[Establishment of BCL-2 Inhibitors-Resistant B-cell Acute Lymphoblastic Leukemia Cell Lines and Study on Their Resistance Mechanisms].

OBJECTIVE

RS4;11 cell line was used to establish BCL-2 inhibitor-resistant cell lines of B-cell acute lymphoblastic leukemia (B-ALL) and explore the possible mechanisms of drug resistance.

METHODS

RS4;11 cell line was continuously induced and cultured by low and ascending concentrations of BCL-2 inhibitors navitoclax and venetoclax to construct navitoclax-resistant cell line RS4;11/Nav and venetoclax-resistant cell line RS4;11/Ven. The cell viability was detected by MTT assay, and the cell apoptosis was detected by flow cytometry. Differentially expressed genes (DEGs) between RS4;11 drug-resistant cell lines and parental cell line were detected by transcriptome sequencing technology (RNA-seq), and mRNA expression levels of DEGs between drug-resistant cell lines and parental cell line were detected by real-time PCR (RT-PCR). Western blot was used to detect the expression levels of BCL-2 family anti-apoptotic proteins in drug-resistant cell lines and parental cell line.

RESULTS

The drug-resistant cell lines RS4;11/Nav and RS4;11/Ven were successfully established. The resistance index (RI) of RS4;11/Nav to navitoclax and RS4;11/Ven to venetoclax was 328.655±47.377 and 2 894.027±300.311, respectively. The results of cell apoptosis detection showed that compared with the drug-resistant cell lines, RS4;11 parental cell line were significantly inhibited by BCL-2 inhibitors, while the apoptosis rate of drug-resistant cell lines was not affected by the drugs. Western blot assay showed that the expression of anti-apoptotic proteins of BCL-2 family did not increase significantly in drug-resistant cell lines. RNA-seq, RT-PCR and Western blot assays showed that the expression of EP300 in drug-resistant cell lines was significantly higher than that in parental cell line (P <0.05).

CONCLUSION

Drug-resistant B-ALL cell lines could be successfully established by exposing RS4;11 cell line to the ascending concentration of BCL-2 inhibitors, and the drug resistance mechanism may be related to the overexpression of EP300.