Growth inhibition of keratinocytes by TGF-β and the impaired signaling in cancer cells

1. c-myc启动子中TGF-β应答元件的鉴定：在c-myc启动子中鉴定出TGF-β应答元件。该元件与小鼠基质溶素基因中发现的TGF-β抑制元件（TIE）序列一致。c-myc启动子中的TIE与E2F位点部分重叠，并在TGF-β刺激后1小时内结合TGF-β调节的Smad。在鳞状细胞癌细胞系中寻找导致c-myc对TGF-β反应受损的核蛋白：在几种鳞状细胞癌细胞系中，TGF-β不抑制c-myc启动子的转录活性。我们比较了癌细胞和角化细胞细胞系（HaCaT）之间结合TIE/E2F寡核苷酸的核蛋白，发现通过dna亲和沉淀和sds - page在癌细胞中发现了一些增强或丢失的蛋白带。阻断Smad通路赋予HaCaT细胞侵袭性生长活性：我们建立了一种三维培养方法，显示鳞状细胞癌细胞的间质侵袭性。通过这种方法，HaCaT细胞通过表达显性阴性Smad3 （Smad3D407E）获得侵袭性活性。Smad3D407E表达水平与获得的侵袭性活动有良好的相关性。3 .我们通过DNA芯片分析筛选Smad3D407E在HaCaT细胞中诱导的差异表达基因，寻找Smad通路中有助于获得侵袭活性的靶基因。BMP信号和靶基因：我们克隆了小鼠Smad6基因组克隆，并在Smad6启动子中鉴定了BMP应答元件。BMP-responsive element （GC2）的GC-rich sequence与果蝇中的Madresponsive element相似。bmp调控的smad和Co-Smad直接结合GC2。R-Smad增强了GC2元件Smad6的转录。另一方面，Co-Smad抑制了bmp -2/4和BMP-6/7的转录活性，它们属于不同的亚家族，在一些生物学试验中表现出协同效应。BMP-2/4和BMP-6/7分别优先结合ALK-3/6和ALK-2。这些受体的组成活性形式的表达也有额外的作用，这些受体被认为至少部分地激活不同的信号通路。少

1. Identification of a TGF-β responsive element in the c-myc promoter :A TGF-β responsive element was identified in the c-myc promoter. This element had an consensus sequence with TGF-β inhibitory element (TIE) which was identified in the mouse stromelysin gene. TIE in the c-myc promoter partially overlapped an E2F site and bound TGF-β-regulated Smad within 1 h after stimulation by TGF-β.2. Pursuit of nuclear proteins responsible for impaired c-myc-response to TGF-β in squamous cell carcinoma cell lines :Transcriptional activity of the c-myc promoter was not suppressed by TGF-β in several squamous cell carcinoma cell lines. We compared nuclear proteins which bound to the TIE/E2F oligonucleotides between cancer cells and a keratinocyte cell line (HaCaT), and found several protein bands enhanced or lost in cancer cells by DNA-affinity precipitation followed by SDS-PAGE.3. Blocking of Smad pathway confers invasive growth activity on HaCaT cells :We have established a 3-dimensional culture … More method exhibiting stromal invasion of squamous cell carcinoma cell lines. Using this method, HaCaT cells were shown to obtain invasive activity by expression of dominant negative Smad3 (Smad3D407E). There was good correlation between expression levels of Smad3D407E and obtained invasive activities. We screened the differentially expressed genes induced by Smad3D407E in HaCaT cells by DNA chip analyses to search the target genes of Smad pathway which contribute to acquire invasive activity.4. BMP signaling and the target gene :We have cloned mouse Smad6 genomic clones, and identified a BMP-responsive element in the Smad6 promoter. The BMP-responsive element (GC2) had a GC-rich sequence similar to a Madresponsive element in Drosophila. BMP-regulated Smads and Co-Smad directly bound GC2. R-Smad enhanced transcription of Smad6 from GC2 element. On the other hand, Co-Smad suppressed the transcriptional activity.BMP-2/4 and BMP-6/7 belong to different subfamily and exhibit cooperative effects in some biological assays. BMP-2/4 and BMP-6/7 preferentially bound ALK-3/6 and ALK-2, respectively. Expression of constitutively active forms of these receptors also gave additional effects and these receptors were suggested to activate at least partially distinct signaling pathways. Less