Identification of the inhibitor of labelled thymidine incorporation into HeLa cell DNA present in endometrial extract

Europ. J. CancerVol. 11, pp. 657-667. Pergamon Press 1975. Printed in Great Britain

Identification of The Inhibitor of Labelled Thymidine Incorporation into HeLa Cell DNA Present in Endometrial Extract S. CHEVALIER and W. G. VERLY Department of Biochemistry, University of Montreal, Montreal, Canada

Abstract--An extract of bovine endometrium was found to decrease 3H-thymidine incorporation into DNA of HeLa cells, but also, although to a lesser extent, of Ehrlich and Novikoff cells. Thymidine in the extract was responsiblefor about 80 ~o of this inhibition. No inhibitor strictly specific.for HeLa cells could be detected. An extract of human endometrium decreased, while an extract of human myometrium increased 3H-thymidine incorporation into DNA of HeLa cells. The inhibitory effect of the endometrial extract was also observed on Ehrlich and Novikoff cells. The endometrial extract contained thymzdine which was absent from the myometrial extract; thymidine in the endometrial extract was, however, a preparation artefact. Both human endometrial and myometrial extracts possessed an enzymic activity that converted thymidine into thymine; at high doses, the myometrial extract also decreased 3H-thymidine incorporation into D NA of HeLa cells. Addition of unlabelled thymidine to the incubation me&um has not the same inhibitory action in different kinds of cells on SH-thymidine incorporation into D NA. HeLa cells are very sensitive to this dilution effect. The differences in behavior of various cellular systems can be wrongly interpreted as the result of the action of a tissue-specific inhibitor.

INTRODUCTION

own growth by a negative feedback mechanism. On the other hand, the chalones so far described have no species-specificity. Hinderer, Volm and Wayss [3] found that a crude preparation from h u m a n endometrium inhibited the incorporation of 3H-thymidine into DNA of H e L a cells, but not of cells from the amelanotic melanoma of the Syrian hamster or of mouse fibroblasts (L cells) cultured in vitro; it had no effect either on Yoshida sarcoma or Fortner I I I melanoma ascites cells. On the other hand, a crude preparation of h u m a n myometrium stimulated the incorporation of aH-thymidine into DNA of H e L a cells as well as of the other kinds of cells tested; this was attributed to non-specific nutritive influences. Because H e L a cells are cancer cells derived from the cervix epithelium of a h u m a n uterus, Hinderer et al. [3] concluded that the h u m a n endometrium contained

IT HAS been proposed that tissue growth is under the control of "antitemplates" [1] or "chalones" [2]. In plinciple, a chalone is secreted by cells in the extraceUular fluid and exerts an inhibitory effect on the proliferation of cells of the same kind; this tissue-specificity seems to be the most important property of the chalones to enable each tissue to control its Accepted 20 May 1975. *The research was supported by grants from the National Cancer Institute and the Medical Research Council of Canada. S. Chevalier was recipient of a MRC studentship. The authors wish to thank Dr. J. Gagnon from Ste-Justine Hospital, Montreal, who supplied the human endometrium and myometrium and Miss J. Flamand who provided the HeLa cells used in this work. 657

658

S. Chevalier and W. G. Verly

an endometrial chalone. The aim of this work was to isolate, from the endometrial crude extract prepared by the technique of Hinderer et al. [3], the inhibitor of 3H-thymidine incorporation into the DNA of HeLa cells. While this work was under way, Wayss and Volm [4] observed that the crude endometrial extract was also active on human fetal liver and kidney cells; they then revised their previous opinion and concluded that the endometrial inhibitor was not a chalone. MATERIAL

AND

METHODS

Solutions Antibiotic solution. 1-00g of streptomycin sulfate (General Biochemicals) and 0-63 g of penicillin G potassium salt (Calbiochem) are dissolved in water and the volume brought to 100 ml. MEM-antibiotic solution. 10.7 g of minimum essential medium (Eagle) powder with Spinner salts and L-glutamine (F-14, Gibco) and 2.2 g N a H C O 3 are dissolved in water; 4.0 ml of phenyl red solution (0.05 g/l, 510 Gibco) and 10 ml of antibiotic solution are added; the solution is brought to 1000 ml. The solution is sterilized by passing through a 0.45 pm Millipore filter. Trypan blue solution. 0.4% trypan blue (525, Gibco) in Hanks' solution. Hanks' solution. Gibco 406S diluted 10 times and sterilized 15 min at 130°C. To 100 mt of this solution, 2"5 ml of sterile 5% N a H C O 3 are added to have a 0-015M NaHCO3 final concentration. The p H is 7.4 in a 95% air-5% CO2 atmosphere. Cells About 75 x 106 HeLa cells are inoculated into a 1 1. Spinner flask (Bellco Glass Inc.) containing 300 ml of MEM-antibiotic solution; calf serum (Tissue Culture Service Ltd.) is added to obtain a 5% concentration. After a 3-day incubation at 37°C, a 0.5 ml aliquot of the cell suspension is mixed with 0.1 ml of trypan blue solution and, after 5 min, the cells are counted in a hemacytometer. The 300-ml culture usually yields 3.0-3.5 x l0 s HeLa cells; dead cells do not exceed 2% in the population. The culture is transfel red into tubes and centrifuged 5 min at 1000 rev/min; the sediments are then put in Hanks' solution to have about 2"5 x 106 cells/ml. Ehrlich cells are inoculated i.p. into male Swiss white mice (20-25 g) and Novikoff cells

into male Sprague-Dawley rats (150-175 g). The ascites fluid is collected one week later. The fluid is centrifuged and the cells washed 4 times with Hanks' solution. They are finally put in suspension in Hanks' solution to have about 2.5 x 106 cells per ml.

Preparation of tissue extract Bovine endometrium is dissected from adult cow uterus taken immediately after the animal has been killed. H u m a n endometrium and myometrium were provided by Dr. J. Gagnon of Ste-Justine Hospital, Montreal. Tissues were dissected after hysterectomy of 20-45 year-old women, and frozen to - 2 0 ° C until needed. These uterus did not show any major pathology, in particular no cancer. Fresh bovine endometrium or thawed human endometrium and myometrium, kept in crushed ice, are cut into pieces with scissors, minced in an equal volume of cold water, then crushed and sonicated with a Polytron model P T 4 5 / 2 (7000 rev/min, 5000 Hz). From the homogenates, 105,000 g supernatants are prepared by two different methods: - - M e t h o d I (Hinderer et al. [3]): the homogenate is lyophilized and the powder extracted with water (20mg/ml, 4°C, 12h). The suspension is centrifuged at 100,000 rev/min for 15 min in a RC-2B Sorvall centrifuge; the supernatant is centrifuged at 105,000 g for 150 min in a Beckman L2-65 centrifuge. - - M e t h o d II: the homogenate is immediately centrifuged at 10,000 rev/min for 15 rain in a RC-2B Sorvall centrifuge. The 105,000 g supernatant is obtained after centrifugation for 150 min at 105,000 g in a Beckman L2-65 centrifuge. The clear supernatants are kept frozen at - 10oc.

Assay of the action on 3H-thymidine incorporation into D NA The activity of our preparation on 3Hthymidine incorporation into DNA is tested on 107 HeLa cells (3.9 ml of the cell suspension in Hanks' solution obtained previously) incubated into 25 ml Erlenmeyer flasks containing a maximum of 1.0 ml of preparation in the assay or an equal volume of water into the control,* and Hanks' solution to adjust the volume at 4.9 ml. Tritiated thymidine labelled in the * I n some cases, larger volumes of preparations are lyophilized to a powder and dissolved in Hanks' solution ( m a x i m u m 1.0 ml) before the assay; Hanks' solution is then added to the control.

Identification of The Inhibitor of Labelled Thymidine methyl group (4 pCi; 6 Ci/mmol; Bioresearch) in 0.1 ml of Hanks' solution is added bringing the total volume to 5.0 ml. The flasks are incubated for 1 hr at 37°C in an atmosphere of 95% air-5% CO2 (v:v) with continuous shaking. The incubation is stopped by cooling the flasks in ice. In experiments where the activities of our preparations on HeLa, Ehrlich and Novikoff cells are compared, the incubation is carried out for 15 min only; other conditions remain unchanged. For the determination of the DNA specific radioactivity, the flask content is transferred to a 15-ml Ccrex tube and is centrifuged for 10 min at 6000 rev/min in the refrigerated RC-2B Sorvall centrifuge. The incubation medium is pipetted away, then 3.0 ml of 10% cold trichloroacetic acid (TCA) containing unlabelled thymidine ( 100 pg/ml) are added; after vigourous stirring with a Vortex mixer, the tubes are centrifuged again. The supernatant is discarded and the manipulation is repeated on the sediment with 2-5 ml of 10% cold TCA. The last sediment is suspended in 5 ml of 95% ethanol, the suspension is centrifuged as above, and the supernatant discarded; this manipulation is repeated twice on the sediment. The final sediment is suspended in 2.5 ml of 5% TCA and the tube heated at 90°C for 15 min with occasional shaking. After cooling at room temperature, the tube is centrifuged for 15 min at 10,000 rev/min and the supernatant collected [5]. A 1 ml aliquot of this supernatant is used for the determination of the radioactivity, while the DNA content is estimated on another 1 ml aliquot by the diphenylamine method, the absorbance being read at 600 nm [6]. The DNA specific radioactivity (DNA sp. rad) is arbitrarily given in disintegrations per min per 1000A~00; the standard deviation for the control is 6%. The inhibited fraction (I.F.) of 3H-thymidine incorporation into DNA is expressed as: I.F. = DNA sp. rad. (control)DNA sp. rad. (assay) DNA sp. rad. (control) As did Verly et al. [6] with liver chalone, we found a linear relationship between 1/(1--I.F.) and the amount of bovine endometrium supernatant (method I) added in the assay flask. By analogy with the liver chalone unit, we have defined the HeLa unit as the amount of inhibitor necessary to reduce by 50% the amount of 3H-thymidine incorporated into the DNA of HeLa cells in our experimental

659

conditions (1 hr at 37°C). The number of units (U) is given by the equation: U = I . F . / (1--I.F.). To minimize the error in U, we use only I.F. values between 0.30 and 0.70.

Separation of incubation medium, TCA-soluble and TCA-insolublefractions of HeLa cells [7]. After incubation of HeLa cells in the presence of 3H-thymidine, the suspension, cooled at 4°C, is centrifuged and the supernatant (incubation medium) collected. The cells are washed with 5 ml of Hanks' solution containing 500 pg of unlabelled thymidine, then with 5 ml of Hanks' solution; each washing is followed by a centrifugation. After removing the supernatant from the last centrifugation, 2 ml of cold 5% TCA are added to the cells; the mixture is homogenized in a Potter-Elvehjem apparatus before being centrifuged to separate the TCAsoluble and TCA-insoluble fractions.

Chromatography on Dowex-50 [8] The sample is placed on top of a 1 × 26 cm column of Dowex AG 50W X2 equilibrated with 0.1 M ammonium formate buffer pH 3.2. Elution is performed with the same buffer at a rate of 0.5 ml/min and fractions of 3.0 ml are collected. The column was calibrated with thymine, thymidine and thymidine phosphates. Table 1 gives the elution position of the different compounds: thymidine is well separated from thymine and the thymidine phosphates; the three thymidine phosphates (dTMP, d T D P and dTTP) are not retained by the ionexchanger and emerge in the same position.

Dialysis The supernatant is placed in a cellophane bag (Visking Co) with pores permeable to molecules below 4000D, and dialyzed against five volumes of water at 4°C with three changes; the whole process lasts 20 hr. The dialysate is lyophilized and the residue is taken in water; a 10 min centrifugation at 10,000 rev/min in a Sorvall RC-2B, rotor SS-34, allows discarding of the insoluble material.

Chromatography on Sephadex G-15 A column of 2"5 cm dia (Pharmacia) is filled in the cold room at 4°C with Sephadex G-15 up to 45 cm and equilibrated with water. The sample dissolved in 2.5 ml of water is placed on top of the Sephadex column and the elution is performed with water at a rate of 5 ml/hr;

660

S. Chevalier and W. G. Verly

fractions of 2"0 ml are collected. Vo (74 ml) was determined with Pharmacia blue dextran.

Table 1. Chromatography on Dowex-50 of thymine, thymidine and thymidine phosphates

Compound

Maximum (fraction number)

Thymine Thymidine dTMP dTDP dTTP

13-14 11-12 4-5 4-5 4-5

Thymine and thymidine were bought from Sigma; thymidine monophosphate (dTMP), thymidine diphosphate (dTDP) and thymidine triphosphate (dTTP) came from NBC. About 200/2g of the compound in I ml of 5% TCAwere placed on top ofa 1 × 26 cm column o¢ Dowex AG 50W X2. The elution was performed with 0" 1 M ammonium formate buffer, pH 3.2 ; fractions of 3 ml were collected and their absorbance at 260 mm determined. The Table gives the fraction corresponding to the maximum of the elution peak.

Thin layer chromatographies The lyophilized sample is dissolved in 50 #1 of water. Three different systems were used: - - S y s t e m C: cellulose M N 300 plate, Macherey-N~igel; solvent: n-butanol-acetic acidwater (60: 15:25); --System CM : carboxymethylcellulose M N 300 C M plate, Macherey-N~igel; solvent: n-butanol-acetic acid-water (60: 15 : 25) ; --System PEI : polyethyleneiminecellulose MN-polygram cel 300/PEI plate, MachereyN~gel; solvent: 0.25 M NaC1 in water. After chromatography, the plates are examined under a u.v. lamp and the fluorescent spots are delineated. For the detection of the ninhydrin-positive compounds, the chromatograms are cut in two longitudinally and one half-band is immersed in 0-5% ninhydrin in acetone and dried. The other half-band is divided in 1 cm portions; the powder of each portion is removed and eluted with 1 ml of water. The absorbance at 260 nm is determined in a model 130 Hitachi Perkin-Elmer spectrophotometer; 100 t~l aliquots are used for the biological assay.

Ethanol precipitation To the 105,000 g supernatant kept at 4°C, pure ethanol is added dropwise, with continuous stirring, up to a concentration of 60% (v:v); the precipitate (fraction I) is collected by centrifugation at 10,000 rev/min for 15 min in the RC-2B Sorvall centrifuge. To the new supernatant, pure ethanol is further a d d e d up to 87% (v:v); the second precipitate (fraction II) is also collected by centrifugation. The fraction soluble in 87% ethanol (fraction III) is lyophilized. Each fraction is dissolved in a minimum volume of water.

Protein determination The protein content of the various preparations is determined by the method of Lowry et al. [9] using bovine serum albumin as standard. RESULTS

Bovine endometrium The 105,000 g supernatant, prepared from bovine endometrium according to method I, was tested for its action on 3H-thymidine incorporation in the DNA of HeLa, Ehrlich and Novikoff cells. Table 2 shows that a dose containing 0.31 mg of proteins was inhibitory in all cases although the magnitude of the inhibition varied from one cell type to another; the inhibition was greater with H e L a cells.

( 1) Purification of the main inhibitor Bovine endometrium supernatant (150 ml) prepared by method I was submitted to dialysis: more than 70% of the recovered inhibitory activity on H e L a cells appeared in the dialysate; the specific activity (number of inhibitory unit s per mg of proteins) in the dialysate was about 10 times higher than in the supernatant. The 3 1. dialysate was lyophilized and the residue taken in 3 ml of water; the solution was centrifuged to discard the insoluble material. This clear solution from the dialysate (2.5 ml; 1.8 mg of proteins; 3850 inhibitory units) was chromatographed on Sephadex G-15. The inhibitor was eluted in a single peak (Ve/Vo = 2.7) (Fig. 1); the pooled fractions corresponding to this peak were called DS-G15. Table 2 shows that DS-G15 was active on HeLa, Ehrlich and Novikoff cells. Thymidine, which was the main low mol. wt. inhibitor found in a bovine liver extract by Lenfant et al. [10], emerges with a Ve/V o of 2.8 from the same Sephadex G-15 column.

Identification o f The Inhibitor o f Labelled Thymidine Table 2.

661

Inhibitory activities o f bovine and human endometrium preparations on HeLa, Ehrlich and Novikoff cells

I.F. Preparation

Proteins (mg)

HeLa

Ehrlich

Novikoff

0"31 0.0005

0"67 0.41

0.53 0.29

0.39 0.30

0.38 2.47 0.02

0.54 0.59 0.72

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0.47 0.58

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105,000 g supernatant DS-G15 Ethanol precipitation Fraction I (0-60%) Fraction II (60-87%) Fraction III (soluble in 87%) Human endometrium

105,000 g supernatant

The supernatants were prepared by method I; the other endometrial preparations are described in the text. The same dose (expressed in nag of proteins) was added into the incubation medium containing sI-I-thymidine and HeLa, Ehrlich or Novikoff cells. Gontrols without endometrial preparation were carried out. The incubation lasted 15 rain at 37°G before the determination of the DNA specific radioactivity; the inhibited fraction (I.F.) of tritium incorporation into DNA was calculated.

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Fig. 1. Chromatography on Sephadex G-15 of the dialysate from the bovine endometrium supernatant A portion of the concentrated dialysate (1.8 mg of proteins; 3850 inhibitory units) was ¢hromatographed in water on a 2.5 x 45 ¢m Sephadex G-15 column. Fractions qf 2 ml were collected at a rate of 5 ml[hr. The continuous line gives the absorbance at 280 nm, while the graded line limiting the stippled area gives the number ( U) of inhibitory units in each fraction. Vo (74 ml) = void volume.

DS-G15, in parallel with thymidine, was submitted to three successive thin layer chro-

matographies in different systems. A portion (1 ml) of D S - G 1 5 was lyophilized and the residue taken in 50 pl of water. This solution (200 inhibitory units) and 45/~g of thymidine, also dissolved in 50 pl of water, were first chromatographed on cellulose (system C). Thymidine gave one fluorescent spot of R f 0 . 7 5 . By contrast, D S - G 1 5 gave six fluorescent spots and three spots reacting with ninhydrin; analysis of the water eluate from the cellulose powder of 1-cm long portions o f the chromatogram, showed a peak of inhibitory activity associated with the fluorescent, ninhydrin-positive, spot of R f 0.75 (Fig. 2). The pooled eluates of the R f 0.75 spots from several C-chromatograms of thymidine or inhibitor were lyophilized; the residues (called T H Y - C 7 5 or DS-G15-C75) were dissolved in water and 50 #1 of each were chromatographed on carboxymethylcellulose (system CM). T H Y C75 gave one fluorescent spot of R f 0.75, while DS-G15-C75 (400 units) gave three ninhydrinpositive spots and one fluorescent spot; the inhibitor was associated with the fluorescent, partly ninhydrin-positive, spot of R f 0.75 (Fig. 2). After elution from their corresponding spots of two CM-chromatograms, lyophilization and redissolution in a minimum of water, 50 pl of T H Y - C 7 5 - C M 7 5 and 50 pl of DS-G15-C75CM75 (540 units) were chromatographed on PEI-cellulose (system PEI). In each case, there was only one fluorescent, ninhydrinnegative, spot of R f 0.82; the inhibitory activity was associated with this spot (Fig. 2). The eluates from the R f 0 . 8 2 spots of the PEIchromatograms were called respectively T H Y -

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Fig. 2. Purification by thin layer chromatographies of the main bovine endometrium inhibitor. The inhibitor (DS-G 15), in parallel with thymidine (THY), was submitted to three successive thin layer chromatographies:DS-G15 and thymidine were applied on C-plates; the eluates from the Rf0.75 spots of the C-chromatograms were applied on CM-plates ; the eluates from the RfO.5 spots of the CM-chromatograms were applied on PEI-plates. Cm = migrated distance in cm. (a) 50 pl of the aqueous solution of the sample was applied on the plate and developed as described above. Fluorescent spots are encircled; ninhydrin-positive regions, revealed on half-bands, are the black half-spots. (b) absorbance (A) at 260 nm (dotted line) and the number (U) of units of inhibitory activity on HeLa cells (continuous line) were measured on the eIuate from the powder.

C75-CM75-PEI82 and DS-G15-C75-CM75PEI82. Thymidine and inhibitor were compared at each step of purification. The u.v. spectra of THY-C75-CM75-PEI82 and DS-G15-C75CM75-PEI82 are identical (Fig. 3). Equivalent amounts (A~60 x volume) of THY-C75-CM75PEI82 and DS-G 15-C75-CM75-PEI82 have the same inhibitory action on 3H-thymidine incorporation into DNA of H e L a cells (Table 3). Table 3. Comparison of the inhibitory activity of thymidine and thefractions containing the bovine endometrium main inhibitor taken in the course of purification

(2) Thymidine content o f the endometrium supernatant Ten millilitres of 105,000 g supernatant were lyophilized. The residue, dissolved in 2.5 ml of water, was placed (211 units) on a 2.5 x 45 cm column of Sephadex G-15 and eluted with water. The absorbance at 280 nm of the collected fractions and their biological activity on 3H-thymidine incorporation into DNA of HeLa cells were measured. Stimulatory factors were separated from 357 units of inhibitory factors. The greater part of the inhibitory factors was in a peak having the position of thymidine (Fig. 4).

Preparation

I.F.

(3) Search f o r an inhibitor specific f o r H e L a cells

DS-G15 Thymidine DS-G15-C75 THY-C75 DS-G 15-C75-CM75 THY-C75-CM75 DS-G 15-C75-CM75-PEI82 THY-C75-CM75-PEI82

0.42 0.64 0.64 0.68 0.68 0.73 0.56 0.58

The 105,000 g supernatant was submitted to fractional precipitation with ethanol. Table 2 shows that fraction I (0-60% ethanol), fraction II (60-87% ethanol), and fraction I I I (soluble in 87% ethanol) contained inhibitors, but that the inhibitory action of none of these fractions was specific for H e L a cells.

Equivalent amounts (pl x A26o) of thymidine or inhibitor solutions were added to the incubation medium containing HeLa cells and 3H-thymidine; the control received an equal amount of water. After 1 hr at 37°C, the DNA specific radioactivities were measured and I.F. values calculated.

(4) Action o f endometrial supernatant on D N A replication H e L a cells (10 7) were incubated in the presence of 4 pCi of 3H-thymidine (6 Ci/mmol) with 0.1 ml of endometrial supernatant in a

Identification of The Inhibitor of Labelled Thymidine

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Fig. 3. Comparison of the u.v. spectra of thymidine and of the inhibitory fractions in the course of the purification of the bovine endometrium main inhibitor The spectra were recorded with the Gary 14R spectrophetometer. The continuous lines give the absorbanee (A) of the fractions containing the main inhibitor, while the dotted lines give the absorbance of the thymidine solutions. (a) DS-GI5 and thymiaine (b) DS-G15-C75 and THY-G75 (c) D$-G15-C75-GM75 and THY-G75-GM75 (d) DS-GI5-C75-CM75-PEI82 and THY-G75-GM75PEI82.

total volume of 5 ml. After 1 hr at 37°C, the cells were separated from the incubation medium and treated with TCA. Analysis on Dowex-50 of the incubation medium showed that the radioactivity was mainly in the position of thymidine; the presence of the endometrial supernatant increased by 25% the amount of 3H-thymidine that was not used by the cells (Fig. 5). Analysis on Dowex-50 of the TCA-soluble fraction showed that the radioactivity was mainly in the position of the thymidine phosphates. Incubation with the endometrial supernatant strongly decieased this radioactivity (Fig. 5) ; by analogy with the procedure followed to calculate the inhibition of 3Hthymidine incorporation into DNA, an I.F. of 0.75 can be calculated. The TCA-insoluble fraction was utilized to estimate t h e DNA specific radioactivity. The

Chromatography on Sephadex (3-15 of the bovin endometrium supernatant Lyophilized bovine endometrlum supernatant (211 units) in 2.5 ml of water was placed on a 2.5 x 45 ¢m column of Sephadex G-15 and eluted with water. The continuous line gives the absorbanee at 280 nm, while the graded line limiting the stippled area gives the number ( U) of units of inhibitory activity. There is, after 230 ml, a region containing stimulatoty factors. On this column, thymidine is eluted around 200 ml.

endometrial supernatant was responsible for an I.F. of 0.77.

Human endometrium and m.yometrium The 105,000 g supernatant from human endometrium was prepared using either method I or method II; a 105,000 g supernatant was prepared from human myometrium using only method I. The action of increasing amounts of these supernatants on 3H-thymidine incorporation into DNA of HeLa cells was investigated (Fig. 6). At low doses, containing less than 2 mg of proteins, the endometrial supernatant method I stimulated the incorporation; inhibition appeared only at larger doses. The endometrial supernatant method II was tested only at doses containing more than 2 mg of proteins; its inhibitory effect was similar although lower than that of the endometrial supernatant method I. The myometrial supernatant was mainly stimulatory, although inhibition appeared at doses containing more than I0 mg of proteins. The same dose (4.6 mg of proteins) of the endometrial supernatant method I was assayed on HeLa, Ehrlich and Novikoff cells. The results are analogous to those obtained with

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Fig. 5. Action of bovine endometrium supernatant on 3Hthymidine uptake by HeLa cells and on the radioactivity of the intracellular thymidine phosphates HeLa cells were incubated in the presence of 3H-thymidine for 1 hr at 37°C with bovine endometrium supernatant (3.4 units) and a control without supernatant. A 1-ml (liquot of the incubation medium (a) or of the cellular TCA-soluble fraction (c) were chromatographed on Dowex-50 and the radioactivity measured in the eluted fractions. The continuous lines correspond to the incubation with the supernatant and the dotted lines to the control. A chromatography with commercial 3H-thymidine is presented in (b) ; note the impurity eluted before the thymidine peak. N =fraction number; R = dis/min x 1 0 - 5

bovine endometrial supernatant: the inhibition of 3H-thymidine incorporation into DNA was observed with the three kinds of cells, although the effect was more important with HeLa cells (Table 2). To see whether human endometrial and myometrial supernatants contained thymidine, they were chromatographed on Dowex-50; a trace amount (0.04 #Ci) of purified 3H-thymidine* was added to locate the elution peak of * C o m m e r c i a l 3 H - t h y m i d i n e very often c o n t a i n s a n i m p u r i t y w h i c h is eluted f r o m Dowex-50 i m m e d i a t e l y before t h e t h y m i d i n e peak. T h i s i m p u r i t y c a n be seen in Fig. 5(b). Purified 3 H - t h y m i d i n e is p r e p a r e d f r o m c o m m e r c i a l 3 H - t h y m i d i n e b y t a k i n g only the fractions c o r r e s p o n d i n g to t h y m i d i n e after a c h r o m a t o g r a p h y o n Dowex-50.

Fig. 6. Action of human endometrium and myometrium supernatants on 3H-thymidir~ incorporation into D N A of HeLa cells LF. is given as a function of the amount of supernatant (rag of proteins). O, endometrial supernatant method I ; x , endometrial supernatant method H ; Q, myometrial supernatant method L

this nucleoside. The collected fractions were assayed for radioactivity and for their action on HeLa cells. Figure 7 shows that, when the endometrial supernatant method I was chromatographed, the greater part of the inhibitory activity migrated with thymidine. By contrast, no inhibitory activity could be eluted from the Dowex-50 when the endometrial supernatant method II or the myometrial supernatant method I were chromatographed. 3H-thymidine was incubated for 1 hr at 37°C with the endometrial (method I or II) or myometrial (method I) supernatants, then chromatographed on Dowex-50. In every case, the peak of radioactivity was no longer in the position of thymidine; 3H-thymidine had been converted into 3H-thymine (Fig. 8).

Thymidine The action of increasing amounts of unlabelled thymidine on the 3H-thymidine incorporation into DNA was studied with HeLa, Ehrlich and Novikoff cells. Figure 9 shows a linear relationship between 1/(1-I.F.) and the amount of unlabelled thymidine added. The slopes are the same for HeLa and Ehrlich cells, but it is 2.3 times smaller with Novikoff cells.

Identification of The Inhibitor of Labelled Thymidine 30

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Action on thymidine of human endometrium and

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Dowex-50 ehromatographies of human endometrium and myometrium supernatants. The lyophilized supernatants were taken in 2 ml of water; after removal of the insoluble material by centrifugation, 0.04/~Ci of purified 3H-thymidine was added to the clear solution which was placed on a 1 x 26 cm column of Dowex AG 50 W X2 at 4°C. The elution was performed with O. 1 M ammonium formate buffer, p H 3.2, and 3 ml fractions were collected; 0.5 ml was used to determine the radioactivity (dotted line), while the remaining 2.5 ml were lyophilized and the residue taken in 1 ml of Hanks' solution for the assay on HeLa cells (LF.; continuous line). N =fraction number; R = dis /min x l O- 3. ( a } purified 3H-thymidine alone; (b) endometrium supernatant method I (42 mg of proteins); (c) endometrium supernatant method I I (92 mg of proteins) ; (d) myometrium supernatant method I (46 mg of proteins).

The supernatant (1 ml) was added to 4 ml of H a n ~ ' solution containing 4 pCi of 3H-thymidine. After 1 hr of incubation at 37°C, the solutions were cooled at 4°C and 1 ml aliquots were chromatographed on Dowex AG 5 0 W X 2 (continuous line). Controls of 3H-thymidine incubated without supernatant were run on the same column (dotted line). N = fraction number; R = dis/min × 1 0 - s . (a) endometrial supernatant method I (6.9 mg of proteins) ; (b) endometrial supernatant method I I (13.8 mg of proteins); (c) myometrial supernatant method I (36.0 mg of proteins).

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DISCUSSION

T o have enough material to isolate the inhibitor, we first used bovine endometrium. A supernatant was prepared following the method of Hinderer, V o l m and Wayss [3]: the homogenate was lyophilized, the resulting powder was extracted with water and the extract centrifuged at 105,000 g for 150 rain. Bovine endometrium supernatant inhibits the incorporation of 3H-thymidine into D N A of H e L a cells, but it is also active on Ehrlich and Novikoff cells. T o purify the inhibitor, the supernatant was first dialyzed: more than 70% of the recovered inhibitory activity was found in the dialysate. W h e n chromatographed on Sephadex G-15, E*

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Fig. 9. Action of thymidine on 3H.thymidine incorporation into D N A of HeLa, Ehrlich and Novihoff cells. Increasing amounts of unlabelled thymidine were added to cells incubated with 4 pCi of 3H-thymidine (6 Ci]mmol) in a total volume of 5 ml. After 15 rain at 37°C, the D N A specific radioactivity was determined. The graph gives 1/(1-LF.) against the amount (fig) of unlabelled thymidine. 0 , HeLa cells; ×, Ehrlieh cells; Q, Novikoff eells.

666

S. Chevalier and

the dialysable fraction gave a single peak of inhibitory activity with a Vo/Vo = 2.7, i.e., in the elution position of thymidine. Further purification of the dialysable inhibitor in three successive thin layer chromatographies showed that it was indeed thymidine: the spectrum of the pure inhibitor was identical to that of thymidine; equivalent amounts (A260 × volume) of inhibitor and thymidine had the same action on 3H-thymidine incorporation into DNA of HeLa cells. The supernatant of bovine endometrium was directly chromatographed on Sephadex G-15 to determine what pa~ t of its inhibitory activity was due to thymidine: 211 inhibitory units were placed on the column; stimulatory factors were separated from 357 units of inhibitory factors of which 78% were in the position of thymidine. An attempt to separate an inhibitor specific for HeLa cells was made by fractional precipitation of the supernatant with ethanol. Three fractions (0-60%; 60-87%; soluble in 87%) were examined: none was specific for HeLa cells. Because our results with bovine endometiium seemed to differ from those of Hinderer et al. [3] with h u m a n endometrium, we decided to repeat the work with h u m a n tissues. Supernatants of h u m a n endometrium and myometrium were prepared following the procedure of these authors. In agreement with them, we found that, at the doses they used, the endometrial supernatant inhibited while the myometrial supernatant stimulated the incorporation of 3H-thymidine into DNA of HeLa cells. However, our endometrial supernatant was also inhibitory for Ehrlich and Novikoff ceils; the relative effects wel e exactly the same as with the supernatant of bovine endometrium. Using Dowex-50 chromatography, the h u m a n endometrial supernatant was found to contain thymidine which was absent from the h u m a n myometrial supernatant. Moreover, the h u m a n endometrial supernatant contained an enzyme that hydrolyzed thymidine into thymine. The h u m a n myometrial supernatant also possessed this enzymic activity and, indeed, this supernatant was inhibitory at high doses; it is likely that, at high doses, the destruction of 3H-thymidine was more important than the nutritive effect of the extract. The presence of thymidine in liver extract has been reported by Lenfant et al. [10]. The occurrence of thymidine in endometrial but not in myometrial supernatant could be explained by the fact that endometrium possesses more cell nuclei per unit weight than

w . c . Ver myometrium and that the cell turnover is high in the endometrium so that this tissue might contain necrotic cells. It was, however, still possible that thymidine was a preparation artefact: the beef liver homogenate of Lenfant et al. [10], which was heated at 50°C before addition of ethanol, contained thymidine, while the rabbit liver homogenate of Deschamps and Verly [7], which was kept at 4°C during all the manipulations, did not contain this nucleoside. To investigate this point, a h u m a n endometrial supernatant was prepared in a more direct way: the homogenate was immediately centrifuged at 105,000g. This supernatant (method II) had a similar but lower effect on 3H-thymidine incorporation into DNA of HeLa cells than the supernatant prepared according to the method of Hinderer et al. [3]; both supernatants contained an enzymatic activitythatdestroyed 3H-thymidine, but thymidine was not found in supernatant method II. Thymidine is thus a preparation artefact of method I. The salvage pathway, which utilizes exogenous thymidine in these in vitro experiments, is a minor route compared to the endogenous synthesis of thymidine phosphates. Deschamps and Verly [7] calculated that, with regenerating liver slices and in their experimental conditions, less than 1/5000 of the thymine in the synthesized DNA derived from the 3H-thymidine of the incubation medium. A decrease of 3H-thymidine incorporation into DNA is not necessarily indicative of an inhibition of DNA synthesis. Various causes can decrease 3H-thymidine incorporation into cellular DNA: (1) presence of thymidine in the extract which lowers the specific radioactivity of the labelled precursor in the incubation m e d i u m [10] ; (2) addition of enzymes which convert thymidine into thymine [11-14] that can no longer be used by the cell [15]; (3) decrease of the plasma membrane permeability to thymidine or decrease of the phosphorylation of this deoxynucleoside [8]; (4) variation in the size of the intracellular thymidine phosphate pool; (5) decrease of the conversion of the thymidine phosphates into DNA. Only the latter cause corresponds to an inhibition of DNA synthesis. To demonstrate that it is really the case, it is necessary to measure the specific radioactivity of the deoxynucleoside phosphates: one is then in a position to calculate the amount of DNA synthesized from the radioactivity incorporated into this macromolecule. Using this technique, Deschamps and Verly [7] showed that their purified liver inhibitor decreased DNA

Identification of The Inhibitor of Labelled Thymidine synthesis in regenerating liver slices. A totally different situation was observed with bovine endometrium supernatant a n d HeLa cells: when incubated with 3H-thymidine in the presence of the supernatant, the radioactivity in the cellular thymidine phosphates was decreased by the same factor as the radioactivity in DNA. It must be concluded that the rate of DNA synthesis remained unchanged: if the endometrial supernatant inhibits 3H-thymidine incorporation into DNA, it has no action whatsoever on DNA synthesis in HeLa ceils. The effect of the addition of unlabelled thymidine to the incubation medium on 3H-thymidine incorporation into DNA was studied with HeLa, Ehrlich and Novikoff cells. The incubation medium contained 0.16 #g of 3H.thymidine; 0.2 7/~g of unlabelled thymidine had to be added to have a 50% reduction of tritium incorporation into DNA with HeLa and Ehrlich cells, and 0.62 #g with Novikoff ceils. The dilution factors are respectively 2"7 and 4.9; Lenfant et al. [10] found a factor of 5.8 with regenerating liver slices and one much greater with kidney slices. These differences can be interpreted wrongly as the result of the action of a tissue-specific inhibitor. HeLa cells are, among the biological systems so far studied, one of the most sensitive to the inhibition, by unlabelled thymidine, of 3H-thymidine incorporation into cellular DNA.

Thymidine has the same action on HeLa and Ehrlich cells, while bovine or h u m a n endometrial supernatants were more active on HeLa cells than on Ehrlich cells. But we have seen that thymidine is not the only nonspecific inhibitor in these supernatants: h u m a n endometrium also contains enzymes that destroy the 3H-thymidine of the incubation medium; moreover stimulatory factors are present in those crude preparations. We are thus unable to confirm the conclusion of Hinderer, Volta and Wayss [3] that the factor in endometrial extract which inhibits thymidine incorporation into HeLa cell DNA is a chalone. Indeed, these authors observed later [4] that their crude endometrial extract had no tissue specificity since it was also active on h u m a n fetal liver and kidney cells; Volm and Wayss [16] even came to the conclusion that the chalone concept, developed for the epidermis, could perhaps not be extended to other tissues. This is likely too pessimistic; it might well be that HeLa cells are just not the right cells to use to discover the endometrial chalone. Besides, two kinds of chalones have been described: those interfering with S phase and those inhibiting mitosis; the assay used in the work of Hinderer et al. [3] and in our work would only enable to find an inhibitor of DNA replication.

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1.

667

P. WEISS, In Biological Specificity and Growth, G. Butler, Princeton University Press, Princeton (1955). 2. W.S. BULLOUOH,C. O. HEWEXT and E. B. LAURENCE,Exp. Cell Res. 36, 182 (1964). 3. H. HINDERER, M. VOLM and K. WAYSS,Exp. Cell Res. 59~ 464 (1970). 4. K. WAYSSand M. VOLM, Verh. dtsch. Ges. Path. 54, 496 (1970). 5. W . C . SCHemER, Methods in Enzymology. (Edited by S. P. COLOWlCKand N. O. KAPLAN) Vol. III, p. 680. Academic Press, New York (1957). 6. W.G. VERL'Z,Y. DESCHAMPS,J. PUSHPATHADAMand M. DESROSmRS,Canad. J. Biochem. 49, 1376 (1971). 7. Y. DESCH~PS and W. G. VERLY, Biomedicine, In press (1975). 8. G. NILSSON,Exp. Cell Res. 59, 207 (1970). 9. O . H . LOWRY, N. J. ROSEBROUOH,A. F. FARR and R. T. RANDALL,J. biol. Chem. 193, 265 (1951). 10. M. Lm'~FANT, L. KREN-PRosCHEK, W. G. VF.RLY and H. DUOAS, Canad. J. Biochem. 51, 654 (1973). 11. M. FmEDKIN and D. ROBERTS,d. biol. Chem. 207, 245 (1954). 12. S. PERRY andJ. C. MARSH, Proc. Soc. exp. Biol. (N.Y.) 115, 51 (1964). 13. W . C . DEWEY, R. M. HUMPHRSYand B. A. DEDETA, Exp. Cell Res. 80, 349 (1968). 14. M. MIYAMOTOand H. TERAYAMA,Biochim. Biophys. Acta. (Amst.) 228, 324 (1971). 15. A.A. PLEm'L and R. SCHOENHEI~mR,J. biol. Chem. 153, 203 (1944). 16. M. VOLM and K. WAYss, Naturwissenschaften 58, 458 (1971).