Effects of cell density and cell growth alterations on cyclic nucleotide levels in cultured human diploid fibroblasts

Printed 3” Sweden Copyright @ 1978 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-48?7/78/1141-0101$0?.00/0

Experimental Cell Research 114 (1978) 101-l 10

EFFECTS

OF CELL ON CYCLIC

DENSITY

AND CELL

NUCLEOTIDE

HUMAN

DIPLOID

LEVELS

GROWTH

ALTERATIONS

IN CULTURED

FIBROBLASTS

HO SAM AHN,’ SARA G. HOROWITZ,’ HARRY EAGLE* and MAYNARD ‘Departments

H. MAKMAN’

of Biochemistry and Molecular Pharmacology, and 2Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA

SUMMARY cGMP and CAMP concentrations were studied in cultures of two strains of normal human diploid lung tibroblasts, WI38 and KL-2, under various conditions which alter growth rate. Higher levels of CAMP were found in fibroblasts grown in medium with low (0. l-l .O%) serum concentration and thus exhibiting a decreased rate of growth. A rise in CAMP also preceded the decreased growth rate when medium was not changed for 4 days or longer (starvation). The reinitiation of cell growth by addition of fresh medium containing the standard 10% serum to either starved or serumrestricted cells was preceded by a rapid drop in CAMP level. Cellular CAMP levels increased to a moderate extent as sparse cultures first increased in density, but did not continue to rise as the culture approached saturation density. cGMP levels were inversely related to cell density: much higher cellular cGMP levels were found at low density than at higher cell density, whether cells were rapidly proliferating under standard growth conditions or had their growth arrested by omission of medium change or restriction of serum. Thus, under these conditions the steady state levels of cGMP appear to be related to cell density rather than rate of cell proliferation. However, a transient but appreciable increase in cGMP did occur upon the addition of fresh medium containing 10% serum to starved or serum-restricted cells, a condition leading to reinitiation of cell proliferation. Smaller but significant increases in cGMP were also evident following routine addition of fresh medium with serum to growing cells fed every other day and following mild EDTA-trypsin treatment of confluent WI38 fibroblasts. Thus, at least dual control mechanisms appear to be involved in the regulation of cGMP levels. Comparison of mid- and late-passage-WI38 cells revealed no significant differences either in the levels of cGMP at snarse densities or in the density-dependent change in levels. These results suggest that levels of both CAMP and cGMP are influenced by cell density and also by conditions which alter the rate of cell proliferation.

Although the basic mechanisms by which proliferation of non-transformed mammalian fibroblasts in culture is regulated are poorly understood much information has accumulated concerning influences on their growth by a variety of environmental factors [I, 21 such as serum [3, 41, cell-cell contact [S], nutrients [6], pH of medium [7, 81 and hormones or growth-stimulating agents [9-181.

Studies with mouse embryo fibroblasts and human lymphocytes have indicated possible opposing influences of adenosine 3’,5’-cyclic monophosphate (CAMP) and guanosine 3’,5’-cyclic monophosphate (cGMP) on the regulation of cell proliferation [19-211. Thus, the induction of fibroblast proliferation by growth-promoting agents such as insulin, serum proteases, and fibroblast growth factor (FGF) is asExp

Cd

Res

114 (1978)

102

Ahn et al.

sociated with early decreases in intracellular CAMP, and in some instances by increases in cGMP [13, 19, 22-321. On the other hand, the addition of CAMP or its analogues to cultures of libroblasts and lymphocytes counteracted the growthstimulating effects of cGMP and other agents [12, 26, 27, 33, 341. A transient increase (lo-fold) in cGMP and decrease in CAMP (Zfold) was observed in growth-arrested 3T3 fibroblasts upon the restoration of certain amino acids to a medium deficient in these amino acids [28, 351. For at least some types of fibroblasts, the basal CAMP levels are higher in slowly growing than in more rapidly growing cells. Levels of CAMP were found to be higher and levels of cGMP lower in fibroblasts at confluency than at sparse densities [22, 23, 35-381. However, in other studies no cGMP elevation was detected following the addition of growth-promoting agents to human peripheral lymphocytes [39], mouse 3T3 fibroblasts [40], and chick embryo fibroblasts [4 11. In addition to the fact that the findings reported in some of the above mentioned studies are contradictory, their relevance to human diploid fibroblasts is complicated by the fact that these cells exhibit a finite life span in culture [42]. Consistent with results with other cells the addition of fresh serum to WI38 human diploid fibroblasts decreased cellular CAMP levels, while changing the culture medium to a salt solution resulted in increased cellular CAMP levels [43]. However, there are also conflicting reports concerning the responsiveness of human fibroblasts to the inhibitory action of CAMP and CAMP-elevating agents, as well as the occurrence and nature of density-dependent changes in CAMP levels in these cells [22, 26, 32, 36, 43-481. Further, there is no reported study of

cGMP in relation to cell proliferation in normal diploid fibroblasts. The present study was initiated to delineate the CAMP and cGMP levels in relation to cell density and/ or growth rate in two strains of normal human diploid lung fibroblasts (WI38 and KL-2) under various culture conditions. cGMP levels of mid- and late-passage WI38 Iibroblasts were also determined, and the influence of EDTA-trypsin treatment on cellular cGMP levels assessed. A preliminary report of these studies has been presented [49].

MATERIALS

AND METHODS

Cell cultures Two strains of human diploid fibroblasts derived from human embryonic lung, WI38 (obtained from Dr L. Hayflick) at passage 20 to 30 unless otherwise mentioned, and KL-2 [8] were used. WI38 cells were grown in Eagle’s basal medium for diploid cells with Earle’s balanced salts containing 25 mM Hepes (N2-hydroxy-ethylpiperazine-N’-2-ethanesulfonic acid) buffer (pH 7.4), 100 NJ/ml of penicillin and 100 pg/ ml of streptomycin, supplemented with 10% fetal calf serum (FCS) (Flow Laboratories). KL-2 cells were grown in the same minimum essential medium supplemented with 5% calf serum and 5% FCS. KL-2 cells were fed every day and WI38 cells every other day. The cells were grown as a monolayer and maintained at 37°C in a humidified 95 % air, 5 % CO1 atmosphere. Cells in stock cultures were subcultured by treatment with EDTA-trypsin solution (Gibco; 0.5 g trypsin and 0.2 e EDTA ner liter of isotonic saline). The cells were monitore’d periodically for the absence of mycoplasma.

Experimental

procedures

Cells were plated into 100 mm Falcon plastic dishes (approx. 2-3 x lo6 WI38 cells) or into T-75 Falcon plastic flasks (approx. 34~ lo6 KL-2 cells) containing the complete medium and harvested at different intervals after subculture. In serum restriction and starvation exneriments cells were mated and initiallv cultured in the complete medium ior at least 1-2 days prior to modification of the culture conditions. Triplicate cultures were used for each experimental point. Medium was aspirated and the cells were quickly washed once with isotonic saline at 5°C. This washing did not significantly alter the results. The cells were scraped with a rubber policeman in 1.5 ml 10% trichloroacetic acid (TCA).

Human jibroblasts

Table 1. Effect of varying concentration and cyclic nucleotide level

of serum on WI38 and KL-2 cell proliferation Cells transferred on day 3 from medium with 10% serum to medium with 0.1 (for W138) or 0.2 %(forKL-2)serum

Cells grown in IO% Serum

Cells” WI38 Day 4 Day 6 KL-2 Day 5 Day 6

103

cyclic nucleotides

I % Serum

Protein cGMP (pmoles/mg bd cm*) protein)

CAMP (pmoles/mg protein)

10.7 15.7

0.52+0.04 0.3420.02

11.5t3.0 11.3+1.3

4.0 9.2

1.25+_0.05 18.1+2.1 0.55+_0.07 l5.3k2.5

2.8 3.5

1.87kO.31 10.6+6.0 2.48kO.35 22.5k4.4

24.0 32.0

0.42&O. I5 0.05+0.01

3.9k2.6 9.2f0.90

12.1 16.0

1.6kO.43 9.3k2.2 0.90*0.40 22.Ok2.1

6.1 9.2

4.Okl.5 2.1f0.18

Protein cGMP

CAMP

Protein cGMP

CAMP

16.O+_l.l 15.6k6.7

a After subculture in complete medium for 3 days, cells were incubated in medium containing varying concentrations of serum and harvested on designated day. Each value represents the mean + SD. for 6 determinations. For further details see Materials and Methods.

Cyclic nucleotide

assays

[3H]~AMP and [3H]cGMP were added to monitor recovery of intracellular cyclic nucleotides after each purification step. Final recovery was approx. 70% for both CAMP and cGMP. TCA precipitates were removed by centrifugation and TCA supematant solution was washed three times with ethyl ether to remove TCA. After removal of residual ether by a stream of N2, the washed supemate was applied to prewashed AC I X8 (formate) column (0.7X5 cm). The CAMP was eluted with 8 ml of 1 N HCOOH, and cGMP with 4 N HCOOH, and the lyophilized residue was taken up either in 1 ml distilled H,O for CAMP protein binding assay, or in 0.5 to 1.0 ml of 50 mM sodium acetate buffer pH 6.2 for CAMP and cGMP radioimmunoassays [50]. Similar results were obtained either by the CAMP protein binding assay or the CAMP radioimmunoassay. The reconstituted final samples were acetylated prior to cGMP radioimmunoassay [51], increasing the sensitivity of the procedure about 5-fold. Duplicate determinations were made for each purified sample.

RESULTS Cyclic nucleotide concentrations and cell densities

Cell cultures were grown in the complete medium with frequent changes of medium to prevent possible changes in cyclic nucleotide concentrations due to serum depletion. Under the culture conditions, the WI38 and KL-2 fibroblasts exhibited den-

sity dependent inhibition of growth; the WI38 cells reached saturation densities of 2.6 to 2.9 mg cell protein per 100 mm dish (60 cm* surface area) whereas the KL-2 attained much higher cell densities of 11-13 mg cell protein per T-75 flask (75 cm* surface area). The growth rate of WI38 and KL-2 as measured by cell protein increase per culture vessel per day did not slow down until cultures approached terminal densities. The cellular CAMP concentration of WI38 cells was 14+2 pmoles CAMP per mg protein at a sparse density (approx. 0.8 mg protein per 100 mm dish) and increased to 24+_3 pmoles CAMP per mg protein at a density of initial cell contact (approx. 1.5 mg per 100 mm dish or about one-half the maximum density) with no further significant change as maximum density was approached. The KL-2 cells showed a similar pattern of limited density-dependent CAMP change; CAMP level rose from 6 pmoles to 10.5 pmoles per mg protein as cell density increased from a sparse (1.2 mg protein per T-75 flask) to a density of initial cell contact (2.6 mg protein per T-75 flask) with no furExp Cell Res I14 (1978)

104

Ahn et al.

1.6

- 25 - 20

3.4

-

I5

- IO

Fig. 2. Abscissa: )2

-5

1.5

- 12

b

- IO 1.0

- 6 -6

I.5

-4 -2 i

I

2

3

4

5

6

7

cell density (a) mg protein/60 cm*; (6) mg protein/75 cmZ; ordinate: pmoles cGMP/mg protein. Inverse relationship between intracellular cGMP levels and density of WI38 and KL-2 fibroblasts. (a) WI38 fibroblasts; (b) KL-2 fibroblasts. Data are pooled from at least 10 separate experiments involving cells grown under various conditions (standard medium, serum-depleted medium, omission of daily medium changes).

6

Fig. 1. Abscissa:

(a, b) time in culture (days); ordinate: (a) (left) mg protein/l00 mm dish (O-O); (b) (lefr) mg protein/T75 flask (O-O); (a, b) (right) pmoles cGMP/mg protein (O-O); (a. b) Cfurther righf) pmoles cAMP/mg protein (A-A). Effect of cell density on intracellular CAMP and cGMP levels in WI38 and KL-2 fibroblasts. (a) WI38 tibroblasts; (6) KL-2 tibroblasts. The WI38 and KL-2 cells used were passages 28 and 5, respectively.

ther increase in CAMP levels at maximum density. Representative data for density dependent changes of CAMP levels in WI38 and KL-2 cells are shown in fig. 1. In contrast to the results obtained for CAMP levels intracellular cGMP levels were found to change markedly in both WI38 and KL-2 cells (figs 1, 2). cGMP levels were initially high at low cell density and decreased steadily as cell density increased. At confluency, cGMP levels were less than one-third (WI38 fibroblasts) or one twelfth (KL-2 fibroblasts) those found in cells at low density with a striking inverse

relationship between cGMP concentration and cell density. Furthermore, this inverse relationship was observed not only in fibroblasts grown in complete medium with frequent change of medium, but also in libroblasts cultured under various conditions such as restricted serum, omission of medium change, etc. (see the following section and also table 1 and figs 2, 3). Cellular cyclic nucleotide concentrations in relation to concentrations of serum in medium With both WI38 and KL-2 the amount of cell growth correlated with the concentration of serum in the medium (table 1). Higher levels of cGMP and in most instances also of CAMP were noted at low serum concentrations (1.0, 0.2 or 0.1%). Whether at low or high serum concentration, cGMP levels decreased with increasing cell density. When serum concentration was changed in midstream 24 h before final harvest, both cell protein and cGMP level were intermediate between those of cells grown throughout at the initial and those at the final serum concentrations (table 2). A

Human fibroblasts

Table 2. Effect of changing serum concentration Serum concentration during culture days 2-4 Serum concentration during final 24 h Protein @g/cm’) cGMP (pmoleslmg protein) CAMP (pmoles/mg protein)

10 10 24.0 0.65 3.9

on cyclic nucleotide 1 1 12.1 1.6 9.3

0.2 0.2 6.1 4.0 16.0

105

cyclic nucleotides

1 10 23.3 0.70 4.9

levels of KL-2 cells

0.2 10 16.7 1.08 5.4

10 1 17.3 1.05 11.0

10 0.2 13.9 1.70 7.2

Cells were initially plated and allowed to grow in medium with 10% serum for 24 h. This medium was then replaced with fresh medium containing the indicated concentrations of serum for 48 h, followed by a final medium change as indicated 24 h before cell harvest for determination of cyclic nucleotide and protein levels. The standard errors are within 12% of the experimental mean values.

shift-up of serum concentration caused increases in cell protein and decreases in both CAMP and cGMP levels, whereas a shiftdown led to reduction in cell protein and elevation in both CAMP and cGMP levels 24 h later. The alteration in serum concentration had a rapid and marked effect on the growth rate of KL-2 cells.

major difference in cell protein per culture dish (fig. 3). In both fed and starved cells the cellular cGMP levels diminished as cell density increased, but were somewhat lower in the starved cultures than in fed cultures at the same density; cGMP levels were estimated to be about one-half the levels of fed cultures of the same density based on data of figs 2 and 3.

Effect of starvation on cyclic nucleotide concentrations

KL-2 cells were first plated and cultured in complete medium with daily medium change. Beginning day 3, daily medium change was discontinued in some of the cultures in order to investigate relationship between the gradual growth halt induced by depletion of nutrients and the cell cyclic nucleotide levels. The starved cells continued to grow at a rate similar to that of daily-fed cells up to day 6 (4 days after the last medium change). Thereafter, their growth rate was greatly decreased, and they reached a plateau in cell density about one half the final density of daily-fed cells between day 7 and 9 (fig. 3). The daily-fed cells continued to grow rapidly, reaching their terminal density between day 9 and day Il. The CAMP levels in the starved cells increased by day 6 to values about twice those of the fed cells, even though as yet starved and fed cells showed no

50 40 30 20 IO

c

d

20 16 I2 646

9

II

nnn, 6

7

9

Fig. 3. Abscissa: time in culture (days); ordinate: (a) pmoles cAMP/mg protein; (b) pmoles cGMP/mg protein; (c, d) mg protein/T75 flask. Effect of omission of daily medium change on KL-2 cell proliferation and cyclic nucleotide levels. (a. c) Daily fed cells; (b, d) starved cells. Slashed bars, pmoles cAMP/mg protein; dotted bars, pmoles cGMP/ mg protein. The first group of cells (designated as fed cells) received the standard medium change every day; the second group (designated as starved cells) received a medium change only 24 h after seeding. Em Cell Res 114 f 197P’

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Ahn et al.

increase detected within the first 24 h (fig. 4). No such changes were found in continuously fed or starved cultures. When WI38 cells grown in 0.2% serum were refed with fresh medium containing 0.2% serum, cells showed no significant change in cGMP levels. However, when cells were changed to a medium containing 10% serum, a j-fold increase in cGMP levels occurred within 2 min (table 3). Within 5 min cGMP levels then declined to a level which was about twice that prior to Fig. 4. Abscissa: time after refeeding (hours); ordithe medium change and which was mainnate: (a) (left) pmoles cAMP/mg protein (A-A or A---A); (b) (left) mg protein/T75 flask; (a) (right) tained up to 5 h (table 3). A similar but pmoles cGMP/mg protein (0-U or q -- -0). Effect of refeeding on cell protein and cyclic nu- slower increase in cGMP was noted in cells cleotide levels in starved KL-2 fibroblasts. -, Fed grown in medium containing 10% serum cells; ---, starved cells. All cells were kept without further medium change from the second day after after refeeding with fresh medium (conseeding until initiation of the experiment 4 days later. taining 10% serum); no detectable increase Starved cultures represent those still without medium change and fed cultures those refed with fresh medium in cGMP level was noted at 2 min but a containing serum at zero time. Initial values are those 5-fold increase was evident by 1 h and obtained at zero time with subsequent values plotted thereafter the cGMP level began to decline on the abscissa using an exponential scale. (table 3). The acute effect of EDTA-trypsin treatment on cGMP levels was also examined. Acute changes in cyclic nucleotide Confluent WI38 (passage 28) cells were concentrations following addition of treated with EDTA-trypsin at concentrafresh medium or EDTA-trypsin to tions used for subcultivation (see Materials cultures grown under various conditions and Methods) for 5 min. With this time of A time course study was carried out in exposure cell rounding but no cell detachorder to investigate kinetics of cyclic nu- ment occurred. This treatment resulted in cleotide changes in response to growth in- an increase in cGMP levels from 0.031+ duction after refeeding of starved KL-2 cells. Upon addition of the complete medium to 4 day starved KL-2 cells, the previously elevated CAMP level declined significantly within 1 h and maintained this diminished level over a 48 h period (fig. 4). On the other hand, the GMP levels went up markedly within 1 h after the feeding continued to rise for 24 h, and thereafter began Fig. 5. Abscissa: cell density (mg protein/60 cm’); to decline to levels which were somewhat ordinate: pmoles cGMP/mg protein. Inverse relationship between cell density and cGMP higher than those of starved non-refed culconcentration in both mid-passage and late-passage tures (fig. 4). After refeeding, cell protein WI38 tibroblasts. O-O, Passage 28 cells; CO, increased over this 48 h period, with the passage 54 cells. Exp Cell RESI14 (1978)

Human fibroblasts

Table 3. Acute effect of changing

serum concentration on cGMP levels in WI38 fibroblasts, expressed as pmoles cGMPlmg protein Time after final medium change Final medium change

0 min”

2 min

Ih

5h

IO%+ 10% 0.2%-+0.2% 0.2%+ 10%

1.7 9.5 9.5

1.7 8.9 50.4

9.4 10.0 17.9

5.6 9.4 16.1

One day after subcultivation in medium with 10% serum, WI38 fibroblasts (passage 31) were incubated for 2 days in medium containing either 0.2% or 10% serum. The cells (sparse cultures) were harvested at various times after a final medium change as indicated (e.g. 0.2%+ 10%: cells incubated in medium containing 0.2% serum were switched to medium containing 1% serum). The standard errors are within 15% of the experimental mean values. a Before final medium change.

cyclic nucleotides

107

of the density-dependent decrease in cGMP level between mid and late passages. No significant differences in cGMP levels were found between passage 28 cells and passage 54 cells (fig. 5); and both cultures showed similar inverse relationships between cGMP level and cell density (fig. 5). Essentially similar results were obtained when passage 27 and passage 51 cells were compared (results not shown). DISCUSSION

The elevation of CAMP levels in human libroblasts upon initial cell-cell contact, with no further elevation at saturation density, as shown here is in an essential agreement with a report on 3T3 fibroblasts [54]. Our results do not support a mediation by 0.01 to 0.18+0.02 pmoles/mg protein CAMP of inhibition of growth at saturation (a mean + S.E.M. for triplicate samples), density in WI38 and KL-2 human diploid tibroblasts. Also, in the majority of studies about a 6-fold increase. of other cultured cells there appeared to be cGMP levels in mid- and late-passage no significant increase in CAMP levels at WI38Jibroblasts confluency [22-24, 35-37, 43, 44, 55, 561. The human lung fibroblast WI38 has been While some results have differed from this extensively used for study of cell aging finding this may at least in part stem from in vitro since it has limited capacity for differences in the cell type studied or in doubling and maintains normal cell function the manner of handling the cells. For exand diploid karyotype throughout most of ample, lack of medium change leading to its life span [42]. Although there are sev- depletion of serum factors in the confluent eral reports [43, 44, 521 indicating changes cultures might itself have been responsible in CAMP levels or in hormonal stimulation for elevation of CAMP levels in some inof levels with cell senescence, no report stances [23, 24,27,57]. In studies of adenyexists to our knowledge on the relationship late cyclase activity of cell homogenates, between cell aging and the cGMP system. with increasing cell density, increases in Since intracellular cGMP levels are corre- basal as well as PGE1-, epinephrine- and lated both with cell densities in stabilized NaF-stimulated activities were reported by cultures, and with the rate of cell proliferathis laboratory for several cell lines [37, tion in WI38 cells when growth is re-initi58-601 including human lung fibroblasts ated, and since the cell growth rate de- [61]. The magnitude of the increase in basal creases with increasing passage numbers adenylate cyclase activity appreciably ex[53], an attempt was made to detect any ceeded that reported here for CAMP levels difference in cGMP level or in the pattern in the intact human fibroblasts. However, Exp Cd Rr\ / 14 I lY78~

108

Ahn et al.

in these cells CAMP phosphodiesterase activity also increases with cell density [62] and therefore one would not expect the results for intracellular CAMP levels to parallel those for adenylate cyclase activity. In contrast to CAMP, cGMP levels were closely and inversely correlated with cell density in both WI38 and KL-2 fibroblasts (figs 1, 2). Similar changes in cGMP levels have also been reported by our laboratory for cultures of rabbit lens epithelial cells [63]. Less dramatic changes but in the same direction were found by others for mouse 3T3 fibroblasts [30]. Since in the present study cGMP levels decreased much more markedly with increasing cell density than did the growth rate (fig. l), and since levels remained elevated in cells with growth arrest at low cell density by serum restriction, it appears that cGMP levels are not necessarily related directly to growth rate per se. These results do not rule out the possibility that decreased cGMP concentration might be required for density-dependent inhibition of growth [6, 301. Also, as found in the present study and discussed later, certain conditions which re-initiated cell growth do lead to large albeit transient increases in cGMP levels. Examination of the effect of EDTA-trypsin on cGMP levels in an effort to find out the possible source and origin of high levels of cGMP at sparse cell densities revealed an appreciable acute stimulatory effect of EDTA-trypsin on cGMP level in confluent KL-2 cells. Even after EDTA-trypsin treatment, however, the absolute level of cellular cGMP was lower than that found in cells at sparse densities. In addition, the elevated cGMP levels in human fibroblasts at low density represent a stable change that can be maintained for at least several days by restricting growth of well attached and proliferating cells by serum restriction. E.rp Cell

Res

I14 (19781

Unlike mouse 3T3 fibroblasts [lo] and chick embryo fibroblasts [64], confluent human diploid skin and lung fibroblasts are relatively insensitive to the growth stimulatory effect of proteolytic enzymes (e.g., trypsin, pronase, etc.) [7, 651. Hence, the physiological significance of the rapid elevation of cGMP levels of confluent cultures by trypsin is not clear. A similar increase in cGMP levels has been reported to occur when confluent chick embryo fibroblasts are released from contact inhibition by trypsin [41]. The higher levels of cGMP reported here in both WI38 and KL-2 diploid fibroblasts after serum restriction are in marked contrast with a recent report [30] showing greatly reduced cGMP levels in 3T3 Iibroblasts but are in agreement with the findings for 3T3 cells of Miller et al. [40]. The higher levels of cGMP in the serum-restricted cells in this study appear to be due to lower densities of the serum-restricted cells since control and serum restricted cells at similar densities (table 1) did not show any significant difference in cGMP levels. This points out the importance of taking into consideration any density difference between control and experimental cells when comparing cGMP levels. In the present study, refeeding of starved KL-2 fibroblasts caused a rapid increase in cellular cGMP as well as a decrease in CAMP levels. The addition of medium containing 0.2% serum to cells grown in that medium had no effect, but the addition of medium containing 10% serum resulted in striking elevation of cGMP levels, indicating that serum was reponsible for the observed rise. The rapid and dramatic increase in cGMP levels observed in the serum-restricted or the starved human fibroblasts after addition of serum (table 2 and fig. 4) is in agreement with most previous

Human fibroblasts

studies of mouse 3T3 fibroblasts and other cell types [19, 28-30, 32, 351 although Miller et al. [40] reported a drop in cGMP levels in 3T3 fibroblasts after addition of serum. It has been reported using 3T3 cells that increases in cGMP occur upon reinitiation of proliferation of cells in GOphase or when synchronized cells pass through the Gl phase of the cell cycle, but do not occur following reactivation of randomly arrested or exponentially growing cells [28, 351. Since WI38 cells are reversibly arrested at the Gl (or GO) phase by deprivation of serum [26, 661, it may be that the rapid and transient increases in cGMP levels due to addition of serum to these cells serves in some as yet unknown manner as a signal for further progression of this diploid cell through the cell cycle. These studies, together with those indicating an influence of cell density on cGMP levels discussed earlier indicate that at least dual control mechanisms are involved in the regulation of cGMP levels in human tibroblasts. This work was supported by NIH Grants 2POl AGOO374,5ROl CA 13176and P30-CA 13330-06.

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Received September 19, 1977 Revised version received December 30, 1977 Accepted January 16, 1978