Changes in polyamine metabolism in WI38 cells stimulated to proliferate

Printed in Sweden Copyright 0 1975 by Acadmnic Press, Inc. All rights gf reproduction in any form reserved

Experimental Cell Research 90 (1975) 8-14

CHANGES

IN POLYAMINE STIMULATED

0. HEBY,r*

L. J. MARTON,

METABOLISM

IN

WI38

CELLS

TO PROLIFERATE

I** L. ZARDL2

D. H. RUSSELLI***

and R. BASERGA2

‘Laboratory of Pharmacology, Baltimore Cancer Research Center, National Cancer Institute, NIH, Baltimore, Md 21211, and 2Department of Pathology and Fels Research Institute, Temple University School of Medicine, Philadelphia, Pa 19140, USA

SUMMARY Quiescent confluent monolayers of WI38 human diploid fibroblasts were stimulated to proliferate by replacement of the exhausted medium with fresh medium containing 10 % fetal calf serum. The cellular content of the polyamines, putrescine, spermidine, and spermine was studied at various intervals after the nutritional change. The putrescine content increased during the pre-replicative phase of the cell cycle, whereas the content of spermidine and spermine did not increase until after the initiation of DNA synthesis. By varying the composition of the stimulating medium it was possible to alter the percentage of cells that were stimulated to proliferate. Measurement of the cellular polyamine content and 3H-thymidine (3H-TdR) incorporation into DNA at the time of the maximal rate of DNA synthesis showed that the magnitude of putrescine accumulation depended on the percentage of cells that were stimulated to proliferate. These results indicate that there may be a connection between polyamine synthesis and subsequent DNA replication.

The rate of polyamine biosynthesis has been shown to increase markedly in several models of stimulated growth both in vitro and in vivo (for references see Heby et al. [l]) resulting in accumulation of putrescine, spermidine and spermine. In many of those models of stimulated growth, e.g. phytohemagglutinin (PHA)- and concanavalin A (con A)-stimulated lymphocytes, estrogen-stimulated uterus, regenerating liver, etc., the changes in polyamine metabolism may be re* Reprint requests: Dr 0. Heby, Naffziger Laboratories for Neurosurgical Research, Department of Neurological Surgery, University of California Medical Center, San Francisco, Calif. 94143, USA. ** Present address: Naffziger Laboratories for Neurosurgical Research, Department of Neurological Surgery, University of California Medical Center, San Francisco, Calif. 94143, USA. * * * Present address: Department of Pharmacology, Arizona Medical Center, University of Arizona, Tucson, Ariz. 85721, USA. Exptl Cell Res 90 (1975)

lated to cellular hypertrophy as well as preparation for DNA synthesis and mitosis. The present investigation was designed (1) to provide information about the cellular polyamine content in a model of stimulated growth in which the changesobserved cannot be related to tissue hypertrophy, but more directly to DNA synthesis and cell division; (2) to determine whether there is a positive quantitative correlation between increasing cellular polyamine content and increasing fraction of cells stimulated to proliferate from a stationary phase. Quiescent confluent monolayers of WI38 human diploid fibroblasts were stimulated to initiate DNA synthesis and cell division by changing the medium [2]. The cellular content of putrescine, spermidine, and spermine were determined at various intervals after stimula-

Polyamines in cellular proliferation tion. By varying the composition of the stimulating medium, the degreeof stimulation, i.e. the percentage of cells that were stimulated to proliferate, was altered [3]. The possible correlation between cellular polyamine content and degree of stimulation was examined by measuring polyamine content and 3HTdR incorporation at the time of the maximal rate of DNA synthesis.

MATERIALS

AND METHODS

Cell culture WI38 human diploid fibroblasts, purchased from Flow Laboratories (Rockville, Md), were grown in roller vessels (Bellco Glass, Inc., Vineland, N.J.) in Basal Medium Eagle supplemented with 10 % fetal calf serum (Flow Labs), streptomycin (50 !/g/ml), and 2 mM glutamine. Between the 24th and 28th generations, the cells were transferred for the actual experiments to plastic Falcon flasks (surface of 75 cm%; 250 ml) containing 25 ml of medium. The culture medium was changed 24 h after plating and the experiments were performed 7 days later when the monolayers were confluent and when a Falcon flask contained 2 x 10” cells (variation i 10 %). At 7 days after plating, confluent monolayers of WI38 cells show a very low rate of DNA synthesis and mitosis, with 0.1-0.5 % of the cells being labeled by a 20-25 h exposure to 3H-TdR [4]. When WI38 cells had reached confluence they were stimulated to proliferate by replacing the conditioned medium with fresh medium containing 10 % fetal calf serum. Under these conditions 60-80% of the quiescent cells were stimulated to synthesize DNA and divide. In some experiments 10 % fetal calf serum was added to the conditioned medium or the conditioned medium was replaced with fresh medium containing only 0.3 % fetal calf serum. Under these latter conditions 30-40 % and O-5 %, respectively, of the quiescent cells were stimulated to proliferate. Following stimulation, the individual cultures were arrested at the indicated times (figs 1-3, table 1) by washing the cells twice with 10 ml of ice-cold phosphate-buffered saline [5]. The cells were subsequently harvested from the culture flasks in 10 ml of phosphate-buffered saline by scraping with a rubber policeman and sedimented in a refrigerated International centrifuge at 2 000 g at 0°C for 5 min. The cells were kept frozen until analysis.

Preparation of cell extracts and polyamine analysis Cellular HCIO, 1 000 g by the

pellets were sonicated in 400 /il of 0.2 M at 0°C. The homogenates were centrifuged at for 15 min and the supernatants were analysed method of Seiler & Wiechmann [6, 71. This

9

method is sensitive enough to allow for a quantitative analysis of the polyamines including putrescine, in spite of its low cellular concentration, in a confluent monolayer of WI38 cells grown in one Falcon flask; i.e. 2 x lo6 cells. The details of the method as applied have been described elsewhere by Heby et al. [S].

Incorporation of 3H- TdR For these experiments WI-38 cells were grown in Falcon flasks as described above, At 19 h after change of medium, i.e. the time of the maximal rate of DNA synthesis [2], the cells were pulse-labeled with 3HTdR (5 &i/ml in Balanced Salt Solution) for 30 min to determine the incorporation of 3H-TdR into DNA. 3H-TdR (thymidine-methyl-3H, spec. act. 6.7 Ci/ mmole) was obtained from New England Nuclear Co., Boston, Mass. Unstimulated confluent monolayer cells were also pulse-labeled. The incorporation was stopped by washing the cells with ice-cold phosphate-buffered saline containing an excess of unlabeled TdR. The amount of 3H-TdR incorporated into acid-insoluble material [2] was measured in a Packard Tri-Carb (Model 3380) liquid scintillation spectrometer using the Triton-toluene scintillation cocktail [9] at an efficiency of 30 “,, for 3H.

RESULTS Stimulation of cell proliferation Seven days after plating, confluent:monolayers of WI38 cells show a very low rate of 3Hthymidine incorporation into DNA and very few mitoses are seen [2]. Practically all the cells are in the GO phase of the cell cycle [3, 10, II]. Replacement of the exhausted medium with fresh medium containing 10% fetal calf serum stimulates the cells to synthesize DNA and divide [2]. The incorporation of 3H-TdR into DNA begins to increase at 12 h and reaches a peak between 15 and 21 h after stimulation [2, 121.The cells start dividing 24 h after stimulation and the cumulative mitotic index reaches a peak at 27-33 h [2, 121. The percentage of cells that are stimulated to proliferate varies with the batch of fetal calf serum and with the age (number of generations) of the culture [4]. Cultures older than 30 generations showed poor stimulation and were not used routinely. To study the degree of stimulation in the Exptl

Cell Res 90 (1975)

10 Heby et al. Table 1. Polyamine content in confluent monolayers

of WI38 human diploid fibroblasts

after

various degrees of stimulation The figures shown are the means & S.E.M. of 3 Falcon flaska cultures Hours after stimulation

Experimental conditions A Unstimulated

cells

0

B Cells stimulated by the addition of fresh medium containing 0.3 % fetal calf serum

1 8 19

C Cells stimulated by the addition of 10 % fetal calf serum to the conditioned medium

A 19

D Cells stimulated by the addition of fresh medium containing 10 % fetal calf serum

1 8 19

3H-TdR incorporation into DNAb

SpermineC

86+13

502i.11

15os+130

6 122+l

61+10 89k 6 531-11

52Oi28 538f21 483 2 22

1588_+ 47 1564+ 92 14871 65

3951622219

73& 6 156i 8 263 i 14

533161 503 ) 28 523 & 43

1458j- 86 1 363+ 41 1418k114

80246i9863

120+11 236&27 448+41

558 k27 388f23 460 & 29

1449+132 1113-t 77 1238$-134

actual experiments we measured the incorporation of 3H-TdR at 19 h after change of medium, i.e. at the time of the maximal rate of DNA synthesis [2, 121. Table 1 shows the rates of 3H-TdR incorporation into DNA after the addition of media of various composition. Fresh medium supplemented with 10 % fetal calf serum induced maximal stimulation, 10% fetal calf serum added directly to the conditioned medium resulted in half maximal stimulation, and fresh medium supplemented with 0.3 Y0fetal calf serum caused no or only minimal stimulation of DNA synthesis (table 1). Polyamine content in confluent monolayers of WI38 human diploid fibroblasts after change of medium

Figs l-3 show the changes in polyamine content that occurred in cultures of WI38 Cell Res 90 (1975)

SpermidineC

2 600+417

a Each Falcon flask contained 2 x lo6 cells (variation b Cpm/Falcon flask culture. ’ Pmoles/Falcon flask culture.

Exptl

PutrescineC

979

< 10 %).

”

” dL0

10

20

y1

4.

y,

Figs 1-3. Abscissa: time after change of medium (hours); ordinate: nmoles/Falcon flask. Fig. 1. Putrescine content in confluent monolavers of WI38 human diploid fibroblasts after change of medium. Fresh medium supplemented with 10% fetal calf serum was added at time 0. Each plastic Falcon flask (surface area of 75 cm2; 250 ml) contained 2 x lo6 cells (variation < 10 X). Means + S.E.M. of 3-6 Falcon flask cultures.

Polyamines ill cellular proliferation

11

At 48 h after stimulation, i.e., when 6080 % of the cells have traversed the cell cycle a single time, the amount of putrescine, spermidine, and spermine per Falcon flask culture was 5.9, 2.4, and 1.8 times the amount in an unstimulated culture. When these values were adjusted, to account for the increase in cellularity, they showed that the approximate cellular content of putrescine, spermidine, and spermine at 48 h after stimulation was 3.3-3.7, 1.4-1.5, and 1.0-1.1 times the content of unstimulated WI38 cells. Fig. 2. Spermidine content in confluent monolayers of WI38 human diploid fibroblasts after change of medium, Caption is as in fig. 1.

Polyamine content in confluent monolayers of WI38 human diploid fibroblasts after various degreesof stimulation Table 1 shows the content of putrescine, spermidine, and spermine in confluent monolayers of WI38 fibroblasts 1, 8, and 19 h after various degreesof stimulation. When 10% fetal calf serum in fresh medium is added to confluent monolayers, 60-80 % of the cells are stimulated to proliferate [3] and 19 h after change of medium the cellular putrescine content was 5.2-fold that of unstimulated cells. When 10% fetal calf serum is added directly to the old conditioned medium only 30-40 % of the cells are stimulated [3] and the putrescine content at 19 h was 3.1-fold that of unstimulated cells. When 0.3 % fetal calf serum in fresh medium is

cells after stimulation with fresh medium supplemented with 10% fetal calf serum. Spermine was the quantitatively dominating polyamine, and in unstimulated WI38 cells the cellular content was 3.9-fold and 18.2fold that of spermidine and putrescine, respectively. By 6 h after stimulation the cellular putrestine content was significantly increased and by 19 h it was 5.5-fold that of unstimulated WI38 cells (fig. 1). A marked decreasein the putrescine content occurred 24 h post-stimulation. Very shortly after stimulation, there was an increase in the cellular spermidine content. It was already evident within 30-60 min after stimulation; the content was 50 % higher than in unstimulated cells (fig. 2). Subsequently, however, the spermidine content decreased and during the remainder of the pre-replicative phase it was similar to that of unstimulated WI38 cells. At 24 h after stimulation 0.8. the spermidine content started to increase. The cellular spermine content was relatively constant during the pre-replicative phase (fig. 3). It seemedas if spermine accumulation was Fig. 3. Spermine content in confluent monolayers of WI38 human diploid fibroblasts after change of initiated approx. 24 h after stimulation. medium. Caption is as in fig. 1. Exptl

Cell Res 90 (1975)

12 Heby et al. added to monolayers only a few cells are stimulated [3] and at 19 h no increase in the putrescine content was observed. Compared with the changes in cellular content of spermidine (and spermine) that occurred in cells stimulated maximally, those of cells stimulated to a lesser extent were negligible. It seems likely that the increase in putrescine content during the pre-replicative phase is related to stimulation of DNA synthesis since its magnitude is dependent on the fraction of cells that are stimulated to proliferate. DISCUSSION When cells in culture reach a saturation density, which is specific for each cell type [13-161, the rate of cellular proliferation is markedly reduced. Upon replacement of the exhausted medium with fresh medium containing fetal calf serum, cellular proliferation resumes [2]. The biochemical events which follow the stimulation of DNA synthesis and division of confluent monolayers of WI38 human diploid fibroblasts have been described in detail [2-4, 12, 17-201. An almost immediate increase in the activity of ornithine decarboxylase, the enzyme that catalyses the conversion of ornithine to putrescine, has been observed to follow stimulation of cellular proliferation in several experimental systems (for references see Heby et al. [l]). However, in WI38 cells no change was observed in the ornithine decarboxylase activity within the first few hours after stimulation [I], nor did the putrescine content increase during that time period. Not until 6 h after stimulation was an increase in the putrescine content observed. In a careful time study Hiiltta & Janne [21], and more recently Gaza et al. [22], have shown that the increase in putrescine synthesis in regenerating rat liver is biphasic. Exptl

Cell Res 90 (1975)

The time between partial hepatectomy and occurrence of the first peak was not related to the age of the animal; it was 4 h in all age groups [21]. The time between operation and occurrence of the second peak, however, was related to the age of the animals; i.e., the older the animal, the later the occurrence of the peak of putrescine synthesis [21]. Previously, Bucher et al. [23] had been able to correlate the length of the pre-replicative phase in regenerating liver with the age of the animal; i.e., the older the animal the later the occurrence of the peak of DNA synthesis. Therefore, by combining these results we conclude that only the second peak of putrescine synthesis is likely to be related to DNA synthesis. The first peak may be caused by a stimulus unrelated to the cell’s preparation for DNA synthesis and division. In fact, agents that do not stimulate hepatic DNA synthesis and division (i.v. injection of hypertonic glucose; i.p. injection of Celite) have been shown to cause a marked increase in ornithine decarboxylase activity within 4 h [24]. Contrary to the long duration of the increased ornithine decarboxylase activity after partial hepatectomy, only a brief increase in enzyme activity, with a peak within 4 h, was produced by these agents even after an extended duration of treatment. The results of the previously mentioned studies on regenerating rat liver [21, 23, 241 were compared with those obtained using WI38 human diploid fibroblasts stimulated to proliferate by nutritional changes. Since the duration of the pre-replicative phases are almost equal in these two systems, the time of the occurrence of putrescine synthesis in WI38 cells corresponds to that of the second peak of putrescine synthesis in regenerating liver. Because of this fact, and since the magnitude of putrescine accumulation was related to the fraction of WI38 cells stimulated to synthesize DNA, we propose that

Polyamines

there may be a connection between the increase in cellular putrescine content and subsequent DNA replication. The fact that there was no peak in putrescine synthesis in WI38 cells stimulated to proliferate corresponding in time to that of the first peak in regenerating liver, further corroborates the hypothesis that only the second peak of putrescine synthesis in regenerating liver is related to DNA synthesis. Further support for a connection between increased putrescine synthesis and DNA replication comes from studies in which factors that delay the S phase (e.g. hypophysectomy [25], aging [26], 8-azaguanine [26], and 5-azacytidine [26]) were shown to delay the induction of ornithine decarboxylase activity. The increase in putrescine synthesis, which preceded the initiation of DNA synthesis in WI38 cells stimulated to proliferate from a quiescent state, coincides with the increased rate of ribosomal RNA synthesis, observed by Zardi & Baserga [27]. As with ribosomal RNA synthesis,only those nutritional changes that stimulated cellular proliferation caused an increased synthesis of putrescine. The fact that putrescine and ribosomal RNA synthesis show a temporal correlation does not necessarily mean that these two events are also causally related. However, the studies by Kay & Cooke [28] and Kay & Lindsay [29] provide further circumstantial support for the idea that the increase in putrescine synthesis is associated with the increase in ribosomal RNA synthesis. The decrease in putrescine concentration in the WI38 cells which occurred between 19 and 24 h after the nutritional change is probably caused by the increased synthesis of spermidine and spermine which started at this time. A transient increase in the cellular spermidine content occurred as early as 30 to 60 min after the change of medium. It seems

in cellular

proliferation

13

as if this early increase in cellular spermidine content is due to synthesis rather than to uptake of spermidine from the medium, since the activity of putrescine-activated S-adenosylmethionine decarboxylase, the enzyme that catalyses spermidine formation, paralleled the changes in spermidine content [l]. This early stimulation of spermidine synthesis coincides with the increased synthesisof nonhistone chromosomal proteins [12], the marked rise in chromatin template activity for RNA synthesis [4], and the increased incorporation of 3H-uridine into the RNA of whole cells [4]. Fetal calf serum, and therefore the culture medium, contained putrescine as well as spermidine but these exogenous polyamines did not seemto enter the WI38 cells to any appreciable extent, not even after stimulation, as determined after addition of l”C-labelled polyamines to the culture medium and subsequent determination of the amount of incorporation into washed cells (data not shown). Exogenous spermidine did not affect the cellular proliferation when added to the culture medium instead of fetal calf serum in a concentration equal to that in fresh medium supplemented with 10 “iofetal calf serum. The effect of spermidine addition on 3H-TdR incorporation into DNA of WI38 cells was evaluated 19 h after the addition of polyamine-supplemented medium. Pohjanpelto & Raina [30] have identified a growth factor in conditioned medium of human fibroblast cultures as putrescine. However, cellular proliferation was stimulated only within a strikingly small concentration range, and growth stimulation was not expressed until 2 to 3 days after the addition of putrescine.

This work was supported in part by USPHS grants CA-08373 and CA-12923 from the NCI, NIH, and gifts from the Phi Beta Psi Sorority and the Joe Gheen Medical Foundation. Exptl

Cell Res 90 (1975)

14 Heby et al. REFERENCES 1 Heby, 0, Marton, L J, Zardi, L, Russell, D H & Baserga, R, The cell cycle in malignancy and immunity (ed J C Hampton). NTIS, Springfield, Va. In press. Wiebel, F & Baserga, R, J cell physiol 74 (1969) 191. Rovera, G & Baserga, R, Exptl cell res 78 (1973) 118. Farber, J, Rovera, G & Baserga, R, Biochem j 122 (1971) 189. Merchant, D J, Kahn, R H & Murphy, W H, Handbook of cell and organ culture, p. 217. Burgess Publishing Co, Minneapolis (1964). 6. Seiler, N & Wiechmann, M, Hoppe-Seyler’s Z physiol Chem 348 (1967) 1285. 7. - Progress in thin-layer chromatography and related methods (ed A Niederwieser & G Pataki) vol. 1, p. 94. Humphrey Science Publishers, Ann Arbor, Mich. (1970). 8. Heby, 0, Sarna, G P, Marton, L J, Omine, M, Perry, S & Russell, D H, Cancer res 33 (1973) 2959, 9. Patterson, M S & Greene, R C, Anal them 37 (1965) 854. 10. Costlow, M & Baserga, R, J cell physiolS2 (1973) 11. Baskrga, R, Costlow, M & Rovera, G, Fed proc 32 (1973) 2115. 12. Rovera, G & Baserga, R, J cell physiol 77 (1971) 201. 13. Borek, C & Sachs, L, Proc natl acad sci US 56 (1966) 1705.

Exptl

Cell Res 90 (1975)

14. Kruse, P F Jr, Whittle, W & Miedema, E, J cell biol 42 (1969) 113. 15. Ceccarini, C & Eagle, H, Nature new biol 233 (1971) 271. 16. Agrell, I P S & Gabbay, M, Exptl cell res 82 (1973) 131. 17. Rovera, G, Farber, J & Baserga, R, Proc natl acad sci US 68 (1971) 1725. 18. Baserga, R, Rovera, G & Farber, J, In vitro 7 (1971) SO. 19. Tsuboi, A & Baserga, R, J cell physiol SO (1972) 107. 20. Stein, G, Chaudhuri, S & Baserga, R, J biol them 247 (1972) 391s. 21. Holtta, E & Janne, J, FEBS lett 23 (1972) 117. 22. Gaza, D J, Short, J & Lieberman, I, FEBS lett 32 (1973) 251. 23. Bucher, N L R, Swaffield, M N & DiTroia, J F, Cancer res 24 (1964) 509. 24. Schrock, T R, Oakman, N J & Bucher, N L R, Biochim biophys acta 204 (1970) 564. 25. Russell, D H & Snyder, S H, Endocrinology 84 (1969) 223. 26. Cavia, E & Webb, T E, Biochim biophys acta 262 (1972) 546. 27. Zardi, L & Baserga, R, Exptl molec pathol 20 (1974) 69. 28. Kay, J E & Cooke, A, FEBS lett 16 (1971) 9. 29. Kay, J E & Lindsay, V J, Exptl cell res 77 (1973) 428. 30. Pohjanpelto, P & Raina, A, Nature new biol 235 (1972) 247. Received May 8, 1974 Revised version received July 15, 1974