We propose to study the physiological and chemical regulation of two melanoma associated antigens (MAAs) of biological and possible clinical significance using molecular clones that we plan to isolate based on our interspecific transfer of the genes for the MAA. The MAAs """"""""376.96 and """"""""203.7"""""""", glycoproteins of 100kD molecular weight, are tumor-specific in their cellular display. Each is modulated by the stage of tumor cell malignancy. Both antigens are induced by interferons (IFNs). Melanoma cell 376.976 antigenicity is positively regulated by human and IFNs, while 203.7 antigenicity is induced by human IFN. The MAAs are being considered as possible cancer immunotherapeutic and immunodiagnostic """"""""targets"""""""" due to their tumor specificity and IFN regulation. We used an efficient, unique mouse melanoma recipient cell B78H1 and a specific, convenient red cell immunorosetting assay of viable transfected colonies to isolate primary human melanoma DNA-mediated gene transferents of B78H1 displaying each MAA. We then added DNA from the primary MAA transfectants to B78H1 thus generating secondary (2 degree) transfectants. DNAs of 2 degree transfectants for each gene contain human alu-type repeat fragments (probably MAA gene-associated). We will increase the MAA gene representation in 2 degree B78H1 transfectants by means of a selective co-amplification strategy and will further physically enrich for the genes. We will then construct libraries for the amplified enriched genomic DNAs in cloning vector EMBL 3B, and will isolate MAA gene clones based on nucleic acid hybridization with a human alu-specific probe. We will use the initial MAA gene clones and subclones derived from them to analyze structure and transcriptional expression of endogenous MAA genes in human test cells that do and that do not express the MAA, thus deriving molecular information about operative biological regulatory mechanism. We will use gene transfer and ultimately reverse genetic approaches, to analyze MAA gene linked sequences important for regulated MAA expression. We expect that these studies will yield useful information that will permit the use of the MAA and the antibodies that recognize them in cancer immunodiagnosis and therapy.
| Nowak, J; Cohen, E P; Graf Jr, L H (1991) Cytotoxic activity toward mouse melanoma following immunization of mice with transfected cells expressing a human melanoma-associated antigen. Cancer Immunol Immunother 33:91-6 |
| Rosenberg, C D; Ferrone, S; Hamby, C V et al. (1990) Interspecific DNA-mediated transfer and amplification of a gene specifying a Mr 100,000 human melanoma-associated cell surface glycoprotein. Cancer Res 50:1559-65 |
| Graf Jr, L H; Rosenberg, C D; Mancino, V et al. (1988) Transfer and co-amplification of a gene encoding a 96-kDa immune IFN-inducible human melanoma-associated antigen. Preferential expression by mouse melanoma host cells. J Immunol 141:1054-60 |
