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。在低浓度下，它可以作为一种促氧化剂，可能是通过与超氧化物形成过氧亚硝酸盐，而在高浓度下，它的抗氧化作用占主导地位。有令人信服的证据表明，细胞促氧化状态——即例如，活性氧、有机过氧化物和自由基浓度的增加可以促进初始细胞的肿瘤生长。另一方面，细胞抗氧化防御系统的酶可以阻止或抑制促氧化状态，许多抗氧化剂是抗促进剂和抗癌物质。因此，NO被证明参与细胞转化和刺激肿瘤生长并不奇怪，而其他研究表明NO对肿瘤细胞具有细胞抑制和/或细胞毒性作用。要测试的总体假设是“NO对人类乳腺癌细胞系有双相作用，即在低稳态浓度下，它会增加细胞增殖，而在高稳态浓度下，它会减少细胞增殖。这种双相作用是由于其作用于细胞周期蛋白d1 -细胞周期蛋白依赖性激酶(CDK)-视网膜母细胞瘤蛋白（pRG）级联。一氧化氮在雌二醇导致细胞增殖的某些作用中也起到中介作用。”这一假设将在4个具体目标下进行检验。具体目标：我们将测定不同浓度NO对人乳腺癌细胞系细胞周期、增殖和DNA合成的影响。具体目标2。我们将确定低稳态浓度NO对HBC细胞系中pRB磷酸化的调节作用，增加细胞周期蛋白D1的表达，并阐明NO可能作用于细胞周期蛋白D1- cdk -pRB级联的步骤。具体目标3。我们将确定高稳态浓度NO（在特定目的1下确定）对调节pRB磷酸化的影响，并阐明NO可能作用于HBC细胞系中细胞周期蛋白D1-CDK-pRB级联的步骤。具体目标我们将在体外确定NO合成抑制剂调节雌二醇诱导的乳腺癌细胞增殖的能力；缩短细胞周期的G0/G1期及其分子机制。

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.