Department of Microbiology, College of Natural Sciences, Chungbuk National University, Cheongju 361-763, Republic of Korea.
J Microbiol Biotechnol. 2013 Nov 28;23(11):1627-35. doi: 10.4014/jmb.1306.06023.
Mitochondria often play central roles in apoptotic pathways, and disruption of the mitochondrial transmembrane potential (ΔΨm) has been observed in various cells undergoing apoptosis. Human cytomegalovirus (HCMV) infection induces apoptosis in permissive cells; however, investigations of mitochondria-targeted apoptosis in HCMV-infected human foreskin fibroblast (HFF) cells have been limited. Here, we investigated the mitochondrial apoptosis pathway in HCMV-infected HFF cells. Flow cytometry analysis using JC-1 revealed that HCMV infection induces disruption of ΔΨm in HFF cells when administered 24 h postinfection (hpi), and this disruption was maximized at 48 hpi. Moreover, cytochrome c, normally a mitochondrial inner membrane protein, was detected in cytoplasmic extracts of HCMV-infected cells, but not mock-infected cells, by western blot analysis at 24 hpi. A caspase activity assay based on fluorescence spectrophotometry using a fluorogenic substrate revealed an increase in caspase-3 activity at 48 hpi in HCMV-infected cells. Caspase-8 activity was increased at 72 hpi in HCMV-infected cells. These results imply that HCMV infection induces mitochondria-mediated apoptosis in HFF cells.
线粒体在凋亡途径中经常起着核心作用，并且在各种经历凋亡的细胞中观察到线粒体跨膜电位（ΔΨm）的破坏。人巨细胞病毒（HCMV）感染诱导允许细胞发生凋亡；然而，对 HCMV 感染的人包皮成纤维细胞（HFF）细胞中线粒体靶向凋亡的研究受到限制。在这里，我们研究了 HCMV 感染的 HFF 细胞中的线粒体凋亡途径。使用 JC-1 的流式细胞术分析表明，HCMV 感染在感染后 24 小时（hpi）诱导 HFF 细胞中 ΔΨm 的破坏，并且这种破坏在 48 hpi 时最大化。此外，通过在 24 hpi 时进行的western blot 分析，在 HCMV 感染的细胞中但不在 mock 感染的细胞中检测到细胞色素 c，正常情况下它是线粒体内膜蛋白。使用荧光分光光度法基于荧光底物的半胱天冬酶活性测定显示在 HCMV 感染的细胞中在 48 hpi 时 caspase-3 活性增加。在 HCMV 感染的细胞中，caspase-8 活性在 72 hpi 时增加。这些结果表明 HCMV 感染诱导 HFF 细胞中线粒体介导的凋亡。
