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Cell Cycle Disturbances and Mitotic Catastrophes in HeLa Hep2 Cells following 2.5 to 10 Gy of Ionizing Radiation

Authors' Affiliations: Departments of ¹ Immunology, ² Radiation Physics, and ³ Diagnostic Radiology, Umeå university, Umeå, Sweden

Requests for reprints: Torgny Stigbrand, Department of Immunology, Umeå University, 90185 Umeå, Sweden. Phone: 46-90-785-2671; Fax: 46-90-785-2250; E-mail: torgny.stigbrand{at}climi.umu.se.

Purpose: Experimental radioimmunotherapy delivering absorbed doses of 2.5 to 10 Gy has been shown to cause growth retardation of tumors. The purpose of this study was to elucidate the sequential molecular and cellular events occurring in HeLa Hep2 cells exposed to such doses.

Methods: Dose-response curves, activation of cell cycle checkpoints, and mitotic behavior were investigated in HeLa Hep2 cells following 2.5- to 10-Gy irradiation by carrying out 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, Western blots, fluorescence-activated cell sorting analysis, and immunofluorescence stainings. Terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling staining was used to detect apoptosis.

Results: A G₂-M arrest was shown by fluorescence-activated cell sorting analysis. p53 and p21 were found to be up-regulated but were not immediately related to the arrest. The G₂-M arrest was transient and the cells reentered the cell cycle still containing unrepaired cellular damage. This premature entry caused an increase of anaphase bridges, lagging chromosomal material, and multipolar mitotic spindles as visualized by propidium iodide staining and immunofluorescence staining with -tubulin and -tubulin antibodies. Furthermore, a dose-dependent significant increase in centrosome numbers from 12.6 ± 6.6% to 67 ± 5.3% was identified as well as a dose-dependent increase of polyploid cells from 2.8 ± 1.3% to 17.6 ± 2.1% with the highest absorbed dose of 10 Gy. These disturbances caused the cells to progress into mitotic catastrophe and a fraction of these dying cells showed apoptotic features as displayed by terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling staining 5 to 7 days after irradiation.

Conclusion: An absorbed dose of 2.5 to 10 Gy was shown to force HeLa Hep2 cells into mitotic catastrophe and delayed apoptosis. These might be important cell death mechanisms involved in tumor growth retardation following radioimmunotherapy of solid tumors.