Alterations in gamma-actin and tubulin-targeted drug resistance in childhood leukemia.
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 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.


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 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.


RESULTS
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).


CONCLUSIONS
These data provide functional and associative clinical evidence of a novel form of drug resistance that involves interactions between gamma-actin and microtubules.
Verrills
Nicole M
NM
Children's Cancer Institute Australia for Medical Research, PO Box 81, Randwick NSW 2031, Australia.
Po'uha
Sela T
ST
Liu
Marjorie L M
ML
Liaw
Tracy Y E
TY
Larsen
Martin R
MR
Ivery
Michael T
MT
Marshall
Glenn M
GM
Gunning
Peter W
PW
Kavallaris
Maria
M
D000199:Actins / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002648:Child; D004252:DNA Mutational Analysis; D019008:Drug Resistance, Neoplasm / Q000187:drug effects*; D015180:Electrophoresis, Gel, Two-Dimensional; D034261:Epothilones / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D020868:Gene Silencing / Q000187:drug effects; D006801:Humans; D007930:Leucine; D013058:Mass Spectrometry; D051379:Mice; D008870:Microtubules / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D009154:Mutation / Q000187:drug effects; D009447:Neuroblastoma / Q000188:drug therapy / Q000378:metabolism; D010957:Plasmids; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism*; D011392:Proline; D034741:RNA, Small Interfering / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014404:Tubulin / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D050257:Tubulin Modulators / Q000494:pharmacology*; D014410:Tumor Stem Cell Assay; D014633:Valine; D014747:Vinblastine / Q000494:pharmacology
D000199:Actins; D000970:Antineoplastic Agents; D034261:Epothilones; D034741:RNA, Small Interfering; D014404:Tubulin; D050257:Tubulin Modulators; D014747:Vinblastine; D011392:Proline; D007930:Leucine; D014633:Valine; C114026:desoxyepothilone B