Johnsson A, Zeelenberg I, Min Y, Hilinski J, Berry C, Howell S B, Los G
Departments of Surgery, University of California, San Diego, La Jolla, CA, 92037-0058, USA.
Br J Cancer. 2000 Oct;83(8):1047-54. doi: 10.1054/bjoc.2000.1420.
The goal of this study was to identify genes whose mRNA levels are differentially expressed in human cells with acquired cisplatin (cDDP) resistance. Using the parental UMSCC10b head and neck carcinoma cell line and the 5.9-fold cDDP-resistant subline, UMSCC10b/Pt-S15, two suppressive subtraction hybridization (SSH) cDNA libraries were prepared. One library represented mRNAs whose levels were increased in the cDDP resistant variant (the UP library), the other one represented mRNAs whose levels were decreased in the resistant cells (the DOWN library). Arrays constructed with inserts recovered from these libraries were hybridized with SSH products to identify truly differentially expressed elements. A total of 51 cDNA fragments present in the UP library and 16 in the DOWN library met the criteria established for differential expression. The sequences of 87% of these cDNA fragments were identified in Genbank. Among the mRNAs in the UP library that were frequently isolated and that showed high levels of differential expression were cytochrome oxidase I, ribosomal protein 28S, elongation factor 1alpha, alpha-enolase, stathmin, and HSP70. The approach taken in this study permitted identification of many genes never before linked to the cDDP-resistant phenotype.
本研究的目的是鉴定在获得性顺铂（cDDP）耐药的人类细胞中mRNA水平差异表达的基因。使用亲本UMSCC10b头颈癌细胞系和5.9倍cDDP耐药亚系UMSCC10b/Pt-S15，制备了两个抑制性消减杂交（SSH）cDNA文库。一个文库代表在cDDP耐药变体中水平升高的mRNA（UP文库），另一个代表在耐药细胞中水平降低的mRNA（DOWN文库）。用从这些文库中回收的插入片段构建的阵列与SSH产物杂交，以鉴定真正差异表达的元件。UP文库中共有51个cDNA片段，DOWN文库中有16个cDNA片段符合差异表达的既定标准。这些cDNA片段中87%的序列在Genbank中得到鉴定。在UP文库中经常分离且差异表达水平高的mRNA中，有细胞色素氧化酶I、核糖体蛋白28S、延伸因子1α、α-烯醇化酶、微管相关蛋白和热休克蛋白70。本研究采用的方法能够鉴定许多以前从未与cDDP耐药表型相关联的基因。
