Department of Hematology, The First (No. 1) Hospital of Baoding, Baoding, P R China.
Leuk Lymphoma. 2012 Jul;53(7):1383-9. doi: 10.3109/10428194.2011.650695. Epub 2012 Jan 31.
The phosphatase and tensin homology deleted on chromosome 10 (PTEN) gene is a novel tumor suppressor gene of the phosphatase family. Studies have shown that the PTEN gene is probably involved in human malignant disease pathogenesis, multidrug resistance, angiogenesis and extramedullary infiltration. This study was designed to investigate the effect of wild-type PTEN gene transfection on drug resistance reversal in K562/ADM leukemia cells in vitro and the possible mechanism. A recombinant adenovirus containing green fluorescent protein gene and wild-type PTEN gene (Ad-PTEN-GFP) or a recombined adenovirus containing green fluorescent protein gene only (Ad-GFP) was transfected into K562/ADM cells. These cells were then treated with different concentrations of adriamycin, cytarabine or arsenic trioxide, respectively. The half-maximal inhibitory concentration (IC(50)) of each drug was detected by MTT assay and the drug resistance reversal factor (RF) was calculated. The proliferation inhibition rate of these K562/ADM cells treated with or without the above-mentioned drugs was determined by MTT assay and the apoptosis rate was evaluated by flow cytometry. PTEN, nuclear factor-κB (NF-κB), I-κB, p53, multidrug resistance genes MDR1 and MRP, and apoptosis related genes Bcl-2, Bcl-xL and Bax mRNA levels were detected by real-time fluorescence relative-quantification reverse transcription polymerase chain reaction (FQ-PCR). PTEN, Akt, p-Akt and NF-κB (p65) protein levels were detected by Western blot. Results showed that PTEN gene transfection could increase the sensitivity of K562/ADM cells to chemotherapeutic drugs. The drug resistance reversal index of adriamycin, cytarabine and arsenic trioxide was 3.8-fold, 2.65-fold and 2.64-fold, respectively, after PTEN gene transfection. NF-κB, MDR1, Bcl-2 and Bcl-xL mRNA levels as well as p-Akt and NF-κB (p65) protein levels were down-regulated, while p53 and Bax mRNA levels were up-regulated in K562/ADM cells after transfection with Ad-PTEN-GFP. Therefore, wild-type PTEN gene transfection might increase drug sensitivity or reverse drug resistance via inhibiting the PI3K/Akt pathway and regulating downstream molecules of the cell signaling transduction pathway in K562/ADM cells, such as down-regulating NF-κB, MDR1, Bcl-2 expression but up-regulating the expression of p53 and Bax.
第 10 号染色体缺失的磷酸酶及张力蛋白同源物（PTEN）基因是一种新型的磷酸酶家族抑癌基因。研究表明，PTEN 基因可能参与人类恶性疾病的发病机制、多药耐药性、血管生成和髓外浸润。本研究旨在探讨野生型 PTEN 基因转染对体外 K562/ADM 白血病细胞耐药逆转的影响及其可能的机制。将含有绿色荧光蛋白基因和野生型 PTEN 基因的重组腺病毒（Ad-PTEN-GFP）或仅含有绿色荧光蛋白基因的重组腺病毒（Ad-GFP）转染至 K562/ADM 细胞中。分别用不同浓度的阿霉素、阿糖胞苷或三氧化二砷处理这些细胞，通过 MTT 法检测每种药物的半抑制浓度（IC50），并计算耐药逆转因子（RF）。通过 MTT 法测定上述药物处理前后 K562/ADM 细胞的增殖抑制率，并通过流式细胞术评价细胞凋亡率。采用实时荧光相对定量逆转录聚合酶链反应（FQ-PCR）检测 PTEN、核因子-κB（NF-κB）、I-κB、p53、多药耐药基因 MDR1 和 MRP 以及凋亡相关基因 Bcl-2、Bcl-xL 和 Bax mRNA 水平。采用 Western blot 法检测 PTEN、Akt、p-Akt 和 NF-κB（p65）蛋白水平。结果显示，PTEN 基因转染可增加 K562/ADM 细胞对化疗药物的敏感性。PTEN 基因转染后，阿霉素、阿糖胞苷和三氧化二砷的耐药逆转指数分别为 3.8 倍、2.65 倍和 2.64 倍。转染 Ad-PTEN-GFP 后，K562/ADM 细胞中 NF-κB、MDR1、Bcl-2 和 Bcl-xL mRNA 水平以及 p-Akt 和 NF-κB（p65）蛋白水平下调，而 p53 和 Bax mRNA 水平上调。因此，野生型 PTEN 基因转染可能通过抑制 PI3K/Akt 通路并调节细胞信号转导通路下游分子，如下调 NF-κB、MDR1、Bcl-2 表达，上调 p53 和 Bax 表达，增加 K562/ADM 细胞的药物敏感性或逆转其耐药性。
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