A hepatic membrane-associated factor stimulates nuclear DNA synthesis in cultured fibroblastic cells

Biochimica et Biophysica A cta, 696 (1982) 134-138

134

Elsevier Biomedical Press BBA 91015

A H E P A T I C MEMBRANE-ASSOCIATED FACTOR S T I M U L A T E S NUCLEAR DNA S Y N T H E S I S IN CULTURED FIBROBLASTIC CELLS SUBAL BISHAYEE and MANJUSRI DAS

Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104 (U.S.A.) (Received June 23rd, 1981)

Key words: Plasma membrane," Growth factor," Nuclear DNA synthesis (Mouse liver)

Nuclear DNA replication in cultured mouse fibroblasts is stimulated by isolated hepatic plasma membranes in a time- and concentration-dependent manner. The plasmalemmal activity is susceptible to trypsin treatment, and to treatment with protein modifying agents, N-ethylmaleimide, N-bromosuccinimide, and 2-hydroxy-5-nitro-henzylbromide.

Introduction

Materials and Methods

Growth of normal animal cells is known to be influenced by various humoral factors including hormones, growth factors and nutrients [1] and also by non-humoral factors such as cell density and direct cell-cell contact [2,3]. A number of soluble humoral factors have been purified [4-7] and their interactions with responsive cells have been well characterized [8-15]. In contrast, the mechanisms for cell-contact mediated growth regulation are unclear. Cell contact can act as an antimitogenic signal as in contact inhibition [16], or in special cases, it can act as a mitogenic signal [17]. Characterization of the cell surface regulatory molecules responsible for these contact-mediated phenomena may be an important step towards understanding how cell-cell interactions influence proliferation or differentiation in vivo. In the present paper we report a preliminary characterization of a plasma membrane-associated growth factor from mouse liver.

The procedure of Aronson and Touster [18] described for isolation of rat liver plasma membranes was used without any further modification for preparation of liver plasma membranes from Swiss white mice. The 33000-78000 × g pellet was suspended in 57% sucrose/5 mM Tris-HC1, pH 8.0, and subjected to sucrose gradient equilibrium centrifugation as described [18]. The fraction P2 (8.5-34% sucrose) contained about 25% of the input 5'-nucleotidase activity and less than 0.1% of the input N-acetyl-fl-D-glucosaminidase activity present in the original tissue homogenate. This fraction, referred to as mouse liver membrane, was used for the stimulation experiments described in this paper. For preparation of membranes from NR-6 cells, the procedure of Whittenberger et al. [16] described for isolation of 3T3 plasma membranes was used without any further modification. All membrane preparations were sterilized by ultraviolet irradiation (5 min) before use in cell experiments. Membrane protein was determined by using the procedure of Lowry et al. [19], with bovine serum albumin as the standard.

135

Results Addition of purified mouse liver membranes to density-inhibited cultures of fibroblastic cells (Swiss mouse 3T3 or NR-6) resulted in a stimulation of [3H]thymidine incorporation into D N A (Table I). The stimulatory activity was associated with a plasma membrane particulate fraction. Incubation of cells with increasing quantities of murine hepatic membranes resulted in an increasing stimulation of D N A synthetic rate upto a

TABLE I MEMBRANE-INDUCED SYNTHESIS

STIMULATION

OF

DNA

Monolayer cultures of Swiss mouse 3T3 cells and mouse NR-6 cells [20] were grown and maintained as described [13] in Dulbecco's modified Eagle's medium containing 10% fetal calf serum (Gibco). Confluent monolayers of Swiss 3T3 cells or NR-6 cells in 16 m m dishes were incubated at 37°C for 24 h with l ml Dulbecco's modified Eagle's medium containing 2% fetal calf serum (DME-2% FCS). The cells were then incubated at 37°C for 22 h with 0.3 ml of this conditioned medium containing either no membrane or the indicated a m o u n t s of m e m b r a n e protein. At the end of incubation, [3H]thymidine incorporation into cellular D N A was measured as described [12]. The cell monolayers were incubated at 37°C for l h with [3H]thymidine (New England Nuclear, l / ~ C i / m l , 0.65/~M) in 0.5 ml DME-2% FCS. At the end of incubation, the cell monolayers were washed twice with 0.15 M N a C l / 1 0 m M Tris-HC1, p H 7.4, and incubated at 4°C for 20 rain with l ml 5% trichloracetic acid. The trichloracetic acid-insoluble radioactivity sticking to the culture dishes was washed once with 5% trichloracetic acid and twice with methanol. The insoluble material was then solubilized with 0.5 M N a O H , neutralized with HCI, and then counted for radioactivity using a xylenebased scintillation fluid. Cell type

Swiss 3T3

NR-6

NR-6

Membrane addition

None Mouse liver membrane (76/tg) None Mouse liver membrane (76/~g) None NR-6 membrane (42/t g) NR-6 membrane (7/~g)

[ 3H]Thymidine incorporation (cpm) 3 700 17200 3 800 16 100 3 200 3 980 3 100

point, and then the effect gradually plateaued (Fig. 1). Isolated membranes from rabbit liver also produced a similar stimulation (data not shown), but membranes prepared from NR-6 cells lacked this stimulatory effect (Table I). These suggest that the stimulation may be specific for hepatic membranes, but the effect is not species specific. The stimulatory effect of hepatic membranes could also be measured by autoradiographic nuclear labeling (Table II). There was an increase in percent-labeled nuclei with increasing amounts of hepatic membranes, and at near-maximal stimulation (69% nuclei labeled) there was a 4-5-fold increase in nuclear labeling index (Table II). The observed stimulation of D N A synthesis was not an artifact of membrane cytotoxicity. Viable cell counts (95% of total), as measured by trypan blue exclusion, did not decrease during incubation (0-20 h) with increasing quantities of membranes (upto 200 #g membranes/16 mm dish). The extent of stimulation of DNA synthesis was dependent upon the time of contact of membranes with the cells (Fig. 2). Stimulation appeared to increase in proportion to contact time until at least 10h. The extent of stimulation was also

1500C

v

8

E

IO00C

500C

I--

L 6

260 360 46o 560 Mernbrone Protein per dish (/.Lg)

Fig. l. Membrane concentration dependence of stimulation of D N A synthesis. Confluent monolayers of mouse NR-6 cells in 16 m m dishes were incubated at 26°C for 6 h in 0.3 ml conditioned DME-2% FCS medium containing the indi~:ated a m o u n t s of mouse liver membrane protein. Then the cells were washed free of membranes and incubated at 37°C for 15 h with 0.3 ml membrane-free conditioned medium. At the end of incubation [3H]thymidine incorporation into D N A was measured as described in the legend for Table I.

136 TABLE II NUCLEAR AUTORADIOGRAPHIC O F M E M B R A N E ACTIVITY

1600(

DETERMINATION

NR-6 cells (in D M E medium containing 10% FCS) were plated onto glass coverslips in 16 mm dishes at a density of 3-104 ceUs/sq, cm. After 24 h, the medium was changed to D M E medium containing 0.5% FCS, and the cells were incubated at 37°C in this low-serum medium for 48 h. These cells were then incubated at 37°C for 6 h with the indicated amounts of mouse liver membranes in 0.3 ml of conditioned DME-0.5% FCS medium. At the end of incubation, the cells were washed with conditioned medium, and then further incubated with 0.5 ml conditioned medium containing [3H]thymidine (New England Nuclear, 1 # C i / m l , 0.65 #M). After 20 h incubation at 37°C, the monolayers were washed twice with 0.15 M NaCI/10 m M Tris-HC1, p H 7.4, and fixed in methanol at 4°C for 5 rain. The coverslips were air-dried, mounted on glass slides, and then treated with NTB2 emulsion (Kodak). After a 24 h incubation in the dark at 4°C, the slides were developed and fixed. Labeled nuclei were visualized as dark spots containing at least five dark grains. The unlabeled nuclei were stained with Papanicolaou hematoxylin Harris stain (Fisher). For each determination of percent-labeled nuclei, at least 400 cells were counted. Increasing the development time from 24 to 48 h did not significantly increase the nuclear labeling index under these experimental conditions. Hepatic membranes

Labeled unlabeled

% Nuclei labeled

None 66/~g membranes 200/xg membranes

82 : 430 239 : 231 293:132

16% 51% 69%

.B ,2ooc

8000

i

J

4O00

6

' ' i'o/j Membrane Incubation Time (Hours)

Fig. 2. Effect of increasing time of contact with membranes upon [3H]thymidine incorporation rate. Density-inhibited monolayers of NR-6 cells in 16 mm dishes were incubated at 37°C for the indicated time periods with 76 #g mouse liver membrane proteins in 0.3 ml conditioned DME-2% FCS. At the end of incubation, the monolayers were washed free of membranes and incubated with 0.3 ml membrane-free conditioned DME-2% FCS medium at 37°C. At 21 h after the addition of membranes, [ 3H]thymidine incorporation into DNA was measured as described in the legend for Table I.

~ 12ooo dependent upon the temperature at which contact was made with membranes. Membrane contact at 37°C was about 1.3-fold better than that at 26°C (data not shown). A study on the time course of appearance of D N A replicative activity after a fixed 6 h incubation with membranes showed that replicative activity reached its maximum at about 2 1 - 2 6 h after the addition of membranes (15-20 h after removal of membranes), and there was a sharp decline in activity after this time (Fig. 3). The peak of D N A synthetic activity in Fig. 3 probably represents a round of S phase D N A replication. A second peak was not observed at a later point (data not shown), suggesting that the cells do not undergo more than one cycle of replication after a single membrane treatment.

Z oT ooo 6

'

ib

'

2'o

go

'

Time After Membrane Removal (Hours)

Fig. 3. Time course of appearance of DNA replicative activity after membrane-treatment. Density-inhibited monolayers of NR-6 cells in 16 mm dishes were incubated at 26°C for 6 h with 140 #g membrane protein in 0.3 ml conditioned DME-2% FCS medium. Then the monolayers were washed free of membranes and incubated with 0.5 ml conditioned DME-2% FCS at 37°C for the indicated time periods. At the end of incubation [3H]thymidine incorporation into DNA was measured as described in the legend for Table I.

137

In order to determine the nature of the active components in liver membranes, the membranes were subjected to several treatments prior to incubation with cells (Table III). Treatment with DNAase did not reduce activity, but treatment with trypsin led to a drastic reduction in activity, suggesting that the active components in membranes are most probably proteins. The activity was reduced to about 50% of control by treatment with 10 mM N-ethylmaleimide, suggesting the inTABLE III CHARACTERIZATION OF THE ACTIVITY IN THE EXTRACT The following chemicals were from Sigma Chemical Company: twice-crystallized trypsin (7600 BAEE units/mg protein), DNAase-I (1400 Kunitz units/mg protein), N-ethylmaleimide, 2-hydroxy-5-nitro-benzylbromide, N-bromosuccinimide, and soybean trypsin inhibitor. Mouse liver membranes (3.5 mg) in 1 ml of 20 mM Tris-HCl, pH 7,4 were treated at 25°C for 30 min with the indicated reagents. In the case of trypsin treatment, the incubation mixture also contained 5 mM CaC12. At the end of the incubation, the tube containing trypsin received soybean trypsin inhibitor at a final concentration of 174 # g/ml. After 10 min incubation at 25°C the reaction mixtures were diluted with 5 ml of 10 mM Tris-HCl, pH 7.4 and centrifuged to recover the membranes. The washed membranes were then assayed for activity as follows. Treated membranes (140 #g) in 0.3 ml conditioned DME-2% FCS were incubated at 26°C for 6 h with density-inhibited monolayers of T3 cells in 16 mm dishes. At the end of incubation, the cells were washed free of membranes and incubated at 37°C for 15 h with 0.3 ml membrane-free conditioned DME-2% FCS. At the end of incubation, [3H]thymidine incorporation into DNA was measured as described in the legend for Table I. Control, membrane-induced [3 H]thymidine incorporation (100%) was about 4900 cpm, after subtraction of the base-line incorporation (I 500 cpm) in quiescent unstimulated 3T3 cells. Treatment of membranes

% control stimulation of [ 3H]thymidine incorporation

None DNAase (200/~g/ml) Trypsin (58 gg/ml) Soybean trypsin inhibitor (50 ttg/ml) Trypsin (58 gg/ml) plus soybean trypsin inhibitor (50 ttg/ml) N-Ethylmaleimide (10 mM) 2-Hydroxy-5-nitro-benzylbromide (10 mM) N-Bromosuccinimide (10 raM)

100 95 60 99 98 50 5

10

volvement of sulfhydryl groups in the maintenance of active structure in the protein(s), Table III shows that the activity was also severely inhibited by 2-hydroxy-5-nitro-benzylbromide, a modifying reagent specific for tryptophan residues in proteins [21], and by N-bromosuccinimide, a reagent specific for tryptophan and tyrosine residues [22]. Since a number of proteases have been shown to possess mitogenic activity [23,24], we were interested in examining whether treatment of membranes with protease inhibitors would lead to a reduction in mitogenic activity. However, treatment with soybean trypsin inhibitor (Table lII), N-a-p-tosyl-L-lysine-chloromethyl ketone (0.56 mM) or L- 1- tosylamide- 2- phenylethyl- chloromethylketone (0.56 mM) did not reduce activity below control (data not shown), suggesting that a trypsin or chymotrypsin-like protease is probably not responsible for the DNA replication stimulatory activity. Conclusion

The present work shows, thus, that addition of purified hepatic membranes to quiescent cultures of fibroblastic cells results in a stimulation of nuclear DNA replication, and the results strongly suggest that the activity is associated with a protein. This stimulation of DNA synthesis by membranes is not a simple consequence of its particulate nature, since NR-6 membranes are devoid of activity. In addition, treatment of membranes with trypsin or protein-modifying agents do not cause any breakdown of particles, but brings about a drastic reduction in mitogenic activity. Thus, this membrane-associated mitogenic activity raises some pertinent questions regarding cell proliferation in vivo. Growth control in vivo is mediated by various types of long and short range interactions. The long range control is seen best with hormones or humoral factors which interact with various organs located at great distances from each other [1]. The second type of control is seen only between neighboring cells, and the effect may be either inhibitory [2,16] or stimulatory [17]. Stimulatory interactions between cells could be due to either (a) the provision of a proper substratum or an extracellular matrix by the supporting cell [25]; or (b) the production of soluble growth factors

138

which act only within a short range due to their labile nature; or (c) the presence of plasma membrane associated mitogenic factors on the surfaces of supporting cells [17]. The results reported here strongly suggest a role for plasma membraneassociated growth factors in stimulatory shortrange interactions between cells. Since the activity is plasmalemmal and incapable of exerting its influence beyond a fixed boundary, anatomic location will be the critical determining factor in the choice of target cells. Therefore, it is of interest to examine the cellular localization and target cell specificity within liver of this plasmalemmal mitogenic mediator. Acknowledgments We thank Mark Pittenger for assistance in the preparation of plasma membrane-enriched fraction from mouse liver. This work was supported by Research Grants (AM-25819 and AM-25724) awarded to M.D. from the National Institutes of Health. M.D. is a Research Career Development Awardee of the National Institutes of Health (AM -00693). References l Sato, G. and Ross, R. (1979) in Hormones and Cell Culture, Cold Spring Harbor Conference on cell proliferation series, Vol. 6 2 Dulbecco, R. and Stoker, M.G.P. (1970) Proc. Natl. Acad. Sci. USA 66, 204-210 3 Frazier, W. and Glaser, L. (1979) Annu. Rev. Biochem 48, 491-523

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