Article Figures & Data
Figures
- Fig. 1.
Regulated levels of VDR target genes in MCF-12A and MDA-MB-231 cells treated with 1α,25(OH)2D3. Cells (2 × 104/cm2) were plated into six-well plates and allowed to grow for 36 hours to ensure that cells were in mid-exponential phase. Cells were treated with 1α,25(OH)2D3 (100 nmol/L) or left untreated (control). Total RNA was isolated after the indicated time periods, reverse transcribed, and the target genes amplified and the fold increase calculated as described in Materials and Methods. Points, mean of three separate experiments amplified in triplicate wells; bars, SE. A, CYP24; B, GADD45α. C, VDUP-1. *, P < 0.05, treatments that were significantly greater than control. All points in (A) were significantly different from control, and between MCF-12A and MDA-MB-231.
- Fig. 2.
Fold elevation of nuclear corepressor mRNA levels in breast cancer cell lines. A, VDR and corepressor mRNA levels measured by quantitative reverse transcription-PCR in T47-D, ZR-75-1, MCF-7, and MDA-MB-231 compared with MCF-12A nonmalignant breast epithelial cells. Total mRNA was isolated from triplicate cultures in mid-exponential phase, reverse transcribed, and the target genes amplified in triplicate as described in Materials and Methods. *, P < 0.05; **, P < 0.01; ***, P < 0.001. B, protein was isolated from parallel subconfluent (S) and confluent (C) MCF-7 and MDA-MB-231 cells (Materials and Methods) and resolved by SDS-PAGE and probed with antibody to cyclin E. Representative blots are shown with the position of the proteins indicated on the left. Blots were subsequently stripped and reprobed for β-actin. C, the fold reduction in the mRNA levels of NCoR1, NCoR2/SMRT, and TRIP15/Alien was measured in confluent cultures of T47-D, ZR-75-1, MCF-7, and MDA-MB-231, compared with subconfluent controls, by quantitative reverse transcription-PCR. Points, mean of three separate experiments amplified in triplicate wells; bars, SE.
- Fig. 3.
Expression and altered ratio of VDR to corepressor mRNA levels in matched primary cultures. A, the relative expression of VDR levels in ERα-positive (n = 14) and ERα (n = 7) tumors compared with matched controls. Levels were normalized to expression of the mammary epithelial markers cytokeratin 19. B to D, the ratio of corepressor mRNA to VDR, after normalization to cytokertain 19, in matched tumor and normal pair as measured by quantitative reverse transcription-PCR as described in Materials and Methods.
- Fig. 4.
Regulation of GADD45α, VDUP-1, and CYP24 mRNA in MDA-MB-231 cells treated with RO-26-2198 plus TSA. Cells (2 × 104/cm2) were plated into six-well plates and allowed to grow for 36 hours to ensure that cells were in mid-exponential phase. Cells were treated either with RO-26-2198 (100 nmol/L), TSA (15 nmol/L), the combination of RO-26-2198 plus TSA, or left untreated (control). Total RNA was isolated after the indicated time periods, reverse transcribed, and the target genes amplified and the fold increase calculated as described in Materials and Methods. Points, mean of three separate experiments amplified in triplicate wells; bars, SE. Gene regulation in cells treated with RO-26-2198 plus TSA. A, GADD45α; B, VDUP-1; C, YP24. *, P < 0.05, treatments that were significantly greater than control (for GADD45α and VDUP-1). *, P < 0.0001, treatments that were significantly greater than control (for CYP24).
Tables
- Table 1.
Cell sensitivities towards 1α,25(OH)2D3, TSA, and 5-aza-dCyd
Cell line ED50CP 1α,25(OH)2D3 (nmol/L) ED50LP 1α,25(OH)2D3 (nmol/L) ED25LP TSA (nmol/L) ED50LP TSA (nmol/L) ED25LP 5-aza-dCyd (nmol/L) ED50LP 5-aza-dCyd (nmol/L) MCF-12A 20 300 25 40 85 130 T47-D 15 >1,000 10 45 110 >8,000 ZR-75-1 100 >1,000 35 100 740 >8,000 MCF-7 100 >1,000 25 45 65 1,825 MDA-MB-231 >100 >1,000 15 30 85 8,000 NOTE: Cellular responses were screened on two different assay formats, clonal proliferation (CP) in soft agar in 24-well plates and liquid proliferation (LP) in 96-well plates. The ED50 and ED25 were interpolated from dose-response graphs.
- Table 2.
Combinations of vitamin D3 compounds plus either TSA or Aza enhance antiproliferative responses in breast cancer cells
NOTE: 1α,25(OH)2D3 or RO-26-2198 (100 nmol/L) was combined with TSA (25 nmol/L for MCF-12A and 15 nmol/L for cancer cell lines) or 5-aza-dCyd (250 nmol/L). Proliferation inhibition was measured in liquid media after 96 hours, with redosing after 48 hours. The predicted (P) values represent the inhibition of vitamin D3 compound alone added to the inhibition from TSA alone, 5-aza-dCyd alone, or TSA with 5-aza-dCyd together. The observed (O) values represent the actual inhibition of proliferation obtained. All experiments were carried out thrice independently in triplicate wells. Interactions are represented as follows: bold type, strong additive; normal type, additive; shaded box, subadditive.
Volume 12, Issue 7
