title: Expression cloning of the cDNA encoding a melanoma-associated Ag recognized by mAb HMB-45. Identification as melanocyte-specific Pmel 17 cDNA.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Klinik und Poliklinik für Dermatologie, Venerologie und Allergologie, Universitätsklinikum, Essen, Germany.
authors.forename: S N
authors.initials: SN
authors.lastname: Wagner
authors.forename: C
authors.initials: C
authors.lastname: Wagner
authors.forename: H
authors.initials: H
authors.lastname: Höfler
authors.forename: M J
authors.initials: MJ
authors.lastname: Atkinson
authors.forename: M
authors.initials: M
authors.lastname: Goos
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D000951:Antigens, Neoplasm / Q000235:genetics / Q000276:immunology*; D015152:Blotting, Northern; D003001:Cloning, Molecular / Q000379:methods; D018076:DNA, Complementary / Q000032:analysis; D004273:DNA, Neoplasm / Q000032:analysis* / Q000235:genetics; D006801:Humans; D008545:Melanoma / Q000235:genetics / Q000276:immunology*; D008562:Membrane Glycoproteins; D009363:Neoplasm Proteins / Q000032:analysis / Q000235:genetics / Q000276:immunology; D011506:Proteins / Q000032:analysis* / Q000235:genetics* / Q000276:immunology; D017422:Sequence Analysis, DNA; D012878:Skin Neoplasms / Q000276:immunology; D014407:Tumor Cells, Cultured; D058951:gp100 Melanoma Antigen
chemical_list: D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D018076:DNA, Complementary; D004273:DNA, Neoplasm; D008562:Membrane Glycoproteins; D009363:Neoplasm Proteins; C548637:PMEL protein, human; D011506:Proteins; D058951:gp100 Melanoma Antigen