melphalan and Mast-Cell-Sarcoma

We have previously shown that exposure of P815 tumor cells to melphalan (L-phenylalanine mustard; L-PAM) leads to up-regulation of B7-1 surface expression, and this L-PAM-induced up-regulation requires de novo RNA synthesis and is associated with accumulation of B7-1 mRNA. Here we show that the effect of L-PAM on B7-1 surface expression can be mimicked by exposing P815 tumor cells to oxidative stress but not to heat shock. Moreover, the antioxidant N-acetyl-L-cysteine prevented the L-PAM-induced accumulation of B7-1 mRNA in P815 tumor cells, suggesting that reactive oxygen species are involved in the transcriptional regulation of L-PAM-induced B7-1 gene expression. Although AP-1 and NF-kappaB are regarded as redox-sensitive transcription factors and the promoter/enhancer region of the B7-1 gene contains an AP-1 and an NF-kappaB binding site, exposure of P815 tumor cells to L-PAM led to rapid and transient activation only of NF-kappaB, but not AP-1, that bound specifically to a probe containing the respective binding site in the murine or human B7-1 gene. Moreover, exposure of P815 tumor cells to a cell-permeable peptide that selectively inhibits NF-kappaB activation by blocking the activation of the IkappaB-kinase complex was found to inhibit the L-PAM-induced B7-1 mRNA accumulation, indicating that NF-kappaB activation is essential for the L-PAM-induced B7-1 gene expression. Taken together, these results indicate that L-PAM leads to activation of B7-1 gene expression by activating NF-kappaB via a pathway that involves reactive oxygen species.

Topics: Acetylcysteine; Amino Acid Sequence; Animals; Antigens, CD; Antineoplastic Agents, Alkylating; Antioxidants; B7-1 Antigen; B7-2 Antigen; Binding, Competitive; Cell Membrane; Cell Membrane Permeability; Cell Nucleus; Enhancer Elements, Genetic; Gene Expression Regulation, Neoplastic; Hot Temperature; Humans; Hydrogen Peroxide; I-kappa B Kinase; Macromolecular Substances; Mast-Cell Sarcoma; Melphalan; Membrane Glycoproteins; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Multigene Family; NF-kappa B; Oligonucleotide Probes; Peptides; Promoter Regions, Genetic; Protein Binding; Protein Serine-Threonine Kinases; RNA, Messenger; Transcription Factor AP-1; Tumor Cells, Cultured; Up-Regulation

In this study, we show that administration of low-dose melphalan (l -PAM, l -phenylalanine mustard) to mice bearing a large MOPC-315 plasmacytoma led to a rapid up-regulation of B7-1 (CD80), but not B7-2 (CD86), expression on the surface of MOPC-315 tumor cells. This l -PAM-induced preferential up-regulation of B7-1 surface expression was due, at least in part, to a direct effect of l -PAM on the tumor cells, as in vitro exposure of MOPC-315 tumor cells to l -PAM led to the preferential up-regulation of B7-1 surface expression. Moreover, in vitro exposure of MOPC-315 tumor cells to two other anticancer modalities, gamma-irradiation and mitomycin C, resulted in the preferential up-regulation of B7-1 surface expression. This effect was not restricted to MOPC-315 tumor cells, as preferential up-regulation of B7-1 surface expression was observed also following in vitro exposure of the P815 mastocytoma (that is negative for both B7-1 and B7-2 surface expression) to any of the three anticancer modalities. The up-regulation of B7-1 surface expression following in vitro exposure of tumor cells to l -PAM, gamma-irradiation, or mitomycin C required de novo protein and RNA synthesis, and was associated with the accumulation of mRNA for B7-1 within 4-8 h, indicating that the regulation of B7-1 expression is at the RNA transcriptional level. These results have important implications for an additional immune-potentiating mechanism of these anticancer modalities in clinical setting.

Topics: Animals; Antibiotics, Antineoplastic; Antigens, CD; Antineoplastic Agents, Alkylating; B7-1 Antigen; B7-2 Antigen; Cell Membrane; Drug Administration Schedule; Gamma Rays; Gene Expression Regulation, Neoplastic; Injections, Intraperitoneal; Mast-Cell Sarcoma; Melphalan; Membrane Glycoproteins; Mice; Mice, Inbred BALB C; Mitomycin; Neoplasm Transplantation; Plasmacytoma; Protein Biosynthesis; Proteins; RNA; Tumor Cells, Cultured; Up-Regulation