Wagner S N, Wagner C, Höfler H, Atkinson M J, Goos M
Klinik und Poliklinik für Dermatologie, Venerologie und Allergologie, Universitätsklinikum, Essen, Germany.
Lab Invest. 1995 Aug;73(2):229-35.
mAb HMB-45 recognizes a melanocyte lineage-associated Ag present in "activated melanocytes" and malignant melanoma cells. Despite its important practical significance in diagnostic pathology and its potential role as an "activation marker" of melanocytic cells, the HMB-45-reactive Ag remained undefined. Molecular characterization of the HMB-45-reactive Ag may help in analyzing underlying mechanisms of melanocyte activation and melanoma tumor progression.
A cDNA library constructed from the HMB-45-immunoreactive human melanoma cell line SK-MEL-28 was screened for expression of the cDNA encoding the HMB-45-reactive protein. Screening was performed by expression in COS-7 cells and subsequent immunocytochemical screening. Correlation between HMB-45 immunoreactivity and mRNA expression of the cloned cDNA was analyzed by antisense mRNA expression in HMB-45-immunoreactive SK-Mel-28 cells, by Northern blot hybridization, and by comparative immunohistochemistry and in situ hybridization.
A cDNA clone encoding the HMB-45-reactive Ag was isolated and shown to be homologous to the Pmel 17 cDNA. Expression of either HMB-45 or Pmel 17 cDNA in COS-7 cells induced cytoplasmic HMB-45 immunoreactivity as described for melanoma cells. Conversely, constitutional HMB-45 immunoreaction in SK-MEL-28 cells could be markedly reduced by expression of antisense Pmel 17 RNA. In several tissues, Pmel 17 mRNA content conformed to the known expression pattern of HMB-45-reactive Ag. Comparative in situ hybridization and immunohistochemistry demonstrated a consistent co-localization of Pmel 17 transcripts and HMB-45-reactive Ag at the cellular level, with strong expression in "activated" melanocytes and melanoma cells and significantly less expression in normal adult melanocytes.
We conclude that Pmel 17 cDNA encodes the HMB-45-reactive Ag. Pmel 17 has been postulated to be involved in melanin synthesis and expression of melanoma-peptide epitopes recognized by CTLs. Differential expression of Pmel 17/HMB-45 may have considerable effects in the stepwise process of melanoma progression by aberrant pigment formation and expression of CTL-reactive epitopes.
单克隆抗体HMB - 45识别存在于“活化黑素细胞”和恶性黑色素瘤细胞中的一种与黑素细胞谱系相关的抗原。尽管其在诊断病理学中具有重要的实际意义，且作为黑素细胞的“活化标志物”具有潜在作用，但HMB - 45反应性抗原仍未明确。对HMB - 45反应性抗原进行分子特征分析可能有助于分析黑素细胞活化和黑色素瘤肿瘤进展的潜在机制。
从HMB - 45免疫反应性人黑色素瘤细胞系SK - MEL - 28构建的cDNA文库中筛选编码HMB - 45反应性蛋白的cDNA表达。通过在COS - 7细胞中表达并随后进行免疫细胞化学筛选来进行筛选。通过在HMB - 45免疫反应性SK - Mel - 28细胞中进行反义mRNA表达、Northern印迹杂交以及比较免疫组织化学和原位杂交，分析HMB - 45免疫反应性与克隆cDNA的mRNA表达之间的相关性。
分离出一个编码HMB - 45反应性抗原的cDNA克隆，显示其与Pmel 17 cDNA同源。如黑色素瘤细胞中所述，在COS - 7细胞中表达HMB - 45或Pmel 17 cDNA均可诱导细胞质HMB - 45免疫反应性。相反，通过表达反义Pmel 17 RNA可显著降低SK - MEL - 28细胞中的固有HMB - 45免疫反应。在几种组织中，Pmel 17 mRNA含量符合HMB - 45反应性抗原的已知表达模式。比较原位杂交和免疫组织化学显示，在细胞水平上Pmel 17转录本与HMB - 45反应性抗原一致共定位，在 “活化” 黑素细胞和黑色素瘤细胞中表达强烈，而在正常成人黑素细胞中表达明显较少。
我们得出结论，Pmel 17 cDNA编码HMB - 45反应性抗原。Pmel 17被认为参与黑色素合成以及被细胞毒性T淋巴细胞识别的黑色素瘤肽表位的表达。Pmel 17/HMB - 45的差异表达可能通过异常色素形成和细胞毒性T淋巴细胞反应性表位的表达，在黑色素瘤进展的逐步过程中产生相当大的影响。
