Abstract
The expression of the gp100 antigen is generally thought to be confined to cells of the melanocytic lineage, which makes the protein a suitable melanoma-specific marker. Strikingly, after screening a panel of normal tissues, tumour samples and cell lines of non-melanocytic origin, we found transcripts encoding gp100 in virtually every tissue and cell line tested. In contrast, tyrosinase and MART-1/MelanA transcripts were detected only in cells of the melanocytic lineage. However, no gp100 protein could be detected by either Western blotting or cytotoxicity assays. Therefore, at the protein level, gp100 remains exclusive for cells of melanocytic origin despite its transcription in many cell types. The major implication of this finding is that screening of patient material for gp100 expression should preferrably be performed by antibody staining. Reverse transcriptase polymerase chain reaction (RT-PCR) can be employed, provided that it is performed in a tightly controlled, semiquantitative setting.
Highlights
The renal cell carcinoma cell lines (RCC) LE-9104-RCC, LE921 l-RCC and LE-9415-RCC and the melanoma cell line Mel 603 were established in our laboratory
In this study we show that the melanocyte lineage-specific antigen gplOO can be detected by Reverse transcriptase polymerase chain reaction (RT-PCR) in tumour cell lines originating from tissues other than those of the melanocytic lineage
Using RT-PCR, we show that there is a low level of transcription of gplOO in virtually every cell type
Summary
The renal cell carcinoma cell lines (RCC) LE-9104-RCC, LE921 l-RCC and LE-9415-RCC and the melanoma cell line Mel 603 were established in our laboratory. The RCC cell line SK-RC-7 was kindly provided by Dr E Oosterwijk (Department of Urology, Nijmegen University, The Netherlands). The melanoma cell line Mel 624 and TIL 1200 lymphocytes were kindly provided by Dr Y Kawakami (NCI, NIH, Bethesda, MD, USA). Synthesis, RNA from the cell lines was treated with DNAase I (Gibco BRL, Breda, The Netherlands) for 30 min at 37°C followed by phenol extraction and precipitation. For semi-quantitative analysis 0.2 ,l of [32P]-dCTP was added to the PCR mixture For both gplOO and ,B-actin we observed that amplification was linear at 25 and 21 cycles respectively. The PCR products of ,B-actin (top) or gplOO (lower) were separated on acrylamide gels and quantified by phosphor-imaging. Antibody blocking of cytotoxicity was performed by incubation of the chromiumlabelled target cells with W6/32 ascites in 1:100 dilution at 20°C for 30 min.
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