dma alkali-labile sites induced by incorporation of 5-aza-2

[CANCER RESEARCH 45, 3197-3202, July 1985]

DMA Alkali-labile Sites Induced by Incorporation of 5-Aza-2'-deoxycytidine into

DMA of Mouse Leukemia L1210 Cells

Maurizio D'Incaici,1 Joseph M. Covey, Daniel S. Zaharko, and Kurt W. Kohn2

Laboratory of Molecular Pharmacology, [M. D., K. W. K.], and Laboratory of Medicinal Chemistry and Pharmacology [J. M. C., D. S. Z.], Developmental TherapeuticsProgram, Division of Cancer Treatment, National Cancer Institute, NIH, Bethesda, Maryland 20205

ABSTRACT

The effects of 5-aza-2'-deoxycytidine on DMA in mouse L1210

leukemia cells were investigated using the alkaline elution technique. By comparing the DNA elution rate at pH 12.1 and 12.6,it was found that the drug produced DNA alkali-labile lesions.Alkali-labile sites were present only in DNA strands that were

synthesized in the presence of the drug. They persisted for atleast 48 h after drug treatment, and only after 72 h did thenumber of alkali-labile sites decline, thus suggesting a slow repairprocess. The production of alkali-labile sites was found to be

concentration dependent and observable at concentrationswhich were effective in inhibiting the clonogenic viability of L1210cells and which are attainable in vivo. 5-Aza-2'-deoxycytidine did

not cause other DNA lesions such as DNA double-strand breaksor DNA-protein cross-links. Two hypotheses were considered toexplain the origin of alkali-labile lesions in DNA that has incorporated 5-aza-2'-deoxycytidine: (a) the production of apyrimi-

dinic sites by a glycosylase that recognizes and removes aza-cytosine from DNA and (b) the alkali-catalyzed decomposition ofazacytosine residues to ring-opened products which could leadto alkali-induced DNA strand scission through a /3-elimination

mechanism. The second hypothesis was considered to be themore probable and suggests that the alkali lability may be ameans by which one could determine the extent of substitutionand precise location of azacytosine residues or their ring-opened

products in DNA.

INTRODUCTION

aza-dCyd3 (19) is an antineoplastic drug with significant activity

against some murine (18, 23, 24) and human (21) leukemias.Biochemical studies demonstrated that aza-dCyd is incorporated

into DNA (24) and inhibits DNA methylation in a manner similarto that of azacytidine (3, 10). The mechanisms of the cytotoxicand antineoplastic action of aza-dCyd have yet to be elucidated.It has been suggested that the antileukemic activity of aza-dCyd

may be related to its ability to inhibit DNA methylation (25). Eventhough many studies have shown that DNA methylation is involved in the control of gene expression (10, 17, 20), the mechanisms by which aza-dCyd induced hypomethylation of DNA

would lead to cytotoxicity remain entirely speculative. The effectsof aza-dCyd incorporation on the integrity of cellular DNA has

not been investigated. It is, however, conceivable that DNAbreakage could result from aza-dCyd incorporation due to either

1On leave from Istituto di Ricerche Farmacologiche "Mario Negri," Milan, Italy.2To whom requests for reprints should be addressed.3The abbreviations used are: aza-dCyd, 5-aza-2'-deoxycytidine (NSC 127716);

MENU, methylnitrosourea; PBS, phosphate-buffered saline (0.15 M NaCI with 10.9mM KHuPC-4and 5.6 rriM Na2HPOÂ«).

Received 10/1/84; revised 4/9/85; accepted 4/11/85.

the chemical instability of the drug (14) or to an enzymatic repairprocess. In the present study, we examined the DNA damageproduced by aza-dCyd in L1210 leukemia cells using the alkaline

elution technique. We report herein that DNA strands synthesized in the presence of aza-dCyd acquired alkali-labile lesions

that were converted by alkali to strand breaks.

MATERIALS AND METHODS

Cell Culture. Mouse L1210 leukemia cells were grown at 37Â°C in

suspension culture in RPM11630 medium (HEM Research, Inc.) supplemented with 20% heat-inactivated horse serum (Grand Island BiologicalCo., Grand Island, NY), 0.84 mw L-glutamine, penicillin (82 units/ml),

streptomycin (82 Mg/ml) (all from the Media Unit, NIH), and 50 UMmercaptoethanol (Sigma Chemical Co., St. Louis, MO). Stock cultureswere maintained in exponential growth at a density between 0.5 x 106and 1 x 106 cells/ml.

Labeling. For experiments in which cells were labeled for 24 h, [14C]-

thymidine (specific activity, 52 mCi/mmol; New England Nuclear) wasadded to the culture medium at a concentration of 0.02 ÃŸC\/m\.Forexperiments where cells were labeled for 3 or 4 h, a concentration of 0.1iiCi/ml [14C]thymidine was used. L1210 cells that were used as an

internal standard were grown in a medium supplemented with 0.05 MCi/ml [3H]thymidine (specific activity, 20 Ci/mmol; New England Nuclear)and 10~6 M unlabeled thymidine for 20 h.

Drug Treatment. aza-dCyd was kindly provided by Dr. D. DeVos,

Pharmachemie B. V. (Haarlem, Holland) and by the Drug Synthesis andChemistry Branch, National Cancer Institute (Bethesda, MD). aza-dCyd

is known to be unstable in aqueous solution and decomposes in atemperature- and pH-dependent manner (14) to form several noncytc-toxic products (2). In RPMI 1630 medium at 37Â°C, aza-dCyd decom

posed in a first-order fashion with a f1/2of 17.5 h (2). To minimize theeffects of drug decomposition, aza-dCyd was always dissolved in sterile

PBS immediately before use.aza-dCyd was dissolved and diluted in PBS (pH 7.4) and added to cell

suspensions (2.5 x 105 to 7.0 x 10s cells/ml) to give a final drug

concentration between 0.1 and 100 ng/m\. After incubation with drug,the cells were washed 3 times by centrifugation and resuspended infresh medium. Cell density was determined using a Coulter Counter(Model 2BI; Coulter Electronics, Hialeah, FL).

Alkaline Elution. The method of alkaline elution was recently reviewedin detail (11). In brief, approximately 106 cells were resuspended in cold

PBS and layered on polycarbonate filters, 0.8 ^m pore size and 25 mmdiameter (Nuclepore Corp., Pleasanton, CA). Cells were then lysed witha solution containing 2% sodium dodecyl sulfate-0.02 M Na2EDTA-0.1 M

glycine, pH 10.0 (lysis solution), which was allowed to flow through thefilter by gravity. After connecting the outlet of the filter holders to thepumping system, proteinase K, 2 ml of 0.5 mg/ml (EM Laboratories,Darmstadt, West Germany), dissolved in the lysis solution were addedto a reservoir over the polycarbonate filters and pumped for approximately 1 h at a rate of 0.35 ml/min. DNA was eluted from the filters bypumping 0.02 M EDTA solution adjusted to pH 12.1 or 12.6 withtetrapropylammonium hydroxide (RSA Corp., Elmsford, NY) containing0.1% sodium dodecyl sulfate through the filters at approximately 2 ml/

CANCER RESEARCH VOL. 45 JULY 1985

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MODE OF ACTION OF aza-dCyd

hr. Three-h fractions were collected, with fractions and filters processed

as described previously (11).For the DMA double-strand breaks assays, cells were lysed with only

2 ml of the lysis solution containing proteinase K, 0.5 mg/ml, and theelution buffer was adjusted to pH 9.6.

For the DNA-protein cross-linking assay, cells were layered on poly-

vinyl chloride filters, 2 Â¿impore size and 25 mm diameter (Millipore Corp.,Bedford, MA) and then lysed with 5 ml of the lysis solution. The detergentwas then washed away with 5 ml of 0.02 M Na2EDTA solution. Theelution buffer was the same as that used for DMA single-strand breaks,

pH 12.1, except that no sodium dodecyl sulfate was added.

RESULTS

Chart 1 shows the elution kinetics at pH 12.1 and 12.6 of[14C]DNA from L1210 cells exposed to aza-dCyd, 0 to 10 ng/m\,

for 24 h. A dose-dependent increase in elution rate was observed. The elution curves were not linear, as seen in X-irradiated

cells, but were curved downward, signifying an increase in elutionrate with time in alkali. This observation suggests the presenceof alkali-labile lesions (11). The alkali-labile origin of the DMA

strand breaks was further supported by the substantially greaterelution rates at pH 12.6 as compared to pH 12.1. (The elution ofDMA from cells irradiated with 300 rads was only slightly fasteratpH 12.6 than at pH 12.1.)

In Chart 2, the elution of L1210 DMA obtained from cellsexposed to aza-dCyd for 24 h was found to increase linearlywith drug concentration. The amount of DMA eluted at pH 12.6exceeded that eluted at pH 12.1 by a nearly constant ratio. Thedose response observed after 24 h incubation of cells in drug-

free medium was similar to that obtained immediately after drugwashout.

Alkali lability was quantitatively evaluated in terms of the ratioof DNA elution at pH 12.6 relative to that at pH 12.1 (Chart 3).The ratios immediately after and 24 h after drug exposure were1.53 and 1.52, respectively. As a reference standard, cellstreated with MENU were similarly assayed (Chart 3C). The pH

elution ratio was 1.49, a value nearly identical to that obtainedwith aza-dCyd.

The colony-forming ability of cells treated with aza-dCyd for

24 h in the dose range used in Charts 1 and 2 was 2.8 to 0.1%of control (1, 2). To exclude the possibility that the alkali-labile

lesions were indirect products in dying cells, similar experimentswere performed using 2-h treatments with aza-dCyd, 10 ^g/ml(Chart 4) which allowed more than 10% survival of colony-forming ability. Alkali-labile lesions were again observed, as

demonstrated by the faster elution from the filter at pH 12.6relative to pH 12.1. Although the elution curves in these experiments were linear rather than curved downward, this effect maybe explained by the fact that labeling of DNA is not uniform aftera short incubation period.

In order to determine whether alkali-labile lesions occurredexclusively in DNA regions where aza-dCyd was incorporated, 2labeling protocols were compared. Cells were labeled with [14C]-

thymidine either at the time of the 2-h aza-dCyd treatment("simultaneous" protocol) or at an earlier time ("sequential" pro

tocol). The details of the protocols as well as the results aregiven in Chart 4 and Table 1. In the sequential protocol, theproduction of alkali-labile lesions was negligible. Thus, alkali-

labile sites occurred only in DNA that was synthesized at thetime of aza-dCyd treatment. This suggests that these lesionsoccur only in DNA single-strand segments that have incorporated

the drug.The number of alkali-labile sites observed after a 48-h incu

bation of treated cells in drug-free medium was similar to that

found 24 h after the end of drug treatment (Table 1). Thisindicates that these lesions are either not repaired or are repairedvery slowly. In order to further evaluate this possibility, experiments were conducted in which the presence of alkali-labile sites

in DNA was assessed at several different times after treatment.Chart 5 shows that 4 h after the end of a 4-h aza-dCyd treatment,there was a significant number of alkali-labile sites. These lesions

appeared to increase slightly up to 12 h, reaching a plateau

0.90.8

^ 0.7Â»

Â£ 0.6c

I 0.5

Chart 1. Alkaline elution profiles of [14C]DNA

of L1210 cells treated for 24 h with aza-dCyd,0 (x), 0.1 (â€¢),1.0 (â€¢),or 10 iig/ml (A). Elutionswere carried out at pH 12.1 (A) and pH 12.6(B). Labeling with ["CJthymidine, 0.02 xCi/ml,and treatment with aza-dCyd were simultaneous. Each point, mean of 3 values; oars, range., elution of [3H]DNA of L1210 after irra

diation with 300 rads in the cold; 0.1 ^Ci [3H]-

thymidine for 24 h was used to label these cells.Each point, mean of 60 values; bara, SE.

o-oJD

S3Ã³

0.4

0.3

0.2

0.1

pH 12.1300 rads

pH 12.6

300 rads

0 2.5 5.0 7.5 10 12.5 15 0 2.5 5.0 7.5 10 12.5 15

Time of Elution


3198




which persisted for up to 48 h. At 72 h, substantially fewer alkali-labile sites were observed.

Experiments were also performed to determine whether aza-dCyd produced other DNA lesions such as double-strand breaksor DNA-protein cross-links. As shown in Table 2, no DNAdouble-strand breaks were found after 24 h incubation with aza-dCyd

0.5

0.4

g 0.3S

S0.2

0.1

pH 12.6

pH 12.1

0.1 1.0aza-dC (Â¿tg/ml)

10

Chart 2. Log (retention) of [14C]DNA as a function of aza-dCyd (aza-dC) con

centration. The retention values represent the fraction of the DNA retained on filterafter 15 h elution (5 fractions) considering the retention after collection of the firstfraction to be 1.0. The duration of treatment was 24 h, and elutions were carriedout at pH 12.1 and pH 12.6 immediately after treatment (O, A) or after 24 hpostincubation in drug-free medium (â€¢,A). Bars, range.

at concentrations between 0.1 and 10 ^g/ml. In addition, 24-hexposure of L1210 cells to aza-dCyd, 10 pg/m\, yielded a DNA-protein cross-link frequency of 13.2 rad equivalents (the X-raydose which would give a single-strand break frequency equivalent to the DNA-protein cross-links present) (11) indicating nosignificant formation of DNA-protein interactions by this compound.

DISCUSSION

aza-dCyd treatment of L1210 cells produced alkali-labilesitesin the DNA that was synthesized during exposure to the drug.This was demonstrated by using the alkaline elution method andcomparing the elution rate of DNA from polycarbonate filters atpH 12.1 and 12.6. After a 24-h exposure of cells to aza-dCyd,alkali-labile sites were demonstrated at 0.1 nQÂ¡m\,and theseincreased in a concentration-dependent manner. The aza-dCydconcentrations used were in the range of those achievable inplasma of L1210-bearing mice treated with therapeutic doses(1), and such concentrations are highly effective in inhibiting theclonogenic ability of L1210 cells (i.e., 0.1 /^g/rnl for 24 h causeda 1.5-log cell kill, and 1 or 10 /Â¿g/mlresulted in approximately 3-log cell kill) (1, 2). The production of alkali-labile sites in DNAoccurred exclusively in DNA that incorporated the drug. Thiswas shown by the observation that there were no alkali-labilelesions in [14C]thymidine-labeledDNA when the labeling period

preceded drug treatment.Formation of alkali-labilesites in DNA has been demonstrated

for other compounds (4, 5) and has been attributed either tobase-free sites in the DNA or to the formation of phosphotries-ters. The alkali-labilesites produced by aza-dCyd treatment hada pH sensitivity ratio (ratio of elution rates at pH 12.6 and 12.1)of 1.53, similar to the value (1.49) obtained in cells treated withMENU. MENU is a potent alkylating agent known to produce

<D

Â£ 0.2ICL

C

S 0.4S

CO)

_>

SoK

0.6

0.8

24 hr a/d cIC

Slope = 1.53

24 hr aza dC24 hr drug-free

Slope = 1.52

MI-NU

Slope = 1.49

1.0 0.8 0.6 0.4 0.2 O 1.0 0.8 0.6 0.4 0.2 O 1.0 08 0.6 0.4 0.2

Relative Retention (pH 12.1)

Charta. Alkali-lability of DNA strand breaks observed by alkaline elution in aza-dCyd (aza-cfC)-treated cells. L1210 cells were treated for 24 h with aza-dCyd, 0.1,1.0, or 10 ng/ml and assayed immediately (A) and after 24 h postincubation in drug-free medium (B). For reference, cells were treated with 25, 50, or 100 Â¡Mof MENU(MENU) for 1 h and analyzed immediately (C). Data include 3 to 4 experiments under each treatment condition. Elution rates at pH 12.1 and pH 12.6 are compared.Internal standards, consisting of [3H]thymidine-labeled L1210 cells irradiated with 300 rads in the cold were included in each assay. Relative retention of ["C]DNA was

evaluated with respect to these internal standards. Hence, corrections are implicitly included for the small effect of pH on the elution of DNA strands not bearing alkali-

labile lesions.


3199




Chart 4. Alkali-labile lesions were confinedto DMA segments that were synthesized duringexposure of cells to aza-dCyd. Cells were exposed to aza-dCyd and [14C]thymidine either

simultaneously ( ) or sequentially ( ).Simultaneous treatment consisted of aza-dCyd,10 ^g/ml, for 2 h and ["CJthymidine, 0.1 nCi/

ml, for 3 h beginning 0.5 h before addition ofaza-dCyd. Sequential treatment consisted of["Cjthymidine for 3 h, followed by chase withfresh medium for 3 h, followed by aza-dCyd for2 h. In both protocols, cells were incubated indrug-free medium for 24 h after drug treatmentand labeling. Alkaline elution assays were at pH12.1 (A) and pH 12.6 (B). â€¢,control, simultaneous; O, control, sequential; â€¢aza-dCyd, simultaneous; D, aza-dCyd, sequential; A, 300rads 7-rays in the cold; oars, range.

0.90.8

0.7

- 0.6jS

Ã•Â°-5o

l 0.4

0.3

0.2

0.1

pH 12.1

300 rads

pH 12.6

300 rads

2.5 7.5 10 12.5 15 0 2.5

Time of Elution

7.5 10 12.5 15

Table 1Alkali-labile lesions in [ÃŒ4C]DNAofL1210 cells treated tor 2 h with aza-dCyd, 10 ng/ml, and labeled with ["CJthymidine,

either at the time of treatment (simultaneous protocol) or before treatment (sequential protocol)

Details of protocols are the same as in the legend to Chart 4, except that cells were analyzed after both 24 and 48 hof postdrug incubation.

24 hpostincubationProtocolSimultaneous

Controlaza-dCyd300radsSequential

Controlaza-dCyd

300 radspH

12.10.047a

(0.042-0.058)"

0.23 (0.22-0.24)0.79(0.77-0.81)0.038

(0.034-0.041)0.058 (0.046-0.073)0.59 (0.54-0.63)pH

12.60.074

(0.072-0.076)0.47 (0.44-0.487)0.86(0.81-0.92)0.07

(0.06-0.08)0.13 (0.108-0.151)0.72 (0.68-0.76)48

hpostincubationpH

12.10.037

(0.034-0.043)0.273(0.266-0.277)0.058

(0.056-0.06)0.064 (0.064-0.065)0.77 (0.75-0.79)pH

12.60.067

(0.066-0.067)0.451(0.418-0.513)0.107(0.106-0.107)

0.113(0.103-0.128)0.84 (0.74-0.93)

* Mean of 3 determinations of -log (retention) of DMA after 15 h of elution (fifth fraction) considering the retention after

collecting the first fraction to be 1.0.6 Numbers in parentheses, range.

Table 2Assay for DNA double-strand breaks in L1210 treated for 24 h with Aza-dCyd, 0.1 to WO ng/ml

Elution was carried out at pH 9.6 after lysing the cells on polyvinyl chloride filters. Values are the -log (retention) of DNA after 12 h of elution

(fourth fraction) considering the retention after collecting the first fraction as 1.0.

Aza-dCyd (jig/ml)

Control 3000 rads 0.1 1 10 1000.024a (0.02-0.03)6 0.11 (0.10-0.12) 0.025(0.01-0.03) 0.04(0.02-0.05) 0.025(0.02-0.03) 0.036(0.03-04)

3 Mean of 3 replications.b Numbers in parentheses, range.

alkali-labile sites by both of the processes mentioned above (4).

Although the concentrations and exposure times for MENU usedin these experiments produced a substantial number of alkali-

labile sites, the same conditions have been shown to reduceL1210 soft-agar colony formation by less than 1 log (6).

Two hypotheses will be considered to explain the origin ofalkali-labile lesions in DNA that has incorporated aza-dCyd. It ispossible that these lesions result from the formation of apyrimi-

dinic sites in DNA, perhaps by the action of a glycosylase which

may recognize and remove incorporated azacytosine residuesfrom DNA. This process would be analogous to the removal ofuracil from DNA by uracil glycosylase (15).

Since a prolonged steady-state level of alkali-labile lesions wasobserved following aza-dCyd treatment (up to 48 h; Chart 5),

the above hypothesis would require that the glycosylase recognize and remove azacytosine residues very slowly. This may bereasonable if the enzyme in question normally acts on a differentbase (e.g., uracil). The steady-state level observed would corre-


3200




05

0.3

Â§ 0.2

0.1

24 32 40 48 56

Time After Aza-dC Washout Ihr)

64 72

Chart 5. Frequency of alkali-labile lesions during postincubation period followingtreatment with aza-dCyd (Aza-dC). Cells were simultaneously treated with aza-dCyd, 10 fjg/ml, and labeled with [14C]thymidine, 0.1 nCi/ml, for 4 h. After various

times of postincubation, cells were analyzed by alkaline elution at pH 12.6. Cellswere diluted with fresh medium after 24 and 48 h in order to keep the cellconcentration between 3 and 9 x Mf/m\.

NHj NH.,

l ? ?

Chart 6. Proposed chemical mechanism for the production of alkali-inducedDMA strand breaks due to the presence of incorporated azacytidine residues (fop).The proposed mechanism is analogous to the alkali-induced DMA strand scissionat base-free sites (bottom).

spond to roughly one alkali-labile site per 3 x 106 nucleotides

(12). Assuming a persistance time of 1 h (an ad hoc value), theextent of removal of alkali-labile lesions over a period of 48 hwould be approximately 16/106 nucleotides. For the steady state

to exist, the initial frequency of aza-dCyd substitutions wouldhave to be large compared to the latter value, at least on theorder of one per 10" nucleotides. Glazer and Knode (9) reportedthe incorporation of aza-dCyd, 214 pmol/106 HT-29 cells follow

ing 24 h of drug exposure at a concentration of 10 MM(3 ^g/ml).This corresponds to an incorporation of one aza-dCyd residue

per 167 nucleotides, which is 1 to 2 orders of magnitude greaterthan that required to account for the observed steady state.Thus, the glycosylase hypothesis is quantitatively plausible, provided that aza-dCyd residues are poorly recognized by the

enzyme.Since base-free sites can produce replication errors, aza-dCyd

might, according tp this hypothesis, be expected to be mutagenic(22). Although azacytidine is weakly mutagenic in bacteria (8),neither this compound nor aza-dCyd appears to be mutagenic

in mammalian cells (7,13).The other hypothesis is that the alkali-labile sites are a mani

festation of the chemical instability of the azacytosine ring inalkali (14). This compound undergoes ring opening between C-6

and N-1 to form an AMormyl compound; the formyl group is thenlost, yielding 1-/3-D-ribofuranosyl-3-guanylurea (Chart 6). The

subsequent reactions in Chart 6 represent a hypothetical sequence to indicate how the presence of the guanylurea moietyin DNA could lead to a strand break by 0-elimination of the 3'-

phosphate. This reaction sequence is analogous to the base-catalyzed /3-elimination of 3'-phosphate which accounts for the

alkali lability of base-free sites in DNA (Chart 6, Line 2) (15).

Kinetics studies by Lin ef a/. (14) show that the decompositionrate of aza-dCyd increases with pH, and that about 80% isdecomposed in 1 h at pH 10.4 and 24Â°C.Under our conditions

of alkaline elution at pH 12.1 to 12.6, the decomposition wouldbe considerably faster, and we would expect that essentially allof the azacytosine rings would be opened in less than 1 h at pH12.1. The fact that we find a substantially slower rate of strandcleavage at pH 12.1 than at pH 12.6 over the 15-h period of

alkaline elution (Chart 2) could be explained by the assumptionthat the rate-limiting step in strand scission occurs at a step

subsequent to ring opening. The finding that the pH 12.1:pH12.6 ratio of elution rates is similar for cells treated with aza-dCyd or MENU (Chart 3) would indicate that the /3-elimination

rates for the 2 pathways shown in Chart 6 are similar.Of the 2 plausible explanations for the alkali lability of DNA

that has incorporated aza-dCyd, the second appears to be

simpler and therefore the more probable hypothesis. If this lattermechanism is shown to be correct, the formation of alkali-

dependent DNA strand breaks could be used as a means todetermine the extent and precise location of azacytosine substitutions in DNA. The presence of an alkali-labile site, however,would not distinguish between an azacytosine residue, a ring-

opened residue, or an apyrimidinic site in DNA. Nevertheless,the present results suggest that the incorporated azacytosinesor their degradation products must persist in the DNA for at least48 h.

Additional experiments will be required to elucidate further themechanism of alkali-labile site formation. Demonstration of glycosylase activity in vitro may be possible using [14C]aza-dCyd to

produce a labeled DNA substrate. In addition, the conversion ofputative apyrimidinic sites into frank breaks prior to alkalineelution using either an apyrimidinic site endonuclease (15) orputrescine plus Mgz+ treatment (16) would provide confirming

evidence. If the guanylurea moiety leads to an intrinsically alkali-

labile site in the absence of enzymatic activity, isolated DNA orsynthetic polynucleotides containing aza-dCyd should show

spontaneous cleavage upon alkaline incubation.

ACKNOWLEDGMENTS

We wish to thank Susan Hurst-Calderone for her technical assistance and MadieTyler for typing the manuscript.

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1985;45:3197-3202. Cancer Res Maurizio D'Incalci, Joseph M. Covey, Daniel S. Zaharko, et al. -deoxycytidine into DNA of Mouse Leukemia L1210 Cells

′DNA Alkali-labile Sites Induced by Incorporation of 5-Aza-2

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dma alkali-labile sites induced by incorporation of 5-aza-2

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