Article Figures & Data
Figures
- Fig. 1.
Relative expression of UGT1A1 mRNA in colon cancer cell lines. Columns, mean of relative expression of UGT1A1 (arbitrary units) determined by QRT-PCR. The housekeeping gene was used as an endogenous control and showed a coefficient of variation of 15%.
- Fig. 2.
UGT1A1 expression following 5-Aza-dC (5 μmol/L) and/or trichostatin A (TSA; 300 nmol/L) treatment in colon cancer cell lines. Results for all three low UGT1A1 mRNA expression cell lines: (A) HCT-116, (B) COLO-320DM, and (C) HCT-15. All expression results were assessed by QRT-PCR using β2-microglobulin as an endogenous control. DMSO and PBS did not modify UGT1A1 mRNA expression. Columns, mean of two separated experiments done in triplicate; bars, SD. *, P < 0.05.
- Fig. 3.
Repression of UGT1A1 promoter activity by DNA methylation in luciferase constructs. A, UGT1A1 promoter constructs of variable lengths and their associated CpG. B, digestion of plasmid DNA with the methylation-sensitive restriction enzyme HpaII, which cuts CCGG but not CmCGG sites. Luciferase activity in fully methylated (black) and unmethylated (white) promoter constructs for all three low UGT1A1 mRNA expression cell lines: (C) HCT-116, (D) COLO-320DM, and (E) HCT-15. Columns, mean of two separate experiments done in triplicate; bars, SD. LUC, luciferase; M, methylated; U, unmethylated. **, P < 0.01.
- Fig. 4.
Schematic representation of the UGT1A1 gene methylation profile. A, schematic map of the CpG-rich regions (regions A-F) in UGT1A1. Vertical ticks, CpG sites. Amplicons 1 to 8 produced by PCR for bisulfite sequencing are also indicated. Arrow, direction of the bisulfite sequencing (for each amplicon). Nucleotide position of the C moiety of CpG regions relative to the ATG are as follows: F (−5,000 to −4,885), E (−1,157 to −892), D (−704 to −642), C (−513 to −432), B (−330 to −4), and A (+21 to +233) based on the UGT1A1 reference sequence AF297093. B, methylation profiles of colon cancer cell lines in regions A to E. Methylation status is presented as the mean of the methylation percentages of CpG for the indicated region and includes all CpG in that region. Comparison was done between percentages for a specific region in UGT1A1-negative and UGT1A1-positive cell lines. For significantly different methylation percentages between low-expressing and high-expressing cell lines, regions were reamplified and resequenced multiple times (at least thrice) for confirmation. *, P < 0.05; **, P < 0.01.
- Fig. 5.
Demethylation treatment increases protein levels and inactivation of SN-38 through glucuronidation. A, Western blot analysis of UGT1A1 protein after treatment with 5-Aza-dC alone or in combination with trichostatin A for the HCT-116 cell line. B, enzymatic assays for SN-38 glucuronidation with the isolated microsomal preparations from cells treated with both 5-Aza-dC and trichostatin A. Percentage of SN-38 glucuronide formation.
Tables
- Table 1.
Primers sequences and probes
Name Primer sequence* Constructs primers Common primer (R) 5′-TGGCAGGAGCAAAGGCGCCGCTCGAGCGG-3′ P-269bp (F) 5′-CGAGCTCGGAGGTTCTGGAAGTACTTGGC-3′ P-540bp (F) 5′-CGACGCGTGTGCTGGACTCAATAAATATTG-3′ P-1316bp (F) 5′-CGACGCGTGGATCTTGGGCCAGTGGAATG-3′ P-2218bp (F) 5′-CGACGCGTGGCCTAGTTCCTAGCATAGTG-3′ Bisulfite sequencing 1 (F) 5′-ATTAGGGAGTTATAGTTTTTGG-3′ 1 (R) 5′-CAACTAACCCTTTACTTACTACA-3′ 2 (R) 5′-GGGGAAGATTTTTGTTTATATG-3′ 2 (F) 5′-AATACCCACTCCACAACTCC-3′ 3 (F) 5′-TGTGAGTTTGGTTTATTTTATGG-3′ 3 (R) 5′-TACTTCCAAAACCTCAAAATCC-3′ 4 (F) 5′-TTGGTGTAT(CT)GATTGGTTTTTG-3′ 4 (R) 5′-CAATAACTACCATCCACTAAAATC-3′ 5 (F) 5′-GGTTTTGGAAGTATTTTGTTGTG-3′ 5 (R) 5′-CAACCATAAC(AG)CCTTTACTCCTA-3′ 6 (F) 5′-GGGTTTAGTGGTGTTTTATGTT-3′ 6 (R) 5′-ATAACATCAAAACTACTTTCTACC-3′ 7 (F) 5′-GATTTTGTTATGTTTTTGTTTGG-3′ 7 (R) 5′-CAAAACTCAATAAATCCTAAACA-3′ 8 (F) 5′-GTAGGTTTTAGTTATTTGTTTGAA-3′ 8 (R) 5′-CAATCCCAAAAACACTACATC-3′ Primer for automatic sequencing 1 (F) 5′-GTAATGAAGGTGAGTTTTATAG-3′ 1 (R) 5′-CTAAATATCCTAAAAACCTATAC-3′ 2 (R) 5′-ATACTACCTACTCACTTATATC-3′ 3 (F) 5′-TGTGAGTTTGGTTTATTTTATGG-3′ 4 (R) 5′-CAATAACTACCATCCACTAAAATC-3′ 5 (F) 5′-GGTTTTGGAAGTATTTTGTTGTG-3′ 6 (F) 5′-GGGTTTAGTGGTGTTTTATGTT-3′ 7 (R) 5′-CAAAACTCAATAAATCCTAAACA-3′ 8 (R) 5′-CAATCCCAAAAACACTACATC-3′ QRT-PCR UGT1A1 QRT-PCR (F) 5′-GACTGTCCAGGACCTATTGAGCTC-3′ QRT-PCR (R) 5′-CATTAATGTAGGCTTCAAATTCCTGG-3′ QRT-PCR (P) 5′-(FAM)ATCATGCCCAATATGGTT(MGB)-3′ Abbreviations: F, forward; R, reverse; P, probe; FAM, carboxyfluorescein; MGB, minor groove binder.
↵* Accession no. for UGT1A1 reference sequence: AF297093.
- Table 2.
Relative UGT1A1 expression compared with the control group for each cell line
Colon adenocarcinoma cell lines Control for 5-Aza-dC (PBS) Control for TSA (DMSO) 5-Aza-dC (fold increased) TSA (fold increased) 5-Aza-dC + TSA (fold increased) HCT-116 1 1 19 (19) 1 (1) 128(128) COLO-320DM 1 1 15 (15) 1 (1) 75 (75) HCT-15 309 244 2,132 (7) 293 (1) 4,075 (13) NOTE: Cells were treated as described in Materials and Methods with 5-Aza-dC alone (5 μmol/L) or in combination with trichostatin A (300 nmol/L).
Volume 12, Issue 6
