Abstract
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.
Highlights
Flow cytometric analysis of propidium iodide stained cells (Table II) demonstrated no significant (p Ͼ 0.05) cell cycle redistribution at the time of maximum CR133 or KCREB expression (48 h after transfection); similar results were obtained when pCMV-proliferating cell nuclear antigen (PCNA) and CR133 were co-transfected. This demonstrates that radiosensitization by DN-CREB expression is at least in part mediated by PCNA and its effect on DNA repair. These studies were initiated because we have previously shown that the transcription factors CREB, early growth response, ETS, and STAT3 are activated by ionizing radiation via the epidermal growth factor receptor (EGFR)/mitogen-activated protein kinase (MAPK) pathway [8], and inhibition of either EGFR tyrosine phosphorylation or MAPK activity radiosensitizes a variety of human tumor cells [2,3,4,5]
Because PCNA is both transcriptionally regulated by CREB (10 –12) and involved in repair of radiation-induced DNA damage (14 –18), we examined the effects of DN-CREB on PCNA promoter activation and expression of PCNA protein
These experiments show that DN-CREB expression significantly reduces basal levels of both PCNA-chloramphenicol acetyl transferase (CAT) reporter construct activity and PCNA protein, affirming the role of CREB on PCNA gene expression
Summary
These results expand on previous studies with other cell types demonstrating by gel-shift analysis radiation-induced stimulation of CREB binding [9] Other transcription factors, such as CCAATT/enhancer-binding protein- and STAT1, only partially depend on signals from the EGFR/. 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. The activation of epidermal growth factor receptor (EGFR) by ionizing radiation induces in autocrine growth regulated (and progression through the S-phase of the cell cycle) (19 –20), depletion of PCNA could potentially modify cellular radiosensitivity through both repair inhibition and cell cycle redistribution mechanisms. We have investigated the mechanism of DN-CREB-mediated radiosensitization by defining the effects of CREB on PCNA function and expression
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