Polymerization of tubulin in apoptotic cells is not cell cycle dependent

Prominent, specific tubulin structures were identified in human leukemic cells undergoing apoptosis following treatment with cytotoxic drugs. In order to determine whether tubulin reorganization was dependent upon the stage of the cell cycle at which apoptosis was induced, the human leukemic T-cell line CCRF-CEM was treated with cytotoxic doses of drugs known to arrest cells at different stages of the cell cycle. Apoptosis was confirmed by the detection of characteristic single and multiple nucleosome-sized fragments by agarose gel electrophoresis of isolated DNA. Cells were treated with vincristine, methotrexate, and dexamethasone, which have been shown to induce cell cycle arrest at G2-M, S-phase, and G1, respectively. Treated and untreated cells were analyzed by immunocytochemistry for beta-tubulin or Ki-67 antigen (to confirm cell cycle phase) and scored for apoptotic morphology. Dual staining for cellular tubulin and DNA content, measured by flow cytometry, was used to confirm the stage at which the cycling cells arrested. Increased total cellular tubulin immunofluorescence was observed in treated compared to untreated cells. Our results indicate that CCRF-CEM cells undergo apoptosis (identified morphologically) at all stages of the cell cycle except mitosis. We conclude that the reorganization of cellular tubulin that we have observed in apoptotic cells is independent of the tubulin involvement in cell division and thus may be an integral part of the apoptotic process.