Eberle J, Garbe C, Wang N, Orfanos C E
Department of Dermatology, University Medical Center Benjamin Franklin, Free University of Berlin, Germany.
Pigment Cell Res. 1995 Dec;8(6):307-13. doi: 10.1111/j.1600-0749.1995.tb00679.x.
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.
在早期研究中发现两个在黑色素瘤细胞系中表达受抑制的克隆，其序列分析表明9F2与TRP - 1基因相同，6F5与含有长3'非翻译区的TRP - 2相同。为了进一步研究酪氨酸酶基因家族在正常和恶性黑素细胞中的表达，通过Northern印迹法和逆转录聚合酶链反应（RT - PCR）分析了一系列黑色素瘤细胞系和培养的黑素细胞。用TRP - 1、TRP - 2和酪氨酸酶特异性的cDNA片段对Northern印迹进行杂交，对于巢式酪氨酸酶PCR，外侧引物扩增出284 bp片段，内侧引物扩增出207 bp片段。对在不同培养基中生长的14个已建立的黑色素瘤细胞系与7个人正常黑素细胞（NHM）培养物进行研究发现，所有三个色素基因在NHM中均有表达，而在几乎所有黑色素瘤细胞系中色素基因表达均受抑制，14个样本中有4个完全不表达。特别是，酪氨酸酶和TRP - 2基因总是一起表达，4个黑色素瘤细胞系中单独缺失TRP - 1 mRNA。巢式RT - PCR证实培养的黑色素瘤细胞酪氨酸酶mRNA呈阴性，基因组Southern印迹排除了基因缺失。基因表达似乎与用于培养的培养基类型无关。这些发现表明黑色素瘤细胞系中色素基因表达受抑制或缺失，很可能是由于调控机制，并且酪氨酸酶和TRP - 2与TRP - 1之间可能存在差异。总体而言，酪氨酸酶的RT - PCR在识别黑色素瘤患者外周血中的黑色素瘤细胞方面价值有限；可能需要TRP - 1、TRP - 2和其他额外的标志物。
