ESTRADIOL, NITRIC OXIDE AND CYCLIN D1 IN BREAST CANCER

NO是一种自由基，现在被认为是一种重要的细胞内信号分子。在低浓度时，它可能通过与超氧化物形成过氧亚硝酸盐而起到促氧化剂的作用，而在高浓度时，它的抗氧化作用占主导地位。有令人信服的证据表明，细胞的氧化促进剂状态--即，增加活性氧物种、有机过氧化物和自由基的浓度，可以促进启动的细胞向肿瘤生长。另一方面，促氧化状态可以被细胞抗氧化防御系统的酶阻止或抑制，许多抗氧化剂是抗启动剂和抗癌物质。因此，NO被证明参与细胞转化和刺激肿瘤生长也就不足为奇了，而其他研究表明NO对肿瘤细胞具有细胞抑制和/或细胞毒作用。有待检验的总体假说是：“NO对人乳腺癌细胞株具有双相作用，即在低稳态浓度时，它促进细胞增殖，而在高稳态浓度时，它抑制细胞增殖。这种双相作用是由于它作用于细胞周期蛋白D1-细胞周期蛋白依赖性蛋白(CDK)-视网膜母细胞瘤蛋白(PRG)级联反应。NO还在雌二醇导致细胞增殖的某些作用中起中介作用。”这一假设将在4个具体目标下进行检验。具体研究内容如下：1.检测不同浓度的NO对人乳腺癌细胞株细胞周期、增殖和DNA合成的影响。具体目的2.研究低稳态浓度的NO对HBC细胞中pRb蛋白磷酸化的调节作用，增加细胞周期蛋白D1的表达，阐明一氧化氮可能作用于细胞周期蛋白d1-细胞周期蛋白依赖性蛋白依赖性蛋白激酶-蛋白酪氨酸酶的级联反应的步骤(S)。特定目的3.我们将确定高稳态浓度的NO(在特定目的1下测定)对pRb磷酸化的调节作用，并阐明在HBc细胞系中，NO可能作用于细胞周期蛋白D1-CDK-pRb级联反应的步骤(S)。具体目的4.检测NO合成抑制剂对雌激素诱导的乳腺癌细胞体外增殖的调节作用；缩短细胞周期的G0/G1期，并探讨该现象的分子机制。

NO is a free radical and is now thought to be an important intracellular signaling molecule. It in low concentrations, can act as a pro-oxidant presumably by forming peroxynitrite with superoxide, whereas at higher concentrations its antioxidant action predominates. There is convincing evidence that cellular pro-oxidant states-i.e., increased concentrations of active oxygen species and organic peroxides and radicals can promote initiated cells to neoplastic growth. On the other hand, pro- oxidant states can be prevented or suppressed by the enzymes of the cellular antioxidant defense system and many antioxidants are anti-promoters and anti-carcinogens. It is, therefore, not surprising that NO has been shown to involve cellular transformation and stimulation of neoplastic growth, whereas other studies indicate that NO has a cytostatic and/or cytotoxic effect on tumor cells. The overall hypothesis to be tested is that "NO has a biphasic action on human breast cancer cell lines, i.e. at low steady state concentrations, it increases cell proliferation, whereas at high steady state concentrations, it decreases cell proliferation. This biphasic action is due to its actions on cyclin D1-cyclin dependent kinase (CDK)-retinoblastoma protein (pRG) cascade. NO also acts as an intermediary in some of the actions of estradiol leading to cell proliferation." This hypothesis will be tested under 4 specific aims. They are as follows: Specific Aim 1. We will determine the effects of different concentrations of NO on cell cycle and proliferation and DNA synthesis of human breast cancer (HBC) cell lines. Specific Aim 2. We will determine the effects of low steady state concentrations of NO to modulate phosphorylation of pRB in HBC cell lines, increase cyclin D1 expression, and elucidate the step(s) at which NO may be acting on the cyclin D1-CDK-pRB cascade. Specific Aim 3. We will determine the effects of high steady state concentrations of NO (determined under specific aim 1) to modulate the phosphorylation of pRB and elucidate the step(s) at which NO may be acting on the cyclin D1-CDK-pRB cascade in HBC cell lines. Specific Aim 4. We will determine the ability of inhibitors of NO synthesis to modulate the estradiol induced increase in breast cancer cell proliferation in vitro; shorten the G0/G1 phase of the cell cycle and the molecular mechanisms involved in this phenomenon.