Cancer Therapy: Preclinical

Effect of and Decitabine in Combination in Human Leukemic Cell Lines Tai c hun Q in , 1Emile M.Youssef,1Jaroslav Jelinek,1Rong Chen,1 Allen S. Yang,2 Guillermo Garcia-Manero,1andJean-PierreJ. Issa1

Abstract Purpose: 1-h-D-Arabinofuranosylcytosine (cytarabine; ara-C) is the most active agent in myeloid leukemia. 5-Aza-2¶-deoxycytidine (DAC) is a cytosine analogue that inhibits DNA methylation and also has activity in myeloid leukemia.Therefore, we investigated combining these two drugs in human leukemia cell lines in vitro. Experimental Design: We initially examined the effects of ara-C and DAC on human leukemia cell lines HL60, ML-1,RAji, andJurkat.We measured IC50 of DAC and ara-C in these cell lines and calculated a combination index of these two drugs given either simultaneously or sequentially. In searching for mechanisms relative to epigenetic regulation for this effect, we examined DNA methylation of LINE and Alu repetitive elements as a surrogate for global genomic DNA methyla- tion. In addition, we sorted Annexin V positive and negative cells and measured differences in LINE methylation between them. Results: The combination of DAC and ara-C showed additive induction of cell death in ML-1 and synergistic induction in HL60, Raji, and Jurkat. Sequentially, DAC followed by ara-C was a synergistic combination in all cell lines. Low-dose DAC induced more hypomethylation than high doses of the drug, whereas ara-C had no effects on methylation. The combination of ara-C with DAC either together or DAC followed by ara-C resulted in inhibition of LINE demethylation in HL60. The RIL gene, which is silenced by DNA hypermethylation, was activated by DAC, but the addition of ara-C to DAC reduced RIL gene activation. DAC treatment increased H3 Lys9 acetylation of Alu elements, whereas ara-C had no effect, and the addition of ara-C to DAC inhibited this effect. Finally, we showed that after DAC exposure, Annexin V positive cells were more hypomethylated than AnnexinV negative cells. Conclusion: The combination of DAC and ara-C showed additive or synergistic effects on cell death in four human leukemia cell lines in vitro, but antagonism in terms of epigenetic effects. One possible explanation for these paradoxical observations is that hypomethylated cells are sensi- tized to cell killing by ara-C. These data suggest that DAC used in combination with ara-C has clinical potential in the treatment of .

Epigenetics have acquired increased recognition as a driving traps DNA methyltransferase in the form of a covalent force in human leukemia over the past few years, leading protein–DNA adduct.As a result, cellular DNA methyltrans- to the revival of interest in DNA methylation inhibitors as ferase is rapidly depleted, and concomitantly genomic DNA is antineoplastic agents (1).5-Aza-2 ¶-deoxycytidine (DAC) is a hypomethylated during continued DNA replication, resulting potent that incorporates into DNA and in replication-dependent DNA hypomethylation.Gene inacti- vation (‘‘silencing’’) of tumor-suppressor, growth-inhibitory, DNA repair, and several apoptosis genes can be mediated by DNA hypermethylation of gene promoters (2–5).DAC- Authors’ Affiliations: 1Department of Leukemia, The University of Texas M. D. induced hypomethylation is associated with reactivation of Anderson Cancer Center, Houston,Texas and 2Department of Medicine, University multiple genes, and it is thought that this effect on gene of Southern California, Norris Comprehensive Cancer Center, Los Angeles, expression contributes to the mechanism of responding to the California Received 11/21/06; revised 4/4/07; accepted 5/9/07. drug.DAC is active in myeloid leukemia (6) and was recently Grant support: NIH grants CA100632 and CA108631. approved by the Food and Drug Administration for the The costs of publication of this article were defrayed in part by the payment of page treatment of MDS. charges. This article must therefore be hereby marked advertisement in accordance The deoxycytidine analogue 1-h-D-arabinofuranosylcytosine with 18 U.S.C. Section 1734 solely to indicate this fact. Note: J-P.J. Issa is an American Cancer Society Clinical professor. (cytarabine, ara-C) is the most commonly used drug in the Requests for reprints: Jean-Pierre J. Issa, Department of Leukemia-428, treatment of acute myeloid leukemia (7).Ara-C exerts its University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, cytotoxicity by inhibiting DNA polymerase or by terminating @ Houston,TX 77030. Phone: 713-745-2260; Fax: 713-745-2261;E-mail: jpissa chain elongation upon incorporation into newly synthesized mdanderson.org. F 2007 American Association for Cancer Research. DNA (8, 9).DAC and ara-C have similar structures and meta- doi:10.1158/1078-0432.CCR-06-2762 bolic pathways.Once inside the cell, both are phosphorylated

www.aacrjournals.org 4225 Clin Cancer Res 2007;13(14) July 15, 2007 Downloaded from clincancerres.aacrjournals.org on October 4, 2021. © 2007 American Association for Cancer Research. Cancer Therapy: Preclinical to the monophosphate form by the rate-limiting enzyme, Materials and Methods deoxycytidine kinase.Subsequently, they are converted to diphosphate and triphosphate forms by other cellular kinases. Cell culture and treatment protocols. The human leukemic cell lines The fact that both DAC and ara-C are active in myeloid HL60, ML-1, Raji, and Jurkat were obtained from American Type leukemia through different mechanisms of action has trig- Collection.The cells were grown in RPMI 1640 plus 10% heat- gered interest in combining them.However, the effect of inactivated FCS in plastic tissue culture plates in a humidified j combining these two nucleoside analogues remains contro- atmosphere containing 5% CO2 at 37 C.For the growth inhibition  5 versial.Combination of the two drugs showed antagonism in assay, cells were placed at a density of 1 10 /mL in 5 mL of medium L1210 mouse leukemia, murine leukemic L5178 Y cell lines, and split 24 h before treatment.Different concentrations of DAC and and pediatric acute myeloid leukemia cells in vitro (10).The ara-C alone or in combination were added to the medium simulta- neously.To measure IC , fresh DAC and ara-C was added every mechanism was presumed to be either that both of these 50 24 h without changing medium.The doses that inhibited 50% drugs competed for deoxycytidine kinase (11–13) or that proliferation (IC50) were analyzed by the median-effect method ara-C reduced DAC incorporation through inducing (CalcuSyn software, Biosoft). In vitro cytotoxicity was assayed in arrest.A synergistic effect was found in a deoxycytidine triplicate by the following experimental conditions: DAC alone, ara-C kinase–deficient HL-60 cell line, and in pediatric patients alone, DAC + ara-C, and DAC followed by ara-C.The proportion of live with refractory acute lymphocytic leukemia by sequentially cells in treated plates was measured by trypan blue exclusion.The adding ara-C to DAC (14–16).The mechanism was proposed effects of combinations were estimated using the CalcuSyn software, to be that ara-C induced deoxycytidine kinase silencing by which was developed based on the median-effect method created by hypermethylation, which could be reactivated by DAC. Chou and Talalay (17). However, these studies used high doses of DAC, which may Pyrosequencing-based methylation analysis. Genomic DNA was prepared from cells and bisulfite conversion of genomic DNA was not be relevant to the clinic.There is little data on the carried out.The Alu and LINE element PCR assay was used to measure epigenetic effects of combination of these drugs in human global methylation and was modified for pyrosequencing-based leukemia cell lines.This study investigated further the methylation analysis (18).PCR cycling conditions were 96 jC for combination of ara-C and DAC in four human leukemic cell 90 s, 43jC for 60 s, and 72jC for 120 s for 40 cycles.The PCR product lines. was bound to Streptavidin Sepharose HP (Amersham Biosciences) and

Fig. 1. Effects of DAC and ara-C on LINE methylation and cell viability. A, effects of DAC on cell growth in leukemia cell lines. Cells were plated inT-25 plates at a density of  5 1 10 /mL and treated with different doses of DAC from 30 nmol/L to 30 Amol/L. Cell viability was measured by trypan blue exclusion and IC50 was calculated by the median-effect method. B, effects of ara-C on cell growth in leukemia cell lines. Cells were treated with different doses of ara-C from 10 nmol/L to 10Amol/L. Cell viability was measured. C, low-dose DAC induced optimal hypomethylation in HL60. Cells were treated with different doses of DAC from 20 nmol/L to 50 Amol/L. Cell pellets were collected and DNA was extracted for bisulfite treatment. LINE methylation was measured by pyrosequencing-based analysis. D, recovery of LINE methylation.We treated HL60 with DAC 0.2 and 2 Amol/L, and then washed the medium away.The recovery of LINE methylation was measured at days 3, 5, and 20 after that.

Clin Cancer Res 2007;13(14) July 15, 2007 4226 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on October 4, 2021. © 2007 American Association for Cancer Research. Cytarabine and Decitabine in Human Leukemic Cell Lines the Sepharose beads containing the immobilized PCR product were purified, washed, denatured using a 0.2 mol/L NaOH solution, and Table 1. Measurement of IC50 of DAC and ara-C in washed again.Then, 0.3 Amol/L pyrosequencing primer was annealed human leukemia cell lines to the purified single-stranded PCR product and pyrosequencing was done using the PSQ HS 96 Pyrosequencing System (Pyrosequen- Cell lines IC50 cing, Inc.). Methylation quantification was done using the provided DAC (nmol/L) Ara-C (nmol/L) software.Primers were as follows: Alu forward primer (5 ¶-GGGACACC- HL60 200 F 54 37 F 6.1 CCTGATCGTATATTTTTATTAAAAATATAAAAATTAGT-3¶), reverse ¶ ¶ ML-1 98 F 27 17 F 1.9 primer (5 -CCAAACTAAAATACAATAA-3 ), biotinylated universal primer Raji 54 F 21 16 F 3.2 ¶ ¶ (5 -GGGACACCGCTGATCGTTTA-3 ), and pyrosequencing primer Jurkat 1,200 F 180 72 F 9.1 (5¶-AATAACTAAAATTACAAAC-3¶); LINE forward primer (TTTTT- TGAGTTAGGTGTGGG), biotin-labeled reverse universal primer (GGGA- NOTE: Values are presented as mean F SE of three independent CACCGCTGATCGTTTATCTCACTAAAAAATACCAAACAA), and experiments. pyrosequencing primer (GGGTGGGAGTGAT). Annexin V staining and sorting. For this experiment, cells were treated with fresh drugs and medium daily for 4 days.Cells were collected, washed, and stained with 100 AL Annexin V incubation reagents (TACS kits, TREVIGEN).Apoptosis was analyzed by the BD blue exclusion.At high doses, ara-C resulted in virtually no FACSCalibur assay and Annexin V positive and negative cells were viable cells, whereas a fraction of cells was still alive at the sorted with the BD FACSVantage Hi-Speed Flow Cytometer.Annexin V highest dose of DAC (Fig.1A and B).Global methylation using FITC and propidium iodide emission was collected with 530/30 and the DNA repetitive element LINE as a marker was measured by 575/26 nm filters, respectively.DNA was extracted from both Annexin pyrosequencing-based analysis.LINE was heavily methylated V positive and negative cells for bisulfite treatment.LINE methylation in HL60. We treated HL60 cells with DAC at 0.02, 0.2, 1, 2, 5, was analyzed by pyrosequencing-based analysis. 20, and 50 Amol/L daily for 4 days.Low-dose DAC induced Quantitative reverse transcription-PCR. Real-time quantitative hypomethylation and this effect was lost at high doses reverse transcription-PCR was done with the ABI 7700 Sequence (Fig.1C).The methylation level was almost normal after Detector (Applied Biosystems).We used commercially available 50 Amol/L DAC treatment.Ara-C treatment at different doses primers sets with minor groove binder probe for PDLIM4 (RIL) and h-actin as an internal control (Applied Biosystems).Total cellular RNA had no effect on global methylation (data not shown). was extracted by TRIzol reagent (Life Technologies).RNA was eluted Hypomethylation of LINE was not stable, as the methylation with RNase-free water, quantified at an absorbance at 260/280 nm, and level quickly recovered in drug-free medium back to normal used for first-strand cDNA synthesis.Reactions for quantitative reverse levels.Interestingly, we observed that low-dose DAC (0.2 Amol/L) transcription-PCR were done with the TaqMan universal PCR Master treated cells had a slower recovery of methylation level than Mix kit (Applied Biosystems) in 96-well plates.Each sample was those treated at a higher dose (DAC 2 Amol/L; P < 0.01 on measured in triplicate.Assembled plates were then covered and run day 3 and P < 0.05 on day 5; Fig. 1D). j using the following conditions: an initial denaturation step of 95 C for Synergism of combining DAC and ara-C in leukemia cell j j 10 min followed by 45 cycles at 95 C for 15 s and 60 C for 1 min.The lines. We first measured IC of DAC and ara-C in different resulting data were analyzed with ABI Prism 7000 SDS software 50 cell lines.IC 50 for DAC varied from 54 nmol/L in Raji to 1,200 (Applied Biosystems).The threshold cycles ( CT) were determined and nmol/L in Jurkat, whereas IC50 for ara-C varied from 16 nmol/L the differences in the CT values for b-actin and RIL were calculated. Chromatin immunoprecipitation assays. The protocols for chromatin in Raji and 72 nmol/L in Jurkat (Table 1).To evaluate the immunoprecipitation have been described previously (19).Briefly, cells effects of combining ara-C and DAC on cell viability, we used a were treated with formaldehyde to cross-link histones to DNA.After fixed ratio of IC50 for DAC and ara-C in all the groups, which washing, the cells were sonicated.The lysate was then divided into three is equivalent to 1/8, 2/8, 4/8, 6/8, and 8/8 IC50 of DAC and fractions; the first and second ones were diluted in lysis buffer, and the ara-C, respectively.The combination of DAC and ara-C simul- third one was used for input control.The first lysate was incubated with taneously or sequentially was synergistic in most cell lines, A 9 10 L of anti-Lys acetylated histone H3 antisera (Upstate Biotechnol- indicated by combination index (CI) values of <0.8 (Fig. 2). In j ogy) at 4 C overnight.To collect the immunoprecipitated complexes, ML-1 only, combining DAC and ara-C was additive rather than protein A-Sepharose beads (Pharmacia Biotech) were added and synergistic.For example, The CI value in Raji cells ranged from incubated for 1 h at 4jC.After washing, the beads were treated with RNase for 30 min at 37jC and then proteinase K overnight.The cross- 0.35 to 0.87 with DAC and ara-C simultaneously, and from links were then reversed by heating the sample at 65jC for 6 h.DNA 0.19 to 0.46 with ara-C after DAC. No antagonism (CI > 1.2) was extracted, and PCR amplification of DNA was carried out on was observed in any cell line.To determine whether apoptotic diluted DNA aliquots, using Alu forward primer (GCACCCAGCT- cell death is responsible for ara-C– and DAC-induced decrease GAATTTTCCA) and Alu reverse primer (CTGGGTTTGGCTTTTGAA- in cell viability, we did flow cytometry analysis (fluorescence- TTG).The PCR products we revisualized by agarose gel electrophoresis activated cell sorting) and Annexin V staining.Cells were and quantitated by capillary electrophoresis using the Agilent 2100 treated with ara-C 50 nmol/L in combination with DAC 0.2 bioanalyzer (Agilent Technologies). and 2 Amol/L.Fresh medium and drugs were added every 24 h. The synergistic effect of DAC plus ara-C on apoptosis was Results observed.(Fig.3A). Ara-C inhibits DAC-induced epigenetic effects. In search for Low-dose DAC induces optimal hypomethylation. We initially mechanisms of synergistic or additive effects of combining examined effects of DAC and ara-C on global genomic DAC and ara-C, we examined the effects of ara-C and DAC on methylation and cell viability.We treated with DAC and ara- DAC-induced epigenetic modulation.We used LINE and Alu C at different doses daily for 4 days.DAC and ara-C induced as markers of global methylation (20).First, we studied LINE cell death in a dose-dependent manner as measured by trypan methylation after ara-C alone and found that ara-C 50 or

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Fig. 2. CI plots of DAC ^ ara-C combinations in human leukemia cell lines. A, CI plots treated with DAC and ara-C simultaneously. The effect of the combinations was assessed by trypan blue exclusion 4 d after cells were incubated with drugs: DAC alone, ara-C alone, and DAC + ara-C. B, CI plots treated with ara-C after DAC. Cells were divided into three groups: DAC alone, ara-C alone, and DAC followed by ara-C. Cells were treated with DAC for 4 d, maintained in fresh medium for 1d, and then treated with ara-C for 4 d. The combinations were in a fixed molar ratios based on the IC50 values of each drug. The effects of combinations were estimated using the CalcuSyn software, which was developed based on the median-effect method. CI < 0.8 indicates synergy; CI = 0.8 to 1.2 is additive; and CI > 1.2 means antagonism.

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Fig. 3. Ara-C inhibits DAC-induced epigenetic effects.A, the combination of DAC and ara-C simultaneously was synergistic on apoptosis. HL60 cells were treated with DAC 0.2 and 2 Amol/L and ara-C 50 nmol/L alone or simultaneously for 4 d. Cells were harvested, fixed with cold 70% ethanol, stained with propidium iodide, and analyzed by fluorescence-activated cell sorting. Apoptosis was assayed by the appearance of a sub-G1population. B, combination of ara-C with DAC reduced LINE demethylation. After HL60 cells were treated with DAC and ara-C, LINE methylation was measured. C, combination of ara-C and DAC reduced Alu demethylation. D, RIL gene methylation in HL60. Aberrant methylation was detected by bisulfite-PCR followed by restriction enzyme digestion. Unmethylated bands (top) and methylated bands (bottom, arrow)werequan- titated by densitometry. E, RIL gene expression. RIL gene expression was assayed by RT-PCR using S-14 internal control or real-time PCR using h-actin as internal control (data not shown). F, Alu H3 Lys9 acetylation. Chromatin immunoprecipitation assay was conducted using antibodies against H3 Lys9. PCR of Alu repetitive elements was done. The intensity of the PCR bands was quantitated byAgilent 2100 bioanalyzer. Columns, ratios of precipitated DNA over input calculated as a relative precipitated fold.

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Fig. 4. Ara-C preferentially kills hypomethylated cells. A, ara-C after DAC treatment was synergistic on apoptosis. HL60 cells were treated with DAC 0.2 Amol/L for 4 d; cells were centrifuged and maintained in drug-free medium for 1d before the addition of ara-C for 4 d.The sub-G1fraction of cells were determined by fluorescence-activated cell sorting analysis. B, ara-C after DAC treatment reduced LINE demethylation. After treatment, LINE methylation was measured. C, AnnexinV staining for apoptosis assay in HL60 cells. Apoptosis was assayed byAnnexinV staining and fluorescence-activated cell sorting analysis.The percentage of apoptotic cells (AnnexinV positive) was indicated in the top right gradient. D, LINE methylation in AnnexinV positive and negative HL60 cells. Cells were treated and centrifuged everyday to remove the debris during treatment. At the end of the treatment, cells were stained with AnnexinV; AnnexinV positive and negative cells were sorted using a BDFACSVantage Hi-Speed Flow Cytometer. LINE methylation was measured. Columns, mean of three separate experiments undertaken in triplicate plates. Student’s t test was used to compare the significance between two groups (*, P < 0.05). E, a model to explain how ara-C reduces LINE demethylation. DAC-induced hypomethylation in HL60 is not homogeneous. Cells with less methylation are prone to death or to be AnnexinV positive, whereas cells with higher methylation are AnnexinV negative. The addition ofara-CtoDACkilled more hypomethylated cells, leading to elevation of LINE methylation in both AnnexinV positive and negative cells.

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500 nmol/L had no effects on methylation.After DAC 0.2and (26.4% versus 30%, data P < 0.05). When ara-C 50 nmol/L was 2 Amol/L alone, the methylation decreased from 64% to 18% added to DAC 0.2 Amol/L, LINE methylation of Annexin V and 22%, respectively (P < 0.01 compared with control). When positive and negative cells was decreased to 32% and 35%, ara-C 50 nmol/L was added, LINE methylation decreased to respectively (P < 0.01 and P < 0.01 compared with DAC alone, 31% and 42%, respectively (P < 0.01 compared with DAC respectively; Fig.4D).These are minimal estimates of the alone; Fig.3B).High-dose ara-C (500 nmol/L) added to DAC differences because dead cells would not be sorted in this 0.2 and 2 Amol/L was more efficient at inhibiting hypometh- experiment.Our data are consistent with a model whereby ylation (42% and 57%, respectively) than low-dose ara-C. hypomethylation sensitizes cells to ara-C, leading to cell death Similarly, ara-C treatment inhibited Alu hypomethylation by at a higher LINE methylation threshold (Fig.4E). DAC (Fig.3C).The ara-C effects were observed with DAC at A 0.2 and 2 mol/L, which induced optimal hypomethylation.To Discussion investigate whether ara-C affects DAC-induced gene reactiva- tion, we studied RIL, a gene previously found methylated in DAC and cytarabine are two of the drugs that have the leukemia and silenced by DNA hypermethylation in HL60. highest single-agent activity in myeloid neoplasms, making a DAC treatment at 0.2, 2, and 5 Amol/L induced hypometh- combination of the two an obviously attractive clinical ylation (Fig.3D) and gene activation (Fig.3E).Ara-C alone had proposition.However, both are cytosine analogues that use no effect on gene expression.However, the addition of ara-C to deoxycytidine kinase as a first step toward DNA incorporation DAC inhibited RIL gene expression by reverse transcription- (23, 24).Consequently, there has been serious theoretical and PCR (Fig.3E) and real-time PCR ( P < 0.01, data not shown). experimental concern for antagonism between the two drugs DNA methylation targets histone modifications, which also (11).However, we show clearly that, at low but clinically play critical roles in epigenetic silencing (21, 22).Therefore, we relevant doses of DAC, no antagonism is observed; rather, examined the effects of inhibition of DNA methylation on Alu synergistic effects are seen in most experimental conditions H3-Lys9 acetylation (a mark of active chromatin).To this end, with additivity seen in one.Interpretation of these data and we did chromatin immunoprecipitation on HL60 cells treated extrapolation to the clinic are complicated by the limitations of with DAC 2 Amol/L daily for 4 days.As shown in Fig.3F, Alu the assays used.Although cytotoxicity and induction of cell elements showed an increasing level of H3-Lys9 acetylation death are appropriate end points for cytarabine, which works after DAC.Ara-C alone had no effects on acetylation, but the primarily by interrupting DNA synthesis, the clinical effects of addition of ara-C to DAC reduced H3-Lys9 acetylation. DAC are thought to reflect its epigenetic mechanism of action Ara-C preferentially kills hypomethylated cells. The data (reactivation of genes), which may not be well represented in discussed above show that ara-C inhibited DAC-induced epi- cytotoxicity assays.Indeed, clinical responses to this agent genetic activation (hypomethylation, gene expression, histone in vivo take a long time, often requiring multiple exposures, and acetylation).This could be due to direct inhibition of DAC have been hypothesized to include differentiation and immune activity (e.g., reduced incorporation into DNA), to the fact that activation components (1), all effects that are difficult to model both drugs work on the same subset of cells (dividing cells), or in vitro.Nevertheless, our data show that DAC does not lessen to preferential killing of hypomethylated/gene-activated cells cytarabine cytotoxicity and indeed may potentiate it in most by ara-C.To test these possibilities, we first examined the effects conditions tested.It is reassuring enough that a of a of ara-C administered after DAC treatment, reasoning that combination of the two agents can now be seriously envisaged. ara-C given after DAC should not interfere with incorporation Interestingly, although the combination of cytarabine and or direct activity of DAC.HL60 cells were treated with DAC DAC showed greater apoptosis induction than either agent (0.2 Amol/L) daily for 4 days, followed by 1 day of rest.After alone, we also observed antagonism in terms of epigenetic that, cells were exposed to control medium or to ara-C at 50 effects (hypomethylation, gene reactivation).Given that cytar- and 500 nmol/L.As shown in Fig.4A, ara-C 50 nmol/L after abine has no epigenetic effects alone, and the antagonism is DAC 0.2 Amol/L was synergistic on apoptosis.DAC induced seen when giving the agents together or when cytarabine hypomethylation at day 4 (from 59% to 20%) and cells follows DAC (after a period of drug wash out), it is likely that exposed to control medium showed recovery of methylation the antagonism is apparent but not real and reflects killing (from 20% to 42%; Fig.4C).When cells were treated with of the most hypomethylated cells.In other words, if cytara- ara-C 50 and 500 nmol/L, methylation seemed to increase bine were truly antagonistic, the antagonism should not significantly (47% with ara-C 50 nmol/L; 52% with ara-C have been observed when it was given after DAC.Rather, 500 nmol/L, P < 0.01 and P < 0.05 compared with DAC alone, our data are most consistent with chemosensitization by respectively; Fig.4C).Because ara-C alone has no effects on hypomethylation.Thus, if the most hypomethylated cells are methylation, we interpret this result as suggesting that ara-C is killed most readily by cytarabine, the apparent molecular killing preferentially the most hypomethylated cells. effect (which is determined on surviving cells) is of reducing To directly test the hypothesis that global genomic hypo- the amount of measured hypomethylation.Indeed, we show methylation enhances sensitivity to ara-C, we sorted Annexin V that cells undergoing apoptosis (based on Annexin V stain- positive and negative cells treated with DAC 0.2 and 2 Amol/L, ing) have measurably lower levels of methylation.Although and ara-C 50 nmol/L, alone or simultaneously for 4 days using the difference is low, it is an underestimate of the true dif- a BD FACSVantage Hi-Speed Flow Cytometer, and investigated ference given that cells that already died cannot be sorted by the LINE methylation levels of each fraction.LINE methylation Annexin V and therefore do not contribute to the measured levels in Annexin V positive (dying) cells were lower compared results. with Annexin V negative cells after DAC 0.2 Amol/L treatment It is interesting to consider possible mechanisms for chemo- (24.1% versus 26.4%, P = NS), and DAC 2 Amol/L treatment sensitization to cytarabine by DAC.One possibility relates to

www.aacrjournals.org 4231 Clin Cancer Res 2007;13(14) July 15, 2007 Downloaded from clincancerres.aacrjournals.org on October 4, 2021. © 2007 American Association for Cancer Research. Cancer Therapy: Preclinical the additive effects of DNA adducts induced by both cytarabine mechanism of action of this drug—epigenetic at low doses and and DAC.However, the sequential studies suggest that cytotoxic at high doses (via DNA adduct formation).Clinically, hypomethylation sensitizes cells to cytarabine in the absence the data stress the importance of using low doses of DAC to of active drug (DAC) in the medium.A simple hypothesis to maximize its epigenetic effects, a strategy that has had some explain this effect relates to activation of proapoptotic genes by success in myeloid malignancies (29–31).Following up on the epigenetic effects of DAC.Indeed, multiple genes such as this, the current data suggest that low-dose DAC may potentiate TMS1, HRK, and others that directly affect the process of the activity of cytarabine in myelogenous leukemia, a concept apoptosis are silenced in cancer and their activation by DAC that deserves testing in the clinic. could be one of the key determinants of chemosensitization by the drug (25–27). The finding of a U-shaped dose-response curve for hypo- Acknowledgments methylation induced by DAC is of considerable clinical interest. We thank Dr. Randall Evans for the Flow Cytometry analysis and Annexin V It recapitulates previous data on differentiation induced by sorting and Drs.William Plunkett and Rong Chen for general advice and help in using the drug in vitro (28), and it stresses the dual nature of the the CalcuSyn software.

References 1. Issa JP. DNA methylation as a therapeutic target in comitant treatment with cytarabine. CancerTreat Rep 22. Jackson-Grusby L, Laird PW, Magge SN, Moeller cancer. Clin Cancer Res 2007;13:1634 ^ 7. 19 8 6;70 :14 51 ^ 3. BJ, Jaenisch R. Mutagenicity of 5-aza-2¶-deoxycyti- 2. Toyota M, Kopecky KJ,Toyota MO, Jair KW,Willman 12. Evans JS, Mengel GD. The reversal of cytosine ara- dine is mediated by the mammalian DNA methyltrans- CL, Issa JP. Methylation profiling in acute myeloid leu- binoside activity in vivo by deoxycytidine. Biochem ferase. Proc Natl Acad Sci U S A 1997;94:4681 ^ 5. kemia. Blood 2001;97:2823 ^ 9. Pharmacol 1964;13:989 ^ 94. 23. Galmarini CM, Mackey JR, Dumontet C. Nucleoside 3. Plumb JA, Strathdee G, Sludden J, Kaye SB, Brown 13. Graham FL, Whitmore GF. The effect of h-D-arabi- analogues: mechanisms of drug resistance and rever- R. Reversal of drug resistance in human tumor xeno- nofuranosylcytosine on growth, viability, and DNA sal strategies. Leukemia 2001;15:875 ^ 90. grafts by 2¶-deoxy-5-azacytidine-induced demethyla- synthesis of mouse L-cells. Cancer Res 1970;30: 24. Townsend AJ, ChengYC. Sequence-specific effects tion of the hMLH1gene promoter. Cancer Res 2000; 2627 ^ 35. of ara-5-aza-CTP and ara-CTP on DNA synthesis by 60:6039^44. 14. GomyoY,SasakiJ,BranchC,RothJA, purified human DNA polymerases in vitro :visualiza- 4. Fulda S, Kufer MU, Meyer E, van Valen F, Dockhorn- Mukhopadhyay T. 5-Aza-2¶-deoxycytidine upregu- tion of chain elongation on a defined template. Mol Dworniczak B, Debatin KM. Sensitization for death re- lates caspase-9 expression cooperating with p53- Pharmacol1987;32:330^9. ceptor-or drug-induced apoptosis by re-expression of induced apoptosis in human cells. 25. Moriai R,Tsuji N, Kobayashi D, et al. A proapoptotic caspase-8 through demethylation or gene transfer. Oncogene 2004;23:6779 ^ 87. caspase recruitment domain protein gene, TMS1, is Oncogene 2001;20:5865 ^ 77. 15. Kong XB,TongWP, ChouTC. Induction of deoxycy- hypermethylated in human breast and gastric cancers. 5. Santi DV, Norment A, Garrett CE. Covalent bond for- tidine kinase by 5-azacytidine in an HL-60 cell line re- Anticancer Res 2002;22:4163 ^ 8. mation between a DNA-cytosine methyltransferase sistant to arabinosylcytosine. Mol Pharmacol 1991;39: 26. Liu WM, Stimson LA, Joel SP.The in vitro activity and DNA containing 5-azacytosine. Proc Natl Acad 250 ^ 7. of the tyrosine kinase inhibitor STI571 in BCR-ABL Sci U S A1984;81:6993^ 7. 16. Richel DJ, Colly LP, Willemze R. The antileukaemic positive chronic myeloid leukaemia cells: synergistic 6. Yang AS, Doshi KD, Choi SW, et al. DNA methyla- activity of 5-Aza-2-deoxycytidine (Aza-dC) in interactions with anti-leukaemic agents. Br J Cancer tion changes after 5-aza-2¶-deoxycytidine therapy patients with relapsed acute leukaemia. Bone Marrow 2002;86:1472 ^ 8. in patients with leukemia. Cancer Res 2006;66: Transplant 1989;4 Suppl 3:64. 27. Nakamura M, Ishida E, Shimada K, Nakase H, Sakaki 5495 ^503. 17. Chou TC, Talalay P. Quantitative analysis of dose- T, Konishi N. Defective expression of HRK is associat- 7. Johnson SA. Nucleoside analogues in the treatment effect relationships: the combined effects of multiple ed with promoter methylation in primary central ner- of haematological malignancies. Expert Opin Phar- drugs or enzyme inhibitors. Adv Enzyme Regul 1984; vous system lymphomas. Oncology 2006;70:212^ 21. macother 2001;2:929 ^ 43. 22:27^ 55. 28. Jones PA, Taylor SM. Cellular differentiation, cyti- 8. Hileman EO, Liu J, Albitar M, Keating MJ, Huang P. 18. Yang AS, Estecio MR, Doshi K, Kondo Y,Tajara EH, dine analogs and DNA methylation. Cell 1980;20: Intrinsic oxidative stress in cancer cells: a biochemical Issa JP. A simple method for estimating global DNA 85^93. basis for therapeutic selectivity. Cancer Chemother methylation using bisulfite PCR of repetitive DNA ele- 29. Frost P, Abbruzzese JL, Hunt B, Lee D, Ellis M. Pharmacol 2004;53:209^19. ments. Nucleic Acids Res 2004;32:e38. Synergistic cytotoxicity using 2¶-deoxy-5-azacyti- 9. Sampath D, Rao VA, Plunkett W. Mechanisms of ap- 19. Kondo Y, Shen L, Issa JP. Critical role of histone dine and or 4-hydroperoxycyclophospha- optosis induction by nucleoside analogs. Oncogene methylation in silencing in co- mide with human tumor cells. Cancer Res 1990;50: 2003;22:9063^74. lorectal cancer. Mol Cell Biol 2003;23:206 ^ 15. 4572^7. 10. Hubeek I, Peters GJ, Broekhuizen AJ, et al. Potenti- 20. Hwu HR, Roberts JW, Davidson EH, Britten RJ. In- 30. La Rosee P, Johnson K, Corbin AS, et al. In vitro ation of in vitro ara-C cytotoxicity by ribonucleotide sertion and/or deletion of many repeated DNA efficacy of combined treatment depends on the un- reductase inhibitors, cyclin-dependent kinase modula- sequences in human and higher ape evolution. Proc derlying mechanism of resistance in imatinib-resistant tors and the DNA repair inhibitor aphidicolin in paedi- Natl Acad Sci U S A 1986;83:3875 ^ 9. Bcr-Abl-positive cell lines. Blood 2004;103:208 ^ 15. atric acute myeloid leukaemia. Br J Haematol 2005; 21. Fuks F, Burgers WA, Brehm A, Hughes-Davies L, 31. Cameron EE, Bachman KE, Myohanen S, Herman 131:219^ 22. Kouzarides T. DNA methyltransferase Dnmt1 associ- JG, Baylin SB. Synergy of demethylation and histone 11. ColomboT, Rossi C, D’Incalci M. Antagonism of 5- ates with histone deacetylase activity. Nat Genet deacetylase inhibition in the re-expression of genes si- aza-2¶-deoxycytidine antileukemic activity by con- 2000;24:88^91. lenced in cancer. Nat Genet 1999;21:103 ^ 7.

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