The basic FGF-saporin mitotoxin (bFGF-SAP and a recombinant fusion protein, fp-bFGF-SAP) is a hybrid cytotoxic protein composed of basic FGF covalently coupled to a ribosomal toxin which offers a novel approach for the treatment of melanoma. The applicant has demonstrated the bFGF-SAP and fp-bFGF-SAP provide a novel chemotherapeutic modality for melanoma and for a number of other human tumors, growing both in vitro and as xenografts in nude mice. However, tumors eventually recur in xenografted mice, and importantly, 10-40 percent appear resistant to further therapy with bFGF-SAP. As bFGF-SAP moves closer to clinical trials, it becomes increasingly important to understand resistance and to devise strategies for combating resistance. Such strategies might be widely applicable to other toxin-conjugates as well. To that end, he has recently isolated a clone, FSB4, of SK-MEL-5 human melanoma cells that is resistant to bFGF-SAP in vitro. Resistance is due to autocrine FGF down-regulating high affinity receptors and thereby reducing binding of bFGF-SAP. In vivo, FSB4 nude mouse xenografts maintain resistance to bFGF-SAP and have served as a model for his initial investigation of therapeutic modalities to circumvent resistance in which animals are treated first with agents which neutralize autocrine growth factors and then with mitotoxins. To most effectively block the emergence of resistance or to circumvent existing resistance, it is essential to determine the molecular and cellular events responsible for increased autocrine activity and to determine if this mode of resistance can be generalized to other SK-MEL-5 resistant clones and to resistant subclones of other melanomas. The studies proposed here will: 1. subclone cell lines from the existing fp-bFGF-SAP resistant SK-MEL 5 tumors; 2. characterize the biochemical and biological mechanisms responsible for resistance in FSB4, FSB7, and the other resistant subclones of SK-MEL-5 derived in Aim 1, above; 3. explore in vitro and in vivo therapeutic modalities designed to prevent or circumvent fp-bFGF-SAP resistance; 4. determine whether findings with SK-MEL-5 in Specific Aims 1-3 can be generalized to other melanoma lines. A long term goal of this work, beyond the scope of funding sought here, will be to generalize findings to bFGF-SAP mitotoxin therapy of prostatic, ovarian, bladder, and breast carcinomas.
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