POLYMERIZATION OF TUBULIN IN APOPTOTIC CELLS IS NOT CELL-CYCLE DEPENDENT

Prominent, specific tubulin structures were identified in human leukem ic cells undergoing apoptosis following treatment with cytotoxic drugs . In order to determine whether tubulin reorganization was dependent u pon the stage of the cell cycle at which apoptosis was induced, the hu man leukemic T-cell line CCRF-CEM was treated with cytotoxic doses of drugs known to arrest cells at different stages of the cell cycle. Apo ptosis was confirmed by the detection of characteristic single and mul tiple nucleosome-sized fragments by agarose gel electrophoresis of iso lated DNA. Cells were treated with vincristine, methotrexate, and dexa methasone, which have been shown to induce cell cycle arrest at G(2)-M , S-phase, and G(1), respectively. Treated and untreated cells were an alyzed by immunocytochemistry for beta-tubulin or Ki-67 antigen (to co nfirm cell cycle phase) and scored for apoptotic morphology. Dual stai ning for cellular tubulin and DNA content, measured by flow cytometry, was used to confirm the stage at which the cycling cells arrested. In creased total cellular tubulin immunofluorescence was observed in trea ted compared to untreated cells. Our results indicate that CCRF-CEM ce lls undergo apoptosis (identified morphologically) at all stages of th e cell cycle except mitosis. We conclude that the reorganization of ce llular tubulin that we have observed in apoptotic cells is independent of the tubulin involvement in cell division and thus may be an integr al part of the apoptotic process. (C) 1994 Academic Press, Inc.