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

Abstract

Proteomic investigations have revealed alterations in cytoskeletal proteins expressed in human acute lymphoblastic leukemia cells that are resistant to microtubule-disrupting agents. We characterized gamma-actin expression in antimicrotubule drug-resistant leukemia and examined the effect of altered gamma-actin in resistance of acute lymphoblastic leukemia to antimicrotubule agents. 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 gamma-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 gamma-actin gene expression in human neuroblastoma SH-EP cells. Expression of gamma-actin (normalized to that of beta2-microglobulin [beta2M]) 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 gamma-actin to beta2M expression between diagnosis and relapse samples was determined by two-sided unpaired Student's t tests. We identified novel mutant forms of gamma-actin and the concomitant loss of wild-type gamma-actin in CCRF-CEM/VLB100 cells and CCRF-CEM/dEpoB140 cells. Mouse NIH/3T3 cells that expressed the mutant gamma-actin proteins were more resistant to antimicrotubule agents than cells transfected with empty plasmid. Human neuroblastoma SH-EP cells transfected with gamma-actin siRNA displayed higher relative resistance to paclitaxel (P<.001), vinblastine (P = .04), and epothilone B (P = .045) than mock-transfected cells. No gamma-actin gene mutations were identified in 37 samples of primary leukemia cells (eight from patients at diagnosis, 29 from patients at relapse). Gamma-actin gene expression was lower in acute lymphoblastic leukemia samples collected at clinical relapse (n = 25; mean gamma-actin/beta2M = 0.53) than in samples collected at diagnosis (n = 44; mean gamma-actin/beta2M = 0.68; difference = 0.15, 95% confidence interval [CI] = 0.04 to 0.27, P = .01). These data provide functional and associative clinical evidence of a novel form of drug resistance that involves interactions between gamma-actin and microtubules.