title: Cytotoxic effects of temozolomide and radiation are additive- and schedule-dependent.
abstract: OBJECTIVE
  Despite aggressive therapy comprising radical radiation and temozolomide (TMZ) chemotherapy, the prognosis for patients with glioblastoma multiforme (GBM) remains poor, particularly if tumors express O(6)-methylguanine-DNA-methyltransferase (MGMT). The interactions between radiation and TMZ remain unclear and have important implications for scheduling and for developing strategies to improve outcomes.
  
  
  METHODS
  Factors determining the effects of combination therapy on clonogenic survival, cell-cycle checkpoint signaling and DNA repair were investigated in four human glioma cell lines (T98G, U373-MG, UVW, U87-MG).
  
  
  RESULTS
  Combining TMZ and radiation yielded additive cytotoxicity, but only when TMZ was delivered 72 h before radiation. Radiosensitization was not observed. TMZ induced G2/M cell-cycle arrest at 48-72 h, coincident with phosphorylation of Chk1 and Chk2. Additive G2/M arrest and Chk1/Chk2 phosphorylation was only observed when TMZ preceded radiation by 72 h. The ataxia-telangiectasia mutated (ATM) inhibitor KU-55933 increased radiation sensitivity and delayed repair of radiation-induced DNA breaks, but did not influence TMZ effects. The multiple kinase inhibitor caffeine enhanced the cytotoxicity of chemoradiation and exacerbated DNA damage.
  
  
  CONCLUSIONS
  TMZ is not a radiosensitizing agent but yields additive cytotoxicity in combination with radiation. Our data indicate that TMZ treatment should commence at least 3 days before radiation to achieve maximum benefit. Activation of G2/M checkpoint signaling by TMZ and radiation has a cytoprotective effect that can be overcome by dual inhibition of ATM and ATR. More specific inhibition of checkpoint signaling will be required to increase treatment efficacy without exacerbating toxicity.
authors.affiliation: Brighton and Sussex Medical School, University of Sussex, Falmer, UK. a.j.chalmers@sussex.ac.uk
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Chalmers
authors.forename: Elliot M
authors.initials: EM
authors.lastname: Ruff
authors.forename: Christine
authors.initials: C
authors.lastname: Martindale
authors.forename: Nadia
authors.initials: N
authors.lastname: Lovegrove
authors.forename: Susan C
authors.initials: SC
authors.lastname: Short
mesh_terms: D018906:Antineoplastic Agents, Alkylating / Q000008:administration & dosage / Q000009:adverse effects*; D064007:Ataxia Telangiectasia Mutated Proteins; D002110:Caffeine / Q000494:pharmacology; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors; D045744:Cell Line, Tumor / Q000528:radiation effects; D000071877:Checkpoint Kinase 1; D064447:Checkpoint Kinase 2; D003131:Combined Modality Therapy / Q000009:adverse effects; D004260:DNA Repair / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors; D003606:Dacarbazine / Q000008:administration & dosage / Q000009:adverse effects / Q000031:analogs & derivatives*; D004334:Drug Administration Schedule; D016195:G2 Phase / Q000187:drug effects* / Q000528:radiation effects*; D005909:Glioblastoma / Q000201:enzymology / Q000532:radiotherapy*; D006801:Humans; D009025:Morpholines / Q000494:pharmacology; D009363:Neoplasm Proteins / Q000378:metabolism; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011753:Pyrones / Q000494:pharmacology; D011836:Radiation Tolerance / Q000187:drug effects; D000077204:Temozolomide; D014410:Tumor Stem Cell Assay; D025521:Tumor Suppressor Proteins / Q000037:antagonists & inhibitors
chemical_list: C495818:2-morpholin-4-yl-6-thianthren-1-yl-pyran-4-one; D018906:Antineoplastic Agents, Alkylating; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D009025:Morpholines; D009363:Neoplasm Proteins; D011753:Pyrones; D025521:Tumor Suppressor Proteins; D002110:Caffeine; D003606:Dacarbazine; D019853:O(6)-Methylguanine-DNA Methyltransferase; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; C578634:CHEK2 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases; D000077204:Temozolomide