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DNA Damage and Cellular Stress Responses

Lack of P-Glycoprotein Expression by Low-Dose Fractionated Radiation Results from Loss of Nuclear Factor-B and NF-Y Activation in Oral Carcinoma Cells

¹ Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania; Departments of ² Radiation Medicine and ³ Surgery (Division of Otolaryngology), University of Kentucky, Lexington, Kentucky; ⁴ Geisinger-Fox Chase Cancer Center, Geisinger Clinic, Wilkes-Barre, Pennsylvania; ⁵ Department of Radiation Oncology, Fox Chase Cancer Center, Philadelphia, Pennsylvania; ⁶ Department of Surgery, Central Arkansas Veterans Healthcare Services, and ⁷ Department of Otolaryngology-Head and Neck Surgery, University of Arkansas for Medical Sciences, Little Rock, Arkansas

Requests for reprints: Paul M. Spring, Basic Research, Otolaryngology-Head and Neck Surgery, University of Arkansas for Medical Sciences, Little Rock, AR 72205. Phone: 501-686-5020; Fax: 501-526-7153. E-mail: pspring{at}uams.edu

Multidrug resistance (MDR) is associated with the overproduction of the 170-kDa transmembrane protein P-glycoprotein (MDR1) caused by transcriptional activation. However, the activity of the MDR1 promoter in response to different doses of ionizing radiation has not been investigated. In this study, two squamous cell carcinoma oral cavity cell lines, T-167 and T-409, were exposed to either a standard clinical dose of 2 Gy or low-dose fractionated radiation therapy (LDFRT), delivered as 0.5 Gy in four fractions. MDR1 gene expression and degree of cell death were assessed. Clinically relevant 2-Gy dose of radiation resulted in increased expression of MDR1 by reverse transcription-PCR and luciferase reporter assays in both cell lines (T-167 and T-409), whereas LDFRT did not. LDFRT caused enhanced apoptosis when compared with the 2-Gy dose in T-167 and T-409 cells as assessed by terminal nucleotidyl transferase–mediated nick end labeling (TUNEL) assays. Transcription of the MDR1 gene is regulated by numerous transcription factors, which include nuclear factor-B (NF-B), NF-Y, SP1, YB1, MEF1 (MDR1 promoter-enhancing factor 1), p53, and NF-R1. Interestingly, 2 Gy robustly induced both NF-B and NF-Y in T-167 and T-409 cells, but did not show induction when exposed to LDFRT. Silencing the expression of the DNA binding subunit of NF-B, p50, by small interfering RNA vector resulted in a decrease of MDR1 function by rhodamine 123 efflux assay in T167 cells exposed to 2 Gy. Together, these results provide evidence for the lack of induction of P-glycoprotein expression by LDFRT, which has important implications in combinatorial cancer therapy, including the use of LDFRT as an adjuvant for chemotherapy. (Mol Cancer Res 2008;6(1):89–98)