Jiang S Y, Jordan V C
Department of Human Oncology, University of Wisconsin Comprehensive Cancer Center, Madison 53792.
J Natl Cancer Inst. 1992 Apr 15;84(8):580-91. doi: 10.1093/jnci/84.8.580.
The growth of estrogen receptor (ER)-positive breast cancer cells is hormonally regulated, but the majority of breast cancers are ER negative and unresponsive to hormonal therapy.
To test whether hormonal control over replication can be re-established in ER-negative cells, we transfected ER-negative MDA-MB-231 (clone 10A) cells with sense and antisense constitutive ER expression vectors containing the gene for either wild-type or mutant ER linked to the gene for neomycin resistance aminoglycoside phosphotransferase (neo). A Northern blot analysis was done on total RNA from eight of the 10 transfectant clones produced to detect messenger RNA coding for ER and neo, and a Western blot analysis was done on protein extracted from the cells of one mutant and two wild-type ER sense transfectant clones to determine the molecular weight of the ER in transfectants. Levels of ER in transfectants were measured both by enzyme immunoassay and by ligand-binding methods. To ascertain whether the ER in wild-type and mutant sense transfectants was functional, we tested the effects of 17 beta-estradiol (E2) and/or an antiestrogen, ICI 164,384, on 1) ER-activated gene regulation (by transient transfection of these cells a second time with a reporter plasmid containing an estrogen response element linked to the chloramphenicol acetyl transferase [CAT] gene), 2) induction of progesterone receptor, 3) DNA replication, and 4) cell cycle kinetics.
Messenger RNA coding for ER and for neo was detectable in both sense and antisense transfectant clones. Sense transfectants (both mutant and wild-type) expressed ER protein with a molecular weight similar to that found in ER-positive control cells. By the ligand-binding method high levels of ER were detected in both wild-type and mutant transfectants, although by the enzyme immunoassay method lower levels were detected in mutant transfectants. ER from both wild-type and mutant sense transfectants appeared functional, since E2 stimulated the expression of reporter-linked CAT and of progesterone receptor in these transfectants. E2 inhibited DNA replication in wild-type sense transfectants at a concentration of 10(-10) M and mutant sense transfectants at a concentration of 10(-8) M, and ICI 164,384 blocked this effect.
ER-negative breast cancer cells stably transfected with either a mutant or wild-type ER gene regain hormonal responsiveness; however, E2 inhibits rather than stimulates cell growth.
Reactivation of quiescent ER may provide a novel therapeutic approach for controlling ER-negative breast cancers.
雌激素受体（ER）阳性乳腺癌细胞的生长受激素调节，但大多数乳腺癌为ER阴性，对激素治疗无反应。
为了测试在ER阴性细胞中是否可以重新建立对复制的激素控制，我们用含有与新霉素抗性氨基糖苷磷酸转移酶（neo）基因相连的野生型或突变型ER基因的有义链和反义链组成型ER表达载体转染ER阴性的MDA-MB-231（克隆10A）细胞。对产生的10个转染克隆中的8个的总RNA进行Northern印迹分析，以检测编码ER和neo的信使RNA，并对从1个突变型和2个野生型ER有义链转染克隆的细胞中提取的蛋白质进行Western印迹分析，以确定转染体中ER的分子量。通过酶免疫测定法和配体结合法测量转染体中的ER水平。为了确定野生型和突变型有义链转染体中的ER是否具有功能，我们测试了17β-雌二醇（E2）和/或抗雌激素ICI 164,384对以下方面的影响：1）ER激活的基因调控（通过用含有与氯霉素乙酰转移酶[CAT]基因相连的雌激素反应元件的报告质粒再次瞬时转染这些细胞），2）孕酮受体的诱导，3）DNA复制，以及4）细胞周期动力学。
在有义链和反义链转染克隆中均可检测到编码ER和neo的信使RNA。有义链转染体（突变型和野生型）表达的ER蛋白分子量与ER阳性对照细胞中发现的相似。通过配体结合法在野生型和突变型转染体中均检测到高水平的ER，尽管通过酶免疫测定法在突变型转染体中检测到的水平较低。野生型和突变型有义链转染体中的ER似乎都具有功能，因为E2刺激了这些转染体中报告基因连接的CAT和孕酮受体的表达。E2在浓度为10^(-10) M时抑制野生型有义链转染体中的DNA复制，在浓度为10^(-8) M时抑制突变型有义链转染体中的DNA复制，而ICI 164,384阻断了这种作用。
用突变型或野生型ER基因稳定转染的ER阴性乳腺癌细胞恢复了激素反应性；然而，E2抑制而不是刺激细胞生长。
静止ER的重新激活可能为控制ER阴性乳腺癌提供一种新的治疗方法。
