Paterson Ian C, Davies Maria, Stone Andrea, Huntley Suzy, Smith Emily, Pring Miranda, Eveson John W, Robinson C Max, Parkinson E Kenneth, Prime Stephen S
Department of Oral and Dental Science, University of Bristol, Bristol, BS1 2LY, UK.
Oncogene. 2002 Feb 28;21(10):1616-24. doi: 10.1038/sj.onc.1205217.
This study examined the role of TGF-beta1 in human keratinocyte malignancy. Two carcinoma-derived human oral keratinocyte cell lines, BICR 31 and H314, were selected on the basis of their known resistance to TGF-beta1-induced G(1) arrest, the presence of wild type TGF-beta cell surface receptors and normal Ras. Smad 4 protein was undetectable in both cell lines, but Smad 2 and Smad 3 were expressed at levels comparable with a fully TGF-beta responsive cell line, and treatment of the cells with TGF-beta1 resulted in the phosphorylation of Smad 2. Treatment with exogenous TGF-beta1 resulted in a failure to induce transcription from an artificial Smad-dependent promoter and a failure to down-regulate c-myc, but resulted in an up-regulation of AP-1 associated genes (Fra-1, JunB and fibronectin). Transient transfection of Smad 4 into BICR 31 restored TGF-beta1-induced growth inhibition and Smad-dependent transcriptional activation. Protracted treatment of cells with exogenous TGF-beta1 resulted in the attenuation of cell growth in vitro. To over-express TGF-beta1, both cell lines were transfected with latent TGF-beta1 cDNA; neutralization studies of conditioned media demonstrated that whilst the majority of the peptide was in the latent form, a small proportion was present as the active peptide. Cells that over-expressed endogenous TGF-beta1 grew more slowly in vitro compared to both the vector-only controls and cells that did not over-express the peptide. Orthotopic transplantation of cells that over-expressed endogenous TGF-beta1 to the floor of the mouth in athymic mice resulted in marked inhibition of primary tumor formation compared to controls. Expression of a dominant-negative TGF-beta type II receptor in cells that over-expressed endogenous TGF-beta1 resulted in enhanced cell growth in vitro and diminished the tumor suppressor effect of the ligand in vivo, indicating that the endogenous TGF-beta1 was acting in an autocrine capacity. The results demonstrate that over-expression of endogenous TGF-beta1 in human malignant oral keratinocytes leads to growth inhibition in vivo and tumor suppression in vitro by mechanisms that are independent of Smad 4 expression and TGF-beta1-induced G(1) arrest.
本研究检测了转化生长因子β1（TGF-β1）在人角质形成细胞恶变中的作用。基于已知的对TGF-β1诱导的G1期阻滞的抗性、野生型TGF-β细胞表面受体的存在以及正常的Ras，选择了两种源自癌组织的人口腔角质形成细胞系，即BICR 31和H314。在这两种细胞系中均未检测到Smad 4蛋白，但Smad 2和Smad 3的表达水平与完全对TGF-β有反应的细胞系相当，用TGF-β1处理细胞导致Smad 2磷酸化。用外源性TGF-β1处理导致无法从人工的Smad依赖性启动子诱导转录，且无法下调c-myc，但导致AP-1相关基因（Fra-1、JunB和纤连蛋白）上调。将Smad 4瞬时转染到BICR 31中可恢复TGF-β1诱导的生长抑制和Smad依赖性转录激活。用外源性TGF-β1对细胞进行长期处理导致体外细胞生长减弱。为了使TGF-β1过表达，两种细胞系均用潜伏性TGF-β1 cDNA转染；对条件培养基的中和研究表明，虽然大多数肽处于潜伏形式，但有一小部分以活性肽形式存在。与仅转染载体的对照细胞和未过表达该肽的细胞相比，过表达内源性TGF-β1的细胞在体外生长更慢。将过表达内源性TGF-β1的细胞原位移植到无胸腺小鼠的口腔底部，与对照相比，导致原发性肿瘤形成受到明显抑制。在过表达内源性TGF-β1的细胞中表达显性负性TGF-β II型受体导致体外细胞生长增强，并减弱了配体在体内的肿瘤抑制作用，表明内源性TGF-β1以自分泌方式起作用。结果表明，人恶性口腔角质形成细胞中内源性TGF-β1的过表达通过独立于Smad 4表达和TGF-β1诱导的G1期阻滞的机制导致体内生长抑制和体外肿瘤抑制。
