C13 cells

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Experimental Cell Research III (1978) 23 l-236

POLYAMINE

METABOLISM

IN BHK21/C13

CELLS

Loss of Spermidine from Cells Following Transfer to Serum-depleted Medium MAUREEN

A. L. MELVIN

and H. M. KEIR

Department of Biochemistry, University of Aberdeen, Marischal College, Aberdeen, AB9 IAS, Scotland, UK

SUMMARY The growth rate of BHK21/C13 cells in culture was slowed down by transferring growing cells to serum-depleted medium. Following deprivation of serum, the intracellular concentration of polyamines decreased. The amount of spermidine relative to spermine decreased, and this change was the result of the spermidine content per cell decreasing more than the spermine content. The decrease in cell content of polyamines was accompanied by release of polyamines from the cells into the culture medium. The polyamines released were examined using cells whose polyamines had been labelled by prior incubation of the cells with radioactive putrescine. Almost all of the radioactivity released into the medium was found in spermidine, even though the cells contained most of their radioactivity in spermine. It is suggested that specific release of spermidine may be an important mechanism by which these cells can regulate their intracellular content of polyamlnes.

In mammalian cells, one of the earliest responses to a growth stimulus is a dramatic increase in the activity of ornithine decarboxylase (EC 4.1.1.17), the enzyme which catalyses the rate-limiting step in polyamine biosynthesis [ 11.The rise in activity of omithine decarboxylase (a) can be prevented by administration of putrescine or spermidine simultaneously with the growth stimulus [2-51 suggesting that the function of omithine decarboxylase is to ensure that the cell contains suffkient polyamines for its requirements, and (b) is usually followed by a significant increase in intracellular amine content implying that these amines have some function(s) important in cell growth [6-8]. We examined the changes in polyamine metabolism that occur when the growth rate of mammalian cells in culture is slowed down. A quiescent cell population can be

obtained by lowering the concentration of serum in the culture medium abruptly from 10% (v/v) to 1% (v/v) or less [9-l I]. This treatment arrests growth of the cells in an ‘early’ phase of the cell cycle, within 12 h of serum deprivation. We aimed to determine whether intracellular polyamine levels in BHK211C13 cells can be controlled by mechanisms involved in the degradation or excretion of polyamines, or whether they are controlled only by their rates of biosynthesis. MATERIALS

AND METHODS

Solutions TD buffer contains NaCl at 137 mM, KC1 at 5 mM, NapHPOl at 0.7 mM and Trizma base at 25 mM adjusted to pH 7.4 with HCI. Buffered trypsin solution is 0.25% (w/v) trypsin in TD buffer, with dextrose [O.1% (w/v)] Crystamycin (160 n&l) and phenol red [O.OOlS% (w/v)], sterilised by filtration. PBS is phosphate-buffered saline (NaCl at 137 mM, KC1 at 3 mM, N%HPO, at 8 mM, KHzPOl at 1.5 mM, adjusted to Exp CeNRes 111 (1978)

232

Melvin and Keir

pH 7.2 and sterilised by autoclaving). Ten percent formal saline is 10 ml of 40% (w/v) formaldehyde added to 90 ml of a solution containing 9 mM NaCl and 100 mM Na,SO,.

Materials 1,4(n)-[3H]putrescine dihydrochloride (16-25 Gil mmol). [“C]Spermidine n-i-hydrochloride (122 mCi/ mmol), [W]spermine tetrachloride (115 mCi/mmol) and [VH]thymidine (29.4 Cilmmol), were purchased from the Radiochemical Centre, Amersham, Bucks. Non-radioactive putrescine, spermidine and spermine, and dansyl chloride, were obtained from Sigma (London) Chemical Co. Ltd., Kingston-upon-Thames, Surrey; L-proline was from BDH Biochemicals Ltd., Poole, Dorset; tissue culture materials including horse serum screened for mycoplasma, from Flow Laboratories Ltd., Irvine, Ayrshire; and Crystamycin from Glaxo Laboratories Ltd, Greenford, Middlesex. Silica gel G thin layer chromatography plates (20x20 cm) were from E. Merck, Darmstadt, BRD.

Cell culture BHK211C13 cells [12] were grown in monolayer culture at 37°C in a 5% CO* atmosphere, in 2.24 litre roller bottles in Eagle’s medium, Glasgow modification [ 131containing 10% (v/v) horse serum and 10% (v/v) tryptose phosphate broth (medium ETH&, until about two generations short of confluence. The cell sheet was then washed with buffered trypsin solution, which was immediatelv poured off. and the cells left at room temperature untilthey detached from the glass. The cells were washed off the glass with ETHlo, and after determination of cell number [ 1l] were dispensed into elastic Petri dishes in ETH,.. Thev were then incubated at 37°C in a 5% CO,‘atmosphere for l&24 h after which the medium was replaced with fresh medium, either ETHlo or EHr, which is Eagle’s medium, Glasgow modification, containing 1% (v/v) horse serum. Cells were routinely checked for mycoplasma contamination. The number of cells in S phase was estimated by the incorporation of E3H]thymidine into acid-insoluble material [ 111.

Determination and DNA

and separated on thin-layer chromatography plates [ 171.Fluorescent regions corresponding to each of the amines were scraped from the plates and estimated auantitativelv bv their fluorescence in methanol/ ammonia (93 : SF, or their radioactivity determined by liquid scintillation spectrometry using a Tritontoluene-based fluor mixture.

Radioactive polyamines

labelling

of intracellular

Non-confluent cells growing in roller bottles were incubated for 24 h in ETH,, containing [3Hlputrescine at 1.0 @X/ml. The radioactive medium was poured off and the cell sheet washed twice with ETHr, prior to harvesting. The cells were dispensed into 90 mm diam. plastic Petri dishes at a density of 0.8~ 108cells/dish, and incubated overnight in ETHlo as described above.

Estimation of radioactive in cells and medium

polyamines

The cell sheet was washed twice in situ with ETHlo or EH, medium at 37”C, and 8 ml of fresh medium added. After incubation for the required time, the medium was carefully removed and c&rifuged at 200 g,, for 5 min to sediment cell debris. Ice-cold 2 M HClO, was added to the medium to a final concentration of 0.2 M. The samules were kept at 4°C for 15 min with occasional mixing on a vortex mixer, then the acidinsoluble material was removed by centrifugation and polyamines in the supematant fraction dansylated and analysed as before [17] using 1.0 ml aliquots of each sample for dansylation. The cell sheet was washed three times with ice-cold PBS to remove any labelled amines that were absorbed to the cell surface [S]. The cells were scraped off the dishes with ice-cold PBS, and the polyamines extracted and analysed as described above.

RESULTS Inhibition

of cell growth

of protein, RNA

Non-confluent (growing) monolayer cultures of BHK21/C13 cells were transferred Cells in 90 mm diam. plastic Petri dishes wer6 washed from medium that contained 10% (v/v) sethree times in situ with ice-cold PBS, scraped off the dishes in PBS, and collected by centrifugation at 200 rum to medium that contained 1% (v/v) g,, for 5 min. 0.4 ml of ice-cold 0.2 M HClO, was serum. Within 24 h there was significant inadded to the cells from each dish, and the sample homogenised at 4°C bv 10 strokes of a alass micro- hibition of the cell growth rate compared homogeniser with a close-fitting pestle. -The homo- with control cultures in 10% serum megenate was left at 4°C for 15 min before centrifuaation dium, as determined by cell number, DNA, at 200 g,, for 5 min at 4°C. Protein, RNA and-DNA were extracted from the precipitate as described by RNA and protein content (table 1).

Munro & Fleck [ 141and estimated respectively by the method of Lowry et al. [15], by extinction at 260 nm (one extinction unit=32 pg of RNA/ml), and by Burton’s diphenylamine method [16]. The putrescine, spermidine and spermine extracted from cells by 0.2 M HClO, were converted to their dansyl derivatives, Exp Cell Res 1I I (1978)

Polyamine

content of cells

The intracellular concentrations of polyamines decreased within 24 h of transfer of

Polyamine metabolism in BHK21 lC13 cells Table 1. Amounts of cellular constituents in growing and serum-deprived BHK21 cells Cell culture

Cell no. (cells x10-6/ dish)

Protein (md dish)

DNA (l-4 dish)

RNA (l.4 dish)

1.5

0.61

37

82

6.4

1.63

112

250

1.6

0.65

40

80

Initial culture 24hin ETHIo 24 h in EH,

growing cells to serum-depleted medium (table 2). The concentration of spermidine fell dramatically within 24 h, whereas the spermine content showed a less marked decrease. In some experiments a slight increase in the spermine content per cell was noted, but the concentration of spermidine and the total polyamine content always decreased substantially. The putrescine content of the cells was relatively low; consequently, changes in the intracellular content of putrescine following serum deprivation were insignificant compared with those of spermidine and spermine. A decrease in the molar ratio of spermidine to spermine could be detected as early as 6 h after deprivation of serum; by 24 h the ratio had decreased substantially but showed little further change thereafter. Fate of intracellular polyamines The conversion of putrescine to spermidine and spermine occurs quite rapidly in

233

BHK21 cells (fig. 1). When cells were incubated for 2 h in medium containing radioactive putrescine and 10% (v/v) serum, then transferred to fresh medium without label, only about 2% of the intracellular label was found in putrescine 4 h later, while about 77% was in spermidine and 20 % in spermine. Cells were labelled for 24 h using r3H]putrescine. The cells were then incubated in fresh medium without label for 24 h to allow the label to equilibrate with intracellular polyamine pools. After this time approx. 50% of the intracellular label was in spermidine and 50 % in spermine. The cells were then transferred to fresh medium containing either 10 or 1% (v/v) serum. There was an immediate efflux of labelled material from the cells into the medium (fig. 2). Much more label was lost from the cells when the medium contained 1% serum than when it contained 10% serum. In order to determine the nature of the polyamines that were released from the cells following their transfer to serumdepleted medium, [3H]putrescine-labelled cells were deprived of serum and the acidsoluble fractions from the cells and from the medium in which they were incubated were analysed to determine in what compounds the radioactivity was located. The acidsoluble radioactive material in the cells (fig. 3A) consisted of spermidine and spermine, the proportion of the total that was in spermine increasing with time. In contrast, at all

Table 2. ESfect of serum deprivation on the polyamine content of BHK21 cells Cell culture

Spermine (nmoles/lW cells)

Spermidine (nmoles/lOB cells)

Putrescine (nmoles/lW cells)

Ratio spermidine spermine

Initial culture 24 h in ETHto 24 h in EH,

1.33 1.57 1.25

2.20 2.03 0.95

0.29 -

1.65 1.29 0.76 Exp Cell RPS I II (1978)

234

Melvin and Keir

0

24

40

Fig. 1. Abscissa: time in non-radioactive medium (hours); ordinate: intracellular radioactivity (% of total). O-O, Spermidine; O-O, spermine; A-A, putrescine. Formation of spermidine and spermine from putrestine in BHK21 cells. The cells were incubated for 2 h in medium containing 10% (v/v) serum and rH]putrescine (2 pCi/ml), before transfer to non-radioactive medium.

times following transfer of cells to serumdepleted medium the acid-soluble radioactivity in the medium (fig. 3B) was located exclusively in spermidine. Neither labelled putrescine nor labelled spermine was found in significant amount in the medium. The results presented in fig. 3 suggest that when the growth rate of BHK21 cells is arrested by deprivation of serum, the cells specifically excrete spermidine into the medium. Spermine is not excreted then converted to spermidine in the medium, because cells incubated in medium containing 10% (v/v) serum and [‘*C]spermine take up the label, which is then found almost exclusively in intracellular spermine (table 3, line 3). Some conversion of spermine to spermidine takes place intracellularly however, as has been described for other mammalian cells [lg211. Assessment of the viability serum-deprived cells

of

It was important to show that loss of spermidine from serum-deprived cells was not a consequence of cell death or of detachment of cells from the monolayer. Most of the experiments were conducted using cells culExp CellRes

III

(1978)

I 24

01 0

I 46

Y 0

I 24

I 48

Fig. 2. Abscissa: time in fresh medium (hours); ordinafe: radioactivity (% of total) in (A) cells; (B) medium after transfer of [3H]putrescine-labelled cells to fresh medium containing 10% serum (0-O) or 1% (v/v) serum (O-O). In this experiment, total radioactivity in cells (i.e. 100%) at 0 h was 12.2~10~ cpmldish. Cells were incubated for 24 h in ETHlo medium containing [SH]putrescine (1.0 j&i/ml) then incubated in fresh medium, without label, for 24 h to allow equilibration of labelled polyamines with intracellular polyamine pools. Cells were then transferred to fresh medium containing either 10% or 1% (v/v) serum.

tured in plastic Petri dishes. However, similar results were obtained using glass dishes, to which the cells adhere most tenaciously [22]. Microscopic examination of particulate matter in the medium 24 h after serum deprivation, when the cells were cultured in plastic dishes, indicated that less

1A

+

t

9

01 0

I

I

I

24

40

72

0

L

12

24

40

12

Fig. 3. Abscissa: time in 1% serum medium (hours); ordinate: radioactivity (% of total) located in spermidine (0-O) and spermine (O-O) in (A) cells; (B) medium. In this experiment, total radioactivity in cells at 0 h was 5.0~ 1W cpm/dish. Identification of [JH]spermine in cells and medium after transfer of pH&utrescinelabelled cells from medium containing 10% (v/v) serum to medium containing 1% serum.

Polyamine

Table 3. Uptake of exogenous spermidine and spermine, and their intracellular interconversions in BHK21 cells deprived of serum % Total radioactivity Exogenous radioactive polyamine Spermidine Spermine

Cell culture

Spermidine

Spermine

Initial After Initial After

83 55 5 16

17 45 95 84

culture 24 h in EHr culture 24 h in EH,

Cells were incubated for 2 h in medium containing 10% (v/v) serum and [Wlspermidine or [“C]spermine at 0.15 &i/ml, before transfer to non-radioactive medium containing 1% serum. The cells were harvested at zero time or after 24 h in the ‘low-serum’ medium.

than 5% of the cells present in the original culture had dislodged from the monolayer, whereas approx. 70% of the initial 3Hlabelled polyamines was lost from the cells during this time. The cells which remained attached to the monolayer after 48 h in ‘lowserum’ medium could be stimulated to grow by incubating them in fresh medium containing 10% (v/v) serum, as described elsewhere [Ill, and DNA synthesis recommenced within 12 h, as estimated by the uptake of [3H]thymidine into acid-precipitable material. Within 2ti8 h almost all of the cells in the monolayer had re-initiated DNA Table 4. RNA, DNA and polyamine serum to serum-deprived cultures Cell culture Initial culture 24 h in 10% serum 48hin l%serum 24 h after readdition of 10% serum to serum-deprived (48 h) cultures

metabolism

in BHK21 /Cl3 cells

235

synthesis, as determined by autoradiography. The DNA, RNA, spermidine and spermine content per culture increased significantly within 24 h of readdition of serum, and the molar ratio of spermidine to spermine increased to a value typical of ‘growing’ cells (table 4). DISCUSSION These studies provide support for the hypothesis that intracellular amounts of polyamines, particularly spermidine, have an important correlation with cell growth rate. When the growth rate of BHK21/C13 cells is slowed down by depriving the cells of serum, the intracellular polyamine content decreases within 24 h and the molar ratio of spermidine to spermine falls significantly (table 2). The change in the spermidinel spermine ratio after serum deprivation is due primarily to a decrease in the intracellular concentration of spermidine; the spermine content changes less dramatically (table 2). Examination of the fate of intracellular polyamines , labelled by incubating the cells in medium containing [3H]?utrestine (fig. l), reveals that following deprivation of serum large amounts of polyamines are lost from the cells into the culture medium (fig. 2). We have shown recently [22] that when the growth rate of BHK21/C13

content of BHK21

cells 24 h after re-addition

RNA (&lish)

DNA &g/dish)

Spermine (nmoles/dish)

Spennidine (nmoles/dish)

Ratio spermidinel sperrnine

76 275 70

39 180 41

1.5 11.4 3.2

3.0 15.7 1.5

2.0 1.4 0.5

206

107

11.8

14.0

1.1

of

Exp Cell Res I I I (I 978)

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Melvin and Keir

cells in culture is slowed down by depriving the cells of isoleucine [23], or by allowing the monolayer to approach confluence, intracellular polyamines are lost from the cells into the medium. Thus it seems that excretion of polyamines from BHK21/C13 cells is a response of these cells specifically to signals for cessation of growth. Co-ordinate with the release of intracellular polyamines there is depletion of the intracellular polyamine content. Similar results have been obtained recently from Chinese hamster ovary cultures after hyperthermic exposure [24]. Identification of the labelled polyamines in BHK21/C13 cells and in the incubation medium after deprivation of serum reveals that the label in the medium is located almost exclusively in spermidine, whereas the cells contain both labelled spermidine and spermine (fig. 3). This suggests that specific release of spermidine may be an important mechanism whereby BHK2 l/C 13 cells can regulate their intracellular polyamine content. Inside the cells the conversion of putrescine to spermidine is very rapid (fig. 1) and the intracellular concentration of putrescine is very low compared with the concentrations of spermidine and spermine (table 2); so it is likely that these cells dispose of putrescine excess to their requirements by first converting it to spermidine. Moreover, the conversion of spermidine to spermine is reversible in serumdeprived BHK21/C 13 cells (table 3), so spermine excess to a cell’s requirements may be metabolised via its conversion to spermidine, as has been suggested previously [21, 251. The present results indicate that in BHK21/C13 cells the control of intracellular polyamine concentrations is not confined to pathways of polyamine biosynthesis [21], but is mediated to a significant extent by Exp Cell Res 111 (1978)

excretion of polyamines, particularly spermidine, from the cells. We thank the Medical Research Council (Grant No. G975/38C) for supporting this research, and Alan Y. Charles of Aberdeen for a gift of equipment. A brief preliminary report of the work has been published [26].

REFERENCES 1. Tabor, C W & Tabor, H T, Ann rev biochem 45 (1976) 285. 2. Kay, J E & Lindsay, V J, Biochem j 132 (1973) 791. 3. Janne, J & Hiillt;i, E, Biochem biophys res commun 61 (1974) 449. 4. Theoharides, T C & Canellakis, Z N, Nature 255 (1975) 773. 5. (I&k, J L & Fuller, J L, Biochemistry 14 (1975) 6. Bachrach, U, Functions of the naturally occurring polyamines. Academic Press, London and New York (1973). 7. Clark, J L & Duffy, P, Arch biochem biophys 172 (1976) 551. 8. Russell, D H & Taylor, R T, Endocrinology 88 (1971) 1397. 9. Howard, D K, Hay, J, Melvin, W T & Durham, J P, Exp cell res 86 (1974) 31. 10. Biirk, R R, EXD cell res 63 (1970) 309. 11. Craig, R K, Costello, P A & Keh, H M, Biochem j 145(1975) 233. 12. McPherson, I A & Stoker, M G P, Virology 16 (1962) 147. 13. Busby, D W G, House, W & McDonald, J R, Virological technique. Churchill, London (1964). 14. Munro, H N & Fleck, A, Methods in biochemical analysis (ed D Glick) vol. 14, pp. 159-160. Interscience, New York (1966). 15. Lowry, 0 H, Rosebrough, N J, Farr, A L & Randall, R J, J biol them 193 (1951) 265. 16. Burton. K, Biochem i 62 (19561315. 17. Herbst, E J & Dion, A S, Fed proc 29 (1970) 1563. 18. fiirres, M, Acta physiol Stand 71 (1%7) suppl. 19. S&es, M & Janne, J, Acta them Stand 21 (1%7) 815. 20. Pett, D M & Ginsberg, H S, Fed proc 27 (1%8) 615. 21. Kano, K & Oka, T, J biol them 251 (1976) 2795. 22. Melvin, M A L & Keir, H M. Unpublished observations. 23. Tobey, R A & Ley, K D, Cancer res 31 (1971) 46. 24. Gemer, E W &Russell, D H, Cancer res 37 (1977) 482. 25. Williams-Ashman, H G, Pegg, A E & Lockwood, D H, Advances in enzyme regulation 7 (1%9) 291. 26. Melvin, M A L & Keir, H M, Biochem sot trans 5 (1977) 711. Received May 3 1, 1977 Revised version received September 2, 1977 Accepted September 9, 1977