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The author s declare that they have no competing interests. SRA generated the resistant cell lines, assisted in performing the microarray experiments, and performed the data subset analysis and selection of the validation genes. RN performed and analyzed the qPCR validation experiments for the selected genes. BG carried out the microarray experiments and the initial data analysis to identify genes with significantly different expression in the resistant cell lines.
AP contributed to the conception of the study, reviewed the manuscript and oversaw the microarray experiments. KLM performed the proliferation analysis of the cell lines and analyzed the data. SC carried out the immunoblots and generated the graphs. CL contributed to the conception of the study, oversaw the selection of the cell lines, oversaw the proliferation analysis, contributed to the final data analysis and drafted the manuscript. All authors read and approved the final manuscript.
Table S2. Determination of significant difference between the parent and resistant cell lines according to QPCR analysis. Figure S1. Comparison of gene expression changes between the resistant cell lines. The log fold change in gene expression is shown for each resistant cell line.
We thank the Genetics Laboratory at the Sudbury Regional Hospital for the use of the microarray scanner. National Center for Biotechnology Information , U. Journal List J Ovarian Res v. J Ovarian Res. Published online Nov Stephen R Armstrong 1 Dept. Rashmi Narendrula 1 Dept.
Amadeo M Parissenti 1 Dept. Author information Article notes Copyright and License information Disclaimer. Corresponding author. Stephen R Armstrong: ac. Received Jul 30; Accepted Oct This article has been cited by other articles in PMC. Primer sequences and melting temperatures. Additional file 2 Table S2.
Additional file 3 Figure S1. Abstract Background Current protocols for the treatment of ovarian cancer include combination chemotherapy with a platinating agent and a taxane. Methods The A epithelial endometrioid ovarian cancer cell line was used to select for isogenic carboplatin, docetaxel and dual drug resistant cell lines. Results Three isogenic cell lines were developed and resistance to each drug or the combination of drugs was confirmed.
Conclusions Ovarian tumor cells can acquire resistance to both carboplatin and docetaxel when selected in the presence of both agents. Determination of IC 50 The number of colonies growing was recorded by taking photomicrographs of five random fields X magnification per drug concentration and counting the colonies in each field.
Cell line selection Cell line selection was performed as described in a previous study by Guo et al. Cell line growth rate analysis Cells were plated at a density of 2. Statistical analysis of changes in gene expression For the microarray data, significant differences in fold change expression between co-cultured controls and resistant lines was determined using the Partek Genomics Suite program Partek Inc.
Table 1 Resistance values determined by clonogenic assay expressed as IC 50 for each cell line and drug s. Open in a separate window. Figure 1. Figure 2. Figure 3. Figure 4. Figure 5. Figure 6. Figure 7. Selection of validation gene sets Unique gene signals based on the microarray results were selected from each of the three resistant cell lines for validation by QPCR and immunoblotting.
Table 2 Validation gene set , genes with significant changes in one or more of the resistant cell lines according to microarray analysis. Table 3 Validation gene set , genes present in all three cell lines according to microarray analysis.
Significant differences confirmed between parent and resistant cell lines Significant differences in transcript levels from the microarray data were confirmed by QPCR for all transcripts in at least one cell line except the GSTO1 transcript, which was not found to be significantly different from the parent in any of the cell lines, despite the microarray results Additional file 2 : Table S2. The dual resistant line contains specific differences in gene expression To examine if the expression level of the selected genes was significantly different between the resistant cell lines, one-way ANOVA was performed on the log of the fold change as determined by QPCR.
Changes in protein expression determined by immunoblotting Further confirmation of the changes in gene expression, at the protein level, was attempted by immunoblotting. Figure 8. Figure 9. Discussion Selection of resistant cell lines In this study, a set of three isogenic drug-resistant ovarian cancer cell lines has been generated from the A ovarian cancer cell line.
Characterization of levels of resistance During the selection for single or dual drug resistance in our study, the gradual increase in drug concentration, beginning with a dose fold below the IC 50 of the parental A cell line, generated populations of resistant cells and avoided selection of a few drug resistant clones.
Immunoblot confirmation of changes in protein expression Immunoblots were performed to determine if changes in gene expression at the transcript level could be confirmed at the protein expression level. Conclusions In this study, we report the establishment of a novel cell line with documented resistance to both carboplatin and docetaxel.
Competing interests The authors of this paper have no competing interests to declare. Supplementary Material Additional file 1: Table S1. Click here for file 47K, doc. Additional file 2: Table S2. Click here for file 44K, doc.
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