CYSTATIN C EFFECT ON B16 MELANOMA METASTASIS

癌症的转移性扩散是该病难治性的主要原因。用化学或生物制剂干预癌症转移可能会延长癌症患者的生存时间。该项目的总体目标是阐明天然半胱氨酸蛋白酶抑制剂--半胱氨酸蛋白酶抑制剂--作为抗肿瘤转移剂的步骤。长期目标是将半胱氨酸蛋白酶作为恶性肿瘤传统治疗的辅助治疗手段。这些研究中使用的模型是高转移性的B16小鼠黑色素瘤细胞系，这些细胞系已经被基因修饰以过量产生cystatin C。此外，一些实验将探索纯化的cystatin C对转移性黑色素瘤细胞的运动、体外侵袭、体内生长和转移特性的影响。运动性和体外侵袭将用改良的Boyden小室进行。转移研究(体内)将通过尾静脉注射标记细胞或皮肤注射黑色素瘤细胞来进行，以便测量肿瘤的生长和转移。用针对半胱氨酸蛋白酶C和组织蛋白酶B的抗体进行定位研究，以及组织蛋白酶B活性的测量，将有助于确定半胱氨酸蛋白酶在转移中的作用。将创建涉及胱抑素C的特定遗传结构，以允许标记活动帮助本地化研究。常规荧光显微镜和共聚焦显微镜都将用于定位研究。本项目的具体目的是确定：L。过表达胱抑素C是否抑制早期肺部定植？2.无论是在高表达胱抑素C的克隆中还是在体内给药治疗黑色素瘤时，胱抑素C是否抑制肿瘤的生长。3.外源性Cystatin C在体外对B16黑色素瘤细胞有何影响？4.将基因融合或修饰后的Cystatin基因导入黑色素瘤细胞表达载体，对Cystatin C进行定位。这些特定的目的将有助于我们加深对半胱氨酸蛋白酶抑制剂作为抗转移药物的作用的了解。这类抑制剂的治疗价值将在这种性质的研究中揭示出来，因为靶酶和抑制剂被很好地定义，而B16黑色素瘤是最好的特征转移细胞系之一。

The metastatic spread of cancer is primarily responsible for the intractable nature of the disease. Interference of cancer metastasis with chemical or biologic agents could perhaps bring about increased survival times in cancer patients. The overall goal of this project is to elucidate steps by which natural cysteine proteinase inhibitors, the cystatins, may act as antimetastatic agents. The long term goal is to target cysteine proteinases as a therapeutic adjunct to traditional treatments for malignant neoplasms. The model to be used in these studies are highly metastatic B 16 mouse melanoma cell lines which have been genetically modified to overproduce cystatin C. In addition, a number of experiments will explore the effect of purified cystatin C on metastatic melanoma cell characteristics such as motility, invasion in vitro, growth and metastasis in vivo. Motility and in vitro invasion will be conducted with modified Boyden chambers. Metastasis studies (in vivo) will be carried out by tail-vein injections of labeled cells or dermal injections of melanoma cells so that tumor growth and metastasis can be measured. Localization studies with antibodies specific for cystatin C and cathepsin B, together with cathepsin B activity measurements will help define the role of cysteine proteinases in metastasis. Specific genetic constructs involving cystatin C will be created to allow marker activities to aid in localization studies. Both conventional fluorescence microscopy and confocal microscopy will be used in localization studies. The specific aims for this project are to determine: l. Does cystatin C overexpression inhibit an early stage of lung colonization? 2. Whether cystatin C inhibits tumor growth, either in clones overexpressing cystatin C or when cystatin C is administered to melanoma tumors in vivo. 3. What effects occur upon exogenous addition of cystatin C to B16 melanoma cells in vitro? 4. Localization of cystatin C by transfection of melanoma cells with gene fusions or modified cystatin in expression vectors. These specific aims will help advance our knowledge of the role of cysteine proteinase inhibitors as anti-metastatic agents. The therapeutic value of this class of inhibitors will be revealed in studies of this nature because the target enzyme and inhibitor are well defined and the B16 melanoma are among the best of characterized metastatic cell lines.