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Genome-wide modulation of gene transcription in ovarian carcinoma cells by a new mithramycin analogue.

Vizcaíno C, Núñez LE, Morís F, Portugal J - PLoS ONE (2014)

Bottom Line: Nanomolar concentrations of DIG-MSK abrogated the expression of genes involved in a variety of cell processes including transcription regulation and tumor development, which resulted in cell death.The effect of DIG-MSK in the control of gene expression by other transcription factors was also explored.DIG-MSK affected several biological processes and molecular functions related to transcription and its cellular regulation in A2780 cells, including transcription factor activity.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Biología Molecular de Barcelona, Consejo Superior de Investigaciones Cientificas, Barcelona, Spain.

ABSTRACT
Ovarian cancer has a poor prognosis due to intrinsic or acquired resistance to some cytotoxic drugs, raising the interest in new DNA-binding agents such as mithramycin analogues as potential chemotherapeutic agents in gynecological cancer. Using a genome-wide approach, we have analyzed gene expression in A2780 human ovarian carcinoma cells treated with the novel mithramycin analogue DIG-MSK (demycarosyl-3D-β-D-digitoxosyl-mithramycin SK) that binds to C+G-rich DNA sequences. Nanomolar concentrations of DIG-MSK abrogated the expression of genes involved in a variety of cell processes including transcription regulation and tumor development, which resulted in cell death. Some of those genes have been associated with cell proliferation and poor prognosis in ovarian cancer. Sp1 transcription factor regulated most of the genes that were down-regulated by the drug, as well as the up-regulation of other genes mainly involved in response to cell stress. The effect of DIG-MSK in the control of gene expression by other transcription factors was also explored. Some of them, such as CREB, E2F and EGR1, also recognize C/G-rich regions in gene promoters, which encompass potential DIG-MSK binding sites. DIG-MSK affected several biological processes and molecular functions related to transcription and its cellular regulation in A2780 cells, including transcription factor activity. This new compound might be a promising drug for the treatment of ovarian cancer.

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Effects of the treatment of A2780 cells with 8 nM or 80 nM DIG-MSK on a panel of genes characterized as being related to “ovarian neoplasm” and containing at least one putative Sp1-binding site in the proximal promoter region.(A) Hierarchical clustering of changes in gene expression for each treatment. Dendrograms show average-linkage hierarchical clustering based in Pearson correlation coefficients. For the sake of comparison, lowercase letters at the right side indicate “clusters” with shared characteristics that are detailed in Results. (B) Network generated by the Genomatix Pathway System (GePS) representing bibliographic relationships for Sp1 co-expressed gene profiles (p = 1.48E-03). Dashed lines indicate genes associated by co-citation, while continuous lines indicate genes associated by expert-curation. Filled diamonds and triangles indicate the promoter of gene “B” (the gene with the diamond/triangle) has a corresponding experimentally validated binding site for the transcription factor encoded by gene “A”. Open triangles indicate that the binding of a particular transcription factor to the gene promoter has not been described unambiguously. The sequence logo for the consensus Sp1 binding site, retrieved from JASPAR, is shown at the top right of panel B.
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pone-0104687-g004: Effects of the treatment of A2780 cells with 8 nM or 80 nM DIG-MSK on a panel of genes characterized as being related to “ovarian neoplasm” and containing at least one putative Sp1-binding site in the proximal promoter region.(A) Hierarchical clustering of changes in gene expression for each treatment. Dendrograms show average-linkage hierarchical clustering based in Pearson correlation coefficients. For the sake of comparison, lowercase letters at the right side indicate “clusters” with shared characteristics that are detailed in Results. (B) Network generated by the Genomatix Pathway System (GePS) representing bibliographic relationships for Sp1 co-expressed gene profiles (p = 1.48E-03). Dashed lines indicate genes associated by co-citation, while continuous lines indicate genes associated by expert-curation. Filled diamonds and triangles indicate the promoter of gene “B” (the gene with the diamond/triangle) has a corresponding experimentally validated binding site for the transcription factor encoded by gene “A”. Open triangles indicate that the binding of a particular transcription factor to the gene promoter has not been described unambiguously. The sequence logo for the consensus Sp1 binding site, retrieved from JASPAR, is shown at the top right of panel B.

Mentions: The effects of DIG-MSK on gene expression were also examined by clustering analysis of a selected set of genes. A hierarchical clustering, based on Pearson correlation coefficients, of 32 differentially expressed genes is shown in Fig. 4A. This set of genes was obtained after filtering the 278 up- and down-regulated genes influenced by both DIG-MSK concentrations (intersections in the Venn diagrams shown in Fig. 3A) using the Genomatix Pathway System (GePs). GePs identified 72 genes as belonging to “Ovarian Neoplasm” category within “Diseases/MeSH”. Ten of these genes were eliminated from the following analysis because their interaction with others was not evident. The remaining genes were then interrogated for the presence of putative Sp1 binding sites in their promoters using TELiS. Of them, 32 genes contained at least one Sp1-binding consensus sequence. Therefore, the clustered heat-map (Fig. 4A) summarizes the relationship between drug activity at each concentration and changes in the expression of Sp1-responsive genes relevant to ovarian cancer progression. Figure 4A offers a convenient way to visualize patterns of dissimilarity in the effects of either drug concentration. Dendrograms, showing average-linkage hierarchical clustering, clustered together several genes involved in common cellular pathways. For the sake of comparison, we have labeled those clusters using lowercase letters (Fig. 4A). Cluster labeled “a” contains genes up-regulated by either treatment, although 80 nM DIG-MSK had a superior enhancing effect. It encompassed genes related to various cell functions, as cell adhesion, migration, and proliferation, including genes involved in the control cell-cycle progression such as CDKN1A (p21WAF), which up-regulation was consistent with the transient halt of cells in G1 phase, described below. Most of these up-regulated genes, listed in Table 2, also contain putative Sp1-binding sites, although, in this case, Sp1 and DIG-MSK did not seem to compete directly for binding to consensus promoter sequences. Cluster “b” contains not only several genes that have been described to be highly expressed in ovarian cancer, but also DDB2, which enhanced expression would correlate with an augmented sensitivity of ovarian cancer cells to some chemotherapeutic agents [37]. Cluster “c” contains genes usually found highly expressed in ovarian carcinoma. DIG-MSK did not abrogate the expression of that particular group of genes. Cluster “d” contains genes whose expression can be induced in cellular stress conditions, as we may expect after drug treatment. We marked as “e” a single gene (GRN) that, although it clustered near others, it was peculiar in being up-regulated by 8 nM DIG-MSK and down-regulated by 80 nM DIG-MSK. GRN is a prognostic marker in epithelial ovarian carcinoma [38]. The large cluster “f” consists of genes that underwent a dose-dependent down-regulation. Among the genes clustered there was E2F1, a transcription factor, as well as genes that may contribute to tumorigenesis, including invasive ovarian carcinoma [39]. At the very edge of cluster “f”, but still closely related to it, KCNMA1 was labeled as “g” (Fig. 4A). Amplification of this gene, which was strongly inhibited by DIG-MSK, has been associated with high cell proliferation and poor prognosis [40].


Genome-wide modulation of gene transcription in ovarian carcinoma cells by a new mithramycin analogue.

Vizcaíno C, Núñez LE, Morís F, Portugal J - PLoS ONE (2014)

Effects of the treatment of A2780 cells with 8 nM or 80 nM DIG-MSK on a panel of genes characterized as being related to “ovarian neoplasm” and containing at least one putative Sp1-binding site in the proximal promoter region.(A) Hierarchical clustering of changes in gene expression for each treatment. Dendrograms show average-linkage hierarchical clustering based in Pearson correlation coefficients. For the sake of comparison, lowercase letters at the right side indicate “clusters” with shared characteristics that are detailed in Results. (B) Network generated by the Genomatix Pathway System (GePS) representing bibliographic relationships for Sp1 co-expressed gene profiles (p = 1.48E-03). Dashed lines indicate genes associated by co-citation, while continuous lines indicate genes associated by expert-curation. Filled diamonds and triangles indicate the promoter of gene “B” (the gene with the diamond/triangle) has a corresponding experimentally validated binding site for the transcription factor encoded by gene “A”. Open triangles indicate that the binding of a particular transcription factor to the gene promoter has not been described unambiguously. The sequence logo for the consensus Sp1 binding site, retrieved from JASPAR, is shown at the top right of panel B.
© Copyright Policy
Related In: Results  -  Collection

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pone-0104687-g004: Effects of the treatment of A2780 cells with 8 nM or 80 nM DIG-MSK on a panel of genes characterized as being related to “ovarian neoplasm” and containing at least one putative Sp1-binding site in the proximal promoter region.(A) Hierarchical clustering of changes in gene expression for each treatment. Dendrograms show average-linkage hierarchical clustering based in Pearson correlation coefficients. For the sake of comparison, lowercase letters at the right side indicate “clusters” with shared characteristics that are detailed in Results. (B) Network generated by the Genomatix Pathway System (GePS) representing bibliographic relationships for Sp1 co-expressed gene profiles (p = 1.48E-03). Dashed lines indicate genes associated by co-citation, while continuous lines indicate genes associated by expert-curation. Filled diamonds and triangles indicate the promoter of gene “B” (the gene with the diamond/triangle) has a corresponding experimentally validated binding site for the transcription factor encoded by gene “A”. Open triangles indicate that the binding of a particular transcription factor to the gene promoter has not been described unambiguously. The sequence logo for the consensus Sp1 binding site, retrieved from JASPAR, is shown at the top right of panel B.
Mentions: The effects of DIG-MSK on gene expression were also examined by clustering analysis of a selected set of genes. A hierarchical clustering, based on Pearson correlation coefficients, of 32 differentially expressed genes is shown in Fig. 4A. This set of genes was obtained after filtering the 278 up- and down-regulated genes influenced by both DIG-MSK concentrations (intersections in the Venn diagrams shown in Fig. 3A) using the Genomatix Pathway System (GePs). GePs identified 72 genes as belonging to “Ovarian Neoplasm” category within “Diseases/MeSH”. Ten of these genes were eliminated from the following analysis because their interaction with others was not evident. The remaining genes were then interrogated for the presence of putative Sp1 binding sites in their promoters using TELiS. Of them, 32 genes contained at least one Sp1-binding consensus sequence. Therefore, the clustered heat-map (Fig. 4A) summarizes the relationship between drug activity at each concentration and changes in the expression of Sp1-responsive genes relevant to ovarian cancer progression. Figure 4A offers a convenient way to visualize patterns of dissimilarity in the effects of either drug concentration. Dendrograms, showing average-linkage hierarchical clustering, clustered together several genes involved in common cellular pathways. For the sake of comparison, we have labeled those clusters using lowercase letters (Fig. 4A). Cluster labeled “a” contains genes up-regulated by either treatment, although 80 nM DIG-MSK had a superior enhancing effect. It encompassed genes related to various cell functions, as cell adhesion, migration, and proliferation, including genes involved in the control cell-cycle progression such as CDKN1A (p21WAF), which up-regulation was consistent with the transient halt of cells in G1 phase, described below. Most of these up-regulated genes, listed in Table 2, also contain putative Sp1-binding sites, although, in this case, Sp1 and DIG-MSK did not seem to compete directly for binding to consensus promoter sequences. Cluster “b” contains not only several genes that have been described to be highly expressed in ovarian cancer, but also DDB2, which enhanced expression would correlate with an augmented sensitivity of ovarian cancer cells to some chemotherapeutic agents [37]. Cluster “c” contains genes usually found highly expressed in ovarian carcinoma. DIG-MSK did not abrogate the expression of that particular group of genes. Cluster “d” contains genes whose expression can be induced in cellular stress conditions, as we may expect after drug treatment. We marked as “e” a single gene (GRN) that, although it clustered near others, it was peculiar in being up-regulated by 8 nM DIG-MSK and down-regulated by 80 nM DIG-MSK. GRN is a prognostic marker in epithelial ovarian carcinoma [38]. The large cluster “f” consists of genes that underwent a dose-dependent down-regulation. Among the genes clustered there was E2F1, a transcription factor, as well as genes that may contribute to tumorigenesis, including invasive ovarian carcinoma [39]. At the very edge of cluster “f”, but still closely related to it, KCNMA1 was labeled as “g” (Fig. 4A). Amplification of this gene, which was strongly inhibited by DIG-MSK, has been associated with high cell proliferation and poor prognosis [40].

Bottom Line: Nanomolar concentrations of DIG-MSK abrogated the expression of genes involved in a variety of cell processes including transcription regulation and tumor development, which resulted in cell death.The effect of DIG-MSK in the control of gene expression by other transcription factors was also explored.DIG-MSK affected several biological processes and molecular functions related to transcription and its cellular regulation in A2780 cells, including transcription factor activity.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Biología Molecular de Barcelona, Consejo Superior de Investigaciones Cientificas, Barcelona, Spain.

ABSTRACT
Ovarian cancer has a poor prognosis due to intrinsic or acquired resistance to some cytotoxic drugs, raising the interest in new DNA-binding agents such as mithramycin analogues as potential chemotherapeutic agents in gynecological cancer. Using a genome-wide approach, we have analyzed gene expression in A2780 human ovarian carcinoma cells treated with the novel mithramycin analogue DIG-MSK (demycarosyl-3D-β-D-digitoxosyl-mithramycin SK) that binds to C+G-rich DNA sequences. Nanomolar concentrations of DIG-MSK abrogated the expression of genes involved in a variety of cell processes including transcription regulation and tumor development, which resulted in cell death. Some of those genes have been associated with cell proliferation and poor prognosis in ovarian cancer. Sp1 transcription factor regulated most of the genes that were down-regulated by the drug, as well as the up-regulation of other genes mainly involved in response to cell stress. The effect of DIG-MSK in the control of gene expression by other transcription factors was also explored. Some of them, such as CREB, E2F and EGR1, also recognize C/G-rich regions in gene promoters, which encompass potential DIG-MSK binding sites. DIG-MSK affected several biological processes and molecular functions related to transcription and its cellular regulation in A2780 cells, including transcription factor activity. This new compound might be a promising drug for the treatment of ovarian cancer.

Show MeSH
Related in: MedlinePlus