Dong Mei, Feng Feng-yi, Lin Chen, Zhang Xue-yan, Fu Ming, Liang Xiao, Zha Yuan-yuan, Lu Hai-yan, Wu Min
Department of Medical Oncology, Cancer Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100021, China.
Zhonghua Yi Xue Za Zhi. 2004 Feb 17;84(4):323-8.
To investigate the mechanisms of the drug resistance of gemcitabine resistance variant of the human lung adenocarcinoma cell line A549-Gem.
Immunohistochemistry and RT-PCR were used to tested the expression of P-glycoprotein and transcription of mRNA of multidrug resistance gene and deoxycytidine kinase. Using a cDNA microarray compared the expression profiles between the resistant A549-Gem and the parent cell line A549.
The A549-Gem shown slight positive expression of P-glycoprotein compared with positive control by immunohistochemistry, but A549 was negative for P-gp. RT-PCR amplified mRNA, using specific dCK and multidrug resistant gene 1 (mdr1) primers, demonstrated that A549-Gem express amplicon of mdr1 but no products of mdr1 was detected in A549. In the parent cell line, dCK mRNA amplication product could be detected, but did not found in resistant cells. About 18.8% of the total DNA elements had substantially altered level of expression in resistant A549-Gem compared with A549 by cDNA microarray. The biochemical functions of the different expressed genes are diverse and include oncogene and tumor suppressor gene, cell cycle regulator, heat shock protein, apoptotic and antiapoptotic factors, DNA transcription factors, DNA repair and recombination factors, signal transduction genes, protein translation genes, as well as a large number of metabolic genes. Several cluster genes overexpressed in resistant cell line, including ubiquitin-proteasome system, zinc finger protein, glutaredoxin and heat shock protein, may be related to the mechanisms of gemcitabine resistance.
The mechanisms of resistance to gemcitabine are multifactorial, may include multidrug resistance and dCK deficiency. Differential expression genes monitored by cDNA microarray between A549-Gem and A549 may be related to the mechanisms of gemcitabine resistance in human lung cancer and potentially suggest the prevalence of expression of these genes in drug resistance. The use of cDNA microarray has provided a global view of the response of lung cancer cells to gemcitabine at the genomic level, it should be suitable for examining the development of drug resistance in cancer.
研究人肺腺癌细胞系A549-Gem吉西他滨耐药变异体的耐药机制。
采用免疫组织化学和逆转录-聚合酶链反应（RT-PCR）检测P-糖蛋白的表达以及多药耐药基因和脱氧胞苷激酶mRNA的转录情况。利用cDNA微阵列比较耐药的A549-Gem与亲本细胞系A549之间的表达谱。
免疫组织化学结果显示，与阳性对照相比，A549-Gem的P-糖蛋白呈轻度阳性表达，而A549的P-糖蛋白为阴性。使用特异性脱氧胞苷激酶（dCK）和多药耐药基因1（mdr1）引物进行RT-PCR扩增mRNA，结果表明A549-Gem表达mdr1扩增子，而在A549中未检测到mdr1产物。在亲本细胞系中可检测到dCK mRNA扩增产物，但在耐药细胞中未发现。与A549相比，cDNA微阵列显示耐药的A549-Gem中约18.8%的总DNA元件表达水平发生了显著改变。差异表达基因的生化功能多样，包括癌基因和抑癌基因、细胞周期调节因子、热休克蛋白、凋亡和抗凋亡因子、DNA转录因子、DNA修复和重组因子、信号转导基因、蛋白质翻译基因以及大量代谢基因。耐药细胞系中几个簇基因的过表达，包括泛素-蛋白酶体系统、锌指蛋白、谷氧还蛋白和热休克蛋白，可能与吉西他滨耐药机制有关。
吉西他滨耐药机制是多因素的，可能包括多药耐药和dCK缺乏。cDNA微阵列监测的A549-Gem与A549之间的差异表达基因可能与人肺癌吉西他滨耐药机制有关，并可能提示这些基因在耐药中的表达普遍性。cDNA微阵列的使用在基因组水平上提供了肺癌细胞对吉西他滨反应的全局视图，适用于研究癌症耐药性的发展。
