HA117 endows HL60 cells with a stem-like signature by inhibiting the degradation of DNMT1 via its ability to down-regulate expression of the GGL domain of RGS6.

Shuangshuang Li,Shaoyan Liang,Yi Wang,Huan Wu,Xiaoqing Li,Xianqing Jin,Yuxia Guo,Aamir Ahmad

doi:10.1371/journal.pone.0180142

Shuangshuang Li, Shaoyan Liang + Show 6 more

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https://doi.org/10.1371/journal.pone.0180142

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Abstract

All-trans retinoic acid (ATRA) induces complete remission in almost all patients with acute promyelocytic leukemia (APL) via its ability to induce the in vivo differentiation of APL blasts. However, prolonged ATRA treatment can result in drug resistance. In previous studies, we generated a multi-drug-resistant HL60/ATRA cell line and found it to contain a new drug resistance-related gene segment, HA117. In this study, we demonstrate that ATRA induces multi-drug-resistant subpopulations of HL60 cells with a putative stem-like signature by up-regulating the expression of the new gene segment HA117. Western blot analysis and quantitative real-time PCR demonstrated that HA117 causes alternative splicing of regulator of G-protein signaling 6 (RGS6) and down-regulation of the expression of the GGL domain of RGS6, which plays an important role in DNA methyltransferase 1 (DNMT1) degradation. Moreover, DNMT1 expression was increased in multi-drug resistance HL60/ATRA cells. Knockdown of HA117 restored expression of the GGL domain and blocked DNMT1 expression. Moreover, resistant cells displayed a putative stem-like signature with increased expression of cancer steam cell markers CD133 and CD123. The stem cell marker, Nanog, was significantly up-regulated. In conclusion, our study shows that HA117 potentially promotes the stem-like signature of the HL60/ATRA cell line by inhibiting by the ubiquitination and degradation of DNMT1 and by down-regulating the expression of the GGL domain of RGS6. These results throw light on the cellular events associated with the ATRA-induced multi-drug resistance phenotype in acute leukemia.

Highlights

Complete remission is induced by all-trans retinoic acid (ATRA) in almost all patients with acute promyelocytic leukemia (APL) by inducing the differentiation of APL blasts in vivo
We found no significant difference in DNA methyltransferase 1 (DNMT1) messenger RNA (mRNA) expression in any of the cell lines (Fig 4a and S1 Dataset)
We found that multi-drug-resistant HL60/ATRA cells showed higher expression of the cancer stem cell marker CD133 [32] and the leukemia stem cell marker Interleukin-3 receptor alpha chain (CD123) [19] than wild-type HL60 cells

Summary

Introduction

Complete remission is induced by all-trans retinoic acid (ATRA) in almost all patients with acute promyelocytic leukemia (APL) by inducing the differentiation of APL blasts in vivo. Prolonged ATRA treatment can cause drug resistance[1]. Better understanding of the molecular basis of ATRA-induced drug resistance is warranted to exploit the markers and mechanisms underlying this drug-resistant phenotype. We used ATRA to select drug-resistant HL60 cells, which led to the generation of the multi-drug-resistant cell line, HL-60/ATRA. Suppression subtractive hybridization[2] and microarray analysis of differentially expressed sequences HL-60/ATRA cells enabled us identify a highly expressed sequence, which we refer to as HA117 (GenBank accession number: CB214920)[3]. Bioinformatics analysis of human genomic sequences identified the human gene fragment encoding HA117. The gene is located on the reverse strand of chromosome 14q24.2 in an intergenic region between Regulator of G-protein signaling 6 (RGS6) and Double PHD Fingers Family, Member 3 (DPF3)

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