Expression of liver phenotypes in cultured mouse hepatoma cells: Synthesis and secretion of serum albumin

DEVELOPMENTAL

Expression

BIOLOGY

35.83-96

(1973)

of Liver Phenotypes in Cultured Mouse Hepatoma Synthesis and Secretion of Serum Albumin H. P. BERNHARD,

Department

of Biology,

G. J. DARLINGTON

Yale University,

Kline

Accepted

Biology May

Cells:

AND F. H. RUDDLE

Tower,

New

Haven,

Connecticut

06520

24, 1973

A permanent cell line, designated Hepa, has been isolated from a mouse hepatoma, BW 7756. The cell line synthesizes and secretes albumin at rates appreciably higher than previously reported hepatomas adapted to in vitro conditions. Monospecific antimouse serum albumin was produced in rabbits, and mouse serum albumin secreted by the hepatoma cells was identified by double diffusion, immunoelectrophoresis, and radioimmunodiffusion. A quantitative immunoassay was used to measure albumin secretion and to study the effects of culture conditions on albumin secretion. A subclonal analysis was performed to study the homogeneity and stability of cloned hepatoma lines in respect to albumin secretion. Different secretion rates were observed during the culture cycle. Significant clonal variation in respect to albumin secretion was found among ten subclones. The significance of clonal variation is discussed in relation to the study of epigenetic control of albumin expression in somatic hybrid cells.

In these combinations, the human chromosomes will be preferentially segregated. We believe it should then be possible to test the influence of particular human chromosomes, derived from cells of a known epigenetic constitution on the expression of specific differentiated phenotypes native to the rodent parental cells. The system also permits us to test the influence of the intact mouse genome on the expression of human genes which code for specialized phenotypes. In order to construct this type of experimental system, it is necessary to isolate and characterize rodent parental cell lines which continue to express differentiated phenotypes. It will be the purpose of this report to describe the capacity of a murine hepatoma adapted to tissue culture to synthesize and secrete serum albumin. The production of albumin is a characteristic function of liver cells. Albumin synthesis and secretion has been studied extensively in uioo (review by Rothschild et al., 1972), and in recent years an increasing number of reports have been published describing the production of albumin and other serum proteins by liver and hepa-

INTRODUCTION

Somatic cell populations in oitro can be used for genetic analysis in mammalian organisms by exploitation of parasexual mechanisms such as cell fusion and chromosome segregation (Ruddle, 1973). Somatic cell genetic systems of analysis can also be combined with experimental systems which seek to reveal mechanisms of developmental regulation. Such systems depend on the fusion of cells of different or identical epigenetic states. Experiments already reported have demonstrated the modulation of numerous differentiated traits representative of a number of differentiated cell types (Davidson, 1971). Unfortunately, many of these experiments cannot be analyzed in genetic terms, because of their intraspecific composition. We are concerned with the formulation of systems which allow a combined developmental and genetical approach to the analysis of development. These systems make use of established rodent cell lines of defined epigenetic types. These will be hybridized to normal diploid human cell strains also of defined differentiated states. 83 Copyright All rights

0 1973 by Academic Press. Inc. Of reproduction in any form reserved.

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toma cells in culture. Albumin producing permanent cell line designated Hepa was primary liver cultures have been estabestablished in our laboratory in 1970 by lished from fetal rat liver by Leffert and Darlington. A clonal line Hepa-l and ten Paul (1972) and from fetal human liver by subclones have been derived (see Fig. 1). Bissel and Tilles (1971). Recently, Borek Hepa-l is heteroploid, has a modal chro(1972) isolated a transformed hepatomamosome number of 69 and possesses 4-6 like liver line from adult rat liver, which biarmed marker chromosomes. The doualso secretes serum albumin. Kaighn and bling time is 28 hr. Hepa-l expresses sevPrince (1971) were successful in establisheral liver-associated functions in vitro, and ing several human cell lines from fetal its characteristics have been described in previous reports (Bernhard et al., 1971; liver, which produce albumin and various Darlington et al., 1972, 1973; Benedict et combinations of other serum proteins. With few exceptions (Coon, 1969; Wayal., 1973). mouth et al., 1971), permanent cell lines Culture techniques. The cells were propmedium MAB 8713 which produce serum proteins have been agated in Waymouth (Grand Island Biological Company, Grand isolated so far only from hepatomas (Bancroft et al., 1969; Ohanian et al., 1969; Island, New York). The medium was supRichardson et al., 1969; Tashjian et al., plemented with 10% fetal calf serum (GIB1970; Bernhard et al., 1971; Peterson and CO, Grand Island, New York). This medium contains 10 pg of crystalline porcine Weiss, 1972). The production and secretion of albumin in hepatomas and cultured insulin per liter. Streptomycin (50 mu/ml) hepatoma lines compared to that of intact and penicillin (50 pg/ml) were added rouliver is significantly reduced (Schreiber et tinely to the medium. The cells were grown al., 1970; Weigand et al., 1971). This report in Falcon tissue culture dishes or flasks describes the study of albumin secretion by (TC dishes 3002, 60 x 15 mm, TC flasks a permanent mouse hepatoma cell line 3012 25 cm2 Falcon Plastic, Los Angeles, California) at 37” C in a humidified atmoswhich produces serum albumin at a rate higher than previously found in cultured phere which contained 5% CO,. The pH hepatoma cells. In our study, cloning and was maintained at 7.2 with bicarbonate subcloning was performed to investigate buffer. Cloning was performed after the the stability and homogeneity of the cell method of Puck et al. (1956). More detailed line in respect to albumin production. information on cloning and culture techExperiments were designed to establish niques will be given in the paper by Darculture conditions which favored the maxilington et al. (1972). mal production of albumin and which Preparation of antisera. Mouse serum elucidated factors controlling albumin synalbumin (MSA) Cohn fraction V was obthesis and secretion in vivo. As stated tained from Mann Research Laboratory, above, the hepatoma line was originally derived and then characterized as part of a Mouse hepatoma BW 7756 carried in C57L/J mice program of developmental studies based on / 1 cell hybridization. Subsequent reports will in oitro uncloned Hepa describe those aspects of our research. I clone MATERIALS

AND

Hepa-l I

METHODS

Cell line. Cells derived from the BW 7756 hepatoma (Jackson Laboratory, Bar Harbor, Maine) carried in C57 leaden mice have been serially propagated in vitro. A

subclones II la FIG.

lb

1. Clonal

II Ic

Id history

I le

Hepa-l II If

Ig

of mouse

I Ih

I1 Ii

hepatoma

Ik lines.

BERNHARD,

DARLINGTON

AND RUDDLE

New York. It contained 4% a-globulins. The Cohn fraction V was further purified by TCA precipitation and solubilization in acidic ethanol following the method of Korner and Debro (1956). The purified albumin recovered was electrophoretically homogeneous as demonstrated by agarose electrophoresis and diffusion against goat antimouse serum and was used for the immunization of rabbits (male, white New Zealand rabbits, 8 weeks old, 2 kg body weight). Five milligrams of MSA emulsified in 0.25 ml of phosphate-buffered saline (PBS) and 0.5 ml of complete Freund’s adjuvant was injected intramuscularly. Ten days later the injection was repeated with the same amount of MSA using incomplete adjuvant. A booster of 5 mg MSA in 0.5 ml PBS was injected intramuscularly at day 17. Sera were collected starting at day 24 at 7-day intervals. The sera were processed according to standard procedures as described by Williams and Chase (1967). Purification of proteins from hepatoma culture medium. Supernatant medium was

collected from cultures in the logarithmic phase of growth. The medium was dialyzed and concentrated by pressure ultrafiltration through a Diaflow membrane PM 30 (Amicon, Lexington, Massachusetts) which retains more than 98% albumin. A glycine-citrate buffer described by Kaighn and Prince (1971) was used for dialysis. Concentrated samples were lyophilized and dissolved in distilled water for double diffusion and immunoelectrophoretic analytical procedures. Double diffusion (Ouchterlony) and immunoelectrophoretic analytical procedures. Double diffusion and electrophoresis

were performed in 1% agarose in a sodium barbital buffer 0.1 ionic strength, pH 8.6, 50 mM sodium diethylbarbiturate, 10 mM diethylbarbituric acid, 50 mM sodium acetate, 2 mM calcium lactate. For double diffusion the agarose contained in addition 8% dextran 500,000 MW (Pharmacia, Pis-

Albumin

Synthesis

by Cell

Clones

85

cataway, New Jersey). Electrophoretic separation was carried out at 8 V/cm for 60 min. Precipitin patterns were developed overnight at room temperature. They were then photographed by incident light. Dried slides were stained with Amido Black, destained in 2% acetic acid, and photographed. Radioimmunoassay. Logarithmically growing cells were incubated for a period of 24 hr with serum-free medium, which contained 10 &i/ml uniformly labeled leucine (nL-‘4C-leucine (U), S.A. 25.4 mCi/mmole, Schwarz and Mann, New York). The supernatant medium was collected, dialyzed by ultrafiltration, and lyophilized. Dissolved samples representing 300-fold concentration of the supernatant were assayed by immunoelectrophoresis. Diffusion was performed against rabbit antimouse serum and against monospecific rabbit antimouse serum albumin. The dried agarose slides were coated with nuclear track emulsion (Kodak, NTB 2) diluted 1: 1 with water and developed after 7-10 days exposure. Quantitative

immunoassay

of MSA.

MSA was assayed by the electroimmunodiffusion method of Laurel1 (1966). The advantage of this method is that a quantitative assay can be performed with accuracy on unpurified samples from supernatant medium without any concentration. For the assay of intracellular albumin, the cells were harvested with trypsin (Viokase, Gibco Grand Island, New York) and washed with isotonic PBS, the intracellular albumin was released by sonication as described by Campbell et al. (1960); 5 ~1 samples were applied to agarose gels which contained 0.5-2.0% monospecific rabbit anti-MSA. The sodium barbital buffer described for the Ouchterlony technique was used as gel and separation buffer. Separation was carried out at lo-15 V/cm for 6-12 hr depending on the concentration of antibodies in the gel. A Shandon electrophoresis apparatus MOD. U77 with chilled platen was used for electrophoresis

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(Shandon, Sewickley, Pennsylvania). The temperature was kept between 4 and 8” C during the run to prevent excessive condensation at the gel surface. The gels were washed in several changes of 1% saline for 2 days to remove all the unreacted antiserum. After drying, the slides were stained with amidoblack as described before and magnified lo-fold with a photographic enlarger. The’measurements of the peak heights were then made on the enlarged images (Fig. 2). Different amounts of purified MSA were run for each assay as reference standards. Linear quantitation was obtained for samples ranging from 50 ng to 5000 ng MSA. No interference by heterologous albumin was observed when the samples were run in the presence of lo-fold excess bovine serum albumin (BSA). Protein determination. Protein was measured by the method of Lowry et al. (1951) and by the modified Lowry procedure described by Oyama and Eagle (1956). Cell numbers. The trypsin disaggregated cells were counted with a Coulter Counter model B, equipped with a lOO-pm aperture (Coulter Electronics, St. Hialeah, Florida).

VOLUME

35, 1973

precipitin line was found, identical with purified MSA. The identity of the hepatoma protein with MSA was further con-

RESULTS

Identification

of

Mouse

Serum Albumin

The anti-MSA obtained from rabbits has been shown to be monospecific for MSA by double diffusion and immunoelectrophoresis. No cross reaction with bovine serum albumin and other bovine proteins present in the culture medium was observed within the range of concentrations used for the immunological assays. If concentrated samples of supernatant medium from mouse hepatoma cultures were subjected to double immunodiffusion, identity between the precipitin lines of hepatoma proteins and purified MSA or MSA present in mouse liver extracts was demonstrated (Fig. 3). If the samples were tested against monospecific rabbit anti-MSA, a single

FIG. 2. Quantitative immunoassay for mouse cerum albumin. Electroimmunodiffusion was performed in 1% sodium barbital buffered agarose at pH 8.6 (LaurelI, 1966). Antimouse serum albumin (AMSA) was added to the agarose at a final concentration of 0.5% AMSA. Separation was carried out for 6 h at 10 V/cm. The dried slides were stained with Amido Black and photographed at lo-fold magnification for the measurement of the peak heights. AMSA = Antimouse serum albumin (rabbit) MSA = Mouse serum albumin, ethanol extract of Cohn fraction V. 1 = 500 ng MSA, 2 = 250 ng MSA.

BERNHARD,

DARLINGTON

AND RUDDLE

Albumin

Synthesis

by Cell

Clones

FIG. 3. Identification of mouse serum albumin by double diffusion (Ouchterlony). Double diffusion was performed in 1% sodium barbital buffered agarose at pH 8.6. Dextran (mw 500,000) was added to the agarose at a final concentration of 8%. Precipitin lines were photographed by incident light after 2 days’ diffusion at room temperature. AMS = Antimouse serum (rabbit). MSA = mouse serum albumin, ethanol extract Cohn fraction V; ML = mouse liver. Homogenized mouse liver was centrifuged for 15 min at 40,000 g, and the clear supernatant was assayed. Hl = Mouse hepatoma (Hepa-1). Culture medium dialyzed and concentrated 300-fold by ultrafiltration. The sample volume is 5 ~1 each.

firmed by immunoelectrophoresis and diffusion against monospecific rabbit antiMSA (Fig. 4). Leucine-‘T Incorporation Serum Albumin

into

Mouse

The incorporation of “C labeled leucine was followed by radioimmunoelectrophoresis. Studies on protein biosynthesis using this method must be interpreted cautiously. Labeling of serum proteins can occur by several ways not related to protein biosynthesis: by binding to the amino acid itself, by coprecipitation with newly made non-

specific proteins, or serum proteins already present in the medium can bind the labeled amino acid (Philips and Thorbecke, 1965). The latter possibility was ruled out by incubating cell free medium which contained 10% BSA or MSA with the labeled leucine. No labeled precipitin fractions were observed when concentrated medium was tested against anti-MSA by double immunodiffusion. Further it has been demonstrated by immunoelectrophoresis that the labeled fraction was identical with the precipitin line formed between purified MSA and anti-MSA (Fig. 5). These results

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hours later the supernatant was saved for the MSA assay and the cells were harvested for the determination of cell protein and intracellular MSA. This procedure was repeated with further replicate bottles at l-day intervals, until the cultures reached confluency. All MSA secretion values are Secretion of MSA during Culture Growth corrected for the increase of cell protein The accumulation of MSA in the super- during the accumulation period. The highnatant medium was measured quantitaest secretion rates were found during lag tively by the Laurel1 electroimmunodiffuphase (Fig. 6). A small but significant drop sion method. In the first experiment, MSA of the MSA secretion rate was observed secret.ion was studied during culture consistently when the cultures entered loggrowth. The results provided the basis for arithmic growth, usually at day three. One kinetic assays of MSA secretion and for the might also notice the significant increase of quantitative comparison of different cell the standard deviation at this point. No populations in respect to MSA secretion decrease of albumin secretion was observed rates. Replicate cultures were plated out in in the stationary phase as it has been small Falcon bottles at 5 x 10’ cells per reported by others (Ohanian et al., 1969; bottle and the cells were allowed to attach Tashjian et al., 1970). To investigate the in medium containing 10% FCS. After 24 possibility that differences of secretory achr the first set of three bottles was har- tivity exist between cells in lag and logavested for protein determinations. In an- rithmic phase, MSA present intracellularly other set of three bottles the medium was was followed throughout the culture cycle. replaced with serum free medium and One might expect to find accumulation of MSA was allowed to accumulate. Twelve MSA in the cells for short periods of time, together with the data obtained from the kinetic assays of MSA secretion provide conclusive evidence that the protein accumulated in the supernatant medium is mouse serum albumin, synthesized and secreted by the hepatoma cells.

AMSA

MSA FIG. 4. Identification of mouse serum albumin by immunoelectropboresis. Electropboresis was performed in 1% sodium barbital-buffered agarose at pH 6.6. Separation was carried out for 60 min at 6 V/cm. The dried agarose slides were stained with Amido Black. AMSA = Antimouse serum albumin (rabbit). MSA = mouse serum albumin, ethanol extract Cohn fraction V; Hl = mouse hepatoma (Hepa-1) culture medium concentrated 300.fold by ultrafiltration. The sample volume is 5 ~1 each. Identity of the electrophoretically separated H-l protein with MSA is demonstrated by the fusion of the precipitin lines.

BERNHARD, DARLINGTONAND

Albumin Synthe& by Cell Clones

RUDDLE

89

AMSA

AMSA FIG. 5. Radioimmunoelectrophoresis of mouse serum albumin. Immunoelectrophoresis was performed in 1% sodium barbital buffered agarose at pH 8.6. Separation was carried out for 60 min at 6 V/cm. The dried agarose slides were coated with nuclear track emulsion (Kodak NTB-2) and exposed for 7-10 days. AMS = Antimouse serum (rabbit); Hl = mouse hepatoma (Hepa-1) culture medium dialyzed and concentrated 300-fold by ultrafiltration. The sample volume is 5 ~1. The cultures were incubated for 24 hours with 10 &i/ml oL-“C-leucine, which was added to the culture medium.

-I4 - 13 -

12

-I1

-10 -9 -8 -7O

= !!? e Q ii : = J ii

-6 -5 -4

0

I

2

3

4

5

6

7

8

9

DAYS

FIG. 6. Mouse serum albumin (MSA) secretion of mouse hepatoma cells (Hepa-1) during culture growth. MSA secreted into the supernatant medium and intracellular MSA released by sonication was assayed by electroimmunodiffusion (Laurell, 1966). Three determinations were performed from triplicate samples, the values are given as average mean * standard deviation. The sample volume is 5 ~1 for each determination. Cell protein was measured by the method of Lowry et al. (1951). Cell numbers were counted with a Coulter Counter. 0, MSA released into the supernatant medium is expressed as nanograms per milligram of cell protein secreted in 1 hour; A, intracellular MSA released by sonication (Campbell et al., 1960) is expressed as nanograms per milligram of cell protein; 0, cell number per culture dish.

90 particularly

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at

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during

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After 2 days the medium

was replaced by

serum-free medium, and54 aliquots of growthwhichcoincide with decreased secretion rates. If intracellular albumin was assayed over one growth cycle, no changes in MSA present in the cells were observed. The ratio of intracellular MSA to total cell protein remained fairly constant (Fig. 6). In particular, no increase of intracellular MSA was detectable at the point where MSA secretion rates dropped and the cultures entered logarithmic growth. Serum albumin detected intracellularly represents approximately one-fourth of the amount which is secreted per hour during the logarithmic phase of the growth cycle. If we assume a rather slow degradation of the protein in uiuo as well as in vitro (Rothschild et al., 1972; Tashjian et al., 1970), we estimate that albumin, once synthesized, is exported within 15-20 minutes. This is in good agreement with estimates reported by Tashjian et al. (1970) for a rat hepatoma line, the absolute amounts of albumin secreted per hour being quite different. Similar data have been reported by several authors. Based on the incorporation of labeled amino acids in vim, Schreiber et al. (1970) have calculated that the period between intracaval injection of the radioactive amino acid and the appearance of labeled albumin in the blood was on the order of 15 min. Detailed time course studies on the transport of newly synthesized albumin have been reported by Glaumann and Ericsson (1970) and by Peters et al. (1971). Fifteen to 20 minutes after injection of the tracer, the Golgi apparatus contained albumin of the highest specific activity. Kinetics

of MSA Secretion

The rate of MSA secretion was determined more accurately by short time accumulation experiments (Fig. 7); 1 to 5 x lo5 cells were plated out in small petri dishes (35 mm diameter) containing 1 ml of medium. The cells were allowed to attach in medium supplemented with 10% FCS.

supernatant were taken at 2-hour intervals. The accumulation of MSA was measured during the following 24 hours. A linear increase of MSA in the supernatant was measured during the first 14 hours; after that the accumulation began to level off, and the standard deviations were significantly increased. Similar observations have been reported by Bissel and Tilles (1971). Identical results were obtained when the accumulation of MSA was followed in culture medium containing 10% FCS. It has been shown in uiuo and in perfused liver systems that osmotic pressure regulates albumin secretion and probably its synthesis (Rothschild et al., 1972). One might therefore consider the possibility that MSA secreted by the cells affects albumin secretion by increasing the osmotic pressure of the medium. This is not the case. It has been reported by Rothschild et al. (1972) that albumin per se did not depress its synthesis and secretion in vitro. We have performed various experiments to determine whether the accumulation of MSA into the supernatant medium can be manipulated by increasing the osmolarity of the culture medium. We found no indication that BSA (40 mg/ml), the average normal plasma concentration for albumin in uiuo, or sucrose (10 mg/ml) added to the cultures during the accumulation period affected the accumulation of MSA in the supernatant medium. We are left with the observation that linear accumulation persists only for a limited period of time, and we think it is important to assure that the accumulation is linear, if albumin secretion rates are calculated from such an experiment. It is therefore not possible to compare the secretion rates presented here with values determined previously in other hepatoma cell lines (Bancroft et al., 1969; Richardson et al., 1969; Tashjian et al., 1970; Peterson and Weiss, 1972). The secretion rates given here are

BERNHARD,

DARLINGTON 10000

AND RUDDLE

Albumin

Synthesis

by Cell

Clones

91

r

V 0

I 2

, 4

I 6

a

I 10

I 12

I 14

I 16

HOURS

FIG. 7. Kinetics of mouse serum albumin (MSA) secretion by mouse hepatoma cells (Hepa-1). MSA secreted into supernatant medium was assayed by electroimmunodiffusion (Laurell, 1966). The assay was performed while the cultures were in logarithmic growth. MSA secreted is given as differential of cell protein. Sample volume is 5 ~1 for each determination. Mean and standard deviations are calculated from 10 determinations for each point.

expressed as nanograms of MSA secreted per milligram of protein per hour, calculated from the linear increase during the first 12 hr after the medium had been replaced. Routinely the assays were performed without FCS present in the medium. Effects

of Drugs

on Albumin

Production

The fact that MSA can be assayed in serum-free medium allowed us to study the effect of different drugs on albumin production in vitro under defined conditions. The effects of several hormones, insulin, cortisone, and thyroxine, on serum albumin production in vivo have been reviewed by Rothschild et al. (1972). Several authors have studied the stimulation of albumin secretion by hydrocortisone in a rat hepatoma cell culture (Bancroft et al., 1969; Richardson et al., 1969; and Tashjian

et al., 1970). We have performed preliminary experiments with clone Hepa-1. Hepa-l has been demonstrated to be inducible for tyrosine aminotransferase activity (TAT) by the synthetic steroid dexamethasone. Dexamethasone concentrations ranging from 10m5 M to lo-’ M have been found to induce TAT maximally (Bernhard et al., 1971; Darlington et al., 1972). The same dexamethasone concentrations have been chosen to test its effect on MSA synthesis in Hepa-l cultures. Thyroxine was tested at concentrations between 10m5 M and 10m8 M. The cells were plated out as described for the routine assay of MSA. The medium was replaced with serum-free medium containing the hormones at various concentrations and the effect of the hormone was studied during the following 24 hours. The effect of the hormones was tested during lag, log, and stationary phase of culture

92

DEVELOPMENTAL

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growth. Both hormones proved to be ineffective in respect to MSA secretion under the conditions tested. Continuous exposure to dexamethasone 10m5 M in medium supplemented with 10% FCS for 2 weeks did not produce any change of MSA secretion. The effect of insulin on MSA production by Hepa-l cells is at present under investigation. The parental population Hepa as well as subclones derived from Hepa-l will be included in further studies on the hormonal stimulation of MSA production in vitro.

Analysis

of Subclones

MSA secretion rates were analyzed in subclones derived from the clonal line Hepa-l (Fig. 1). The secretion rates were determined following the standard procedure as described under “Kinetics of MSA secretion.” No significant difference was found between Hepa and clone Hepa-l (Table 1). Upon subcloning of clone Hepa-l significant differences in the amounts of MSA produced were observed among the ten subclones tested. The subclones also differ remarkably in their growth capacity. Therefore, one might argue that the secretion differences found are due to the fact that in the course of the standard assay for MSA, fast growing clones might have already entered logarithmic growth, while the slow ones were still in lag phase. Secretion rates determined by the standard procedure would then mimic differences in MSA secretion capacity between the different clones. This was not the case. Cell protein and cell number were measured in each experiment to assure that the cultures were in logarithmic growth during the experiments. In addition, the different MSA secretion rates in lag and logarithmic growth as observed in Hepa-l (Fig. 6) would not account for the differences observed between some of the subclones. The values assayed repeatedly in individual subclones were reproducible. These determinations were performed within a relatively short period of a few weeks, the

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35, 1973 TABLE

1

SERUM ALBUMIN SECRETION RATES OF CULTURED MOUSE HEPATOMA CELLS= Cell line

Hepa Hepa-l b Hepa-lab b : e f g h i k

MSA

(ng/mg/hr)

Mean

SD

720 420 230 2200 loo0 900 700 550 450 260 250 230

150 200 100 150 loo 150 50 200 100 100 100 150

Molecules/ cell/min

57,600 33,600 18,400 176,ooo 80,000 72,000 56,000 44,000 36,000 20,800 20,000 18,400

Sample number

6 18 18 6 6 6 6 6 6 6 6 6

’ Mouse serum albumin (MSA) was accumulated in the supernatant medium during logarithmic culture growth. MSA in the medium was assayed by electroimmunodiffusion (Laurell, 1966). MSA secreted per hour is given as corrected for the increase of cell protein or cell number during the accumulation. Protein was measured by the method of Lowry et al. (19511. Cell numbers were determined with a Coulter counter. MSA molecules secreted per minute per cell are calculated by assuming a molecular weight of 69,000 for MSA. b Means and SD are calculated from three experiments, which were performed over a period of 4 months. The cells were in continuous culture for that period.

cells being in continuous culture. To study further the stability of the function, clone Hepa-l and subclone Hepa-la were assayed repeatedly over an extended period in continuous cultures. The values given for these two populations (Table 1) are the pooled results of three experiments performed within 4 months. Hepa-l and Hepa-la proved to be quite stable in respect to MSA secretion, under routine culture conditions. The standard deviations were not significantly increased if compared with the SD, which were obtained from measurements performed within a few days. DISCUSSION

The availability of a hepatoma line, which produces serum albumin has already

BERNHARD,

DARLINGTON

AND

RUDDLE

proved to be useful for studying the modulation of specialized functions in hybrid somatic cells (Peterson and Weiss, 1972; Darlington et al., 1973). Information on the stability of this trait and on the homogeneity of cultured cells and their clonal derivatives contributes significantly to the analysis of phenotypic expression in vitro. The mouse hepatoma line reported in this study secretes albumin at a rate of 57,600 molecules/cell per minute. Similar values have been reported by Ohanian et al. (1969) for a rat hepatoma. Other rat hepatomas reported in the literature produced considerably less albumin as demonstrated by measuring secretion rates (Bancroft et al., 1969; Peterson and Weiss, 1972). The presence of insulin in our selection and maintenance media might have been important for the isolation of cells which produce high amounts of albumin. It has been reported by John and Miller (1970) that insulin is required for the maximal production of albumin in perfused liver. Upon cloning and subcloning of the mouse hepatoma line extensive variation was found among the populations isolated. One subclone (Hepa-lb) was isolated which produces 176,000 molecules/cell/minute. This is ten times more compared to the secretion rate of the parental clone (Hepa-la) (Table 1). As liver and possibly hepatomas consist of several cell types, it is not unexpected that clonal lines can be isolated which produce higher amounts of albumin if the secretion rates are calculated on a per cell basis. Liver parenchyma1 cells are presumably the only ones that produce serum albumin. They represent approximately 60% of the total liver cell mass (Munro, 1969). Hamashima et al. (1964) demonstrated by immunofluorescent methods that in normal human liver only a small percentage of cells is engaged in albumin production at a given time. Using fluorescein coupled antimouse serum albumin, we have studied the production of albumin in several lines derived from the mouse hepatoma. Our results

Albumin

Synthesis

by Cell

Clones

93

confirm the observations reported by Hamashima et al. (1969). In addition, we have compared Hepa and its clonal derivatives, and we found no apparent differences in the frequency and distribution of albuminproducing cells. However, as we did not use synchronized cell cultures, the method does not allow us to distinguish whether this is due to a stable epigenetic heterogeneity in the cell population or to the fact that the cells were in transient physiological states, in the case of dividing cells in different compartments of the cell cycle. If it is assumed that in the clonal lines all the cells are involved in the production of serum albumin, the rate of secretion per cell does not compare favorably to that calculated from perfused rat liver systems. The highest secretion value (175,000 molecules/cell/minute) (Table 1) determined in the mouse hepatoma represents approximately 10% of the secretion rate reported for perfused rat liver (Gordon and Humphrey, 1960). This estimate is based on the assumption that 1 g of liver contains 1.7 x 10’ cells (Weibel et al., 1969), and approximately lOa cells per gram of liver are parenchymal cells (Munro, 1969). The perfused liver reported by Gordon and Humphrey (1960) secretes, therefore, about 2 x 10’ molecules/cell/minute serum albumin. Several reports, including our own, have shown that albumin, once synthesized, is secreted within a few minutes (Glaumann and Ericsson, 1970; Schreiber et al., 1970; Peters et al., 1971). The half-life of albumin in viva is on the order of 20 days (Rothschild et al., 1972). Very little information is available on the degradation of albumin secreted into the tissue culture medium but preliminary studies suggest that turnover occurs at a low rate in comparison to synthesis (Tashjian et al., 1970). If we consider the rapid secretion process and the slow degradation of the secreted albumin it seems to us that the assay of secretion is a useful means to determine the synthesizing capacity of cells in vitro. The assay of secretion rates

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or hereditary nonhas been chosen for its simplicity and somal rearrangements, chromosomal factors. It has been reported because measurement of albumin synthesis by means of labeled amino acid incorporepeatedly that particular, specialized ration requires extensive purification. It functions are extinguished in hybrid clones has been pointed out by several authors (see review by Ephrussi, 1972). Peterson that data on albumin synthesis can be and Weiss (1972) have reported that in rather misleading, if the labeled albumin is hybrids between albumin producing rat not processed to radiochemical purity hepatoma cells and mouse fibroblasts, rat (Schreiber et al., 1970; Peters et al., 1971; albumin production was reduced or lost in several hybrid clones. The recovery of low Rotermund et al., 1970). The immunoassay used in this study is less laborious and or nonalbumin producers in hybrid clones allows the quantitation of albumin producmight be an alternative explanation for the tion with good reproducibility. We have “modulation” of the function observed. demonstrated that with this technique it is The same interpretation might be applied possible to characterize cell populations by to the cases in which the restoration of their specific secretion rates, if the cell temporarily lost functions in hybrids has density is carefully controlled. Our data been explained on the basis of the segregafrom the clonal analysis provide useful tion of repressor elements (Klebe et al., information on the homogeneity of cul- 1970; Weiss and Chaplain, 1971; Bertured hepatoma cells in respect to albumin tolotti and Weiss, 1971). However, in the production as well as on the stability of this case of the kidney specific esterase-2 excell function under various culture condipression the evidence for genome-genome tions. The fact that subclones have not interaction is possibly strengthened by the been found which lack albumin secretion fact that the restoration of the esterase-2 completely demonstrates the stability of expression may be correlated with the this phenotype in cultured hepatoma cells. segregation of human chromosomes (Klebe However the data from the clonal analysis et al., 1970). The demonstration of hetclearly indicate that the extent of albumin erogeneity in the expression of specialsecretion might vary considerably between ized phenotypes as presented in our alsubclones derived from a clonal cell line. bumin studies and in the study of TAT, as Further cloning of subclone Hepa-la which recently reported by Aviv and Thompson has been proved to be stable over an (1972), introduces an additional source of extended period in continuous culture, variation in phenotype expression in hymight answer the question whether this brid cells. It is our view that without an variation is due to physiological instability adequate characterization of phenotype or genetic heterogeneity within clones. The variation in the parental cell populations, fact that a cell population derived from a hybridization experiments cannot be propsingle cell is not necessarily homogeneous erly evaluated. The characterization of in respect to a specific trait has recently population heterogeneity in the several been reported by Aviv and Thompson mouse hepatoma cell populations serves as (1972) in the case of inducible tyrosine a basis for the experimental analysis of aminotransferase (TAT) in the rat hepa- phenotype modulation in various hybrid toma line HTC. The possibility that they cell combinations. Subsequent reports will were analyzing a heterogenous “clone” is deal with these experiments, which are very unlikely because of the cloning proce- currently in progress. dure they used. They conclude that the The authors would like to express their appreciation variability observed is related rather to the to Mrs. Mae Reger for her excellent secretarial work. instability of this function, due to extraorThis study was supported by NIH grant USPHS dinary high rates of mutation, chromo5-ROl-BM 09966 and NSF grant GB 34303. and a

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