Pak Brian J, Lee Jane, Thai Boun L, Fuchs Serge Y, Shaked Yuval, Ronai Ze'ev, Kerbel Robert S, Ben-David Yaacov
Molecular and Cellular Biology, Sunnybrook and Women's College Health Sciences Centre and Toronto Sunnybrook Regional Cancer Centre, University of Toronto, Toronto, Ontario, Canada.
Oncogene. 2004 Jan 8;23(1):30-8. doi: 10.1038/sj.onc.1207007.
While melanomas are resistant to the cytotoxic effects of radiotherapy, little is known about the molecular mechanisms underlying this intrinsic resistance. Here, we describe the utilization of retroviral insertional mutagenesis to facilitate the analysis of genetic changes that are associated with radioresistance in human melanoma. A radial growth phase human melanoma cell line, WM35, was infected with a replication-defective amphotropic murine retrovirus and subsequently selected for X-ray radiation-resistant variants. Several radiation-resistant clones were independently isolated and characterized. Interestingly, these clones also displayed resistance to ultraviolet radiation and to the chemotherapeutic drug cis-diamminedichloroplatinum(II) (CDDP). By Northern and Western analyses, we showed that the expression of DOPAchrome tautomerase (DCT), also known as tyrosinase-related protein 2 (TYRP2), an enzyme that functions in eumelanin synthesis, was significantly elevated in the radiation-resistant clones relative to the parental WM35 cells. Moreover, the levels of DCT in a variety of human melanoma cell lines correlated with their relative levels of radioresistance and the enforced expression of DCT conferred increased resistance to UV(B) treatment. An analysis of stress signaling induced by radiation as well other cytotoxic stressors showed that resistance associated with DCT overexpression applied specifically to treatments that activate the ERK/MAPK pathway. Indeed, DCT overexpression in a melanoma cell line resulted in increased ERK activity. Moreover, ectopic expression of dominant-active MEK in this melanoma cell line conferred UV(B) resistance suggesting that the ERK/MAPK pathway downstream of DCT may play a critical role in radiation and drug resistance. Overall, given that each gamma- and UV(B)-resistant cell line also exhibited resistance to CDDP and that CDDP-resistant clones showed increased resistance to UV(B) irradiation, these results suggest a common mechanism underlying radio- and chemoresistance, which is mediated by DCT expression.
虽然黑色素瘤对放射治疗的细胞毒性作用具有抗性，但对于这种内在抗性背后的分子机制却知之甚少。在此，我们描述了利用逆转录病毒插入诱变来促进对与人类黑色素瘤放射抗性相关的基因变化的分析。将处于辐射生长阶段的人类黑色素瘤细胞系WM35用复制缺陷型双嗜性鼠逆转录病毒感染，随后选择对X射线辐射具有抗性的变体。独立分离并鉴定了几个抗辐射克隆。有趣的是，这些克隆对紫外线辐射和化疗药物顺二氯二氨铂（II）（CDDP）也具有抗性。通过Northern和Western分析，我们发现多巴色素互变异构酶（DCT），也称为酪氨酸酶相关蛋白2（TYRP2），一种在真黑色素合成中起作用的酶，在抗辐射克隆中的表达相对于亲本WM35细胞显著升高。此外，多种人类黑色素瘤细胞系中DCT的水平与其相对放射抗性水平相关，并且DCT的强制表达赋予了对UV（B）处理的抗性增加。对辐射以及其他细胞毒性应激源诱导的应激信号的分析表明，与DCT过表达相关的抗性特异性地适用于激活ERK/MAPK途径的处理。实际上，黑色素瘤细胞系中DCT的过表达导致ERK活性增加。此外，在该黑色素瘤细胞系中异位表达显性活性MEK赋予了对UV（B）的抗性，表明DCT下游的ERK/MAPK途径可能在辐射和药物抗性中起关键作用。总体而言，鉴于每个抗γ和UV（B）的细胞系也表现出对CDDP的抗性，并且抗CDDP的克隆对UV（B）照射的抗性增加，这些结果表明由DCT表达介导的放射抗性和化学抗性存在共同机制。
