ARTICLE |
Alterations in 
-Actin and Tubulin-Targeted Drug Resistance in Childhood Leukemia
Affiliations of authors: Children's Cancer Institute Australia for Medical Research, Randwick, Australia (NMV, STP, MLML, TYEL, GMM, MK); Australian Proteome Analysis Facility, Macquarie University, Sydney, Australia (NMV); School of Women's and Children's Health, University of New South Wales, Sydney, Australia (STP, TYEL); Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark (MRL); Faculty of Pharmacy, University of Sydney, Sydney, Australia (MTI); Centre for Children's Cancer and Blood Disorders, Sydney Children's Hospital, Randwick, Australia (GMM); Oncology Research Unit, The Children's Hospital at Westmead, Westmead, and Discipline of Paediatrics and Child Health, University of Sydney, Sydney, Australia (PWG)
Correspondence to: Maria Kavallaris, PhD, Experimental Therapeutics Program, Children's Cancer Institute Australia for Medical Research, PO Box 81, Randwick NSW 2031, Australia (e-mail: m.kavallaris{at}ccia.unsw.edu.au).
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 
-actin expression in antimicrotubule drug–resistant leukemia and examined the effect of altered -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 -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 -actin gene expression in human neuroblastoma SH-EP cells. Expression of -actin (normalized to that of 
2-microglobulin [2M]) 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 -actin to 2M expression between diagnosis and relapse samples was determined by two-sided unpaired Student's t tests. Results: We identified novel mutant forms of -actin and the concomitant loss of wild-type -actin in CCRF-CEM/VLB100 cells and CCRF-CEM/dEpoB140 cells. Mouse NIH/3T3 cells that expressed the mutant -actin proteins were more resistant to antimicrotubule agents than cells transfected with empty plasmid. Human neuroblastoma SH-EP cells transfected with -actin siRNA displayed higher relative resistance to paclitaxel (P<.001), vinblastine (P = .04), and epothilone B (P = .045) than mock-transfected cells. No -actin gene mutations were identified in 37 samples of primary leukemia cells (eight from patients at diagnosis, 29 from patients at relapse). -Actin gene expression was lower in acute lymphoblastic leukemia samples collected at clinical relapse (n = 25; mean -actin/2M = 0.53) than in samples collected at diagnosis (n = 44; mean -actin/2M = 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 -actin and microtubules.
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Can Mutations in -Actin Modulate the Toxicity of Microtubule Targeting Agents?
- Tito Fojo
J Natl Cancer Inst 2006 98: 1345-1347.[Extract] [Full Text] [PDF]
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