Incomplete expression of the tyrosinase gene family (Tyrosinase, TRP-1, and TRP-2) in human malignant melanoma cells in vitro
Incomplete expression of the tyrosinase gene family (Tyrosinase, TRP-1, and TRP-2) in human malignant melanoma cells in vitro
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DOI：
10.1111/j.1600-0749.1995.tb00679.x
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发表时间：
1995-12-01
期刊：
影响因子：
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通讯作者：
Orfanos, CE
中科院分区：
文献类型：
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作者：
Eberle, J;Garbe, C;Orfanos, CE
早期研究中发现黑色素瘤细胞系中受到抑制的两个克隆的序列分析表明，9F2 与 TRP-1 基因相同，6F5 与含有长非翻译 3' 末端的 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印迹和逆转录酶聚合酶链反应（RT-PCR）分析了一系列黑素瘤细胞系和培养的黑素细胞。用特异于 TRP-1、TRP-2 和酪氨酸酶的 cDNA 片段探测 Northern 印迹，对于巢式酪氨酸酶 PCR，外部引物指定 284 bp 片段，嵌套引物指定 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 个正常人黑色素细胞 (NHM) 培养物进行比较后发现，所有三种色素基因均在 NHM 中表达，而色素基因表达在几乎所有黑色素瘤细胞系中均受到抑制，并且在 14 个样本中的 4 个样本中完全不存在。特别是，酪氨酸酶和 TRP-2 基因被发现总是一起表达，并且在四种黑色素瘤细胞系中单独缺乏 TRP-1 mRNA。通过巢式 RT-PCR 证实培养的黑色素瘤细胞的酪氨酸酶 mRNA 呈阴性，并通过基因组 Southern 印迹排除基因缺失。基因表达似乎与培养所用培养基的类型无关。这些发现表明黑色素瘤细胞系中色素基因的表达受到抑制或缺乏，很可能是由于调节机制所致，并且一方面酪氨酸酶和 TRP-X 之间可能存在差异，另一方面 TRP-1 之间可能存在差异。总体而言，酪氨酸酶 RT-PCR 对于识别黑色素瘤患者外周血中的黑色素瘤细胞的价值有限；可能需要 TRP-1、TRP-2 和其他附加标记。
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-X 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.