Article Figures & Data
Figures
- Figure 1.
A, cells were treated twice over a 96-h period with ethanol (lane 1), RA (1 μmol/L; lane 2), VPA (250 μmol/L; lane 3), RA + VPA (lane 4), Aza-dC (2 μmol/L; lane 5), RA + Aza-dC (lane 6), and RA + Aza-dC + VPA (lane 7) and RT-PCR was done as described in Materials and Methods. PCR products were analyzed by electrophoreses on 1.5% TAE-agarose gel and stained with ethidium bromide. B and C, ChIP assays using an antibody to acetylated histone H3 were used to assess the ability of 1 μmol/L RA (B, lane 1) or 2 μmol/L Aza-dC (B, lane 2) in combination with differing VPA concentrations (C, lanes 1-5) to restore histone H3 acetylation to the RARβ2 P2 promoter. Cells were treated twice over a 96-h period with RA and Aza-dC in combination with 5 mmol/L VPA (C, lane 1), 3 mmol/L VPA (C, lane 2), 1 mmol/L VPA (C, lane 3), 500 μmol/L VPA (C, lane 4), 250 μmol/L VPA (C, lane 5), RA + Aza-dC (C, lane 6), and vehicle control (C, lane 7). Immediately before the ChIP assay, RA (1 μmol/L) and β-estradiol (100 nmol/L) were added to the cells for 30 minutes before formaldehyde cross-linking and histone H3 acetylation assessed at the RARβ2-RARE. The promoter regions of PS2 and GAPDH act as internal positive controls. Thirty-two (B) and 33 (C) cycles of PCR were used in the ChIP experiments. PCRs were analyzed by electrophoresis on a 2% TAE-agarose gel and repeated yielding similar results. βE2, β-estradiol; C, Aza-dC; IP, immunoprecipitation with anti–histone H3 antibody; R, RA; V, VPA.
- Figure 2.
RT-PCR was used to detect expression of RARα (314 bp), RARβ (256 bp), and RARγ (394 bp) in MCF-7 (A), MDA-MB-231 (B), MDA-MB-453 (C), SK-BR3 (D), and HS578T (E) breast cancer cells. Amplification of β-actin (379 bp) was used to confirm cDNA integrity. Cells were treated twice over a 96-h period with ethanol (lane 1), RA (1 μmol/L; lane 2), VPA (250 μmol/L; lane 3), RA + VPA (lane 4), Aza-dC (2 μmol/L; lane 5), RA + Aza-dC (lane 6), and RA + Aza-dC + VPA (lane 7) and RT-PCR was done as described in Materials and Methods. A nonspecific amplification product was observed in control and VPA-treated MDA-MB-231 cells (B) and control MDA-MB-453 cells (C). The identity of the PCR product of each primer pair was confirmed by automated DNA sequencing. PCR products were analyzed by electrophoreses on 1.5% TAE-agarose gel and stained with ethidium bromide.
- Figure 3.
Real-time PCR (A-D) was done as described in Materials and Methods. Cells were treated twice over a 96-h period with RA (1 μmol/L), VPA (250 μmol/L), RA + VPA, Aza-dC (2 μmol/L), RA + Aza-dC, and RA + Aza-dC + VPA. The expression of RARβ2 was measured relative to cellular β-actin levels and quantified relative to calibrator sample (asterisks). Real-time PCR analysis was done in triplicate for each sample. Columns, mean; bars, SD. No signal indicates that RARβ2 was not detected under the conditions used. R, RA (1 μmol/L); V, VPA (250 μmol/L); C, Aza-dC (2 μmol/L); Con, control.
- Figure 4.
Proliferation of MCF-7 (A-C) and MDA-MB-231 (D-F) breast cancer cells was monitored in cells treated twice with combinations of VPA (250 μmol/L), RA (1 μmol/L), and Aza-dC (2 μmol/L) during the 96-h growth experiment. Growth inhibition expressed as percentage of untreated control MCF-7 (C) and MDA-MB-231 (F) cells is displayed to permit comparison between experiments done on different occasions. Growth assays were done on a minimum of four occasions. Points, mean; bars, SE.
- Figure 5.
RT-PCR was used to detect expression of RARβ in MCF-7 (A), MDA-MB-231 (C), MDA-MB-453 (E), and SK-BR3 (G) breast cancer cells. Amplification of β-actin was used to confirm cDNA integrity. Cells were treated twice over a 96-h period with ethanol, RA (1 μmol/L), and Aza-dC (2 μmol/L) with decreasing VPA concentrations (lane 1); RA + Aza-dC + VPA (250 μmol/L; lane 2); RA + Aza-dC + VPA (100 μmol/L; lane 3); RA + Aza-dC + VPA (50 μmol/L; lane 4); and RA + Aza-dC (lane 5). RT-PCR was done as described in Materials and Methods. PCR products were analyzed by electrophoreses on 1.5% TAE-agarose gels and stained with ethidium bromide. Proliferation of MCF-7 (B), MDA-MB-231 (D), MDA-MB-453 (F), and SK-BR3 (H) breast cancer cells was monitored in cells treated twice with combinations of RA (1 μmol/L) with Aza-dC (2 μmol/L) and varying VPA concentrations as indicated during the 96-h growth experiment. Cell number was measured after 96-h growth. Growth assays were done in quadruplicate. Columns, mean; bars, SE.
Tables
- Table 1.
Sequences of primer pairs used to assess mRNA expression using RT-PCR
Gene Sense primer (5′-3′) Antisense primer (5′-3′) Product (bp) RARα GTCTGTCAGGACAAGTCCTCAGG GCTTTGCGCACCTTCTCAATGAG 314 RARβ2 GACTGTATGGATGTTCTGTCAG ATTTGTCCTGGCAGACGAAGC 255 RARγ AATGACAAGTCCTCTGGCTACCAC CAGATCCAGCTGCACGCGGTGGTC 394 β-actin GCTCGTCGTCGACAACGGCTC GTACATGGCTGGGGTGTTGAAGG 379 NOTE: The identity the product of each primer pair was confirmed by automated DNA sequencing.
Volume 4, Issue 3
