Wu S P, Theodorescu D, Kerbel R S, Willson J K, Mulder K M, Humphrey L E, Brattain M G
Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77030.
J Cell Biol. 1992 Jan;116(1):187-96. doi: 10.1083/jcb.116.1.187.
Transforming growth factor-beta 1 (TGF-beta 1) has previously been implicated as a potential negative autocrine or paracrine growth regulator of certain cell types (Arteaga, C. L., R. J. Coffey, Jr., T. C. Dugger, C. M. McCutchen, H. L. Moses, and R. M. Lyons. 1990. Cell Growth & Differ. 1:367-374; Hafez, M. M., D. Infante, S. Winawer, and E. Friedman. 1990. Cell Growth & Differ. 1:617-626; Glick, A. B., K. C. Flanders, D. Danielpour, S. H. Yuspa, and M. B. Sporn. 1989. Cell Regulation. 1:87-97). This is based mainly on experiments assessing the effects of exogenous TGF-beta 1 or neutralizing antibodies to TGF-beta 1 on normal or tumor cell proliferation in vitro. However, direct evidence demonstrating such a negative regulation of tumor cell growth in vivo is still lacking. To overcome this problem we have constructed and used an antisense expression vector for TGF-beta 1 as a means of regulating endogenous TGF-beta 1 expression in tumor cells. Antisense-transfected FET human colon carcinoma cells showed a fivefold reduction in TGF-beta 1 mRNA and 15-fold reduction in TGF-beta 1 secretion. Antisense mRNA was detected in transfected cells by an RNase protection assay. Compared to control cells, cultured antisense-transfected cells showed a reduction in lag phase time rather than a change in doubling time. Cloning efficiencies of transfected cells were four times greater than control cells in anchorage-independent assays. Control cells did not form tumors at 5 x 10(5) in athymic nude mice. Antisense-transfected cells formed tumors in 40% of animals injected. At higher inocula (1 x 10(6) cells) antisense-transfected cells formed tumors in 100% of animals injected, but control cells still failed to form tumors. These results show that TGF-beta 1 acts as a negative growth regulator of human colon carcinoma cells in vivo as well as in vitro. Acquisition of partial or full resistance to such inhibitory effects may therefore contribute to tumor development and progression.
转化生长因子-β1(TGF-β1)此前被认为是某些细胞类型潜在的负性自分泌或旁分泌生长调节因子(阿特亚加,C.L.,R.J.科菲,Jr.,T.C.达格,C.M.麦卡琴,H.L.摩西,和R.M.莱昂斯。1990年。《细胞生长与分化》1:367 - 374;哈菲兹,M.M.,D.因方特,S.维纳韦尔,和E.弗里德曼。1990年。《细胞生长与分化》1:617 - 626;格利克,A.B.,K.C.弗兰德斯,D.丹尼尔普尔,S.H.尤斯帕,和M.B.斯波恩。1989年。《细胞调控》1:87 - 97)。这主要基于评估外源性TGF-β1或TGF-β1中和抗体对体外正常或肿瘤细胞增殖影响的实验。然而,仍缺乏在体内证明肿瘤细胞生长受到这种负性调节的直接证据。为克服这一问题,我们构建并使用了TGF-β1的反义表达载体,作为调节肿瘤细胞内源性TGF-β1表达的一种手段。反义转染的FET人结肠癌细胞TGF-β1 mRNA减少了五倍,TGF-β1分泌减少了15倍。通过核糖核酸酶保护试验在转染细胞中检测到了反义mRNA。与对照细胞相比,培养的反义转染细胞的延迟期时间缩短,而倍增时间没有变化。在非贴壁依赖性试验中,转染细胞的克隆效率比对照细胞高四倍。对照细胞在无胸腺裸鼠中以5×10⁵个细胞接种时未形成肿瘤。反义转染细胞在40%的注射动物中形成了肿瘤。在更高接种量(1×10⁶个细胞)时,反义转染细胞在100%的注射动物中形成了肿瘤,但对照细胞仍然未能形成肿瘤。这些结果表明,TGF-β1在体内和体外均作为人结肠癌细胞的负性生长调节因子。因此,对这种抑制作用获得部分或完全抗性可能有助于肿瘤的发生和发展。