ANGIOSTATIC FUNCTION OF HUMAN 16K PROLACTIN

血管生成在乳腺癌中起着至关重要的作用，是肿瘤发生和转移的重要组成部分。生长中的肿瘤会产生调节内皮细胞功能的血管生成因子和血管抑制因子。许多血管抑制因子，如血管抑制素和血管内皮抑制素，是肝素结合的大蛋白的裂解产物，通过抑制新生血管形成而表现出抗肿瘤活性。催乳素（PRL）是一种23 kDa的垂体激素，其主要作用靶点是乳房。其n端片段命名为16K PRL，在体内和体外均具有血管抑制活性，其功能在物种间是保守的。然而，16K PRL抑制肿瘤生长和转移的能力尚未得到证实。我们已经证实，蛋白水解裂解大鼠PRL （rPRL）抑制基础和fgf刺激的人类和牛内皮细胞的增殖。我们发现转移性的MDA-MB-435人乳腺癌细胞在短暂转染哺乳动物表达载体后，产生并分泌16K和23K的人PRL (hPRL)，并且杆状病毒感染的昆虫细胞分泌具有血管抑制活性的16K PRL。还建立了用于检测16K PRL血管抑制作用的小鼠体内肿瘤模型。我们的假设是：16K PRL是一种有效的血管抑制因子，通过不同的受体作用于内皮细胞。用16K PRL治疗乳腺癌可以通过减少原发肿瘤和转移瘤的血供来抑制其生长。第一个具体目的是确定与表达23K hPRL或含有空载体的MDA-MB-435细胞相比，16K hPRL是否能抑制稳定表达16K hPRL的移植物细胞的生长。第二个具体目的是检测16K hPRL是否由人类肿瘤异种移植物产生，并确定肿瘤产生的16K hPRL对血管密度、细胞增殖和凋亡的影响。第三个具体目标是在昆虫细胞中生成重组16K hPRL，并研究其生化特性、血管抑制活性以及与肿瘤和内皮细胞的结合。第四个特定目的是确定16K hPRL是否抑制移植胸腺小鼠转移瘤的生长，使用三种可选择的递送方法：每日注射重组16K PRL，通过肌肉注射16K hPRL质粒进行基因转移，或植入中空纤维包裹的表达16K PRL的COS细胞。

Angiogenesis plays a critical role in breast cancer and is an integral part of tumorigenesis and metastasis. Growing tumors produce both angiogenic and angiostatic factors that regulate endothelial cell function. Many angiostatic factors, e.g., angiostatin and endostatin, are heparin-binding, cleaved products of large proteins which exhibit anti-tumorigenic activity by inhibiting neovascularization. Prolactin (PRL) is a 23 kDa pituitary hormone whose main target is the breast. Its N-terminal fragment, named 16K PRL, possesses angiostatic activity both in vitro and in vivo and its function is conserved across species. However, the ability of 16K PRL to arrest tumor growth and metastases has not been demonstrated. We have confirmed that proteolytically-cleaved rat PRL (rPRL) suppresses basal and FGF-stimulated proliferation of human and bovine endothelial cells. We showed that the metastatic MDA-MB-435 human breast cancer cells, transiently transfected with mammalian expression vectors, produce and secrete 16K and 23K human PRL (hPRL), and also demonstrated that baculoviral-infected insect cells secreted 16K PRL with angiostatic activity. In vivo tumor models for testing the angiostatic action of 16K PRL, using athymic mice, have also been established. Our hypothesis is: 16K PRL is a potent angiostatic factor which acts on endothelial cells via distinct receptors. Treatment of breast cancer with 16K PRL should suppress growth of primary tumors and metastases by reducing their blood supply. The first specific aim will determine whether 16K hPRL arrests growth of grafted MDA-MB-435 cells stably expressing 16K hPRL compared with those expressing 23K hPRL or containing empty vector. The second specific aim will examine whether 16K hPRL is generated by human tumor xenografts and determine the effects of tumor-produced 16K hPRL on blood vessel density, cell proliferation and apoptosis. The third specific aim will generate recombinant 16K hPRL in insect cells and examine its biochemical properties, angiostatic activity and binding to tumors and endothelial cells. The fourth specific aim will determine whether 16K hPRL suppresses growth of metastases in grafted athymic mice, using three alternative delivery methods: daily injections of recombinant 16K PRL, gene transfer via intramuscular injections of 16K hPRL plasmid, or implantation of 16K PRL-expressing COS cells encapsulated in hollow fibers.