Mechanisms of the drug resistance of a 2', 2-difluorodeoxycytide (gemcitabine)-resistant variant of the human lung adenocarcinoma cell line

Abstract

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.