Arsenic trioxide induces depolymerization of microtubules in an acute promyelocytic leukemia cell line

BACKGROUND: Arsenic trioxide (As2O3) is a well-known and effective treatment that can result in clinical remission for patients diagnosed with acute promyelocytic leukemia (APL). The biologic efficacy of As2O3 in APL and solid tumor cells has been explained through its actions on anti-proliferation, anti-angiogenesis, and apoptotic signaling pathways. We theorize that As2O3 activates a pathway that disrupts microtubule dynamics forming abnormal, nonfunctioning mitotic spindles, thus preventing cellular division. In this study, we investigated how As2O3 induces apoptosis by causing microtubule dysfunction. METHODS: Cultured NB4 cells were treated with As2O3, paclitaxel, and vincristine. Flow cytometric analysis was then performed. An MTT assay was used to determine drug-mediated cytotoxicity. For tubulin polymerization assay, each polymerized or soluble tubulin was measured. Microtubule assembly-disassembly was measured using a tubulin polymerization kit. Cellular microtubules were also observed with fluorescence microscopy. RESULTS: As2O3 treatment disrupted tubulin assembly resulting in dysfunctional microtubules that cause death in APL cells. As2O3 markedly enhanced the amount of depolymerized microtubules. The number of microtubule posttranslational modifications on an individual tubulin decreased with As2O3 concentration. Immunocytochemistry revealed changes in the cellular microtubule network and formation of polymerized microtubules in As2O3-treated cells. CONCLUSION: The microtubules alterations found with As2O3 treatment suggest that As2O3 increases the depolymerized forms of tubulin in cells and that this is potentially due to arsenite's negative effects on spindle dynamics.

CKbeta8-1 alters expression of cyclin E in colony forming units-granulocyte macrophage (CFU-GM) lineage from human cord blood CD34 + cells

A C6 beta-chemokine, CKbeta8-1, suppressed the colony formation of CD34 + cells of human cord blood (CB). Molecular mechanisms involved in CKbeta8-1-medicated suppression of colony formation of CD34 + cells are not known. To address this issue, the level of various G1/S cell cycle regulating proteins in CKbeta8-1-treated CD34 + cells were compared with those in untreated CD34 + cells. CKbeta8-1 did not significantly alter the expression of the G1/S cycle regulation proteins (cyclin D1, D3, and E), CDK inhibitor (p27and Rb), and other cell proliferation regulation protein (p53) in CB CD34 + cells. Here we describe an in vitro system in which CB CD34 + cells were committed to a multipotent progenitor lineage of colony forming units-granulocyte/macrophage (CFU-GM) by a simple combination of recombinant human (rh) GM-CSF and rhIL-3. In this culture system, we found that cyclin E protein appeared later and disappeared faster in the CKbeta8-1-treated cells than in the control cells during CFU-GM lineage development. These findings suggested that cyclin E may play a role in suppressing the colony formation of CFU-GM by CKbeta8-1.