CYSTATIN C EFFECT ON B16 MELANOMA METASTASIS

癌症的转移性扩散是导致疾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增加癌症患者的生存时间。本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，可作为抗转移剂的步骤。长期目标是靶向半胱氨酸蛋白酶作为恶性肿瘤的传统治疗的治疗辅助。这些研究中使用的模型是高转移性B 16小鼠黑素瘤细胞系，该细胞系已被遗传修饰以过量产生半胱氨酸蛋白酶抑制剂C。此外，许多实验将探索纯化的半胱氨酸蛋白酶抑制剂C对转移性黑色素瘤细胞特性如运动性、体外侵袭、体内生长和转移的影响。将使用改良的Boyden室进行运动性和体外侵袭。转移研究（体内）将通过尾静脉注射标记细胞或皮肤注射黑素瘤细胞进行，以便测量肿瘤生长和转移。半胱氨酸蛋白酶抑制剂C和组织蛋白酶B特异性抗体的定位研究，以及组织蛋白酶B活性的测定，将有助于确定半胱氨酸蛋白酶在转移中的作用。将创建涉及半胱氨酸蛋白酶抑制剂C的特定遗传构建体，以允许标记物活性来帮助定位研究。传统的荧光显微镜和共聚焦显微镜将用于定位研究。该项目的具体目标是确定：l.半胱氨酸蛋白酶抑制剂C过表达是否抑制肺定植的早期阶段？2.半胱氨酸蛋白酶抑制剂C是否抑制肿瘤生长，无论是在过表达半胱氨酸蛋白酶抑制剂C的克隆中还是当半胱氨酸蛋白酶抑制剂C在体内施用于黑素瘤肿瘤时。3.在体外将半胱氨酸蛋白酶抑制剂C外源性添加到B16黑色素瘤细胞中会产生什么影响？4.通过基因融合或表达载体中修饰的胱抑素转染黑色素瘤细胞定位胱抑素C。这些具体的目标将有助于推进我们的知识的作用，半胱氨酸蛋白酶抑制剂作为抗转移剂。 这类抑制剂的治疗价值将在这种性质的研究中揭示，因为靶酶和抑制剂是明确定义的，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.