ANGIOSTATIC FUNCTION OF HUMAN 16K PROLACTIN

血管生成在乳腺癌中起关键作用，是肿瘤发生和转移的组成部分。 生长中的肿瘤产生血管生成因子和血管生成抑制因子，调节内皮细胞功能。 许多血管抑制因子，例如，血管抑制素和内皮抑制素是肝素结合的大蛋白质的裂解产物，其通过抑制新血管形成而显示出抗肿瘤活性。 催乳素（PRL）是一种23 kDa的垂体激素，其主要靶点是乳房。 其N-末端片段，命名为16 K PRL，在体外和体内都具有血管抑制活性，并且其功能在物种间是保守的。 然而，16 K PRL阻止肿瘤生长和转移的能力尚未得到证实。我们已经证实，蛋白水解裂解的大鼠PRL（rPRL）抑制基础和FGF刺激的人和牛内皮细胞的增殖。 我们表明，转移性MDA-MB-435人乳腺癌细胞，瞬时转染哺乳动物表达载体，产生和分泌16 K和23 K人PRL（hPRL），也证明了杆状病毒感染的昆虫细胞分泌16 K PRL与血管抑制活性。 还建立了使用无胸腺小鼠测试16 K PRL的血管抑制作用的体内肿瘤模型。 我们的假设是：16 K PRL是一种有效的血管生成抑制因子，通过不同的受体作用于内皮细胞。用16 K PRL治疗乳腺癌应通过减少其血液供应来抑制原发性肿瘤和转移瘤的生长。第一个具体目标将确定与表达23 K hPRL或含有空载体的那些相比，16 K hPRL是否阻止稳定表达16 K hPRL的移植MDA-MB-435细胞的生长。 第二个具体目标将检查16 K hPRL是否由人肿瘤异种移植物产生，并确定肿瘤产生的16 K 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产生重组16 K hPRL，并检查其生化特性，血管抑制活性以及与肿瘤和内皮细胞的结合。 第四个具体目标将确定16 K hPRL是否抑制移植无胸腺小鼠中转移瘤的生长，使用三种替代递送方法：每日注射重组16 K PRL，通过肌肉注射16 K hPRL质粒进行基因转移，或植入封装在中空纤维中的表达16 K 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.