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

1. c-myc启动子中TGF-β响应元件的鉴定：在c-myc启动子中鉴定了TGF-β响应元件。该元件与在小鼠基质溶解素基因中鉴定的TGF-β抑制元件（TIE）具有一致序列。在c-myc启动子中的TIE部分重叠E2 F位点，并在TGF-β刺激后1 h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，c-myc启动子的转录活性不受TGF-β抑制。我们比较了癌细胞和角质形成细胞系（HaCaT）中与TIE/E2 F寡核苷酸结合的核蛋白，通过DNA亲和沉淀和SDS-PAGE发现癌细胞中有几条蛋白条带增强或丢失。阻断Smad通路赋予HaCaT细胞侵袭性生长活性： 关于我们 显示鳞状细胞癌细胞系间质侵袭的方法。使用这种方法，HaCaT细胞显示通过表达显性失活Smad 3（Smad 3D 407 E）获得侵袭活性。Smad 3D 407 E的表达水平与获得的侵袭活性之间具有良好的相关性。利用DNA芯片技术筛选Smad 3D 407 E诱导HaCaT细胞差异表达的基因，寻找Smad通路中与获得侵袭性相关的靶基因. BMP信号传导和靶基因：我们克隆了小鼠Smad 6基因组克隆，并鉴定了Smad 6启动子中的BMP反应元件。BMP反应元件（GC 2）具有与果蝇中的Madresponsive元件相似的富含GC的序列。BMP调节的Smads和Co-Smad直接结合GC 2。R-Smad增强了Smad 6从GC 2元件的转录。BMP-2/4和BMP-6/7属于不同的亚家族，在某些生物学实验中表现出协同作用。BMP-2/4和BMP-6/7分别优先结合ALK-3/6和ALK-2。这些受体的组成型活性形式的表达也提供了额外的效果，并且这些受体被认为至少部分地激活不同的信号传导途径。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