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

1.c-myc启动子中转化生长因子-β反应元件的鉴定：c-myc启动子中存在转化生长因子-β反应元件。该元件与小鼠基质分解素基因中发现的转化生长因子-β抑制元件(TiE)具有相同的序列。C-myc启动子与E2F部分重叠，在转化生长因子-β刺激后1h内与转化生长因子-β调控的Smad结合。鳞状细胞癌细胞系c-myc对转化生长因子-β反应受损的核蛋白追寻：转化生长因子-β不抑制几种鳞癌细胞系c-myc启动子的转录活性。我们比较了癌细胞和角质形成细胞系(HaCaT)之间Tie/E2F寡核苷酸结合的核蛋白，发现通过DNA亲和沉淀和SDS-PAGE.3在癌细胞中有几条蛋白质条带增强或丢失。阻断Smad途径对HaCaT细胞的侵袭性生长活性：我们建立了三维培养的…更多的方法显示鳞状细胞癌细胞系的间质侵袭。用这种方法，HaCaT细胞通过表达显性阴性的SMAD3(Smad3D407E)获得侵袭活性。Smad3D407E的表达水平与获得的侵袭活性有很好的相关性。利用DNA芯片技术筛选Smad3D407E诱导HaCaT细胞差异表达的基因，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，并鉴定了Smad6启动子中的BMP反应元件。BMP反应元件(GC2)具有一个富含GC的序列，类似于果蝇中的MadResponse元件。BMP调节的Smads和Co-Smad直接与GC2结合。R-Smad促进GC2元件中Smad6的转录。BMP-2/4和BMP-6/7属于不同的亚家族，在一些生物检测中表现出协同作用。BMP-2/4和BMP-6/7分别优先结合ALK-3/6和ALK-2。这些受体的组成活性形式的表达也起到了额外的作用，这些受体被认为至少激活了部分不同的信号通路。较少

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