Schäfer A et al. (2010), Gemcitabine functions epigenetically by inhibit...

XB-ART-42442

PLoS One 2010 Nov 19;511:e14060. doi: 10.1371/journal.pone.0014060.

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Gemcitabine functions epigenetically by inhibiting repair mediated DNA demethylation.

Schäfer A , Schomacher L , Barreto G , Döderlein G , Niehrs C .

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Gemcitabine is a cytotoxic cytidine analog, which is widely used in anti-cancer therapy. One mechanism by which gemcitabine acts is by inhibiting nucleotide excision repair (NER). Recently NER was implicated in Gadd45 mediated DNA demethylation and epigenetic gene activation. Here we analyzed the effect of gemcitabine on DNA demethylation. We find that gemcitabine inhibits specifically Gadd45a mediated reporter gene activation and DNA demethylation, similar to the topoisomerase I inhibitor camptothecin, which also inhibits NER. In contrast, base excision repair inhibitors had no effect on DNA demethylation. In Xenopus oocytes, gemcitabine inhibits DNA repair synthesis accompanying demethylation of oct4. In mammalian cells, gemcitabine induces DNA hypermethylation and silencing of MLH1. The results indicate that gemcitabine induces epigenetic gene silencing by inhibiting repair mediated DNA demethylation. Thus, gemcitabine can function epigenetically and provides a tool to manipulate DNA methylation.

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Species referenced: Xenopus
Genes referenced: calr ctrl elk1 gadd45a gal.2 gem mlh1 pou5f3

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	Figure 1. Gemcitabine inhibits Gadd45a mediated gene activation.(A–B) Luciferase reporter assays of HEK293T cells transiently transfected with HpaII in vitro methylated Gal-responsive reporter, together with either Gadd45a (A) or Gal-Elk1 (B, specificity control). Cells were treated with DMSO (control, Ctrl), gemcitabine (Gem), camptothecin (Cpt), etoposide (Eto), β-lapachone (βLap), merbarone (Mer) as indicated. Shown is the fold activation by Gadd45a (A) or Gal-Elk1 (B) over control transfected cells. Error bars represent standard deviation. Significance was assessed via unpaired Student's t-test using the control sample as reference: ** = p<0.01. (C) Western blot analysis of EGFP expression. Whole cell extracts of HEK293T cells transiently transfected with in vitro methylated pOctTK-EGFP reporter with Gadd45a or pBl-KS (control), with or without gemcitabine treatment as indicated.
	Figure 2. Gemcitabine impairs Gadd45a mediated demethylation.(A) Methylation-sensitive Southern blot. HpaII in vitro methylated plasmid pOctTK-EGFP was recovered from HEK293T cells after transient co-transfection with Gadd45a or pBl-KS (control), with 65 h gemcitabine treatment as indicated. Recovered plasmids were digested with the indicated restriction enzyme and the products analyzed by Southern blot using a GFP probe. (B) Bisulfite sequencing analysis of five HpaII sites within the pOctTK regulatory region upon transient transfection and treatment as in (A). White and black circles, unmethylated, methylated CpG, respectively. Arrow marks EGFP translation start site.
	Figure 3. Gadd45a mediated DNA demethylation is unaffected by BER inhibitors.Methylation status of the HpaII site −299 (see Fig. 2B) in the pOctTK-EGFP regulatory region was assayed by methylation sensitive PCR 48 h after transient co-transfection with or without hGadd45a. Cells were treated with the topoisomerase II inhibitor etoposide (Eto), the NER inhibitors gemcitabine (Gem) and camptothecin (Cpt), or the BER inhibitors CRT 0044876 (CRT), betulinic acid (Bet) and ABT-888 (ABT) as indicated. Untransfected unmethylated (non-me) and HpaII in vitro methylated reporter plasmid (me) served as reference. Significance was assessed via unpaired Student's t-test using the untreated Gadd45a transfected sample as reference: * = p<0.05; = p<0.01; * = p<0.001.
	Figure 4. Gemcitabine inhibits unscheduled DNA synthesis of methylated DNA.Methylated oct4 plasmid was injected with or without BrdU into Xenopus oocytes in presence or absence of gemcitabine (Gem) and recovered after incubation (see diagram). To control for equal loading, in vitro BrdU labeled luciferase plasmid (Luc) was added after oocyte lysis. PCR analysis of immunoprecipitated DNA using oct4 or Luc specific primers was carried out.
	Figure 5. Gemcitabine does not affect global methylation levels.(A–B) Methylation of chromosome 1 satellite 2 (C1S2) in HEK293 (A) or MCF7 cells (B) was analyzed by combined bisulfite restriction analysis (COBRA). Note that neither gemcitabine (Gem, 33–134 nM) nor etoposide (Eto, 43 nM) treatment affect C1S2 methylation. C1S2 methylation of RKO cells (A, right) and HCT116 cells (116, B, right) serve as control for high C1S2 methylation. U, undigested (unmethylated) PCR amplicon; M, digested (methylated) restriction fragment. (C) COBRA analysis of C1S2 methylation in HCT116 cells as in (A, B). Cells were transfected with X. tropicalis Gadd45a (xtGadd45a) or treated with 5-aza-2′-deoxycytidine (AZA). U, undigested (unmethylated) PCR amplicon; M, digested (methylated) restriction fragment. (D) Capillary electrophoresis (CE) of DNA 5-methylcytosine (5mC). HCT116 cells were either treated with AZA or transiently transfected with X. tropicalis Gadd45a (xtGadd45a). After 48 h 5mC levels were determined by CE. Error bars represent standard deviation. Significance was assessed via Student's t-test using the untreated sample as reference: * = p<0.05.
	Figure 6. Gemcitabine induces hypermethylation and silencing of MLH1.(A) Methylation sensitive PCR analyzing MLH1 promoter methylation state in MCF7 or HEK293 cells. Cells were treated as indicated with increasing concentrations of gemcitabine (Gem, 33–134 nM) or etoposide (Eto, 43 nM) as control. HpaII restriction is methylation sensitive and allows the quantification of the MLH1 methylation state. As control, samples were also treated with the methylation insensitive isoschizomer MspI. Error bars represent the standard deviation of three biological replicates. Significance was assessed via unpaired Student's t-test using the untreated sample as reference: * = p<0.05; ** = p<0.01. (B) Cells were treated as in (A). Relative expression of MLH1 normalized to GAPDH was monitored by qPCR. Error bars represent the standard deviation of three biological replicates. Significance was assessed via unpaired Student's t-test using the untreated sample as reference: * = p<0.05; = p<0.01; * = p<0.001.

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