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. 2008 Nov 15;68(22):9358-66.
doi: 10.1158/0008-5472.CAN-08-1860.

p53-inducible ribonucleotide reductase (p53R2/RRM2B) is a DNA hypomethylation-independent decitabine gene target that correlates with clinical response in myelodysplastic syndrome/acute myelogenous leukemia

Petra A Link  1 , Maria R Baer, Smitha R James, David A Jones, Adam R Karpf
Affiliations

p53-inducible ribonucleotide reductase (p53R2/RRM2B) is a DNA hypomethylation-independent decitabine gene target that correlates with clinical response in myelodysplastic syndrome/acute myelogenous leukemia

Petra A Link et al. Cancer Res. .

Abstract

While the therapeutic activity of the deoxycytidine analogue decitabine is thought to reflect its ability to reactivate methylation-silenced genes, this agent is also known to trigger p53-dependent DNA damage responses. Here, we report that p53-inducible ribonucleotide reductase (p53R2/RRM2B) is a robust transcriptional target of decitabine. In cancer cells, decitabine treatment induces p53R2 mRNA expression, protein expression, and promoter activity in a p53-dependent manner. The mechanism of p53R2 gene induction by decitabine does not seem to be promoter DNA hypomethylation, as the p53R2 5' CpG island is hypomethylated before treatment. Small interfering RNA (siRNA) targeting of DNA methyltransferase 1 (DNMT1) in wild-type p53 cells leads to genomic DNA hypomethylation but does not induce p53R2, suggesting that DNMT/DNA adduct formation is the molecular trigger for p53R2 induction. Consistent with this idea, only nucleoside-based DNMT inhibitors that form covalent DNA adducts induce p53R2 expression. siRNA targeting of p53R2 reduces the extent of cell cycle arrest following decitabine treatment, supporting a functional role for p53R2 in decitabine-mediated cellular responses. To determine the clinical relevance of p53R2 induction, we measured p53R2 expression in bone marrow samples from 15 myelodysplastic syndrome/acute myelogenous leukemia (MDS/AML) patients undergoing decitabine therapy. p53R2 mRNA and protein were induced in 7 of 13 (54%) and 6 of 9 (67%) patients analyzed, respectively, despite a lack of methylation changes in the p53R2 promoter. Most notably, there was a significant association (P = 0.0047) between p53R2 mRNA induction and clinical response in MDS/AML. These data establish p53R2 as a novel hypomethylation-independent decitabine gene target associated with clinical response.

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Figures

Fig. 1
Fig. 1
p53R2 is a p53-dependent decitabine gene target. A, p53R2 mRNA expression in cancer cell lines following decitabine treatment. p53R2 was measured by Northern blot analysis in the indicated colorectal cancer cell lines five days post decitabine treatment, using the indicated drug concentrations. The p53 status of each cell line is indicated and the cell lines are described in the text. Ethidium bromide staining of total RNA confirmed equivalent RNA loading. B, p53R2 protein expression in cancer cell lines following decitabine treatment. p53R2 was measured by Western blot analysis under the same experimental conditions described in A. C, p53-dependent p53R2 promoter activity in decitabine-treated cells. Left: The activity of a p53R2 luciferase reporter construct containing the intron 1 p53 binding site was determined before and after decitabine-treatment in RKO (p53 wildtype) and HT29 (p53 mutant) cells. Right: activity of the p53R2 intron 1 construct containing a mutated p53 binding site.
Fig. 2
Fig. 2
The p53R2 5′ CpG island is hypomethylated in colon cancer cells in the absence of decitabine treatment. A, Diagram of the 5′ region of the p53R2 gene, showing the CpG island (lower rectangle), intron 1, and exons 1 and 2 (upper rectangles). The position of the p53 binding site (p53 B.S.) in intron 1 is also shown. The bent arrow corresponds to the NCBI-predicted transcriptional start site (TSS). B, MSP analysis of two different regions of the p53R2 5′ CpG island. Testis DNA served as an unmethylated control, and SssI modified DNA served as a methylated control. Cell lines are the same as in Fig. 1A–B. C, Sodium bisulfite sequencing analysis of the central region of the p53R2 5′ CpG island in RKO, HCT116, and LOVO colorectal cancer cell lines. Open and filled circles represent unmethylated and methylated CpG sites, respectively, and rows correspond to individually sequenced clones. SssI modified DNA was analyzed as a positive control for DNA methylation. Numbers indicate nucleotide positions relative to the NCBI-predicted p53R2 TSS.
Fig. 3
Fig. 3
The effect of DNMT1 siRNA knockdown and treatment with other DNMT inhibitors on p53R2 expression in RKO cells. A, RKO cells were treated with the indicated concentrations of wildtype or mutant DNMT1 siRNA for five days. LF2000 indicates cells treated with only the transfection reagent. Alternatively, RKO cells treated with the indicated concentrations of decitabine (DAC) and harvested five days post treatment. DNA extracts were obtained, and genomic 5-methyldeoxycytidine (5mdC) levels were determined as described in Materials and Methods. B, p53R2 expression was measured by qRT-PCR under the same set of conditions described in A. C, The activity of the p53R2 intron 1 construct was measured under the same set of conditions described in A. D, p53R2 expression in RKO cells was measured by qRT-PCR five days post-treatment with the indicated concentrations of decitabine (DAC), 5-azacytidine (5-aza), zebularine, or RG108. RKO cells were treated with the indicated concentrations of drugs at day 0 and day 3 and cells were harvested at day 5 post treatment. PBS serves as the vehicle control for the 1st three drugs, while DMSO serves as the vehicle control for RG108.
Fig. 4
Fig. 4
Decitabine treatment induces p53R2 mRNA and protein expression in MDS/AML patient bone marrow. A, p53R2 expression in bone marrow samples obtained from 13 MDS/AML patients undergoing decitabine therapy. Pre- and post-treatment samples are shown, and the number of cycles of therapy (#c) is listed for post-treatment samples. p53R2 expression was measured by qRT-PCR. For each patient, p53R2 expression was normalized to GAPDH, and pre-treatment samples were each set at one to normalize between patients. Plus signs (+) indicate patients in whom at least one post-treatment sample showed biologically significant (defined as > 4 fold) p53R2 induction compared to the pre-treatment sample. Dashes (−) indicate patients for whom no post-treatment sample showed significant p53R2 induction. p53R2 expression in RKO cells treated with decitabine (DAC) for five days is shown as a positive control. Note that patients #4 and #7 did not yield sufficient quality RNA for analysis and are not shown. B, p53R2 Western blot analysis of cytosolic protein extracts harvested from bone marrow samples from nine MDS/AML patients undergoing decitabine therapy. Pre- and post-treatment samples are shown, and the number of cycles of therapy (#c) is listed for post-treatment samples. Fold induction (or decline) relative to the pretreatment sample is shown beneath the Western blot. Plus signs (+) indicate patients in whom the post-treatment samples showed biologically significant (defined as > 1.5 fold) p53R2 induction. Dashes (−) indicate patients in whom p53R2 induction was not observed. RKO cells treated with decitabine (DAC) for five days are shown as a positive control. Ponceau S staining confirmed equivalent protein loading. Note that patients #10–15 did not yield sufficient quality protein for analysis and are not shown.
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