Endogenous prostaglandin E2 synthesis inhibits growth of polyoma virus-transformed 3T3 fibroblasts

Printed in Sweden Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/79/13ooOl-~5$02.00/0

Experimental

ENDOGENOUS GROWTH

PROSTAGLANDIN

OF POLYOMA

124 (1979) l-5

Cell Research

E2 SYNTHESIS

VIRUS.-TRANSFORMED

INHIBITS

3T3 FIBROBLASTS

JAN AKE LINDGREN, HANS-ERIK CLAESSON and SVEN HAMMARSTRGM Department of Chemistry, Karolinska Institutet, S-10401 Stockholm, Sweden

SUMMARY Indomethacin lowered the cellular content of adenosine 3’: 5’-monophosphate (CAMP) and stimulated growth of polyoma virus-transformed 3T3 fibroblasts. Exogenous prostaglandin E2, at concentrations produced in the absence of inhibitor, reversed the effects of indomethacin on CAMP levels and cell proliferation. Therefore, endogenously produced prostaglandin E2 decreases cell growth and raises the levels of CAMP in these cells.

Polyoma virus-transformed 3T3 fibroblasts (py 3T3) produce high levels of prostaglandins compared to regular 3T3 cells [l]. Furthermore, the endogenously produced PGE, raises the levels of CAMP in these cells [2]. Since CAMP inhibits proliferation of many cells (e.g. [3-51) the effects of the endogenous PGE, production on the growth of py 3T3 cells have now been determined. Indomethacin, a potent inhibitor of prostaglandin biosynthesis, was used for these experiments. The results show that the drug stimulates growth and that the effect can be reversed by physiological concentrations of PGE,. It has previously been shown that indomethacin stimulates growth of HeLa, L-929 and HEP-2 cells [6].

MATERIALS

AND

METHODS

Materials Dulbecco’s modified Eagle’s medium, calf serum, trypsin solution (2.5 %) and penicillin-streptomycin solution (10 000 IU/me-10 mg/ml) were obtained from Gibco Bio-Cult Laboratories (Glasgow). Plastic Petri dishes were from Flow Laboratories (Irvine, UK);

cyclic [3H]AMP (37.7 Cilmmol) from New England Nuclear (Dreieichenhain. W. Germany); CAMP and indomethacin from Sigma Chemical Co. (St Louis, MO); prostaglandins Ez and F= from the Upjohn Company (Kalamazoo, Mich.) and AGl-X8 anion exchange resin from Bio-Rad Laboratories (Richmond, Calif.). [5,6,8,11,12,14,15-3H,]-labeled prostaglandins Fm and F2s were prepared using NaBHe reduction of [5,6,8,11,12,14,15-,H,]prostaglandin E, (178 Cilmmol; from New England Nuclear). Prostaglandin Fts was similarly prepared from PGE,.

Cells BALB/c 3T3 fibroblasts (3T3) and polyoma virustransformed BALB/c 3T3 tibroblasts (py 3T3) were originally provided by Dr M. M. Burger, University of Basel. The cells were maintained in culture as described before [2]. Experiments were carried out in Dulbecco’s modified Eagle’s medium containing 10 % (3T3) or 5% (py 3T3) calf serum. Cells were counted in a Coulter counter (Coulter Electronics Ltd., Harpenden, Hertfordshire) after removal from the dish by treatment with trypsin. Cell culture media and cells were harvested and treated as described [2].

Quantitative determinations of PGEz, CAMP and DNA PGEI concentrations in culture media were assayed by radioimmunoassay after NaBH, reduction to PGFm and PGFsp [7]. The cellular CAMP level was determined using the protein binding technique of Gilman [8]. Prior to assay the samples were purified as described [2]. Cellular DNA contents were assayed according to Burton [9].

2

Lindgren,

Claesson and Hammarstriim

Table 1. DNA content/culture

dish

Experimental procedures see legends to figs 1 and 2 Each DNA value is the mean of two determinations on six (controls) or four culture dishes. The increase in DNA content per group during 48 h was used as a measure of growth rate Days after planting

Cell line py 3T3

3T3

Addition None (control) IM (10 nM) IM (1 PM) IM (1 pM)+PGEz None (control) IM(lOOnM) IM (1 PM) IM (1 pM)+PGE,

Day 1

Day 3

DNA @g/dish

DNA @g/dish

Increase %

27.3f0.65 24.1k1.3 22.9k2.2 21.8k2.0 30.5kl.l 27.5kO.71 28.2k1.5 26.7k1.4

190 156 144 132 163 137 143 130

9.4

11.6

Day 5 Sign.

PI ‘(:I

FJ

/Lg/dish

Increase %

64.4k2.6 60.6k3.5 60.5k1.5 51.4f2.6 49.Ok1.2 48.7k1.2 47.4kO.94 48.7k1.4

136 151 164 136 61 77 68 82

Sign. n.s. n.s. (‘1 n.s. (“I n.s.

Statistical analyses were performed using Student’s r-test: u 0.05>p>O.O1. b O.Ol>p~O.OOl. c pjO.@l (differences in DNA contents @g/dish) between controls and different experimental groups). n.s., not significant.

RESULTS At a concentration of 1 PM, indomethacin totally inhibited PGEz production in py 3T3 and 3T3 cultures (figs 1 a, 2~). The levels of 1.2

CAMP were substantially reduced in indomethacin-treated py 3T3 cells (fig. lb), but unchanged in indomethacin-treated 3T3 cells (fig. 2b). A lower inhibitor concentra-

b

1

I. Abscissa: time (days after planting); ordinate: (a) PCE, (&ml); (b) CAMP (pmol/pg DNA). Py 3T3 cells were planted at a density of 3 200 cells/ cm2 on 90 mm culture dishes. Twenty-four hours later the medium was changed. One set of cultures received fresh medium without additions (control; O-O, a). To other sets of cultures were added fresh media containing 10 nM indomethacin (A-A, 0); 1 PM indomethacin (A-A, n ) or 1 mM indomethacin plus PGE, Fig.

Erp Cell Res I24 (1979)

(O-O, n ). Subsequently, the media were removed every second day throughout the experiments and replaced by fresh media with the same additions. To mimic the PGE, production in control cells, PGE, was added daily to the last set of cells (10, 75, 75, 25, 120, 30, li30, 85, 300 and 250 rig/ml on days l-10, respectively). Each point represents the mean value from duplicate (PGE,) or triplicate (CAMP) determinations on each of six (controls) or four culture dishes.

Inhibition

of cell growth by endogenous PGEz

Day 9

Day 7 DNA pg/dish

Increase %

97.3k3.5 96.0+_ 1.6 103.3k1.6 %3.2+ 1.8 60.5f0.97 6O.Ok1.9 60.0+0.74 63.3kO.83

51 58 71 72 23 23 27 30

Sign. ns. (9 Cb) ns. ns. (*I

3

Day 11

DNA pg/dish

Increase %

128.4k4.2 127.054.4 148.7k2.6 115.4k3.0 61.1kO.72 60.2kO.94 60.5kO.86 62.4+ 1.2

32 32 44 31 1 0 1 0

Sign. n.s. (9 (9 n.s. n.s.

n.s.

DNA pg/dish

Increase %

143.6f3.9 146.9f2.0 182.046.0 132.9f3.0 59.0fl.l 56.5k1.2 57.322.1 63.021.0

12 ;: 15 0 0 0 1

Sign. n.s. CC) (*I ns. ;;’

tion (10 nM) reduced the PGE, synthesis by 60-80% in py 3T3 cells (fig. 1 a) and the CAMP levels by 25-60% (fig. 1 b). Indomethacin (10 nM to 1 PM) retarded growth of both 3T3 and py 3T3 cells be-

tween days appreciable days 3 and growth rate tures treated

1 and 3 (table 1, fig. 3). No effect was observed between 5. Subsequently, an increased was observed in py 3T3 culwith 1 PM indomethacin. The

Fig. 2. Abscissa: time (days after planting); ordinate: (a) PGE, (&ml); (6) CAMP (pmol/pg DNA). 3T3 cells were planted at a density of 3 200 cells/cm2 on 90 mm culture dishes. Twenty-four hours later the medium was changed. One set of cultures received fresh medium without additions (control; O-O, a). To other sets of cultures were added fresh media containing 0. I PM indomethacin (A-A, IY), 1 PM indomethacin

(A-A, n ) or 1 &i’indomethacin plus 300 ng PGE,/ml (m-H, 0). Subsequently, the media were removed every second day throughout the experiment and replaced by fresh media with the same additions. Each point represents the mean value from duplicate (PGE,) or triplicate (CAMP) determinations on each of six (control) or four culture dishes. Exp Cell

Res 124 (1979)

4

Lindgren,

139

Cluesson and Hummarstriim 130-

a

b

Fig. 3. Abscissa:

OJ

(1 01

, 3

, 5

I,, 7

(days after planting); ordinate: DNA (% of control). (a) py 3T3; (b) 3T3. Experimental procedure, see legends to figs 1 and 2. Ten nM (py 3T3) or 0.1 PM (3T3) indomethacin (A-A); 1 PM indomethacin (A-A); 1 PM indomethatin plus PGE, (O-0). 9

11

01

3

5

cell number per culture dish on day 11 was 5.5&0.66x106 in control cultures and 8.6+ 0.71 X 106 in indomethacin-treated cultures. No stimulation of cell growth was observed in cultures treated with 10 nM indomethacin (fig. 3a, table 1). The prostaglandin endoperoxide synthase inhibitor had no effect on the growth of regular 3T3 fibroblasts (fig. 3b, table 1). PGEe was added daily to py 3T3 cells, grown in the presence of 1 PM indomethatin, to mimic the PGEB synthesis in untreated cells (fig. 1 a). This raised the CAMP levels (fig. 1b) and prevented the effect of indomethacin on cell growth (fig. 3a, table 1). Exogenous PGEz (300 rig/ml) did not influence CAMP levels or proliferation in regular 3T3 cells (fig. 3 b, table 1). DISCUSSION The present results show that cellular levels of CAMP increase in py 3T3 cells but remain constant in 3T3 fibroblasts as the cells become confluent (figs 1b, 2 b, table 1). It has previously been shown [2] that the increased CAMP levels in the transformed Exp Cell

Res 124 (1979)

i

4

1'1

cells are caused by elevated prostaglandin E, synthesis. This paper deals with effects of the elevated prostaglandin production in py 3T3 on the growth of these cells. Inhibition of prostaglandin synthesis by indomethacin decreased the content of CAMP in py 3T3 cells (fig. lb). At first (days l-3), the growth rate was decreased. Later, as proliferation diminished in untreated cells, stimulated growth was observed in indomethacin-treated cells (table 1, fig. 3 a). A biphasic effect of indomethatin on proliferation has also been reported for diploid human libroblasts [lo]. The decrease of CAMP levels and the stimulation of cell growth by indomethacin were completely reversed by PGEz (fig. lb, table 1). It is conceivable that the initial inhibitory effect was due to inhibition of PGF, synthesis since this prostaglandin appears to stimulate proliferation of certain cells [lo, 111. Incomplete (70-80%) inhibition of PGE, synthesis by 10 nM indomethacin decreased the CAMP levels in py 3T3 cells by 25-60 % but did not stimulate growth. This suggests that the CAMP levels have to be further

Inhibition lowered before stimulation of proliferation occurs. PGE, does not increase CAMP synthesis in the regular 3T3 fibroblasts ([ 121, fig. 2 b). Since E-type prostaglandins may inhibit nutrient transport in animals cells [ 13, 141 the effects on growth of exogenous PGEz in 3T3 cultures were determined (fig. 36). No decrease was observed. This supports the concept that PGE, inhibits growth of py 3T3 cells by raising the levels of CAMP. Indomethacin did not stimulate growth of 3T3 cells suggesting that their (low) prostaglandin production does not restrict growth (fig. 2a, 3b). The results presented show that endogenous prostaglandin E2 synthesis by polyoma virus-transformed 3T3 fibroblasts decreases the growth rate of these cells in vitro. Py 3T3 cells can also grow as tumors in mice. It is possible that under such conditions PGE, production by these cells has other and/or additional effects, e.g. inhibition of lymphocyte cytotoxicity [ 151. Therefore, the effects of endogenous prostaglandin production on tumor growth in vivo require further investigation.

of cell growth by endogenous PGEz

5

We thank Margareta Hovgard and Kerstin Johansson for expert technical assistance. This work was supported by a grant from the Swedish Cancer Society (1503-B80-01X) and the Swedish Medical Research Council (03X-217).

REFERENCES 1. Hammarstrom, S, Eurj biochem 74 (1977) 7. 2. Claesson, H-E, Lindgren, J A & Hammarstrom, S, Em j biochem 74 (1977) 13. 3. Rvan. W L & Heidrick. M L. Science 162 (1968) 1484: 4. Sheppard, J R, Proc natl acad sci US 68 (1971) 1316. 5. Otten, J, Johnson, G S & Pastan, I, J biol them 247 (1972) 7082. 6. Thomas, D R, Philpott, G W & Jaffe, B M, Exp cell res 84 (1974) 40. 7. Lindgren, J A, Kindahl, H & Hammarstrom, S, FEBS lett 48 (1974) 22. 8. Gilman, A G, Proc natl acad sci US 67 (1970) 305. 9. Burton, K, Anal biochem 59 (1956) 63. 10. Taylor, L & Polgar, P, FEBS lett 79 (1977) 69. 11. De Asua, L J, Clingan, D & Rudland, P S, Proc natl acad sci US 72 (1975) 2724. 12. Claesson, H-E, Lindgren, J A & Hammarstrom, S, FEBS lett 81 (1977) 415. 13. Sheppard, J R & Plagemann, P G W, J cell physiol 85 (1975) 163. 14. Polgar, P & Taylor, L, Biochem j 162 (1977) 1. 15. Droller, H J, Schneider, M U & Perlmann, P, Cell immunol39 (1978) 165. Received December 22, 1978 Revised version received February 19, 1979 Accepted June 6, 1979

Exp CellRes

124 (1979)