Senescence-associated (beta)-galactosidase reflects an increase in lysosomal mass during replicative ageing of human endothelial cells.
Senescence-associated (beta)-galactosidase reflects an increase in lysosomal mass during replicative ageing of human endothelial cells.
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发表时间：
2000-10
影响因子：
4
通讯作者：
D. Kurz;S. Decary;Ying Hong;J. Erusalimsky
中科院分区：
文献类型：
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作者：
D. Kurz;S. Decary;Ying Hong;J. Erusalimsky
衰老相关(β)-半乳糖苷酶被广泛用作复制性衰老的生物标志物。然而，目前尚不清楚这是否是一种独特的酶在pH 6下具有活性，并在衰老过程中差异表达，还是经典的酸溶酶体(β)-半乳糖苷酶增加的表现。在这里，我们通过改变年轻和衰老的人脐静脉内皮细胞的细胞内和溶酶体pH值，研究了衰老相关-(β)-半乳糖苷酶活性的起源，并使用流式细胞术检测了这些操作对活性水平的影响。用氯喹或巴菲霉素A使溶酶体碱化(1)，以及用尼日利亚菌素将细胞内环境平衡到pH 6，导致细胞内(β)-半乳糖苷酶活性的严重抑制（92% -99%）。然而，不受pH值变化的影响，衰老细胞的(β)-半乳糖苷酶活性水平比年轻细胞高3 - 6倍。这种活性的增加与(β)-半乳糖苷酶蛋白水平的增加同时发生。吖啶橙染色显示，随着繁殖年龄的增加，溶酶体含量增加，这与(β)-半乳糖苷酶的增加有关。这些发现表明，衰老相关(β)-半乳糖苷酶是在亚理想pH下残留溶酶体活性的一种表现，由于衰老细胞中溶酶体含量的增加而变得可检测到。
Senescence-associated (beta)-galactosidase is widely used as a biomarker of replicative senescence. However, it remains unknown whether this is a distinct enzyme active at pH 6, and differentially expressed in senescence, or a manifestation of an increase in the classic acid lysosomal (beta)-galactosidase. Here we have investigated the origin of senescence-associated-(beta)-galactosidase activity by modifying the intracellular and lysosomal pH of young and senescent human umbilical vein endothelial cells and examining the effect of these manipulations on the levels of activity, using a flow cytometric assay. Lysosomal alkalinisation with chloroquine or bafilomycin A(1), as well as equilibration of the intracellular milieu to pH 6 with nigericin, caused a profound (92-99%) inhibition of the total intracellular (beta)-galactosidase activity. However, independent of pH alterations, senescent cells showed levels of (beta)-galactosidase activity three- to sixfold higher than young cells. This increase in activity occurred in parallel to an increase in (beta)-galactosidase protein levels. Acridine Orange staining revealed an increase in lysosomal content with replicative age, which correlated with the increase in (beta)-galactosidase. These findings demonstrate that senescence-associated (beta)-galactosidase is a manifestation of residual lysosomal activity at a suboptimal pH, which becomes detectable due to the increased lysosomal content in senescent cells.