Background: Previous studies of patients with acute leukemia showed that plasma cGMP levels were markedly elevated before treatment, fell after successful therapy but increased after relapse. In many cells high concen...Background: Previous studies of patients with acute leukemia showed that plasma cGMP levels were markedly elevated before treatment, fell after successful therapy but increased after relapse. In many cells high concentrations of GMP have an antiproliferative effect. The cellular cGMP extrusion from cancer cells may represent an acquired resistance against an endogenous antiproliferative signal molecule. Multidrug resistance associated protein 5 (MRP5) has been identified as an important cGMP transporter. Methods: A human erythroleukemia cell line (HEL) was used to study the impact of cGMP and cGMP-elevating compounds like theophylline, sodium nitroprusside (SNP) and sodium nitrite (NaNO2). MRP5 was overexpressed in HEL cells by transfection. Concentrations of cGMP were determined with RIA or HPLC and cell densities were determined by cytometry. Results: The concentration ratio between extra- and intracellular cGMP concentrations was >1, meaning that HEL cells extruded cGMP against a concentration gradient and MRP5 was identified in these cells. In some cell types butyrate increases cellular cGMP levels by stimulating soluble guanylyl cyclase (sGC) and thereby the cellular efflux. This effect did not exist for HEL cells. MRP5 transfected HEL cells which were exposed to cGMP was clearly more sensitive to the antiproliferative effect than the wild type. On the other hand, exposing the transfected HEL cells to cGMP-elevating agents (theophylline, SNP and NaNO<sub>2</sub>) showed less sensitivity than the wildtype. Conclusion: This study supports the idea that some cancers acquire resistance against endogenous signal molecules with antiproliferative potency.展开更多
目的探讨线粒体核糖体蛋白S5(mitochondrial ribosomal protein S5,MRPS5)对人膀胱癌细胞的增殖能力及其中干细胞干性特征的影响。方法 Western blot检测人膀胱癌及癌旁正常组织MRPS5的表达;构建靶向干扰MRPS5的慢病毒载体,感染膀胱癌...目的探讨线粒体核糖体蛋白S5(mitochondrial ribosomal protein S5,MRPS5)对人膀胱癌细胞的增殖能力及其中干细胞干性特征的影响。方法 Western blot检测人膀胱癌及癌旁正常组织MRPS5的表达;构建靶向干扰MRPS5的慢病毒载体,感染膀胱癌细胞株T24,以干扰Scramble序列作为对照,Western blot检测MRPS5干扰效率;细胞活力实验检测细胞增殖能力;流式细胞术检测细胞周期;细胞成球实验观察成球能力;Western blot检测肿瘤干性转录因子Nanog,Oct4,c-Myc和Sox2的表达;小鼠皮下移植瘤实验观察T24体内成瘤能力。结果 MRPS5在膀胱癌组织中的表达高于癌旁正常组织;慢病毒干扰载体PLKO.1-sh MRPS5有效,且干扰MRPS5表达后T24细胞增殖能力下降(P<0.05),周期阻滞于S期,干性因子表达均下调,成球率无变化(P>0.05)但成球直径明显减小;同时小鼠体内成瘤体积减小[(0.784±0.278)vs(0.500±0.245)cm3,P<0.05],瘤质量减轻[(0.862±0.372)vs(0.412±0.248)g,P<0.05]。结论 MRPS5在膀胱癌中较高表达,且干扰MRPS5表达能抑制T24增殖并抑制T24中的干细胞生物学特征。展开更多
文摘Background: Previous studies of patients with acute leukemia showed that plasma cGMP levels were markedly elevated before treatment, fell after successful therapy but increased after relapse. In many cells high concentrations of GMP have an antiproliferative effect. The cellular cGMP extrusion from cancer cells may represent an acquired resistance against an endogenous antiproliferative signal molecule. Multidrug resistance associated protein 5 (MRP5) has been identified as an important cGMP transporter. Methods: A human erythroleukemia cell line (HEL) was used to study the impact of cGMP and cGMP-elevating compounds like theophylline, sodium nitroprusside (SNP) and sodium nitrite (NaNO2). MRP5 was overexpressed in HEL cells by transfection. Concentrations of cGMP were determined with RIA or HPLC and cell densities were determined by cytometry. Results: The concentration ratio between extra- and intracellular cGMP concentrations was >1, meaning that HEL cells extruded cGMP against a concentration gradient and MRP5 was identified in these cells. In some cell types butyrate increases cellular cGMP levels by stimulating soluble guanylyl cyclase (sGC) and thereby the cellular efflux. This effect did not exist for HEL cells. MRP5 transfected HEL cells which were exposed to cGMP was clearly more sensitive to the antiproliferative effect than the wild type. On the other hand, exposing the transfected HEL cells to cGMP-elevating agents (theophylline, SNP and NaNO<sub>2</sub>) showed less sensitivity than the wildtype. Conclusion: This study supports the idea that some cancers acquire resistance against endogenous signal molecules with antiproliferative potency.
