SummaryWe report here the identification of a new shared human melanoma antigen recognized by a human leukocyte antigen (HLA)-A*68011-restricted cytotoxic T lymphocyte clone (CTL 128). The cDNA encoding this antigen is composed of a partially spliced form of the melanocyte differentiation antigen tyrosinase-related protein ( TRP )-2 , containing exons 1-4 with retention of intron 2 and part of intron 4 ( TRP-2-INT2 ). The sequence coding for the antigenic epitope is located at the 5 Ј end of intron 2 and is available for translation in the same open reading frame of the fully spliced TRP-2 mRNA. This peptide is also recognized by CTL 128 when presented by the HLA-A*3301, a member of the HLA-A3-like supertype that includes the HLA-A*68011. Quantitative reverse transcription PCR analysis carried out on total and/or cytoplasmic mRNA demonstrated that, in contrast to the fully spliced TRP-2 mRNA expressed in melanomas, normal skin melanocytes, and retina, the TRP-2-INT2 mRNA could be detected at significant levels in melanomas but not in normal cells of the melanocytic lineage. Instead, in these normal samples, both the spliced and the unspliced transcript of gp100 were expressed at high levels. Absence of endogenous TRP-2-INT2 expression in melanocytes was also confirmed by lack of recognition of HLA-A*68011-transduced, TRP-2 ϩ melanocyte lines by CTL 128. These results indicate that a partially spliced form of a differentiation antigen mRNA, present in the cytoplasmic compartment of neoplastic but not normal cells of the melanocytic lineage, can be the source of a melanoma-restricted T cell epitope.

The RanGTPase activating protein RanGAP1 has essential functions in both nucleocytoplasmic transport and mitosis. In interphase, a significant fraction of vertebrate SUMO1-modified RanGAP1 forms a stable complex with the nucleoporin RanBP2/Nup358 at nuclear pore complexes. RanBP2 not only acts in the RanGTPase cycle but also is a SUMO1 E3 ligase. Here, we show that RanGAP1 is phosphorylated on residues T409, S428, and S442. Phosphorylation occurs before nuclear envelope breakdown and is maintained throughout mitosis. Nocodazole arrest leads to quantitative phosphorylation. The M-phase kinase cyclin B/Cdk1 phosphorylates RanGAP1 efficiently in vitro, and T409 phosphorylation correlates with nuclear accumulation of cyclin B1 in vivo. We find that phosphorylated RanGAP1 remains associated with RanBP2/Nup358 and the SUMO E2–conjugating enzyme Ubc9 in mitosis, hence mitotic phosphorylation may have functional consequences for the RanGTPase cycle and/or for RanBP2-dependent sumoylation.

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We previously reported that a melanoma antigen, recognized by tumor-specific cytotoxic T lymphocytes, was encoded by intron sequences retained in a partially spliced transcript of the tyrosinase-related protein-2/DOPAchrome tautomerase gene. At difference with the mRNA encoding tyrosinase-related protein-2, this anomalous transcript was not expressed in melanocytes. This study examined whether neoplastic and/or normal cells of the melanocytic lineage could express additional forms of tyrosinase-related protein-2 mRNA. Screening of a melanoma-derived cDNA library with a tyrosinase-related protein-2 probe allowed identification of two novel isoforms. The first, tyrosinase-related protein-2-long tail, corresponds to the dominant transcript detected on melanomas and melanocytes by northern blot analysis. Tyrosinase-related protein-2-long tail is identical to the tyrosinase-related protein-2-encoding published cDNA sequence except for an extended 3'-untranslated region and is originated by alternative polyadenylation. This novel 3'-untranslated region contains an alternatively spliced, tyrosinase-related protein-2 last exon in the second isoform (tyrosinase-related protein-2-8b). The protein encoded by tyrosinase-related protein-2-8b is identical to tyrosinase-related protein-2 in its first 460 amino acids but possesses a different carboxyl-terminus devoid of transmembrane domain. Tyrosinase-related protein-2-long tail exhibited DOPA-chrome tautomerase activity, when transiently transfected into COS-7 cells. On the contrary, no detectable activity was exhibited by tyrosinase-related protein-2-8b. Reverse transcription-polymerase chain reaction analysis indicated that tyrosinase-related protein-2-long tail and tyrosinase-related protein-2-8b are expressed by tyrosinase-related protein-2-positive melanomas and normal melanocytes. Moreover all cell lines positive for tyrosinase-related protein-2 isoforms expressed tyrosinase and, all but one, tyrosinase-related protein-1. These data show that the human tyrosinase-related protein-2/DOPAchrome tautomerase gene can yield different isoforms by alternative poly(A) site usage or by alternative splicing. The pattern of expression of these isoforms suggest that they might play a part in the normal pathway of melanin biosynthesis.

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Our study was aimed at investigating whether interaction of human melanoma cells with the extracellular matrix (ECM) protein fibronectin (FN) could regulate lymphokine gene expression. Serum-deprived cells (quiescent condition) of a metastatic melanoma cloned line were cultured either on uncoated or on FN- or BSA-coated surfaces. By means of reverse transcriptase- polymerase chain reaction (RT-PCR), we analyzed mRNA expression of 4 cytokines interleukin (IL)-1alpha, IL, 1beta, IL-6 and IL-8-and 9 growth factors-endothelial cell growth factor (ECGF), basic fibroblast growth factor (bFGF), fibroblast growth factor (FGF)-5, HST, keratinocyte growth factor (KGF), transforming growth factor (TGF)-alpha TGF-beta1, TGF-beta2 and TGF-beta3. When cultured on FN, melanoma cells expressed IL-1beta and IL-6 transcripts in addition to IL-1beta, IL-8, ECGF, TGF-beta1, TGF-beta2 and TGF-beta3, already present in quiescent cells. Amplification parameters to achieve semi-quantitative RT-PCR were then determined for each detectable factor, thus allowing us to measure a selective enhancement of mRNA levels for IL-1alpha, IL-6, IL-8 and TGF-beta2 upon interaction with FN by quiescent melanoma cells. This augmented expression was inhibited by an anti-integrin beta1 chain monoclonal antibody (MAb). Moreover, the amounts of IL-6, IL-8 and IL-beta produced in the supernatants, as assessed by ELISA, correlated with the corresponding mRNA expression. Extension of this analysis to the other 5 human primary and metastatic melanoma lines confirmed the ability of FN to selectively up-regulate only IL-6 and IL-8 secretion. Our data indicate that FN is able to modulate expression and secretion of a defined subset of lymphokines in human melanoma.

Human melanomas can express unique tumor antigens, resulting from mutated proteins, and shared epitopes encoded for by normal genes, but these two classes of antigens have not been previously compared for immunogenicity and retention in metastatic cells. Here, we identified a new unique antigen generated by a point mutation in the peroxiredoxin 5 (Prdx5) gene in an HLA-A*0201+ human metastatic melanoma lacking the wild-type allele. An antioxidant assay, with recombinant Prdx5 proteins, and evaluation of peroxide accumulation in transiently transfected cells, indicated that the mutant protein retained its enzymatic activity. The mutation in the Prdx5 protein did not generate a new HLA agretope but yielded an HLA-A*0201–restricted T cell epitope (Prdx5110-119). By HLA-tetramer analysis, in a tumor-invaded lymph node, >50% of mutant Prdx5-specific CD8+ T cells (frequency 0.37%/CD8+) showed a CCR7+/− CD45RA− “TCM” or “TEM” phenotype, as found in Melan-A/MART-1–specific T cells (frequency 0.68%/CD8+) in the same tissue. In agreement with their memory phenotype, the Prdx5-specific T cells readily expanded in vitro in mixed lymphocyte-tumor culture, as did the Melan-/MART-1–specific T cells. By immunohistochemistry of the invaded lymph node, the mutant Prdx5 protein was expressed in all neoplastic cells, in contrast with the heterogeneous expression of shared antigens as Melan-A/MART-1, gp100 and tyrosinase. Thus, a unique tumor antigen can be as immunogenic as the melanoma differentiation antigens but, in contrast to the latter, may be retained in all metastatic cells possibly as result of the relevant cellular function exerted by the mutated protein.