CELL CYCLE, APOPTOSIS AND P53-DEPENDENT GENES EXPRESSION AFTER g-IRRADIATION ARE CORRELATED TO THE P53 STATUS IN FIVE HUMAN HEAD AND NECK SQUAMOUS CELL CARCINOMA CELL LINES

Anne-France Dekairellea,*, Jean-François Rosierb,c, Vincent Grégoireb and Jean-Luc Galaa,d

aLaboratory of Applied Molecular Technology, Center for Human Genetics, and  bRadiobiology Unit, Université Catholique de Louvain (UCL), Bruxelles, Belguim ; cC.H. Jolimont, Tumoral Metabolism Research Unit, Belgium ; dDefense Laboratories Department (DG MR), Brussels, Belgian Armed Forces

* Anne-France.Dekairelle@lbcm.ucl.ac.be

INTRODUCTION. The tumor suppressor p53 protein plays a central role in maintaining the genomic integrity of the cell, among others by the control of the cell cycle and the regulation of apoptosis (1,2) through transcriptional modulation of regulatory genes (3). It is estimated that more than 50% of all human cancers contain a p53 mutation (1,4). In  squamous cell carcinoma of the head and neck (HNSCC) ~80% of the tumors have shown to be p53 mutated (5). In HNSCC discrepant results have been observed between p53 status, tumor response and outcome, making p53 a controversial prognostic indicator in this group of tumors (6). In the current study, we used a functional assay in yeasts to characterize the p53 gene status in five HNSCC cell lines (SCC61, SQD9 , SC173, SC179 and SC263) and compared this to post-irradiation cell cycle arrests, induction of apoptosis and induced expression of p21, GADD45, bax, bcl-2 and cyclinB1.

METHODS. The p53 status of each cell line was determined by immunocytochemistry (ICC) and functional analysis of separated alleles in yeast (FASAY) against p21 and bax promoter regions. Cell cycle analysis was assessed by flow cytometry and apoptosis by determination of caspase-3-like activity as well as cytoplasmic histone-associated DNA fragments. The expression of p53, p21, GADD45, bax, bcl-2 and cyclinB1 was quantified by real-time Taqman PCR.

RESULTS. P53 nuclear overexpression was found in all HNSCC, except SCC63. Expression of a non functional p53 protein was evidenced by FASAY in the five cell lines. Sequence analysis confirmed p53 inactivation in four SCC cell lines due to missense mutations, and the lack of p53 expression in p53 ICC-negative SC263 due to a nonsense mutation. No arrest in G1 and no apoptosis was seen after irradiation, in accordance with the lack of p53-mediated p21 and bax transactivation on FASAY. Whereas no irradiation-induced apoptotic response was observed in the 5 HNSCC cell lines, they all presented a post-irradiation G2/M arrest. By real time PCR, overexpression of p53 and lack of induction of  p21, GADD45, bax, bcl-2 and cyclinB1 were found in the five HNSCC cell lines. 

DISCUSSION. According to the in vitro results, the functional assay is expected to contribute to a better understanding of cellular responses of HNSCC cells to DNA damaging agents, and to fill the gap between the biological, biophysical, pharmacological and clinical fields. These p53-mutated and radioresistant cell lines appear as a good model for assessing the efficacy of apoptotic drugs acting on the G2/M chekpoint or attempts to restore the p53 proapoptotic function.

ACKNOWLEDGMENT. This study was supported by Loterie Nationale (grant N° N4064-1A) and the Fonds National de la Recherche Scientifique (grant F.N.R.S. - Télévie N° 7.4512.02). 

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