The need for dynamic methods for measuring cell cycle perturbations: a study in radiation-treated lymphoblastoid cell lines of varying p53 status.
Reports on the p53-related cell cycle and apoptotic responses of EBV-transformed lymphoblastoid cell lines to DNA damage have led to some confusion. This may be due to differences in the nature of the specific p53 mutations under examination, but it can also be partly attributed to methodological and analytical problems (e.g. the inappropriate use of static DNA histograms for cell cycle analysis). Taking seven lymphoblastoid cell lines derived from both normal individuals and Li-Fraumeni Syndrome/Li-Fraumeni-Like (LFS/LFL) patients of differing p53 status, we completed a detailed study of radiation-induced cell cycle perturbations. Using BrdUrd pulse labelling and flow cytometry it was found that, regardless of p53 status, the cells did not arrest in G1 despite all of the lines showing p53 upregulation 3 hours postirradiation. The irradiated cells did, however, show a general slowing both in S-phase entry from G1 and in movement through S-phase. These facts would not have been apparent from the analysis of static DNA histograms. The problems with the use of static methods to assess changes in the dynamics of cell cycle progression apply not only to studies involving EBV-transformed cell lines, but also to a wide range of investigations into the molecular control of cell proliferation.
Gilchrist
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Richard Dimbleby Department of Cancer Research, UMDS, St Thomas' Hospital, London, UK.
Lomax
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ME
Camplejohn
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RS
D000963:Antimetabolites; D017209:Apoptosis / Q000528:radiation effects; D001402:B-Lymphocytes / Q000737:chemistry* / Q000166:cytology* / Q000821:virology; D015153:Blotting, Western; D001973:Bromodeoxyuridine; D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral; D004273:DNA, Neoplasm / Q000032:analysis; D016193:G1 Phase / Q000502:physiology / Q000528:radiation effects; D016195:G2 Phase / Q000502:physiology / Q000528:radiation effects; D004854:Herpesvirus 4, Human / Q000235:genetics; D006801:Humans; D016196:S Phase / Q000502:physiology / Q000528:radiation effects; D013194:Staining and Labeling; D016159:Tumor Suppressor Protein p53 / Q000032:analysis*
D000963:Antimetabolites; D004273:DNA, Neoplasm; D016159:Tumor Suppressor Protein p53; D001973:Bromodeoxyuridine