[CANCER RESEARCH 42, 1300-1306, April1982]0008-5472/82/0042-0000$02.00

DMA Ligase and DNase Activities in Mouse Erythroleukemia Cells duringDimethyl Sulfoxide-induced Differentiation1

Barbara M. Scher,2 William Scher, Andrew Robinson, and Samuel Waxman

Cancer Chemotherapy Foundation Laboratory, Division of Medical Oncology, Department of Medicine [W. S., A. R., S. W.] and Department of Microbiology ¡B.M.S.], Mount Sinai School of Medicine, New York, New York 10029

ABSTRACT

DNA ligase and DNase levels were measured in cell-freeextracts from untreated mouse erythroleukemia (MEL) cellsand from cells treated with dimethyl sulfoxide (Me2SO) toinduce erythroid differentiation. The DNase activity present inthe extracts was sensitive to inhibition by G-actin and was,

therefore, presumed to be DNase I. When the MEL cells wereinduced to differentiate by culturing in the presence of 1.8%Me2SO for 3 or 4 days, the apparent activity of the DNA ligasedecreased to approximately 12% of the value in untreated MELcells. In contrast, the apparent DNase I activity of the extractsfrom Me2SO-treated cells increased over that in extracts fromuntreated cells by a factor of 2. The activity of acid phospha-tase, a lysosomal enzyme, remained unchanged. When strainDR-10, a mutant of the MEL cells which does not undergoMe2SO-induced differentiation, was treated with Me2SO, the

DNA ligase and DNase activities of extracts from these cellsremained unchanged as compared to extracts from untreatedDR-10 cells. Therefore, the marked decrease in the level of

DNA ligase activity appeared to be related to the process ofdifferentiation in the Me2SO-treated MEL cells.

INTRODUCTION

Friend virus-infected MEL3 cells are cloned cells propagated

in long-term cultures in which nearly every cell can be induced

to synthesize hemoglobin and to mature along the erythroidline by the addition of Me2SO and other chemically definedinducing agents (7, 33). This phenomenon enables the cells tobe used as a model system for the study of the mechanism ofcellular differentiation in general and erythroid differentiationin particular.

When the MEL cells are allowed to undergo terminal maturation, morphological changes occur in the nucleus whichmimic some of the changes seen in vivo during erythrocytematuration. The nuclei become smaller, and the chromatinbecomes highly condensed (27).

In addition to the morphological changes in the nuclei, biochemical changes in the metabolism of the nuclear DNA alsooccur. DNA formed after the addition of the Me2SO or otherinducing agents is hypomethylated (4), and the DNA products

' This work was supported by USPHS Grants AM 16690-06 from the National

Institute of Arthritis, Metabolism, and Digestive Diseases; CA 24402-03 from theNational Cancer Institute; CH-144 from the American Cancer Society; the Chemotherapy Foundation, Inc.; and the Charles E. Merrill Trust. Part of this work waspresented at the 1980 meeting of the American Association for Cancer Research,San Diego, Calif. (30).

2 To whom requests for reprints should be addressed, at Division of Medical

Oncology, Mount Sinai School of Medicine, 1 Gustave L. Levy Place, New York,N. Y. 10029.

3 The abbreviations used are: MEL. mouse erythroleukemia; Me2SO, dimethyl

sulfoxide.Received March 26, 1981; accepted January 7, 1982.

formed at this time when compared to untreated cells have alower single-strand molecular weight as judged by alkaline

sucrose gradient centrifugation (38). Prelabeled DNA formedbefore the addition of Me2SO also decreases in single-strand

molecular weight after the addition of Me2SO. Rotationallyconstrained DNA ("folded genomes" or nucleoids) isolated

from Me2SO-treated cells has a slower sedimentation velocity

(31 ) and lower superhelical density (19) as compared to untreated cells.

In order to further examine the background against whichthese changes occur in the nuclei of maturing MEL cells, thelevels of activity associated with certain enzymes involved inDNA metabolism, DNA ligase (EC 6.5.1.1) and DNase activities,in particular, DNase I (EC 3.1.21.1)-like activity, were analyzedin cell-free extracts of MEL cells grown in the presence and in

the absence of Me2SO.

MATERIALS AND METHODS

Materials. Glasgow Minimal essential medium and fetal bovine serum were purchased from Grand Island Biological Co., Grand Island,N.Y., and Associated Biomedic System, Buffalo, N. Y., respectively;Me2SO, ethidium bromide, agarose (Electrophoretic Grade, type II),and crystalline bovine serum albumin were purchased from SigmaChemical Co., St. Louis, Mo.; EcoRI restriction nuclease and bacterio-phage A DNA was from Miles Laboratories, Inc., Elkhart, Ind.; bacterio-phage PM2 DNA was obtained from Boehringer Mannheim, Indianapolis, Ind.; DNase I was purchased from Worthington Biochemical Corp.,Freehold, N. J.; and T4 DNA ligase was obtained from New EnglandBiolabs, Beverly, Mass. Bacillus subtilis bacteriophage <>105 DNA wasthe generous gift of Dr. A. Garro. G-actin from rabbit muscle, purified

according to the method of Spudich (37) and homogeneous as assayedby polyacrylamide gel electrophoresis (26), was the generous gift ofDr. S. Puszkin.

Cell Culture. MEL cell clone 5-86 (Mo.,SO differentiation-sensitive)(31) and MEL cell clone DR-10 (Me2SO differentiation-resistant) (24),

both derivatives of cell clone 745, were grown as described previously(33). Cell line 5-86 was used in all experiments unless specifically

noted. Where indicated, Me2SO was added to the medium at 1.8% (v/v) just prior to seeding. Cells growing in logarithmic phase were addedto yield 105/ml. Cell number was determined with a hemocytometer.

To estimate the proportion of hemoglobin-containing cells in culture,the cells were stained for benzidine-reactive material (25, 34). Cell

viability was determined by trypan blue exclusion (12).Cell Fractionation. All procedures were carried out at 0-4° unless

otherwise noted. For preparation of cell lysates, approximately 50 to80 ml of each cell type were grown with or without Me2SO for 4 days(unless otherwise noted) at 37°to a cell density of approximately 3 to4 x 106 cells/ml. The cell suspensions were centrifuged at 250 x g

for 20 min, washed twice with 2 ml 0.95% NaCI solution (32), andresuspended in 10 mw Tris-HCI (pH 7.6) containing 0.2 M KCI, 10 mn/tMgCI2, 0.1 mM EDTA, and 5 mM 2-mercaptoethanol (added just priorto use) (Buffer A) to a final cell density of 108 cells/ml. Buffer A was

chosen since buffers of similar salt content were shown to be effectivein the extraction of DNA ligase activity from nuclei (35) and were also

1300 CANCER RESEARCH VOL. 42

DNA Ligase and D/Vase Activities in Erythroleukemia Cells

effective in the stabilization of lysosomes to minimize contamination ofthe extracts by lysosomal contents (2). The cells were homogenized by30 to 40 strokes of a Teflon Potter-Elvehjem homogenizer in a smooth

glass tube attached to a mechanical stirrer rotating at 3000 rpm. Thehomogenates were centrifugea at 250 x g for 10 min, and the supernatant solutions were collected and centrifuged again at 100,000 x gfor 1 hr in a Beckman ultracentrifuge (Beckman Instruments, Inc.,Spinco Division, Palo Alto, Calif.). The clear supernatant solutions wereused as cell extracts for all subsequent operations. Extracts used fornuclease assays were dialyzed for 24 hr against 3 changes of 1 litereach of Buffer A. Protein was determined by the method of Lowry et al.(18) using bovine serum albumin as a standard.

The yield of DNA ligase per untreated cell and the specific activity ofthe extracts from untreated cells varied from approximately 1 to 10pmol cohesive termini ligated per 30 min per 108 cells and 0.3 to 3.7

pmol cohesive termini ligated per 30 min per mg protein, respectively,depending on the mechanical stirrer used. The best results wereobtained with the Standard Servodyne Unit (Cole-Parmer Instrument

Co., Chicago, III.). In all cases, however, the differences between theMe2SO-treated and untreated cells remained the same. The DNase

activities were not as sensitive to these varying extraction conditions.Hemoglobin was determined by a modification of the method of

Weins et al. (39). For the hemoglobin assay, the cell extracts preparedin the high-salt Buffer A were analyzed spectrophotometrically at 415

nm directly after centrifugaron without further treatment. Extractsprepared in this way did not exhibit the marked turbidity previouslynoted in high-speed supernatant solutions prepared from untreated

MEL cells (13).Assay for DNA Ligase. DNA ligase activity was determined accord

ing to the general method of Moore era/. (21) by following the ligationof the cohesive termini of bacteriophage DNA (9). The cohesive terminiof bacteriophage $105 DNA (28) were used as the substrate. Theincubation mixture (40 pi) contained: 10 rnw Tris-HCI (pH 7.6); 0.12M KCI; 10 mM MgCI2; 0.15 mM ATP; 15 to 30 fig G-actin; 2.42 fig (92fmol, as bacteriophage genome) $105 DNA [M,. 2.3 x 106 (3)]; and 3

to 50 fig protein from cell-free extracts. When T4 DNA ligase was usedas the source of enzyme, 0.001 to 0.015 deoxy(adenylate-thymidylate)

circle formation units, as defined by Modrich and Lehman (20), wereadded to each assay. The specific activity of this preparation wasapproximately 103 units/mg. Assays were preincubated at 25° for 5

min prior to the addition of substrate DNA in order to allow for theinactivation of endogenous DNase I by G-actin. After the addition ofsubstrate, reactions were incubated at 37°for 30 min. Reactions wereterminated by heating at 60° for 5 min and then were chilled. The

reaction mixtures were adjusted to a final Tris-HCI concentration of 0.1

M (pH 7.6), and 50 to 100 units of EcoRI restriction nuclease wereadded. The reactions were incubated at 37°for 2.5 hr and then were

terminated by the addition of 0.1 volume 0.25 M EDTA. Proteinase Kwas added to a final concentration of 500 »gml and incubatedovernight at 25° followed by 1 hr at 37°. The mixtures were dilutedwith 1 volume H2O, heated to 75°for 10 min to release cohesive ends,

and quickly chilled in an ice bath. Aliquots of the reaction mixturecontaining approximately 0.6 ¿ig</>l05 DNA were subjected to electro-phoresis on ethidium bromide-agarose slab gels at 1.5 V/cm for 18 hrat 25°(28). The gels were then photographed under UV as described

previously (28). The relative fluorescence intensity of each band ofDNA was determined by directly scanning each gel with a Farrandchromatogram analyzer (Farrand Optical Co., Valhalla, N. Y.). Theareas under the curves were estimated either with a planimeter or byweighing a paper tracing of each area.

Under these assay conditions, up to 91% of the cohesive ends ofthe input DNA could be recovered in a heat-stable form after incubation

with enzyme.DNase Assay A. DNase activity in cell-free extracts was determined

by measuring the cleavage of the cohesive termini of $105 DNA (28).The incubation mixtures (40 /il) contained Buffer A, 2.42 jig (92 fmol,as bacteriophage genome) $105 DNA, and 10 to 100 ¡igprotein from

dialyzed cell-free extracts. The reactions were incubated at 37°for 20min, heated to 60°for 5 min to terminate the reaction, and treated with

EcoRI endonuclease and proteinase K as detailed above. The reactionmixtures were not, however, further diluted, nor were they heated to75°to disrupt cohesive termini. Aliquots of the reaction mixture con

taining 1.2 ¡ig$105 DNA were subjected to electrophoresis as described above.

DNase Assay B. Using PM2 DNA, endodeoxyribonuclease activityin cell-free extracts was determined as previously described (14) bymeasuring the cleavage of Form I DNA of bacteriophage PM2 (super-

coiled DNA) to Form II DNA (nicked, circular DNA). The incubationmixtures (50 fil) containing 0.02 M Tris-HCI (pH 8.0), 0.01 M MgCI2,

1.23 fig (198 fmol as bacteriophage genome, 150 fmol as Form I) PM2DNA [M,, 6.2 x 106 (6)], and 1 to 3 fig protein from dialyzed cell-freeextracts were incubated for 20 min at 37°. Reactions were terminated

by the addition of 0.1 volume 0.25 M EDTA and then were treated withproteinase K (500 pg/ml) as described above. Aliquots of the reactionmixture containing 0.6 jug PM2 DNA were subjected to electrophoresisin ethidium bromide-agarose gels as described above. DNase Assay B

is a more sensitive assay than DNase Assay A. A quantity of enzymethat cleaved 1 fmol cohesive termini of $105 DNA per 30 min usingDNase Assay A cleaved approximately 100 fmol supercoiled PM2 DNAper 30 min to yield the nicked circular PM2 DNA product using DNaseAssay B.

Acid Phosphatase Assay. Acid phosphatase assays were performedusing Phosphatabs from General Diagnostics, Warner-Lambert Co.,

Morris Plains, N. J.

RESULTS

The 0105 genome is a double-stranded DNA with single-

stranded complementary ends (28). These ends are cohesivedue to hydrogen bonding, and they hold the DNA in a circle.There are 6 EcoRI restriction endonuclease sites in this circle.When <j>105DNA is cleaved by EcoRI, 8 fragments (FragmentsA to H) are formed. The fragments can be separated accordingto size by ethidium bromide-agarose gel electrophoresis. The

order of separation is A to H, Fragment A being the largest.Under our assay conditions, only the larger fragments, A to F,can be easily visualized. The order of these fragments in thelinear genome is D, E, G, B, H, F, and C (29). Fragment A isformed by the joining of the cohesive termini of the end fragments, C and D (28).

As can be seen in Chart 1, if the hydrogen bonds of thecohesive termini are disrupted by heating to 75°,Fragment A

is converted to the smaller fragments, C and D. If Fragment Ais reacted with DNA ligase to form a covalently sealed form ofA (noted here as A'), heating will not disrupt this A' fragment,

and little or none of Fragments C and D will be formed. IfFragment A is attacked by nuclease in the region of thecohesive ends, Fragments C and D will be formed at theexpense of Fragment A. These reactions are the basis forassays for DNA ligase and nuclease activities.

Reaction of T4 DNA Ligase with <.105 DNA. The ability ofT4 DNA ligase to react with the cohesive ends of 0105 DNA isshown in Chart 2. Increasing amounts of DNA ligase producedincreasing amounts of material present as ligated, heat-stable

Fragment A relative to the amount of total termini present ineach reaction.

The amounts of material migrating at the positions of Fragments A, C, and D were measured in both experimental andcontrol assays as described in "Materials and Methods." The

fractions of total termini present in the form of heat-stable

Fragment A were calculated from the following expressions:

APRIL 1982 1301

8. M. Scher et al.

b)

e)

DNA heatligase 75°IQmin

nuclease

Chart 1. Schematic representation of the standard assay for DNA ligase andDNase Assay A. When Fragment A, which contains the cohesive termini of o 105DNA, is heated in the presence of Buffer A. partial dissociation into Fragments Cand D occurs (a). When Fragment A is first treated with DNA ligase to formcovalent bonds which join the cohesive termini, heating will no longer dissociatethe covalently sealed, heat-stable form of Fragment A (A') into Fragments C and

D (b). When Fragment A is treated with DNase. it is dissociated into FragmentsC and D even in the absence of heating (c). The proportion of Fragments A, C,and D in a reaction mixture can be quantitated by separating the fragments byethidium bromide-agarose gel electrophoresis and then measuring the fluorescence intensity of each DNA band so produced.

5 IO I5T4 DNA LIGASE (Units» IO3)

Chart 2. Effect of varying amounts of T4 DNA ligase on the ligation of thecohesive termini of <j>105DNA. DNA ligase assay conditions and analysis of thereaction products by agarose gel electrophoresis were as described in "Materialsand Methods." Calculations are described in "Results." T4 DNA ligase [New

England Biolabs; specific activity, 1000 deoxy(adenylate-thymidylate) circle formation units/mg (20)] was added in amounts ranging from 0.001 to 0.015 unitas indicated.

A + C + D

F A°' AO + C0 + Do

where F„is the fraction of total termini present as heat-stableFragment A in a given reaction and F^ is the fraction of totaltermini present as heat-stable Fragment A in control assaysincubated in the absence of enzyme. A, C, and D represent theareas under the curves produced by Fragments A, C, and D,respectively. A0, C0, and D0 represent the area under thecurves produced by Fragments A, C, and D, respectively, inassays incubated in the absence of enzyme.

FA and F^ were used to calculate the mol of cohesive terminiligated according to the following expression:

where 7 is mol of cohesive termini ligated and N is mol of DNAadded to the reaction.

In the reactions shown in Chart 2, 57 fmol of the cohesivetermini of the 92 fmol added were dissociable by heating andcould be scored in the ligation reaction. At this salt concentration, 35 fmol were not dissociable by heating and could not betallied in the assay. However, it is possible that at least someof these 35 fmol were also ligated. Thirty-two fmol were ligatedby saturating amounts of enzyme. This is 56% of the 57 fmolthat were heat dissociable. Twenty-five fmol, comprising ap

proximately 27% of the input DNA, remained heat labile andcould not be ligated. The ends of these DNA fragments mightnot have been ligatable due to damage that occurred, possiblyduring isolation.

As an internal control for each assay, the areas under thecurves generated by all of the fragments were normalized tothe area under the curve generated by Fragment B. When thiswas done, it was seen that the ratios of the areas of internalFragments E and F relative to Fragment B did not changemarkedly while the ratios of the areas of the ligatable FragmentsA, C, and D normalized to internal Fragment B changed markedly. For example, as the T4 DNA ligase added to each assayranged from 0 to 0.015 deoxy(adenylate-thymidylate) circle

formation units, the A:B ratio was gradually altered from 0.5 to0.94, and the C:B and D:B ratios dropped from 0.45 to 0.2 andfrom 0.36 to 0.14, respectively. In contrast, as the T4 DNAligase concentration increased, the E:B and F:B ratios remained at 0.45 to 0.49 and 0.33 to 0.36, respectively, whichis in good agreement with the theoretical ratios of 0.49 and0.3, respectively, based on the apparent molecular weights ofthe internal fragments (28).

Effect of ATP on the DNA Ligase of Cell-free Extracts. TheDNA ligase activity assayed as described above in dialyzedcell-free extracts of untreated MEL cells grown for 4 days was

completely dependent on ATP. In the absence of ATP thecohesive termini ligated equalled less than 3 fmol/30 min/mgprotein, while in the presence of 0.2 mw ATP, the cohesivetermini ligated equalled 89 fmol/30 min/mg protein.

Effect of G-Actin on the DNase Activity of Cell-free Extracts. DNase activity was measured in cell-free extracts ofMEL cells according to DNase Assay A as described in"Materials and Methods." The effect of G-actin on the total

DNase activity was tested. As can be seen in Table 1, G-actincompletely inhibited the total DNase activity of extracts prepared from both untreated and Me2SO-treated cells as determined at neutral pH. This suggested that the major DNaseactivity noted in these preparations was due to DNase I, sincethe inhibition of DNase I by G-actin is a highly specific inter

action (15).DNA ligase and DNase activities in crude cell-free extracts

TabletEffect of G-actin on the DNase activity from MEL cells

MEL cells were grown for 4 days in the presence or in the absence of 1.8%(v/v) Me. SO. and the levels of DNase were measured by DNase Assay A indialyzed cell-free extracts as described in "Materials and Methods," except that

30 /ig of G-actin per assay were added where indicated.

Day1

234DNase

(pmol/30 min/Me2SO G-actin mg protein)0.081

+ <0.003+ - 0.156+ + <0.003

1302 CANCER RESEARCH VOL. 42

DNA Ligase and D/Vase Activities in Erythroleukemia Cells

compete with one another; therefore, the above reaction conditions were utilized to assay each activity in the presence ofthe other. DNase assays were routinely performed with extractsdialyzed to remove endogenous ATP, which abolished all competing DNA ligase activity. DNA ligase assays were routinelyperformed in the presence of an excess of G-actin (15 to 30

/ig/assay) in order to eliminate the major competing DNaseactivity of the preparations (15).

Effect of Me2SO Treatment on DNA Ligase Activity fromMEL Cells. The effect of Me2SO treatment on DNA ligaseactivity in MEL cells that were grown for 3 days in the presenceand in the absence of 1.8% Me2SO was determined. Cell-free

extracts were prepared and tested for DNA ligase activity. TheDNA products of the DNA ligase reactions were separated byethidium bromide-agarose gel electrophoresis and quantitatedby comparing the relative intensities of the fluorescence ofeach DNA band. The level of resolution of the DNA ligasereaction products and the method of quantitation are demonstrated in Chart 3.

These results demonstrated that in assays of extracts fromuntreated cells there was a loss of material from Fragments Cand D and a corresponding increase in the amount of materialin the Fragment A region (Chart 3, middle) when compared toassays lacking cell-free extract (Chart 3, fop). The gel patternof the products from assays containing extracts from Me2SO-

treated cells exhibited only a slight loss of material from the Cand D fragment region and, concomitantly, a slight increase ofmaterial in the Fragment A region (Chart 3, bottom).

The data shown in Chart 3 can be quantitated as follows.Cohesive termini (59 fmol) were ligated in the standard DNAligase assay by 25 /¿gcell extract protein from untreated MELcells, and cohesive termini (6 fmol) were ligated in the standardDNA ligase assay by 25 ¿igcell extract protein from Me2SO-

treated cells. In these assays, the A:B ratios for assays containing no cell-free extract, untreated cell-free extract, andMe2SO-treated cell-free extract were 0.35, 1.4, and 0.45,

respectively, while for all 3 assays, the E:B and F:B ratiosremained at 0.45 and 0.3, respectively.

The effect of increasing protein concentration from cell-free

extracts of MEL cells grown for 4 days in the presence orabsence of Me2SO on DNA ligase activity is summarized inChart 4. Increasing amounts of extract from untreated cells ledto linear increases in the ligation of Fragments C and D to formmaterial migrating with Fragment A up to a protein concentration of 25 jug/assay. The maximum activity occurred at 50 /ig/assay. In this experiment, 29 fmol of cohesive termini, of thetotal 92 fmol added, were dissociable by heating into Fragments C and D and were thus able to be scored for ligation. Ofthe 29 fmol, 21 fmol were ligated in the presence of 50 ¡igofprotein. The remaining 8 fmol (9% of the input DNA) did notparticipate in the ligation reaction. Therefore, as was seen inthe assay with highly purified T4 DNA ligase, not all of the inputDNA was able to be ligated, possibly due to damaged ends.

Much less DNA ligase activity was seen in extracts preparedfrom Me2SO-treated cells. The response of the assay withMe2SO-treated cells remained linear at all concentrations ofprotein tested but the activity seen with 25 /¿gof protein fromMe2SO-treated cells was only 12% of that of the activity seen

with the untreated cells.Control assays were obtained with incubations done in 3

ways: (a) without enzyme; (b) in the presence of EDTA added

COZLU

ü•z.LuO{/)UJDCO

0 l 2 3 4 5 6 7

MIGRATION (CM)Chart 3. Fluorimetrie scans of the reaction products from DNA ligase assays

after ethidium bromide-agarose gel electrophoresis. After growth in culture bothwith and without Me. SO for 3 days, cells were harvested, cell-free extracts wereprepared, and DNA ligase assays were performed as described in "Materialsand Methods" with £105 DNA as substrate. The incubations were done in the

presence of Buffer A alone (top), 25 mg protein from untreated cells (middle); and25 f»gprotein from Me2SO-treated cells (bottom). For visual comparison, aphotograph of each gel is presented above each gel scan. The gel itself, not thephotograph, was scanned. The untreated cultures had less than 1% hemoglobin-containing cells, and the Me2SO-treated cultures had 52% hemoglobin-containing cells, as determined by benzidine-hydrogen peroxide staining. The viabilityof the cells in these and other experiments, as determined by trypan blueexclusion, was 90% for the untreated cells and 97 to 99% for the Me2SO-treatedcells.

prior to the addition of enzyme; or (c) with extracts heated to90°for 5 min. By all 3 methods, FAoremained unchanged.

DNase Assay. The effect of increasing protein concentrations from extracts of untreated and Me2SO-treated MEL cells

on the digestion of the cohesive termini of <>105 DNA can beseen in Chart 5. In contrast to the changes seen in DNA ligaseactivity, the DNase activity, as determined by DNase Assay A,

APRIL 1982 1303

ß.M. Scher et al.

S zo

z(E 10

20 40 60 80 100

PROTEIN ifig)

Chart 4. Effect of varying amounts of cell-free extracts of MEL cells fromuntreated (O) and Me2SO-treated (•)cultures on the ligation of the cohesivetermini of &ÌQ5DNA. After growth in culture for 4 days, cells were harvested,cell-free extracts were prepared, and DNA ligase assays were performed asdescribed in "Materials and Methods." The levels of cohesive termini of <f>105

DNA ligated were determined from the amounts of heat-stable Fragment A formedat the expense of Fragments C and D and were measured by agarose gelelectrophoresis as described in "Materials and Methods." Calculations aredescribed in "Results." The untreated cultures contained 0.3% or less benzidine-

reactive cells while the Me2SO-treated cultures contained 85% benzidine-reactivecells. The hemoglobin content of the extracts from Me2SO-treated cultures was8.8 nmol/ml. This is equivalent to a hemoglobin content of 6.6 pg/benzidine-

reactive cell.

did not decrease after 4 days of treatment of cells with Me2SO,but rather increased slightly. When equivalent amounts ofprotein were used, the activity in extracts of Me2SO-treatedcells was about 2-fold greater than that in extracts from un

treated cells.DNA Ligase and DNase Activities in Cell Line DR-10. The

levels of DNA ligase activity were followed in both the Me2SOdifferentiation-sensitive, wild-type strain of MEL cells, 5-86,and a Me2SO differentiation-resistant strain of MEL cells, DR-

10. After 4 days of culture with and without added Me2SO, thelevels of DNA ligase in the Me2SO-treated 5-86 cultures haddecreased markedly but were unchanged in the Me2SO-treatedDR-10 cultures (Chart 6). The quantitation of these experiments

is shown in Table 2. After growth in the presence of Me2SO,5-86 cultures produced a high level of benzidine-reactive cells

as compared to untreated cultures. At the same time, the levelsof DNA ligase in the cells dropped to about 10 to 20% of thevalues seen in the untreated cells. In contrast, the specificactivity of the DNase increased about 2-fold in the Me2SO-

treated cells as compared to untreated cells, and the levels ofacid phosphatase, an enzyme used as an indicator of lysosomalcontamination (1), did not change. However, when DR-10 cells

- 50o

S 405ud 30

2

l 20

LU

K IOLUIOO

ZO 40 60 80PROTEIN (/jg)

IOO

Chart 5. Effect of varying amounts of cell-free extracts of MEL cells fromuntreated (O) and Me2SO-treated (•)cultures on the cleavage of the cohesivetermini of <j>105 DNA. Cells were grown for 4 days, extracts were prepared, andDNase assays (Assay A) were performed as described in "Materials and Methods." The levels of cohesive termini of (f>105 DNA cleaved were determined from

the amounts of Fragments C and D formed at the expense of Fragment A.Electrophoresis and calculation of results was as indicated in Chart 4.

10 20 30PROTEIN (fig)

Chart 6. Effect of cell-free extracts of different cell lines of MEL cells fromuntreated and Me2SO-treated cultures on the ligation of the cohesive termini of<)>105DNA. Cells were grown for 4 days, extracts were prepared, and DNA ligaseassays were performed as described in "Materials and Methods." The levels of

cohesive termini ligated were determined as in Chart 4. The cell lines used were5-86, grown either in the absence (O) or in the presence (•)of Me2SO, and DR-10, grown either in the absence (A) or in the presence (A) of Me2SO.

Table 2

Effect of growth in Me2SO on the levels of DNA ligase. DNase, and acid phosphatase in cultured MELcells

MEL cells were grown for 4 days as described in "Materials and Methods" in the presence or in the

absence of 1.8% (v/v) Me2SO as indicated. The cells were stained for benzidine-reactive material, and thelevels of the indicated enzymatic activities were measured in dialyzed cell-free extracts as described in"Materials and Methods."

Experiment1IIIIITPIIlinp Growth with1 '••"Me2SO5-86+5-86+5-86+DR-10+(pmol/30

min/mgprotein)B

+a(%)<0.3660.385<0.357<0.3<0.3DNA

ligase0.28<0.0030.300.020.330.070.290.28DNaseAssayA0.070.180.080.23DNaseAssayB16.029.29.09.6Acid

phosphatase

((imol/hr/mg protein)0.620.62

B+, benzidine-reactive cells.

1304 CANCER RESEARCH VOL. 42

DNA Ligase and DNase Activities in Erythroleukemia Cells

Table 3Effect of the addition of cell-free extracts from Me2SO-treated MEL cells on the

DNA ligase activity of cell-free extracts from untreated MEL cells

MEL cells were grown for the number of fir indicated in the presence or in theabsence of Me2SO as described in "Materials and Methods. Cell-free extracts

were prepared, and DNA ligase assays were performed as described in"Materials and Methods."

Protein fromun-Timein culture treatedextracts(hr)

(fig/assay)96

3.16.23.16.2119

6.212.56.212.5Protein

fromMe2SO-treatedextractsQig

/assay)3.16.23.16.26.212.56.212.5DNA

ligaseactivity.cohesiveterminili-gated

(fmol/30min/assay)11.123.6<1.01.011.323.710.222.3<1.0<1.012.524.6

were tested, different results were obtained. Followingtreatment, no benzidine-reactive cells were noted, and the DNAligase and DNase activities remained the same as in untreatedDR-10 cells.

Effect of the Addition of Cell-free Extracts from Me?SO-Treated Cells on the DNA Ligase Activity of Cell-free Extracts

from Untreated Cells. When equivalent amounts of proteinfrom cell-free extracts prepared from Me2SO-treated cells wereadded to cell-free extracts prepared from untreated cells, noinhibition of the DNA ligase activity in the cell-free extracts of

untreated cells was seen. This was noted with cells grown bothfor 96 hr and for 119 hr in the presence or in the absence ofMe2SO (Table 3). This result suggests that the lack of DNAligase activity in Me2SO-treated extracts is not due to the

presence in these extracts of a phosphatase, exonuclease,ATPase, protease, or other inhibitor of the assay or of the DNAligase enzyme itself.

DISCUSSION

The activities of DNA ligase and actin-inhibitable DNase were

assayed in MEL cells. DNA ligase was assayed by followingthe formation of a heat-stable bond in the cohesive termini of

bacteriophage 0105 DNA. The bonds thus formed were stableto heating, sodium dodecyl sulfate treatment (data not shown),and protease digestion (proteinase K was included in all assays). Bond formation was ATP dependent. All of these properties are consistent with those of a eukaryotic DNA ligase(36).

DNase activity was assayed either by following the destruction of the cohesive termini of 0105 DNA or by following theendonucleolytic cleavage of supercoiled PM2 DNA. The DNaseactivity in extracts prepared from both untreated and Me2SO-treated MEL cells was completely inhibited by G-actin in the

assays performed at neutral pH. These results suggested thatthe predominant "neutral" DNase extracted from the cells was

DNase I or DNase l-like. Two other DNases with different

properties than DNase I have been found in Friend leukemiavirus-infected cells. An endonuclease is present in Friend leu

kemia virus propagated in a mouse embryo cell line (23), anda calcium-stimulated nuclease activity has been found in nuclei

of Me2SO-treated MEL cells, but not in untreated MEL cells

(16).Using these assays, the activities of DNA ligase and the

actin-inhibitable DNase were assayed in MEL cells grown for

3 or 4 days in the absence or in the presence of Me2SO, atreatment which induces some steps of terminal differentiationin these cells. In the cells grown for 3 or 4 days in the presenceof Me2SO, the apparent level of DNA ligase activity dropped toabout 12% of that seen in cultures grown in parallel in theabsence of Me2SO.

In contrast, the apparent specific activity of actin-inhibitableDNase increased 2-fold in Me2SO-treated cells as comparedto untreated cells, while the activity of acid (lysosomal-type)

phosphatase remained unchanged.When dialyzed extracts from either untreated or Me2SO-

treated cells were assayed for DNase activity in the presenceof G-actin, no change in the pattern of EcoRI fragments of the

input DNA was seen. There was no breakdown of Fragment Ato Fragments C and D due to actin-resistant endo- or exonu

clease activity, nor was there any change in the yield offragments recovered from the assay.

To test whether Me2SO present in cell-free extracts couldaffect the activity of DNA ligase, Me2SO was added directly toDNA ligase assays. No effect of added Me2SO was seen (datanot shown). When extracts were dialyzed as described in"Materials and Methods" to remove traces of Me2SO, DNA

ligase activities remained the same as those before dialysis(data not shown).

Cell line DR-10 is known to take up Me2SO from the mediumat the same rate as wild-type cell lines, although the uptake in

this case does not lead to induction of erythroid differentiation(24). The levels of DNA ligase and DNase activities wereapproximately the same in DR-10 and 5-86 cells prior to

treatment with Me2SO and, when the cells were grown in thepresence of Me2SO, no changes in enzyme levels were seenin DR-10 cells in comparison to the alterations noted in 5-86

cells (Table 2). These data suggest that the apparent changesin the levels of DNA ligase activity occurred as a result of thedifferentiation process and were not due to the presence ofMe2SO in the cells per se.

Experiments were done in which DNA ligase assays werecarried out in the presence of mixtures of extracts from untreated cells and extracts from Me2SO-treated cells (Table 3).

These assays indicated that there were no components presentin the extracts from Me2SO-treated cells which inhibited theDNA ligase activity of the untreated cells. These results argueagainst the possibility that the apparent lack of DNA ligaseactivity is being caused by the destruction or inactivation ofsubstrates or enzyme by phosphatases, nucleases, proteases,ATPases, or other inhibitors of the DNA ligase assay. Thereason for this apparent deficiency in DNA ligase activity inMe2SO-treated cells is not known. It seems unlikely that theDNA ligase(s) is simply no longer extractable from the nucleiand thus unavailable for the assay. Several chromatin proteinsfrom Me2SO-treated MEL cells have been shown to be moreeasily extractable from the nuclei by aqueous salt solutionswhen compared to chromatin proteins extracted from the nucleiof untreated cells (17). The DNA ligase deficiency could be dueto the sequestering of the DNA ligase activity in some inactiveform in the extracts from Me2SO-treated cells or could be due

to a change in the rate of synthesis or breakdown of the

APRIL 1982 1305

B. M. Scher et al.

enzyme in vivo. Further experiments will be needed to clarifythese points.

In general, when MEL cells are committed to differentiate,the multiplication potential of the cells is impaired, and only alimited number of cell divisions occur (11). However, whenMEL cells were grown in medium with Me2SO, no reductionwas seen in the level of DMA polymerase after 4 days of growth,and only a 50% reduction was seen after 5 days as comparedto cells grown without Me2SO (8). Giri et al. (10) have reportedthat there were no quantitative differences in DMA polymerasea activity between hexamethylene bisacetamide-induced and

uninduced MEL cells and that DMA polymerase ßactivityremained constant throughout the cell cycle in both inducedand uninduced cells. In contrast, as demonstrated here, thelevel of DNA ligase activity extracted from MEL cells grown for3 or 4 days in the presence of Me2SO was only 12% of thatextracted from MEL cells grown without Me2SO. If this decrease of DNA ligase activity reflects the true state of the cellin vivo, it is possible that such a marked reduction of DNAligase activity in the Me2SO-induced cells could contribute to

the loss of proliferative capacity seen in these cells. It is ofinterest that recent work on the activity of DNA ligase in otherdevelopmental systems has described changes in both thelevels and types of DNA ligase present which correlate withdevelopmental changes in the tissues studied (5, 22).

ACKNOWLEDGMENTS

We thank Drs. J. Wetmur and A. Garro for valuable discussions, N. Hellingerand H. Yen for expert technical assistance, and J. Kassouf. Department ofUrology, Mount Sinai School of Medicine, for the acid phosphatase determinations.

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