Maximilian Moergel1,2, Jutta Goldschmitt3, Marcus Stockinger4, Martin Kunkel5. 1. Department of Oral and Maxillofacial Surgery, University of Mainz, Medical Center, Augustusplatz 2, 55131, Mainz, Germany. maximilian.moergel@unimedizin-mainz.de. 2. Department of Oral and Maxillofacial Surgery, Johannes Gutenberg-University, Medical Center, Augustusplatz 2, 55131, Mainz, Rheinland-Pfalz, Germany. maximilian.moergel@unimedizin-mainz.de. 3. Department of Oral and Maxillofacial Surgery, University of Mainz, Medical Center, Augustusplatz 2, 55131, Mainz, Germany. 4. Department of Radiation Oncology, University of Mainz, Medical Center, Langenbeckstr. 1, 55101, Mainz, Germany. 5. Department of Oral and Maxillofacial Surgery, University of Bochum, Medical Center, In der Schornau 23-25, 44892, Bochum, Germany.
Abstract
OBJECTIVES: This study investigated the expression of ΔNp63α in carcinoma cell lines of the upper aerodigestive tract and their potential influence on radioresistance using a small interfering RNA (siRNA) knockdown approach. MATERIALS AND METHODS: Four carcinoma cell lines were investigated for the expression of the ΔNp63 isoform by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) (0, 24, 48 h) with and without single dose irradiation of 6 Gy. Furthermore, all cell lines were transfected with siRNA against the ΔNp63α isoform over 24 h. Knockdown effectiveness was controlled by qRT-PCR and Western blot. Apoptotic events were evaluated by terminal transferase dUTP nick end labeling (TUNEL) assay and cross-checked by a test for cell viability (WST-1, Roche) over 48 h. RESULTS: All cell lines presented varying expression of the ΔNp63α isoform with and without irradiation. A sufficient knockdown rate was established by siRNA transfection. Knockdown of the ΔNp63 isoform showed an effect on radiation sensitivity proven by an increase of apoptotic events detectable by immunofluorescence (TUNEL assay) and likewise a significant reduction of formazan production (WST-1 test) in three cell lines. CONCLUSIONS: We found overexpression of ΔNp63α with and without irradiation in three cell lines, and the knockdown of ΔNp63α led to increased apoptotic events and fewer viable cells. Thus, the overexpression of ΔNp63α might protect carcinoma cells against irradiation effects. CLINICAL RELEVANCE: The present work supports the hypothesis that protein 63 might serve as a negative predictor for irradiation response and survival in a clinical setting and may be a target for future therapeutic strategies.
OBJECTIVES: This study investigated the expression of ΔNp63α in carcinoma cell lines of the upper aerodigestive tract and their potential influence on radioresistance using a small interfering RNA (siRNA) knockdown approach. MATERIALS AND METHODS: Four carcinoma cell lines were investigated for the expression of the ΔNp63 isoform by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) (0, 24, 48 h) with and without single dose irradiation of 6 Gy. Furthermore, all cell lines were transfected with siRNA against the ΔNp63α isoform over 24 h. Knockdown effectiveness was controlled by qRT-PCR and Western blot. Apoptotic events were evaluated by terminal transferase dUTP nick end labeling (TUNEL) assay and cross-checked by a test for cell viability (WST-1, Roche) over 48 h. RESULTS: All cell lines presented varying expression of the ΔNp63α isoform with and without irradiation. A sufficient knockdown rate was established by siRNA transfection. Knockdown of the ΔNp63 isoform showed an effect on radiation sensitivity proven by an increase of apoptotic events detectable by immunofluorescence (TUNEL assay) and likewise a significant reduction of formazan production (WST-1 test) in three cell lines. CONCLUSIONS: We found overexpression of ΔNp63α with and without irradiation in three cell lines, and the knockdown of ΔNp63α led to increased apoptotic events and fewer viable cells. Thus, the overexpression of ΔNp63α might protect carcinoma cells against irradiation effects. CLINICAL RELEVANCE: The present work supports the hypothesis that protein 63 might serve as a negative predictor for irradiation response and survival in a clinical setting and may be a target for future therapeutic strategies.
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