Heruth D P, Zirnstein G W, Bradley J F, Rothberg P G
Children's Mercy Hospital, Molecular Genetics Laboratory, Kansas City, Missouri.
J Biol Chem. 1993 Sep 25;268(27):20466-72.
Elevated expression of the c-myc oncogene is a frequent finding in tumors and cell lines derived from carcinomas of the colon and rectum. In a previous study we demonstrated that the differentiation agent sodium butyrate causes a rapid reduction in the expression of c-myc RNA in the rectal carcinoma cell line SW837. This effect was blocked by inhibitors of protein synthesis, suggesting that butyrate causes the induction of an activity that has a negative effect on c-myc expression. In the present work we demonstrate that the rapid decrease in the level of c-myc RNA, upon treatment of SW837 cells with 2 mM butyrate, is followed by a slower decrease in the level of p53 RNA and an increase in the RNA levels for fibronectin and a placental type alkaline phosphatase. Using in vitro elongation of nascent transcripts to measure transcription and actinomycin D chase experiments to measure RNA stability, we show that the reduction in expression of c-myc RNA is due to an increase in the block to transcriptional elongation, rather than a decrease in transcriptional initiation or an increase in degradation of the RNA. We conclude that sodium butyrate induces an activity that increases the transcriptional block in SW837 cells, and that regulation of transcriptional elongation is an important mechanism for regulating c-myc expression in this cell type. A shift in relative usage of the two major promoters in the c-myc gene accompanies the reduction in expression. The potential significance of this finding with respect to transcriptional elongation is discussed. Mutations in the exon 1/intron 1 boundary region of the c-myc gene cause an increase in transcriptional elongation in Burkitt lymphoma. We sequenced this region in a series of cell lines derived from colorectal carcinomas, all of which had an elevated level of c-myc expression, to determine if a similar mutational mechanism is at work in this disease. All of the lines examined had a normal c-myc DNA sequence, suggesting that the deregulation of c-myc expression in colon cancer is not due to a cis mutation in this region.
c-myc癌基因的高表达在源自结肠和直肠癌的肿瘤及细胞系中很常见。在之前的一项研究中，我们证明分化剂丁酸钠会导致直肠癌细胞系SW837中c-myc RNA的表达迅速降低。这种效应被蛋白质合成抑制剂阻断，这表明丁酸钠会诱导一种对c-myc表达有负面影响的活性。在本研究中，我们证明用2 mM丁酸钠处理SW837细胞后，c-myc RNA水平迅速下降，随后p53 RNA水平缓慢下降，而纤连蛋白和胎盘型碱性磷酸酶的RNA水平升高。通过使用新生转录本的体外延伸来测量转录，并使用放线菌素D追踪实验来测量RNA稳定性，我们表明c-myc RNA表达的降低是由于转录延伸受阻增加，而不是转录起始减少或RNA降解增加。我们得出结论，丁酸钠诱导一种活性，该活性增加SW837细胞中的转录阻断，并且转录延伸的调节是调节该细胞类型中c-myc表达的重要机制。c-myc基因中两个主要启动子相对使用的转变伴随着表达的降低。讨论了这一发现对于转录延伸的潜在意义。c-myc基因外显子1/内含子1边界区域的突变会导致伯基特淋巴瘤中转录延伸增加。我们对一系列源自结肠直肠癌的细胞系的该区域进行了测序，所有这些细胞系的c-myc表达水平都升高，以确定在这种疾病中是否有类似的突变机制起作用。所有检测的细胞系c-myc DNA序列均正常，这表明结肠癌中c-myc表达的失调不是由于该区域的顺式突变。
