Bronzert D A, Greene G L, Lippman M E
Endocrinology. 1985 Oct;117(4):1409-17. doi: 10.1210/endo-117-4-1409.
We have selected and cloned a stable variant of the MCF-7 human breast cancer cell line (LY 2) that is resistant to LY 117018 (LY), a potent antiestrogen that inhibits cell growth at concentrations as low as 10(-10) M. The cell line was selected by increasing the concentration of LY in the growth medium in a stepwise manner from 10(-8) to 10(-6) M as the cells become resistant. LY2 has been cloned in soft agar and carried for over 50 passages with no change in resistance. Other antiestrogens, such as tamoxifen and 40-hydroxytamoxifen no longer inhibit cell proliferation of LY 2. The cell line is still responsive to estrogen in a cell proliferation assay, but contains somewhat less estrogen receptors than MCF-7. The cytosolic estrogen receptor sediments to a 4S position on high salt sucrose density gradient centrifugation and is completely shifted to a denser gradient region when the receptor is incubated with a monoclonal antiestrophilin. The nuclear estrogen receptor when covalently labeled with [3H]tamoxifen aziridine has the same mol wt (62,000) in both MCF-7 and LY2 cells, when determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In a competitive binding assay, LY 117018 competes for [3H]estradiol binding to its cytosol receptor with the same Ki in both MCF-7 and LY2 cells. When the induction of estrogen-specific proteins was examined, no detectable progesterone receptor could be detected in either estrogen-induced or control LY2 cells, in contrast to MCF-7 cells. However, both 52,000- and 160,000-dalton proteins were estrogen inducible in the medium of LY2 and MCF-7 cells, as measured by labeling with [35S]methionine. The phenotypic stability of the antiestrogen resistance in LY2 cells coupled with the cross-resistance the antiestrogens of widely different structures make this cell line an ideal model system for the study of hormone resistance in human breast cancer. In addition, while the mechanism of resistance is currently not elucidated, the selective loss of estrogen-inducible functions in this cell line may provide powerful clues for future study.
我们筛选并克隆了MCF-7人乳腺癌细胞系的一个稳定变体（LY 2），该变体对LY 117018（LY）具有抗性，LY是一种强效抗雌激素药物，在低至10^(-10) M的浓度下就能抑制细胞生长。通过在细胞产生抗性时，将生长培养基中LY的浓度从10^(-8) M逐步提高到10^(-6) M来筛选该细胞系。LY2已在软琼脂中克隆，并传代培养50多次，抗性没有变化。其他抗雌激素药物，如他莫昔芬和4-羟基他莫昔芬，不再抑制LY 2的细胞增殖。在细胞增殖试验中，该细胞系仍对雌激素有反应，但雌激素受体含量比MCF-7略少。在高盐蔗糖密度梯度离心中，胞质雌激素受体沉降到4S位置，当该受体与单克隆抗雌激素亲和蛋白孵育时，会完全转移到密度更高的梯度区域。当用[3H]他莫昔芬氮丙啶进行共价标记时，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测定，MCF-7和LY2细胞中的核雌激素受体具有相同的分子量（约62,000）。在竞争结合试验中，LY 117018在MCF-7和LY2细胞中以相同的Ki值竞争[3H]雌二醇与其胞质受体的结合。当检测雌激素特异性蛋白的诱导情况时，与MCF-7细胞不同，在雌激素诱导的或对照的LY2细胞中均未检测到可检测到的孕酮受体。然而，通过用[35S]甲硫氨酸标记检测发现，LY2和MCF-7细胞培养基中的52,000道尔顿和160,000道尔顿的蛋白均可被雌激素诱导。LY2细胞中抗雌激素抗性的表型稳定性以及对结构差异很大的抗雌激素的交叉抗性，使其成为研究人类乳腺癌激素抗性的理想模型系统。此外，虽然目前抗性机制尚未阐明，但该细胞系中雌激素诱导功能的选择性丧失可能为未来的研究提供有力线索。
