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.