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Biochemical genetic analysis of indanocine resistance in human leukemia
1Department of Medicine and The Sam and Rose Stein Institute for Research on Aging, University of California, San Diego, La Jolla, California 92093-0663, USA.
Abstract:
Indanocine is a potent tubulin-binding drug that is cytotoxic to multidrug-resistant cancer cell lines. We demonstrated that indanocine specifically induces apoptosis in malignant B cells from patients with chronic lymphocytic leukemia. To address the exact biochemical basis for indanocine toxicity, an indanocine-resistant clone was selected from mutagenized CEM human lymphoblastoid cells. The resistant cells displayed a stable indanocine-resistant phenotype for at least 9 months in drug-free culture. The cloned cells are cross-resistant to colchicine and vinblastine, but not to paclitaxel, and do not have increased expression of the multidrug-resistant p170 glycoprotein. In both parental cells and cell extracts, indanocine treatment caused tubulin depolymerization. In contrast, the tubulin in the resistant clone did not depolymerize under identical conditions. Both extract mixing and cell fusion experiments suggested that a stable structural change in microtubules, rather than a soluble factor, was responsible for indanocine resistance. Sequence analysis of parental and resistant cells revealed a single point mutation in the M40 isotype of beta-tubulin at nucleotide 1050 (G-->T, Lys(350)-->Asn) in the indanocine-resistant clone, in a region close to the putative colchicine binding site.
Insights
Indanocine induces apoptosis in chronic lymphocytic leukemia cells. A specific mutation in beta-tubulin causes resistance to indanocine by preventing tubulin depolymerization.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Indanocine is a cytotoxic drug targeting tubulin, effective against multidrug-resistant cancer cells.
- Indanocine induces apoptosis in malignant B cells from chronic lymphocytic leukemia patients.
Purpose of the Study:
- To elucidate the biochemical mechanism underlying indanocine's toxicity.
- To identify the molecular basis of resistance to indanocine.
Main Methods:
- Selection and characterization of an indanocine-resistant cell clone.
- Cross-resistance profiling with other tubulin-binding agents.
- Analysis of tubulin polymerization and depolymerization.
- Cell fusion and extract mixing experiments.
- DNA sequencing of beta-tubulin isotypes.
Main Results:
- The resistant clone exhibited cross-resistance to colchicine and vinblastine, but not paclitaxel.
- Indanocine failed to induce tubulin depolymerization in the resistant clone.
- A point mutation (G-->T, Lys350Asn) was identified in the M40 isotype of beta-tubulin.
- The mutation is located near the colchicine binding site and confers resistance via a stable structural change in microtubules.
Conclusions:
- Indanocine resistance is mediated by a structural alteration in beta-tubulin, not by a soluble factor or P-glycoprotein.
- The Lys350Asn mutation in beta-tubulin directly impacts indanocine's ability to bind and depolymerize microtubules.
- This finding provides critical insight into the molecular mechanisms of drug resistance in cancer therapy.