T Miki

By cDNA expression cloning we have isolated a gene encoding a shared human melanoma antigen recognized by HLA-A2 restricted autologous and allogenic tumor-infiltrating lymphocytes (TILs) from patients with metastatic melanoma. By using both transient and stable expression systems, transfection of this gene into non-antigen-expressing HLA-A2+ cell lines resulted in recognition by the antigen-specific TILs. The sequence of this cDNA revealed a previously undescribed putative transmembrane protein whose expression was restricted to melanoma and melanocyte cell lines and human retina but no other fresh or cultured normal tissues tested or other tumor histologies. Thus, we have identified a gene encoding a melanocyte lineage-specific protein (MART-1; melanoma antigen recognized by T cells 1) that is a widely shared melanoma antigen recognized by the T lymphocytes of patients with established malignancy. Identification of this gene opens possibilities for the development of immunotherapies for patients with melanoma.

The cultured T-cell line TIL1200, established from the tumor-infiltrating lymphocytes (TILs) of a patient with advanced metastatic melanoma, recognized an antigen on most HLA-A2+ melanomas and on all HLA-A2+ cultured neonatal melanocytes in an HLA-A2 restricted manner but not on other types of tissues or cell lines tested. A cDNA encoding an antigen recognized by TIL1200 was isolated by screening an HLA-A2+ breast cancer cell line transfected with an expression cDNA library prepared from an HLA-A2+ melanoma cell line. The nucleotide and amino acid sequences of this cDNA were almost identical to the genes encoding glycoprotein gp100 or Pmel17 previously registered in the GenBank. Expression of this gene was restricted to melanoma and melanocyte cell lines and retina but was not expressed on other fresh or cultured normal tissues or other types of tumor tested. The cell line transfected with this cDNA also expressed antigen recognized by the melanoma-specific antibody HMB45 that bound to gp100. A synthetic 10-amino acid peptide derived from gp100 was recognized by TIL1200 in the context of HLA-A2.1. Since the administration of TIL1200 plus interleukin 2 resulted in regression of metastatic cancer in the autologous patient, gp100 is a possible tumor rejection antigen and may be useful for the development of immunotherapies for patients with melanoma.

A heparin-binding mitogen was isolated from conditioned medium of human embryonic lung fibroblasts. It exhibited broad target-cell specificity whose pattern was distinct from that of any known growth factor. It rapidly stimulated tyrosine phosphorylation of a 145-kDa protein in responsive cells, suggesting that its signaling pathways involved activation of a tyrosine kinase. Purification identified a major polypeptide with an apparent molecular mass of 87 kDa under reducing conditions. Partial amino acid sequence analysis and cDNA cloning revealed that it was a variant of hepatocyte growth factor, a mitogen thought to be specific for hepatic cells and structurally related to plasminogen. Recombinant expression of the cDNA in COS-1 cells established that it encoded the purified growth factor. Its site of synthesis and spectrum of targets imply that this growth factor may play an important role as a paracrine mediator of the proliferation of melanocytes and endothelial cells, as well as cells of epithelial origin.

Multidrug resistance is a major obstacle to cancer treatment. Using an expression cDNA library transfer approach to elucidating the molecular basis of non-P-glycoprotein-mediated multidrug resistance, we previously established that expression of multidrug resistance protein (MRP), an ATP-binding cassette superfamily transporter, confers multidrug resistance (G. D. Kruh et al., Cancer Res., 54: 1649-1652, 1994). In the present study, we generated NIH/3T3 MRP transfectants without using chemotherapeutic drugs to facilitate the pharmacological analysis of the MRP phenotype. MRP transfectants displayed increased resistance to several lipophilic drugs, including doxorubicin, daunorubicin, etoposide, actinomycin D, vincristine, and vinblastine. However, increased resistance was not observed for Taxol, a drug for which transfection of MDR1 confers high levels of resistance. Verapamil increased the sensitivity of MRP transfectants relative to control transfectants, but reversal was incomplete for doxorubicin and etoposide, the drugs for which MRP conferred the highest resistance levels. For the latter two drugs, MRP transfectants, which were approximately 8- and approximately 10-fold more sensitive than control cells in the absence of verapamil, exhibited 3.8- and 3.3-fold relative sensitization with 10 microM verapamil, respectively, but remained approximately 2 and approximately 3-fold more resistant than control cells. Analysis of drug kinetics using radiolabeled daunorubicin revealed decreased accumulation and increased efflux in MRP transfectants. Confocal microscopic analysis of intracellular daunorubicin in MRP transfectants was consistent with reduced intracellular drug concentrations, and also revealed an altered pattern of intracellular drug distribution characterized by the initial accumulation of drug in a perinuclear location, followed by the development of a punctate pattern of drug scattered throughout the cytoplasm. This pattern was suggestive of a process of drug sequestration, possibly followed by vesicle transport. Both increased drug efflux and perinuclear drug accumulation are consistent with the reported localization of MRP in plasma and cytosolic membranes (N. Krishnamachary and M. S. Center, Cancer Res., 53: 3658-3663, 1993; M. J. Flens et al., Cancer Res., 54: 4557-4563, 1994). These results thus indicate that the drug specificity of MRP is quite similar to that of MDR1, but also suggest potential differences in Taxol specificity and the level of verapamil sensitivity. In addition, these results indicate that MRP functions to extrude drug from the cell, but additionally suggest the intriguing possibility that drug sequestration contributes to drug resistance by protecting cellular targets and/or contributing to drug efflux.