一种新型糖皮质激素抵抗性人类B细胞急性淋巴细胞白血病细胞系的特征分析，伴有AMPK、mTOR和脂肪酸合成途径抑制

Characterization of a novel glucocorticoid-resistant human B-cell acute lymphoblastic leukemia cell line, with AMPK, mTOR and fatty acid synthesis pathway inhibition.

作者信息

Li Yuanyuan, Zuo Chuan, Gu Ling

机构信息

Laboratory of Hematology/Oncology, Department of Pediatrics, Key Laboratory of Birth Defects and Related Diseases of Women and Children (Sichuan University), Ministry of Education, West China Second University Hospital, Sichuan University, No.20, Section 3, Renmin South Road, Chengdu, 610041, People's Republic of China.

Joint Laboratory of West China Second University Hospital, Sichuan University and School of Life Science, Fudan University for Pulmonary Development and Disease, Chengdu, 610041, China.

出版信息

Cancer Cell Int. 2021 Nov 25;21(1):623. doi: 10.1186/s12935-021-02335-7.

DOI:10.1186/s12935-021-02335-7

PMID:34823530

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC8614043/

Abstract

BACKGROUND

Acquired glucocorticoid (GC) resistance remains the main obstacle in acute lymphoblastic leukemia (ALL) therapy. The aim of the present study was to establish a novel GC-resistant B-ALL cell line and investigate its biological characteristics.

METHODS

A cell culture technique was used to establish the GC-resistant cell line from the parental cell, NALM-6. Molecular and cellular biological techniques including flow cytometry, MTT assay, western blotting, DNA fingerprinting analysis and whole transcriptome sequencing (WTS) were used to characterize the GC-resistant cell lines. Nude mice were used for xenograft studies.

RESULTS

The GC-resistant cell line, NALM-6/HDR, was established by culturing NALM-6 cells under hypoxia for 5 weeks with a single dexamethasone (Dex) treatment. We subcloned the NALM-6/HDR cell lines, and got 6 monoclone Dex-resistant cell lines, NALM-6/HDR-C1, C3, C4, C5, C6 and C9 with resistance index (RI) ranging from 20,000-50,000. NALM-6/HDR and its monoclone cell line, NALM-6/HDR-C5, exhibited moderate (RI 5-15) to high resistance (RI > 20) to Ara-c; low or no cross-resistance to L-Asp, VCR, DNR, and MTX (RI < 5). STR analysis confirmed that NALM-6/HDR and NALM-6/H were all derived from NALM-6. All these cells derived from NALM-6 showed similar morphology, growth curves, immunophenotype, chromosomal karyotype and tumorigenicity. WTS analysis revealed that the main metabolic differences between NALM-6 or NALM-6/H (GC-sensitive) and NALM-6/HDR (GC-resistant) were lipid and carbohydrates metabolism. Western blotting analysis showed that NALM-6/HDR cells had a low expression of GR and p-GR. Moreover, AMPK, mTORC1, glycolysis and de novo fatty acid synthesis (FAS) pathway were inhibited in NALM-6/HDR when compared with NALM-6.

CONCLUSIONS

NALM-6/HDR cell line may represent a subtype of B-ALL cells in patients who acquired GC and Ara-c resistance during the treatment. These patients may get little benefit from the available therapy target of AMPK, mTORC1, glycolysis and FAS pathway.

摘要

背景

获得性糖皮质激素（GC）耐药仍然是急性淋巴细胞白血病（ALL）治疗的主要障碍。本研究的目的是建立一种新型的GC耐药B-ALL细胞系，并研究其生物学特性。

方法

采用细胞培养技术从亲代细胞NALM-6建立GC耐药细胞系。运用包括流式细胞术、MTT法、蛋白质印迹法、DNA指纹分析和全转录组测序（WTS）在内的分子和细胞生物学技术对GC耐药细胞系进行表征。使用裸鼠进行异种移植研究。

结果

通过将NALM-6细胞在低氧条件下培养5周并单次给予地塞米松（Dex）处理，建立了GC耐药细胞系NALM-6/HDR。我们对NALM-6/HDR细胞系进行亚克隆，获得了6个单克隆Dex耐药细胞系，即NALM-6/HDR-C1、C3、C4、C5、C6和C9，耐药指数（RI）范围为20,000 - 50,000。NALM-6/HDR及其单克隆细胞系NALM-6/HDR-C5对阿糖胞苷表现出中度（RI 5 - 15）至高耐药（RI > 20）；对左旋门冬酰胺酶、长春新碱、柔红霉素和甲氨蝶呤的交叉耐药性低或无（RI < 5）。STR分析证实NALM-6/HDR和NALM-6/H均源自NALM-6。所有这些源自NALM-6的细胞表现出相似的形态、生长曲线、免疫表型、染色体核型和致瘤性。WTS分析显示，NALM-6或NALM-6/H（GC敏感）与NALM-6/HDR（GC耐药）之间的主要代谢差异在于脂质和碳水化合物代谢。蛋白质印迹分析表明，NALM-6/HDR细胞中GR和p-GR的表达较低。此外，与NALM-6相比，NALM-6/HDR中AMPK、mTORC1、糖酵解和从头脂肪酸合成（FAS）途径受到抑制。