METASTASIS SUPPRESSOR GENES IN HUMAN CUTANEOUS MELANOMA

我们最近证明了缺陷基因的替换 转移性人黑色素瘤C8161中人类6号染色体的信息 细胞在不降低致瘤性的情况下抑制转移。我们的目标是 鉴定和定位6号染色体上的致病基因(S) 抑制转移并了解该基因是如何工作的(S)。朝向那个方向 最后，我们将系统地缩小6号染色体上编码的区域 对于候选基因，我们将把我们的观察扩展到其他 转移性人黑色素瘤细胞系。提出了三个具体目标 要实现这些目标： 恶性黑色素瘤转移抑制基因的定位 人类6号染色体。将使用两种实验方法。(1)我们 将使用微细胞介导的染色体转移来引入完整的Short 6号染色体的长臂(6p)和长臂(6q)插入C8161。混合动力车将成为 评估裸小鼠或SCID小鼠的转移率。那些混血儿 这些被抑制转移的基因将识别DNA的区域 将在随后的实验中引入。我们的目标是横断面 DNA序列逐渐变小，直到5Mb区域被鉴定为 编码负责抑制转移的基因(S)。(2)我们会 分离C8161-6号染色体杂交种的转移逆转株。1~2成熟 在小鼠体内注射杂交瘤后发生的转移将是 建立了细胞系并进行了核型分析。损失任何或全部 在转移来源的细胞中增加6号染色体将证明 含有转移抑制基因的区域。 为了确定增加6号染色体是否能抑制其他肿瘤的转移 人黑色素瘤细胞系。将引入正常的人类6号染色体 转移性人皮肤黑色素瘤细胞系A375M和M24Met。 杂交种将在裸或SCID小鼠体内进行转移评估。 选择这些株系是因为：(1)它们在实验中是转移的 (2)它们在6号染色体上有缺陷。 将单个基因导入C8161细胞并测定其对C8161的影响 抑制肿瘤转移。伴随着绘制基因图谱的方法 在具体目标1中，将确定有限数量的候选基因。 基于功能(即，已经牵涉到肿瘤进展或 转移)和定位(即，在6号染色体上)，并转染到 C8161。转基因将接受(1)基因表达增加的测试， 以及(2)评价对裸小鼠淋巴转移的抑制作用 与同时注射亲本细胞相比。 尽管这些实验将集中在恶性黑色素瘤上，但结果 可能会提供有关监管的重要线索 多种实体瘤的转移表型。

We recently demonstrated that the replacement of defective genetic information on human chromosome 6 in metastatic human melanoma C8161 cells suppress metastasis without reducing tumorigenicity. Our goals are to identify and map the gene(s) on chromosome 6 responsible for inhibiting metastasis and understand how the gene(s) works. Toward that end, we will systematically narrow the region on chromosome 6 which codes for candidate genes; and, we will extend our observations to additional metastatic human melanoma cell lines. Three Specific Aims are proposed to accomplish these goals: To define the locus of malignant melanoma metastasis-suppressor genes on human chromosome 6. Two experimental approaches will be used. (1) We will use microcell-mediated chromosome transfer to introduce intact short (6p) and long (6q) arms of chromosome 6 into C8161. Hybrids will be evaluated for metastasis in athymia nude or SCID mice. Those hybrids which are suppressed for metastasis will identify the regions of DNA to be introduced in subsequent experiments. Our goal is to transect progressively smaller DNA sequences until a 5 Mb region is identified as coding the gene(s) responsible for suppressing metastasis. (2) We will be isolate metastatic revertants of C8161-chromosome 6 hybrids. Rare metastases which develop following injection of hybrids in mice will be established as cell lines and karyotyped. Loss of any or all of the added chromosome 6 in the metastases-derived cells will be evidence that the regions containing metastasis-suppressor genes. To determine whether adding chromosome 6 suppress metastasis in other human melanoma cell lines. Normal human chromosome 6 will be introduced into the metastasis human cutaneous melanoma cell lines A375M and M24Met. Hybrids will be assessed for metastasis in athymia nude or SCID mice. These lines were chosen because: (1) they are metastatic in experimental systems and (2) they have defects in chromosome 6. To transect individual genes into C8161 and determine their effect on suppressing metastasis. Concomitant with the approach to map the genes in Specific Aim 1, a limited number of candidate genes will be identified based upon function (i.e., already implicated in tumor progression or metastasis) and location (i.e., on chromosome 6), and transfected into C8161. Transfectants will be (1) tested for increased gene expression, and (2) evaluated for suppression of metastasis in athymia nude mice compared to simultaneously injected parental cells. Although these experiments will focus on malignant melanoma, the results are likely to provide important clues regarding the regulation of the metastatic phenotype in many types of solid tumors.