Stimulation of growth of serum-deprived chick embryo fibroblasts by 3′,5′-phosphodiesterase

Stimulation of growth of serum-deprived chick embryo fibroblasts by 3′,5′-phosphodiesterase

Printed in Sweden Experimental STIMULATION EMBRYO OF Cell Research GROWTH FIBROBLASTS OF BY D. A. LAWRENCE 9.5 (1975) 54-62 SERUM-DEPRIVED ...

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in Sweden




Cell Research





9.5 (1975) 54-62




Fondation Curie, lnstituf du Radium, Section de Biologie, F-91405 Orsay, et Unit6 de Physiologie Cellulaire. INSERM, Paris, France

SUMMARY In chick embryo fibroblasts (CEF) deprived of serum, DNA synthesis is reduced to a basal level in about 12 h, cell division ceases after 24-36 h and their morphology changes to a rounded, less refringent form. During several days without serum the CAMP content of the cells showed a slow increase or a maintenance of the level found before serum was removed. When CEF deprived of serum for 24 h were treated with beef heart 3’,5’-phosphodiesterase (PHD) the CAMP level fell about 40% after 3 h, 3H-thymidine incorporation into DNA was strongly stimulated with a peak of incorporation at 12 h after the start of PHD treatment, cell morphology returned to that observed before serum deprivation, and at 24 h there was an evident growth in cell population, with a parallel increase in protein content. The growth stimulation by PHD is transitory: after cells had been deprived of serum for 4 days the PHD effect was no longer noticeable on the above parameters. Theophylline (1 mM and 4 mM) inhibited the PHD-mediated stimulation of 3H-TdR incorporation, this could well have been due to its general toxic effect on the cells (see Discussion).

Morphological and growth characteristics of cells appear to depend, at least partly, upon the level of cellular CAMP [l-5]. Although the addition of CAMP stimulates the division of some cells of the hematopoietic system, namely pluripotent stem cells [6] and thymocytes [7], CAMP seems, on the contrary, to exert an inhibitory action on the growth of embryonic ‘normal’ fibroblasts or on established cell lines in vitro. Briefly, the salient facts are that (1) normal fibroblasts of several species contain higher levels of CAMP than their transformed counterparts [8, 91; (2) for both cell types during logarithmic growth there is an inverse relationship between growth rate and CAMP content [lo]; (3) at confluency the CAMP content increases in normal fibroblasts but does not do so in their transExptl Cell Res 95


formed derivatives [l 1, 121 and (4) treatment of transformed cells with db-CAMP and inhibitors of 3’,5’-phosphodiesterase induce several changes, such as a return to a normal morphology [ 1,3,4] a reduction of their agglutinability by lectins [13] and an increased sensitivity to an arrest of growth by topo-inhibition [3, 131. CAMP is synthesized by a membranebound enzyme, adenyl cyclase [ 141, and degraded by 3’,5’-phosphodiesterase [15]. Numerous treatments are known to raise or lower intracellular CAMP levels; for example removal of serum from the growth medium increases and its readdition decreases the CAMP content [8, 161. It has long been known that serum deprivation greatly reduces cell growth and DNA synthesis and that readdition of serum trig-


gers the reinitiation of DNA synthesis, permitting cell multiplication anew [17, 181. In view of these points we thought it of interest to look for a possible stimulation of DNA synthesis and cell multilication by reducing the level of cellular CAMP by means 3’S’-phosphodiesterase. of exogenous Here, we show that DNA synthesis and multiplication are stimulated in serumdeprived chicken embryo fibroblasts (CEF) treated with 3’S’-phosphodiesterase preparation.

MATERIAL Cell cultures

and growth

of chick cells


removed, the cells fixed to the plate with 2.0 ml of ice-cold 5% trichloracetic acid (TCA), and 15 min later, they were washed twice with 2.0 ml of ice-cold 5% TCA. The cells were then hydrolysed at 80°C in 2.0 ml of 10% TCA for 90 min [21]. A 200 ~1 aliquot of the hydrolysate was counted in 10 ml of Bray [22] scintillator mixture in a Packard Liquid Scintillation counter. The incorporation of 3H-TdR into DNA was expressed in cpm1106 cells.

Auto-radiography Cells labelled as described above were washed with PBS, detached with trypsin-EDTA, and collected by gentle centrifugation. The cell pellets were resuspended in a drop of serum, and spread on slides. The slides were fixed in Camoy fixative (ethanol 2 parts+ acetic acid 1 part), and then dipped in Ilford K5 emulsion. After 10 days of incubation, slides were fixed and stained with Giemsa.


and media

The chicken cells were CEF prepared from &day-old Brown Leghorn embryos from a lymphomatosis-free strain. which according to their sensitivity to the four antigenic subgroups of Rous virus (A, B; C, D) were presumably of the C/O type [19]. Some embryos could have carried a cryptic leukosis virus since low levels of gs antigen have been detected in some embryos of this strain [20]. The primary cultures were grown in IO cm Falcon dishes, in a standard medium composed of Eagle’s MEM in Earle salts, with double concentration of essential amino acids and vitamins and added nonessential amino acids, plus 10% tryptose phosphate broth (TPB. Difco) 5% calf serum and antibiotics (penicillin, streptomycin, kanamycin, fungizone). In all experiments secondary cultures were used. lo6 cells (unless otherwise stated) were seeded into 3.5 cm Falcon dishes in medium nlus 4% calf serum. but without TPB. After overnight incubation at 37”C, the nlates were thorouehlv washed twice with 3 ml of PBS, and new media without serum or TPB was added. The serum used in these experiments contained 0.0006 U of 3’,5’-phosphodiesterase activity per mg protein (or 0.0352 U/ml; see below for unit definition).

Cell enumeration Cells were detached and dispersed in 1.O ml of 0.06% trypsin containing 10m5 M EDTA. After 15 min of incubation at 37”, 1 ml of serum was added, and cells were collected by gentle pipetting for enumeration in a hemocytometer.

Measurement of thymidine incorporation The incorporation of thymidine into acid-insoluble material was measured bv the addition of I uCi of 3H-TdR (Centre d’Energie- Atomique, Saclay, France; 33 Cilmmol) ner ml of Eaale’s MEM without serum and incubation of the cells for 30 min (longer times are indicated when they occur). The medium was then

Assay of CAMP After removal of the medium the cell layer in each of at least three plates was washed twice with 2.0 ml of ice-cold PBS. then covered with 2.0 ml of ice-cold 5 % TCA for 15 min. The cells were then scraped from the plate, poured into a centrifuge tube, the plate rinsed with 1.O ml of water which was added to the tube and the tube nlaced at -70°C until nroceeding with the extraction of CAMP. To the acid extract were added about 6000 cpm 3H-cAMP (Radiochemical Centre, Amersham, UK; to estimate % recovery of CAMP after extraction) and 0.3 ml in HCl (per plate used in making the extract), after which the mixture was thawed and centrifuged. The precipitate was dissolved in 1 N NaOH for measurement of protein [23]. The sunernatant fluid was added to a column containing 3.6 ml of Dowex 50 (200-400 mesh) equilibrated with 0.1 N HCI. After elution with 5.0 ml of 0.1 N HCI and 1.0 ml of water, to remove TCA, CAMP was eluted with a further 12 ml of water. The fraction containing CAMP was taken to dryness by rotary evaporation under reduced pressure, the residue dissolved in water and aliquots assayed in duplicate, with and without internal standards, for CAMP using an assay kit of the Gilman protein-binding type (Radiochemical Centre, Amersham. UK) and for recovery of ‘H-CAMP. 3 ‘,5 ‘-Ph&phohiesrerase. Beef: heart PHD (3’,5’CAMP phosphodiesterase from beef heart) was obtained from-Sigma (lot number 12OC-7740) and before use in each experiment a suitable aliquot dissolved in Eagle’s MEM without serum was sterilized by a 30 min exposure to a Yo source. Its enzymatic activity was assayed at 37°C for 15 min by the method employing excess snake- venom to convert 5’-AMP to adenosine and AG-I-X-2 (200-400 mesh) anion exchange resin to absorb and quench unreacted substrate while leavine adenosine to be detected bv liauid scintillation [24r Under the conditions of use-in these experiments, i.e., in Eagle’ MEM incubated at 37”c, the activity of the PHD preparation was 0.40 U/mg protein, where 1.O U hydrolyses 1.0 nmole of CAMP to 5’-AMP/min at 37°C at a substrate concentration of 2.0 44. Exprl


Res 95 (1975)


Lawrence and Jullien RESULTS

Evidence for stimulation of cell multiplication by PHD To investigate whether PHD could stimulate the growth of CEF, we chose to test the effect of the enzyme on serum-deprived cells. As shown by Temin [18] the growth of serum-deprived CEF is slowed down, and practically stopped after 24 h. We checked three parameters in order to monitor cell growth and multiplication incorporation of 3H-TdR into DNA, increase of the cell number and total protein content/plate. In our experiments, when CEF were transferred to a serum-free medium, their rate of 3H-TdR incorporation decreased to a basal level in 6 to 12 h, their number stopped increasing or increased only slightly for 24-36 h and thereafter declined slightly, probably because of a moderate sloughing off of cells not compensated by cell multiplication. In addition, within 24 h after transfer to a serum-free medium, there was a change in

Table 1. Effect of PHD treatment on 3HTdR incorporation, cell multiplication and protein content of serum-deprived CEF Control cells A. 3H-TdR incorporation cpm/106 cells (1 &i/ml-24 h) B. No. of cells per 3.5 cm dish (after 24 h) C. Protein content per dish (after 24 h)

PHD-treated cells (0.032 U/ml)

I. 23 750 I. 110300 II. 26 950 II. 106 600 At start of treatment 1.4x106 1.2x106 2.1x106 At start of treatment 105 PLg



Res 95 (1975)




Fig. I. Abscissa: time after addition of PHD (hours); ordinate: cnm 3H-TdR incoroorated ner 1 x lo6 cells. 1

3H-TdR incorporation by CEF in serum-free and in serum-free medium DIUS PHD. 1 x lo6


CEF were seeded in 3.5”cm dishes as described in Materials and Methods. Plates were maintained during 24 h in a serum-free medium, and then refed either with fresh serum-free medium (O-O) or with serum-free medium plus PHD: 0.032 U/ml in expt. I and 2,O.W (A...A) 0.016 (W--m) and 0.032 U/ml (O-.-O) in expt. 3. 3H-TdR incorporation was measuring by labelline with 1 &i/ml of precursor during 30 min. At the time of addition of PHD the cell number/plate was 1.6~ 106 (expt. l), 1.4~106 (expt. 2) and 1.7x LO6(expt. 3). 24 h later, the number of cells in control plates was respectively 1.4~10~ (expt. l), 1.5~10~ (expt. 2), and 1.7~ 1Og(exot. 3). In PHD-treated plates, the number of cells was 2.1 x lo6 (expt. l), 2.3~.1oB (expt. 2) and in exut. 3. resuectivelv 2.1~10~. 2.8~10~ and 3.1~108 in plates stimulated with 0.004; 0.016 and 0.032 PHD U/ml. No rise in cell number was observed 12 h after the beginning of treatment. In all three experiments the protein content paralleled the corresponding cell numbers.


A, I and II, B and C were separate experiments. In all cases, 1 x106 CEF were plated in 3.5 cm dishes in medium with 4% serum, incubated overnight, and then maintained during 24 h in serum-free medium before being used for experiments Expt/


the morphology of the CEF,which became more rounded and less refringent. PHD was added 24 h after serum deprivation (0.032 U/ml). During the next 24 h

3’S’-Phosphodiesterase Table 2. Effect of the period of PHD treatment of CEF on their incorporation of 3HTdR into DNA cpm of 3H-TdR incorporated per 1W cells

Period of treatment of CEF (hours)

Control cells

PHD-treated cells 0.032 U/ml

3 6 12

3 973 4 073 4 746

8 993 16 393 24 023

The cell count at zero time was 1.5~ 106 cells per 3.5 cm dish. After 3 h the medium was removed from duplicate dishes of control and treated cells and replaced with fresh Eagle’s MEM still without serum. This procedure was repeated on other dishes at 6 h. All cultures were labelled with 1 &i/ml 3H-TdR from the 8th until the 12th hour.

there regularly occurred: (a) a reversion to the cellular morphology observed before serum deprivation; (b) an increase of 30-50 % in cell number, although during this same time the cell number in control plates decreased slightly; (c) a parallel increase in protein content per plate; (d) a 34-fold increase of 3H-TdR incorporation, the precursor being added at the start of the experiment and being present until its end 24 h later. Typical results are presented in table 1. Kinetic study These preliminary experiments were followed by a kinetic study of the changes induced in PHD-treated serum-deprived CEF by pulse-labelling with 3H-TdR (1 &i/ml for 30 min), cell enumeration and determination of protein content. On one occasion, we also checked the frequency of labelled cells, i.e. cells in S phase, by radioautography. In all experiments, we observed a peak of 3H-TdR incorporation 12 h after adding PHD (0.032 U/ml) and an increase in cell

and growth of chick cells


number, which began after the 12th h and was evident at 24 h (fig. 1). Results of radio-autography agreed with those obtained by scintillation counting. In control cells, maintained in serum-free medium, the frequency of labelled cells was 2-3 % throughout the experiment. In PHD-treated cells, the frequency of labelled cells increased to 15 % at 8 h, 28 % at 12 h and fell to 2% at 24 h. In addition, an obvious change in cell morphology was detectable at 8-10 h after the start of PHD treatment. The effect of PHD treatment is dosedependent. At lower doses tested (0.016 and 0.004 U/ml), the peaks of 3H-TdR incorporation were respectively lower, and were located earlier, at about 10 and 8 h (fig. 1). Increases in cell number were also reduced. The stimulation of 3H-TdR incorporation became evident about 6 h after the start of

Table 3. Effect of PHD treatment of serum-deprived CEF on cellular levels of CAMP Cell sampling times (hours) -24

0 3 6 12 24

pmoles of cAMPll.0~

lo6 cells in

Control cells

PHD-treated cells

Expt 1

Expt 2

Expt 1

2.4f0.13 2.2kO.20 2.4f0.10 2.9kO.15 2.9kO.07 5.OkO.35

Not done 2.6kO.02 2.8i0.19 2.1f0.11 2.4kO.04 2.1f0.08 2.7kO.25 2.0k0.11 2.5f0.34 1.4f0.16

Expt 2

1.7+0.000 1.7fO.11 1.5kO.21 0.99+0.00

CEF were incubated in serum-free medium for 24 h after which time the cell count was 1.50~ 106 and 1.46x lo6 tells/3.5 cm dish in expt. 1 and 2, respectively. The medium was then replaced with fresh serumfree medium (control) or with this medium plus 0.032 U/ml PHD. At the end of the sampling period the cell count was 1.25~ 108 and 1.95~ 108 cells per 3.5 dish of control cells and PHD-treated cells, respectively in expt. 1 and 1.22x lo6 and 2.10~ 108 ceW3.5 cm dish of control cells and PHD-treated cells, respectively in expt. 2. Each given value represents the arithmetic mean +S.D. of duplicate determinations. Exptl


Res 95 (1975)



and Jullien Variations of cellular cAMP levels

L lxld



, 1 x16


I 0

I 1

I 2

I 3

I 4

Fig. 2. Abscissa: time (days); ordinate: no. of tells/3.5 cm dish. Effect of repeated treatments with PHD on the number of CEF in a serum-free medium. 2x 105 and 1 x lo6 CEF were respectively seeded in 3.5 cm dishes in medium with 4% calf serum. After overnight incubation this medium was replaced with serum-free medium for 24 h (dav - 1). At this time (dav 0). half of the dishes were treated with serum-free medium plus PHD (0.032 U/ml) (O-.-O) or with serum-free medium alone (02Oj. Media’were changed each day and the number of cells/dish counted. Each given value represents the arithmetic mean of four plates; vertical bars show the range.

the treatment in the previous experiments, and was not detectable at 3 h. We now asked the question: what is the minimum period during which PHD needs to be present in the medium for a measurable stimulation of 3H-TdR incorporation to be observed? As shown in table 2, a 3 h treatment provides a clear stimulation of incorporation, measured 8 to 12 h after the beginning of treatment, although a period of treatment longer than 6 h is necessary to obtain the maximum amount of incorporation. It should be noted that PHD activity in the medium decreases during incubation at 37”, and that its half-life time is about 5 h, in presence or in absence of cells in the dishes. Exptl


Res 95 (1975)

Until now it had been implicitly assumed in these experiments that the phosphodiesterase treatment lowered the cellular levels of CAMP. Many reports have shown an inverse relationship between growth rate of fibroblasts and their CAMP levels. Consequently, we measured CAMP levels in CEF maintained in serum-free medium and treated with PHD (0.032 U/ml). In these experiments the recovery of CAMP varied from 65 to 86 %. As shown in table 3, serum deprivation was followed, in control cells, by either a slow rise in, or a maintenance of, the CAMP level, whereas in PHD-treated cells the CAMP level was already lower after 3 h of treatment and continued to decline throughout the experiment. Use of theophylline as PHD inhibitor

As theophylline is frequently employed as a PHD inhibitor [ 1, 151, we checked if theophylline added to serum-deprived CEF together with PHD was able to inhibit the stimulation of 3H-TdR incorporation 6 to 12 h after the start of the treatment. At the tested concentrations (1 and 4 mM) theophylline depressed the 3H-TdR incorporation in CEF treated by PHD, but the same effect was obtained in CEF stimulated by fresh calf serum and also in CEF only maintained in serum-free medium (table 4). Effect on cell growth of several days’ treatment with PHD (fig. 2)

The following experiments were intended to find out whether several successive daily treatments with PHD could sustain a prolonged growth of CEF in serum-free medium. 2x l(r CEF and 1.0~ 108 CEF were respectively seeded in 3.5 cm dishes


Table 4. Effect of theophylline

on 3H-TdR incorporation into DNA of serum-deprived CEF treated with (1) serum-free medium; (2) medium plus serum; and (3) medium plus PHD Incorporation into DNA

of 3H-TdR

Additions to Eagle’s MEM culture medium

cpm/lOB cells

% inhibition by theophylline

None 1 mM theophylline 4 mM theophylline 4 % calf serum 4 % calf serum+ 1 mM theophylline 4 % calf serum+ 4 mM theophylline 0.008 U/ml PHD 0.008 U/ml PHD+ 1 mM theophylline 0.008 U/ml PHD+ 4 mM theophylline 0.032 U/ml PHD 0.032 U/ml PHD+ 1 mM theophylline 0.032 U/ml PHD+ 4 mM theophylline

19 950 10 387 1 945 89 537

48 90

33 733


5 054 31 995


17 666


3 350 54 445


24 308


4 954


CEF were incubated in serum-free medium for 24 h, after which time the cell count was 1.2x 10s tells/3.5 cm dish. The medium was then replaced with fresh serum-free medium containing the above indicated additions. Six hours later the cultures were labeled with 1 @/ml of 3H-TdR and after a further 6 h the amount of label incorporated into DNA was determined as in Methods.

with medium plus 4% serum, incubated overnight and then transferred for a period of 24 h in serum-free medium. PHD (0.032 U/ml) was then added to half of the dishes for each cell concentration, the other half being kept as controls. At intervals of 24 h, plates were refed with either fresh serumfree medium plus PHD, or with only serum-free medium (controls). Each day four dishes of PHD-treated and control cells were used for cell number determinations. In these conditions, PHD was able to sus-

and growth

of chick cells


tain an increase in cell number only during 2 or 3 days (fig. 2). At the lower cell concentration the number of cells almost doubled, from 2.3 x lo5 at the time of the first addition of PHD to 3.8x lo5 48 h later. On days 4 and 5, the cell count decreased, and the cell morphology returned to that of serumdeprived cells, in spite of repeated daily feeding with enzyme. For the higher cell density, the growth was maintained during 3 days, from 1.2~ lo6 cells to 3.0x lo6 cells; then, the number of cells declined slightly and their morphology returned to that of serum-deprived cells. The CAMP levels were also measured at one-day intervals in those cultures seeded at the initial density of 1~10~ cells. There was a significant drop of the CAMP level only on the day following the first additon of the enzyme. Thereafter this level remained constant, below that of control serum deprived cells, indicating that new additions of PHD could not induce a further decrease of CAMP (table 5). Moreover the cells treated daily for 3 days with PHD, and which failed to re-

Table 5. Effect of repeated daily treatments with PHD on CAMP levels of serumdeprived CEF pmoles CAMP per 1 x lo6 cells in Sampling time in days -1 0 : 3 4

Control cells

Cells treated with 0.032 U/ml PHD

1.2kO.16 1.2kO.11 1.4f0.09 2.2kO.21 1.7kO.42 1.8f0.10

0.98+0.07 1.2+0.05 1.2kO.14 1.2+0.04


CEF (1 x 1V ceW3.5 cm dish) were deprived of serum and 24 h later (day 0) the medium was replaced with fresh ‘serum-free medium (controls) or with this medium plus 0.032 U/ml PHD (treated cells). These same treatments were repeated daily for 3 days. Exprl


Res 95 (1975)



and Jullietl

Table 6. Eflect qf serum or PHD serum during 4 days

on cell number and CAMP content oj’ CEF depri\.rd

Culture conditions during first 4 days

Cells maintained in Eagle’s MEM Sampling time Immediately before changing culture conditions after 4 days without serum 1 day after following additions to Eagle’s MEM (a) none (b) PHD (c) serum


-~ Cells maintained in Eagle‘s MEM and treated with PHD during the last 3 days

Cell number per 3.5 cm dish

pmoles CAMP per 1x 10” cells

Cell number per 3.5 cm dish

pmoles cAMP per I x IO”cells

I .0x 10”

I .7f0.42

3.0x IO”


1.0x 106 I .0x 106 1.7x106

I .9&O. 10 1.8kO.08 1. I to.01

2.2x 106 2.6x I06 3.4x 106

1.2~0.04 I .O-tO.OS 0.75 kO.03

After CEF had been deprived of serum for 1 day, on each of the 3 following days the medium was replaced with fresh serum-free medium (controls) or with medium plus 0.032 U/ml PHD (treated cells). After 4 days without serum the medium changes indicated above were effected. Where added, PHD was at 0.032 U/ml and serum at 4%.

spond, at this time, by a further decrease in their CAMP content or by an increase in their number, were still able to do so when the medium was supplemented with 4 % calf serum. Also, control cells maintained for 4 days in a serum-free medium were not stimulated to divide, or to lower their CAMP content, by PHD treatment, but were thus stimulated by addition of serum (table 6).

DISCUSSION The above data show that after addition of PHD to CEF in a serum-free medium the following sequence of events is observed: firstly, a drop in cellular CAMP concentration; secondly, an increase in 3H-TdR incorporation in acid-insoluble material, together with a change in cell morphology; and finally an increase in the cell count, paralleled by an increase in the protein content per plate. Clearly the enzyme Exptl


Res 95 (1975)

preparation stimulates cell growth and division. The most reasonable and likely (but not the only) hypothesis is that PHD triggers DNA synthesis and cell multiplication by a drop in the cellular CAMP concentration. However, the verification of this hypothesis remains to be substantiated, and other explanations can be proposed. For instance the drop in cellular CAMP could be actualIy due to PHD, but the cell multiplication would result from unknown contaminant(s), such as a trypsin-like neutral protease, an insulin-like factor [18], or some other protein present in the commercially purified enzyme preparation; or PHD could stimulate cell multiplication, not through its enzymatic activity, but through some other property peculiar to it as a protein. The experiment performed in the hope of counteracting the effects of PHD with theophylline did not allow a clear-cut interpretation. Theophylline reduced, in like proportions, 3H-TdR incorporation into

3’S’-Phosphodiesterase CEF in serum-free medium, in medium with serum, and in medium supplemented with PHD. If one assumes that theophylline acts only as a PHD inhibitor and exerts no other effects on other cellular enzymes or structural macromolecules, one may develop the following argument: the CAMP level and its enzymatic control direct DNA synthesis in CEF in medium with serum as well as in serum-free medium, and therefore the inhibition by theophylline of cellular PHD or of PHD present in the medium would lead to a reduction of the rate of DNA synthesis. However, data exist which strongly suggest that theophylline, besides being a PHD inhibitor, is able to interfere with other cellular processes. (1) A concentration of 2.5 mM theophylline kills 50% (in terms of colony forming ability) of L cells [25]; (2) theophylline at high concentrations (10 to 20 mM) is also an inhibitor of DNA polymerase [26] and (3) theophylline is an inhibitor of the dark repair of UV-induced lesions [27, 281. Considering the present ignorance about the interactions of theophylline with enzymatic or structural molecules of cells, it appears unreasonable to choose between the following three hypotheses to explain these results: the theophylline-induced depression of DNA synthesis is (a) actually due to PHD inhibition; (b) it is due to some other mechanism(s); (c) it is due to a combination of (a) and (b). This being stated, it remains that PHD treatment stimulates cell multiplication, but this stimulation of CEF maintained in a serum-free medium is moderate and transitory. At 3 days after the beginning of the treatment, the cell response is no longer noticeable. Possibly a proliferative response to PHD-treatment requires a minimum number or amount of serumdependent factors, which progressively dis-

and growth of chick cells


appear, necessarily, during serum deprivation, thereby limiting PHD-mediated effects to the first 72 h following removal of serum. To conclude, our results afford strong arguments in favour of the concept that the changes in CAMP, adenyl-cyclase and 3’,5’-phosphodiesterase observed in vitro in fibroblasts are the cause rather than the consequence of the change in the proliferative state of cell populations. In a wider view it would be of interest to look for an inverse effect of PHD treatment on cells such as pluripotent hemopoietic stem cells and rat thymic lymphocytes, of which the multiplication is stimulated by the dibutyryl analog of CAMP [6, 71. While we admit the difficulty of revealing the actual mechanism responsible for our observations, they are not directly comparable to those [29] where mouse 3T3 cells were stimulated to grow by protease treatment, since these cells were contactinhibited and grown in the presence of serum. We thank Dr R. Latajet and Dr P. Vigier for their helpful advice during this work and in the preparation of the manuscript. This study was supported by contract number NOICP 43219 under the Special Virus Cancer Program of the NCL, NIH-PBS, and with an Action Thematique Concertee 64442236 de I’INSERM.


5. 6. 7. 8. 9. 10.

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Res 95 (1975)



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1313. Carchman, R A, Johnson, G S, Pastan, 1 & Scolnick, E M, Cell 1 (1974) 59. Sheppard, J R, Proc natl acad sci US 68 (1971) 1316. De Robertis, E, Rodriquez de Lores Arnaiz, G, Alberici, M, Butcher, R W & Sutherland, E W, J biol them 242 (1967) 3487. Butcher, R W & Sutherland, E W, J biol them 237 (1962) 1244. Kram, R, Mamont, P & Tomkins, G M, Proc natl acad sci US 70 (1973) 1432. Eagle, H, Science 122 (1955) 501. Temin, H M, Int j cancer 3 (1968) 771. Biquard, J M & Vigier, P, Virology 47 (1972) 444. Vigier, P & Bataillon, G, Virology 45 (1971) 309. Sefton, B M & Rubin, H, Proc natl acad sci US 68 (1971) 3154.


Res 95 (197.5)

22. Bray, G A, Anal biochem 1 (1960) 279. 23. Lowry, 0 H, Rosebrough, J N, Farr, A 1~ & Randall, R J, J biol them 193 (19.51) 265. 24. Brooker, G, Thomas Jr, L J & Aonleman, M M, Biochemistry 7 (1968) 4177. .. 25. Rauth, A M, Radiat res 3 1 (1967) I2 I. 26. Wragg, J B, Carr, J V & Ross, V C. J cell biol 35 (1967) 146A. 27. Lehmann, A R, Eur j biochem 3 1 (1972) 438. 28. Zajdela, F & Latarjet, R, Compt rend acad sci 277 D (1973) 1073. 29. Noonan, K D & Burger, M M, Exp cell res 80 (1973) 405. Received December 2, 1974 Revised version received February 17, 1975