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Vol. 48, No. 3, 1991

Article (PDF 1927 KB)

Original Paper

Detection of Pleiotropic Drug Resistance by the RapidImmunofluorescence Assay of Drug Effects on the Cell SkeletonH. Mujagi, Z. Mujagi

Clinical Oncology Services, Institute of Pathologic Physiology, and Institute ofBiochemistry, Medical Faculty, University of Banja Luka, Bosna, Yugoslavia

Address of Corresponding Author

Oncology1991;48:202-209 (DOI: 10.1159/000226928)

Key Words

Cytoskeleton

Drug resistance

Tubulin-binding agents

Monoclonal antitubulin antibodies

Immunofluorescence

MCF-7 breast cancer cells

Abstract

We describe the development of an immunofluorescent method for the detection ofresistance to agents which affect the integrity of the cellular microtubular network.Three pleiotropic resistant MCF-7 human breast carcinoma cell lines mixed withvaginal adenocarcinoma cells were selected in serially increasing drugconcentrations, and demonstrated a 30-fold increase in resistance to colchicine.

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Transport studies indicated that there was no difference in drug accumulationbetween the sensitive and resistant lines. The colchicine-binding capacity of cellextracts from sensitive and resistant cells was similar (Kd for sensitive cells was 1.9 x10

-6Mand for resistant cells 1.58 x 10

-6M). There were, however, significant

differences in cytoskeletal morphology between sensitive and resistant cells. Drug-sensitive cells were mostly large (about 70 m

2) and flattened. Their cytoplasm was

filled with a microtubular network in which, in most of the cases, single fibers couldbe differentiated. Cells usually had a microtubule-organizing center and paracorticalbundles of microtubules. In contrast, drug-resistant cells were mostly rounded andgrew in clumps. In only 40% of these cells could single microtubular fibers bedifferentiated. Resistant cells lacked a microtubule-organizing center and had noclear paracortical bundles of microtubules. The tubulin-binding agents tested causeda sequence of morphological changes in sensitive cells. These changes includedprecipitation of tubulin and disappearance of cytoskeletal structure. Changesoccurred initially within 3 h of incubation, but were expressed in all cells after 6 h. If,after 3 h of drug exposure, cells were subcultured in drug-free media, thecytoskeletal structure reformed within 10 h. Maximal recovery of cytoskeletalstructure occurred 22 h after drug removal and was sustained up to 36 h. In contrastto changes observed in sensitive cells, drug exposure did not induce changes in themorphology of cytoskeleton in resistant cells. Cells from all three resistant lines

reverted to sensitivity after 7 months of culture in drug-free media. This was firstdetected by immunofluorescence and then confirmed by cloning assay. Since thecytoskeletal disintegration of sensitive cells is readily detectable within a few hoursof in vitro drug treatment, immunofluorescent imaging may have its clinicalapplication in predicting the sensitivity/resistance to microtubule-binding agents.

Copyright 1991 S. Karger AG, Basel

Author Contacts

H. Mujagi, Clinical Oncology Services, Medical Faculty, University of Banjaluka, S.Sabitovica 10, YU-78000 Banjaluka, Bosna (Yugoslavia)

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