hormonal regulation of net dma synthesis in mcf …...dna synthesis relative to controls in mcf-7...

,*r^Ã¯ '

[CANCER RESEARCH 42, 1727-1735, May 1982]

Hormonal Regulation of Net DMA Synthesis in MCF-7 Human Breast CancerCells in Tissue Culture1

Susan C. Aitken and Marc E. Lippman2

Medicine Branch, National Cancer Institute, NIH, Bethesda, Maryland 20205

ABSTRACT

Estrogens and anti-estrogens have striking effects on growth

of target tissues in vivo. In order to examine these effects invitro, experimental methods for the rigorous assessment ofrates of net DMA synthesis in tissue culture systems are required. The quantitation of DNA synthesis with inorganic[32P]orthophosphate is described. The limitations of more conventional [3H]thymidine labeling are illustrated in this hormon-

ally responsive system. Estrogen administration increases thespecific activity of acid-soluble phosphate and increases netDNA synthesis relative to controls in MCF-7 human breastcancer cells. Stimulation is most evident after 36 hr of hormonetreatment. Conversely, tamoxifen produces substantial inhibition of net DNA synthesis after 36 hr of hormone treatment.Thymidine availability modulates the effect of estrogens andanti-estrogens on DNA synthesis and constitutes an independ

ent experimental variable.

INTRODUCTION

Effects of estrogens and anti-estrogens on cell growth, pro

tein synthesis, and DNA synthesis in hormonally responsivebreast cancer cells in tissue culture have been documentedpreviously (22, 32, 34). Although hormonal induction of specific proteins has been demonstrated (26, 29, 53), some investigators have failed to demonstrate estrogen effects on cellgrowth in vitro in cell lines which nonetheless demonstrate anabsolute estrogen requirement for tumor formation in vivo (44,45). Under widely varying growth conditions, the MCF-7 breastcancer cell line is variously reported to be unresponsive toestrogen (8), to demonstrate increased DNA polymerase activity after estrogen treatment (19), and to respond to estrogenwith increased cell number and increased incorporation ofmacromolecular precursors (32, 33). The ZR-75-1 humanbreast cancer cell line requires estrogens for exponentialgrowth in defined serum-free medium (2, 3). However, the

mitogenic role of estrogens in target tissues and breast cancercell lines in particular (40, 47) remains controversial.

In addition to variation in growth conditions which modulateestrogenic effects, the common use of [3H]dThd3 to monitor

DNA synthesis may contribute to the observed disparity inexperimental results. The conditions under which this ligandaccurately reflects growth in cells are severely limited (9, 25).We will present data illustrating the distinction between[3H]dThd incorporation and net DNA synthesis under varying

1Presented in part at the First International Congress on Hormones and

Cancer, Rome, Italy, 1979(31).2 To whom requests tor reprints should be addressed, at National Cancer

Institute, NIH, Building 10. Room 6B02. Bethesda, Md. 20205.3 The abbreviations used are: dThd, thymidine; IMEM, improved Eagle's

minimal essential medium; IMEM-X, improved Eagle's minimal essential mediumwith tu' mol phosphate per liter and without asparagine.

Received April 14, 1981; accepted January 7, 1982.

hormonal conditions. The utility of 32PÂ¡in measuring net DNA

synthesis will be discussed.

MATERIALS AND METHODS

Tissue Culture Techniques. MCF-7 cells, a line of human breast

cancer cells obtained from the Michigan Cancer Foundation, weremaintained in continuous tissue culture (8). This line is known to containestrogen receptors and to respond to estrogens and anti-estrogens in

a variety of biological parameters (10, 19, 20, 26, 32, 33, 49). Cellswere maintained in monolayer culture in IMEM (43), supplemented withtwice the usual concentrations of glutamina, penicillin, and streptomycin (NIH Media Unit) and with 10% fetal calf serum (Grand IslandBiological Co., Grand Island, N. Y.). These cells were shown repeatedlyto be free of Mycoplasma contamination during the course of thisstudy. Two passages prior to a given experiment, cells were placed inIMEM supplemented with 10~7 mol insulin (Lilly Biologicals, Chicago,

III.) per liter and with 2.5% charcoal-treated calf serum shown to be

essentially free of estradici (44). Upon reaching confluence, cells weresuspended in trypsin solution (0.5% trypsin:0.02% EDTA; NIH MediaUnit) and plated replicately in plastic multiwell tissue culture dishes(Flow Laboratories, Inc., Rockville, Md.) in IMEM plus 2.5% charcoal-treated calf serum plus 10' mol insulin per liter. When plates became

60 to 70% confluent, medium was replaced with IMEM containing areduced concentration of phosphate (10~6 mol/liter) and lacking as

paragine. This nonessential amino acid was removed from medium tofacilitate labeling of DNA with anticipated precursors of de novo pyrim-

idine synthesis used in other experiments. This medium will be referredto as IMEM-X. After 4 to 12 hr, medium was again replaced with IMEM-X plus or minus 17/?-estradiol (5 x 10"9 mol/liter) or the anti-estrogentamoxifen (19) (10~6 mol/liter) (23). Labeled precursors were addedat varying intervals as described in "Results." Cells were harvested by

suspension in 0.9% NaCI solution plus 0.02% EDTA (w/v) and collected by centrifugation in a high-speed serofuge (Clay Adams, Parsip-

pany, N. J.). Cell pellets were washed at least once with 0.9% NaCIsolution and recentrifuged. Pellets were stored at â€”20Â°pending further

processing or were used immediately for cell counting.Analytical Methods. For cell counts, cells were suspended in 1.0 ml

phosphate-buffered saline (15 mw sodium phosphate: 140 mM NaCI,pH 7.4):EDTA and vortexed. Aliquots (500-|ul) were dispersed in 10 ml

Isoton (Coulter Electronics, Inc., Hialeah, Fla.) and counted in a cellcounter (Coulter Model E). Alternately, the cell pellet was suspendedin 0.6 to 1.0 ml of double glass-distilled water and disrupted with 51-

sec bursts of a sonicator at its lowest setting. Aliquots were taken fromthe sonicate for protein determination (34) and for collection of totalacid-precipitable material on Millipore filters (29). The acid-soluble

fraction was obtained by treatment with 5% perchloric acid and centrifugation at 1500 x g for 10 min. The supernatant was analyzed fortotal phosphate content by a modification of the Fiske-Subbarow pro

cedure (21). A portion of this fraction was also taken for determinationof incorporation of labeled 32P. These values will provide the basis for

the specific activity of the intracellular phosphate pool. Thin-layerchromatography (11, 41 ) of the acid-soluble extract (50) or the differ

ential acid hydrolysate of the perchloric acid precipitate (38) was usedoccasionally to isolate particular nucleotides. The solvent system chosen |n-butanol:ethanol:5 N HCI (30:20:20)] does not differentiate between the nonphosphorylated or the mono-, di-, or triphosphate forms

MAY 1982 1727

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S. C. Aitken and M. E. Lippman

of a given nucleotide.A second aliquot of the sonicate was incubated (37) (1 hr) with 15

mg pancreatic RNase (type I), 1.5 mg T, RNase (Boehringer MannheimBiochemicals, Indianapolis, Ind.), and 30 mg pronase (Calbiochem-

Behring Corp., Gaithersburg, Md.) per 200 ml of sonicate. Followingincubation, a portion of this material was incubated with ethidiumbromide (5, 6) and analyzed fluorometrically for DNA content. A secondportion was subjected to hydroxylapatite (Bio-Rad Laboratories, Rock-

ville Center, N. Y.) column chromatography (36, 37), precipitated onMillipore filters, and counted as for acid-precipitable material. Occasionally, the column eluate was acid-precipitated, hydrolyzed, andanalyzed by thin-layer chromatography as described above.

Counting Procedures. Radioactivity associated with the above cellular fractions was determined in a Packard liquid scintillation counterwith windows set to isolate '4C, 3H, and 32P channels with maximum

efficiency and minimal cross-over. Aquasol (New England Nuclear,

Boston, Mass.) or Hydrofluor (National Diagnostics, Inc., Somerville,N. J.) was utilized as the counting solution.

Analysis of Data. Statistical analyses of data were conducted usinga TI 59 desk top calculator (Texas Instruments, Inc., Dallas, Texas) orthe Hewlett Packard 9830A computer and appropriate software (Hewlett-Packard Statistical Analysis Package and utility laboratory programs developed in this laboratory). In other cases, MLAB, an on-linemodeling laboratory facility available on the DEC-10 computer system

at NIH (28), was used to analyze labeling kinetics. This processing willbe discussed further in relation to particular experiments. Statisticalmethods and tables are taken from Dixon and Massey (17).

RESULTS

Experimental Conditions

Conditions of Cell Growth. In order to generate measurableincorporation of 32PÂ¡into DNA in MCF-7 cells, the ambient

I0r

2 -,

IMEM IMEM-X

20 40 60 0 20 40 60 80

Time (Hours)

Chart 1. Effect of IMEM-X and hormones on growth of MCF-7 cells. MCF-7cells were plated replicately in multiwell tissue culture dishes in IMEM-X plus2.5% charcoal-treated calf serum plus 10 ' M insulin. At Time 0, medium wasreplaced again and hormones (estradiol. 5 x 10~9 mol/liter; tamoxifen, 10""

mol/liter) were administered. Cells were harvested at the times reported, andaliquots were taken for determination of cell number. O, control; A, tamoxifen;â€¢estradiol.

phosphate concentration in the culture medium was reducedinitially to 10"5 mol/liter. Asparagine was removed additionally

to facilitate potential labeling with de novo pyrimidine precursors.

MCF-7 cells grew for at least 72 hr and the rate of growth foruntreated, estrogen-treated, and tamoxifen-treated cells was

not affected by the lowered ambient phosphate concentrationor by asparagine deprivation (Chart 1). Estrogen-treated cells,however, maintained a higher growth rate than controls.

IMEM-X also had no effect on rates of precursor incorpora

tion into macromolecules (Table 1). We conclude that thechange in medium to IMEM-X does not appreciably alter cellgrowth and that IMEM-X supports estrogenic effects on MCF-

7 cells.Equilibration and Specific Activity of "PL We anticipated

that equilibration of 32PÂ¡with preexisting DNA precursor (de-

oxynucleotide) pools would require much more time than hasbeen observed with an [3H]dThd label. Chart 2A demonstratesthat linear incorporation of 32P into DNA demands approxi

mately 4 hr. Hormonal treatment (control, tamoxifen, estradiolplus tamoxifen) failed to alter this time requirement.

A 2-factor analysis of variance (time and hormone treatment)indicated that the specific activity of the total acid-solublephosphate pool became constant after 6 hr of exposure to 32PÂ¡

(Chart 2B). The amount of label accumulating in estrogen-treated cells was significantly greater than in controls (p <0.05, Q statistic). This experiment was conducted between 20and 32 hr after hormone treatment. Similar results were obtained between 48 and 60 hr after hormone addition (data notpresented). Consequently, we used labeling periods between6 and 8 hr in later experiments.

In order to convert incorporation of label 32Pto mass units of

phosphate in DNA, it is essential to obtain an estimate ofspecific activity of 32PÂ¡in precursor pools for DNA synthesis. It

is, however, technically much easier to extrapolate from thespecific activity of phosphate in the total acid-soluble poolrather than from small deoxyribonucleoside triphosphate poolsrepresenting less than 1% of total phosphate (19). Determinations of specific activity of 32PÂ¡can be obtained directly on theacid-soluble pool as described previously ("Materials andMethods"). In contrast, the routine determination of specificactivity of 32PÂ¡in nucleotides is impossible when dealing with

small replicate samples. We observed that following equilibration of 32P in the acid-soluble fraction the specific activity of

dThd phosphate [10.1 Â±0.7 (S.D.) dpm/pmol PÂ¡)and totalacid-soluble phosphate (10.5 Â±2.4 dpm/pmol) were identical

(Appendix, Part 1).Our results indicate that it is indeed possible to estimate

rates of net DNA synthesis using the specific activity of acid-soluble phosphate as a conversion factor from radioactivity tomass units. We reemphasize that label must have equilibratedwith endogenous phosphate and that specific activity refers to

Table 1Effect of IMEM-X on incorporation of label into acid-precipitable material in MCF-7 cells

Cells were labeled with ['"CJacetate (5 )iCi/ml; 8 hr), [3H]dThd (1 fiCi/ml; 2 hr), [3H]uridine (1 jiCi/ml; 2 hr), or [3H]leucine (1 /iCi/ml; 2 hr)and harvested 48 hr after transfer to either IMEM or IMEM-X.

dpm/mg

MediumIMEM

IMEM-XProtein

(mg)0.48

Â±0.050.47 Â±0.06[3H]dThd189,000

Â±22,187213,680 Â±20,163['"CJacetate4.16

x 106 Â±4.28 x 1054.28 X 10Â°Â±3.61 X 105[3H]Uridine183,000

Â±11,781216,350 Â±13,217["CJLeucine43,200

Â±2.61939,712 Â±4,612

1728 CANCER RESEARCH VOL. 42



TAM

304~o

5 25

I2o.- 20

IÃ•Ã• 15CD

10go

5

Hormonal Regulation of DNA Synthesis In Vitro

B

E2 + TAM

10 12 14

TIMEIhoursI TIMEIhoursI

Chart 2. A, incorporation of 32P, into DNA of MCF-7 cells. Cells were transferred to IMEM-X 32 hr before Time 0. Twenty-four hr before Time 0, cells weretransferred again to IMEM-X plus or minus estradici (5 X 10~a mol/liter; E2) and/or tamoxifen (10~6 mol/liter; TAM). 32P,was added at Time 0, and cells wereharvested at the times indicated. DNA was isolated as described previously ("Materials and Methods"). Values were normalized per unit protein. For reasons of

clarity, standard deviations (bars) are depicted for control samples only although variations in all groups did not exceed 15% of the reported value. B, incorporationof 32P,into acid-soluble pool of MCF-7 cells. Cells were treated as described previously (A). The acid-soluble fraction was obtained as described in "Materials andMethods."

the 32P radioactivity/mass of phosphate, not radioactivity/

mass of nucleotide. Experimental variation due to hormonalregulation of the specific activity of phosphate pools will bediscussed below.

Measurement of DNA Synthesis. Since 32P is incorporated

into a variety of cellular materials and only 1 to 2% of this labelenters DNA, we found it necessary to develop proceduresresulting in highly purified DNA preparations. Hydroxylapatitechromatography and differential enzymatic hydrolysis werechosen to process small samples for isolation of DNA (32, 33).Part 2 of the Appendix demonstrates that hydroxylapatite chromatography and enzymatic hydrolysis together reduced uridineand leucine label to background levels and yielded high recovery of DNA free from contamination with protein or RNA.

In addition, we confirmed that material isolated from hydroxylapatite was indeed DNA by comparing the amount of 32P

radioactivity found in dTMP isolated from an hydroxylapatiteeluate with the total 32P in that eluate. As predicted, approximately 25% of the total 32P was chromatographically isolated

in dTMP (Appendix, Part 3). We concluded that label elutedfrom hydroxylapatite following enzymatic hydrolysis is a validindex of net DNA synthesis. This procedure was followed in allsubsequent experiments.

A complication of the use of radioactive precursors to measure DNA synthesis is that one may overestimate net DNAsynthesis under conditions in which various repair processesconstitute a significant part of incorporated label. This wasconsidered unlikely since, in the absence of induction of DNAdamage, excision repair represents less than 2% of DNA-

synthetic activity in most replicating cells (12, 13, 40). Thisdegree of activity generally cannot be detected against abackground of scheduled DNA synthesis. However, we wereuncertain of the levels of activity in MCF-7 cells and were

additionally concerned over possible DNA damage producedby 32P. Therefore, we compared the incorporation of 32P over

a 72-hr period with estimates of change in mass of DNA asmeasured by ethidium bromide fluorometry (Appendix, Part 4).Accumulation of DNA is similar using both methods. Even afterprolonged exposure to 32P which may induce cell and DNA

damage, the amount of repair is not measurable against thebackground of replicative DNA synthesis. It should be notedthat any repair contribution of less than 5% of incorporationwould not be statistically detectable considering the normalvariability seen under our labeling conditions.

Hormonal Regulation of Phosphate Metabolism

Since phosphate is an important metabolic element, wesuspected that hormonal treatment might alter the intracellularspecific activity of 32P|.Chart 3 shows the effect of estrogen on

intracellular phosphate pools.Cells were exposed to varying concentrations of 17/?-estra-

diol(10~13to 10~5 mol/liter) for 32 hr. All values are expressed

as the ratio to values seen in control (untreated) MCF-7 cells.

Although estrogen treatment did not significantly alter the sizeof phosphate pools (nmol PÂ¡per mg protein), the specificactivity of acid-soluble phosphate (dpm 32P per nmol P,) exhibited a dose-dependent response and was maximal at 10"9

mol/liter.In addition to direct quantitative measurements in MCF-7

cells, we conducted a series of kinetic studies using mathematical models developed by Cooper (15). Utilizing appropriateequations, it is possible to estimate endogenous pool size atthe time of introduction of a labeled precursor as well as thekinetic constants for entry and exit of label. MLAB, a generalized graphic display and curve-fitting program capable of deal

ing with differential equations and available at the NIH computerfacility, was used to fit the data. Chart 4 shows a typical curve(MCF-7 cells under control conditions). The time of exposureto 32PEis represented on the abscissa (time, t). Accumulation

MAY 1982 1729




2.0 r

! IJ

2-oT

Ã•Ã•

0.5 -

Specific Activityl32?/P;)

1.5 o

l

0.5

Iff'2 Iff'" Iff8 IffÂ« Iff*

ESTRADICI (moles/liter)

Chart 3. Effect of varying concentrations of estradici (E..) on phosphate poolsin MCF-7 cells. Eight hr before Time 0, cells were transferred to IMEM-X. AtTime 0, cells were transferred again to IMEM-X Â±varying concentrations ofestradici as indicated. At 24 hr, MP, (1 pCi/ml) was added to wells and cells wereharvested 8 hr after labeling. The radioactivity and phosphate content of acid-soluble pools were determined as described previously. The protein content ofsonicates was also determined. Values were normalized per unit protein and arepresented as the average of at least 2 determinations. Bars, S.D.

25

--â€”â€¢Â°

a Experimental Value

o Predicted ValueLit)-MX (1 - expl-at Ml)

6 9TIME (hours)

12

Chart 4. Incorporation of 32P,into the acid-soluble pool of MCF-7 cells. Data

were obtained as described for Chart 2B. Data were fitted to the function

L(0 = M x [1 - exp(-a(/M)]

using the computer program ML AB (13. 24), where t = time. M = initial poolsize, a â€”rate of entry into the pool and L = accumulation of label in the pool.

of 32Pin the acid-soluble pool (dpm/mg protein, L) is shown

on the ordinate. Data were fitted to the function

/.(f) = Mx[1 -exp(-af/M)]

where a is the rate of uptake of label and M is the pool size.The best fit of the data to the function is shown by the slashedline drawn through predicted points (D). The actual experimental observations (A) are also depicted.

Hormonal effects on the parameters defined by MLAB analysis are shown in Table 2. Data are obtained from MLAB asdpm/unit time and are converted to mass units (nmol PÂ¡)bymultiplying all parameters (M, a, b) by the experimentally

Table 2Kinetic analysis of incorporation of "P, into precursor pools for DNA synthesis

Analysis was conducted utilizing MLAB, a generalized curve-fitting programavailable at NIH. Experimental groups are shown in Column 2. M (pool size) isobtained by dividing the dpm units returned by the computer program by theexperimentally determined specific activity of ':'P, in the acid-soluble pool (dpm32Pper pmol P,). L refers to the amount of label (dpm) accumulating in the acid-

soluble pool, a (rate of uptake of label) and b (rate of loss of label) were similarlyanalyzed by MLAB in dpm units and converted to mass units as described for M.All values are normalized per unit protein and are the mean of 3 determinations.Standard deviations were in the range of 5 to 10% of recorded values. The 24-and 48-hr times (Column 1) refer to independent experiments.

Time(hr)2448TreatmentControlEstradiciTamoxifenControlEstradiciTamoxifenM(nmol

PJ26.829.524.721.027.821.6L(dpm x103)99.0125.187.2135.6189.6141.8nmol

Pi/hra4.365.634.183.617.283.12b3.946.124.023.546.963.10

determined specific activity of 32Pin the acid-soluble pool. Two

experiments, 24 and 48 hr after hormone addition, were conducted. 32Piwas added to cells as described previously, andthe incorporation of label into acid-soluble pools was determined as a function of time. Note that rates of entry (a) andexit (b) of phosphate in the acid-soluble pool are approximatelyequal in any given experimental situation, a result predicted ifconstant specific activity has truly been attained. Neither estrogen nor anti-estrogen affected phosphate pool size (M, Column3) except after 48 hr of estrogen treatment when pools wereelevated relative to controls. However, estrogen produced substantial increases in both rates of uptake (a, Column 5) andexit (b. Column 6) of phosphate at both 24- and 48-hr timepoints. The accumulation of label (L, Column 4) was alsoelevated. Tamoxifen treatment only slightly affected phosphatekinetics at either 24 or 48 hr although decreases in uptake andexit were statistically significant at 48 hr (p < 0.01, T test).

Both direct quantitative measurements (32P/mass PÂ¡)and

kinetic studies (accumulation of label per unit time; MLABcomputer-assisted analysis) indicate that estrogen and anti-estrogen treatments modulate phosphate metabolism in MCF-7 cells. Estrogen increases specific activity and rate of flux ofintracellular phosphate whereas tamoxifen has only slight effects on overall metabolism until late incubation stages. Theexperimental variability in phosphate metabolism reinforces thenecessity for independent determination of the specific activityof phosphate pools if incorporation of 32Pinto DNA is to be

used as an index of net synthetic rate.

Hormonal Regulation of Net DNA Synthesis

Hormonal effects on rates of DNA synthesis are much greaterthan corresponding changes in total phosphate metabolism.Table 3 illustrates the action of hormones on the rate ofincorporation of 32P into DNA. Values were obtained by per

forming least-squares regression analysis on the appearanceof 32Pi in DNA per unit time. The slope of the line (dpm/hr) is

converted to mass units using the specific activity of the acid-

soluble pool as described above. Regardless of experimentaltreatment, incorporation of phosphate into DNA representedonly 3 to 4% of the total exit of phosphate (Table 2b) from theacid-soluble pool. Estrogen significantly increased the rate of





Table 3Effect of hormones on net DNA synthesis in MCF-7 cells

MCF-7 cells were treated with hormones (tamoxifen or estradici plus tamoxi-

fen) and harvested 24 or 48 hr after treatment. At varying times before harvest(1 to 12 hr), 32P;(1 fiCi/ml) was added to wells. The incorporation of label into

DNA was determined as described previously, dpm were converted to mass units(pmol P,) by correcting for specific activity of acid-soluble pools as noted in Table2. r (regression coefficient) was determined by least-squares analysis of the rateof incorporation of label per unit time.

24 hr 48 hr

ControlTamoxifenEstradicipmol

P,152

166257r0.99

0.880.97pmol

PÂ¡155

85285r0.97

0.970.99

incorporation of phosphate into DNA by nearly 2-fold at both

24 and 48 hr. Tamoxifen on the other hand decreased incorporation to approximately 60% of control levels after 48 hr ofhormone administration.

The effect of varying concentrations of estradiol on theincorporation of 32Pi into DNA is shown in Chart 5. Thirty-two

hr of hormone treatment produced a dose-related increase(maximal at 10~9 mol estradiol per liter).

We then questioned whether administration of exogenousdThd could affect the rate of net DNA synthesis in MCF-7 cells.

An experimental protocol to produce and reduce data wasdesigned as follows. In any given experiment, each experimental group is divided into 3 subsets. One subset is labeled for 6hr with 32PÂ¡,a second is labeled for 8 hr, and a third is similarlylabeled with 32Pi for 8 hr and in addition is pulsed for the 2 hrprior to harvest with [3H]dThd. This protocol is necessitated bythe extended period required for equilibration of 32PÂ¡with

intracellular phosphate pools and by the fact that dThd administration, as will be demonstrated later, affects the pattern ofnet DNA synthesis in MCF-7 cells and thus constitutes an

independent variable in experimental design. Final analysisinvolves 3 sets of values: the effect of dThd on the system(dThd-control), the effect of the experimental manipulation(hormone-control), and the effect of dThd coupled with theeffect of hormone (hormone plus dThd-control).

In the following experiments, all estrogen treatment groupsare noted as estrogen plus tamoxifen. Our presentation reflectsthe observation that in multiple experiments 5 x 10~9 molestradiol per liter and 5 x 10"9 mol estradiol per liter plus 10~6

mol tamoxifen per liter produced identical results. The primarypurpose of simultaneous hormone administration was to demonstrate that tamoxifen effects were specifically anti-estrogenic

since estrogen blocked any tamoxifen inhibition. In the interestof simplicity, we choose to present only data from simultaneousestrogen plus tamoxifen treatment as representing estrogenaction.

The time course of the effects of estradiol and tamoxifen onnet DNA synthesis in the absence or presence of exogenousdThd is shown in Chart 6, A and B. The limited ability of MCF-7 cells to survive under serum-free conditions precluded extension of time course studies beyond 60 to 72 hr. Values arenormalized against the values observed after 8 hr of a giventreatment. As shown previously, 8 hr is the minimum labelingtime which can be used in 32P incorporation studies in MCF-7

cells and thus constitutes a zero time point. Controls presenteda highly variable background with one peak of DNA synthesisoccurring between 24 and 36 hr after serum-free transfer anda second peak occurring at about 48 hr. In the absence of

3.0 r

Â° 2.5

2.0

Oo

irOo.ocou

1.5

1.0

0.5

0.0

Phosphate

IO'1 IO'1 106 10" IO2

ESTRADIOL (moles liter!

Chart 5. Effect of varying concentrations of estradiol (Â£2)on DNA synthesisin MCF-7 cells. Cells were treated as described previously (Chart 3). Twenty-fouror 26 hr after hormone addition, 32P,(1 /iCi/ml) was added to wells. Radioactivity

in acid-soluble and DNA fractions was determined as described previously. Theprotein, DNA, and acid-soluble phosphate contents were also determined. Valuesfor incorporation of phosphate (pmol/2 hr) were calculated as described previously and normalized per unit DNA. Results are presented as the ratio to valuesin untreated cells.

exogenously added dThd, tamoxifen is highly inhibitory within18 hr of administration. Concurrent administration of estradioland tamoxifen produced a stimulation of phosphate incorporation well above control levels. Interestingly, the peaks ofincorporation in estrogen-treated cells occurred synchronously

with those in control cells and differed only in magnitude.Repetitions of this experiment (not shown) consistently demonstrated distinct peaks of DNA synthesis in MCF-7 cells. The

time of onset of the first peak varied experimentally between18 and 24 hr after addition of fresh serum-free medium and

extended for approximately 12 hr. A second peak at about 48hr was evident in 3 of 4 experiments in control cells and waspresent invariably in estrogen-treated cells. Addition of dThd(10~7 mol/liter; 5 ftCi/ml) increased the amplitude of the initial

peak of phosphate incorporation into DNA (Chart 6B). Theinhibition due to tamoxifen was partially reversed and valuesapproached baseline levels. dThd administration had little effect on phosphate incorporation during later stages of theincubation in control or estrogen-treated cells although valuesin tamoxifen-treated cells remained elevated over values ob

served in the absence of dThd. It appears that dThd availabilityand hormonal treatment constitute interacting experimentalvariables.

Chart 7 isolates the effects of estradiol and exogenous dThdon net DNA synthesis. Cells were harvested at the times indicated after hormone administration and after labeling as described previously. In this case, values for net DNA synthesisare normalized against the values in control cells at the sametime point. The effects of dThd are clearly restricted to an earlywave of DNA synthesis whereas estrogen effects applied tolate incubation stages.

In summary, estrogens and anti-estrogens exert powerfulregulatory effects on DNA synthesis in MCF-7 cells. Cells

frequently show 2 major peaks of DNA synthesis: an early

MAY 1982 1731




5 r

Tamoxifen -

Estradici

60 72

TIME (hours)

12 24 36 48 60 72

TIME (hours!

Chart 6. A, effect of hormones on net DMA synthesis in MCF-7 cells (minus dThd). Eight hr prior to Time 0, cells were transferred to IMEM, and at Time 0, mediumwas replaced again [no hormone, tamoxifen (2 x 10~e mol/liter), or estradici (10~8 mol/liter) plus tamoxifen (2 x 10~s mol/liter)]. 32P,was added to samples (1 /iCi/

ml) 8 or 6 hr before the indicated time of harvest. Data reduction to mass units of phosphate (pmol/2 hr) incorporated into DNA was performed as describedpreviously. The presented values were normalized against the value observed within the same experimental group after 8 hr of incubation. Standard deviations werein the range of 10 to 15% at all represented points. B, effect of hormones on net DNA synthesis in MCF-7 cells (plus dThd). Cells were treated and data obtained asdescribed previously (A) with the exception that 2 hr prior to the indicated time of harvest |'H]dThd (5 Â«Ci/ ml; 10~7 mol/liter) was added to some of those wells whichwere also labeled with 32P,for 8 hr.

24 36 48

TIME (hours)

60 72

Chart 7. Effect of exogenous dThd and estradiol on net DNA synthesis inMCF-7 cells. Cells were treated as described previously (Chart 6/1) with theexception that estradiol (5 x 10~9 mol/liter) and control (untreated) cells con

stituted major experimental treatments. Presented values were normalizedagainst the values seen in controls at the same time point. Standard deviationswithin any experimental group did not exceed 15%.

wave which is associated with the availability of exogenousdThd (18 to 30 hr) and a second wave of synthesis unaffectedin magnitude by the administration of dThd. The effects ofestrogen alone are manifested in an amplification of thesewaves of DNA synthesis rather than in a change in the timeframe within which cells enter DNA synthesis. Estrogen actionrelative to controls is especially evident during later phases ofthe serum-free incubation. Tamoxifen administration decreases

net DNA synthesis within 18 hr after treatment, and this effectcan be blocked by simultaneous exposure to estradiol. dThdaddition to the medium at concentrations in excess of 1CT7

mol/liter partially reverses the inhibition produced by tamoxi

fen, and this reversal is most apparent during the early wave ofDNA synthesis.

DISCUSSION

MCF-7 cells resemble other vertebrate cell lines in phosphateutilization. The usual size of the soluble phosphate pool inMCF-7 cells (30 nmol/106 cells, Table 2) and the time required

for equilibration of label (6 to 8 hr, Chart 2) are consistent withdata reported for other vertebrate cell types in tissue culture(14, 24, 27, 55). Equilibration of phosphate among varioussoluble pool components is reported to be extremely rapid(41). Cunningham and Pardee (16) noted that approximately50% of organic acid-soluble 32P label could be localized in

nucleotide components in mouse 3T3 fibroblasts within 15 minof addition of label. Estimates of pool size of nucleotide components in other cell lines based on the assumption of aconstant specific activity of 32P in all such pools (51) give

excellent agreement with independent estimates based onenzymatic assay (30, 46). Observations in other tissue culturesystems thus support the evidence in MCF-7 cells that thespecific activity of the acid-soluble phosphate pool is an ac

curate indicator of the specific activity of the deoxynucleotidephosphate pool.

The use of 32PÂ¡to measure rates of DNA synthesis has been

reported in a variety of tissue culture systems. It is generallyaccepted that the incorporation of 2 molecules of phosphateinto DNA represents the synthesis of 1 base pair of nucleotides;hence 2 nmol of incorporated phosphate = 1 nmol of DNA.However, the conditions under which 32P incorporation can be

converted to mass units of DNA synthesized frequently havenot been examined stringently. MCF-7 cells show great varia

bility in the specific activity of precursor phosphate pools (Chart3). Failure to examine labeling conditions results in consider-





able error in the extrapolation from counts incorporated toconclusions concerning DNA synthesis.

Using the techniques described above, the specific regulation of DNA synthesis by estrogens and anti-estrogens can be

examined in vitro. Data in Charts 5 and 6 indicate that estrogenincreases the growth of MCF-7 cells. We cannot state whether

this is due to (a) a decrease in cell death, (b) an increasedgrowth rate (decrease in length of the cell cycle), or (c) anincreased recruitment of nondividing cells arrested in GÃ¬(G0)into the actively dividing population. There is evidence thatestrogen acts in vivo to recruit from G0 into G, as, for example,in the induction of uterine growth (20). It has also been reportedthat estrogen addition to MCF-7 cells decreases cell cycletime. Increased growth was attributed in this case to a specificdecrease in the length of the Gt phase of the cell cycle (52).

MCF-7 cells represent a transformed cell line in which we(39) and others have previously been unable to demonstratean absolute dependence on estrogen as a trophic factor.Conditions of culture and the baseline growth rate of cells arecritical for demonstrating estrogen growth effects. Cells appearto grow equally well in the absence of estrogen provided othertrophic factors (serum, insulin) are present, and it is difficult todemonstrate estrogenic effects in the presence of optimalamounts of these factors (47, 48). In the ZR-75-1 human breast

cancer cell line, it has been possible to establish estrogenstimulation of cell growth under chemically defined conditions(2, 3). Recently, another human breast cancer cell line dependent on estrogen for growth has been described (54).

In these studies, MCF-7 cells show an apparent partial syn

chronization of phosphate incorporation into DNA followingtransfer to serum-free medium (Charts 6, A and B, and 7).

Neither estrogen nor tamoxifen affected the timing of waves ofDNA synthesis with respect to controls. The causes of thispartial synchronization of the cell population are unknown.Transformed fibroblast cells (4, 16, 18) are known to bestimulated by addition of fresh medium with characteristic delayperiods of approximately 24 hr, similar to the delay reportedhere. Other nutritionally arrested cells are known to respond toaddition of serum or critical nutrients in a synchronous fashion(4). Observations in MCF-7 cells could reflect a similar phe

nomenon. Failure of estrogen to decrease the delay periodrequired for DNA synthesis suggests that the primary effect ofestrogen may not be a decrease in cycle time but an entry ofcells not scheduled to divide into the actively growing population. Our results do not, however, preclude the possibility thatestrogen additionally decreases cell cycle time.

The pattern of net DNA synthesis in MCF-7 cells and theeffects of exogenous dThd on that pattern yield some suggestions on the possible role of salvage and de novo pathways ofpyrimidine production in MCF-7 cells and the potential mechanisms by which estrogen may increase the rate of DNAsynthesis in hormonally responsive systems. The early wave ofDNA synthesis in MCF-7 cells is modulated by the availability

of exogenous dThd. dThd administration increases DNA synthesis at early time points in controls, estrogen-treated cells,and tamoxifen-treated cells (Charts 6, A and B, and 7). Tamox-ifen-treated cells which would have been inhibited were res

cued by exogenous dThd during this early stage of the incubation and actually showed a stimulation above control levelsin one experiment. Estrogen-treated cells have a greater ability,

however, to utilize dThd as a trigger for net DNA synthesis than

do controls. The activity of dThd kinase is known to be increased by estrogens and decreased by anti-estrogens inMCF-7 cells (7). The increase in net DNA synthesis in estrogen-

treated populations relative to controls at early incubationstages could therefore be due to increased dThd kinase activity. However, the ability of dThd to reverse tamoxifen inhibitionimplies that, although dThd kinase activity is decreased, thereis no functional impairment of salvage activity, at least at thelevels of dThd used in these experiments (5 x 10~8 to 2 X10~6 mol/liter). During later stages of the incubation, effects of

dThd on net DNA synthesis were not as apparent, possiblyindicating an increased functional impairment of salvage activity and an increased dependency in MCF-7 cells on de novo

pyrimidine production. Such a dependency may reflect (a) adepletion of available salvage sources of dThd in the earlierwave of DNA synthesis or (b) an estrogen-dependent induction

of critical de novo or intermediary enzymes.These data also reinforce the severe limitations of [3H]dThd

incorporation as an indicator of DNA-synthetic rate. It is wellknown that incorporation of [3H]dThd into DNA is greatly influ

enced by endogenous pools of dThd (1). In studies involvingchemotherapeutic agents (fluorouracil, hydroxyurea, metho-

trexate) known to alter availability of nucleotide precursors forDNA synthesis, it may be critical to be able to assess DNAsynthesis independently of specific nucleotide labels. Our dataalso indicate that much of the estrogenic stimulation and tamoxifen inhibition of DNA synthesis would be overlooked experimentally when using [3H]dThd as a sole marker for DNA

synthesis.Under the circumstances outlined here, however, it is pos

sible to accurately measure net DNA synthesis and independently to assess the contribution of the salvage route of pyrimidine utilization. The experimental protocol described earlier,coupled with the ability to precisely measure salvage and denovo pathways of pyrimidine production, may yield significantinformation on the biochemical mechanism by which estrogensact as trophic hormones. In spite of the fact that human breastcancer cells such as the ZR-75 and MCF-7 lines are trans

formed, the ability of estrogenic hormones to alter growth andDNA synthesis in these systems appears to mimic the action ofestrogens in vivo. We conclude that measurements of phosphate incorporation into DNA, when conducted according tothis protocol, can be applied to a variety of experimentalsituations. The MCF-7 cell line can provide a highly useful

system for the investigation of both the mechanism of estrogenand anti-estrogen action and regulation of growth processes

and a model system for the investigation of drug effects.

APPENDIX

1. Specific Activity of Intracellular 32P,.Approximately 10BMCF-7 cells werelabeled with 32P,(1 ^Ci/ml; 12 hr). The acid-soluble fraction was obtained, and

radioactivity and phosphate content were determined as described previously("Materials and Methods"). The remainder of the preparation was neutralized

with 1 N KOH and centrifuged (1000 x g; 10 min). The resulting supernatantwas lyophilized and resuspended in 0.01 N HCI for thin-layer chromatography("Materials and Methods"). The area of the plate corresponding to a dTMP

marker was cut out and eluted with 0.01 N HCI. Radioactivity and phosphatecontent were again determined.

2. Purification of DNA. Approximately 10e cells were pulsed (1 .uCiâ€¢ml; 1 hr)with [3H)dThd, [5-3H]uridine. or [3H]leucine, effective precursors for DNA, RNA,

and protein, respectively. Column 1 describes the step in the fractionationprocess. The remainder of the table presents the percentage of radioactivityrecovered following acid precipitation on Millipore filters. Total acid-precipitablecounts are taken as 100%. Hydroxylapatite chromatography, RNase plus pro-

MAY 1982 1733




nase incubation, or hydroxylapatite chromatography following RNase plus pro-nase treatment all yield excellent recovery of DNA ([3H]dThd, Column 2). How

ever, considerable amounts of uridine and leucine label remained followingRNase plus pronase hydrolysis alone, and uridine was a substantial contaminantwhen samples were applied directly to hydroxylapatite columns without priorenzymatic hydrolysis. All values are reported as the percentage of total acid-precipitable radioactivity (Row 1) and represent the mean Â±S.D. of 3 determinations.

Fraction [3H]dThd [3H]Uridine [3H]Leucine

Total acid precipitableRNase Â±pronase incubationHydroxylapatite chromatographyRNase Â±hydroxylapatite

chromatography

100 Â±18 100 Â±9 100 Â±2282 Â±17 15 Â±4 42 Â±1196 Â±16 11 Â±6 2 Â± 0.794 Â±11 1 Â±0.5 1 Â± 0.2

3. Recovery of "'P, in Cellular Fractions. Cells were labeled simultaneouslywith 32P,(1 fiCi/ml; 8 hr) and [3H)dThd (1 /iCi/ml; 2 hr). An hydroxylapatite eluate

was divided into 2 aliquots, one for total radioactivity and a second for acidprecipitation and hydrolysis to constituent nucleotides.

["CJdTMP (0.1 udÃ¬was added to the acid-precipitable pellet from unlabeled

MCF-7 cells just prior to acid hydrolysis as an index of recovery for chromatog

raphy.Samples were lyophilized and chromatographed as described previously

("Materials and Methods"). The fraction corresponding to a dTMP marker was

cut from the plate and counted directly. Values in the table are presented as thepercentage of recovery (sonicate = 100%) and were of special interest as dpm.Column 2 fl3H]dThd) provides an index of the efficiency of DNA recovery. Virtuallyall acid-precipitable dThd label was recovered again in the hydroxylapatite eluate.The pattern of recovery of 32P is presented in Column 3. Approximately 2% oftotal 32P is recovered in the hydroxylapatite eluate. On further chromatography,approximately 25% of this 32P was then identified in the dTMP position. This

value would be predicted if the hydroxylapatite eluate is indeed only DNA and ifdThd is considered to represent approximately 25% of the total nucleotide basesin DNA. All values are the mean Â±S.D. of 3 determinations and are normalizedper unit protein in the sonicate.

[3H]dThd 12P,

FractionSonicateAcid

precipitableHydroxylapatiteeluate(DNA)Chromatography(dTMP)Recovery

standardd'4C]dTMP)32P

dpm indThd(corrected

forrecovery)32P

in dThd/32PinDNA%

dpm%100

Â±10 213,745 100Â±1381Â±11 171 ,587 28 Â±180Â± 8 170,996 2Â±0.10.5

Â±0.050.814.38

X10"0.26dpm1.71

x4.80x1.67X3.55

X10'10Â«10s104

4. Contribution of Repair to Measurement of net DNA Synthesis. Approximately 10e cells/roller bottle were plated replicately in IMEM plus 2.5% charcoal-treated calf serum plus 10' mol insulin per liter. When cells were subconfluent,

medium was replaced with IMEM-X. At Time 0, medium was again replaced withIMEM-X plus 5 x 10~9 mol 17 ^-estradiol per liter and approximately 1 Ci 32P,

per ml. One group of samples was harvested immediately (Time 0), and theamount of DNA present was set at zero. Other groups were harvested atsuccessive 24-hr intervals. DNA synthesis was quantitated by ethidium bromidefluorometry (Column 3) and by conversion of 32P incorporation to mass unitsbased on the specific activity of 32P in the acid-soluble pool at time of harvest.Isolation of DNA was performed as described previously in "Materials andMethods."

Time(hr)024

4572nmol

DNA(P,)0

18.2 Â±3.629.6 Â±3.732.0 Â±6.2nmol

DNA (ethidiumbromide)Â±

4.422.3 Â±3.229.4 Â±6.134.1 Â±3.6

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hormonal regulation of net dma synthesis in mcf …...dna synthesis relative to controls in mcf-7...

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