Tissue distribution of a melanoma-associated antigen D-1 immunogenic in patients with melanoma

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Abstract

A human melanoma-associated antigen D-1 was recently identified by screening an expression cDNA library derived from mRNA of cultured melanoma cells with sera of melanoma patients. The aim of this study is to present in vivo expression and precise distribution of D-1 in normal tissues and benign or malignant neoplasms. By in situ hybridization, we found that the D-1 mRNA was exclusively expressed in the cytoplasms of melanoma cells, but not in keratinocytes, fibroblasts and lymphocytes adjacent to melanoma nests. Further immunohistochemical studies revealed that the expression of D-1 antigen was distributed to both the surface and cytoplasm of melanoma cells, indicating that D-1 antigen can be recognized by killer T lymphocytes or antibodies in vivo. No significant mRNA nor peptide of D-1 was detected in basal cell carcinoma, squamous cell carcinoma and other benign tumors such as melanocytic nevi and seborrheic keratosis. We also confirmed that D-1 mRNA and peptide were not expressed in normal organs by dot blot hybridization and western blot analysis, respectively. These results will assess the suitability of recombinant D-1 protein to implement active specific immunotherapy against melanoma.

Introduction

The significant increase in the incidence of malignant melanoma in recent years, and the lack of effective therapy for the metastasized melanoma [1], have stimulated interest in developing alternative therapeutic approaches besides anti-cancer chemicals. Malignant melanoma has been known to be an immunogenic tumor on the basis of clinical observations such as spontaneous regression and development of melanoma-associated vitiligo. Many investigators have therefore taken interest in immunotherapy and conducted various trials 3, 4, 5, 6. Active specific immunotherapy is an attractive option, since (a) it prevents melanoma growth in animals [2], (b) cellular and humoral immune responses to the antigens expressed by human melanoma cells have a beneficial effect on the clinical course of the disease 3, 4, 5, 6, and (c) active specific immunotherapy is not associated with major side effects.

To develop useful immunogens for active specific immunotherapy in patients with melanoma, we have screened an expression cDNA library in lambda gt11 constructed from a cultured human melanoma cell line using sera from patients with melanoma. The identified clone, D-1 [7], has 1029 bp and does not have significant homology of nucleic and amino acid sequences with viral and mammalian gene stored in Genetyx (Genetyx, Software Development Co., Tokyo). cDNA D-1 hybridizes to a 2.0 kb mRNA extracted from three different human melanoma cell lines, neuroblastoma, erythroleukemia, B and T lymphoid cell lines, but not to those from renal cell carcinoma, normal fibroblasts or peripheral lymphocytes. D-1 antigen is an approximately 50 kDa molecule which is expressed on the surface of melanoma cells and differs from various melanoma-associated antigens [7]. In this study, we analysed the substantial expression and localization of this novel cDNA clone in vivo.

Section snippets

Tissue preparation

Fresh biopsy specimens from patients with skin tumors (16 cases: malignant melanoma; six cases: squamous cell carcinoma; six cases: basal cell epithelioma; five cases: seborrheic keratosis; eight cases: nevus cell nevus) and from organs of normal adult were fixed in freshly prepared 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) at 4°C overnight. Dehydration was done at 4°C in ascending concentrations of ethanol. Tissues were treated with 100% chloroform and were embedded in paraffin.

Preparation of RNA probes

In situ hybridization of D-1 mRNA

Digoxigenin-labeled single-strand RNA probes were utilized for hybridization. The results from the in situ hybridization analysis are summarized in Table 1. In all of 11 primary lesions and five metastatic lesions of melanoma samples, D-1 mRNA was detected in melanoma cells. Representative pictures are shown in Fig. 1. D-1 mRNA was clearly detected in all of melanoma specimens, including primary (Fig. 1A) and metastatic lesions (Fig. 1C). In the specimens of both primary lesions and metastatic

Discussion

In order to identify human melanoma antigens that are immunogenic in patients, an expression cDNA library of cultured human melanoma cells A375 in lambda gt11 was screened with sera from patients with melanoma. The cloned cDNA (1029 bp), referred to as D-1 [7], was found to be a new melanoma-associated antigen, which encodes a 34 kDa peptide, without significant homology to viral or mammalian amino acid sequences previously reported. The highly hydrophilic profile of D-1 protein was suggested

Acknowledgements

This paper is supported in part by Grants-in Aid for Scientific Research (B04454287, C05807014 and B07457050) from the Ministry of Education, Science and Culture, Japan.

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