Activation of telomerase by ionizing radiation: differential response to the inhibition of DNA double-strand break repair by abrogation of poly (ADP-ribosyl)ation, by LY294002, or by Wortmannin.
OBJECTIVE
Telomerase activity represents a radiation-inducible function, which may be targeted by a double-strand break (DSB)-activated signal transduction pathway. Therefore, the effects of DNA-PK inhibitors (Wortmannin and LY294002) on telomerase upregulation after irradiation were studied. In addition, the role of trans-dominant inhibition of poly(ADP-ribosyl)ation, which strongly reduces DSB rejoining, was assessed in comparison with 3-aminobenzamide.


METHODS
COM3 rodent cells carry a construct for the dexamethasone-inducible overexpression of the DNA-binding domain of PARP1 and exhibit greatly impaired DSB rejoining after irradiation. Telomerase activity was measured using polymerase chain reaction ELISA 1 h after irradiation with doses up to 10 Gy. Phosphorylation status of PKB/Akt and of PKCalpha/beta(II) was assessed by western blotting.


RESULTS
No telomerase upregulation was detectable for irradiated cells with undisturbed DSB rejoining. In contrast, incubation with LY294002 or dexamethasone yielded pronounced radiation induction of telomerase activity that could be suppressed by Wortmannin. 3-Aminobenzamide not only was unable to induce telomerase activity but also suppressed telomerase upregulation upon incubation with LY294002 or dexamethasone. Phospho-PKB was detectable independent of irradiation or dexamethasone pretreatment, but was undetectable upon incubations with LY294002 or Wortmannin, whereas phospho-PKC rested detectable.


CONCLUSIONS
Telomerase activation postirradiation was triggered by different treatments that interfere with DNA DSB processing. This telomerase upregulation, however, was not reflected by the phosporylation status of the putative mediators of TERT activation, PKB and PKC. Although an involvement of PKB in TERT activation is not supported by the present findings, a respective role of PKC isoforms other than alpha/beta(II) cannot be ruled out.
Neuhof
Dirk
D
Laboratory of Radiation Biology, Department of Radiation Oncology, University of Heidelberg, Heidelberg, Germany. dirk.neuhof@med.uni-heidelberg.de
Zwicker
Felix
F
Kuepper
Jan-Heiner
JH
Debus
Juergen
J
Weber
Klaus-Josef
KJ
D000730:Androstadienes / Q000494:pharmacology*; D000818:Animals; D001549:Benzamides / Q000494:pharmacology; D002460:Cell Line; D002867:Chromones / Q000494:pharmacology*; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects*; D004260:DNA Repair / Q000502:physiology*; D003907:Dexamethasone / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects / Q000528:radiation effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D009025:Morpholines / Q000494:pharmacology*; D010766:Phosphorylation; D011064:Poly Adenosine Diphosphate Ribose / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D011506:Proteins / Q000187:drug effects / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D019098:Telomerase / Q000378:metabolism* / Q000528:radiation effects; D015854:Up-Regulation / Q000187:drug effects; D000077191:Wortmannin
D000730:Androstadienes; D001549:Benzamides; D002867:Chromones; D004791:Enzyme Inhibitors; D009025:Morpholines; D011506:Proteins; C024857:poly(ADP)-ribosylated proteins; D011064:Poly Adenosine Diphosphate Ribose; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D003907:Dexamethasone; C025160:3-aminobenzamide; D051057:Proto-Oncogene Proteins c-akt; D011493:Protein Kinase C; D019098:Telomerase; D000077191:Wortmannin