Cell Tissue Kinet. (1979) 12, 153-160.
EFFECT O F A N INHIBITING FACTOR ISOLATED F R O M R A T LIVER O N D N A P O L Y M E R A S E S I N R E G E N E R A T I N G R A T LIVER G . P I E T U , N . M U N S C H , * S. M O U S S E T *A N D C. F R A Y S S I N E T *
U1I de I’INSERM, Hospital P . Brousse, and *Institut de Recherches ScientiJ7ques sur le Cancer, Villejuif,France (Received 11 January 1978; revision received 10 May 1978)
ABSTRACT
We partially purified an inhibitory factor (LIF), isolated from 105,000 g supernatant of a saline adult rat liver homogenate. LIF stopped in uitro cell multiplication by blocking the GI-S transition, and reduced in uiuo [3H]thymidine incorporation into liver DNA in two-thirds hepatectomized rats. This reduction in DNA synthesis was observed at 24 hr after hepatectomy, even when the LIF was injected before the beginning of the S phase, 10hr after hepatectomy, i.e. when DNA polymerase activity had not yet increased. Under these experimental conditions, LIF in uiuo treatment prevented a DNA polymerase activity from increasing after partial hepatectomy, so that enzyme activity at 24 hr in LIF-treated rats decreased compared to the controls. No direct inhibitory effect of LIF on a DNA polymerase was detected. LIF did not affect p DNA polymerase. These results suggest that LIF plays a part in controlling liver growth.
The mechanisms which control liver growth and enable partial hepatectomy to be followed by compensatory hypertrophy and hyperplasia are undoubtedly complex. On the one hand, the existence of stimulating factors has been shown (Short et al., 1972; Nadal & Boffa, 1975; Richman et al., 1976; Bucher & Patel, 1977); on the other, several authors, since Saetren’s work was published (1956), have described inhibiting factors capable of exerting feedback control (Higueret et al., 1975; Nadal, Lombard & Zajdela, 1976; Onda & Yoshikawa, 1975; Vinet & Verly, 1976; Nilsson, 1976). Although the physiological importance of the factors described is difficult to establish, the balance attained between some of them certainly does much to ensure the organ’s homeostasis. During the past few years, we began to purify factors inhibiting cell multiplication. They were isolated from rat liver homogenate. The factors isolated correspond to protein fractions with a molecular weight of 80,000 as measured by ultracentrifugation. Factors of an identical nature are found in the perfusate of the organ, leading to the idea that these products are Correspondence: Dr C. Frayssinet, Unit6 de Pathologie Cellulaire, IRSC BP N 8, 94 800 Villejuif, France. 0008-8730/79/0200-0153%02.00 01979 Blackwell Scientific Publications 153
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excreted by the liver in the bloodstream. Inhibitory activity was estimated by inhibiting the multiplication of L F hepatoma cells in culture and measuring the inhibition by cell counts. Working on synchronized cell cultures in vitro, we showed that the inhibitory action of liver extracts worked by blocking the G, to S phase transition (Aujard, Chany & Frayssinet, 1973). These extracts were also active in vivo: thus when injected 10 hr after hepatectomy into partially hepatectomized rats, they inhibited [3Hlthymidine incorporation into liver D N A at 24 hr (Higueret et af., 1975). In partially hepatectomized rats, the variations in the activity of key enzymes for D N A synthesis were well established. Such activity increased considerably 12 hr after surgery. When the inhibiting factor was injected 10 hr after the operation, it was present for 2 hr before the main enzymes increased. We tried to demonstrate whether under these conditions it was possible to show evidence for an effect on ct and p D N A polymerase activity in animals injected with inhibitor. MATERIALS AND METHODS
Animals, isotopes and reagents Male Wistar-Commentry rats weighing 150-180 g each were used. [3HlThymidine was supplied by the CEA (France) and dATP,* dTTP, d G T P and dCTP, by BoehringerMannheim (West Germany). [Methyl-3Hlthymidine 5'-triphosphate came from TRC, Amersham, England. Estimation of inhibitory activity In vitro activity. We used an L F hepatoma cell culture which acted as a screening test for purification, and applied the technique published earlier (Chany 8c Frayssinet, 197 1). Increasing dilutions in PBS of the sample to be tested were added to the cell culture medium at a proportion of 1 : 10 v/v; the corresponding protein concentrations were expressed as pg/ml of final culture medium. We measured its effect on the multiplication of cultured LF hepatoma cells by counting the cells 24 hr after seeding. Inhibition was then measured by comparing the number of cells in the tubes containing the extract to be tested with the number of cells in control tubes containing PBS buffer alone. In the present experiments the inhibitory activity was obtained with protein concentration of 1.O pg/ml of culture medium containing lo5 cells per ml. The lack of cytotoxicity of the fractions was tested by recording the viability of the cells after changing the culture medium. Fractions which were active in vitro were tested in vivo. In vivo activity. Liver D N A synthesis was measured by ['Hlthymidine incorporation, in two-thirds hepatectomized animals which were given the inhibitory preparation by intravenous injection. Preparation of the inhibitory factor The starting material was the 105,000 g supernatant of a rat liver homogenate in PBS (1 :4, w/v). All purification stages were performed at temperatures of 0 to 4OC, according to the technique published earlier (Higueret et al., 1975). These comprised: (a) acid precipitation at * Abbreviations: ['HITdR, tritiated thyrnidine; dATP, deoxyadenosine triphosphate; dTTP, deoxythyrnidine triphosphate; dGTP, deoxyguanosine triphosphate; dCTP, deoxycytidine triphosphate; PBS, phosphate-buffered saline.
Effectof LIF on DNA polymerases
155
pH 4.8 of the 105,000 g supernatant by 2.0 M acetic acid. The inhibitory fraction remained soluble and the precipitate containing half of the proteins was inactive. (b) Molecular filtration on an LKB ACA-4-4 acrylamide agarose Ultrogel column. Five peaks were obtained by this technique. Only the third, the AC, peak, showed inhibitory activity. Inhibitory activity proved reproducible in successive preparations of AC, fractions, which were the fractions studied in vivo. The active AC, fraction’s molecular weight was estimated at 80,000 daltons.
Biochemical measurements Proteins were measured by Lowry’s method. DNA and RNA estimations were made by the Schmidt and Tannhauser technique. Tritiated thymidine incorporation was counted by liquid scintillation. DNA polymerase was estimated according to Bollurn’s technique: the enzyme activity in the liver of treated animals was measured in the 105,000g post-microsomal supernatant. Incubation conditions were as follows. For a DNA polymerase, 50 mM Tris-HC1 buffer, pH 7 . 5 5 - 0 mM MgCl,, 5.0 mM KC1, 100 or 1 0 0 0 p activated ~ DNA, 2 0 0 dNTP, ~ ~ 1.0 pCi [,H]dTTP and 12 p l of the supernatant to be tested. For p DNA polymerase: 50 mM M DNA, Tris-HC1 buffer, pH 8 . 5 , 5.0 mM MgCl,, 100 mM KCl, 100 or 1000 ~ U activated 200 p~ dNTP, 1.O pCi [,H]dTTP and l2pl of the supernatant to be tested. After incubation for 1 hr at 37OC, the acid-insoluble fraction of an aliquot was precipitated on a Whatman GF/C filter disc as previously described and counted by liquid scintillation. Results are expressed in pmol of deoxynucleotide incorporated into the acid-insoluble fraction per mg protein or per ml of preparation in 60 min at 37OC. Experimental procedure Each series of experiments comprised three batches of animals. At time t = 0, they underwent a two-thirds hepatectomy in accordance with Higgins and Anderson’s traditional technique. In order to avoid diurnal variations, surgery was always performed at the same time: 10.00 hours. Batch 1 was killed 10 hr after hepatectomy. At that time, DNA polymerase activity had not yet increased and thus we measured the initial activity. Batch 2 animals were used as controls. At 10 hr after hepatectomy, they were given either an intravenous injection of 4.0 ml PBS or an injection comprising an amount of non-inhibitory proteins equal to the amount administered to the treated animals. Batch 3 was given, at 10 hr after hepatectomy, an intravenous injection of 20 to 25 mg of purified inhibitory fractions. The animals in batches 2 and 3 were killed 24 hr after hepatectomy. 1 hr before killing, they were given an intraperitoneal injection of 50 pCi tritiated thymidine. Livers were removed immediately after killing and homogenized at 0°C in 200 ml Tris-HC1, 50 mM, pH 7.5, 1.0 mM MgCl,, 6.0 m M KCl, 1.0 mM mercaptoethanol and 0.25 M saccharose. An aliquot of this homogenate was used to measure specific DNA activity and the rest was centrifuged for 2 hr at 105,000 g, and DNA polymerase activity was measured in this supernatant. RESULTS
Inhibition of liver DNA synthesis As described in the Materials and Methods section, treated animals were hepatectomized and 10 hr later were given an amount of inhibiting factor designed to block [,H]thyrnidine incorporation into liver DNA. The inhibitory activity of the factor injected had previously been tested on cell cultures, using the techniques described in the Materials and Methods
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section. The amount of inhibitory fraction injected was expressed in mg of protein. Since the preparation of the active component of the AC, peak was very reproducible, identical quantities of proteins could be considered to contain the same quantity of inhibitor. We conducted four series of experiments. The inhibition of [3Hlthymidine incorporation into D N A is shown in Table 1. It may be seen from this table that DNA-specific activity was considerably reduced. Compared with the controls, the percentage of inhibition in treated animals, which may be large, depended on the quantity of inhibitor injected. In experiment (a), involving a smaller amount of inhibitor, the percentage inhibition was proportionately TABLE1. Effect of inhibitory preparations on in cirro liver D N A synthesis
Experiment
Amount injected per animal (mg)
Acid-soluble fraction ( x
Specific activity DNA ( X lo-’)?
Inhibition (%)
(a) Control (4)$ Inhibited (4)
PBS 4.0 ml 10 mg
44 f 7.0 42 f 4.2
9.8 f 1.7 6.0 f 1.5
38
(b) Control (5) Inhibited (5)
PBS 4.0 ml 20 mg
45 f 3.0 61 f 7.0
12 & 0.4 3.0 f 0.6
14
(c) Control (10) Inhibited (10)
PBS 4.0 ml 20 mg
80 f 10 81 f 6.7
6.8 & 0.95 2.1 & 0.26
69
(d) Control (3) Verification (3)
PBS 4.0 ml Inactive proteins 1 5 me. 15 mg
70 f 7.6 66 f 8.5
3.3 0.9 2.46 f 0.35
65 2 3.5
0.57 ? 0.05
Inhibited (8)
82
Values represent means ? s.e. The animals were hepatectomized at time zero. At 10 hr they were given the different preparations by intravenous injection. At 23 hr, they were injected intraperitoneally with 50 pCi [’Hlthymidine and were killed at 24 hr.
* Expressed in d/min per g liver. t Expressed in d/min per mg DNA. $(
) Number of animals in parentheses.
5 Non-significant variations in relation to the control; P > 0.05.
less. In experiment (d) we injected control animals with proteins which were noninhibitory on LF hepatoma cultured cells. Doses were equivalent to those given to treated animals. In this way we eliminated the risk of non-specific inhibitory action due to protein overloading in the animals at the time of intravenous injection. Inactive proteins had no effect on DNA synthesis. Although a single point in time is not sufficient to establish permanent inhibition we can conclude that the incorporation of [3Hlthymidinewas temporarily inhibited in treated animals. In addition, the kinetics we obtained with synchronous cultures of hepatoma cells (Aujard et al., 1973) showed a similar phenomenon: an inhibition of i3HIthymidine incorporation in treated cells as compared to the controls. The value of the acid-soluble fraction was proportional to the amount of radioactive precursor which penetrated the cells without being incorporated into the DNA. This value was clearly not affected by injection of the inhibition product. Liver D N A synthesis after partial hepatectomy was therefore reduced by injection of the inhibitory factor. Such reduction was not caused by decreased penetration of the radioactive precursor into the cells but by the incorporation of the precursor into the DNA. Our results are in agreement with those published earlier by Higueret et al. (1975).
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157
Effects on a and ,Ll DNA polymerase activity in vivo We observed variations in a and p D N A polymerase activity under the experimental conditions described above. The results concerning a D N A polymerase activity are shown in Table 2. At 10 hr after hepatectomy, the value for a D N A polymerase activity still corresponded, in all experiments, to that of liver tissue in the resting state. At 24 hr after hepatectomy, a D N A polymerase activity in the controls showed a very large increase. This result agreed with the observations in the literature reporting enzymatic induction after 10 hr posthepatectomy . TABLE2. Inhibition of the increase in a DNA polymerase activity produced by inhibitory fractions
DNA polymerase activity*
Experiment
10 hr after hepatectomy (batch 1)
24 hr after hepatectomy : control (batch 2)
24 hr after hepatectomy : treated (batch 3)
Increase in enzyme activity (YO)
Control
Treated
(a) (-38)t
53
* 10
295 f 80
109 f 20
219
33
(b) (-74)
85 ? 4.0
293 L- 30
117 f 30
290
2.6
692 k 66
254 k 50
244
5.0
(c) (-69)
212 f 25
~
~
Values represent means k s,e. The number of animals treated is shown in Table 1. The variations between experiments (b) and (c) as regards DNA polymerase activity were due to the difference in the active DNA used.
* pmol/mg protein per 60 min: Enzyme activity was measured for 60 min at 37OC in 60 p l reaction mixture, under the conditions described in the Materials and Methods section. t Inhibition of DNA specific activity as given in percentages in Table 1.
In the animals injected with the inhibitory factor, 24 hr after hepatectomy, values for a D N A polymerase activity rose little or not at all in experiments (b) and (c) and were close to those of the controls at 10 hr post-hepatectomy. It should be noticed that in experiment (a), the amount of inhibiting factor injected, which was less than in series (b) and (c), was nevertheless sufficient to cause a considerable reduction D N A synthesis, measured by thymidine incorporation (Table 1). In experiment (a) at 24 hr after hepatectomy, the a D N A polymerase activity rose significantly in the treated animals as compared to controls at 10 hr; however, this increase was smaller in the treated animals than in the controls. Although the D N A polymerase activity did not rise significantly (experiments b and c), there was also, however, significant D N A synthesis with respect to the controls. The following explanations may be proposed: (a) beside the a D N A polymerase, acting mainly during the D N A synthesis, the role of D N A polymerase is not negligible. (b) Other factors are involved during in vivo D N A synthesis, particularly at the level of the precursor pool, whose role cannot be evaluated in the standardized conditions used in vitro to measure the a polymerase activity. Taking into account these restrictive considerations, the correlation was satisfactory between the dose of inhibiting factor injected, its effect on D N A synthesis, and its effect on the increase in D N A polymerase. The results concerning p D N A polymerase activity are set out in Table 3. In our experiment, few variations were observed in p D N A polymerase activity between 10 and 24
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158
TABLE3. Absence of effects on B DNA polymerase activity after injection of inhibitory preparations
p D N A polymerase activity*
Experiment
Specific inhibition of DNA activity (%)
10 hr after hepatectomy (batch 1)
24 hr after hepatectomy : control (batch 2)
24 hr after hepatectomy: treated (batch 3)
38 74
79 f 3 . 5 t 107 f 10t
126 k 2 8 t 127 f l l t
119 f 6 . 0 t 142 f 2 1 t
Values represent means k s.e.
* p m o l h g protein per 60 min: enzyme activity was measured for 60 pmin at 37OC in 100 p l reaction mixture, under the conditions described in the Materials and Methods section. t Non-significant variations: P > 0.05. hr, after hepatectomy, and the injection of inhibiting factor did not change this enzyme’s activity. The inhibiting factor therefore has no effect on the activity of p DNA polymerase, at least during the initial stages of liver regeneration.
Absence of a direct effect on a DNA polymerase The absence of an increase in a DNA polymerase activity might be due to many causes. The presence in the medium of an inhibitor exerting direct action by blocking polymerase activity or a reduction in the enzyme content itself may be involved. In a first attempt to solve this problem, we added cytosol from treated animals whose polymerase activity had been inhibited, to cytosol from controls whose polymerase was active. If any polymerase inhibitor had been present in the supernatant from treated animals, the DNA polymerase activities of the mixture measured experimentally would have been less than the arithmetic mean of both activities, measured separately. The results are shown in Table 4, which shows no significant difference between the theoretical and measured means. In another attempt we added the inhibitory fraction directly to cytosols from animals which had been hepatectomized 24 hr previously and whose DNA polymerase activity had already increased. We added two different concentrations of inhibitory factor. The controls were given an inactive protein fraction. Both concentrations were chosen so that the weakest of the two correspond to the maximum amount of inhibitor able to reach the liver in the course of in vivo treatment. Table 5 shows that for the protein concentrations reached, there was no direct effect on cc DNA polymerase in uitro, and enzyme activity in the presence of the inhibitor was TABLE 4. Absence of inhibitory action of liver cytosol of treated animals on a DNA polymerase from hepatectomized rat liver cytosol ~~
Control plus treated Control
Treated
Calculated
Measur ed
3800 k 196
992 & 80
2413 k 94
2679 f 147
Values represent means k s.e. The value given for DNA polymerase activity is expressed in pmol/ml per 60 min. These results are the mean of measurements on fourteen animals.
Eflect of LIF on DNA polymerases
159
TABLE5. Absence of direct action by the inhibitory factor on a DNA polymerase activity in uitro+
No addition
PIUS1.5 pg inactive protein?
PIUS1 . 5 f i g inhibitory protein?
3200
2900 f 44
2700
564
331
Plus 15 pg inactive protein?
Plus 15 pg inhibitory protein?
2430 & 129
2100 f 450
Values represent means & s.e.
* Results in pmol/ml per 60 min. f Amount of protein added to 50 pl medium.
not reduced in comparison to the controls. There was consequently no evidence that a factor which directly inhibits a polymerase was present in the cytosol from treated animals. This leads one to believe that the inhibiting factor prevents the increase of a DNA polymerase normally occurring following partial hepatectomy. DISCUSSION The derepression of ‘key’ enzymes of DNA synthesis-and a D N A polymerase is important in this respect-constitutes an essential stage in liver cell division. This process enables quiescent cells to pass from Go or G, to S phase and requires intervention at the gene level itself. Aujard et af.(1978) showed that an active liver homogenate extract can block the passage of cells in synchronous cultures from G, to S. The present work showed that the increase in a DNA polymerase activity had been blocked, and that the blocking had not been caused by a direct inhibition of the enzyme. It occurred when the inhibitor was injected 10 hr after hepatectomy, that is, before a DNA polymerase derepression and therefore before the period corresponding to the G,-S transition. On the other hand, p polymerase, which has little influence on the mechanisms governing liver regeneration and is not stimulated after partial hepatectomy, did not appear to be sensitive to the inhibitory activity. These results support the idea that the target of the inhibitory fractions is not the polymerases themselves but some previous event. It is thus possible to grasp the biochemical in vivo translation of the facts observed in cell cultures. It should be stressed that these inhibiting factors are free of toxicity. Blocking induced in cell cultures can be reversed by changing the medium and animals treated in viuo recover within 24 hr. Many inhibiting factors have been described. Some have low molecular weight (Deschamps & Verly, 1975; Sekas & Cook, 1976), others act by interfering with DNA polymerization itself. Nakai (1976), who examined the action mechanism of a chalone from the Ehrlich ascites tumour, showed that this instantly active product inhibited a and p polymerases, probably non-specifically. We think our preparations should be compared to those described by Vinet & Verly (1976) who isolated, from rabbit liver, a 40,000 dalton protein capable of blocking thymidine phosphate conversion into DNA in Novikoff hepatoma cells. Working on human liver, Nilsson (1 976) isolated a 90,000 dalton protein fraction capable of quickly inhibiting thymidine incorporation into DNA. Lastly, Nadal et af. (1976) showed the presence in rat liver of a factor with a molecular weight exceeding 10,000 which reduced liver cell sensitivity to the stimuli initiating DNA replication. This factor is certainly very
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G.Pietu et al.
closely related if not identical to the molecules we isolated. Such molecules, synthesized by the liver, are easily excreted into the bloodstream (Molimard et al., 1975) and when injected intravenously, are capable of maintaining liver cells in a resting state after partial hepatectomy. They therefore fulfill the essential requirements which characterize cell growth regulators. ACKNOWLEDGMENTS
This work was financially supported by INSERM, under contract No. 40-76-72. REFERENCES AUJARD, C., CHANY,E. & FRAYSSINET, C. (1973) Inhibition of DNA synthesis of synchronized cells by liver extracts acting in vitro. Exp. Cell Res. 78,476. BUCHER,N. & PATEL,U. (1977) Effects of epidermal growth factor (EGF) and Pancreatic hormones on growth of rat liver. J. Cell Biol. 75, 195. CHANY,E. & FRAYSSINET,C. (1971) Presence dans le foie normal de substances inhibant pricocement la croissance de cultures de cellules cancereuses. C.R. Acad. Sci. (Paris), 272, 2644. DESCHAMPS, Y. & VERLY,W.G. (1975) The hepatic chalone. 11. Chemical and biological properties of the rabbit liver chalone. Biornedecine, 22, 195. HIGUERET,P., CHANY,E., MOUSSET,S., GARDES, M. & FRAYSSINET, C. (1975) Activite inhibitrice de fractions proteiques isolees de foie de rat sur I'hypertrophie compensatrice apres hepatectomie partielle, C.R. Acad. Sci. (Paris), 281, 49. MOLIMARD, R., PIETU,G., CHANY,E., TRINCAL,G . & FRAYSSINET, C. (1975) An inhibitor of hepatoma cell multiplication in the efferent fluid from isolated perfused rat liver. Biomedicine, 23, 434. NADAL,C. & BOFFA, G.A. (1975) Inhibitory and anti-inhibitory factors of rat serum active on the GI-S transition of hepatocyte cell cycle. Cell Tissue Kinet. 8, 297. NADAL,C., LOMBARD, M.N. & ZAJDELA,F. (1976) Inhibition of rat hepatocyte multiplications by serum and liver factors. Virchows Arch.path. Anat. Physiol. 20, 277. NAKAI,G.S. (1976) Ehrlich ascites tumour (EAT) chalone effects on nascent DNA synthesis and D N A polymerase alpha and beta. Cell Tissue Kiner. 9, 553. NILSSON,G. (1976) lnhibition of protein and nucleic acid synthesis of animal cells in vitro mediated by high molecular weight inhibitors in human liver extract. Biochim. Biophys. Acla (Amsl.),418,376. ONDA, H. & YOSHIKAWA, J.I. (1975) Q, glycoprotein as a hepatocyte-specific mitosis-inhibiting protein in regenerating rat liver. Gann. 66, 227. RICHMAN,R.A., CLAUS,T.A., PILKIS,S.J. & FRIEDMAN, D. (1976) Hormonal stimulation of DNA synthesis in primary cultures of adult rat hepatocytes. Proc. narl. Acad. Sci. (Wash.), 73, 3589. SAETREN, H. (1956) A principle of auto-regulation of growth. Exp. CellRes. 11, 229. SEKAS,G. & COOK.R.T. (1976) The isolation of a low molecular weight inhibition of ['HITdR incorporation into hepatic DNA. Exp. Cell Res. 102,422. SHORT,J., BROWN,R.F., HUSAKOVA, A.. GILBERTSON, J.R.,ZEMEL,R. & LIEBERMANN, I. (1972) Induction of deoxyribonucleic acid synthesis in the liver of the intact animal. J. biol. Chem. 247, 1757. VINET,B. & VERLY,W. (1976) Purification of a protein inhibitor of DNA synthesis in cells of hepatic origin. Europ. J . Cancer, 12, 2 I I .
EFFECT O F A N INHIBITING FACTOR ISOLATED F R O M R A T LIVER O N D N A P O L Y M E R A S E S I N R E G E N E R A T I N G R A T LIVER G . P I E T U , N . M U N S C H , * S. M O U S S E T *A N D C. F R A Y S S I N E T *
U1I de I’INSERM, Hospital P . Brousse, and *Institut de Recherches ScientiJ7ques sur le Cancer, Villejuif,France (Received 11 January 1978; revision received 10 May 1978)
ABSTRACT
We partially purified an inhibitory factor (LIF), isolated from 105,000 g supernatant of a saline adult rat liver homogenate. LIF stopped in uitro cell multiplication by blocking the GI-S transition, and reduced in uiuo [3H]thymidine incorporation into liver DNA in two-thirds hepatectomized rats. This reduction in DNA synthesis was observed at 24 hr after hepatectomy, even when the LIF was injected before the beginning of the S phase, 10hr after hepatectomy, i.e. when DNA polymerase activity had not yet increased. Under these experimental conditions, LIF in uiuo treatment prevented a DNA polymerase activity from increasing after partial hepatectomy, so that enzyme activity at 24 hr in LIF-treated rats decreased compared to the controls. No direct inhibitory effect of LIF on a DNA polymerase was detected. LIF did not affect p DNA polymerase. These results suggest that LIF plays a part in controlling liver growth.
The mechanisms which control liver growth and enable partial hepatectomy to be followed by compensatory hypertrophy and hyperplasia are undoubtedly complex. On the one hand, the existence of stimulating factors has been shown (Short et al., 1972; Nadal & Boffa, 1975; Richman et al., 1976; Bucher & Patel, 1977); on the other, several authors, since Saetren’s work was published (1956), have described inhibiting factors capable of exerting feedback control (Higueret et al., 1975; Nadal, Lombard & Zajdela, 1976; Onda & Yoshikawa, 1975; Vinet & Verly, 1976; Nilsson, 1976). Although the physiological importance of the factors described is difficult to establish, the balance attained between some of them certainly does much to ensure the organ’s homeostasis. During the past few years, we began to purify factors inhibiting cell multiplication. They were isolated from rat liver homogenate. The factors isolated correspond to protein fractions with a molecular weight of 80,000 as measured by ultracentrifugation. Factors of an identical nature are found in the perfusate of the organ, leading to the idea that these products are Correspondence: Dr C. Frayssinet, Unit6 de Pathologie Cellulaire, IRSC BP N 8, 94 800 Villejuif, France. 0008-8730/79/0200-0153%02.00 01979 Blackwell Scientific Publications 153
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G. Pietu et al.
excreted by the liver in the bloodstream. Inhibitory activity was estimated by inhibiting the multiplication of L F hepatoma cells in culture and measuring the inhibition by cell counts. Working on synchronized cell cultures in vitro, we showed that the inhibitory action of liver extracts worked by blocking the G, to S phase transition (Aujard, Chany & Frayssinet, 1973). These extracts were also active in vivo: thus when injected 10 hr after hepatectomy into partially hepatectomized rats, they inhibited [3Hlthymidine incorporation into liver D N A at 24 hr (Higueret et af., 1975). In partially hepatectomized rats, the variations in the activity of key enzymes for D N A synthesis were well established. Such activity increased considerably 12 hr after surgery. When the inhibiting factor was injected 10 hr after the operation, it was present for 2 hr before the main enzymes increased. We tried to demonstrate whether under these conditions it was possible to show evidence for an effect on ct and p D N A polymerase activity in animals injected with inhibitor. MATERIALS AND METHODS
Animals, isotopes and reagents Male Wistar-Commentry rats weighing 150-180 g each were used. [3HlThymidine was supplied by the CEA (France) and dATP,* dTTP, d G T P and dCTP, by BoehringerMannheim (West Germany). [Methyl-3Hlthymidine 5'-triphosphate came from TRC, Amersham, England. Estimation of inhibitory activity In vitro activity. We used an L F hepatoma cell culture which acted as a screening test for purification, and applied the technique published earlier (Chany 8c Frayssinet, 197 1). Increasing dilutions in PBS of the sample to be tested were added to the cell culture medium at a proportion of 1 : 10 v/v; the corresponding protein concentrations were expressed as pg/ml of final culture medium. We measured its effect on the multiplication of cultured LF hepatoma cells by counting the cells 24 hr after seeding. Inhibition was then measured by comparing the number of cells in the tubes containing the extract to be tested with the number of cells in control tubes containing PBS buffer alone. In the present experiments the inhibitory activity was obtained with protein concentration of 1.O pg/ml of culture medium containing lo5 cells per ml. The lack of cytotoxicity of the fractions was tested by recording the viability of the cells after changing the culture medium. Fractions which were active in vitro were tested in vivo. In vivo activity. Liver D N A synthesis was measured by ['Hlthymidine incorporation, in two-thirds hepatectomized animals which were given the inhibitory preparation by intravenous injection. Preparation of the inhibitory factor The starting material was the 105,000 g supernatant of a rat liver homogenate in PBS (1 :4, w/v). All purification stages were performed at temperatures of 0 to 4OC, according to the technique published earlier (Higueret et al., 1975). These comprised: (a) acid precipitation at * Abbreviations: ['HITdR, tritiated thyrnidine; dATP, deoxyadenosine triphosphate; dTTP, deoxythyrnidine triphosphate; dGTP, deoxyguanosine triphosphate; dCTP, deoxycytidine triphosphate; PBS, phosphate-buffered saline.
Effectof LIF on DNA polymerases
155
pH 4.8 of the 105,000 g supernatant by 2.0 M acetic acid. The inhibitory fraction remained soluble and the precipitate containing half of the proteins was inactive. (b) Molecular filtration on an LKB ACA-4-4 acrylamide agarose Ultrogel column. Five peaks were obtained by this technique. Only the third, the AC, peak, showed inhibitory activity. Inhibitory activity proved reproducible in successive preparations of AC, fractions, which were the fractions studied in vivo. The active AC, fraction’s molecular weight was estimated at 80,000 daltons.
Biochemical measurements Proteins were measured by Lowry’s method. DNA and RNA estimations were made by the Schmidt and Tannhauser technique. Tritiated thymidine incorporation was counted by liquid scintillation. DNA polymerase was estimated according to Bollurn’s technique: the enzyme activity in the liver of treated animals was measured in the 105,000g post-microsomal supernatant. Incubation conditions were as follows. For a DNA polymerase, 50 mM Tris-HC1 buffer, pH 7 . 5 5 - 0 mM MgCl,, 5.0 mM KC1, 100 or 1 0 0 0 p activated ~ DNA, 2 0 0 dNTP, ~ ~ 1.0 pCi [,H]dTTP and 12 p l of the supernatant to be tested. For p DNA polymerase: 50 mM M DNA, Tris-HC1 buffer, pH 8 . 5 , 5.0 mM MgCl,, 100 mM KCl, 100 or 1000 ~ U activated 200 p~ dNTP, 1.O pCi [,H]dTTP and l2pl of the supernatant to be tested. After incubation for 1 hr at 37OC, the acid-insoluble fraction of an aliquot was precipitated on a Whatman GF/C filter disc as previously described and counted by liquid scintillation. Results are expressed in pmol of deoxynucleotide incorporated into the acid-insoluble fraction per mg protein or per ml of preparation in 60 min at 37OC. Experimental procedure Each series of experiments comprised three batches of animals. At time t = 0, they underwent a two-thirds hepatectomy in accordance with Higgins and Anderson’s traditional technique. In order to avoid diurnal variations, surgery was always performed at the same time: 10.00 hours. Batch 1 was killed 10 hr after hepatectomy. At that time, DNA polymerase activity had not yet increased and thus we measured the initial activity. Batch 2 animals were used as controls. At 10 hr after hepatectomy, they were given either an intravenous injection of 4.0 ml PBS or an injection comprising an amount of non-inhibitory proteins equal to the amount administered to the treated animals. Batch 3 was given, at 10 hr after hepatectomy, an intravenous injection of 20 to 25 mg of purified inhibitory fractions. The animals in batches 2 and 3 were killed 24 hr after hepatectomy. 1 hr before killing, they were given an intraperitoneal injection of 50 pCi tritiated thymidine. Livers were removed immediately after killing and homogenized at 0°C in 200 ml Tris-HC1, 50 mM, pH 7.5, 1.0 mM MgCl,, 6.0 m M KCl, 1.0 mM mercaptoethanol and 0.25 M saccharose. An aliquot of this homogenate was used to measure specific DNA activity and the rest was centrifuged for 2 hr at 105,000 g, and DNA polymerase activity was measured in this supernatant. RESULTS
Inhibition of liver DNA synthesis As described in the Materials and Methods section, treated animals were hepatectomized and 10 hr later were given an amount of inhibiting factor designed to block [,H]thyrnidine incorporation into liver DNA. The inhibitory activity of the factor injected had previously been tested on cell cultures, using the techniques described in the Materials and Methods
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section. The amount of inhibitory fraction injected was expressed in mg of protein. Since the preparation of the active component of the AC, peak was very reproducible, identical quantities of proteins could be considered to contain the same quantity of inhibitor. We conducted four series of experiments. The inhibition of [3Hlthymidine incorporation into D N A is shown in Table 1. It may be seen from this table that DNA-specific activity was considerably reduced. Compared with the controls, the percentage of inhibition in treated animals, which may be large, depended on the quantity of inhibitor injected. In experiment (a), involving a smaller amount of inhibitor, the percentage inhibition was proportionately TABLE1. Effect of inhibitory preparations on in cirro liver D N A synthesis
Experiment
Amount injected per animal (mg)
Acid-soluble fraction ( x
Specific activity DNA ( X lo-’)?
Inhibition (%)
(a) Control (4)$ Inhibited (4)
PBS 4.0 ml 10 mg
44 f 7.0 42 f 4.2
9.8 f 1.7 6.0 f 1.5
38
(b) Control (5) Inhibited (5)
PBS 4.0 ml 20 mg
45 f 3.0 61 f 7.0
12 & 0.4 3.0 f 0.6
14
(c) Control (10) Inhibited (10)
PBS 4.0 ml 20 mg
80 f 10 81 f 6.7
6.8 & 0.95 2.1 & 0.26
69
(d) Control (3) Verification (3)
PBS 4.0 ml Inactive proteins 1 5 me. 15 mg
70 f 7.6 66 f 8.5
3.3 0.9 2.46 f 0.35
65 2 3.5
0.57 ? 0.05
Inhibited (8)
82
Values represent means ? s.e. The animals were hepatectomized at time zero. At 10 hr they were given the different preparations by intravenous injection. At 23 hr, they were injected intraperitoneally with 50 pCi [’Hlthymidine and were killed at 24 hr.
* Expressed in d/min per g liver. t Expressed in d/min per mg DNA. $(
) Number of animals in parentheses.
5 Non-significant variations in relation to the control; P > 0.05.
less. In experiment (d) we injected control animals with proteins which were noninhibitory on LF hepatoma cultured cells. Doses were equivalent to those given to treated animals. In this way we eliminated the risk of non-specific inhibitory action due to protein overloading in the animals at the time of intravenous injection. Inactive proteins had no effect on DNA synthesis. Although a single point in time is not sufficient to establish permanent inhibition we can conclude that the incorporation of [3Hlthymidinewas temporarily inhibited in treated animals. In addition, the kinetics we obtained with synchronous cultures of hepatoma cells (Aujard et al., 1973) showed a similar phenomenon: an inhibition of i3HIthymidine incorporation in treated cells as compared to the controls. The value of the acid-soluble fraction was proportional to the amount of radioactive precursor which penetrated the cells without being incorporated into the DNA. This value was clearly not affected by injection of the inhibition product. Liver D N A synthesis after partial hepatectomy was therefore reduced by injection of the inhibitory factor. Such reduction was not caused by decreased penetration of the radioactive precursor into the cells but by the incorporation of the precursor into the DNA. Our results are in agreement with those published earlier by Higueret et al. (1975).
Effectof LIF on DNA polymerases
157
Effects on a and ,Ll DNA polymerase activity in vivo We observed variations in a and p D N A polymerase activity under the experimental conditions described above. The results concerning a D N A polymerase activity are shown in Table 2. At 10 hr after hepatectomy, the value for a D N A polymerase activity still corresponded, in all experiments, to that of liver tissue in the resting state. At 24 hr after hepatectomy, a D N A polymerase activity in the controls showed a very large increase. This result agreed with the observations in the literature reporting enzymatic induction after 10 hr posthepatectomy . TABLE2. Inhibition of the increase in a DNA polymerase activity produced by inhibitory fractions
DNA polymerase activity*
Experiment
10 hr after hepatectomy (batch 1)
24 hr after hepatectomy : control (batch 2)
24 hr after hepatectomy : treated (batch 3)
Increase in enzyme activity (YO)
Control
Treated
(a) (-38)t
53
* 10
295 f 80
109 f 20
219
33
(b) (-74)
85 ? 4.0
293 L- 30
117 f 30
290
2.6
692 k 66
254 k 50
244
5.0
(c) (-69)
212 f 25
~
~
Values represent means k s,e. The number of animals treated is shown in Table 1. The variations between experiments (b) and (c) as regards DNA polymerase activity were due to the difference in the active DNA used.
* pmol/mg protein per 60 min: Enzyme activity was measured for 60 min at 37OC in 60 p l reaction mixture, under the conditions described in the Materials and Methods section. t Inhibition of DNA specific activity as given in percentages in Table 1.
In the animals injected with the inhibitory factor, 24 hr after hepatectomy, values for a D N A polymerase activity rose little or not at all in experiments (b) and (c) and were close to those of the controls at 10 hr post-hepatectomy. It should be noticed that in experiment (a), the amount of inhibiting factor injected, which was less than in series (b) and (c), was nevertheless sufficient to cause a considerable reduction D N A synthesis, measured by thymidine incorporation (Table 1). In experiment (a) at 24 hr after hepatectomy, the a D N A polymerase activity rose significantly in the treated animals as compared to controls at 10 hr; however, this increase was smaller in the treated animals than in the controls. Although the D N A polymerase activity did not rise significantly (experiments b and c), there was also, however, significant D N A synthesis with respect to the controls. The following explanations may be proposed: (a) beside the a D N A polymerase, acting mainly during the D N A synthesis, the role of D N A polymerase is not negligible. (b) Other factors are involved during in vivo D N A synthesis, particularly at the level of the precursor pool, whose role cannot be evaluated in the standardized conditions used in vitro to measure the a polymerase activity. Taking into account these restrictive considerations, the correlation was satisfactory between the dose of inhibiting factor injected, its effect on D N A synthesis, and its effect on the increase in D N A polymerase. The results concerning p D N A polymerase activity are set out in Table 3. In our experiment, few variations were observed in p D N A polymerase activity between 10 and 24
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TABLE3. Absence of effects on B DNA polymerase activity after injection of inhibitory preparations
p D N A polymerase activity*
Experiment
Specific inhibition of DNA activity (%)
10 hr after hepatectomy (batch 1)
24 hr after hepatectomy : control (batch 2)
24 hr after hepatectomy: treated (batch 3)
38 74
79 f 3 . 5 t 107 f 10t
126 k 2 8 t 127 f l l t
119 f 6 . 0 t 142 f 2 1 t
Values represent means k s.e.
* p m o l h g protein per 60 min: enzyme activity was measured for 60 pmin at 37OC in 100 p l reaction mixture, under the conditions described in the Materials and Methods section. t Non-significant variations: P > 0.05. hr, after hepatectomy, and the injection of inhibiting factor did not change this enzyme’s activity. The inhibiting factor therefore has no effect on the activity of p DNA polymerase, at least during the initial stages of liver regeneration.
Absence of a direct effect on a DNA polymerase The absence of an increase in a DNA polymerase activity might be due to many causes. The presence in the medium of an inhibitor exerting direct action by blocking polymerase activity or a reduction in the enzyme content itself may be involved. In a first attempt to solve this problem, we added cytosol from treated animals whose polymerase activity had been inhibited, to cytosol from controls whose polymerase was active. If any polymerase inhibitor had been present in the supernatant from treated animals, the DNA polymerase activities of the mixture measured experimentally would have been less than the arithmetic mean of both activities, measured separately. The results are shown in Table 4, which shows no significant difference between the theoretical and measured means. In another attempt we added the inhibitory fraction directly to cytosols from animals which had been hepatectomized 24 hr previously and whose DNA polymerase activity had already increased. We added two different concentrations of inhibitory factor. The controls were given an inactive protein fraction. Both concentrations were chosen so that the weakest of the two correspond to the maximum amount of inhibitor able to reach the liver in the course of in vivo treatment. Table 5 shows that for the protein concentrations reached, there was no direct effect on cc DNA polymerase in uitro, and enzyme activity in the presence of the inhibitor was TABLE 4. Absence of inhibitory action of liver cytosol of treated animals on a DNA polymerase from hepatectomized rat liver cytosol ~~
Control plus treated Control
Treated
Calculated
Measur ed
3800 k 196
992 & 80
2413 k 94
2679 f 147
Values represent means k s.e. The value given for DNA polymerase activity is expressed in pmol/ml per 60 min. These results are the mean of measurements on fourteen animals.
Eflect of LIF on DNA polymerases
159
TABLE5. Absence of direct action by the inhibitory factor on a DNA polymerase activity in uitro+
No addition
PIUS1.5 pg inactive protein?
PIUS1 . 5 f i g inhibitory protein?
3200
2900 f 44
2700
564
331
Plus 15 pg inactive protein?
Plus 15 pg inhibitory protein?
2430 & 129
2100 f 450
Values represent means & s.e.
* Results in pmol/ml per 60 min. f Amount of protein added to 50 pl medium.
not reduced in comparison to the controls. There was consequently no evidence that a factor which directly inhibits a polymerase was present in the cytosol from treated animals. This leads one to believe that the inhibiting factor prevents the increase of a DNA polymerase normally occurring following partial hepatectomy. DISCUSSION The derepression of ‘key’ enzymes of DNA synthesis-and a D N A polymerase is important in this respect-constitutes an essential stage in liver cell division. This process enables quiescent cells to pass from Go or G, to S phase and requires intervention at the gene level itself. Aujard et af.(1978) showed that an active liver homogenate extract can block the passage of cells in synchronous cultures from G, to S. The present work showed that the increase in a DNA polymerase activity had been blocked, and that the blocking had not been caused by a direct inhibition of the enzyme. It occurred when the inhibitor was injected 10 hr after hepatectomy, that is, before a DNA polymerase derepression and therefore before the period corresponding to the G,-S transition. On the other hand, p polymerase, which has little influence on the mechanisms governing liver regeneration and is not stimulated after partial hepatectomy, did not appear to be sensitive to the inhibitory activity. These results support the idea that the target of the inhibitory fractions is not the polymerases themselves but some previous event. It is thus possible to grasp the biochemical in vivo translation of the facts observed in cell cultures. It should be stressed that these inhibiting factors are free of toxicity. Blocking induced in cell cultures can be reversed by changing the medium and animals treated in viuo recover within 24 hr. Many inhibiting factors have been described. Some have low molecular weight (Deschamps & Verly, 1975; Sekas & Cook, 1976), others act by interfering with DNA polymerization itself. Nakai (1976), who examined the action mechanism of a chalone from the Ehrlich ascites tumour, showed that this instantly active product inhibited a and p polymerases, probably non-specifically. We think our preparations should be compared to those described by Vinet & Verly (1976) who isolated, from rabbit liver, a 40,000 dalton protein capable of blocking thymidine phosphate conversion into DNA in Novikoff hepatoma cells. Working on human liver, Nilsson (1 976) isolated a 90,000 dalton protein fraction capable of quickly inhibiting thymidine incorporation into DNA. Lastly, Nadal et af. (1976) showed the presence in rat liver of a factor with a molecular weight exceeding 10,000 which reduced liver cell sensitivity to the stimuli initiating DNA replication. This factor is certainly very
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G.Pietu et al.
closely related if not identical to the molecules we isolated. Such molecules, synthesized by the liver, are easily excreted into the bloodstream (Molimard et al., 1975) and when injected intravenously, are capable of maintaining liver cells in a resting state after partial hepatectomy. They therefore fulfill the essential requirements which characterize cell growth regulators. ACKNOWLEDGMENTS
This work was financially supported by INSERM, under contract No. 40-76-72. REFERENCES AUJARD, C., CHANY,E. & FRAYSSINET, C. (1973) Inhibition of DNA synthesis of synchronized cells by liver extracts acting in vitro. Exp. Cell Res. 78,476. BUCHER,N. & PATEL,U. (1977) Effects of epidermal growth factor (EGF) and Pancreatic hormones on growth of rat liver. J. Cell Biol. 75, 195. CHANY,E. & FRAYSSINET,C. (1971) Presence dans le foie normal de substances inhibant pricocement la croissance de cultures de cellules cancereuses. C.R. Acad. Sci. (Paris), 272, 2644. DESCHAMPS, Y. & VERLY,W.G. (1975) The hepatic chalone. 11. Chemical and biological properties of the rabbit liver chalone. Biornedecine, 22, 195. HIGUERET,P., CHANY,E., MOUSSET,S., GARDES, M. & FRAYSSINET, C. (1975) Activite inhibitrice de fractions proteiques isolees de foie de rat sur I'hypertrophie compensatrice apres hepatectomie partielle, C.R. Acad. Sci. (Paris), 281, 49. MOLIMARD, R., PIETU,G., CHANY,E., TRINCAL,G . & FRAYSSINET, C. (1975) An inhibitor of hepatoma cell multiplication in the efferent fluid from isolated perfused rat liver. Biomedicine, 23, 434. NADAL,C. & BOFFA, G.A. (1975) Inhibitory and anti-inhibitory factors of rat serum active on the GI-S transition of hepatocyte cell cycle. Cell Tissue Kinet. 8, 297. NADAL,C., LOMBARD, M.N. & ZAJDELA,F. (1976) Inhibition of rat hepatocyte multiplications by serum and liver factors. Virchows Arch.path. Anat. Physiol. 20, 277. NAKAI,G.S. (1976) Ehrlich ascites tumour (EAT) chalone effects on nascent DNA synthesis and D N A polymerase alpha and beta. Cell Tissue Kiner. 9, 553. NILSSON,G. (1976) lnhibition of protein and nucleic acid synthesis of animal cells in vitro mediated by high molecular weight inhibitors in human liver extract. Biochim. Biophys. Acla (Amsl.),418,376. ONDA, H. & YOSHIKAWA, J.I. (1975) Q, glycoprotein as a hepatocyte-specific mitosis-inhibiting protein in regenerating rat liver. Gann. 66, 227. RICHMAN,R.A., CLAUS,T.A., PILKIS,S.J. & FRIEDMAN, D. (1976) Hormonal stimulation of DNA synthesis in primary cultures of adult rat hepatocytes. Proc. narl. Acad. Sci. (Wash.), 73, 3589. SAETREN, H. (1956) A principle of auto-regulation of growth. Exp. CellRes. 11, 229. SEKAS,G. & COOK.R.T. (1976) The isolation of a low molecular weight inhibition of ['HITdR incorporation into hepatic DNA. Exp. Cell Res. 102,422. SHORT,J., BROWN,R.F., HUSAKOVA, A.. GILBERTSON, J.R.,ZEMEL,R. & LIEBERMANN, I. (1972) Induction of deoxyribonucleic acid synthesis in the liver of the intact animal. J. biol. Chem. 247, 1757. VINET,B. & VERLY,W. (1976) Purification of a protein inhibitor of DNA synthesis in cells of hepatic origin. Europ. J . Cancer, 12, 2 I I .
