Estrogen Receptor Activation of p38 Kinase and Apoptosis

描述：(从申请人的摘要中扫描)稳定转染的ER+ HeLa-ER细胞中，我们发现4-羟基三苯氧胺(OHT)-ER和17b-雌二醇 (E2)-ER，极快地激活p38和Jun N-末端 激酶(JNK)途径。OHT-ER或E2-ER强烈激活p38或INK 与诱导细胞凋亡有关。在MCF-7细胞中，OHT，但不是E2， 诱导p38和细胞凋亡，p38抑制剂阻断p38和 细胞凋亡。P38的激活和诱导细胞凋亡是新的代表 确定了雌激素受体的作用。 具体目标是：(1)确定从OHT-ER(和 En-ER)促进p38和JNK的磷酸化和活化。OHT-ER诱导 P38和JNK在2分钟内磷酸化。暗示了一种非基因组效应。(A)我们 将确定OHT-ER所需的ER结构域(如核定位) P38和JNK的激活；(B)评估p38在p38激活中的可能作用 一种膜相关内质网，使用富含假定膜内质网的细胞。通过 专门针对细胞膜的内质网，并通过研究 Er-Caveolae相互作用：(C)使用显性负MKK来确定作用 OHT-ER和E2-ER对p38和JNK的磷酸化中MKKs的表达。(2)至 探讨OHT-ER对p38的激活及其与细胞周期调控的关系 E2-ER与细胞凋亡的诱导。我们将确定这一过程的动力学 在我们的初步研究中看到了p38和JNK激活的第二个晚期阶段， 确定细胞凋亡是否需要p38和JNK的晚期激活，以及 晚期激活是基因组的还是非基因组的。我们提出了一部小说， 可测试，与乳腺癌化学预防和治疗相关的假说 他莫昔芬()，或许还有其他SERM。在乳腺上皮细胞中， DNA损伤所产生的细胞死亡通路的激活程度往往 负责转型有时可能不足以引发 所有受损细胞的凋亡。在ER水平较低的细胞中，额外的 OHT-ER弱诱导p38和JNK增强DNA诱导的细胞凋亡 损伤，从而刺激癌前和早期的细胞凋亡 乳腺癌细胞。(3)对乳腺上皮细胞DNA有轻微损伤 细胞和ER+细胞系具有低水平的紫外光，并决定是否 激活p38或JNK，促进细胞凋亡。(四)确定 乳腺癌细胞系对耐药的进展是否涉及 对p38或jnk的诱导丧失，或p38或jnk的诱导能力丧失 凋亡，我们将利用现有的耐药乳腺癌细胞株和细胞 我们分离、抑制和控制负突变的品系。 这些研究有助于定义ER作用的新途径，并测试一种新的 SERM如何发挥作用的假设。

DESCRIPTION: (Scanned from the applicant's abstract) In stably transfected ER+ HeLa-ER cells, we find that 4-hydroxytamoxifen (OHT)-ER and l7b-estradiol (E2)-ER, elicit an extremely rapid activation of the p38 and Jun N-terminal kinase (JNK) pathways. Activation of p38 or iNK by OHT-ER or E2-ER is strongly correlated with the induction of apoptosis. In MCF-7 cells, OHT, but not E2, induces p38 and apoptosis and a p38 inhibitor blocks induction of p38 and apoptosis. Activation of p38 and induction of' apoptosis represent newly identified actions of estrogen receptor. The Specific Aims are: (1) To identify the pathway leading from OHT-ER (and En-ER) to the phosphorylation and activation of p38 and JNK. OHT-ER induces phosphorylation of p38 and JNK in 2 min. suggesting a nongenomic effect. (a) We will identify the ER domains (such as nuclear localization) required for OHT-ER activation of p38 and JNK; (b) evaluate the possible role in p38 activation of a membrane-associated ER using cells enriched in a putative membrane ER. by specifically targeting ER to cell membranes, and through studies of potential ER-caveolae interactions: (c) use dominant negative MKKs to determine the role of MKKs in phosphorylating p38 and JNK in response to OHT-ER and E2-ER. (2) To investigate the relationship between the activation of p38 by OHT-ER and by E2-ER and the induction of apoptosis. We will determine the kinetics of the second late stage of activation of p38 and JNK seen in our preliminary studies, determine whether apoptosis requires the late activation of p38 and JNK, and whether the late activation is genomic or non-genomic. We propose a novel, testable, hypothesis relevant to both breast cancer chemoprevention and therapy by tamoxifen (Tam), and perhaps by other SERMs. In mammary epithelial cells, the level of activation of cell death pathways produced by the DNA damage often responsible for transformation may sometimes be insufficient to induce apoptosis of all damaged cells. In cells with low levels of ER, the additional weak induction of p38 and JNK by OHT-ER potentiates apoptosis induced by DNA damage, and thereby stimulates apoptosis of pre-malignant and early stage breast cancer cells. (3) We will slightly damage the DNA of mammary epithelial cells and ER+ cell lines with a low level of UV light and determine whether Tam activates p38 or JNK and potentiates apoptosis of the cells. (4) To determine whether the progression of breast cancer cell lines to Tam resistance involves loss of Tam induction of p38 or JNK, or loss of ability of p38 or JNK to induce apoptosis, we will use existing Tam resistant breast cancer cell lines and cell lines we isolate, inhibitors and dominate negative mutants. These studies help define a new pathway for ER action and test a novel hypothesis for how SERMs function.