Figure 1.
Loss of RGS10 expression in ovarian cancer cells.
A. Ovarian cancer cells were isolated from patient malignant ascites and RGS10 expression levels were compared to IOSE cells via western blotting. B.–C. RGS10 transcript (B) and protein (C) expression levels were compared in CAOV-3 ovarian cancer cell lines and IOSE benign ovarian epithelial cells using qRT-PCR and western blotting. D. Cisplatin dose response curves were determined using CellTiter-Blue viability assays in A2780 and A2780-AD cells. E.–F. RGS10 transcript (E) and protein (F) levels were compared in chemoresistant A2780-AD cells relative to their parental chemosensitive cell line A2780. **: p<0.01, ***: p<0.0001.
Figure 2.
A. The RGS10 gene (geneID: 6001) is located on the negative strand of chromosome 10 (NCBI accession: NC_000010.10) at position −121,302,222 to −121,259,339. Two transcription variants RGS10-1 (accession: NM_001005339) and RGS10-2 (accession: NM_002925) have been reported for RGS10 based on alternate start sites that result in distinct first exons. B. The resulting protein isoforms RGS10a (accession: NP_001005339) and RGS10b (accession: NP_002916) vary by only the first 18 or three amino acids. The conserved RGS domain is underlined. C.–D. The expression of total RGS10 transcript (RGStot), RGS10-1, and RGS10-2 were determined in IOSE and CAOV-3 cells (C) and in parental A2780 cells and chemoresistant A2780-AD cells (D). **: p<0.01, ***: p<0.0001.
Figure 3.
Regulation of RGS genes by DNA methylation.
A. The promoter regions of RGS10-1, RGS10-2, RGS2, and RGS5 were analyzed for CpG content using the website Methprimer. For each promoter, a region of genomic DNA 1000 basepairs 5′ of the transcriptional start site and 500 basepairs 3′ of the start site were evaluated for percent GC content and individual CpG dinucleotides. Nucleotide position is indicated along the x-axis and GC content is graphed on the y-axis; CpG islands are indicated with shading. Each CpG dinucleotide is indicated by a hash mark below the nucleotide numbering, and the transcriptional start site is indicated with an arrow. Amplification regions for four bisulfite sequencing primer pairs are indicated by horizontal bars (BS10-1, BS10-2, BS10-3, BS10-4). B. SKOV-3 cells were treated with vehicle or the DNMT inhibitor 5-Aza for nine days, and the transcript levels of the indicated RGS and GAPDH controls were measured at 3, 5, 7, and 9 days of treatment. The RGS transcript was normalized to GAPDH, and is graphed relative to expression in vehicle treated controls at each time point.
Figure 4.
Bisulfite sequencing of RGS10-1 promoter.
RGS10 promoter genomic DNA was aligned with individual sequences of cloned PCR products from primer pair BS10-2 amplification of bisulfite treated genomic DNA from the indicated cell lines. Sequences were subjected to quality control analysis and aligned using BiQ Analyzer software. In this conventional ‘lollipop’ representation, each CpG site in the region (−121,303,076 → −121,302,726) is indicated with a circle; filled circles are methylated, unfilled circles are unmethylated. Lollipop representations of the methylation status of each CpG site in RGS10-1 promoter regions amplified by BS10-1, BS10-3, and BS10-4 primer sets are available in the Supporting Information.
Figure 5.
Methylated fraction of CpG dinucleotides across the RGS10-1 promoter in ovarian cell lines.
The fraction of clones that were methylated at individual CpG dinucleotides across the RGS10-1 promoter is shown. CpG dinucleotides are labeled by their position on chromosome 10(-). Nucleotides that were not methylated in either cell line are not shown. The complete data set with ratios of sequenced clones is shown in Supplementary Table 1. A. Methylation rates are compared between A2780 and A2780-AD cells. B. Methylation rates are compared between IOSE and CAOV-3 cells. Dotted horizontal bar: region −121,303,155 → −121,303,007. Insets: The cumulative fraction of DNA methylation is shown for the entire RGS10-1 promoter and for the indicated region. **: p<0.01. Bent arrow: transcriptional start site. Arrows below x-axis: sites contained within each primer pair (left to right: BS10-1, BS10-2, BS10-3, BS10-4).
Figure 6.
Histone acetylation and HDAC binding at RGS10-1 promoters in chemoresistant A2780-AD cells and parental A2780 cells.
ChIP assays were carried out in A2780 parental cells and multi-drug resistant A2780-AD. Lysates were immunoprecipitated with control, anti-acetyl histone H3, anti-acetyl H3K18, or anti-HDAC1 antibody. Associated DNA was isolated and analyzed via real time PCR using primers spanning the RGS10-1 and GAPDH promoters. Real-time PCR values were normalized to the total amount of promoter DNA added (input). Input values represent 5% of the total cell lysate. * P<0.05. A. Global levels of Histone H3 acetylation associated with RGS10 and GAPDH promoters in A2780 and A2780-AD ovarian cancer cells. Values represent mean ± SEM of four independent experiments. B. Levels of histone H3 acetylated at lysine 18 associated with RGS10-1 and GAPDH promoters in A2780 and A2780-AD ovarian cancer cells. Values represent mean ± SEM of four independent experiments. C. Levels of HDAC1 associated with RGS10 and GAPDH promoters in A2780 and A2780-AD ovarian cancer cells. Values represent mean ± SEM of three independent experiments. D. Western blot analysis of global HDAC1 levels in A2780 and A2780-AD cells.
Figure 7.
Histone acetylation and HDAC binding at RGS10-1 promoters in IOSE and CAOV-3 ovarian cells.
ChIP assays were carried out in normal ovarian IOSE-80 cells and in CAOV-3 ovarian cancer cells. Lysates were immunoprecipitated with control antibody, anti-acetyl histone H3 antibody, or with anti-HDAC1 antibody. Associated DNA was isolated and quantified via real time PCR using primers spanning the RGS10 and GAPDH promoters. Real-time PCR values were normalized to the total amount of promoter DNA added (input). Input values represent 5% of the total cell lysate. * P<0.05 A. Global levels of Histone H3 acetylation associated with RGS10 and GAPDH promoters in normal and chemosensitive ovarian cancer cells. Values for histone H3 acetylation represent mean ± SEM of two independent experiments. B. HDAC1 levels associated with RGS10 and GAPDH promoters in normal and chemosensitive ovarian cancer cells. Values for HDAC1 binding are representative data. Error bars show deviation between technical errors.
Figure 8.
Inhibition of HDAC and DNMT enzymes in chemoresistant cells enhances cisplatin sensitivity and RGS10 expression.
A. A2780-AD cells were plated in 96 well plates and treated with 500 nM trichostatin A (TSA) or vehicle for 48 hours, with or without 30 µM cisplatin for the final 12 hours. Cell survival was assessed using CellTiter-Blue fluorimetric viability assays. Inset: Cell viability normalized to values in the absence of cisplatin. B. A2780-AD cells were treated with vehicle or 500 nM TSA for 36 hours. Gene expression was assessed with RT-PCR as described, and normalized to RPL-13A gene expression. C. A2780-AD cells were treated with 10 µM 5 Azacytidine (5-Aza) or vehicle for 5 days, with or without 30 µM cisplatin for the final 24 hours. D. RGS10 mRNA expression was assessed following 5 days of vehicle or 5-Aza. ***: p<0.0001, *: p<0.05.