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Global gene expression analysis of early response to chemotherapy treatment in ovarian cancer spheroids.

L'Espérance S, Bachvarova M, Tetu B, Mes-Masson AM, Bachvarov D - BMC Genomics (2008)

Bottom Line: However, the induction of genes linked to mechanisms of DNA replication and repair in cisplatin- and topotecan-treated OC spheroids could be associated with immediate adaptive response to treatment.Finally, multicellular growth conditions that are known to alter gene expression (including cell adhesion and cytoskeleton organization), could substantially contribute in reducing the initial effectiveness of CT drugs in OC spheroids.Results described in this study underscore the potential of the microarray technology for unraveling the complex mechanisms of CT drugs actions in OC spheroids and early cellular response to treatment.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, Laval University, Québec (Québec), Canada. syles@hotmail.com

ABSTRACT

Background: Chemotherapy (CT) resistance in ovarian cancer (OC) is broad and encompasses diverse unrelated drugs, suggesting more than one mechanism of resistance. To better understand the molecular mechanisms controlling the immediate response of OC cells to CT exposure, we have performed gene expression profiling in spheroid cultures derived from six OC cell lines (OVCAR3, SKOV3, TOV-112, TOV-21, OV-90 and TOV-155), following treatment with 10,0 microM cisplatin, 2,5 microM paclitaxel or 5,0 microM topotecan for 72 hours.

Results: Exposure of OC spheroids to these CT drugs resulted in differential expression of genes associated with cell growth and proliferation, cellular assembly and organization, cell death, cell cycle control and cell signaling. Genes, functionally involved in DNA repair, DNA replication and cell cycle arrest were mostly overexpressed, while genes implicated in metabolism (especially lipid metabolism), signal transduction, immune and inflammatory response, transport, transcription regulation and protein biosynthesis, were commonly suppressed following all treatments. Cisplatin and topotecan treatments triggered similar alterations in gene and pathway expression patterns, while paclitaxel action was mainly associated with induction of genes and pathways linked to cellular assembly and organization (including numerous tubulin genes), cell death and protein synthesis. The microarray data were further confirmed by pathway and network analyses.

Conclusion: Most alterations in gene expression were directly related to mechanisms of the cytotoxics actions in OC spheroids. However, the induction of genes linked to mechanisms of DNA replication and repair in cisplatin- and topotecan-treated OC spheroids could be associated with immediate adaptive response to treatment. Similarly, overexpression of different tubulin genes upon exposure to paclitaxel could represent an early compensatory effect to this drug action. Finally, multicellular growth conditions that are known to alter gene expression (including cell adhesion and cytoskeleton organization), could substantially contribute in reducing the initial effectiveness of CT drugs in OC spheroids. Results described in this study underscore the potential of the microarray technology for unraveling the complex mechanisms of CT drugs actions in OC spheroids and early cellular response to treatment.

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A. Example images of compact and aggregate spheroid structures derived from OC cells. B. Hierarchical clustering of OC spheroids following treatment with all used drugs (cisplatin, topotecan and paclitaxel (taxol)), that discriminates between compact spheroids and aggregates. A subset of candidate genes were initially obtained by filtering on signal intensity (2-fold), retaining 527 genes. One-way ANOVA parametric test (Welch t-test, variances not assumed equal, p ≤ 0.03) further selected 85 genes. Clustering analysis based on the 85 gene list was performed using the standard Condition Tree algorithm provided in GeneSpring. The mean appears grey, whereas red signifies up-regulation, and green signifies down-regulation (see legend bar). Compact spheroids are indicated in brown, aggregates are indicated in grey. Each cell line is indicated with different color.
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Figure 7: A. Example images of compact and aggregate spheroid structures derived from OC cells. B. Hierarchical clustering of OC spheroids following treatment with all used drugs (cisplatin, topotecan and paclitaxel (taxol)), that discriminates between compact spheroids and aggregates. A subset of candidate genes were initially obtained by filtering on signal intensity (2-fold), retaining 527 genes. One-way ANOVA parametric test (Welch t-test, variances not assumed equal, p ≤ 0.03) further selected 85 genes. Clustering analysis based on the 85 gene list was performed using the standard Condition Tree algorithm provided in GeneSpring. The mean appears grey, whereas red signifies up-regulation, and green signifies down-regulation (see legend bar). Compact spheroids are indicated in brown, aggregates are indicated in grey. Each cell line is indicated with different color.

Mentions: The six OC cell lines used in this study displayed different morphology when grown as spheroids, forming rather compact spheroids (derived from OV-90, OVCAR-3, SKOV-3), or more loose structures or aggregates (derived from TOV-112, TOV-21, TOV-155; examples for both spheroid structures are shown on Figure 7A). As expected, a higher number of genes displayed differential expression upon CT drugs treatment in the aggregates than in the compact spheroids (data not shown). These structure-associated gene expression differences were further confirmed by cluster analysis. Indeed, supervised clustering based on a selected list of 85 genes revealed formation of two major cluster groups that perfectly distinguish between compact and aggregate structures (Figure 7B). Genes implicated in cell adhesion (CSPG3, ITGAV, MUC1), negative regulation of cell proliferation (GPNMB, MXD4) and metabolism (FGF14, CDC42EP1, IGFBP4, GNA15, ARL7, HRMT1L1, LRDD, OR2A1) were comparatively up-regulated in the compact spheroids, while genes associated with cell proliferation (ERBB4, ADRA1B, BMP6), inflammation (SERPING1, CXCL9) and protein modification (DUSP21, FLJ23356, HSPA1A) were predominantly up-regulated in aggregates [see Additional file 9]. Each cell line displayed a separate gene cluster regardless of the drug used (Figure 7B), while no significant clusters were obtained for each specific drug treatment (data not shown). Interestingly, cell lines displaying quite different responses to cytotoxics when grown as monolayers (for example SKOV-3 and OVCAR-3) now show very similar cluster patterns upon treatment as they cluster adjacent to each other (Figure 7B), indicating that differences in drugs response tend to disappear when these OC cell lines are grown as multicellular spheroids.


Global gene expression analysis of early response to chemotherapy treatment in ovarian cancer spheroids.

L'Espérance S, Bachvarova M, Tetu B, Mes-Masson AM, Bachvarov D - BMC Genomics (2008)

A. Example images of compact and aggregate spheroid structures derived from OC cells. B. Hierarchical clustering of OC spheroids following treatment with all used drugs (cisplatin, topotecan and paclitaxel (taxol)), that discriminates between compact spheroids and aggregates. A subset of candidate genes were initially obtained by filtering on signal intensity (2-fold), retaining 527 genes. One-way ANOVA parametric test (Welch t-test, variances not assumed equal, p ≤ 0.03) further selected 85 genes. Clustering analysis based on the 85 gene list was performed using the standard Condition Tree algorithm provided in GeneSpring. The mean appears grey, whereas red signifies up-regulation, and green signifies down-regulation (see legend bar). Compact spheroids are indicated in brown, aggregates are indicated in grey. Each cell line is indicated with different color.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2279123&req=5

Figure 7: A. Example images of compact and aggregate spheroid structures derived from OC cells. B. Hierarchical clustering of OC spheroids following treatment with all used drugs (cisplatin, topotecan and paclitaxel (taxol)), that discriminates between compact spheroids and aggregates. A subset of candidate genes were initially obtained by filtering on signal intensity (2-fold), retaining 527 genes. One-way ANOVA parametric test (Welch t-test, variances not assumed equal, p ≤ 0.03) further selected 85 genes. Clustering analysis based on the 85 gene list was performed using the standard Condition Tree algorithm provided in GeneSpring. The mean appears grey, whereas red signifies up-regulation, and green signifies down-regulation (see legend bar). Compact spheroids are indicated in brown, aggregates are indicated in grey. Each cell line is indicated with different color.
Mentions: The six OC cell lines used in this study displayed different morphology when grown as spheroids, forming rather compact spheroids (derived from OV-90, OVCAR-3, SKOV-3), or more loose structures or aggregates (derived from TOV-112, TOV-21, TOV-155; examples for both spheroid structures are shown on Figure 7A). As expected, a higher number of genes displayed differential expression upon CT drugs treatment in the aggregates than in the compact spheroids (data not shown). These structure-associated gene expression differences were further confirmed by cluster analysis. Indeed, supervised clustering based on a selected list of 85 genes revealed formation of two major cluster groups that perfectly distinguish between compact and aggregate structures (Figure 7B). Genes implicated in cell adhesion (CSPG3, ITGAV, MUC1), negative regulation of cell proliferation (GPNMB, MXD4) and metabolism (FGF14, CDC42EP1, IGFBP4, GNA15, ARL7, HRMT1L1, LRDD, OR2A1) were comparatively up-regulated in the compact spheroids, while genes associated with cell proliferation (ERBB4, ADRA1B, BMP6), inflammation (SERPING1, CXCL9) and protein modification (DUSP21, FLJ23356, HSPA1A) were predominantly up-regulated in aggregates [see Additional file 9]. Each cell line displayed a separate gene cluster regardless of the drug used (Figure 7B), while no significant clusters were obtained for each specific drug treatment (data not shown). Interestingly, cell lines displaying quite different responses to cytotoxics when grown as monolayers (for example SKOV-3 and OVCAR-3) now show very similar cluster patterns upon treatment as they cluster adjacent to each other (Figure 7B), indicating that differences in drugs response tend to disappear when these OC cell lines are grown as multicellular spheroids.

Bottom Line: However, the induction of genes linked to mechanisms of DNA replication and repair in cisplatin- and topotecan-treated OC spheroids could be associated with immediate adaptive response to treatment.Finally, multicellular growth conditions that are known to alter gene expression (including cell adhesion and cytoskeleton organization), could substantially contribute in reducing the initial effectiveness of CT drugs in OC spheroids.Results described in this study underscore the potential of the microarray technology for unraveling the complex mechanisms of CT drugs actions in OC spheroids and early cellular response to treatment.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, Laval University, Québec (Québec), Canada. syles@hotmail.com

ABSTRACT

Background: Chemotherapy (CT) resistance in ovarian cancer (OC) is broad and encompasses diverse unrelated drugs, suggesting more than one mechanism of resistance. To better understand the molecular mechanisms controlling the immediate response of OC cells to CT exposure, we have performed gene expression profiling in spheroid cultures derived from six OC cell lines (OVCAR3, SKOV3, TOV-112, TOV-21, OV-90 and TOV-155), following treatment with 10,0 microM cisplatin, 2,5 microM paclitaxel or 5,0 microM topotecan for 72 hours.

Results: Exposure of OC spheroids to these CT drugs resulted in differential expression of genes associated with cell growth and proliferation, cellular assembly and organization, cell death, cell cycle control and cell signaling. Genes, functionally involved in DNA repair, DNA replication and cell cycle arrest were mostly overexpressed, while genes implicated in metabolism (especially lipid metabolism), signal transduction, immune and inflammatory response, transport, transcription regulation and protein biosynthesis, were commonly suppressed following all treatments. Cisplatin and topotecan treatments triggered similar alterations in gene and pathway expression patterns, while paclitaxel action was mainly associated with induction of genes and pathways linked to cellular assembly and organization (including numerous tubulin genes), cell death and protein synthesis. The microarray data were further confirmed by pathway and network analyses.

Conclusion: Most alterations in gene expression were directly related to mechanisms of the cytotoxics actions in OC spheroids. However, the induction of genes linked to mechanisms of DNA replication and repair in cisplatin- and topotecan-treated OC spheroids could be associated with immediate adaptive response to treatment. Similarly, overexpression of different tubulin genes upon exposure to paclitaxel could represent an early compensatory effect to this drug action. Finally, multicellular growth conditions that are known to alter gene expression (including cell adhesion and cytoskeleton organization), could substantially contribute in reducing the initial effectiveness of CT drugs in OC spheroids. Results described in this study underscore the potential of the microarray technology for unraveling the complex mechanisms of CT drugs actions in OC spheroids and early cellular response to treatment.

Show MeSH
Related in: MedlinePlus