Incomplete expression of the tyrosinase gene family (tyrosinase, TRP-1, and TRP-2) in human malignant melanoma cells in vitro.
Sequence analysis of two clones found repressed in melanoma cell lines in earlier studies showed 9F2 to be identical with the TRP-1 gene and 6F5 with TRP-2 containing a long untranslated 3' end. For further investigation of the expression of the tyrosinase gene family in normal and malignant melanocytic cells, a series of melanoma cell lines and of cultured melanocytes were analyzed by Northern blotting and by reverse transcriptase-polymerase chain reaction (RT-PCR). The Northern blots were probed with cDNA fragments specific for TRP-1, TRP-2, and tyrosinase, for nested tyrosinase-PCR the outer primers specified a 284 bp and the nested primers a 207 bp fragment. Investigations on 14 established melanoma cell lines grown in different media compared with seven normal human melanocyte (NHM) cultures revealed that all three pigment genes were expressed in NHM, whereas pigment gene expression was found repressed in nearly all melanoma cell lines and was completely absent in 4 of 14 specimen. In particular, tyrosinase and TRP-2 genes were found always to be expressed together, and TRP-1 mRNA alone was absent in four melanoma cell lines. Negativity of cultured melanoma cells for tyrosinase mRNA was confirmed by nested RT-PCR, and gene deletion was ruled out by genomic Southern blots. The gene expression seemed independent from the type of medium used for cultivation. These findings indicate repressed or lacking expression of pigment genes in melanoma cell lines, most likely due to regulatory mechanisms, and that differences may exist between tyrosinase and TRP-2 on one hand and TRP-1 on the other. Overall, it seemed that RT-PCR for tyrosinase has limited value for identifying melanoma cells in the peripheral blood of melanoma patients; TRP-1, TRP-2, and other, additional markers may be required.
Eberle
J
J
Department of Dermatology, University Medical Center Benjamin Franklin, Free University of Berlin, Germany.
Garbe
C
C
Wang
N
N
Orfanos
C E
CE
D001483:Base Sequence; D002478:Cells, Cultured; D004247:DNA / Q000032:analysis; D004273:DNA, Neoplasm / Q000032:analysis; D005091:Exons; D005786:Gene Expression Regulation; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D019746:Intramolecular Oxidoreductases; D007535:Isomerases / Q000235:genetics*; D008544:Melanocytes / Q000201:enzymology; D008545:Melanoma / Q000201:enzymology / Q000235:genetics*; D008562:Membrane Glycoproteins; D008969:Molecular Sequence Data; D014442:Monophenol Monooxygenase / Q000235:genetics*; D005810:Multigene Family; D010088:Oxidoreductases; D010858:Pigmentation / Q000235:genetics; D011506:Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D012334:RNA, Neoplasm / Q000032:analysis; D014407:Tumor Cells, Cultured
D004273:DNA, Neoplasm; D008562:Membrane Glycoproteins; D011506:Proteins; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D004247:DNA; D010088:Oxidoreductases; C489799:TYRP1 protein, human; C064056:tyrosinase-related protein-1; D014442:Monophenol Monooxygenase; D007535:Isomerases; D019746:Intramolecular Oxidoreductases; C042501:dopachrome isomerase