Nakashiro K, Kawamata H, Hino S, Uchida D, Miwa Y, Hamano H, Omotehara F, Yoshida H, Sato M
Second Department of Oral and Maxillofacial Surgery, Tokushima University School of Dentistry, Japan.
Cancer Res. 1998 Feb 1;58(3):549-55.
We have recently isolated TSC-22 (transforming growth factor beta-stimulated clone 22) cDNA as a new anticancer drug (Vesnarinone)-inducible gene in a human salivary gland cancer cell line, TYS. We conducted the present study to examine whether up-regulation or down-regulation of TSC-22 can affect the growth of TYS cells in vitro and in vivo. We constructed an expression vector containing sense- or antisense-oriented human TSC-22 cDNA under the transcriptional control of the SR alpha promoter. We cotransfected TYS cells with the sense or antisense expression vector and pSV2neo and obtained more than 200 G418-resistant colonies in each sense or antisense transfectant. Approximately 80% of representative G418-resistant clones expressed the transcripts from transfected sense or antisense TSC-22 cDNA. To avoid the clonal heterogeneity of the cells, we mixed all of the G418-resistant colonies together in each sense or antisense transfectant and examined the expression of TSC-22 protein, in vitro growth, and the tumorigenicity in nude mice. The expression of TSC-22 protein was examined by solid-phase ELISA using a specific antibody against recombinant TSC-22 protein. The expression of TSC-22 protein was up-regulated in the sense transfectants and down-regulated in the antisense transfectants. Contrary to our expectation, up-regulation of TSC-22 protein did not affect both in vitro and in vivo growth of TYS cells. However, down-regulation of TSC-22 markedly enhanced the growth of TYS cells in vitro and in vivo. Furthermore, we examined the expression of TSC-22 mRNA in several human salivary gland tumors. The mRNA expression of TSC-22 in benign and malignant salivary gland tumors was significantly decreased when compared to that in tumor-free salivary glands (P < 0.05; one-way ANOVA), and in some salivary gland tumors, the expression of TSC-22 mRNA was not detectable by reverse transcription-PCR. These results suggest that down-regulation of TSC-22 may play a major role on salivary gland tumorigenesis.
我们最近从人唾液腺癌细胞系TYS中分离出TSC - 22（转化生长因子β刺激克隆22）cDNA，它是一种新的抗癌药物（维司力农）诱导型基因。我们开展本研究以检测TSC - 22的上调或下调是否会在体外和体内影响TYS细胞的生长。我们构建了一个表达载体，其在SRα启动子的转录控制下含有正义或反义方向的人TSC - 22 cDNA。我们将正义或反义表达载体与pSV2neo共转染TYS细胞，在每个正义或反义转染子中获得了200多个G418抗性克隆。大约80%的代表性G418抗性克隆表达了来自转染的正义或反义TSC - 22 cDNA的转录本。为避免细胞的克隆异质性，我们将每个正义或反义转染子中的所有G418抗性克隆混合在一起，检测TSC - 22蛋白的表达、体外生长情况以及在裸鼠中的致瘤性。使用针对重组TSC - 22蛋白的特异性抗体通过固相ELISA检测TSC - 22蛋白的表达。TSC - 22蛋白的表达在正义转染子中上调，在反义转染子中下调。与我们的预期相反，TSC - 22蛋白的上调并未影响TYS细胞的体外和体内生长。然而，TSC - 22的下调显著增强了TYS细胞的体外和体内生长。此外，我们检测了几种人唾液腺肿瘤中TSC - 22 mRNA的表达。与无肿瘤的唾液腺相比，良性和恶性唾液腺肿瘤中TSC - 22的mRNA表达显著降低（P < 0.05；单因素方差分析），并且在一些唾液腺肿瘤中，通过逆转录PCR检测不到TSC - 22 mRNA的表达。这些结果表明TSC - 22的下调可能在唾液腺肿瘤发生中起主要作用。
