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1042 Preferential cytoplasmic localization of P34CDC2 in recurrent head and neck squamous cell carcinoma after irradiation
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Radiation Oncology, Biology, Physics
Volume 32, Supplement 1
1042 PREFERENTIAL CYTOPLASMIC LOCALIZATION OF P34 coc2 IN RECURRENT HEAD AND NECK SQUAMOUS CELL CARCINOMA AFTER IRRADIATION. Cohen-Jonathan E. 1, Toulas C. 2, Rochaix P. 3, David J.F. 3, Daly-Schveitzer N. 1, Favre G. 2 1 - Radiotherapy Department; 2 - Laboratory of Molecular Oncology, 3 - Histopathoiogic Department - Centre Claudius Regaud - 2024, rue du Pont Saint-Pierre, 31052 Toulouse - France. Purpose/Objective : After exposure to ionizing radiation, eukaryotic cells undergo a G2 delay which contributes to the ability of cells to survive irradiation. Modulation of this delay seems to influence radiation sensitivity Entry of cells into mitosis is regulated by a complex of two proteins p34cdc2 and cyclin B. When this complex is activated, it undergoes a transport from cytoplasm into the nucleus and phosphorylates proteins which lead to mitosis, p34cdc2 kinase is activated by two pathways, one binding to cyclin B and the other by phosphorylation/dephosphorylation of p34cdc2 Since G2 delay after irradiation has been correlated with a rapid inhibition of p34cdc2 activity and an enhanced tyrosine phosphorylation, we hypothized that radioresistant tumors could have a lack in regulation of p34 cdc2 kinase activity that explains a greater G2 delay after irradiation. Materials and methods : In this study, we entered 32 patients treated, from 1983 through 1989 in our Institution , for head and neck squamous cell carcinoma by surgery and standard post-operative 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 which had developed a recurrence in the radiotherapy fields (n=25). Immunohistochemical staining was performed with monoclonal antibodies against p34cdc2 (sc-54) and cyclin B (sc-245). A semi-quantitative score was used. The percentage of cells with p34 cdc2 and/or cyclin B positive either in the cytoplasm or in the nucleus was rated as low (score 1 < 25 %), medium (score 2 = 25 - 75 %), or high (score 3 > 75 %). The statistical significance was assessed using Student's t and Chi square tests. Results : For p34cdc2 analysis, no difference in intensity of staining was seen between long term controlled patients and those wo recurred or, when there was a recurrence, before and after radiotherapy. However, there was a highly significant difference (p<0.001) in p34cdc2 cell localization with a preferential cytoplasmic localization only for the patients who have had a recurrence in the radiotherapy fields. This cytoplasmic localization was seen in the primary tumor before radiotherapy and in the recurrence, too. No preferential localization was seen in long term controlled patients. For cylin B, no difference in intensity of staining was seen anywhere and conversely to p34cdc2, no difference in localization appeared in long term controlled patients nor for patients who have had a recurrence. Iio correlation exists between localization of p34 cdc2 and those of cyclin B in patients who recurred. Conclusion : Our results show that p34 cdc2 has a preferential cytoplasmic localization only in tumors which recurre after radiotherapy. In these tumors, cyclin B is found equally in the cytoplasm or in nucleus, and is not correlated with p34 cdc2 localization. These findings suggest that radioresistant tumors may have an intrinsic inhibition of p34 cdc2 activity or a lack of link between cyctin B and p34cdc2 necessary to p34cdc2 activity which could increase the G2 delay after irradiation. Our results, taken together with litterature data, may contribute to explain part of the mechanism of intrinsic radioresistance in head and neck squamous cell carcinoma.
1043 INFLUENCE OF INTRACELLULAR pH ON THE A P O P T O S I S CAUSED BY HYPERTHERMIA Heonjoo P. Chae, MD., PhD. Carol M Makepeace. B S, John C. Lyons, B.S. Chang W Song, Ph.D. University of Minnesota Medical School. Department of Therapeutic Radiology, 424 Harvard St. S.E., Box 494 UMHC, Minneapolis, MN 55455 Purpose/Objective: Apoptotic cell death has been reported to be the malor mode of cell death by hyperthermia treatment. It is a well-known fact that a low intracellular pH (pHi) enhances the cytotoxicity of hyperthermia. The purpose of this study was to elucidate the role of pHi in the induction of apoptosis by hyperthermia Materials and Methods: Apopotosis in HL-60 human promyelocytic leukemic cells and SCK mammary carcinoma cells of A/J mice was quantitated with the use of agarose gel electrophoresis of DNA and released of 9H from 3H-thymidine labelled cells. The pHi was varied by varying the medium pH and also with the use of nigericin (K+ ionophore) and with the inhibitors of PHi regulatory mechanisms, HMA and DIDS Results: At 37°C, incubation of the cells in pH 6.6 medium, but not pH 7 5 medium, for 4-7 hrs. caused aboptosis. The level of apoptosis progressively increased as the medium pH was lowered until 64, at which the pHi was 6.8. Apoptosis declined when the medium pH was further lowered below 6.4 indicating that apoptosis maximally occurs at PHi 6.8. Apoptosis was increased also when the pHi of the cells in pH 7.5 or pH 6.6 medium was lowered to 6.8 using various combinations of nigericin, HMA and DIDS. However, lowering the pHi to below 6.8 with the drugs diminished the apoptosis. Hyperthermia at 42°C for 60 min. induced apoptosis in both HL-60 cells and SCK cells. The level of apoptosis caused by hyperthermia also increased as the pHi was lowered to 6.8, but it declined when the pHi was lowered further. Contrary to the apoptosis, the decline in total clonogenic cell population by hyperthermia was enhanced by lowering the pHi to below 6.8 probably due to an increase in the heat-induced necrotic cell death in the acidic environment Conclusion: Apoptotic cell death caused by hyperthermia progressively increases until the intracellular pH is lowered to about 6.8. As the pHi is lowered to below 6.8, necrotic cell death becomes the malor mode of cell death by hyperthermia. (This work was supported by NCl grant number CA44114.)
Radiation Oncology, Biology, Physics
Volume 32, Supplement 1
1042 PREFERENTIAL CYTOPLASMIC LOCALIZATION OF P34 coc2 IN RECURRENT HEAD AND NECK SQUAMOUS CELL CARCINOMA AFTER IRRADIATION. Cohen-Jonathan E. 1, Toulas C. 2, Rochaix P. 3, David J.F. 3, Daly-Schveitzer N. 1, Favre G. 2 1 - Radiotherapy Department; 2 - Laboratory of Molecular Oncology, 3 - Histopathoiogic Department - Centre Claudius Regaud - 2024, rue du Pont Saint-Pierre, 31052 Toulouse - France. Purpose/Objective : After exposure to ionizing radiation, eukaryotic cells undergo a G2 delay which contributes to the ability of cells to survive irradiation. Modulation of this delay seems to influence radiation sensitivity Entry of cells into mitosis is regulated by a complex of two proteins p34cdc2 and cyclin B. When this complex is activated, it undergoes a transport from cytoplasm into the nucleus and phosphorylates proteins which lead to mitosis, p34cdc2 kinase is activated by two pathways, one binding to cyclin B and the other by phosphorylation/dephosphorylation of p34cdc2 Since G2 delay after irradiation has been correlated with a rapid inhibition of p34cdc2 activity and an enhanced tyrosine phosphorylation, we hypothized that radioresistant tumors could have a lack in regulation of p34 cdc2 kinase activity that explains a greater G2 delay after irradiation. Materials and methods : In this study, we entered 32 patients treated, from 1983 through 1989 in our Institution , for head and neck squamous cell carcinoma by surgery and standard post-operative 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 which had developed a recurrence in the radiotherapy fields (n=25). Immunohistochemical staining was performed with monoclonal antibodies against p34cdc2 (sc-54) and cyclin B (sc-245). A semi-quantitative score was used. The percentage of cells with p34 cdc2 and/or cyclin B positive either in the cytoplasm or in the nucleus was rated as low (score 1 < 25 %), medium (score 2 = 25 - 75 %), or high (score 3 > 75 %). The statistical significance was assessed using Student's t and Chi square tests. Results : For p34cdc2 analysis, no difference in intensity of staining was seen between long term controlled patients and those wo recurred or, when there was a recurrence, before and after radiotherapy. However, there was a highly significant difference (p<0.001) in p34cdc2 cell localization with a preferential cytoplasmic localization only for the patients who have had a recurrence in the radiotherapy fields. This cytoplasmic localization was seen in the primary tumor before radiotherapy and in the recurrence, too. No preferential localization was seen in long term controlled patients. For cylin B, no difference in intensity of staining was seen anywhere and conversely to p34cdc2, no difference in localization appeared in long term controlled patients nor for patients who have had a recurrence. Iio correlation exists between localization of p34 cdc2 and those of cyclin B in patients who recurred. Conclusion : Our results show that p34 cdc2 has a preferential cytoplasmic localization only in tumors which recurre after radiotherapy. In these tumors, cyclin B is found equally in the cytoplasm or in nucleus, and is not correlated with p34 cdc2 localization. These findings suggest that radioresistant tumors may have an intrinsic inhibition of p34 cdc2 activity or a lack of link between cyctin B and p34cdc2 necessary to p34cdc2 activity which could increase the G2 delay after irradiation. Our results, taken together with litterature data, may contribute to explain part of the mechanism of intrinsic radioresistance in head and neck squamous cell carcinoma.
1043 INFLUENCE OF INTRACELLULAR pH ON THE A P O P T O S I S CAUSED BY HYPERTHERMIA Heonjoo P. Chae, MD., PhD. Carol M Makepeace. B S, John C. Lyons, B.S. Chang W Song, Ph.D. University of Minnesota Medical School. Department of Therapeutic Radiology, 424 Harvard St. S.E., Box 494 UMHC, Minneapolis, MN 55455 Purpose/Objective: Apoptotic cell death has been reported to be the malor mode of cell death by hyperthermia treatment. It is a well-known fact that a low intracellular pH (pHi) enhances the cytotoxicity of hyperthermia. The purpose of this study was to elucidate the role of pHi in the induction of apoptosis by hyperthermia Materials and Methods: Apopotosis in HL-60 human promyelocytic leukemic cells and SCK mammary carcinoma cells of A/J mice was quantitated with the use of agarose gel electrophoresis of DNA and released of 9H from 3H-thymidine labelled cells. The pHi was varied by varying the medium pH and also with the use of nigericin (K+ ionophore) and with the inhibitors of PHi regulatory mechanisms, HMA and DIDS Results: At 37°C, incubation of the cells in pH 6.6 medium, but not pH 7 5 medium, for 4-7 hrs. caused aboptosis. The level of apoptosis progressively increased as the medium pH was lowered until 64, at which the pHi was 6.8. Apoptosis declined when the medium pH was further lowered below 6.4 indicating that apoptosis maximally occurs at PHi 6.8. Apoptosis was increased also when the pHi of the cells in pH 7.5 or pH 6.6 medium was lowered to 6.8 using various combinations of nigericin, HMA and DIDS. However, lowering the pHi to below 6.8 with the drugs diminished the apoptosis. Hyperthermia at 42°C for 60 min. induced apoptosis in both HL-60 cells and SCK cells. The level of apoptosis caused by hyperthermia also increased as the pHi was lowered to 6.8, but it declined when the pHi was lowered further. Contrary to the apoptosis, the decline in total clonogenic cell population by hyperthermia was enhanced by lowering the pHi to below 6.8 probably due to an increase in the heat-induced necrotic cell death in the acidic environment Conclusion: Apoptotic cell death caused by hyperthermia progressively increases until the intracellular pH is lowered to about 6.8. As the pHi is lowered to below 6.8, necrotic cell death becomes the malor mode of cell death by hyperthermia. (This work was supported by NCl grant number CA44114.)