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

Lü, Shuqing; Yang, Jianmin; Chen, Zhilong; Gong, Shenglan; Zhou, Hong; Xu, Xiaoqian; Wang, Jianmin

doi:10.1016/j.exphem.2009.04.001

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Volume 37, Issue 7 , Pages 831-837, July 2009

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

Shuqing Lü
- Department of Hematology, Changhai Hospital, Second Military Medical University, Shanghai, China
Jianmin Yang
- Department of Hematology, Changhai Hospital, Second Military Medical University, Shanghai, China
Zhilong Chen
- Department of Pharmaceutical Science and Technology, Institute of Biological Science and Technology, Donghua University, Shanghai, China
Shenglan Gong
- Department of Hematology, Changhai Hospital, Second Military Medical University, Shanghai, China
Hong Zhou
- Department of Hematology, Changhai Hospital, Second Military Medical University, Shanghai, China
Xiaoqian Xu
- Department of Hematology, Changhai Hospital, Second Military Medical University, Shanghai, China
Jianmin Wang
- Department of Hematology, Changhai Hospital, Second Military Medical University, Shanghai, China
- Offprint requests to: Jianmin Wang, M.D., Department of Hematology, Changhai Hospital, Second Military Medical University, 174 Changhai Road, Shanghai, 200433, P.R. China

Received 4 January 2009; received in revised form 16 March 2009; accepted 6 April 2009. published online 17 April 2009.

Objective

To investigate the relationship between bortezomib resistance and mutations in the proteasome β5 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-Tyr−7-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 >2 months, no significant difference in PSMB5 messenger RNA levels was detected between these JurkatB cells and parental Jurkat cells. JurkatB-G322A, JurkatB-C323T, and JurkatB-G322A/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.