Journal Article

Alterations in γ-Actin and Tubulin-Targeted Drug Resistance in Childhood Leukemia

Nicole M. Verrills, Sela T. Po'uha, Marjorie L. M. Liu, Tracy Y. E. Liaw, Martin R. Larsen, Michael T. Ivery, Glenn M. Marshall, Peter W. Gunning and Maria Kavallaris

in JNCI: Journal of the National Cancer Institute

Volume 98, issue 19, pages 1363-1374
Published in print October 2006 | ISSN: 0027-8874
Published online October 2006 | e-ISSN: 1460-2105 | DOI:

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Background: Proteomic investigations have revealed alterations in cytoskeletal proteins expressed in human acute lymphoblastic leukemia cells that are resistant to microtubule-disrupting agents. We characterized γ-actin expression in antimicrotubule drug–resistant leukemia and examined the effect of altered γ-actin in resistance of acute lymphoblastic leukemia to antimicrotubule agents. Methods: Two-dimensional polyacrylamide gel electrophoresis and mass spectrometry were used to identify actin proteins in human acute lymphoblastic leukemia cell lines resistant to vinblastine (CCRF-CEM/VLB100 cells) and desoxyepothilone B (CCRF-CEM/dEpoB140 cells). Fluorescence-based cycle sequencing was used to detect gene mutations. Site-directed mutagenesis was used to generate mutant γ-actin expression plasmids, which were used to transfect mouse NIH/3T3 cells. Clonogenic analysis was used for drug sensitivity studies. A small interfering RNA (siRNA) was used to block γ-actin gene expression in human neuroblastoma SH-EP cells. Expression of γ-actin (normalized to that of β2-microglobulin [β2M]) in primary leukemia cells obtained from patients at diagnosis (n = 44) and relapse (n = 25) was examined using semiquantitative reverse transcription–polymerase chain reaction. Statistical significance of changes in the ratio of γ-actin to β2M expression between diagnosis and relapse samples was determined by two-sided unpaired Student's t tests. Results: We identified novel mutant forms of γ-actin and the concomitant loss of wild-type γ-actin in CCRF-CEM/VLB100 cells and CCRF-CEM/dEpoB140 cells. Mouse NIH/3T3 cells that expressed the mutant γ-actin proteins were more resistant to antimicrotubule agents than cells transfected with empty plasmid. Human neuroblastoma SH-EP cells transfected with γ-actin siRNA displayed higher relative resistance to paclitaxel (P<.001), vinblastine (P = .04), and epothilone B (P = .045) than mock-transfected cells. No γ-actin gene mutations were identified in 37 samples of primary leukemia cells (eight from patients at diagnosis, 29 from patients at relapse). γ-Actin gene expression was lower in acute lymphoblastic leukemia samples collected at clinical relapse (n = 25; mean γ-actin/β2M = 0.53) than in samples collected at diagnosis (n = 44; mean γ-actin/β2M = 0.68; difference = 0.15, 95% confidence interval [CI] = 0.04 to 0.27, P = .01). Conclusions: These data provide functional and associative clinical evidence of a novel form of drug resistance that involves interactions between γ-actin and microtubules.

Journal Article.  9848 words.  Illustrated.

Subjects: Medical Oncology

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