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. 2009 Jan 15;113(3):659-67.
doi: 10.1182/blood-2008-02-140038. Epub 2008 Oct 17.

Mechanisms of resistance to 5-aza-2'-deoxycytidine in human cancer cell lines

Taichun Qin  1 , Jaroslav Jelinek, Jiali Si, Jingmin Shu, Jean-Pierre J Issa
Affiliations

Mechanisms of resistance to 5-aza-2'-deoxycytidine in human cancer cell lines

Taichun Qin et al. Blood. .

Abstract

5-aza-2'-deoxycytidine (DAC) is approved for the treatment of myelodysplastic syndromes, but resistance to this agent is common. In search for mechanisms of resistance, we measured the half maximal (50%) inhibitory concentration (IC(50)) of DAC and found it differed 1000-fold among a panel of cancer cell lines. The IC(50) was correlated with the doses of DAC that induced the most hypomethylation of long interspersed nuclear elements (LINE; R = 0.94, P < .001), but not with LINE methylation or DNA methyltransferase 1 (DNMT1), 3a, and 3b expression at baseline. Sensitivity to DAC showed a low correlation (R = 0.44, P = .11) to that of 5-azacytidine (AZA), but a good correlation to that of cytarabine (Ara-C; R = 0.89, P < .001). The 5 cell lines most resistant to DAC had a combination of low dCK, hENT1, and 2 transporters, and high cytosine deaminase. In an HL60 clone, resistance to DAC could be rapidly induced by drug exposure and was related to a switch from heterozygous to homozygous mutation of DCK. Transfection of wild-type DCK restored DAC sensitivity. DAC induced DNA breaks as evidenced by H2AX phosphorylation and increased homologous recombination rates by 7- to 10-fold. These results suggest that in vitro resistance to DAC can be explained by insufficient incorporation into DNA.

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Figures

Figure 1
Figure 1
Dose-dependent hypomethylation induction by DAC in different cell lines. (A) IC50 of DAC, AZA, and Ara-C in human cancer cell lines. We measured IC50 of DAC, AZA, and Ara-C in a panel of human cancer cell lines, and correlated IC50 of DAC versus IC50 of Ara-C, IC50 of DAC versus IC50 of AZA, respectively. (B) Dose-dependent hypomethylation induction by DAC in different cell lines. After treatment with DAC for 4 days, cells were collected, and DNA was extracted. LINE methylation was measured by bisulfite pyrosequencing analysis. In each cell line, except the most resistant cells (bottom graph), the dose-dependent curve was U-shaped. (B) Absence of correlation of the IC50 of DAC with LINE methylation at baseline (R = 0.05, P = .97). (C) Correlation between the IC50 of DAC with the doses of DAC required for the maximum hypomethylation of LINE (R = 0.94, P < .001).
Figure 2
Figure 2
Pharmacologic mechanisms of resistance to DAC. (A) DNMT1 protein expression was cell replication–dependent. We measured RKO cell growth curve, and DNMT1 protein expression on days 1, 3, and 5 by Western blot analysis. β-Actin was used as a control. (B) DNMT1, 3a, and 3b protein expression was independent of sensitivity to DAC and LINE methylation in different cancer cell lines. We collected exponentially growing cancer cells, extracted protein, and performed Western blot analysis of DNMT1, 3a, and 3b. β-Actin was used as a control. (C) dCK protein expression in several cell lines. dCK protein expression was measured by Western blot analysis. (D) Correlation of different nucleoside metabolic gene expression with the IC50 of DAC. DCK, CDA, hENT1, and hENT2 expressions were measured by real-time PCR using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a control. R and P values reflect Spearman correlation analysis of the IC50 of DAC with the relative gene expression.
Figure 3
Figure 3
dCK deficiency in resistant HL60R cells. (A) DAC hypomethylation induction in HL60D and DAC-resistant HL60R. We treated the cells with DAC (0.2-50 μM) and measured LINE methylation by bisulfite pyrosequencing analysis. (B) RILgene expression. (C) Inhibition of AZA of cell growth. Cells were treated with AZA (0.25-25 μM), and cell viability was measured by trypan blue exclusion. (D) LINE hypomethylation after AZA treatment. (E) Ara-CTP production, as measured by HPLC analysis using [3H] Ara-C as a substrate, was lost in HL60R-resistant cells. (F) dCK activity, as measured by phosphorylation of [3H]Ara-C in cell extracts, was also lost in resistant cells. (G) dCK protein expression was measured by Western blot analysis. β-Actin served as a control. (H) DCK mRNA expression was measured by quantitative PCR.
Figure 4
Figure 4
Spontaneous origin of resistance to DAC. (A) Two subclones of the HL60 cell line. HL60D developed a heterozygous 454C>G point mutation of DCK. This mutation was absent in another batch of HL60 obtained from ATCC. (B) The HL60D cell line had a growth advantage over the HL60 cell line. Cell number was counted from day 1 to 7. (C) Heterozygous deletion in exon 1 of DCK in HL60R cells. (D) Deletion was preexisting in HL60D cells and absent in HL60 cells. We designed a set of primers that spanned the deleted region and amplified that region from parental HL60D cells. (E) HL60R cells transfected with DCK restored sensitivity to DAC. We transfected wild-type DCK into HL60R and selected stably transfected cells by G418. HL60R and DCK-transfected (HL60T) cells were treated with DAC at 0.02, 0.2, 2, and 20 μM, respectively. Cell viability was counted. (F) Transfection of DCK cDNA restored dCK protein expression. dCK protein expression was measured in HL60R and HL60T cells by Western blot analysis.
Figure 5
Figure 5
DAC-induced origin of DAC resistance. (A) Induction of the phosphorylation of the histone H2AX by DAC treatment. HL60D cells were treated with DAC (0.2 and 2 μM) for 3 days, and H2AX phosphorylation was measured by Western blot analysis. β-Actin was used as a control. (B) DAC treatment increased HRR rates. Cells with stable PLNCX-GZ transfection were treated with DAC for 4 days and maintained in drug-free medium for 1 week. Colonies resistant to Zeocin were selected and maintained in methylcellulose medium for 2 weeks. HRR rates were calculated as described in “Measurement of homologous recombination repair.” (C) Verification of intrachromosomal recombination in zeocin-resistant colonies by PCR with recombination-specific primers. PCR-amplified DNA from G418-resistant colonies produced a 2.2-kb fragment diagnostic for the nonrecombined tandem repeats. PCR-amplified DNA from G418- and zeocin-resistant colonies generated a diagnostic 1.1-kb fragment.
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