在多种肿瘤中，生长阻滞特异性转录因子5(growth-arrest specific transcript 5，GAS5)高表达与患者的良好生存期相关。GAS5存在多种剪接变异体，本研究探讨GAS5部分剪接变异体在髓系白血病细胞系中的表达模式。并基于对凋亡诱导敏感的慢性髓系白血病(chronic myeloid leukemia，CML)细胞系K562，研究GAS5部分剪接变异体对K562细胞自身增殖、凋亡以及对靶向药物伊马替尼(imatinib，IM)的敏感性的影响。反转录PCR结果显示，GAS5剪接模式在髓系白血病细胞系HL-60、K562、NB4和KASUMI-1之间未见明显差异。相较于指数增长期，GAS5_AE表达水平在细胞密度引起的生长停滞期有一定程度的累积。通过核转染，将GAS5剪接变异体(pcDNA3-GAS5_1B、-GAS5_2B、-GAS5_3A、-GAS5_4A、-GAS5_O1 或-GAS5_AE)转入 K562 细胞的单克隆株中。通过台盼蓝染色、吖啶橙染色后进行细胞计数，克隆形成实验检测细胞集落形成能力。结果与对照组相比，瞬时转染GAS5剪接变异体并无抑制细胞的基础增殖或增强细胞对药物的敏感性。建立GAS5_O1的稳定转染株，延长培养时程并计算倍增时间，发现GAS5_O1的表达水平和倍增时间成正比(R2=0。5692)。在稳转株O1-C8中观察到GAS5_O1可促进IM引起的细胞生长抑制(56。94％±2。84％vs。36。07％±2。32％，P＜0。05)和凋亡(29。33％±1。30％vs。52。00％± 2。52％，P＜0。05)。上述结果表明，GAS5_O1的上调可能增加CML细胞的倍增时间，并使CML细胞对IM处理敏感。
Growth-arrest Specific Transcript 5 Splicing Variant GAS5_O1 Enhances the Sensitivity of Chronic Myeloid Leukemia Cells to Imatinib
High expression of growth-arrest specific transcript 5(GAS5)is associated with better survival in various tumors.Gas5 has several alternate splicing variants.In this study,we investigated the expression patterns of some of the splicing variants in myeloid leukemia cell lines.Based on chronic myeloid leukemia(CML)cell line K562 that is sensitive to apoptosis induction,the effects of some splicing variants on the proliferation,apoptosis,and sensitivity to the targeted drug imatinib(IM)was studied.The results of reverse transcription PCR showed that there was no significant difference in the splicing pattern of GAS5 among myeloid leukemia cell lines HL-60,K562,NB4,and KASUMI-1.Compared to the exponential phase,the expression level of GAS5_AE was accumulated during the stationary phase.By nuclear transfection,GAS5 splicing variants pcDNA3-GAS5_1B,-GAS5_2B,-GAS5_3A,-GAS5_4A,-GAS5_O1,or-GAS5_AE were transferred into a monoclonal strain of K562 cells.Cell counting was performed after staining with trypan blue or acridine orange,and colony-forming ability was examined through the clonogenic assay.Transient expression of each GAS5 splicing variant did not show any consistent inhibition on basic cell proliferation or enhance cell sensitivity to drugs when compared to the control group.The establishment of GAS5_O1 stably transfected strains allowed for prolonged-culture duration.The doubling times of GAS5_O1 stable clones were found positively associated with GAS5_O1 expression levels(R2=0.5692).GAS5_O1 can promote IM induced cell growth inhibition(56.94％±2.84％vs.36.07％±2.32％,P＜0.05)and apoptosis(29.33％± 1.30％vs.52.00％±2.52％,P＜0.05)in the stable clone O1-C8.These results above suggested that GAS5_O1 upregulation might increase the doubling time of CML cells and sensitize CML cells to IM treatment.
growth-arrest specific transcript 5(GAS5)splicing variantchronic myeloid leukemia(CML)apoptosis
张斯玮、Gwyn T.Williams
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西安医学院基础医学部生物化学与分子生物学教研室,西安 710021
西安医学院西安市病原微生物与肿瘤免疫重点实验室,西安 710021
School of Life Sciences,Keele University,Staffordshire,UK
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