Inhibiting effect of plasma from normal and tumour bearing mice on the mitotic rate of regenerating liver.

Plasma from normal mice and from mice bearing the ES2 transplantable malignant tumour was injected intraperitoneally at a dose of 0.01 ml/g body weight in partially hepatectomized mice. Control animals were injected with a solution of sodium citrate in saline. The recipients were killed at the first (14:00 hours/48 h). These times are the time of day and the number of h after partial hepatectomy and second (14:00 hours/72 h) peak times after partial hepatectomy. The number of colchicine metaphases per 1000 nuclei was determined for hepatocytes and litoral cells. A different effect was obtained with plasma from tumour-bearing compared with normal mice. Plasma from both sources when injected 26 h after partial hepatectomy (16:00 hours/26 h) inhibited the mitotic activity of hepatocytes at the next peak of regenerative activity (14:00 hours/48 h). The plasma from tumour-bearing mice also inhibited the peak on the following day (14:00 hours/72 h), whereas plasma from normal mice had no inhibitory effect and, indeed, a compensatory wave was observed at this time. Furthermore, plasma from tumour-bearing mice also showed an inhibitory effect at the first peak (14:00 hours/48 h) when injected at the time of partial hepatectomy (14:00 hours/00 h) or at 22 h before partial hepatectomy (16:00 hours/-22 h) whereas the injection of plasma from normal mice at these times had no inhibitory effect. In the litoral cells the injection of plasma from tumour-bearing mice made 22 h before hepatectomy (16:00 hours/-22 h) led to a stimulation of mitotic activity which was controlled at 14:00 hours/48 h.(ABSTRACT TRUNCATED AT 250 WORDS)


Introduction
We have recently reported an inhibitory effect of malignant tumour extracts on the mitotic rate of regenerating liver (Echave Llanos et al. 1985;Badr/m et al. 1985b) and the detailed time pattern of this inhibitory effect for one of these tumours (Echave Llanos et al. 1986).The plasma from mice bearing these tumours also showed a clear inhibitory effect on the mitotic rate of regenerating liver (Badr/m et al. 1985 a).
The effect of plasma from normal or hepatectomized mice, injected or delivered by parabiosis or cross circulation, has been reviewed by Echave Llanos (1963) Croisille and Le Douarin (1965) and Bucher (1963) and more recently by Nadal (1979).
In the present paper we report differences between the time patterns of the inhibitory effect on the mitotic rate of regenerating liver, shown by plasma obtained from normal and tumour-bearing mice.

Materials and methods
Tumour.The tumour used for the present experiments is the ES2 transplantable malignant tumour described previously (Echave Llanos et al. 1986).
Plasma.The plasma was prepared by collecting blood from ten normal and ten tumour-bearing mice in 3% sodium citrate (final dilution 9:1) followed by centrifugation at 4,000 rpm at 0~ for 10 min.The plasma was immediately injected intraperitoneally into the recipients.All manipulations were performed under aseptic conditions and all the instruments were cooled before use.
Injections of plasma and saline.The plasma was administered in a dose of 0.01 ml plasma/g body weight.The controls received a solution of sodium citrate in saline only.
According to the results obtained previously with the tumour extract (Echave Llanos et al. 1986) either plasma or saline were given as a single intraperitoneal injection at three different times relative to partial hepatectomy, i.e. at 22 h before hepatectomy, at hepatectomy, or 26h after hepatectomy.The animals were killed at the first (14:00 hours/48 h).These times are the time of day and the number of h after partial hepatectomy and second (14:00 hours/72 h) peak times of mitotic activity in the regenerating liver after partial hepatectomy.
Recipients.A total of 134 C3HS male mice, aged 90 days, were used as recipients.The selection, standardization and the procedure for partial hepatectomy of the animals, and the methods of determination of the mitotic activity were as described previously (Echave Llanos et al. 1986).

Results
The results are given in Fig. 1 and Table 1.Fig. 1 shows the mitotic rates in hepatocytes and litoral cells at the times of the first two peaks after partial hepatectomy and after the injection of normal and tumour-bearing mouse plasma at the three times described above.
Plasma from normal mice produced no effect on the mitotic rate of hepatocytes when injected at the time of partial hepatectomy (14:00 hours/ 00 h) or 22 h before partial hepatectomy (16:00 hours/-22 h), but led to significant inhibition of the mitotic rate at the first peak (14:00 hours/48 h) when injected 26 h after partial hepatectomy (16:00 hours/26 h).At the time of the second peak (14:00 hours/72 h) animals injected with normal plasma at 16:00 hours/26 h showed much higher mitotic rates than control animals.
The injection of plasma from tumour-bearing mice led to significant inhibition of the mitotic rate of hepatocytes when given at any of the three times employed.When the injection was made 26 h after partial hepatectomy (16:00 hours/26 h) the inhibitory effect was still present at the second peak (14:00 hours/72 h).No change was observed in the mitotic rate of litoral cells at 14:00 hours/ 48 h or at 14:00 hours/72 h after injection of plasma from normal mice at any of the three times employed.
The injection of plasma from tumour-bearing mice made 22 h before partial hepatectomy (16:00 hours/-22 h) produced significant stimulation of mitotic activity in litoral cells at 14:00 hours/48 h whereas injection at the time of partial hepatectomy (14:00 hours/00 h) led to significant inhibition.No effect was observed on the mitotic rate of litoral cells at 14:00 hours/48 h when the injection was given 26 h after partial hepatectomy but there was significant inhibition at the second peak (14:00 hours/ 72 h).

Discussion
Our experiments have shown differences between plasma from tumour-bearing compared with normal mice in the pattern of their inhibitory effect on the mitotic rate of hepatocytes and litoral cells in regenerating liver.
Based on the different time patterns of reactions obtained, we believe that several explanations are possible.The lack of an apparent circadian time-dependance in the effect of the injections (Echave Llanos et al. 1986) does not exclude a possible circadian-time dependence in the effects either of several short-lived factors acting in given phase of the cell cycle, or of a single long-lived factor remaining inactive in the circulation until the appropriate sensitive phase of cell cycle arrives.
The effect of plasma from tumour-bearing mice on regenerating liver is similar to that of crude tumour extract (Echave Llanos et al. 1986), not only in the time patterns observed but also in the maximum lag of 60 h for these effects to occur.This is true for hepatocytes as well as for litoral cells, and we believe that this similarity supports the hypothesis that the responsible factors present in the plasma of tumour-bearing mice originate in the tumours.
The inhibitory effects of extracts from transplanted hepatomas have been described recently (Badrfin et al. 1985b) and we have documented similar inhibitory properties in extracts of other transplanted hepatomas of different degrees of differentiation as well as some mammary carcinomas (unpublished results).Similar effects have been reported for human rhabdomyosarcoma (Fryling et al. 1985;Iwata et al. 1985) and in a human colonic carcinoma cell line (Levine et al. 1985).It would be interesting to determine whether plasma from animals bearing these other tumours shows an inhibitory effect on the mitotic rates of cell populations in regenerating liver.We have started this line of research and our preliminary results support this possibility (Badr/tn et al. 1985a).Experiments designed to analyze the cell population specificity of the inhibitory effect of tumour extracts and of plasma from tumour-bearing mice are now under way in our laboratory.
To analyze the experimental conditions of the "in vivo" assay system, it is essential to have a thorough knowledge of the system when studying the effect of purified growth factors.This is particularly true with respect to the times when the factors are injected and the controls are made, which must be related to the well-known circadian variations in growth variables (Echave Llanos 1967; Clausen and Elgjo 1984).These variables behave normally (Echave Llanos et al. 1971;Surur et al. 1985) only when partial hepatectomy is made at the optimal time in a circadian period (Souto and Echave Llanos 1985).The results obtained with normal plasma are a good example of this requirement.The apparent stimulation obtained at the second peak (14:00 hours/72 h) when the injection is made 26 h after partial hepatectomy, is, in fact, a compensatory wave following the previous inhibition observed at the first peak (14:00 hours/48 h).This result reproduces that of Weinbren (1959) and was recognised as a possible source of error as early as 1962 (Echave Llanos 1967).