low, to non-detectable, whereas 293Art extracts contained high levels of Artemis protein, visualized as an intense band migrating at ~100 kDa on Western blots (Figure1A inset). Cells stably retain our inducible Artemis expression plasmid and tolerate high levels of induced Artemis expression with no detectable changes in growth, unlike what has been reported for constructs constitutively producing high Artemis levels[18]. Artemis protein levels were similarly validated for all subsequent experiments.


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)

Elevated Artemis levels confer a radioprotective effect against High and Low LET radiation. Western blots of lysates from human HEK293 cells carrying integrated vectors harboring tetracycline-inducible Artemis expression constructs (293Art), or control vector (HEK293), probed with antibodies recognizing Artemis (top) or tubulin for loading controls (bottom) (1A-inset). Results from a clonogenic survival experiment using HEK293 (open circles) and 293Art Artemis over expressing cells (solid triangles), stained and colonies scored 14 days after exposure to varied doses of X-rays (A) compared to a BrdU proliferation experiment set up in parallel and harvested 48 h post exposure (B). Results from BrdU proliferation experiments following high LET exposures in the form of iron nuclei (C) or titanium ions (D) carried out using the same dose range as X-ray experiments and processed at the same time points.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Elevated Artemis levels confer a radioprotective effect against High and Low LET radiation. Western blots of lysates from human HEK293 cells carrying integrated vectors harboring tetracycline-inducible Artemis expression constructs (293Art), or control vector (HEK293), probed with antibodies recognizing Artemis (top) or tubulin for loading controls (bottom) (1A-inset). Results from a clonogenic survival experiment using HEK293 (open circles) and 293Art Artemis over expressing cells (solid triangles), stained and colonies scored 14 days after exposure to varied doses of X-rays (A) compared to a BrdU proliferation experiment set up in parallel and harvested 48 h post exposure (B). Results from BrdU proliferation experiments following high LET exposures in the form of iron nuclei (C) or titanium ions (D) carried out using the same dose range as X-ray experiments and processed at the same time points.
Mentions: To examine the influence of Artemis levels on cellular survival after exposure to high and low LET radiation, we used an extensively characterized human expression embryonic kidney cell line. The wild type line, T-Rex-293 (hereafter, HEK293), expresses native levels of Artemis and the derivative cell line carries a stably integrated doxycycline-inducible Artemis cDNA (hereafter, 293Art)[16] which can be induced to express high levels of Artemis. The T-Rex-293 cell line was chosen for this work due to its ease of transfection, however our studies have shown survival responses after radiation treatments comparable to primary fibroblasts when measured by a standard clonal survival assay and the BrdU proliferation assay (unpublished data). Artemis protein levels were evaluated twenty-four hours after doxycycline treatment by Western blots of whole cell extracts. Artemis protein levels in HEK293 were very low, to non-detectable, whereas 293Art extracts contained high levels of Artemis protein, visualized as an intense band migrating at ~100 kDa on Western blots (Figure1A inset). Cells stably retain our inducible Artemis expression plasmid and tolerate high levels of induced Artemis expression with no detectable changes in growth, unlike what has been reported for constructs constitutively producing high Artemis levels[18]. Artemis protein levels were similarly validated for all subsequent experiments.

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