Dominant-negative cAMP-responsive element-binding protein inhibits proliferating cell nuclear antigen and DNA repair, leading to increased cellular radiosensitivity.
Selective inhibition of the epidermal growth factor receptor or mitogen-activated protein kinase (MAPK) results in radiosensitization of cancer cells. One potential mechanism involves cAMP-responsive element-binding protein, which is activated by radiation via the epidermal growth factor receptor/MAPK pathway and which regulates synthesis of proliferating cell nuclear antigen (PCNA), a protein involved in repair of ionizing radiation-induced DNA damage. To test for a role of CREB in cellular radiosensitivity, CHO cells were transfected with plasmids expressing dominant-negative CREB mutants (CR133 or KCREB), and various end-points were measured 48 h later. Basal levels of PCNA-CAT reporter construct activity were reduced by 60 and 40% following expression of CR133 and KCREB, respectively; similar decreases were observed in PCNA protein levels. Pulsed-field gel electrophoresis measurements showed that CR133 inhibited the repair of radiation-induced DNA double-strand breaks, and this effect was reversed by over-expression of PCNA; dominant-negative CREB also significantly inhibited split-dose recovery. Clonogenic assays were used to determine surviving fraction; the dose enhancement ratios for dominant-negative CREB-expressing cells compared with control (vector alone) were 1.5 and 1.3 for CR133 and KCREB, respectively. Importantly, co-transfection of mutant CREB and a construct constitutively expressing PCNA protein restored radiosensitivity of CHO cells back to wild-type levels. Moreover, cells expressing either CREB mutant showed no significant cell cycle redistribution. These data demonstrate that genetic disruption of CREB results in radiosensitization, and that this effect can be explained by a mechanism involving decreased PCNA expression and inhibition of DNA repair.
Department of Radiation Oncology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298, USA.
George P
GP
Amorino
Ross B
RB
Mikkelsen
Kristoffer
K
Valerie
Rupert K
RK
Schmidt-Ullrich
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002453:Cell Cycle; D002455:Cell Division; D006224:Cricetinae; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics* / Q000502:physiology*; D004249:DNA Damage; D004260:DNA Repair; D004307:Dose-Response Relationship, Radiation; D005434:Flow Cytometry; D005799:Genes, Dominant; D017930:Genes, Reporter; D005822:Genetic Vectors; D007700:Kinetics; D020935:MAP Kinase Signaling System; D009154:Mutation; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D011485:Protein Binding; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection
D017362:Cyclic AMP Response Element-Binding Protein; D018809:Proliferating Cell Nuclear Antigen