Different mutants of PSMB5 confer varying bortezomib resistance in T lymphoblastic lymphoma/leukemia cells derived from the Jurkat cell line

 Shuqing ü, Jianmin Yang, Zhilong Chen et al.  Experimental Hematology 2009;37:831?837

Department of Hematology, Changhai Hospital,
Second Military Medical University,
174 Changhai Road, 200433 Shanghai, China

Objective. To investigate the relationship between bortezomib resistance and mutations in the上海长海医院血液内科吕书晴
proteasome b5 subunit (PSMB5) gene.
Materials and Methods. Various bortezomib-resistant lymphoblastic lymphoma/leukemia
lines were established by repeated cycles of bortezomib selection. Mutations were detected
by sequencing the complementary DNA of the PSMB5 gene. Mutated clones were selected
by limited dilution and cultured without bortezomib. Messenger RNA expression levels of
PSMB5 in these mutated clones were measured by quantitative reverse transcription polymerase
chain reaction. The degree of resistance was determined by cytotoxicity at various
bortezomib concentrations. The chymotrypsin-like activities were assayed by measuring the
release of the fluorescent 7-amido-4-methylcoumarin from the substrate N-succinyl-Leu-
Leu-Val-TyrL7-amido-4-methylcoumarin.
Results. In addition to the previously reported PSMB5 G322A mutant (Ala49Thr), a C323T
mutant (Ala49Val), and G322A, C326T conjoined mutant (Ala49Thr and Ala50Val) were
selected and clones containing these mutations (JurkatB-G322A, JurkatB-C323T, and
JurkatB-G322A/C326T) were obtained. After being cultured without bortezomib for O2
months, no significant difference in PSMB5 messenger RNA levels was detected between these
JurkatB cells and parental Jurkat cells. JurkatB-G322A, JurkatB-C323T, and JurkatBG322A/
C326T clones displayed 22.0-fold, 39.4-fold, and 66.7-fold resistance, respectively, to
bortezomib compared to Jurkat cells. There were no significant differences between the
chymotrypsin-like activities of these mutants and Jurkat cells. The inhibitory effect of bortezomib
on chymotrypsin-like activity was the weakest in JurkatB-G322A/C326T cells, and the
strongest in JurkatB-G322A cells, with JurkatB-C323T cells falling in between.
Conclusion. Mutations of the PSMB5 gene resulting in substitutions of Ala49 and Ala50 of
PSMB5 protein can confer varying bortezomib resistance.