METASTASIS SUPPRESSOR GENES IN HUMAN CUTANEOUS MELANOMA

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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.