Onwuegbusi B A, Aitchison A, Chin S-F, Kranjac T, Mills I, Huang Y, Lao-Sirieix P, Caldas C, Fitzgerald R C
MRC Cancer Cell Unit, Hutchison/MRC Research Centre, Hills Rd, Cambridge, UK.
Gut. 2006 Jun;55(6):764-74. doi: 10.1136/gut.2005.076430. Epub 2005 Dec 20.
Transforming growth factor beta (TGF-beta) is frequently involved in gastrointestinal carcinogenesis although its contribution to oesophageal adenocarcinoma (AC) and its precursor Barrett's oesophageal epithelium (BE) metaplasia are unclear.
Expression of TGF-beta signalling components was assessed by reverse transcription-polymerase chain reaction (PCR), western blot, and immunohistochemistry in oesophageal endoscopic biopsies and cell lines. Genomic alterations in SMAD4 were characterised by fluorescence in situ hybridisation, methylation specific PCR, and sequencing. Functional integrity of TGF-beta signalling was assessed by characterisation of p21 and proliferation status. Smad4 negative BIC-1 cells were transiently transfected with smad4 and TGF-beta responsiveness evaluated.
smad4 mRNA expression was progressively reduced in the metaplasia-dysplasia-adenocarcinoma sequence (p<0.01). A quarter of AC samples displayed an abnormal Smad4 protein isoform, with no corresponding changes in gene sequence or organisation. Methylation of smad4 has not been described previously but we found promoter methylation in 70% of primary AC samples. In 6/8 oesophageal cell lines, chromosomal rearrangements affected the smad4 locus. Lack of smad4 expression in BIC-1 cells occurred secondary to loss of one copy and extensive deletion of the second allele's promoter region. TGF-beta dependent induction of p21 and downregulation of minichromosome maintenance protein 2 was lost in >80% of BE and AC. TGF-beta failed to inhibit proliferation in 5/8 oesophageal cell lines. In BIC-1, the antiproliferative response was restored following transient transfection of smad4 cDNA.
In BE carcinogenesis, downregulation of Smad4 occurs due to several different mechanisms, including methylation, deletion, and protein modification. Frequent alterations in TGF-beta signalling lead to a functionally significant impairment of TGF-beta mediated growth suppression.
转化生长因子β（TGF-β）常参与胃肠道癌变过程，但其在食管腺癌（AC）及其前体巴雷特食管上皮（BE）化生中的作用尚不清楚。
通过逆转录聚合酶链反应（PCR）、蛋白质印迹法和免疫组织化学法，对食管内镜活检组织和细胞系中TGF-β信号通路成分的表达进行评估。采用荧光原位杂交、甲基化特异性PCR和测序技术对SMAD4的基因组改变进行特征分析。通过检测p21和增殖状态来评估TGF-β信号通路的功能完整性。对Smad4阴性的BIC-1细胞进行smad4瞬时转染，并评估TGF-β反应性。
在化生-发育异常-腺癌序列中，smad4 mRNA表达逐渐降低（p<0.01）。四分之一的AC样本显示出异常的Smad4蛋白异构体，基因序列或结构无相应变化。此前尚未报道smad4的甲基化情况，但我们发现70%的原发性AC样本存在启动子甲基化。在8个食管细胞系中的6个中，染色体重排影响了smad4基因座。BIC-1细胞中smad4表达缺失是由于一个拷贝丢失以及第二个等位基因启动子区域的广泛缺失所致。在超过80%的BE和AC中，TGF-β依赖的p21诱导和微小染色体维持蛋白2的下调消失。在8个食管细胞系中的5个中，TGF-β未能抑制细胞增殖。在BIC-1细胞中，瞬时转染smad4 cDNA后恢复了抗增殖反应。
在BE癌变过程中，Smad4下调是由多种不同机制引起的，包括甲基化、缺失和蛋白质修饰。TGF-β信号通路的频繁改变导致TGF-β介导的生长抑制功能显著受损。
