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. 2011 Nov 21;24(11):2040-3.
doi: 10.1021/tx2003849. Epub 2011 Nov 1.

Cyclophosphamide perturbs cytosine methylation in Jurkat-T cells through LSD1-mediated stabilization of DNMT1 protein

Jing Zhang  1 , Bifeng Yuan, Fan Zhang, Lei Xiong, Jiang Wu, Sriharsa Pradhan, Yinsheng Wang
Affiliations

Cyclophosphamide perturbs cytosine methylation in Jurkat-T cells through LSD1-mediated stabilization of DNMT1 protein

Jing Zhang et al. Chem Res Toxicol. .

Abstract

Aberrant cytosine methylation is known to be associated with cancer development. Here, we assessed how common cancer chemotherapeutic agents perturb cytosine methylation in Jurkat-T acute lymphoblastic leukemia cells. We tested six antitumor agents and found that cyclophosphamide induced the most pronounced increase in global DNA cytosine methylation after a 24-h treatment. Long-term treatment with cyclophosphamide led to a time-dependent increase in cytosine methylation level with up to 4 days of treatment, and the extent of cytosine methylation returned to normal level after 8 days. The trend of change in DNA methylation level paralleled that of the expression level of DNMT1 protein, whereas no significant increase in DNMT1 mRNA level was observed. Previous studies showed that the stability of endogenous DNMT1 protein is regulated by lysine methylation through histone lysine methyltransferase Set7 and lysine-specific demethylase 1 (LSD1), with the methylated DNMT1 being the target for proteasomal degradation. We observed that the elevated expression of DNMT1 protein at 4 days of treatment was correlated with the increased expression of LSD1 protein and with the decreased frequency of K142 methylation in DNMT1. Taken together, our results showed that cyclophosphamide perturbed temporarily global cytosine methylation in Jurkat-T cells via regulation of the lysine methylation level in DNMT1.

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Figures

Figure 1
Figure 1
(A) The HPLC trace for the separation of nucleoside mixture from the enzymatic digestion of genomic DNA isolated from untreated Jurkat-T cells. Shown in the insets are the tandem mass spectrum of the [M+H]+ ion of 5-mdC (m/z 242) and the expanded chromatogram to visualize better the 5-mdC peak. “G” represents guanosine. (B) The level of cytosine methylation in Jurkat-T cells that were exposed to the indicated concentrations of anticancer drugs for 24 hr. Results are reported as the percentages of cytosine being methylated (see Experimental Procedures). (C) Cyclophosphamide-induced alteration of 5-mdC levels in drug-exposed Jurkat-T cells was monitored for up to 10 days. The culture medium was changed and freshly prepared drug was added daily at a final concentration of 10 µM. Results shown in (B) and (C) are the means and standard deviations of data from three independent drug exposure and HPLC measurements.
Figure 2
Figure 2
Western blot results for DNMT1 (A), LSD1 (B), and Set7 (C) and DNMT1 with monomethylation on K142 (D) in the whole cell lysate of Jurkat-T cells treated with 10 µM cyclophosphamide for the indicated periods of time. The culture medium was changed and freshly prepared drug was added daily at a final concentration of 10 µM. β-actin was used a loading control.
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