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Primary hepatocyte cultures have been prepared from normal adult rat liver and from rat liver at 4, 8, 12, 24, and 48 h following partial hepatectomy (removal of 70% of the liver). Cells were maintained in minimal essential medium alone or supplemented with hormones. Comparing DNA synthesis in normal adult rat hepatocytes with DNA synthesis in hepatocytes isolated from regenerating livers, we found with minimal essential medium alone little DNA synthesis in normal adult rat hepatocytes and in hepatocytes isolated 4, 8, or 12 h after 70% hepatectomy. In hepatocytes isolated 24 h after partial hepatectomy, however, the incorporation of [3H]thymidine was 3 times the rate of normal hepatocytes. The addition of insulin to minimal essential medium had minimal effect on DNA synthesis in all hepatocytes. Addition of epidermal growth factor alone or in combination with insulin resulted in a dramatic increase in DNA synthesis in hepatocytes from regenerating rat liver. Increased incorporation was detectable as early as 4 h after partial hepatectomy and reached a maximum at 24 h after the operation. Results obtained with [3H]thymidine incorporation were confirmed by autoradiography and by direct DNA determinations in hepatocyte cultures. Epidermal growth factor binding to the hepatocytes was determined and agreed with previously reported binding studies. Binding of epidermal growth factor in hepatocytes isolated at 4 h after partial hepatectomy was the same as in normal hepatocytes but was undetectable in hepatocytes isolated from rats at 12 and 24 h after partial hepatectomy.
In the past several years, a number of investigators have demonstrated replicative DNA synthesis in hepatocytes isolated from adult rats and have maintained monolayer cultures for several days (1–9). A variety of hormones such as insulin, EGF,3 triiodothyronine, and glucagon as well as other compounds such as pyruvate, calcium concentration, amino acids, and as yet undefined growth factors have been found to be essential or beneficial for replicative DNA synthesis in such cultures (3, 5, 6, 8–14). Hepatocytes have been maintained and grown on a variety of modified surfaces such as fibronectin (15), a connective tissue biomatrix (16), and in most cases collagen gels and membranes or collagen-coated dishes (7, 17, 18).
Attempts have also been made to use this primary hepatocyte system to identify and isolate substances that might elicit and/or maintain the regenerative response following partial hepatectomy. Michalopoulos et al. (7, 19) reported a dramatic stimulation of DNA synthesis in the presence of serum obtained from rats following partial hepatectomy. These results were also confirmed by Nakamura et al. (20). Relatively low-cell density and high serum concentrations resulted in the best stimulation. Strain et al. (21), on the other hand, found normal rat serum to be stimulatory but did not detect significant additional stimulation with serum from partially hepatectomized rats.
Although some investigators have prepared primary hepatocyte cultures from regenerating rat liver (8, 22, 23), no thorough study has been made to compare DNA synthesis in primary cultures prepared from adult rat liver and from regenerating rat liver. Several studies on EGF binding to hepatocytes have also been reported (24–28). Liver membranes and isolated hepatocytes bind EGF specifically and exhibit EGF-dependent phosphorylation of the Mr, 170,000 EGF receptor (26, 28). In addition, EGF receptor numbers have been shown to decrease after partial hepatectomy, suggesting that some humoral factor or factors that regulate(s) liver regeneration “down regulate” the EGF receptor (24).
We have grown hepatocytes on Falcon “Primaria” dishes which have a specific modified plastic substratum for primary cell culture, while at the same time suppressing fibroblast growth (29). Hepatic DNA synthesis has been compared in collagen-coated and Primaria dishes. We have further studied the ability of hepatocytes, isolated from regenerating rat liver at various times after 70% hepatectomy, to synthesize DNA in the absence or presence of various hormones and have compared EGF binding in hepatocytes isolated from normal and regenerating rat liver.
Male Fischer (F344) rats were obtained from Hilltop Lab Animals, Scottdale, PA. The animals were kept in a temperature-and light-controlled room and received food and water ad libitum. Partial hepatectomy and sham hepatectomy were performed according to the method of Higgins and Anderson (30), and all operations were performed between 7:30 and 9:00 a.m.
Collagenase type 1 (125–250 units/mg) was obtained from Worthington Diagnostic Systems, Freehold, NJ. MEM and FCS were purchased from GIBCO Laboratories, Grand Island, NY. Insulin, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, and sodium pyruvate were purchased from Sigma Chemical Company, St. Louis, MO. [methyl-3H]Thymidine (50–80 μCi/mmol) and 125I-EGF (150–200 μCi/μg) were obtained from New England Nuclear Corp., Boston, MA.
Hepatocytes were isolated from rats, weighing between 150 and 250 g, by a modification of the in situ two-step collagenase perfusion technique of Seglen (31) as described by Jirtle et al. (32). The liver was perfused with a cannula in the inferior vena cava with 250 ml of buffer [142 mM NaCl:6.7 mM KCl:10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.4) followed by 250 ml of the same buffer containing in addition 5.7 mM CaCl2 and collagenase (0.5 mg/ml) The hepatocytes were dispersed and washed twice with cold Ca2+-free perfusion buffer and resuspended in basal medium [MEM supplemented with pyruvate (1 mM), aspartate (0.2 mM), serine (0.2 mM), Gentamycin (40 μg/ml)] and for attachment the addition of insulin (10−7 M) and 5% FCS. Viability was determined by trypan blue exclusion, and only preparations having greater than 90% viability were used. Cell number was determined with a hemocytometer.
The cells were plated at a cell density of 2 × 105 per 35-mm Falcon Primaria tissue culture dish in 1.5 ml medium and maintained at 37°C and a 5% CO2 atmosphere. Following a 3-h attachment period, serum-free basal medium was added, and additions were made as indicated. EGF and insulin, when added, were at a concentration of 10 ng/ml and 10−7 M, respectively. Hepatocytes were also plated on Collagen-coated dishes for a comparison with Primaria dishes.
Rat tail collagen solution was prepared from Fischer 344 rats according to the method of Michalopoulos and Pitot (17). One ml of sterile collagen (0.01% acetic acid in H2O, 0.33-mg equivalent of dry UV-sterilized collagen fibers) was added to each non-Primaria 35-mm tissue culture dish. The dishes were allowed to dry for 48–72 h at 37°C and used without any further treatment.
Following the 3-h period to allow for attachment of the hepatocytes, the medium was changed, the appropriate additions were made, and the cells were left for 48 or 72 h. [3H]Thymidine, 7.5 μCi/dish, was present for 24 h prior to harvest. Each experimental group comprised four dishes. At 48 or 72 h, the cells were harvested, one dish was used to determine DNA using the microfluorometric method of Setaro and Morley (33), and the three remaining dishes of each group were treated as described by Michalopoulos et al. (19) to determine [3H]thymidine incorporation.
Cells plated on Primaria dishes were fixed directly on the dishes. Cultures had been exposed to [3H]thymidine (7.5 μCi/dish) for 24 h prior to fixation. Cells in dishes were covered with emulsion (Kodak NTB3), dishes were developed after 8–10 days, and the labeling index was determined by counting a total of 1000 cells.
EGF binding studies were performed essentially as previously described (34). Cells were incubated for 3 h in attachment medium only or an additional 24 h in basal MEM plus insulin. Dishes were washed 3 times with MEM plus insulin and 0.1% bovine serum albumin (binding medium) and cooled to 4°C. Cells were then incubated in 1 ml of the above binding medium in the presence of 0.125–16 nmol 125I-EGF at 4°C for 1 h. After a 45-min incubation, binding equilibrium had been fully established. After the incubation, the dishes were washed 5 times with the above binding medium and then treated for 6 min with 0.7 ml of 0.2 M acetic acid (pH 2.5) containing 0.5 M NaCl to remove cellular bound EGF. This treatment was repeated with 0.4 ml. It has previously been shown that this treatment removes over 90% of the cell-bound radioactivity, and it does not remove the iodine from the EGF (34). DNA determination on selected dishes from each hepatocyte preparation indicated no loss of cells by this treatment. The acetic acid washes were combined, and radioactive content was determined in a gamma counter spectrometer. Nonspecific binding was determined by measuring 125I-EGF binding in the presence of excess unlabeled EGF (1 μg/ml) which was added either 30 min before or simultaneously with 125I-EGF and was approximately less than 1% of the total binding with both methods. Protein was determined by the method of Lowry et al. (35). The results are presented as Scatchard analyses (36).
The majority of reports agree that hepatocytes in primary culture need a special surface for maximal attachment and growth. A collagen substrate has been the preferred method (5, 7, 17, 18). Recently the Falcon Primaria culture dishes have come onto the market. These dishes have a surface-modified plastic substratum specifically for primary cell cultures which also suppresses fibroblast growth. Since it would be more convenient to use such dishes, we compared [3H]thymidine incorporation in collagen-coated and Primaria dishes. The results in Table 1 indicate that Primaria dishes are at least as good or better for the study of hepatocytes in primary culture as collagen-coated dishes. In all subsequent experiments, Primaria dishes were used.
The viability of the hepatocyte preparations was between 90 and 95%. Viability varied slightly from batch to batch but was not related to the physiological state of the animals. The plating efficiency was between 70 and 80% and was the same for hepatocytes from normal and regenerating liver. Flattening of the cells was monitored by microscopic observations. Only hepatocytes from 24-h regenerating liver appeared to have flattened more and had slightly more processes and fat droplets after 24 h in culture than normal cells and those isolated at 4, 8, 12, and 48 h after partial hepatectomy or from sham-operated animals. By 48 h after plating, the appearance of all hepatocytes was the same.
Incorporation of [3H]thymidine into hepatocytes from normal rats and 70% hepatectomized rats isolated 24 h after the operation is shown in Table 2. Hepatocytes were compared in basal MEM with and without hormones, and [3H]thymidine incorporation was compared at two different times, 48 and 72 h in culture. In each case, the regenerating hepatocytes incorporated substantially more [3H]thymidine than the normal cells. Incorporation between 48 and 72 h was in all cases approximately 2.5 times more than between 24 and 48 h. The difference in incorporation between normal and regenerating hepatocytes was the same at 48 and 72 h. A comparison of hepatocytes from sham-operated rats with those from normal rats indicated no detectable differences in morphology or DNA synthesis.
We have recently repeated some of these experiments in the presence of 0.26 mM proline. As reported (10, 37), proline enhances DNA synthesis in hepatocytes in primary culture. We found a 1.5-fold increase in [3H]thymidine incorporation in both normal hepatocytes and hepatocytes isolated from regenerating liver whether the cells were incubated in the presence or absence of EGF plus insulin.
To convince ourselves that the [3H]thymidine incorporation represents largely replicative DNA synthesis, we determined DNA in the cultures directly. With MEM alone at 72 h, the DNA content remained relatively constant in cultures with hepatocytes from normal rat liver and regenerating rat liver. With the addition of insulin plus EGF, however, there was a substantial increase in DNA content in both normal and regenerating hepatocytes as can be seen in Table 3. Addition of 10 mM hydroxyurea, a level that inhibits replication but not repair DNA synthesis (38), resulted in a 40–50% inhibition of [3H]thymidine incorporation in MEM alone, while in MEM supplemented with insulin and EGF, the inhibition was 95%. The inhibition was the same in normal and regenerating hepatocytes.
These results with normal and regenerating hepatocytes isolated at 24 h were not surprising, since maximal DNA synthesis in vivo following partial hepatectomy occurs between 20 and 24 h, and some increase in DNA synthesis can be detected as early as 16 h after partial hepatectomy. We wanted to see whether hepatocytes isolated at various earlier times after 70% hepatectomy (at which time there is no detectable increase in DNA synthesis in vivo) would respond differently in primary cultures. The results of this study are shown in Fig. 1.
With MEM alone or with MEM plus insulin, [3H]thymidine incorporation was similar in normal hepatocytes and in regenerating hepatocytes isolated at 4, 8, 12, and 48 h after partial hepatectomy. Hepatocytes isolated at 24 h after partial hepatectomy were the only ones that showed a 3- to 4-fold increase in DNA synthesis. However, when EGF was added to the medium but particularly EGF plus insulin, there was a significant increase in [3H]thymidine incorporation in hepatocytes isolated as early as 4 h after partial hepatectomy, reaching a maximum at 24 h after partial hepatectomy and declining somewhat at 48 h.
To provide evidence that the [3H]thymidine incorporation represents replicative DNA synthesis and to show that differences in [3H]thymidine incorporation between hepatocytes from normal and partially hepatectomized rats were not due to differences in the precursor pool size or rate of transport, we also determined the labeling index. The results of this determination can be seen in Fig. 2. There was a good correlation between [3H]thymidine incorporation and the number of labeled cells.
Examples of labeled cells can be seen in Fig. 3. In addition to labeled hepatocytes from normal rats in MEM (A) and normal cells in MEM plus insulin plus EGF (B), labeled hepatocytes isolated from rats at 24 h after partial hepatectomy and grown in MEM plus insulin plus EGF are shown (C). In Fig. 4 we show that the labeled cells are hepatocytes. The cells were stained for glucose-6-phosphatase (39) and then processed for autoradiography. The labeled cells also stain for glucose-6-phosphatase and are clearly hepatocytes.
Results of EGF binding to hepatocytes in primary culture are shown in Fig. 5. There was no significant difference in the binding of EGF when normal hepatocytes were compared to hepatocytes isolated 4 h following partial hepatectomy, with binding studies being performed after 24 h in culture, or after 3 h in attachment medium with nonspecific binding determined with labeled and unlabeled EGF present simultaneously. For normal hepatocytes the Kd was 5 × 10−9 M with Bmax, 2.8 × 10−10 M/mg protein binding sites determined after 24 h in culture, and after 3 h in attachment medium, the Kd was 5.5 × 10−9 M with Bmax of 2.4 × 10−10 M/mg protein binding sites. For hepatocytes isolated 4 h after partial hepatectomy, we found a Kd of 6.6 × 10−9 M with Bmax of 3.3 × 10−10 M/mg protein binding sites, determined at 24 h in culture, and a Kd of 6.5 × 10−9 M with Bmax of 3.8 × 10−10 M/mg protein binding sites determined after attachment. The Kd values are similar to those reported by Lin et al. (26) for hepatocytes in primary culture (Kd 7.3 × 10−10 M) and by Moriarity and Savage (25) (Kd 4.4 × 10−9 M). High affinity binding of EGF to hepatocytes isolated from rats 12 and 24 h following partial hepatectomy, however, was drastically reduced, with only very low affinity binding in evidence. This was the case whether binding studies were done after 3 h in attachment medium or after 24 h in culture. We have shown that cells from control animals and cells from 4-h posthepatectomy liver have equivalent affinities and binding capacities for EGF. However, at 12 and 24 h after hepatectomy, only low affinity EGF binding is observed. This seems to confirm results reported by Earp and O’Keefe (24) indicating a decrease in the EGF receptor number following partial hepatectomy. Our results were obtained with intact cells in culture and are not directly comparable.
Our findings show that we measure primarily replicative DNA synthesis in normal hepatocytes and hepatocytes isolated from regenerating rat liver. The results also show that regenerating hepatocytes become, possibly as early as 4 h but definitely at 8 h, able to respond in culture to the addition of EGF with increased DNA synthesis over that induced by EGF in normal hepatocytes. At this time, there is no detectable increase in hepatic DNA synthesis in vivo following partial hepatectomy.
Newly isolated rat hepatocytes in monolayer cultures underwent replicative DNA synthesis in a defined medium without the addition of serum. The principal hormone necessary for DNA synthesis was EGF. Insulin by itself did not enhance [3H]thymidine incorporation over that found with our modified MEM but was stimulatory in combination with EGF. For most of our determinations, we have used the Falcon Primaria dishes instead of collagen-coated dishes. Primaria dishes compared favorably with collagen-coated dishes. They are more convenient to use and reportedly inhibit growth of fibroblasts.
An interesting observation was made with the hepatocytes isolated at various times after partial hepatectomy and incubated in differently supplemented MEM. When insulin and EGF were present, there was a steady increase in [3H]thymidine incorporation and labeling index up to 24 h in the cells isolated from regenerating liver over that of normal liver.
That hepatocytes prepared from regenerating liver 24 h after partial hepatectomy can be stimulated by the addition of EGF to synthesize DNA to a greater extent than hepatocytes from normal rats was first shown by Richman et al. (8). Their assays were done in the presence of 10% calf serum, however, and did not include determinations on hepatocytes isolated from regenerating liver at times when no increased DNA synthesis is detectable in vivo.
The labeling index and the experiments done in the presence of hydroxyurea, as well as direct DNA determinations, indicate that at least the majority of the [3H]thymidine incorporated into the cells is due to replicative DNA synthesis and not to a high level of repair synthesis. It is well known that the labeling index is not affected by severalfold differences in the thymidine pool, which affects instead only the number and size of grains over each labeled nucleus.
An interesting aspect of our finding is the increased incorporation of [3H]thymidine into hepatocytes isolated at 4 and 8 h after partial hepatectomy. At this time there is no detectable increase in DNA synthesis in vivo, but in the presence of EGF these cells in vitro respond with increased DNA synthesis as compared to normal hepatocytes. It appears that these cells, or at least some of the cells, are already committed possibly by a shift in number or rearrangement of EGF receptors. To investigate such a possibility, we studied EGF binding to hepatocytes in culture isolated from normal or regenerating liver. For the determination of nonspecific binding, we followed the method described for binding to hepatocytes by Lin et al. (26). This method preincubates the cells for 30 min with cold EGF before the radioactive EGF is added. It was pointed out by an expert on binding studies that for most binding studies with other cells nonspecific binding is determined by incubating the cells with labeled and unlabeled EGF simultaneously (34). We found that both methods resulted in the same values for nonspecific binding in our system. Our results with EGF binding confirm previous findings that there seems to be a “down regulation” of EGF binding concomitant with increased DNA synthesis and that this decrease in EGF binding is maintained for at least 24 h in culture. The lack of a significant difference in EGF binding when normal hepatocytes were compared with hepatocytes from rats 4 h after partial hepatectomy is not surprising. Despite an increase in EGF-induced [3H]thymidine incorporation, changes in EGF receptor binding affinity are probably minimal at this time after partial hepatectomy. More difficult to explain is the difference in DNA synthesis and labeling index observed with hepatocytes isolated 12 and 24 h after partial hepatectomy and incubated for 72 h in the presence of EGF (10 ng/ml). These cells exhibit more proliferative activity when compared with cells isolated from nonoperated or sham-operated rats. According to the Scatchard analysis (Fig. 5) of EGF binding to hepatocytes for 24 h in culture after they had been isolated from regenerating liver 24 h after partial hepatectomy, binding affinity was so low as to suggest a virtual absence of high affinity EGF receptors. These data confirm the results reported by Earp and O’Keefe (24) in membrane preparations from rats at 24 h after partial hepatectomy. This decline of EGF binding in the cells at 24 h after partial hepatectomy is consistent with their increased sensitivity to EGF It has been shown that, in many instances when cell proliferation is stimulated, EGF binding is decreased (40–43). This phenomenon could be due to internalization of the EGF receptor pool after partial hepatectomy and a repopulation of the surface with receptors from this pool as the hepatocytes continue growing in culture (44).
Whatever the mechanism, our findings are important for several reasons: (a) they clearly demonstrate that hepatocytes are committed to synthesize DNA very early after partial hepatectomy; (b) EGF stimulation of DNA synthesis may be useful to study cell commitment; (c) as reported for fibroblasts, EGF can also exert its mitogenic activity in hepatocytes independently of the membrane receptor state (45); and (d) hepatocytes in culture isolated at different times after partial hepatectomy represent a good system to study the relationship between EGF, mitogenic activity, and receptor state.
We are grateful to Dr. Patricia Eagon for valuable advice on the binding studies.
1Supported by research grants from the Veterans Administration, Project Grant AM-29961 from the NIH, Bethesda, MD, and by Grant 83/003 1096 from Consiglio Nazionale delle Ricerche, Italy.
3The abbreviations used are: EGF, epidermal growth factor; MEM, Eagle’s minimal essential medium; FCS, fetal calf serum