Cytoskeletal proteins distribution patterns in HL-60 leukemia cells treated with doxorubicin

A Grzanka PhD

Department of Clinical Pathomorphology, University School of Medical Sciences and Institute of Biology and Environment Protection, University of Kazimierz Wielki Bydgoszcz, Poland

AIM: The present study characterizes reorganization of actin, vimentin and tubulin in HL-60 leukemia cell line after treatment with doxorubicin using fluorescence microscopy and further define distribution pattern of actin at the ultrastructural level. METHODS: F-actin was labeled with phalloidin conjugated to rhodamine (TRITC), vimentin and tubulin by indirect immunofluorescence methods. TRITC-labeled goat anti rabbit IgG for tubulin and FITC-labeled goat antimouse IgG for vimentin were used. To reduce the lose of soluble proteins during permeabilization, cells were prefixed with bifunctional protein crosslinking reagent DSP (dithiobis succnimidylpropionate). Cells for estimation in fluorescence microscope were collected by using a cytocentrifuge directly onto microscope slide and fixed with 4% paraformaldehyde in microtubule stabilizing buffer (MTSB). To demonstrate actin at the ultrastructural level, a postembedding streptavidin-gold method was used. RESULTS: Doxorubicin treatment of HL-60 cells induced reorganization of cytoskeleton proteins dependent on concentration of doxorubicin. Significant changes in morphology of the cells and in proteins distribution occurred after treatment with 5 and 10 µM doxorubicin. In comparison with control cells, the number of cells decreased and cells were larger, almost all treated cells had irregular surfaces. It was found that vimentin was retracted from cell periphery but there were also cells with budding staining for vimentin, whereas F- actin and tubulin was rather seen at the periphery of the cells. There were labeling of F-actin microfilaments and tubulin at the surface of cells with features of apoptosis. Immunogold labeling of actin was observed in control cells and treated with doxorubicin. Labeling was found in the nucleus and also in the cytoplasm. At the ultrastructural level, cells treated with doxorubicin in a range 5-10µM showed increased positivity for actin in relation with blebbing, margination of nuclear chromatin and bodies containing recognizable nuclear fragments. CONCLUSIONS: The results seemed to indicate that the reorganization of 3 studied cytoskeletal proteins in HL-60 cells treated with doxorubicin is dose-dependent and related rather with features of apoptosis, especially distribution of actin is seemed to be connected with apoptotic body formation.

For more information, contact

Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Paris, France; February 9 - 12, 2002; in the section on Apoptosis - Molecular Mechanisms.