GROWTH-REGULATION OF ESTROGEN RECEPTOR-NEGATIVE BREAST-CANCER CELLS TRANSFECTED WITH COMPLEMENTARY DNAS FOR ESTROGEN-RECEPTOR
GROWTH-REGULATION OF ESTROGEN RECEPTOR-NEGATIVE BREAST-CANCER CELLS TRANSFECTED WITH COMPLEMENTARY DNAS FOR ESTROGEN-RECEPTOR
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DOI：
10.1093/jnci/84.8.580
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发表时间：
1992-04-15
影响因子：
10.3
通讯作者：
JORDAN, VC
中科院分区：
文献类型：
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作者：
JIANG, SY;JORDAN, VC
背景资料：雌激素受体（ER）阳性乳腺癌细胞的生长受到激素调节，但大多数乳腺癌是ER阴性的，对激素治疗无反应。目的和方法：为了测试是否可以在ER阴性细胞中重新建立对复制的激素控制，我们用正义和反义组成型ER表达载体转染ER阴性MDA-MB-231（克隆10A）细胞，所述表达载体含有与新霉素抗性氨基糖苷磷酸转移酶（neo）基因连接的野生型或突变型ER基因。对产生的10个转染子克隆中的8个的总RNA进行北方印迹分析，以检测编码ER和neo的信使RNA，并对从一个突变体和两个野生型ER正义转染子克隆的细胞中提取的蛋白质进行Western印迹分析，以确定转染子中ER的分子量。用酶免疫法和配体结合法测定转染子中雌激素受体的水平。为了确定野生型和突变型正义转染子中的ER是否有功能，我们测试了17-β-雌二醇（E2）和/或抗雌激素ICI 164，384对1）ER激活的基因调控的影响（通过用含有与氯霉素乙酰转移酶[CAT]基因连接的雌激素反应元件的报道质粒第二次瞬时转染这些细胞），2）孕酮受体的诱导，3）DNA复制，和4）细胞周期动力学。结果：在正义和反义转染克隆中均可检测到编码ER和neo的信使RNA。正义转染子（突变体和野生型）表达的ER蛋白的分子量类似于ER阳性对照细胞中发现的。通过配体结合法，在野生型和突变体转染子中检测到高水平的ER，尽管通过酶免疫测定法，在突变体转染子中检测到较低水平。ER从野生型和突变体的正义转染子出现的功能，因为E2刺激的表达，在这些转染子中的孕激素受体的CAT和。E2在浓度为10（-10）M时抑制野生型正义转染子中的DNA复制，在浓度为10（-8）M时抑制突变正义转染子中的DNA复制，ICI 164，384阻断了这种作用。结论：ER阴性乳腺癌细胞稳定转染突变或野生型ER基因恢复激素的反应性，然而，E2抑制而不是刺激细胞生长。意义：静息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。
Background: 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. Purpose and Methods: 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. Results: 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. Conclusion: 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. Implication: Reactivation of quiescent ER may provide a novel therapeutic approach for controlling ER-negative breast cancers.