820 Immunohistochemical analysis of p34 cdc2 and cyclin B cell localization in recurrent head and neck squamous cell carcinoma after irradiation

Abstract

After exposure to ionizing radiation, eukaryotic cells undergo a G2 delay which contributes to the ability of cells to survive irradiation. For some radioresistant cell lines, this delay is prolonged. Entry of cells into mitosis is regulated by a complex of two proteins cyclin B, and the serine-threonine p34^cdc2 kinase. When this complex is activated, it undergoes a transport from cytoplasm into the nucleus and phosphorylates proteins which lead to mitosis. P34<i>cdc2</i> kinase is activated by binding to cyclin B and by phosphorylation/dephosphorylation of p34<i>cdc2</i>. Since G₂ delay after irradiation has been correlated with a rapid inhibition of p34<i>cdc2</i> activity and an enhanced tyrosine phosphorylation, we hypothesized that radioresistant tumors could have a lack in regulation of p34<i>cdc2</i> kinase activity. In this study, we entered 32 patients treated, from 1983 through 1989 at the Claudius Regaud Center, for head and neck squamous cell carcinoma by surgery and standard post-operative doses of radiotherapy. The paraffin embedded tumor specimens had been sampled before radiotherapy for long term controlled patients (n=7) and before and after radiotherapy for patients who had developed a recurrence in the irradiation fields (n=25). Immunohistochemical staining was performed with monoclonal antibodies against p34<i>cdc2</i> (sc-54) and cyclin B (sc-245). A semi-quantitative score was used. For p34^cdc2 analysis, no difference in intensity of staining was observed between long term controlled patients and those who recurred or, when there was a recurrence before and after radiotherapy. However, there was a highly significant difference (<i>P</i><0.001) in p34<i>cdc2</i> cell localization with a preferential cytoplasmic localization only for the patients who have a recurrence in the radiotherapy fields. This cytoplasmic localization was present in the primary tumor before radiotherapy and in the recurrence, too. No preferential localization was observed in long term controlled patients. For cyclin B, no difference in intensity of staining was observed anywhere and conversely to p34<i>cdc2</i>, no difference in localization appeared in long term controlled patients nor for patients who have had a recurrence. No correlation existed between localization of p34<i>cdc2</i> and those of cyelin B in patients who recurred. Our results suggest a probably intrinsic abnormality of p34<i>cdc2</i> activity and a lack of association between eyelin B and p34<i>cdc2</i> in head and neck radioresistant squamous cell carcinoma.