Aasen Trond, Hodgins Malcolm B, Edward Michael, Graham Sheila V
Division of Cancer Sciences and Molecular Pathology, University of Glasgow, Glasgow G12 8QQ, UK.
Oncogene. 2003 Sep 11;22(39):7969-80. doi: 10.1038/sj.onc.1206709.
Disruption of gap junctional intercellular communication (GJIC) and/or connexins (gap junction proteins) is frequently reported in malignant cell lines and tumours. Certain human papillomaviruses (HPV) associated with the development of cancers, especially of the cervix, have previously been reported to downregulate GJIC in vitro. There is also evidence for reduced gap junctions in cervical dysplasia. However, many squamous hyperproliferative conditions, including HPV-induced warts, often show extensive upregulation of certain connexins. The association between HPV and GJIC, and the mechanism and consequence of deregulated GJIC in cervical tumour progression, remains unclear. Therefore, using a variety of nonmalignant and malignant cell lines and an organotypic raft-culture system, we investigated the relationship between HPV, gap junctions and tumour progression. Established cervical tumour cell lines carrying HPV were unable to communicate via gap junctions (when assayed by dye-transfer techniques). This correlated with lack of connexin protein expression, while transfection with connexins 26 or 43 led to functional gap junction membrane plaques. On the other hand, immortal but nonmalignant cell lines that contained episomal or integrated HPV-16, but required feeder-layer and growth-factor support, were consistently well coupled, and expressed multiple connexins at membrane junctions. In vitro selection of feeder-layer and growth-factor-independent variants eventually lead to loss of GJIC, which correlated with loss of membrane and increased cytoplasmic connexin 43 localization. However, this was preceded by loss of differentiation and stromal invasion, as assayed on the organotypic raft-culture model. Using this model, a comparison between noncoupled, well-coupled and connexin-transfected cell lines revealed no firm correlation between GJIC and dysplasia, but GJIC appeared to favour increased stratification. These findings demonstrate that loss of GJIC is frequent, but appears to occur more as a consequence of, rather than being the cause of, epithelial dysplasia, and may be influenced by, but is not directly attributable to, HPV.
间隙连接细胞间通讯（GJIC）和/或连接蛋白（间隙连接蛋白）的破坏在恶性细胞系和肿瘤中经常被报道。先前有报道称，某些与癌症尤其是宫颈癌发生相关的人乳头瘤病毒（HPV）在体外可下调GJIC。也有证据表明宫颈发育异常中间隙连接减少。然而，许多鳞状上皮过度增殖性疾病，包括HPV诱导的疣，常常显示某些连接蛋白大量上调。HPV与GJIC之间的关联，以及GJIC失调在宫颈肿瘤进展中的机制和后果仍不清楚。因此，我们使用多种非恶性和恶性细胞系以及一种器官型筏式培养系统，研究了HPV、间隙连接与肿瘤进展之间的关系。携带HPV的已建立宫颈肿瘤细胞系无法通过间隙连接进行通讯（通过染料转移技术检测）。这与连接蛋白表达缺失相关，而用连接蛋白26或43转染可导致功能性间隙连接膜斑形成。另一方面，含有游离型或整合型HPV - 16、但需要饲养层和生长因子支持的永生化非恶性细胞系始终具有良好的耦联性，并在膜连接处表达多种连接蛋白。体外选择不依赖饲养层和生长因子的变体最终导致GJIC丧失，这与膜的丧失和细胞质中连接蛋白43定位增加相关。然而，在器官型筏式培养模型上检测发现，在此之前就已出现分化丧失和基质浸润。使用该模型，对非耦联、良好耦联和连接蛋白转染的细胞系进行比较，结果显示GJIC与发育异常之间没有确定的相关性，但GJIC似乎有利于增加分层。这些发现表明，GJIC丧失很常见，但似乎更多是上皮发育异常的结果而非原因，并且可能受HPV影响，但并非直接归因于HPV。
