Human melanocytes and melanomas express novel mRNA isoforms of the tyrosinase-related protein-2/DOPAchrome tautomerase gene: molecular and functional characterization.

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.

Translation of a retained intron in tyrosinase-related protein (TRP) 2 mRNA generates a new cytotoxic T lymphocyte (CTL)-defined and shared human melanoma antigen not expressed in normal cells of the melanocytic lineage.

We 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' 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(+) 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.

Immunohistochemical analysis of the T-cell receptor beta-chain variable regions expressed by T lymphocytes infiltrating primary human melanoma.

The T-cell receptor beta-chain variable (TCRBV) region repertoire expressed by tumor-infiltrating lymphocytes was characterized by immunohistochemical analysis using a panel of 18 monoclonal antibodies on cryosectioned specimens of 14 primary vertical growth phase (VGP) melanomas with a T-cell infiltrate histopathologically defined as brisk or nonbrisk. T lymphocytes present in the VGP of all patients displayed a restricted T-cell receptor usage, with a pattern of reactivity similar in brisk versus nonbrisk infiltrates. No evidence of restriction was found in the extra-VGP lymphocytic infiltrates, when available, within the same specimen. Furthermore, the repertoire of TCRBV expressed in nodal metastases was similar to that of the corresponding primary melanomas in the two cases tested. The results obtained by this in situ analysis indicate that the TCRBV repertoire in VGP is determined by a preferential migration of T lymphocytes, possibly indicative of an immune response to melanoma-associated antigens.

Clonal expansion of T lymphocytes in human melanoma metastases after treatment with a hapten-modified autologous tumor vaccine.

Metastatic melanoma patients treated with an autologous DNP-modified tumor cell vaccine develop inflammatory responses in metastatic tumors characterized by infiltration of CD8+ T cells. To further define this immune response, we analyzed T cell receptor beta-chain variable (TCRBV) region repertoire in biopsy specimens and peripheral blood lymphocytes of six patients. After administration of DNP vaccine, a restricted set of TCRBV gene families was found to be expanded compared with prevaccine metastases. In several postvaccine lesions of one patient, obtained over a 2-yr period, TCRBV14+ T cells were clonally expanded and identical T cell clonotypes could be detected. Two major recurring clones were biased toward the use of TCRBJ1S5. Furthermore, T cell lines derived from two such infiltrated skin lesions and, enriched in TCRBV14+ T cells, displayed HLA-class I-restricted lysis of the autologous melanoma cells. Clonal expansion of T cells was demonstrated in the T cell-infiltrated, postvaccine metastasis of a second patient as well. These results indicate that vaccination with autologous, DNP-modified melanoma cells can expand selected clones of T cells at the tumor site and that such clones are potentially destructive to the tumor.

Interaction with fibronectin regulates cytokine gene expression in human melanoma cells.

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.

Expression of interleukin 1 alpha, interleukin 6, and tumor necrosis factor alpha genes in human melanoma clones is associated with that of mutated N-RAS oncogene.

To assess whether RAS oncogenes may affect the expression of cytokines in tumor cells, the presence of interleukins (IL) 1 alpha, 1 beta, 4, 6, 7, and 8, tumor necrosis factor (TNF) alpha and interferon gamma mRNA has been analyzed by reverse transcriptase-polymerase chain reaction in 19 melanoma clones derived from the metastatic cell line 665/2 and previously characterized for RAS mutation and expression. Five of these clones and the parental cell line showed a mutation at codon 61 of N-RAS that resulted in Gln-->Arg substitution (N-RAS/61+), while in the remaining 14, only the wild-type allele for N-RAS was present (N-RAS/61-). With the exception of interferon gamma and IL-4, all the cytokines tested were expressed by the parental 665/2 cell line, whereas IL-1 alpha, IL-6, and TNF-alpha were coordinately transcribed only in the subset of the clones bearing the mutated N-RAS gene. The other cytokine genes studied (IL-1 beta, IL-4, IL-7, and IL-8) displayed a variable degree of expression, and such an heterogeneity was not correlated to the N-RAS phenotype of the clones. The association between N-RAS oncogene and IL-1 alpha, IL-6, and TNF-alpha expression was also found in a 665/2 subline (665/2/5) in which loss of mutated N-RAS genes simultaneously occurred with the loss of IL-1 alpha, IL-6, and TNF-alpha expression. Direct evidence that N-RAS oncogene could influence the pattern of cytokine expression was provided by the coordinate induction of IL-1 alpha, IL-6, and TNF-alpha messenger RNA achieved in N-RAS/61+ transfectants of the N-RAS wild-type melanoma clone 2/21. Furthermore, IL-1 alpha, IL-6, and TNF-alpha could be detected by enzyme-linked immunosorbent assay in the culture medium obtained from N-RAS/61+ melanoma clones as well as from positive transfectants, indicating that lymphokine mRNA expression triggered by the activated N-RAS oncogene lead to a secreted protein. In an N-RAS/61+ melanoma clone, by adding specific antibodies against each cytokine, it was found that soluble IL-1 alpha exerted a positive control on IL-6 mRNA and a negative one on its own expression. In addition, IL-1 alpha and IL-6 were negatively regulated by soluble IL-6 and TNF-alpha.

N-RAS mutations and susceptibility to lymphokine-activated killer (LAK) cells in human melanoma.

RAS oncogene expression has been reported to affect several biological features of rodent tumours cells, including lysability by activated natural killer cells. In order to examine whether expression of mutated RAS genes in human melanoma cells alters their susceptibility to lysis by LAK cells, seven melanoma lines were assessed for the presence of Ki- and N-RAS genes bearing all possible mutations at codons 12, 13 and 61. A panel of 21 clones deriving from the metastatic lesion Me665/2, which had a Gln-->Arg substitution at codon 61 of N-RAS (N-RAS/61+), were also examined. Melanoma cells and clones were used as targets of allogeneic LAK in a 4-h 51Cr-release assay. LAK showed a higher lysis on melanoma lines and clones harbouring a mutated RAS compared with counterparts bearing no RAS mutations. In addition, LAK-mediated lysis drastically decreased on Me665/2 sublines progressively selected by exposure to LAK. This loss was paralleled by a reduction or even disappearance of N-RAS/61+ mRNA signal in Me665/2 sublines. To evaluate whether N-RAS could directly modulate LAK susceptibility to lysis, N-RAS/61+ gene was transfected in two N-RAS wild type (N-RAS/61-) 665/2 melanoma clones by a cosmid vector. In contrast to the high lysability of melanoma cells constitutively expressing the mutationally active N-RAS oncogene, N-RAS/61+ transfectants did not show a consistent high lysability by LAK, compared with some control pSV2neo transfectants.(ABSTRACT TRUNCATED AT 250 WORDS)

Involvement of somatically acquired ecotropic viruses in the immunogenicity of nude-transplanted NIH/3T3 transformed cell lines.

Immunogenic tumor variants were previously derived after transplantation in vivo into nude mice of NIH/3T3-transformed cell lines. Nude-passaged cell lines were rejected by immunocompetent H-2q NIH mice, were recognized by specific CTL clones, and expressed new retroviral Ag. The aim of the present work was to investigate whether somatically acquired proviral sequences were present in the genome of nude-passaged cells and to test directly for a causative relationship between murine leukemia virus (MuLV) expression and immunogenicity. Southern blot analysis of PstI-digested DNA indicated that in contrast to the parental NIH/3T3 transformed cell lines (pT, T12N/5a, NS-1) all the nude-passaged immunogenic variants (pT-nude, T12N/5a-nude, NS-1-nude) contained newly acquired ecotropic-related proviruses. Immediately after in vitro establishment, these tumors displayed multiple integration sites as assessed by analysis of 3' proviral-cellular junctions. Long term in vitro culture of one of the cell lines (pT-nude) resulted in a cell line (pT-nude/vitro) that was clonal or oligo-clonal with respect to viral integration. Northern blot analysis established that the new proviruses were actively transcribed in all the immunogenic variants. To assess whether the somatically acquired ecotropic proviral sequences encode for target structures recognized by specific CTL, obtained after immunization of NIH mice with pT-nude, the parental cell line pT was transfected with plasmids containing the entire AKV MuLV genome, the cloned AKV gag or env genes. Screening of transfectants for their ability to stimulate the production of TNF by anti-pT-nude effectors indicated that cells transfected with the entire ecotropic virus or with MuLV-env gene products could be recognized by an NIH anti-pT-nude CTL line and NIH anti-pT-nude Kq-restricted CTL clones as well as the immunizing target pT-nude.