effects of ethylenediaminetetraacetic acid and 1,10...

(CANCER RESEARCH 40, 4092-4099, November 1980)0008-5472/80/0040-OOOOS02.00

Effects of Ethylenediaminetetraacetic Acid and 1,10-Phenanthroline onCell Proliferation and DMA Synthesis of Ehrlich Ascites Cells1

Chitra Krishnamurti, Leon A. Saryan,3 and David H. Petering4

Department of Chemistry, University of Wisconsin-Milwaukee. Milwaukee. Wisconsin 53201

ABSTRACT

The interactions of ethylenediaminetetraacetic acid (EDTA)and 1,10-phenanthroline with Ehrlich cells have been studied.Both compounds inhibit proliferation of cells in culture. Afterlong-term incubation of cells with these metal-chelating agentsunder conditions in which cell viability is not impaired butproliferation is retarded, the rate of DNA synthesis of EDTA-exposed cells is the same as that of untreated controls,whereas the rate of DNA synthesis of 1,10-phenanthroline-

exposed cells is markedly reduced. This is in agreement withthe inhibition of short-term DNA synthesis by similar concentrations of 1,10-phenanthroline as well as the lack of effect ofEDTA upon DNA synthesis. The rapid direct effect of 1,10-

phenanthroline upon cells in S phase is consistent with thefinding that a large fraction of this metal-binding ligand but not

of EDTA can be readily taken up by cells. These results differin significant ways from the response of Ehrlich cells in vivo tohost zinc deficiency. Finally, titration of ligand-treated cellcultures with zinc reverses the inhibition of DNA synthesis andcell proliferation in a linear manner, suggesting that the removalof ligand by complexation and the addition of a previousunavailable essential metal is occurring in these reactions.

INTRODUCTION

The importance of trace minerals in biological systems hasbeen increasingly recognized in recent years, but comparatively little effort has been directed towards the role of theseelements in cancer (21, 25). It has been found that zinc, inparticular, is required for the optimal proliferation of transplant-

able rodent tumors (7, 14,15,18). Recent studies have definedsome of the growth characteristics and biochemical lesions ofthe Ehrlich tumor in hosts maintained on a zinc-deficient dietary

regimen (15, 19, 24).Previous efforts to induce metal-deficient states in various

cell lines have resorted to the use of chelating agents such asEDTA and 1,10-phenanthroline (5,8,10,12,13, 22, 28). Suchstudies have suggested that these compounds interact specifically with zinc and that their effects on cell cultures parallelthose of natural zinc deficiency imposed by removal of zincfrom the medium supplying cells with nutrients. However, suchmaterials are not necessarily specific for zinc but may bindother divalent cations. Further, the metal complexes which

1Contribution 123 from The Laboratory for Molecular BiomÃ©dical Research.

This work was supported in part by Grant CA 16156-03 from the National CancerInstitute, NIH.

' Present address: Department of Biochemistry, Medical College of Wisconsin,

Milwaukee, Wisconsin 53201.' Recipient of National Cancer Institute Individual Postdoctoral Fellowship CA-

05528. under which a portion of this work was completed.4 To whom requests for reprints should be addressed.

Received April 11. 1980; accepted July 28, 1980.

form may have distinct biological properties. For example, workon the effects of thiosemicarbazone metal-chelating agents

and their metal complexes on the properties of cancer cellshave shown that such ligands may be activated in vivo throughmetal complexation, that more than one metal may activate theligand, and that that the net stability of metal complexes in vivois a function of their reactions with many biomolecules (1, 16-18, 23).

Therefore, the question has been raised as to the relationshipof studies of the properties of cells from zinc-deficient animals

with those involving cells incubated in culture with EDTA and1,10-phenanthroline. Accordingly, in this study, we begin an

examination of the properties of Ehrlich tumor cells grown inculture in the presence of these chelating agents for comparison with results obtained using Ehrlich cells grown in zinc-

deficient mice.

MATERIALS AND METHODS

Reagents and Radioactive Precursors. All reagent chemicals were of the highest purity commercially available. Zincsulfate was obtained from Matheson, Coleman & Bell (Norwood, Ohio). 1,10-Phenanthroline monohydrate was obtained

from Eastman Organic Chemicals (Rochester, N. Y.) and dispensed as a dimethyl sulfoxide solution (gold label grade;Aldrich Chemical Co., Milwaukee, Wis.). EDTA was obtainedfrom Aldrich and dispensed in phosphate-buffered saline [0.01

M potassium phosphate:0.15 M NaCI], pH 7.4. Media andchemicals used for cell culture were obtained from GrandIsland Biological Co. (Grand Island, N. Y.), except 4-morpholi-

nopropanesulfonic acid which was obtained from Aldrich andsodium bicarbonate which was obtained from Mallinckrodt, Inc.(Paris, Kentucky).

Radioactively labeled compounds were purchased as follows: [mef/7y/-3H]thymidine (2.0 /iCi/mmol; 1.0 mCi/ml; Amer-sham/Searle Corp., Arlington Heights, III.); [acef/c-2-'4C]EDTA

(2.76 mCi/mmol; ICN Pharmaceuticals, Inc., Irvine, Calif.).Animals and Ehrlich Ascites Tumor. Female HA/ICR mice

(6 to 8 weeks old) were obtained from ARS/Sprague-Dawley

(Madison, Wis.) and acclimated to our laboratory conditions forapproximately 1 month prior to use. Animals were housed asdescribed previously and fed ad libitum with Purina rodentchow and tap water (15).

Ehrlich ascites tumor was maintained in vivo by weekly i.p.transplantation of approximately 5 x 106 cells into recipient

mice. Animals were sacrificed by cervical dislocation, and thei.p. contents (cells and fluid) were harvested by syringe puncture. Suspensions showing significant contamination by eryth-

rocytes were not used. Cells were separated from ascites fluidby centrifugation at moderate speed using a tabletop clinicalcentrifuge, and the resulting pellet was resuspended in anappropriate medium. Viable cell counts were obtained micro-

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Cellular Effects of EOTA and 1,10-Phenanthroline

scopically using a Spencer Bright-Line hemocytometer (American Optical Corp., Buffalo, N. Y.) at X100 magnification. Cellviability was determined by trypan blue dye (0.4% in 0.15 MNaCI) exclusion test. Protein concentration of cell suspensionswas determined using the Biuret procedure, corrected forsample turbidity with the aid of a scattered transmission attachment on a Beckman Acta V spectrophotometer. Bovineserum albumin was used as the standard.

Ehrlich Cell Cultures. Mouse Ehrlich ascites tumor cells(obtained from Dr. Edvardas Kaminskas, Mt. Sinai MedicalCenter, Milwaukee, Wis.) were propagated in suspension culture in modified Eagle's MEM.5 The medium was prepared asfollows. One package of powdered Eagle's MEM with Earle's

salts (No. 410-1400; Grand Island Biological Co.) was dissolved in 5 liters of glass-distilled water supplemented with 18g 4-morpholinopropanesulfonic acid (final concentration,

0.017 M), 11 g sodium bicarbonate (final concentration, 0.025M), penicillin G (50 mg/liter), and streptomycin sulfate (50 mg/liter) and sterilized by filtration. Medium so prepared wassupplemented with 2.5% (v/v) sterile fetal bovine serum priorto use.

For cell proliferation experiments, cells were plated at aconcentration of 1 x 105 cells/ml into replicate Retri dishes

containing 5 to 10 ml of medium and the appropriate chelatingagent. Plates were incubated at 37Â° in a humidified 5% CO2

incubator for the duration of the experiment, after which theywere harvested by transfer pipet, counted, and saved for assayof DMA synthesis. Manipulations were carried out in a laminarflow hood to minimize the possibility of contamination. Cellcounts and viability were determined as noted above.

Short-Term Uptake of EDTA by Ehrlich Cells. Ehrlich cellsharvested from tumor-bearing animals were suspended in MEMat 37Â°at a concentration of approximately 6.5 x 106 cells/ml.

At times up to 40 min following the addition of approximately0.21 jiiCi of [14C]EDTA, aliquots of the suspension were re

moved, rapidly chilled on ice slush, and centrifuged rapidly ona clinical centrifuge. The supernatant was quantitatively decanted and reserved for radioactivity determination. The cellpellet was resuspended in chilled medium and recentrifuged.The wash was carefully decanted and reserved for radioactivitymeasurement. The cell pellet was dissolved in 0.1 M KOH, andthe 3 compartments were counted by liquid scintillation techniques. Recovery of radioactive label was between 99 and100% with a counting efficiency of greater than 90%.

Long-Term Uptake of EDTA by Ehrlich Cultures. Long-termuptake studies were conducted in suspension culture for periods up to 96 hr. Cells at an initial concentration of approximately 1 x 105/ml were exposed to [">C]EDTA at a concentration of 120 nm EDTA per mg cell protein (3.02 x 10~6 M). At

various times, multiple 1-ml aliquots of culture were removedand filtered through a 12-port manifold filtration apparatus(Millipore Corp., Bedford, Mass.) equipped with Metricel 5-/impore size filters (Gelman Instrument Co., Ann Arbor, Mich.).Filters laden with cells were washed 3 times with chilled MEM(1 ml/wash). The entire filter as well as a 50-fil aliquot of thecombined supernatant and wash fluids was counted for 14C

activity by liquid scintillation. Recovery and counting efficiencywere comparable to the short-term uptake experiments. Cell

5 The abbreviation used is: MEM, minimum essential medium.

counts and viability were determined for the culture at eachtime point studied.

Short-Term Uptake of 1,10-Phenanthroline by EhrlichCells. Ehrlich cells harvested from tumor-bearing animals weresuspended in phosphate-buffered saline, pH 7.4, at 37Â°at aconcentration of approximately 5 x 106 cells/ml. At times upto 40 min following the addition of 1,10-phenanthroline to the

suspension, aliquots were removed, chilled on ice slush, andcentrifuged rapidly on a clinical centrifuge. The supernatantwas quantitatively decanted and treated with an excess offreshly prepared ferrous ammonium sulfate solution (typically5 to 10 julof a 0.14 M solution), mixed well, and read at 510 nmin a scattered transmission chamber attached to a BeckmanActa V spectrophotometer. The stable (1,10-phenanthroline)3-Fe(ll) complex formed was found to have an e5io = 9700 M~1.

Loss of 1,10-phenanthroline from the medium under theseconditions was attributed to uptake of the compound by cells.

[3H]Thymidine Utilization by Ehrlich Cells. Our assay for

short-term inhibition of DMA synthesis in drug-treated tumorcells measures both [3H]thymidine incorporation into acid-pre-cipitable material (containing DMA) and [3H]thymidine uptake

by cells and has been described previously (23). Drug concentrations required for 50% inhibition of DMA synthesis relativeto untreated control and 25% inhibition of thymidine uptake(75% of control) were determined from graphs summarizingthe concentration dependence of the inhibition of these processes by EDTA and 1,10-phenanthroline.

Substantially the same assay was used to determine the levelof DMA synthesis in cultures treated with chelating agents, withthe additional precaution that cell pellets were precipitated withperchloric acid in the presence of 20 to 50 fil of bovine serumalbumin solution (8.6 mg/ml) to prevent inadvertent loss ofacid-insoluble radiolabel during the workup.

Liquid Scintillation Counting. The techniques for the extraction and estimation of radioactively labeled molecules havebeen described previously (24). In these experiments, thecocktail formula was changed to toluene:ethanol (8:2), with aconsequent increase in 3H counting efficiency to approximately

45%.

RESULTS

Uptake of EDTA by Ehrlich Cells. EDTA is not significantlyaccumulated by Ehrlich ascites cells in either short- or long-

term incubations carried out under a variety of experimentalconditions. Table 1 shows that [14C]EDTA is not appreciablyassociated with the cell pellet after a 30-min incubation at 37Â°.A slight molar excess of zinc premixed with the [14C]EDTA had

no effect on the level of uptake of radioactivity by cells. Usingthe calculation of Spencer and Lehninger (26) for the water-

permeable space o_fthe Ehrlich cell (3 jul/mg cell protein), theinternahexternal concentration ratio for both EDTA and zinc:EDTA was found to be approximately 0.68 after a 30-min

incubation. This supports the conclusion that neither EDTA norits zinc complex is markedly taken up by Ehrlich cells underthese conditions. Table 2 shows that increasing the total extracellular concentration of EDTA did not increase the fractiontaken up by cells after 40 min, nor did it differ from the amounttaken up after 10 to 20 min. In this experiment, the internal:external concentration ratio calculated as above was found to

NOVEMBER 1980 4093



C. Krishnamurti et al.

Table 1Short-term uptake ofl"CÂ¡EDTA and Â¡"C]EDTA:zinc by Ehrlich ase/fes ce//s

Conditions of incubation were: 2x10' cells/3 ml incubation; final [EOTA],2.44 x 10 5M or 15.1 nmol/mg cell protein; phosphate-buffered saline medium,

pH 7.4.% of distribution of recovered'"CTime

after(min)[MC]EDTA

102030("ClEDTA:zinc

102030Medium96.796.196.296.797.495.4cpmWash2.93.23.53.02.44.2Cell

pellet0.30.70.30.30.20.4

Table 2Concentration dependence of short-term uptake of Â¡"CJEDTA by Ehrlich

ascites cellsConditions of incubation were: 4X107 cells/3 ml incubation; MEM (pH 7.8)

at 37Â°.

Total[['"CJEDTA]In

suspension (M) nmol/mg cellprotein2.4x 10~5

1.2 x 10 "2.2 X 10~44.1 X 10""7.7

38.469

130%

of recovered'"Ccell

pellet at 40min0.6

0.60.70.6

be approximately 0.63 at each concentration of EDTA used,substantially in agreement with the previous experiment.

Longer incubation in suspension culture (up to 4 days)showed very little change in the percentage of total recoveredradioactivity associated with cells (Chart 1). About 1% of therecovered 14Ccpm is bound to washed cells, with little signifi

cant increase or decrease over the course of the experiment.Uptake of EDTA per 106 viable cells shows an increase only up

to 24 hr, after which an approximately exponential decrease isobserved. However, the culture did not reach a maximum celldensity until 72 hr (viability, >90%). At f = 24 hr, there is anapparent 100-fold concentration excess of EDTA inside cellswhen calculated as above, suggesting that under culture conditions some accumulation of EDTA occurs.

Uptake of 1,1O-Phenanthroline by Ehrlich Cells. In comparison with EDTA, 1,10-phenanthroline is taken up muchmore readily by Ehrlich cells after a short-term incubation.Chart 2 illustrates uptake (measured as loss from the externalmedium) and shows that approximately 20% of the added1,10-phenanthroline is taken up after a 40-min incubation. The

ratio of internahexternal concentrations calculated as for EDTAwas found to be 46.4 (for 150 nmol 1,10-phenanthroline permg) and 60.9 (for 300 nmol 1,10-phenanthroline per mg) andshows a significant accumulation of 1,10-phenanthroline bycells that is related to the external concentration of this compound.

Response of Ehrlich Cultures to EDTA and Zinc:EDTATreatment. Cells were exposed to EDTA or zinc:EDTA (1:1) attime zero and harvested after a 72-hr incubation with theseagents for cell counts and measurement of [3H]thymidine utili

zation. Results are shown in Chart 3 for replicate cultures ateach concentration. Whereas EDTA itself is inhibitory to cellgrowth at concentrations as low as 1.5 x 10~5 M (300 nmol/106 cells or 1.2 jumol/mg) and nearly totally inhibitory at 6 and9 x 10~5 M, zinc:EDTA causes a slight stimulation of cell

s2 30

oI

iÂ¡,0

r

24 48 71TIME IHRI AFTER ADDITION OF WC-EDTA

Chart 1. Long-term uptake of [MC]EDTA in Ehrlich cell cultures. Initial cellconcentration was 1.0 x I0s/ml in MEM supplemented with 2.5% fetal calfserum; [EDTA] was 3.02 x 10~6 M; incubation temperature was 37Â°. O, percentage of recovered 14Ccpm associated with washed cells at each time. â€¢,14Cactivity per 106 viable cells in culture. Bars, range of data at each time.

30

I9 2 20r- 5

IILU tfÃ¬

O DÂ£ "

uj Ou teoc "-

10

10 20 30 40

TIME (MIN) AFTER 1. 10 - PHEN ADDITION

Chart 2. Short-term uptake of 1.10-phenanthroline (1.10-PHEN) by Ehrlichcells. Ehrlich ascites cells were suspended at 37Â°in phosphate-buffered saline,pH 7.2. at concentration of 3.8 x 106 cells/ml (O) and 7.6 x 10" cells/ml (A)and exposed to 3.25 x 10"" M 1,10-phenanthroline (300 nmol/mg cell protein

(O) and 150 nmol/mg (A)]. Aliquots taken at times up to 40 min after treatmentwere analyzed for the remaining [1.10-phenanthroline] in the supernatant.

growth at 1.5 and 3 x 10 5 M and causes only a gradualdecrease in proliferative capacity between 6 and 9 x 10~5 M.Even at 9 x 10~5 M, the highest concentration tested, zinc:

EDTA causes no significant inhibition of cell proliferation withrespect to controls. In contrast, EDTA causes a 100-fold decrease in cell number relative to control and a 2-fold reductionwith respect to the number of cells initially placed in culture.Cell viabilities (not shown) in all zinc:EDTA cultures and allEDTA cultures up to and including 3 x 10~5 M were >98%. At6 x 10~5 M EDTA, viability had decreased to an average of55% and, at 9 x 10~5 M EDTA, to an average of 24%,

indicating that significant cell death was occurring under theseconditions.

Incorporation of [3H]thymidine per culture approximately fol

lows the pattern for cell growth (Chart 3). The efficiency ofthymidine incorporation for EDTA-treated cultures at 0, 1.5,and 3x10~5 M is approximately unchanged, ranging between




Cellular Effects of EOTA and 7,10-Phenanthroline

Table 3Stimulation of EDTA-treated cultures by addition ol Â¿ine

Relative viable cell number per culture and relative incorporated cpm per 106viable cells were evaluated in replicate cultures harvested after 5 days' exposureto EDTA and identical cultures to which ZnSO* (final concentration. 10 * M) had

been added on Day 3.

1 23456789

[EDTA] OR [Zn EDTA] x IO5. M

Chart 3. Response of Ehrlich cultures to EDTA and zinc:EDTA (Zn EDTA)treatment. Cultures plated at an initial concentration of 4.7 x 104 cells/ml (arrow)

were exposed to either EDTA or zinc:EDTA (1:1 ) at the time of plating (f = 0 hr)and harvested 72 hr later for cell enumeration and [3HJthymidine incorporation

studies. Results for replicate plates at each concentration are indicated A.comparison of 3H incorporation into acid-precipitable material for cultures treated

with EDTA (O) and ZnrEDTA (A). B. viable cell number per ml of culture

160,000 and 135,000 cpm/106 cells. Thus, despite the reduc

tion of cell proliferation caused by EDTA over the 72-hr period,

by the end of this time all these cultures are synthesizing newDMA at approximately the same rate. By contrast, at 6 and 9x 10~5 M EDTA, incorporation drops to approximately 20,000

cpm/106 viable cells. Thus, almost no incorporation of radio-label is observed at EDTA concentrations of 6 and 9 x 10~5

M. When examined on a per viable cell basis, thymidine incorporation per cell is almost 10 times lower at these EDTAconcentrations than for untreated cultures. Over the concentration range studied, efficiency of thymidine incorporation percells for zinc:EOTA-treated cultures declines very graduallyfrom 160,000 cpm/106 cells for untreated cultures to 100,000cpm/106 cells for cultures exposed at 9 x 10~6 M zinc:EDTA.

Stimulation of EOTA-treated Cultures by Addition of Zinc.Cells cultivated in cultures which had been exposed to EDTAfor 3 days could be stimulated by addition of ZnSO^ on Day 3.

Initial [EDTA](M)0

1.5 x 10~53 X 10 56 X 1CT5Viable

cells +ZnSO,,Viable

cellscontrol0.84

0.981.252.81[3H]Thymidine

incorporation

into DMA +ZnSOÂ«[3H)Thymidineincorporation

into DMAcontrol0.36

2.582.216.46

Results shown in Table 3 show that cell growth and DNAsynthesis were stimulated by the addition of zinc to EDTA-

treated cultures. These data indicate that the stimulation isgreatest at high concentrations of EDTA, which in themselvesare inhibitory to cell growth as shown in Chart 3. Cultures nottreated with EDTA showed a relative decrease in cell numberand DNA synthesis when exposed to zinc, indicating a toxiceffect of the metal under these conditions. Zinc has beenshown to be inhibitory to DNA synthesis in Ehrlich cells inshort-term incubations as well (24). Stimulation of DNA synthesis was more marked than increase in cell number, reflectingthe need for long-term synthesis before appreciable cell prolif

eration is observed.A similar experiment was performed in which various con

centrations of zinc were added to EDTA-treated cultures.Changes in cell number and [3H]thymidine incorporation were

evaluated. The data are presented in Chart 4. Replicate cultures were exposed to EDTA at time zero. Forty-eight hr thereafter, ZnSC-4 was added and cultures were harvested at 72 hr.

A modest increase in cell number was observed as a result ofzinc addition for cultures exposed at either concentration ofEDTA. A much stronger stimulation of DNA synthesis wasnoted. An approximate 1:1 relationship between the concentration of EDTA used and the amount of zinc needed formaximal stimulation of DNA synthesis was seen particularly atan EDTA level of 4 x 10~5 M. In agreement with Chart 3, EDTAis more inhibitory to cell growth at 4 x 10~5 M than at 2 x1Q-5M.

Inhibition of Ehrlich Cultures by 1,10-Phenanthroline. Inhibition of cell proliferation was observed for Ehrlich cell cultures exposed to 1,10-phenanthroline. Cells originally plated

on Retri dishes were exposed to varying concentrations of1,10-phenanthroline at time zero and harvested 48 hr later for

examination. Results are shown for replicate cultures in Chart5. 1,10-Phenanthroline was found to be inhibitory to cell proliferation and DNA synthesis at a concentration of 5 x 10"6 M

(55 nmol/106 cells or 220 nmol/mg cell protein) with 85%

retention of cell viability. This is 10 to 20% of the concentrationneeded to produce a similar effect with EDTA as shown inChart 3. Cultures incubated at 2.5 x 10~6 M showed little Â¡f

any inhibition when compared to untreated cultures. Efficiencyof [3H]thymidine incorporation (cpm incorporated into DNA per106 cells) at 5 x 10~6 M 1,10-phenanthroline is approximately

one-half that of untreated cultures and the lower 1,10-phenan

throline concentrations tested. When calculated on the basisof percentage of untreated control, an extremely abrupt doseresponse is observed. DNA synthesis occurs at 88% of controlrate at 2.5 x 10~5 M and approximately 10% of control values

NOVEMBER 1980 4095




02468

ADDED [Zn] x IO5, M

Chart 4. Stimulation of EDTA-treated cultures by zinc addition. Cultures wereexposed to EDTA at t = 0 hr at concentrations of 2 x 10~5 (A) and 4 x 1CT5

M (O). ZnSO< was added at f = 48 hr, and all plates were harvested at ( = 72 hrfor cell enumeration and [3H]thymidine incorporation studies. A, comparison ofDMA synthesis levels (cpm of 3H incorporated per 1O6cells); B, viable cells per

ml of culture at f = 72 hr. Replicate plates were evaluated at each concentration.

at 5x10 6M1,10-phenanthroline. This Â¡sa markedly different

result from that found with EDTA, since with that chelatingagent no decrease in normalized rate of DNA synthesis accompanied the observed decrease in cell proliferation.

Restimulation of 1,10-Phenanthroline-treated Cultures byZinc. Various experiments were performed which demonstrated that the inhibitory effect of 1,10-phenanthroline treat

ment could be reversed by subsequent administration of zinc.Ehrlich cell cultures cultivated as in the previous experimentwere exposed to varying levels of 1,10-phenanthroline for 48

hr, at which time zinc sulfate was added to a final concentrationof 5 x 10~6 M. Cells were harvested 24 hr later for DNA

synthesis and cell enumeration. The results are summarized inChart 6. Chart 6A shows that treatment with zinc restored someDNA synthetic activity in cultures partially inhibited by 1,10-

phenanthroline to approximate control values. In Chart 66,however, it can be seen that there is no concomitant increasein cell number resulting from such stimulation. This is in contrast to the results observed for EDTA under similar conditions,suggesting that the effects of 1,10-phenanthroline are more

12345

[1, K>- PHEN) Â«X)6. M

Chart 5. Inhibition of cell proliferation and DNA synthesis in Ehrlich culturesby 1,10-phenanthroline (1,10-PHEN). Cultures were exposed to 1,10-phenanthroline at time zero after plating at an initial concentration of 9 x 104 cells/ml(arrow) and harvested 48 hr later for cell enumeration and [3H]thymidine utilizationstudies. A, 3H cpm incorporated into acid-precipitable material per plate for

replicate plates. O, viable cell number per plate.

[1.10 - PHEN] x WB

Chart 6. Restimulation of 1,10-phenanthroline ( /, 10-PHENMreated cultureswith zinc. Cultures plated at 9 x 10* cells/ml were exposed to varying levels of

1,10-phenanthroline (as in Chart 5) for 48 hr, at which point ZnSO4 (finalconcentration, 5 x 10 ' M) was added. Cells were harvested 24 hr later for cell

enumeration and DNA synthesis studies. A. percentage of recovered cpm incorporated into DNA for zinc-treated cultures (A) and untreated controls (O) forreplicate plates. B, viable cell number per plate for zinc-treated (A) and untreated

(O) cultures.

profound and require a longer period of time for the cultures torecover.

In a second experiment, cultures exposed to 2.5 x 10~6 M




Cellular Effects of EDTA and 1,10-Phenanthroline

1,10-phenanthroline at time zero were treated with varyinglevels of zinc beginning at f = 48 hr and harvested at f = 96

hr instead of 72 hr as done in Chart 6. Results shown in Chart7 illustrate that cell number can be increased by approximately30% above control level by allowing cultures to grow for anadditional day, with the stimulation showing a concentration-dependent response similar to that observed for EDTA-treated

cultures.Effects of EDTA and 1,10-Phenanthroline on DMA Synthe

sis. 1,10-Phenanthroline but not EDTA rapidly inhibits DMAsynthesis in Ehrlich cells, as measured by relative levels ofincorporation of [3H]thymidine into perchloric acid-insoluble

material in short-term in vitro assays (Table 4). The assayenabled simultaneous determination of any effects on thymi-

dine uptake as well. EDTA shows little if any effect on DMAsynthesis or thymidine uptake in cells after a 1-hr incubationwith chelating agent at 37Â°at concentrations up to 3 rriM EDTA(375 nmol/mg cell protein and 94 nmol/106 cells). Under

similar incubation conditions, 0.8 rriM 1,10-phenanthroline(100 nmol/mg cell protein and 25 nmol/106 cells) inhibits DMA

synthesis to 50% of control. Thymidine uptake is diminished to75% of control levels at a much higher level of 1,10-phenanthroline. Hence, at concentrations of 1,10-phenanthroline

which inhibit cell growth (Chart 5), the chelating agent is alsorapidly able to inhibit DMA synthesis.

DU

6-

4

3

2

1

[Zn SO4]x106.M

Chart 7. Restimulation of 1,10-phenanthroline-treated cultures with zinc. Cultures plated at 5.6 x 104 cells/ml were exposed at time zero to 1,10-phenanthroline (final concentration, 2.5 x 10~6 M). At f = 48 hr, cultures were exposedto varying levels of Zn2* and harvested for cell enumeration at ( = 96 hr. Results

for 1,10-phenanthroline-treated cultures (O) are compared to control not treatedwith the chelating agent (A).

Table 4Short-term inhibition of DNA synthesis and thymidine uptake by 1.10-

phenanthroline and EDTA

Concentration (nmol/mg cell proteinin incubation)

DNA synthesisThymidine up

take"

1,10-PhenanthrolineEDTA

100C

>375280

>375a Concentration required to inhibit DNA synthesis by 50% relative to untreated

control after a 1-hr incubation.b Concentration required to inhibit thymidine uptake by cells to 75% relative

to untreated controls after a 1-hr incubation.cÂ±10.

The levels of chelating agent are relatively high compared toother ligands tested previously. For example, the chelatingligand, 2-formylpyridine thiosemicarbazone, inhibits DNA syn

thesis to 50% of control levels at a concentration of 1.4 nmol/mg and is 90% effective at 8 nmol/mg (23).

DISCUSSION

The suggestion that zinc ion is intimately involved in DNAsynthesis and cell division stems from the studies of Fujiokaand Lieberman (10), Lieberman ef al. (12), and Lieberman andOve (13). Exposing cell cultures or perfusing liver with EDTA,they showed that the observed inhibition of DNA synthesiscould only be reversed by Zn2+ among the many metal ions

tested (10, 12). Further, they found a decrease in DNA polym-erase and thymidine kinase activities (12). Because the inhibition of DNA synthesis and the lowering of these enzyme activities have since been noted in tissues of zinc-deficient animals,there has been an identification of the results of EDTA treatment with specific zinc-related processes in the cell (11, 20,27). Similarly, the effects of 1,10-phenanthroline on DNA synthesis and cell proliferation have commonly been ascribed toits effects on zinc-dependent processes (8, 28, 29). Moreover,it has been assumed that EDTA or 1,10-phenanthroline maybe used in cell culture studies to mimic a condition of zincdeficiency (5, 8, 10, 22, 28, 29). This present study, as well asan examination of previous research in the area, can be usedto assess these hypotheses.

There are superficial similarities between the interaction ofEDTA and 1,10-phenanthroline with a variety of cell culture

systems, although these chelating agents have not previouslybeen studied together in the same cell line. Both agents appearto block the progression of cells from d into S phase (8, 22,29). There appears to be a selective reduction of DNA synthesisrelative to RNA and protein synthesis in some systems. However, in the phytohemagglutinin-stimulated lymphocyte model,EDTA inhibits both DNA and RNA synthesis (5). In addition,however, 1,10-phenanthroline can have a direct effect on cells

in S phase to inhibit DNA synthesis (2, 8, 29). Interestingly, theloci of the cell cycle effects are different from those in a modelalgal system, Euglena gracilis, which can be grown underrigorously zinc-deficient conditions (9). These cells, inhibitedby zinc deficiency, accumulate in S and G2 phases. The Ehrlichcells grown in zinc-deficient host mice are thought to have a

block in d phase, leading to selective inhibition of DNA synthesis (15, 19, 24).

One might identify this result with the behavior of the chelating agents described above. However, when the growth ofEhrlich cells in culture is inhibited by EDTA, there is no decrease in the rate of DNA synthesis per viable cell until theagent is frankly toxic to the cells and markedly decreasesviability. This is in marked contrast to the behavior of Ehrlichcells from zinc-deficient mice (4). Here, there is a decrease inproliferation and also a large reduction in the rate of DNAsynthesis per cell. Furthermore, as discussed below, 1,10-phenanthroline has rapid effects on the DNA synthesis ofEhrlich cells in S phase at concentrations which lead to adecrease in cell proliferation. Hence, one cannot identify itseffects with the slow depletion of cellular zinc to produce adeficient state.

Although EDTA and 1,10-phenanthroline similarly reduce

NOVEMBER 1980 4097




cell proliferation, the results of Tables 1 and 2 and Charts 1and 2 show that EDTA and 1,10-phenanthroline can have

different possible sites for reaction in cell culture. Relativelylittle EDTA enters the cells, but a significant fraction of 1,10-

phenanthroline is taken up. This difference is expected considering the ionic character of EDTA and the uncharged nonpolarnature of 1,10-phenanthroline. Thus, EDTA may be restrictedin its activities to the external medium, whereas 1,10-phenan

throline may enter cells and interact with cellular constituents.This is consistent with the inability of EDTA to inhibit short-term

assays of DMA synthesis at concentrations 4 to 5 times thatwhich yields 50% inhibition with 1,10-phenanthroline (Table4).

In a number of previous studies, it has been assumed thatthese chelating agents act either to bind essential zinc, makingit unavailable to cells, or interact with coordinatively unsatu-rated metal sites in proteins such as may exist in the zincmetalloenzymes, DMA and RNA polymerase, to inhibit theircatalytic activity. The basis for the view that EDTA is specificfor zinc comes from the observation that EDTA inhibition of cellproliferation and DMA synthesis can only be reversed by zincamong a number of metal ions tested. However, in reviewingthese experiments, metal ions were added to EDTA-inhibited

cultures in metal:EDTA ratios of 1:25 to 1:120 (5, 10, 22).Clearly, the reversal cannot be due to the complete titration ofEDTA with zinc, leaving excess zinc to enter and activate cells.The titration of 1 to 4% of the EDTA in these experimentshardly changes the effective concentration of free EDTA, andthus, its inhibitory potential is unchanged. Perhaps there is aspecific effect of zinc:EDTA upon the cell cultures. Chart 3shows a definite stimulation of Ehrlich cell cultures by zinc:EDTA. This is an interesting observation, which is, however,not explicable at present. The ineffectiveness of other metalions tested is probably due to the very low concentrations usedso that free EDTA remains in the cultures.

In the present study, the restimulation of EDTA-inhibitedcultures by Zn2+ is stoichiometric (Chart 4). Thus, there is a

break point at 1:1 zinc:EDTA concentrations. However, smallerconcentrations of zinc partially relieve the inhibition as if theremoval of free EDTA and not the addition of free zinc were thekey event. This conclusion finds support in the work of Rubin(22) in which EDTA but not a closely related chelating agent,ethylene glycol bis(/?-aminoethylether)-A/,/V-tetraacetic acid,depressed cell growth and DNA synthesis. This occurred despite the fact that both ligands have very large conditionalstability constants at pH 7.4 for zinc. There is suggested herea specifity of EDTA in its reactions with cells, unrelated to itschelating potential.

In similar studies with 1,10-phenanthroline, others have rou

tinely used stoichiometric or greater concentrations of metalions in experiments to antagonize the effects of the chelatingagent (2, 8, 28). In one such study, Berger ef al. (2) showedthat the degree of antagonism by a series of metal ions addedwith 1,10-phenanthroline correlates directly with their relativebinding affinity with 1,10-phenanthroline. Furthermore, stoi

chiometric or greater concentrations of metal ions are neededto produce the effect (8). Thus, the metal ion is seen to form aninactive metal:1,10-phenanthroline complex, removing the freeligand from reaction with the cells. In the present study, Zn2+

was added after 48 hr of incubation of Ehrlich cells with 1,10-phenanthroline. A titration-like response occurs as seen with

EDTA (Chart 7). Again, it is suggested that the removal ofligand, not the addition of zinc ion, is critical to restimulation ofthese cells.

Other recent studies expand the complexity of the 1,10-

phenanthroline effect. Although it is tempting to assign theimpact of this agent on S-phase events in the cell cycle to thedirect inhibition of DNA polymerase, cellular DNA synthesis inlymphocytes is much more sensitive to this compound than isDNA polymerase activity in homogenates of lymphocytes (28).Furthermore, DNA but not RNA synthesis is inhibited in L1210cells, although both DNA and RNA polymerases are presumedto be zinc metalloproteins (2). In this study, there was noevidence of inhibition of uptake of thymidine or its conversioninto dTTP (2). Thus, 1,10-phenanthroline does not appear to

react directly with thymidine kinase, an enzyme which diminishes in activity under conditions of natural zinc deficiency(20). In in vivo studies of the response of spleen and liver DNAsynthesis to 1,10-phenanthroline, Chang ef al. (4) suggestedfirst that the zinc complex of 1,10-phenanthroline is responsible

for the observed inhibition. In another paper (3), however, theypresented data that a nonchelating analog of 1,10-phenanthroline, 1,7-phenanthroline, is also effective in reducing splenicDNA synthesis. In contrast, others (8) have not observed suchanalogs to be effective against cells in culture. Hence, it issuggested that the in vivo and in vitro effects of 1,10-phenan

throline action may be different.Further, D'Aurora et al. (6) have argued recently that the in

vitro experiments showing the inhibition of DNA and RNApolymerases by 1,10-phenanthroline involve not the directbinding of 1,10-phenanthroline to the enzymes but the intermediate formation of the Cu(l) complex of 1,10-phenanthroline,

which then reacts with the proteins. Hence, as has beencarefully studied with thiosemicarbazones, the addition of chelating agents or metal complexes to cells can lead to a varietyof reactions (16-18, 23). These include chelation of essential

metals to produce cytotoxic agents, the exchange of metalsamong complexes, and reactions of ligands independent oftheir metal-binding properties. The possible removal of essen

tial metals from interaction with cells as may occur with EDTAor the binding of ligands to metal-containing structures in thecell as can be postulated for 1,10-phenanthroline are only

some of the potential reactions.Therefore, it is evident that the effects of EDTA and 1,10-

phenanthroline upon cells are suggestive of mechanisms involving metal ions. However, it is not clear that these arefocused on zinc biochemistry. In this and previous studies,EDTA and 1,10-phenanthroline are obviously different in their

mode of action. In Ehrlich cells, neither can be identified withproduction of a zinc-deficient state as defined in studies ofEhrlich cells grown in zinc-deficient mice (19, 24).

ACKNOWLEDGMENTS

The authors are happy to acknowledge the assistance of Dr. EdvardasKaminskas, Mt. Sinai Medical Center, Milwaukee, Wis., in helping us establishour cell culture facility.

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NOVEMBER 1980 4099



1980;40:4092-4099. Cancer Res Chitra Krishnamurti, Leon A. Saryan and David H. Petering Ehrlich Ascites Cells1,10-Phenanthroline on Cell Proliferation and DNA Synthesis of Effects of Ethylenediaminetetraacetic Acid and

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