Department of Clinical Neuroscience and Center for Molecular Medicine, Karolinska Institutet, SE_17176 Stockholm, Sweden.
Department of Medicine, Solna and Center for Molecular Medicine, Karolinska Institutet, SE_17176 Stockholm, Sweden.
Int J Oncol. 2018 Apr;52(4):1317-1327. doi: 10.3892/ijo.2018.4286. Epub 2018 Feb 26.
Among all brain tumors diagnosed in children, medulloblastomas (MBs) are associated with a poor prognosis. The etiology of MB is not fully understood, yet the impact of epigenetic alterations of oncogenes has previously been established. During the past decade, the human cytomegalovirus (HCMV) has been detected in several types of cancer, including MB. Since DNA methylation occurs in the cell nucleus and this is considered a host defence response, we studied the impact of HCMV infection on DNA methyltransferase (DNMT‑1) in MB (D324) cells, human umbilical vein endothelial cells (HUVECs) as well as in MB tissue sections. We hypothesized that infection and DNMT‑1 intracellular localization are linked. Uninfected and HCMV‑infected D324 cells and HUVECs were analyzed for HCMV immediate early (HCMV‑IE) protein, HCMV‑glycoprotein B (HCMV‑gB) and DNMT‑1 using immunofluorescence staining and quantitative ELISA. DNMT‑1 localized to the nucleus of uninfected and HCMV‑IE- expressing D324 cells and HUVECs, but accumulated in the extra nuclear space in all HCMV‑gB-positive cells. Inhibition of HCMV late protein expression by Cymevene® (ganciclovir) prevented the cytoplasmic localization of DNMT‑1. Treatment of HCMV‑ infected D324 cells and HUVECs with the methylation inhibitor 5-Azacytidine (5AZA), significantly increased HCMV‑IE and HCMV‑gB gene transcription and protein expression. Immunohistochemical staining of DNMT‑1 and HCMV proteins in MB cancer tissue sections revealed both nuclear and cytoplasmic DNMT‑1 localization. In conclusion, DNMT‑1 resides in the cytoplasm of HCMV‑gB-expressing HUVECs and D324 cells. Increased viral protein synthesis in 5AZA-treated cells suggests that HCMV replication may benefit from a DNA methyltransferase-free cellular environment. Our findings emphasize the importance of assessing potential viral activation in the treatment of MB patients with epigenetic drugs.
在所有诊断为儿童的脑肿瘤中，髓母细胞瘤（MB）的预后较差。MB 的病因尚未完全阐明，但先前已经确定了癌基因表观遗传改变的影响。在过去的十年中，人巨细胞病毒（HCMV）已在多种类型的癌症中被检测到，包括 MB。由于 DNA 甲基化发生在细胞核中，这被认为是一种宿主防御反应，因此我们研究了 HCMV 感染对 MB（D324）细胞、人脐静脉内皮细胞（HUVEC）以及 MB 组织切片中 DNA 甲基转移酶（DNMT-1）的影响。我们假设感染和 DNMT-1 细胞内定位是相关的。使用免疫荧光染色和定量 ELISA 分析未感染和 HCMV 感染的 D324 细胞和 HUVEC 中的 HCMV 早期（HCMV-IE）蛋白、HCMV 糖蛋白 B（HCMV-gB）和 DNMT-1。DNMT-1 定位于未感染和表达 HCMV-IE 的 D324 细胞和 HUVEC 的核内，但在所有 HCMV-gB 阳性细胞中积累在核外空间。用 Cymevene®（更昔洛韦）抑制 HCMV 晚期蛋白表达可防止 DNMT-1 的细胞质定位。用甲基化抑制剂 5-氮杂胞苷（5AZA）处理 HCMV 感染的 D324 细胞和 HUVEC，可显著增加 HCMV-IE 和 HCMV-gB 基因转录和蛋白表达。MB 癌组织切片中 DNMT-1 和 HCMV 蛋白的免疫组织化学染色显示 DNMT-1 的核内和细胞质定位。总之，DNMT-1 位于表达 HCMV-gB 的 HUVEC 和 D324 细胞的细胞质中。5AZA 处理细胞中病毒蛋白合成增加表明，HCMV 复制可能受益于无 DNA 甲基转移酶的细胞环境。我们的研究结果强调了在使用表观遗传药物治疗 MB 患者时评估潜在病毒激活的重要性。
