Abstract
We employed cDNA microarrays to identify the differentially expressed genes in a cisplatin-sensitive parental (2008) human ovarian carcinoma cell line and its cisplatin-resistant variant (2008/C13*). Differential expression of five genes was found in the 2008/C13* cells, a result confirmed by semi-quantitative reverse transcription-PCR. The five genes were identified as fibroblast muscle-type tropomyosin and skeletal muscle-type tropomyosin, dihydrodiol dehydrogenase, apolipoprotein J and glucose-6-phosphate dehydrogenase variant-A. Treatment of the 2008 cells with cisplatin (at its IC(50) concentration of 2 microm) induced expression of these genes, as determined by semi-quantitative reverse transcription-PCR analysis using gene-specific primers. In contrast, treatment of the drug-resistant 2008/C13* cells with cisplatin (at its IC(50) concentration of 20 microm) did not lead to the induction of any of the aforementioned genes. Most importantly, constitutive overexpression of dihydrodiol dehydrogenase (but not the other genes) in the 2008 cells led to induction of cisplatin resistance, clearly indicating its role in the development of the resistance phenotype in the 2008/C13* cells. The development of cisplatin resistance in the transfected cells was associated with an increase in the dihydrodiol dehydrogenase enzyme activity. Although at present it is not clear how dihydrodiol dehydrogenase is involved in cisplatin resistance, the identification of this gene as a causal factor suggests the existence of a hitherto undefined pathway resulting in cisplatin resistance.
Highlights
We employed cDNA microarrays to identify the differentially expressed genes in a cisplatin-sensitive parental (2008) human ovarian carcinoma cell line and its cisplatin-resistant variant (2008/C13*)
At present it is not clear how dihydrodiol dehydrogenase is involved in cisplatin resistance, the identification of this gene as a causal factor suggests the existence of a hitherto undefined pathway resulting in cisplatin resistance
A gene was considered as being differentially expressed only if its comparative signal demonstrated a greater than 5-fold increase/decrease. In addition to these quality control features, an experiment was considered valid only if the control genes spotted on the microarray and ϳ100 housekeeping genes) gave a composite cyanine-3 to cyanine-5 ratio of 1, i.e. the expression levels of these genes had to be similar in both cell lines, indicating that the amount of input total RNA from the two cell lines used for the differential expression analysis was similar
Summary
Materials—Cisplatin, camphorquinone, and (S)-(ϩ)-1-indanol were purchased from Aldrich. 1-Acetonaphthenol, dicumarol, NADPϩ, and NADPH were obtained from Sigma. Total RNA extracted in parallel from the parental (2008) and the cisplatin-resistant cells (2008/C13*) were reverse-transcribed into cDNA (complementary DNA) labeled with either biotin or dinitrophenyl using avian myeloblastosis virus RT. To assess the efficiency of the reverse transcription reaction, the incorporation of the biotin and dinitrophenyl into cDNA was compared with previously labeled, control cDNAs of known concentration, as described in detail in the protocol provided by the manufacturer of the kit (PerkinElmer Life Sciences). Hybridization was performed with radiolabeled fulllength dihydrodiol dehydrogenase cDNA overnight This was followed by 1ϫ low stringency wash and 2ϫ high stringency washes at 55 °C as described previously [20]. Each batch had a positive and a negative control to ensure the staining quality
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