(CANCER RESEARCH 47, 3672-3678,
`July 15, 1987]
`and Expression of
`Effect of 5-Azacytidine and Congeners on DNA Methylation
`Deoxycytidine Kinase in the Human Lymphoid Cell Lines CCRF/CEM/0
`and
`CCRF/CEM/dClT1
`
`Bruno E. Antonsson, Vassilios I. Avramis,2 Jonathan Nyce, and John S. Holcenberg
`
`Department of Pediatrics. School of Medicine, University of Southern California, Childrens Hospital of Los Angeles, Division of Hematology-Oncology,
`Los Angeles, California 90027; and University of Southern California, Comprehensive Cancer Center, Los Angeles, California 90033
`
`ABSTRACT
`
`and the deoxycytidine
`lines CCRF/CEM/0
`The human lymphoid cell
`kinase (dCkHeflcient
`CCRF/CEM/dCk"
`were treated with various 5-
`azacytidine
`(5-aza-C)
`nucleosides
`and the effect on DNA methylation
`and dCk activity were examined. 5-Azacytidine
`(5-aza-C), 5,6-dihydro-5-
`azacytidine
`(DHAC),
`5-aza-2'-deoxycytidine
`(5-aza-Cdr),
`and 1-/8-D-
`arabinofuranosyl-5-azacytidine
`(ara-Ai
`') reduced the DNA 5-methylcy-
`tosine level
`in the ( 'KM/0 cells, down to approximately
`10% of the level
`
`in untreated cells. The dCk activity was increased after treatment with
`the 5-aza-C nucleosides
`approximately
`10% compared to untreated cells.
`In ( I M/d( 1
`cells DNA hypomethylation
`between 50 and 25% of
`control was
`seen only after treatment with DHAC and 5-aza-C. No
`decrease
`in the methylation
`level was seen after treatment with 5-aza-
`Cdr or ara-AC. The dCk activity was increased up to 37% after treatment
`with DHAC or 5-aza-C but no increase was observed after treatment
`with 5-aza-Cdr or ara-AC. CEM/dCk" cells treated with DHAC showed
`
`frequency to cells expressing dCk activity of between 0.1 and
`a revertant
`0.6%. Goned revertant < I M/iK V cells isolated from soft agar had dCk
`activity between 31 and 113% compared to the activity
`in untreated
`CEM/0
`cells. This
`in vitro study indicates
`that DHAC and 5-aza-C
`induced dCk re-expression
`in the CEM/dCk~
`cells whereas 5-aza-Cdr
`
`and ara-AC did not.
`
`INTRODUCTION
`
`transfer of a
`The cn/y m¡uic;i11y mediated posttranscriptional
`methyl group from 5-adenosylmethionine
`to the S-carbon atom
`of cytosine results
`in the formation of 5-methylcytosine,
`the
`only modified base known to occur in mammalian DNA (1,2).
`Although the biological
`function of DNA methylation
`is im
`perfectly understood, methylation
`is considered to be one of
`several mechanisms
`involved in differentiation
`(3, 4) and regu
`lation of gene transcription
`(5, 6).
`enzymes
`restriction
`Analyses
`using methylation-sensitive
`in their 5'
`have shown that many genes are hypomethylated
`flanking regions when being actively transcribed and methylated
`when in the nontranscribed
`chromatin conformation
`(7-9). For
`example the /3-globin gene is methylated in cells not expressing
`the gene product whereas in cells expressing /8-globin the gene
`is hypomethylated
`(7, 8). The hypoxanthine
`phosphoribosyl-
`transferase gene has been found to have different methylation
`levels on the active and inactive X-chromosome,
`the gene on
`the active X-chromosome
`being relatively hypomethylated
`(9).
`Further evidence in support of an inverse relationship between
`DNA methylation and active gene transcription
`has come from
`
`revised 4/14/87; accepted 4/20/87.
`Received 12/29/86;
`The costs of publication of this article were defrayed in part by the payment
`of page charges. This article must
`therefore be hereby marked advertisement
`in
`accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
`1This work was supported by ROI ÇA3890S,
`the M. E. Early Foundation,
`The Upjohn Co. grants, and a Childrens Hospital of Los Angeles Postdoctoral
`Research Fellowship to B. A.
`' To whom requests for reprints should be addressed, at Childrens Hospital of
`Los Angeles, Division of Hematology-Oncology,
`4650 Sunset Boulevard, Los
`Angeles, CA 90027.
`
`agent such as 5-aza-C3 (10-
`
`studies utilizing a hypomethylating
`12).
`5-aza-C is a derivative of cytosine with a nitrogen in the 5
`position of the pyrimidine
`ring (13). The nucleoside is phos-
`phorylated in cells to the biologically active triphosphate
`and
`incorporated
`into the nucleic acids (14). When incorporated
`into DNA it induces hypomethylation
`(15). 5-aza-C is unstable
`in aqueous solution at neutral pH and decomposes
`to a com
`pound of unknown biological activity ( 16). DHAC is a deriva
`tive of 5-aza-C with pharmacological
`activity and extensive
`stability in aqueous solution (17, 18).
`ara-C is a nucleoside derivative which has received consider
`able attention as a result of its clinical efficacy against a spec
`trum of human malignancies
`(19-21). However,
`its therapeutic
`usefulness is hindered by the frequent development of resistance
`in tumor cell populations
`after
`repeated ara-C exposure
`(22,
`23). The resistance is often associated with a decrease in dCk
`activity. In vitro work by several authors has shown that ara-C
`is capable of inducing enzymatic hypermethylation
`of DNA
`(24, 25).
`It
`therefore
`becomes an attractive
`hypothesis
`that
`resistance to ara-C is related to hypermethylation
`at
`the dCk
`gene locus, occurring as a consequence of inhibition of DNA
`synthesis during therapy.
`If so, resistance
`to ara-C might be
`reversible by exposure of resistant
`tumor cell populations
`to
`hypomethylating
`agents.
`Indeed, preliminary results from our
`laboratory have suggested that 5-aza-C treatment
`is capable of
`re-establishing
`sensitivity toward ara-C in pediatrie
`patients
`resistant
`to this drug (26-28).
`The present study was undertaken to define on the biochem
`ical level the mechanisms by which 5-aza-C and its congeners
`re-establish ara-C sensitivity in resistant human tumor cells. To
`this end,
`the effects of treatment with 5-carbon-substituted
`cytidine analogues upon DNA methylation and dCk re-expres
`sion were examined in a dCk-deficient
`human leukemia cell
`line.
`
`MATERIALS AND METHODS
`
`RPMI 1640 medium and heat-inactivated fetal calf serum were from
`Irvine Scientific, Irvine, CA, [6-3H]uridineand [5,6-3H]ara-Cwere from
`Moravek Biochemicals Inc., Brea, CA. DE 81 filter paper discs were
`from Whatman, ara-C was from the Upjohn Co., and 5-aza-C and 5-
`aza-Cdr were purchased from Sigma. DHAC and ara-AC were kindly
`provided by the Drug Development Branch, National Cancer Institute,
`NIH. The CCRF/CEM/0 and CCRF/CEM/dCfc- cells were generous
`gifts from Dr. Arnold Fridland, St. Jude's Children's Research Hospi
`tal, Memphis, TN. All other chemicals were of analytical or HPLC
`quality.
`Radioactiveara-C "Substrate Mixture". The [5,6-3H]ara-Cwas puri
`fied by passage through a DEAE-Sepharose column equilibrated in
`
`3The abbreviations used are: 5-aza-C, 5-azacytidine; DHAC, 5,6-dihydro-5-
`azacytidine; ara-AC,
`l-/3-arabinofuranosyl-5-azacytidine;
`5-aza-Cdr, 5-aza-2'-de-
`oxycytidine; ara-C,
`l-/3-arabinofuranosylcytosine;
`5-mC, 5-methylcytosine; dCk,
`deoxycytidine kinase; PBS, phosphate-buffered
`saline; HPLC, high-performance
`liquid chromatography;
`1C»,inhibitory concentration
`for 50% control growth;
`ara-CTP,
`l-/3-arabinofuranosylcytidine
`triphosphate.
`CELGENE 2066
`3672
`APOTEX v. CELGENE
`IPR2023-00512
`
`

`

`DNA HYPOMETHYLATION
`
`AND dCk RE-EXPRESSION
`
`IN CEM/dClr
`
`CELLS
`
`water. This purification step decreased the background in the niter
`assay about 5 times. The Substrate Mixture contained 250 ¿AI[3H]ara-
`C in buffer A (50 mM Tris-HCl, 20 HIM MgCl2, 10 mM KF, 20 HIM
`ATP, 0.10 mM tetrahydrouridine,
`pH 7.6); the specific activity was 120
`dpm/pmol. The counting efficiency was 50 ±2%.
`lines CCRF/CEM/0
`Culture Condition. The human leukemia cell
`and CCRF/CEM/dCk"
`were grown in RPMI medium supplemented
`with 10% heat-inactivated
`fetal calf serum and 10 HIM4-(2-hydroxy-
`methyl)-l-piperazineethanesulfonic
`acid, pH 7.2. The cell cultures were
`incubated at 37°Cin a humidified air atmosphere containing 5% CO2
`(NAPCO Model 6100) and were subcultured every 3-4 days keeping
`the cell density between 1 x 10* and 1 x IO6cells/ml.
`Determination of the IQo Concentrations. The cells were suspended
`in culture medium at a density of 3 x 10s cells/ml and incubated with
`increasing concentrations
`of the nucleosides over a range of 3 logs.
`After a 24-h incubation at 37'C the cultures were washed twice with
`culture medium and resuspended in drug-free medium to the original
`volume. After 24,48, and 72 h the cells were counted and the nucleoside
`concentration
`causing 50% of control growth inhibition was deter
`mined.
`Treatments with Nucleosides. The cells were suspended in culture
`medium at a density of 3 x 10s cells/ml. The various nucleosides,
`dissolved in PBS, were added to desired concentrations
`and the cultures
`were incubated at 37"C for 24 h. At the end of the incubation period
`the cells were collected by centrifugation, washed twice with culture
`medium, and resuspended in culture medium to the original volume.
`At various time intervals during or after the incubation, samples were
`withdrawn for determination of DNA methylation and dCk activity.
`Determination of DNA Methylation Level. In the presence of 10 /iCi
`[6-3H]uridine 3 x 10s cells were incubated in 2 ml of culture medium
`at 37°Cfor 24 h. At the end of the incubation period the cells were
`washed once with PBS and lysed by incubation in 0.3 M NaOH,
`containing 0.1% sodium dodecyl sulfate, at 37'C for 24 h. During this
`incubation the RNA was hydrolyzed. After adjustment of pH to 7.6 the
`samples were incubated with proteinase K (100 «ig/ml)at 65°Cfor one
`h. After cooling the samples
`to 4"( ' the DNA was precipitated by
`
`of 10%.
`acid to a final concentration
`addition of 50% trichloroacetic
`The precipitate was washed consecutively with 5% ice-cold trichloroa
`cetic acid and 70% ice-cold ethanol and finally dried. The purified
`DNA was hydrolyzed to its bases in 88% formic acid at 180°Cfor 50
`min. After drying and reconstitution with PBS the bases were separated
`isocratically by HPLC (Waters) on an SCX column at room tempera
`ture in 60 HIMphosphate buffer, pH 2.5, at a constant
`flow rate of 0.6
`ml/min (Fig. 1). The eluate was monitored at 280 nm and collected in
`fractions of 0.6 ml. The fractions were measured for radioactivity by
`scintillation counting. The amount of radioactivity eluted with cytosine
`(fR 9 min) and 5-mC (/R 17 min) were determined and the percentage
`of 5-mC was calculated according to the following formula.
`
`5-mC =
`
`cpm 5-mC x 100
`cpm 5-mC + cpm cytosine
`
`002
`
`ao
`
`O.OS
`
`llxio'cpm
`
`Fig. 1. HPLC chromatogram of nucleoside bases. The bases were separated
`isocratically on an SCX column at room temperature in 60 mM phosphate buffer,
`pH 2.5, at a constant
`flow rate of 0.6 ml/min. The eluate was monitored at 280
`nm and collected in fractions of 0.6 ml. Upper part, separation of a standard
`solution;
`lower part,
`separation of hydrolyzed DNA from CEM/0 cells after
`incubation with [6-3H]uridine for 24 h. Bars, distribution of the radioactivity. T,
`G, C, A, and mC, thymidine, guanine, cytosine, adenine, and 5-mC, respectively.
`
`allowed to dry for 10 min and then immersed into and washed 5 times
`in deionized water. After the washing procedure the discs were trans
`ferred to scintillation vials, 1 ml of a solution of 0.2 M KC1/0.2 M HC1
`was added, and the samples were incubated for 30 min. Finally 10 ml
`of scintillation fluid was added and the radioactivity was measured by
`scintillation counting. The dCk activity was calculated and expressed
`as pmol phosphorylated ara-C/mg protein x min.
`In Vitro Cloning in Selective Agar of dCk Expressing Cells. The cells
`were treated with 0, 20, or 100 MMDHAC for 24 h, washed with PBS,
`and grown in drug-free medium for 72 h prior
`to plating. The cells
`were plated in 60-mm IViri dishes in RPMI 1640 medium containing:
`10% heat inactivated calf serum; agar; 30, 60, or 100 MMthymidine; 10
`or 30 //\i deoxycytidine; combinations of thymidine and deoxycytidine
`or no nucleosides. The dishes were prepared with two agar layers; the
`lower layer contained 3 ml medium with 0.5% sea plaque agar, and the
`upper
`layer contained the cells in 2 ml medium with 0.25% agar.
`Preliminary results showed plating efficiencies for CEM/0 cells of 35
`to 100% in medium with deoxycytidine or no added nucleosides and
`less than 9 and 5% in 30 and 60 MMthymidine,
`respectively. The cells
`were completely rescued from the thymidine effect by 10 or 30 MM
`deoxycytidine. The CEM/dCk~ cells had similar plating efficiencies of
`35 to 100% in deoxycytidine or without nucleosides. However,
`they
`were much more sensitive to thymidine
`and were not
`rescued by
`deoxycytidine. The number of plated cells per dish varied from 200 to
`5 x IO4,depending on the medium,
`to optimize sensitivity and counting
`of colonies. Several thymidine concentrations were used to ensure that
`suppression of growth would be sufficient
`to detect revenant colonies
`only. After 14 to 21 days incubation at 37°Cthe colonies on the various
`dishes were counted, 5 to 10 dishes were counted for each condition,
`and the revenant
`frequency was calculated according to the following
`equation
`
`Assay of Deoxycytidine Kinase Activity. Cells (2 x 107)were collected
`by centrifugation
`and washed once with PBS. The washed cells were
`resuspended in 200 n\ buffer B (50 mM Tris-HCl, 10 mM MgCl2, 1 mM
`dithiothreitol, pH 7.6) and the cells were lysed by sonication for 30 s.
`Cell debris was removed by centrifugation at 15,000 rpm for 1 h in a
`Sorvall RC2-B refrigerated centrifuge using a SS-34 rotor. The super
`natant was collected and protein concentration was determined spec
`trophotometrically
`according to the method of Ehresmann et al. (29).
`The samples were diluted with buffer B to a protein concentration of
`0.6 mg/ml
`for the CEM/0 cells and to between 10 and 15 mg/ml
`for
`
`the Cl-:M •'<!('k cells. After preheating of the sample dilutions and the
`where TC, C, T, and N were the plating efficiencies in the agar plates
`[3H]ara-C Substrate Mixture at 37"C for 10 min 20 ¿tiof sample dilution
`containing thymidine plus deoxycytidine, deoxycytidine alone,
`thymi
`were mixed with 20 M'Substrate Mixture and further incubated at 37°C
`dine alone, and no nucleoside, respectively.
`As a control, 0.1 to 5% CEM/0 cells were mixed with CEM/dCk^
`for between 15 min and 2 h (30). At the end of the incubation period
`cells and between 4.5 x IO3and 5.8 x 10* of these cells were plated in
`the reaction was quenched by addition of 10 fil of 0.2 M EDTA solution.
`Any precipitate was removed by centrifugation and 45 /J of the super
`agar plates containing 60 MMthymidine and 30 MMdeoxycytidine. A
`natant was applied to a DE 81 filter paper disc. The filter discs were
`linear relationship between added CEM/0 cells and colonies on the
`3673
`
`Revenant
`
`frequency = TC/C - T/N
`
`

`

`DNA HYPOMETHYLATION
`
`AND dCk RE-EXPRESSION
`
`IN CEM/dClr
`
`CELLS
`
`to the nucleosides
`level after exposure
`agar plates was seen. This shows that revenant cells can be detected ¡nDNA methylation
`and that the large number of CEM/dClr cells do not interfere with the were compared to dCk activity
`plating efficiencyof CEM/0 cells
`ICso Concentrations. The IC5'„concentrations
`of the 5-aza-C
`
`cells are shown in
`nucleos¡desfor the CEM/0 and CEM/dCk'
`Colon.es were removed from the thym.dme plus deoxycytidme agar
`¿-.^m.»«
`.
`_ , ,
`, _ _,. ¥ '
`.
`plates and were grown up in suspension. In order to determine the
`for the CEM/0 cells
`Tables 1 and 2" The IC*> concentrations
`amount of dCk in the clones the following assay was performed. Cells
`(1 x IO7)were suspended in 2 ml culture medium and incubated in the were between 0.3 and 100 „Mfor the different nucleosides, ara-
`presence of 200 UMara-C for l h at 37'C. At the end of the incubation AC being the most and DHAC the least toxic. For CEM/dCk
`period the cells were extracted with perchloric acid and the nucleotide
`cells the IC50 concentrations were 200 MMfor DHAC and 40
`containing acid soluble fraction was analyzed by HPLC. Separation of
`fiM for 5-aza-C. 5-aza-Cdr and ara-AC showed very low toxicity
`the nucleosideswas performed at room temperature on a SAX column
`on the CEM/dCk"
`cells with ICso concentrations
`of 500 MM
`developedwith a linear gradient of 0.05 MNKUHjPQ»,pH 2.8, to 0.75
`and over 1 mM, respectively.
`and
`levels in CEM/0
`M NH4H2P04, pH 3.5, at a constant flow rate of 2.0 ml/min. The
`DNA Methylation. The methylation
`eluate was monitored at 280 nm and the amount of formed ara-CTP
`CEM/dCk-
`cells at various time points after a 24-h treatment
`was calculated from the ,ntegrat,on area of the elution peak correspond- wkh DHAC m shown ¡np¡g -, The methylation
`,eve,s ¡n
`untreated cells were for CEM/0, 3.72 ±0.07% 5-mC, and for
`CEM/dCk",
`3.06 ±0.09% 5-mC. The nadir
`in methylation
`level was seen in both cell
`lines 24 h after
`the end of the
`exposure to DHAC.
`In the CEM/0 cells the methylation level
`decreased to 1.49% 5-mC or 40% of the methylation
`level in
`
`The effect of DHAC and the other 5-aza-C derivatives on
`DNA methylation and dCk expression was examined. Changes
`
`RESULTS
`
`t methylation levels in CEM/0 cells at different time points after a 24-H treatment with various 5-aza-C nucleosides
`l>\
`Table l
`The methylation level in untreated CEM/0 cells was 3.72 ±0.07%. Percentage of 5-mC and methylation levels expressed as percentage of control,
`I(nr,, are given for various time points. Each data point
`is the average of at least 3 determinations ±SO.
`Methylation levels at various time points from beginning oftreatmentNucleosideDHAC5-aza-C5-aza-Cdrara-ACConcentration"
`
`
`i.e., 3.72%
`
`OiM)1
`
`10
`20
`100
`2003
`
`6
`100.1
`
`0.3
`0.540.5
`
`3524
`
`h
`(%5-mC)2.23
`
`±0.28
`1.73±0.232.29
`
`±0.23
`1.24 ±0.13
`1.20 ±0.20
`
`0.78 ±0.082.63
`
`±0.13
`1.75 ±0.21
`0.40 ±0.05
`
`0.26 ±0.041.06
`
`33
`32
`2171
`
`47
`11
`7»28
`
`±0.04
`0.97 ±0.37%604762
`
`2648
`
`h
`(%5-mC)3.83
`
`±0.06
`3.06 ±0.13
`2.99 ±0.02
`1.49 ±0.28
`
`0.72 ±0.203.21
`
`±0.08
`2.22 ±0.04
`1.32±0.021.99
`
`±0.07
`
`0.41 ±0.062.84
`
`±0.06
`0.96 ±0.02
`0.96 ±0.02%103
`
`3.82 ±0.06
`15
`ara-C
`' 1C»values were 100, 3, 1, and 0.25 MMfor DHAC, 5-aza-C, 5-aza-Cdr, and ara-AC, respectively.
`* 5-mC cpm were 7-fold higher than background.
`
`h
`
`
`(% 5-mC)%3.60
`
`±0.13
`2.75 ±0.12
`
`1.99 ±0.06532.92
`
`97
`74
`
`
`
`±0.06781.37
`
`82
`80
`40
`1986
`
`60
`3553
`
`1176
`
`
`
`±0.02372.63
`
`±0.04
`
`71
`
`26
`2679
`
`103
`
`time points after a 24-h treatment with various 5-aza-C nucleosides
`cells at different
`Table 2 DNA methylation levels in CEM/dCk'
`Themethylationlevelin untreatedCEM/dCk~cellswas3.06±0.09%.Percentageof 5-mCandmethylationlevelsexpressedas percentageofcontrol,i.e.,3.06%
`5-mC- 100%,aregivenforvarioustimepoints.Eachdatapointis theaverageof at least3 determinations±SD.
`
`treatment24
`
`levels at various time points from beginning of
`
`h (%5-mQ3.09
`h (%5-mC)2.77
`NucleosideDHAC5-aza-C5-aza-Cdrara-ACara-CConcentration"10501002005001000150536051000150500Methylation
`±0.082.05
`
`±0.082.27
`
`
`±0.311.94
`±0.131.40
`±0.101.37
`±0.181.39
`±0.232.94
`
`±0.031.65
`
`±0.101.48
`±0.041.28
`±0.162.93
`
`±0.040.56
`
`
`±0.193.02
`
`±0.103.16
`
`±0.063.04
`±0.093.14
`
`
`±0.080.72
`
`
`±0.123.00
`
`±0.042.84
`
`
`±0.183.01
`
`±0.273.06
`
`±0.123.06
`
`h (%5-mQ2.55
`
`±0.062.44
`
`±0.122.35
`±0.112.45
`
`
`±0.252.17
`
`±0.103.12
`
`±0.06%8380778071102
`
`±0.103.45
`±0.10%101675447429618991039910348
`±0.04%917463464545962498939810010011379
`11C»values were 200, 40, 500, and >1000 IÕMfor DHAC, 5-aza-C, 5-aza-Cdr, and ara-AC, respectively.
`
`3674
`
`

`

`DNA HYPOMETHYLATION AND dCk RE-EXPRESSION IN CEM/dCk" CELLS
`
`Treatment with ara-C produced no change in the methylation
`levels (Tables 1 and 2).
`Deoxycytidine Kinase Expression. The dCk activity in the
`cells before and after
`treatment with the various 5-aza-C nu
`cleosides was measured
`as described under
`"Materials
`and
`Methods."
`In untreated CEM/0 cells the dCk activity was 102
`pmol/mg
`x min. This activity was increased after
`treatment
`with all 5-aza-C nucleosides. The enzyme activity 48 h from
`the beginning of the 24-h treatments was increased about 10%
`compared to untreated cells; 48 h later the activity had returned
`to control
`level. No significant difference between the nucleo
`sides could be detected. Treatment with 100 MMDHAC in
`creased the activity to 112 ±9 pmol/mg x min or to 110% of
`control.
`Increasing the concentration
`of DHAC from 100 to
`200 MMdid not enhance the dCk activity. 5-aza-C, 5-aza-Cdr,
`and ara-AC increased the activity to 111, 106, and 111%,
`respectively, compared to the activity in untreated CEM/0 cells.
`CEM/0
`cells treated with ara-C showed a decrease in dCk
`activity to about 70% of the activity found in untreated cells.
`The CEM/dCk-
`cell line is deficient
`in dCk and untreated
`cells had a dCk activity of between 0.3 and 0.7 pmol/mg x min
`or less than 1% of the activity in CEM/0 cells. At this very low
`level of enzyme activity the variation between assays on differ
`ent days was somewhat
`larger
`than the variation within an
`experiment. The reproducibility within an experiment had stan
`dard deviations of 0.01 to 0.05 pmol/mg x min.
`After treatment with DHAC the dCk activity increased up to
`26% compared to the activity in untreated CEM/dCk"
`cells
`(Table 3). Treatment with 100 MMDHAC increased the dCk
`activity about 16% and treatment with 500 MMor 1 HIMabout
`25%, showing a dose-response
`relationship. Treatment with 50
`MM 5-aza-C increased the dCk activity approximately
`35%
`compared to untreated cells. On the other hand,
`treatment with
`5-aza-Cdr or ara-AC did not
`increase the dCk activity in the
`CEM/dCk"
`cells. ara-C had no effect on the dCk activity (Table
`3). Repeated treatments
`of the CEM/dCk~ cells with DHAC
`did not increase dCk activity compared to a single treatment.
`After
`treatment with 100 and 200 MMDHAC CEM/dCk"
`cells were grown in suspension
`and at various
`time points
`samples were withdrawn and assayed for dCk activity. As shown
`in Table 3 the increase in enzyme activity was persistent
`and
`essentially constant over at least a period of 8 days.
`In vitro Cloning in Selective Agar of dCk Expressing Cells.
`Three separate experiments were performed with CEM/0 and
`CEM/dCk~ cells. Considerable variation was seen among the
`
`three experiments. Nevertheless,
`
`in all three experiments CEM/
`
`cells after treatment with
`Table 3 Deoxycytidine kinase activity in CEM/dCk'
`the various 5-aza-C nucleosides
`The cells were treated with the nucleosides for 24 h and the enzyme activity
`was determined at various time points after the treatments. Each data point
`is
`the average of at least 5 determinations ±SD.
`dCk activity at various time points from beginning of
`treatment
`
`CEM/0
`
`CEM/dCk"
`
`408
`2408
`Time, da/s
`Time, days
`Fig. 2. DNA mcthylation levels at 24-h intervals in CEM/0 and CEM/dCk-
`cells from the beginning of a 24-h treatment with various DHAC concentrations.
`The methylation levels in untreated cells were 3.72 ±0.07% and 3.06 ±0.09%
`in CEM/0 and CEM/dCk-
`cells, respectively.
`
`treatment with 100 UM DHAC and to
`cells after
`untreated
`0.72% 5-mC or 19% of control after
`treatment with 200 MM
`DHAC. With 1 MMDHAC no effect of the methylation level
`was seen in the CEM/0
`cells; 10 MM DHAC decreased the
`methylation level to 82% of the level in untreated cells (Table
`1). Treatment
`of CEM/dCk~
`cells with 100 MM DHAC de
`creased the methylation
`level
`to 1.94% 5-mC or 63% of the
`level in untreated cells; 200 MMDHAC gave a methylation level
`of 1.40% 5-mC or 46% compared to untreated cells. DHAC
`concentrations
`of 500 MMor l m M did not significantly further
`decrease the methylation
`level. However,
`the induced hypo-
`methylation was of transient nature in both cell lines and the
`methylation
`levels had returned by day 7 almost
`to control
`levels (Fig. 2).
`increased in
`effect of DHAC was not
`The hypomethylating
`either of the two cell lines by repeated exposures to the nucleo-
`side. The cells were treated with DHAC every fifth day for 24
`h and the methylation
`levels were determined
`24 h after
`the
`end of each treatment. The methylation
`levels after
`the first
`exposure to DHAC were the lowest levels obtained; additional
`treatments
`could not
`fully achieve the same extent of hypo-
`methylation. After the first exposure to 100 and 200 n\i DHAC
`the methylation
`level decreased to 1.49 ±0.28% and 0.72 ±
`0.20% in the CEM/0 cells and to 1.94 ±0.13% and 1.40 ±
`0.10% in the CEM/dCk~ cells, respectively. However, after the
`
`level was 1.98 ±0.04% and
`the methylation
`third exposure
`1.30 ±0.11% in the CEM/0 cells and 2.28 ±0.05% and 1.82
`±0.17% in the CEM/dCkr
`cells, respectively.
`The effect of the other 5-aza-C nucleosides on the DNA
`methylation levels in the two cell lines are presented in Tables
`1 and 2. All nucleosides caused DNA hypomethylation
`in the
`CEM/0 cells whereas in the CEM/dCk"
`cells hypomethylation
`was seen after
`treatment with DHAC and 5-aza-C only.
`In
`CEM/0 cells 5-aza-Cdr was the most potent hypomethylating
`agent. Half the ICso concentration,
`0.5 MM,decreased the meth
`ylation level to 0.40% 5-mC or 11% of the methylation level in
`untreated CEM/0 cells; this is an exceptionally low level of 5-
`mC. Treatment of CEM/dCk~ cells with 5-aza-Cdr or ara-AC
`
`did not have any effect on the methylation level even at 1 HIM
`(Table 2). The nadir
`in methylation
`level for DHAC was seen
`24 h after the end of exposure to the nucleoside whereas for
`the other three 5-aza-C nucleosides the nadir was at the end of
`the 24-h treatment period.
`
`3675
`
`48 h
`dCk*
`
`96h
`dCk*
`
`192 h
`dCk-
`
`Nucleoside con
`centration
`100 0.66 ±0.05
`0.73 ±0.06 100 0.66 ±0.02
`Control
`DHAC 100 MM 0.85 ±0.02 116 0.71 ±0.02 108 0.76 ±0.05
`DHAC 200 MM 0.86 ±0.03 117 0.75 ±0.03
`114 0.78 ±0.08
`DHAC 500 MM 0.92 ±0.03 126
`DHAC 1 mM
`0.88 ±0.04 121
`5-aza-C 50 MM
`1.00 ±0.14 137
`5-aza-Cdr 360 MM 0.66 ±0.13
`90
`ara-AC 1 mM
`0.73 ±0.02
`100
`ara-C 150 MM
`0.78 ±0.04
`106
`ara-C 500 MM
`0.71 ±0.06
`97
`" dCk activity (pmol/mg x min).
`* Percentage of activity compared to untreated cells.
`
`100
`115
`117
`
`

`

`DNA HYPOMETHYLATION
`
`AND dCk RE-EXPRESSION
`
`IN CEM/dClT CELLS
`
`Table 4 Revenant frequency ofCEM/dCk'
`
`cells after treatment with DHAC
`
`The cells were treated with DHAC for 24 h, grown in suspension for 72 h, and
`plated in for dCk expression selective soft agar plates. After 14 to 21 days of
`incubation the colonies on the plates were counted and the revertan!
`frequency
`was calculated.
`
`Experiment123Treatment
`
`* Reversion frequency calculated according to text.
`* Two different CEM/dCk"
`lines were used in the experiment.
`
`Table 5 Deoxycytidine kinase activity measured as ara-CTP formation in
`CEM/0 and CEM/dCk'
`clones isolated from soft agar plates selective for cells
`expressing dCk
`The clones were removed from the agar plates, grown up in solution, and
`assayed for ara-CTP formation.
`
`populations of cells during tumor progression would occur by
`mutational mechanisms. Recent advances in our understanding
`of the control of eukaryotic gene expression, however, suggest
`that epigenetic mechanisms may also play an important
`role in
`the diversification
`of
`tumor
`cell populations
`during tumor
`frequency*
`DHAC0«M)0»1000*10002010020100Revenant
`progression (32, 33). Among these epigenetic phenomena may
`x10-'0611605850181290360
`be the loss of stable patterns of DNA methylation (34).
`The CEM/dCk"
`cells have been made resistant
`to ara-C by
`repeated exposures
`to the nucleoside. The CEM/dCk~ cells are
`deficient
`in dCk,
`the enzyme required for phosphorylation
`of
`deoxy- and arabinosylcytosine
`derivatives
`(35). Other ara-C-
`resistant cells have also shown deficiency in dCk activity (36,
`37), which would indicate that loss of dCk activity is a common
`mechanism of resistance to this nucleoside drug. However,
`total
`lack of dCk activity has not been observed in clinically resistant
`cell populations
`isolated from patients (38, 39).
`We have shown in the present study that the DNA hypometh-
`ylating agents 5-aza-C and DHAC induce a genome-wide hy-
`pomethylation
`in both CEM/0 lymphoid cells and their CEM/
`d( 'k mutants. Consistent with our previous finding that 5-aza-
`Cdr and ara-AC are not phosphorylated
`to their triphosphates
`or incorporated into DNA of CEM/dCk~ cells, we found these
`
`of ara-CTP
`(nmol of ara-CTP 1 x
`of CEM/0
`only in CEM/0 cells (40).
`agents induced hypomethylation
`10-'cells)1.121.200.850.02°0.08"0.04"1.100.581.271.080.35%
`control100102681.86.93.893491139731
`CellsCEM/0ControlCloneCloneCEM/dCk-ControlControlControlRevertan!Revertan!ReverÃ-an!ReverÃ-an!Revenan!Formation
`Treatment periods of 24 h, corresponding
`to approximately
`one cell cycle for CEM cells, ensured S-phase exposure of the
`total cell population to S-aza-C and its tested derivatives (41).
`While maximum hypomethylation
`following DHAC treatment
`occurred 24 h after cessation of treatment,
`it occurred imme
`diately at the end of treatment
`for 5-aza-C and its other deriv
`atives. This difference is probably a reflection of the increased
`stability of DHAC compared to the other nucleosides (16, 18).
`DHAC and 5-aza-C induced a transient DNA hypomethyla
`tion in both cell lines; 7 days after the treatments
`the methyla
`tion levels had returned to the control
`levels (Fig. 2). However,
`under the experimental
`conditions
`specific changes in methyl
`ation at a small number of sites would not have been detected
`and may have been of a persistent nature. This would permit
`changes in methylation of the dCk gene in the CEM/dCk"
`cells
`
`" Quantilaled manually.
`
`dCk~ cells treated with DHAC gave more colonies on the agar
`plates with combinations of thymidine and deoxycytidine com
`pared to untreated controls or the treated cells grown in thy
`midine only. Table 4 shows the revenant
`frequencies, calculated
`as described under
`"Materials
`and Methods,"
`for
`the three
`experiments. The values of 0 to 0.2% seen in untreated cells
`probably indicate a fraction of revenant cells in the CEM/dCk"
`
`increased
`cells at the time of the experiment. DHAC treatment
`the revenant
`frequency up to 0.6%. The treated CEM/0 cells
`grew normally in the thymidine plus deoxycytidine agar.
`Ten colonies of CEM/dCk"
`revenants
`from DHAC treat
`ments were removed from agar plates containing
`thymidine
`and deoxycytidine
`and individually grown up in suspension.
`The dCk activity in these clones was determined
`from their
`ability to phosphorylate
`ara-C to ara-CTP (27, 28). The clones
`of treated CEM/dCk~
`cells isolated from the selective agar
`plates formed between 31 to 113% ara-CTP compared to un
`treated CEM/0 cells (Table 5). This activity was 8- and 27-fold
`higher
`than in the untreated CEM/dCk~
`cells plucked from
`
`control agar plates.
`
`DISCUSSION
`
`to remain undetected.
`Although in the CEM/0 cells 5-aza-Cdr and ara-AC were the
`most potent
`inducers of hypomethylation,
`these nucleosides
`would not be useful in treatment of cells resistant
`to ara-C due
`to lack of dCk activity. 5-aza-Cdr and ara-AC showed very low
`toxicity toward the CEM/dCk'
`cells (Table 2) with ICso con
`
`than for the CEM/0 cells
`of drugs had no effect on
`
`over 500 times higher
`centrations
`and even these high concentrations
`the methylation level.
`There are several reports that specific genes have been reac
`tivated after treatment with hypomethylating
`agents like 5-aza-
`C (6). We have shown that murine LI210 leukemia cells resist
`ant to ara-C can regain sensitivity to ara-C after treatment with
`DHAC (28). This was also shown in 2 patients who were
`clinically resistant
`to high-dose ara-C and who received 5-aza-
`C followed by high-dose ara-C (28). Here we show that after
`exposure
`to 5-aza-C and DHAC a small proportion
`of the
`resistant
`human leukemia
`cells reexpress dCk activity. Our
`results show a direct
`relationship
`between induced DNA hy
`pomethylation
`and reexpression of dCk activity in the CEM/
`dCk" cell line (Tables 2 and 3). The increase in enzyme activity
`
`has been called
`The problem of acquired drug resistance
`perhaps the most
`important problem currently facing research
`ers interested in cancer chemotherapy
`(28, 31). Classical ge
`netics would suggest
`that
`the evolution
`of
`resistant
`clonal
`
`was up to 0.2 pmol/mg x min, which is 0.2% of the activity in
`the CEM/0 cells. Reexpression
`of dCk activity in the CEM/
`dCk" cells was seen only after
`treatment with DHAC and 5-
`aza-C,
`the two nucleosides phosphorylated
`by uridine-cytidine
`
`3676
`
`

`

`DNA HYPOMETHYLATION AND dCk RE-EXPRESSION IN CEM/dCk" CELLS
`
`but not after treat
`
`kinase and inducing DNA hypomethylation,
`ment with 5-aza-Cdr, ara-AC, or ara-C.
`One simple explanation for our results suggests that repeated
`ara-C exposure results in the development of one or more clonal
`populations
`of cells which have become hypermethylated
`and
`therefore transcriptionally
`inactive at the dCk gene locus, ara-
`C treatment has been shown to lead to genome-wide hypermeth-
`ylation in other
`systems (24, 25). Chemotherapeutic
`regimes
`that result in cytostasis may therefore initiate cellular resistance
`to their presence by hypermethylation-induced
`inactivation of
`a gene locus (or loci), the product of which is required to achieve
`the antitum