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Injection of a substantially purified hepatomitogen into recipient rats that had 40% of their liver removed resulted in a significant stimulation of hepatic DNA synthesis as determined by the labeling index and the mitotic index. Normal or sham-operated rats did not respond to the injection of the mitogen. The extraction and partial purification of this hepatomitogen have previously been reported (A. Francavilla et al., Cancer Res., 47: 5600–5605, 1987). Addition of the factor to an epithelial-like liver-derived cell line in culture (clone 9) or to a hepatoma cell line (HTC-SR) resulted in a dose-dependent stimulation of DNA synthesis. Hepatocytes in primary culture, on the other hand, were not stimulated by the addition of the factor. However, when the mitogen was added to hepatocytes in primary culture, together with conditioned medium, obtained from the responsive cell lines, a significant stimulation of DNA synthesis could be demonstrated in hepatocytes in culture. The stimulation was dose dependent with respect to the mitogen, was abolished by 10 mM hydroxyurea, and was independent of epidermal growth factor. The conditioned medium could be replaced by a protein factor extracted from the two cell lines as previously reported (P. Ove et al., J. Cell. Physiol., 131: 165–174, 1987). It appears that a cofactor is provided by the conditioned medium or by the cell extract, enabling the hepatomitogen to act on hepatocytes in primary culture.
Ever since Higgins and Anderson (1) first demonstrated the remarkable regenerative ability of the liver, investigators have been trying to understand the regulation of growth of hepatocytes. That humoral factors were at least partially involved in this regulation became clear from the parabiosis experiments reported by Moolten and Bucher (2). Identification and purification of potential growth factors were initially tedious, since no good in vitro assays were available. In recent years, when hepatocytes could be maintained in primary culture, there have been numerous reports describing the identification and purification of hepatomitogens stimulating various activities, including DNA synthesis of hepatocytes in primary culture (3–15). Almost all of these studies concentrated on stimulating hepatocytes in culture (3–11) with only a few attempts at the stimulation of DNA synthesis in vivo by means other than surgical or chemical injury to the liver (12–16). A few laboratories, including primarily that of LaBrecque and coworkers (12, 17) as well as our own (16), have isolated and substantially purified a hepatomitogen (HSS4) capable of stimulating DNA synthesis of hepatocytes in vivo. In order to achieve this stimulation it was necessary, however, to initiate the hepatocytes, thus making them responsive to HSS. This initiation was accomplished by inducing a low level of DNA synthesis in recipient rats to be injected with the hepatomitogen (HSS) or equal amounts of nonmitogenic protein such as rat albumin or BSA. These low levels of DNA synthesis, a 1.5- to 3-fold increase in DNA synthesis over control or sham-operated rats, were induced by a 40% hepatectomy (12, 16). Furthermore, HSS did not stimulate DNA synthesis when injected into normal or sham-operated rats. Initially HSS did not stimulate DNA synthesis of hepatocytes in primary culture. This report shows that, similarly to hepatocytes in vivo, hepatocytes in primary culture need to be initiated before they can respond to HSS.
Male Fischer (F344) rats, (180 to 200 g) and weanling rats (60 to 90 g), were purchased from Zivic-Miller Laboratories, Inc., Zelienople, PA, and were kept in temperature- and light-controlled rooms. The animals received food and water ad libitum. Partial hepatectomy and sham hepatectomy were performed according to the method of Higgins and Anderson (1), and all operations were performed between 7:30 and 9:00 a.m.
Ham's F-12 medium, DME, MEM, FBS, antibiotic, and trypsin were obtained from GIBCO Laboratories, Grand Island, NY. Collagenase type 1 (125 to 250 units/mg) was purchased from Worthington Diagnostic Systems, Freehold, NJ, and epidermal growth factor from Collaborative Research, Bedford, MA. Aqueous counting scintillant (ACS) was from Amersham Corporation, Arlington Heights, IL, and [methyl-3H]thymidine (50 to 80 Ci/mmol) was purchased from New England Nuclear Corporation, Boston, MA. All other chemicals were obtained from Sigma Chemical Company, St. Louis, MO.
HTC-SR rat hepatoma cells (a clonal line from Morris hepatoma 7288C) were a generous gift from Dr. H. Baumann, Department of Tumor Biology, Roswell Park Memorial Institute, Buffalo, NY. Stock cultures were maintained in DME, supplemented with 10% FBS and antibiotic/antimycotic in 100-mm dishes at 37°C and a 5% CO2 atmosphere.
Clone 9 normal rat liver cells were obtained from the American Type Culture Collection, Rockville, MD. Stock cultures were grown in Ham's F-12 medium, 10% FBS, and antibiotic/antimycotic at 37°C and a 5% CO2 atmosphere.
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 (18) as previously described (19). Following dispersion and washing of the hepatocytes in cold Ca2+-free perfusion buffer, the cells were resuspended in basal MEM supplemented with nonessential amino acids, pyruvate (1 mM), and gentamycin (40 μg/ml), and, for attachment, insulin (10−7 M) and 5% FBS were added. 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 7 × 104 cells per well in Falcon Primaria 24-well multiwell plates in 0.5 ml of medium per well and maintained at 37°C and a 5% CO2 atmosphere. After a 3-h attachment period the medium was removed, and serum-free basal MEM plus insulin (10−7 M) was added. EGF when present was at a concentration of 10 ng/ml.
To obtain conditioned medium, HTC cells and clone 9 normal rat liver cells were plated at a density of 104 cells/cm2 in 100-mm dishes in regular growth medium for each cell line and incubated. After 48 h, the cells were washed twice with serum-free DME and then incubated in serum-free DME. After 48 h, the medium was collected, centrifuged 10 min at 10,000 × g to remove floating cells, and sterilized by filtration using a 0.22-μm syringe filter.
HSS-F150 was extracted from weanling rat livers by a modification of the method of LaBrecque et al. (12, 20, 21) as previously described. Briefly, the factor was partially purified by successive steps, involving ethanol precipitation, ultrafiltration through an Amicon PM 30 membrane, and finally FPLC, resulting in a 38,000-fold increase in specific activity over that in the original cytosol (16). The active fractions (HSS-F150) from FPLC were dialyzed, lyophilized, and stored at −70°C until used. The activity eluted at 150 mM NaCl. The fraction was dissolved in 5 mM PBS (pH 7.4) for in vivo testing and in MEM and sterilized by syringe filtration for in vitro bioassay with isolated hepatocytes, HTC, or clone 9 cells.
The extraction and purification of the autocrine factor have been described (22). Briefly, the growth factor has been purified from a postmicrosomal supernatant of HTC hepatoma cells by successive steps involving ethanol precipitation, heating at 80°C for 10 min, and chromatography on a DEAE-Bio-Gel A column. The peak fractions, as determined by absorbance at 280 nm, were combined, lyophilized, dissolved in H2O, and dialyzed for 18 h versus H2O. Fractions were sterilized by UV irradiation before assay.
Activity of HSS-F150 in vivo was carried out according to the method of LaBrecque and Pesch (12). A heightened background of DNA synthetic activity in vivo was induced in host rats by a 40% PH. Six h after PH the rats were given i.p. injections of 2 ml of 5 mM PBS (control), pH 7.4, BSA (protein control), or the active FPLC fraction (F150). Eighteen h later, 24 h after the operation, 50 μCi of [3H]-thymidine were injected i.p., and the animals were sacrificed 1 h later.
The animals were killed by cervical dislocation, and the livers were rapidly removed and frozen. Citric acid nuclei were prepared (23). The nuclear pellet was suspended in 1.0 ml of 100 mM citric acid and 2.5 ml of 5% trichloroacetic acid, and the precipitated DNA was collected on celite filter aid-covered filter paper circles as described (24). The celite pad was transferred to a test tube containing 5 ml of 10% perchloric acid and heated at 80°C for 20 min. After centrifugation at 5000 × g, 1 ml of the supernatant was counted in 10 ml of ACS for radioactivity determination, and 1 ml was used for the determination of DNA by the diphenylamine method of Burton (25).
For the routine cell bioassay, stock cultures were subcultured when cells were not yet confluent and distributed at a cell density of 5 × 104 cells per 35-mm tissue culture dish in 2 ml of complete medium required for that particular cell type. The medium was supplemented with 10% FBS unless indicated otherwise.
For the HTC cells a 2.5-h attachment period in 10% FBS:DME was sufficient, whereas 20 h were required for the clone 9 cells to become firmly attached. Those dishes to be assayed in the presence of serum-free medium were given the medium change at 2.5 h for HTC and at 20 h for clone 9 cells. Fractions, previously sterilized by UV irradiation, were added at the time of medium change to all indicated dishes and the cells left for 24 or 48 h. The cells were exposed to [3H]thymidine, 0.4 μCi/dish, for 2 h prior to harvest.
For harvesting, the cells were scraped into the medium, and the dishes were scraped again with 1 ml of PBS. The rinse was combined with the cell suspension and centrifuged for 10 min at 5000 × g. The cell pellet was solubilized in 1 ml of 1 M NaOH by heating at 80°C for 10 min. DNA was precipitated by 5% trichloroacetic acid and collected on celite filter aid-covered filter paper, and radioactivity was determined as previously described (24).
Following the 3-h attachment period, the medium was removed, 0.5 ml of serum-free basal MEM plus insulin (10−7 M) were added, and the cells were left for 24 h. Twenty-four h later the medium was changed again and additions were made. Those wells receiving conditioned medium received 0.25 ml of conditioned medium and 0.25 ml of MEM ± fractions. Cells were incubated for 48 h following this second feeding. In some experiments the conditioned medium or Bio-Gel F4 was left for 24 h on the cells, with no HSS present. Twenty-four h after feeding with conditioned medium, which is 48 h after the initial plating, medium was again changed to basal MEM + insulin and HSS-F150 added. Cells were harvested after an additional 24 h of incubation. They were exposed to 3 μCi of [3H]thymidine/well for 24 h before harvest. Incorporation of [3H]thymidine was determined as described by Michalopoulos et al. (8).
The results in Fig. 1 show DNA synthesis of hepatocytes in primary culture. HSS-F150 had no effect on DNA synthesis in the cultured hepatocytes. This lack of stimulatory activity was observed whether cells were isolated and plated from normal rats or from 40% hepatectomized rats or whether they were maintained in the presence or absence of EGF. Addition of HSS-F150 at different times or to cells isolated from 70% hepatectomized rats similarly was ineffective. These results are not shown in the figure.
Some established liver-derived cell lines, on the other hand, responded with a significant increase in DNA synthesis to the addition of HSS-F150 as well as to the addition of some cruder HSS fractions. The hepatoma cell line, HTC-SR, responded in a dose-dependent manner between 0.25 μg/ml and 1 μg/ml with an increase in DNA synthesis. The response was apparent whether the cells were exposed for 24 or 48 h to the mitogen, and the growth medium was supplemented with FBS. These results are shown in Table 1. These cells did not respond to HSS in the absence of serum. We have previously shown (22) that the cells produce an autocrine factor. In the absence of serum the production and secretion of this autocrine factor are increased. The preferential response of the HTC cells to the autocrine factor would most likely diminish a response to any other mitogen.
The addition of HSS-F150 also stimulated DNA synthesis in a liver-derived epithelial-like cell line which is not tumorigenic. This clone 9 cell line responded in a dose-dependent manner to the same concentrations of HSS-F150 as did the HTC cells. These results are shown in Table 2. Clone 9 cells, unlike the HTC cells, responded only in serum-free medium and not in serum-supplemented medium. These cells are much more dependent on serum for growth than the HTC cells. Although clone 9 cells synthesize and secrete small amounts of the same autocrine factor produced by HTC cells, they do not respond to the autocrine factor, as previously shown (22), thus retaining responsiveness to other mitogens.
The results that established cell lines could respond to HSS were not entirely unexpected. LaBrecque et al. (17, 20, 21) had reported on the purification of HSS using an HTC line for his bioassay. The results with the cell lines in conjunction with our in vivo results indicated to us that the cell lines had properties or produced factors which made them responsive to the hepatomitogen. Our results indicate that this is indeed the case. When hepatocytes in primary culture were exposed to conditioned medium from these cell lines, they could respond to HSS with increased DNA synthesis.
The results shown in Fig. 2 indicate that, in the presence of 50% conditioned medium from clone 9 or HTC cells, as little as 0.025 μg/ml of HSS-F150 stimulated DNA synthesis, and the stimulation was dose dependent. Also shown are the results indicating that equivalent concentrations of HSS-F150 did not stimulate hepatic DNA synthesis when conditioned medium was omitted. Not shown are our findings that conditioned medium from hepatocytes in primary culture did not overcome this lack of a response.
As mentioned earlier, we have previously reported on the isolation and purification of an autocrine factor from HTC-SR cells (22). Although most of the work with autocrine factor was done with an extract from HTC cells, we provided evidence that the same factor could also be obtained from conditioned medium and was also produced by clone 9 cells. The results in Table 3 show that conditioned medium can be replaced by small amounts of the autocrine factor. Two μg of a substantially purified fraction of the autocrine factor, Bio-Gel F4 (22) per ml of MEM, were effective as an initiation factor for the mitogenic activity of HSS-F150. As was the case with conditioned medium, F150 stimulated DNA synthesis in the presence of autocrine factor, whether EGF was present or absent. The table also provides evidence that the mitogenic activity of HSS is additive to the EGF-induced DNA synthesis of hepatocytes in primary culture, suggesting that HSS is a specific mitogen and does not compete for EGF receptors. That EGF-induced [3H]thymidine incorporation in the hepatocyte system represents replicative DNA synthesis has previously been shown by determining the labeling index as well as the mitotic index (16, 19).
The results in Table 4 show that conditioned medium does not need to be present for the entire incubation period nor is it necessary to add the hepatomitogen together with conditioned medium. Hepatocytes can be primed to respond to HSS by a preincubation in conditioned medium. The preincubation period with conditional medium is the most effective in stimulating DNA synthesis when it is present from 24 to 48 h after plating. Stimulation is observed in the presence or absence of EGF. If the conditioned medium is present from 3 to 24 h after plating, stimulation by HSS-F150 was only observed when EGF was present. These latter results are not shown in the table. Preincubation in MEM plus insulin with autocrine factor (Bio-Gel F4) was also effective in making the hepatocytes responsive to HSS. The latter results are not shown in Table 3. We do not know the reason for this finding but can only speculate that hepatocytes are somewhat traumatized during the isolation procedure. Exposure to conditioned medium from 3 to 24 h after isolation is not quite sufficient for initiation, unless EGF is added in addition at 24 h, together with HSS-F150. When the hepatocytes were exposed to conditioned medium from 24 to 48 h after isolation, cells might have had sufficient time during the first 24 h to recover from isolation stress, and a consequent exposure to conditioned medium for 24 h might be enough to initiate these cells for the further stimulatory action of HSS.
That [3H]thymidine incorporation induced by EGF and/or by HSS represents replicative DNA synthesis is indicated by the results shown in Fig. 3. Incorporation of [3H]thymidine due to the presence of EGF and HSS was completely abolished by 10 mM hydroxyurea. In the absence of EGF and HSS there was only a 33% inhibition of [3H]thymidine incorporation, but the stimulation due to HSS alone was also completely abolished. Hydroxyurea has been shown to inhibit DNA replication selectively without affecting DNA repair synthesis (26).
There have been a number of reports in recent years dealing with growth factors for hepatocytes (3–15). In addition to hormones and other already well-characterized factors (3, 5, 6, 27, 28), most investigators have used sera (7–11, 29–31) or liver tissues (13, 16, 17) as the source for potential hepatomitogens. The majority of investigators have used hepatocytes in primary culture or liver-derived epithelial cells in culture as their assay system (3–6, 8, 21). A few attempts have been made to test growth factors in vivo (12–15). We are reporting on a factor (HSS) capable of stimulating DNA synthesis, both in vivo and in vitro.
In our recent report on the isolation and substantial purification of HSS (16), we only described the in vivo system. At that time we were unable to demonstrate any mitogenic activity of HSS for hepatocytes in primary culture. Furthermore, it was essential that the target organ in the recipient animals, the liver, be manipulated. No stimulation was achieved when HSS was injected into normal or sham-operated rats. The liver was manipulated by inducing a low level (2 to 3 times that found in normal rat liver) of DNA synthesis by performing a 40% hepatectomy. A similar assay system had previously been described for dogs (14, 15) and rats (12). It was apparent that a partial surgical removal of the liver primed the remaining cells for the action of HSS. In an earlier abstract we had reported some in vivo and in vitro stimulation of hepatic DNA synthesis with a less purified preparation. It is known that the injection of crude preparations of proteins can cause inflammatory reactions resulting in increased hepatic DNA synthesis. The presence of trace amounts of EGF in crude preparations might stimulate DNA synthesis of hepatocytes in primary culture. Similarly LaBrecque and Bachur had earlier shown in vivo (20) and in vitro (32) stimulation of hepatic DNA synthesis by a relatively crude HSS preparation. Fleig et al. (33) found increased DNA synthesis in rats given injections of a rabbit HSS preparation.
With our highly purified HSS-F150 and also with the 30K fraction we were unable to stimulate in vitro DNA synthesis without the presence of conditioned medium or the Bio-Gel F4 fraction.
Despite our inability to stimulate DNA synthesis of hepatocytes in primary culture by the addition of HSS, we found that two established cell lines, a hepatoma HTC-SR and a Iiver-derived epithelial-like clone 9 cell line, responded well to the mitogenic potential of HSS. LaBrecque et al. (21, 32), as well, had used an HTC hepatoma cell line in their attempts to isolate and purify HSS. It appeared that these cell lines, unlike the hepatocytes in primary culture, possessed properties which allowed them to respond to HSS. Our attempts to make use of this ability and transfer it to the hepatocytes were successful. As can be seen in Fig. 2, the conditioned medium by itself did not stimulate hepatic DNA synthesis; on the contrary, there was substantial inhibition due to 50% conditioned medium in the absence of EGF. It is likely that some essential components were used up from the conditioned medium when it was sup porting HTC or clone 9 cells prior to being used with hepatocytes in primary culture. The presence of the conditioned medium, however, affected the hepatocytes in such a way that they could now respond to HSS, as shown in Fig. 2.
We had previously reported on the isolation and purification of an autocrine factor from the medium or from the HTC or clone 9 cells directly (22). As can be seen in Table 3, the addition of this factor to the hepatocytes in primary culture can replace the need for the conditioned medium.
Despite the fact that both factors, HSS and the autocrine factor, have not been purified to that extent and in sufficient quantities to do a full chemical characterization, they have been substantially purified and behave in this system as competence and progression or initiation factors, similar to the model proposed by Pledger et al. (34) and Stiles et al. (35–37) for the initiation of DNA synthesis in quiescent BALB/c-3T3 cells.
Similarities to the “competence-progression” model are also indicated by the results shown in Table 4. The conditioned medium or the autocrine factor needs to be present only for 24 h, and it is most effective during the 24 to 48 h in order for the hepatocytes to respond to the subsequent addition of HSS. When present during the first 24 h after plating, the addition of EGF was required to bring about stimulation of DNA synthesis in response to HSS-F150. As yet, we have not determined how short an exposure to conditioned medium is sufficient to make the hepatocytes competent.
That HSS stimulates replicative DNA synthesis is indicated by our finding that 10 mM hydroxyurea completely inhibits the HSS-induced DNA synthesis. Epidermal growth factor-induced DNA synthesis was also inhibited as reported earlier (19). In the absence of either EGF or HSS, there was only a 33% inhibition, reflecting most likely a background level of repair synthesis in hepatocytes in primary culture. Evidence for replicative DNA synthesis in vivo, due to the injection ofHSS, has been provided in a previous communication by determining the labeling index and mitotic index (16).
A number of serum factors have recently been found by several authors (7–11, 29–31). So far, the only factor similar or possibly identical to our factor is the HSS described by LaBrecque et al. (12, 17, 20, 21). There are, however, a report describing the isolation of two serum factors which stimulate hepatocyte DNA synthesis in culture in synergy (8) and another report describing the stimulatory effect of cocultures of nonparenchymal liver cells on growth of primary cultured hepatocytes (38). This latter stimulation was to a great extent dependent on cell-cell interaction. Another report describes the isolation and purification of a DNA synthesis promoter from plasma of partially hepatectomized rats (11). This factor, with an apparent molecular weight of 64,000, has been shown to be active in mice in vivo as well as with rat hepatocytes in primary culture.
Except for the HSS described by LaBrecque et al. (12, 17, 20, 21), none of the growth factors described appears to be similar to our hepatomitogen. Whether the mechanism that makes hepatocytes in vivo competent by partial removal of the liver is similar in nature to the in vitro induction of competence by humoral factors is at present not known. The importance of our findings is the demonstration that DNA synthesis and growth of hepatocytes seem to be regulated by more than one factor, and that this interaction resembles the “competence-progression” model advanced for the interaction of some other well-defined growth factors.
We are grateful to John Prelich for technical assistance.
This study was supported by a research project grant from the Veterans Administration; by Project Grant AM-29961 from the NIH, Bethesda, MD; by Grant 885/0216544 from Consiglio Nazionale delle Ricerche, Italy; and by Central Research Development Fund, Category 1, University of Pittsburgh, Pittsburgh, PA 15260.
4The abbreviations used are: HSS, hepatic stimulator substance; DME, Dulbecco's modified Eagle's medium; MEM, minimal essential medium, Earle's salts; EGF, epidermal growth factor; FPLC, fast protein liquid chromatography; PH, partial hepatectomy; PBS, phosphate-buffered saline; BSA, bovine serum albumin; FBS, fetal bovine serum; F150, Fraction 150.