ΔNp63 expression in four carcinoma cell lines and the effect on radioresistance--a siRNA knockdown model.
OBJECTIVE
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.


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.


CONCLUSIONS
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.
Department of Oral and Maxillofacial Surgery, University of Mainz, Medical Center, Augustusplatz 2, 55131, Mainz, Germany. maximilian.moergel@unimedizin-mainz.de.|Department of Oral and Maxillofacial Surgery, Johannes Gutenberg-University, Medical Center, Augustusplatz 2, 55131, Mainz, Rheinland-Pfalz, Germany. maximilian.moergel@unimedizin-mainz.de.
Maximilian
M
Moergel
Department of Oral and Maxillofacial Surgery, University of Mainz, Medical Center, Augustusplatz 2, 55131, Mainz, Germany.
Jutta
J
Goldschmitt
Department of Radiation Oncology, University of Mainz, Medical Center, Langenbeckstr. 1, 55101, Mainz, Germany.
Marcus
M
Stockinger
Department of Oral and Maxillofacial Surgery, University of Bochum, Medical Center, In der Schornau 23-25, 44892, Bochum, Germany.
Martin
M
Kunkel
D002294:Carcinoma, Squamous Cell / Q000235:genetics* / Q000473:pathology; D045744:Cell Line, Tumor; D055785:Gene Knockdown Techniques; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D008957:Models, Genetic; D020033:Protein Isoforms / Q000235:genetics* / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics*; D011836:Radiation Tolerance / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
C081876:CKAP4 protein, human; D008565:Membrane Proteins; D020033:Protein Isoforms; D034741:RNA, Small Interfering