Extinction of Peroxisomal Functions in Hepatoma Cell-Fibroblast Hybrids
El Bachir Bioukar, I Sophie Sarrazin] Marc Conti, 1 Eric Rabetafika, 1 Jean-Paul Carreau, 1 Sophie Dhorne-Pollet, 1 Nicole Raynaud, I and Jean Deschatrette ~,2
Received 16 Mar. 1995--Final 2 Jan. 1996
Although peroxisomes are ubiquitous, differences in the number of organelles and in the expression of associated metabolic activities are observed, depending on the cell type. To investigate the control o f peroxisomal activity in connection with cell differentiation, we constructed hybrids between two types of cells whose histogenetic origins dictate significant differences in peroxisomal activities: hepa- toma cells and fibroblasts, with high and low expression, respectively, o f peroxi- somal functions. In these hybrids, extinction of the elevated activities that characterize liver cells is observed, in parallel with the well-documented extinc- tion of differentiated functions. This suggests the existence in fibroblasts of a negative trans-acting regulation.
Peroxisomes are cellular organelles that carry out a series of catabolic and anabolic functions, some of which are fullfilled mainly or exclusively in these structures. These include 13-oxidation of very long-chain fatty acids (VLCFA) (Lazarow, 1978; Singh et al., 1987; Jakobs and Wanders, 1991), bile acid biosynthesis (Kase et al., 1983), a part of plasmalogen synthesis (Hajra et al., 1979), and glyoxylate transamination (Noguchi and Takada, 1984). In
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addition, peroxisomes are involved in cholesterol synthesis (Thompson et aL, 1987), hydrogen peroxide-based respiration (de Duve et al., 1960), and the metabolism of purine fatty acids, long-chain dicarboxylic acids, prostaglan- din, and xenobiotics. The import of peroxisomal proteins into preexisting organelles leads to the growth of these structures, which divide to form new ones. Thus, the biogenesis of peroxisomes depends on the production of both constituents and preexisting organelles (Borst, 1989; Lazarow, 1988).
Studies of the regulation of peroxisomal activities have focused on the proliferation of these organelles triggered in rodent liver by xenobiotic such as hypolipidemic agents (Lazarow and de Duve, 1976; Osumi and Hashi- moto, 1984). Data have also been collected on the developmental depen- dence of peroxisomes genesis and structures (Keller et aL, 1993). Although the peroxisomes are ubiquitous, present in all cells other than mature erythrocytes (Hruban et aL, 1972; Novikoff et al., 1973), their number and shape and the associated metabolic activities vary, depending on the cell type. The extent of these variations suggests that the biogenesis of peroxi- somes is under the control of the regulatory mechanisms that establish the differentiated state of the cells. Yet whether or not the organelle should be considered as an entity regulated by the program of differentiation is poorly understood.
To investigate this further, we undertook a genetic approach of the phenotypic traits of somatic hybrids between cells of different histogenetic origins. An abundant literature has led to the establishment of rules of expression of these specific functions in such hybrids (Ephrussi, 1972; Ringertz and Savage, 1976; Weiss, 1982). Basically, extinction of differenti- ated traits is observed in products of fusion, while housekeeping functions encoded by both parental genomes are expressed. Furthermore, the associa- tion between chromosome segregation that occurs in the course of cell division and reexpression of specific functions illustrates the stability of epigenotypes in hybrid cells.
Our aim was to determine whether or not peroxisomes are submitted to the same type of regulations in growing hybrids between liver cells and fibroblasts. Hepatocytes are rich in peroxisomes and exhibit high levels of expression of most of the associated functions compared to fibroblasts. We report here clear extinction of peroxisomal functions in hybrids between rat hepatoma ceils and transformed or untransformed human skin fibroblasts.
MATERIALS AND METHODS
Cell Lines. Fao and C2rev7 are well-differentiated subclones (Deschatrette and Weiss, 1974; Deschatrette et al., 1980) of the H4IIEC3 line (Pitot et aI., 1964) derived from the rat hepatoma Reuber H35 (Reuber, 1961). A strain
Peroxisomal Functions in Hepatoma Cell-Fibroblast Hybrids 79
of diploid human skin fibroblasts (hSF) was initiated from explants. A transformed clone was isolated after transfection with the plasmid pAS (Benoit and Chambon, 1981) containing the SV40T antigen. This trans- formed cell line (thSF) has been registered under the name HGK1-SV40T1. Two series of hybrid clones were used. The first series corresponds to products of fusion between Fao or C2Rev7 hepatoma cells and hSF or thSF fibroblasts (see Table 1 for nomenclature). The second series also corre- sponds to hybrids between fibroblasts and rat hepatoma cells. These hybrids clones (WIF12-1, WIF12-6, WIF12-9, WIF12-A, WIF12-E, and FHA7), isolated after extensive segregation of human chromosomes, were previously analyzed by other authors (Griffo et al., 1993).
Culture Conditions. All ceils were cultured in modified (Coon and Weiss, 1969) Ham (1965) F12 medium supplemented with 5% fetal calf serum and equilibrated with 10% CO2 in air. Cells were detached for transfer in trypsin (0.5 g/L)- and EDTA (0.2 g/L)-containing saline solution.
Cell Hybridization. Cell fusion was carried out using a 50% (w/v) polyethylene glycol (PEG) 1000 solution in serum-free medium (Mevel- Ninio and Weiss, 1981). Six hours after PEG treatment, cells were detached, inoculated at a low density, and submitted to the appropriate selective medium the next day. Fao and C2rev7 hepatoma ceils are HGPRT- and resistant to 2 mM ouabain. Selection of hybrids was performed in HAT (hypoxanthine, aminopterin, thymidine) medium containing 1 mM ouabain. Determination of the rat and human chromosome content of hybrids was performed by fluorescence in situ hybridization (FISH) on colcemid- arrested metaphases. Pretreatment of slides, probe labeling, and hybridiza-
Table I. Nomenclature for the Parental and Hybrid Cell Lines
Well-differentiated rat hepatoma clones Nontransformed human skin fibroblasts Human skin fibroblast cell line transformed by SV40T antigen Fao x hSF hybrid clones Fao x thSF hybrid clones C2rev7 x thSF hybrid clone
Chromosomally reduced hybrids between Fao and human fibroblasts. All clones are segregants of a unique product of fusion, WIF12 (Griffo et al., 1993).
Chromosomally reduced hybrids between Faza967 and human fibroblasts. Faza967 is a rat hepatoma clone, progenitor of Fao (Griffo et al., 1993).
80 Bioukar et al.
tion were performed according to published methods (Desmaze et al., 1992). Sonicated total human DNA labeled by nick translation with biotin 11- dUTP (Tropix) was used as a probe. Hybridization signals were amplified and visualized using avidin conjugated to fluorescein (avidin-FITC) and biotinylated goat antiavidin (Vector Laboratories) according to a published method (Driesen et aL, 1991). Analysis was performed with a Leitz Aristo- plan microscope equipped for FITC detection.
Immunocytofluorescence Analysis. Two days after inoculation in plastic wells, the cells were washed with phosphate-buffered saline (PBS) and fixed in 3% cold formaldehyde and methanol as described (Bioukar et aL, 1994). After washing with PBS, fixed cells were incubated for 20 min at 37°C with an antiserum raised against purified rat liver fatty acyl-CoA oxidase (EC 1.3.3.3) (Osumi et al., 1980). This antiserum has been shown to crossreact with the human protein. Slides were then rinsed with PBS, treated with rhodamine-associated anti-rabbit antibodies for 20 min at 37°C, rinsed with PBS, and mounted in buffered glycerin for observation.
Use of Erucic Acid for Tests of Cell Sensitivity and Metabolic Studies. Erucic acid (C22:1~o9) was added to the culture medium as a complex with albumin. Preparation of the complex was described previously (Bioukar et aL, 1994).
Isolation and Analysis of Cell Lipids. Extractions of lipids from cells treated with [14-14C]erucic acid (Dositek, Orsay, France) were performed as described (Bioukar et aL, 1994), as well as separation of the methyl ester derivatives of fatty acide by thin-layer chromatography (Carreau et aL, 1980). The radioactive spots were detected by autoradiography. Quantifica- tion of the signals was performed by densitometric scanning in a linear response range.
Preparation and Analysis of Cellular RNA. The method used to prepare total cellular RNA has been described (Auffray and Rougeon, 1980). Electrophoresis of RNA was performed through agarose gels containing formaldehyde (Maniatis et aL, 1982) and the RNA transferred on nylon membranes. A 1100-bp fragment of the rat 3-ketoacyl-CoA thiolase encod- ing cDNA (clone pMJ203) (Hijikata et aL, 1987) and a 2800-bp fragment of the rat acyl-CoA oxidase encoding cDNA (clone pMJ115) (Miyazawa et al., 1987) were used as molecular probes. The DNA sequences used as probes were 32p-labeled by nick translation. A 30-base oligonucleotide exhibiting 100% identity with the cDNA of both human and rat [3-actin was end labeled. Glyceraldehyde-3-phosphate dehydrogenase cDNA (GAPDH, Clon- tech) was 32p-labeled by the mega prime procedure. Both 13-actin and GAPDH probes were used for quantification of RNA samples. Quantifica- tion of the signals revealed on the autoradiograms of Northern blot analysis was performed by densitometric scanning in a linear response range.
Pcroxisomal Functions in Hepatoma Cell-Fibroblast Hybrids 81
Catalase Activity. Subconfluent cells were harvested using a rubber policeman and homogenized by ultrasonic disruption: Catalase assay was performed as described previously (Johansson and Borg, 1988). Sample enzymatic activities were calculated from a standard curve using catalase (EC 1.11.1.6) purchased from Sigma.
Dihydroxyacetone Phosphate Acyltransferase (DHAP-AT; EC. 2.3.1.42) Activity. The enzymatic activity was measured as described by Singh et al. (1989).
RESULTS
Cell Hybridization and Selection of Hybrids
Hybrids were constructed using as parental ceils well-differentiated rat hepatoma cells of the Fao and C2rev7 clones and transformed and untrans- formed human skin fibroblasts. Six hybrid clones were isolated from these fusion experiments after selection in HAT medium supplemented with Ouabain (see Table I for the nomenclature of these hybrids). To establish the constitution of the hybrids in rat and human chromosomes, FISH experiments with total human DNA as a probe were performed on recta- phases of the isolated hybrid clones. The mean chromosome number of the rat hepatoma lines is 51.9 (range, 50-55), while the untransformed human fibroblasts are diploid. The transformed fibroblasts contain 42-44 chromo- somes with constant abnormalities. Both series of hybrids (with transformed and nontransformed fibroblasts) contained the whole chromosome sets of parental ceils.
Extinction of Liver-Specific Functions in the Hybrids
The selected hybrids did not exhibit the typical hepatocyte-like morphology of the parent hepatoma cell line. No albumin or aldolase B mRNA was detected in these hybrids (data not shown). Overall, the well-documented extinction of tissue-specific functions in somatic cell hybrids was observed in these rapidly growing products of fusion.
Analysis of Expression of Peroxisomal Functions in the Hybrids
Detection of Peroxisomes by Immunocytofluorescence. Peroxisomes were visualized in the parental and hybrid cells using antibodies raised against fatty acyl-CoA oxidase, a peroxisomal matrix enzyme involved in J3-oxidation of VLCFA. As shown in Fig. la, the hepatoma cells were characterized by multiple dots of fluorescence revealing the presence of numerous organelles
~Z
o
Peroxisomal Functions in Hepatoma Cell-Fibroblast Hybrids 83
in these liver ceils. In contrast, the fibroblasts (transformed and untrans- formed; Fig. ld) contained relatively few peroxisomes. The hybrids re- sembled the fibroblast phenotype with fewer peroxisomes compared to the hepatoma ceils (Figs. lb and c).
Resistance to Erucic Acid. Some metabolic functions are exclusive to peroxisomes, including [3-oxidation of very long-chain fatty acids. We have demonstrated previously that the resistance of cells in culture to erucic acid (C22:1), a monounsaturated very long-chain fatty acid, depends on peroxi- somal [3-oxidation (Bioukar et al., 1994). In the absence of [3-oxidation, C22:1 accumulates, with a toxic effect on the cells. Killing curves were established for the parental and hybrid cells, and as shown in Fig. 2, the results indicate that the resistance of the hybrids to this fatty acid was close to that exhibited by the fibroblasts and generally much lower than the level
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EA (#mol/I) Fig. 2. Killing curves of parental cells and hybrids treated with erucic acid (EA). Media containing various concentrations of erucic acid were applied to ceils seeded the previous day at a density of 2 • 10 5 cells per 10-cm-diameter dish. Four days later, living cells were counted. On the ordinate are the data corresponding to the percentage of living cells compared to that in cultures treated with s tandard medium (without erucic acid). Cell line nomenclature as in Table I.
84 Bioukar et al.
of resistance of the hepatoma cells. One hybrid clone (Ftf12) exhibited an intermediate level of resistance. This partial extinction may be linked to quantitative differences in expression of peroxisomal functions among the Fao clone constituting cells.
[3-Oxidation of Erucic Acid. Cells were inoculated in culture dishes (106 cells per 10-cm diameter dish), and the next day a medium containing 25 gM 14-14C-labeled erucic acid (sp act, 2.2 ~Ci/Ixmol) was applied to the cul- tures. The cells were harvested after 24 hr of treatment with labeled C22:1, the lipids were extracted, and monoethylenic fatty acid methyl esters were fractionated on thin-layer chromatography. The autoradiogram (Fig. 3) revealed labeled C20:1 and C18:1, products of B-oxidation and erucic acid (C:22), the starting material, in all three types of cells. In the hepatoma cells the C18:C22 ratio was clearly greater than in the fibroblasts and hybrid cell lines. Similarly the C20:C22 ratio was obviously greater in the hepatoma cells. The quantitative estimations (Table II) confirmed this observation.
Expression of mRNAs Encoding Enzymes of PeroxisomaI [3-Oxidation. Total RNA was prepared from the different cell types and analyzed by electrophoresis, transfer, and hybridization to 32p-labeled rat acyl-CoA oxidase and thiolase cDNAs. Strong signals were detected with both probes in rat hepatoma cells, compared with the very weak signals in human fibroblasts (Fig. 4, lanes 1 and 2 and lanes 3 and 4, respectively). This is relevant both to the poor level of expression of the genes in the fibroblasts and to the rat origin of the probes, mRNA encoding acyl-CoA oxidase was
1 C24:1,,,.~, C22:1.--.~ C20:1.--~ C 18:1.-~-
2 3 4
Fig. 3. Autoradiogram of thin-layer chromatography of fatty acids. Fatty acids were extracted from cells incubated for 24 hr with [14-14C]erucic acid (sp act, 2.2 ~Ci/ ~mol). Fractionation of methyl ester derivatives was performed by thin-layer chroma- tography (see Materials and Methods). Radioactive material was revealed on X-ray films exposed to the plates. Lanes: (1) Fao; (2) Ftf12; (3) Ftf13; (4) thSF1. Nomencla- ture as in Table I.
Peroxisomal Functions in Hepatoma Cell-Fibroblast Hybrids 85
Table I1. Quantification of Labeled Fatty Acids in Cells Treated with [14-14C]Erucic Acid a
aAutoradiograms of the thin-layer chromatography plates revealing labeled erucic acid and its products of [3-oxidation (see Fig. 3 and Materials and Methods) were scanned in a linear re- sponse range. The data are ratios of the collected data for eruric acid (C22), eicosanoic acid (C20), and oleic acid (C18).
also detected in hybrids (Fig. 4A) but the signals correspond to only about 5-10% of the signal detected in the hepatoma cells using the signals for [3-actin as the control (Fig. 4C). The thiolase mRNA was only faintly detected in the hybrid clones (Fig. 4B).
kb 1 2 3 4 5 6 7 8 9 10 A
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Fig. 4. Detection of RNA transcripts encoding peroxisomal radiolabeled pro- teins in parental and hybrid cells. Total RNA was prepared, fractionated in an agarose gel, transferred on nylon membranes, and probed with radiolabeled probes: (A) rat acyl-CoA oxidase cDNA; (B) rat thiolase cDNA; (C) mouse 13-actin cDNA. Before exposure to X-ray films, the filters were washed in 2x SSC, 0.1% NaDodSO4 at 65°C. Lanes: (1) Fao; (2) C2rev7; (3) thSF; (4) hSF; (5) Ftf12; (6) Ftf13; (7) Rtfl6; (8) FfA; (9) FfB; (10) FfD. Nomenclature as in Table I.
86 Bioukar et al.
Catalase Activity. Hepatoma cells are characterized by relatively high levels of catalase activity compared to both transformed and untransformed fibroblasts (Table II). In the hybrids, the activity is generally low, ranging from about 5 to 30% of the level detected in the parental liver cells.
Expression of Peroxisomal Functions in Chromosomally Reduced Hybrids
Expression of tissue-specific functions in somatic hybrids has been shown to be correlated with segregation of specific chromosomes of the extinguishing parental cells. Griffo et al. (1993) reported a detailed phenotypic and karyological characterization of hybrids between Fao hepatoma cells (or Faza967, progenitor of Fao) and diploid human fibroblasts. Three classes of hybrids with significant loss of human chromosomes were identified. Clones of the first class (WIF12-1, WIF12-6) reexpressed most of the hepatic functions, while clones of the second class (WIF12-A and WIF12-E) did not. Finally, WIF12-9 and FHA7 were intermediate, as only a subset of liver functions was fullfilled in these cells.
We have tested the resistance to erucic acid, amount of rat acyl-CoA oxidase mRNA, and catalase activity in this series of hybrid lines. As shown in Fig. 5, all the tested clones exhibit a high level of resistance to the fatty acid. Yet clear differences in the amounts of acyl-CoA oxidase mRNA and catalase activity were detected between the classes of hybrids. Compared to a control transcript, the signal for acyl-CoA oxidase mRNA is about 10-fold higher in the well-differentiated cells (WIF12-1 and WlF12-6; see Fig. 6, lanes 1 and 2) than in the cells where extinction of hepatic functions is observed (WIF12-E; see Fig. 6, lane 3) and intermediate in FHA7 (Fig. 6, lane 4). Also, catalase activity was high in the differentiated hybrids, intermediate in WlF12-9 and FHA7, and low in the extinguished clones (see Table Ill).
DHAP-AT Activities in the Hybrids. To test the involvement of the difference in species of origin of the cell lines in the expression of peroxi- somal functions, we tested DHAP-AT activity, slightly different in both parental cells (400 and 250 pmol/min/mg of protein in Fao and thSF1, respectively). The specific activity for the 10 hybrid clones tested (3 Ff, 2 Ftf, and 5 WIF12) ranged from 380 to 600 pmol/min/mg of proteins.
DISCUSSION
The tissue-specific regulation of peroxisome biogenesis and activity is still very poorly understood. We report here an investigation at the genetic level of the regulation of peroxisomal activities, considered as an entity.
Altogether, the results show an extinction of peroxisomal functions.
Peroxisomal Functions in Hepatoma Cell-Fibroblast Hybrids
1201
87
100
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0 6O c- >
> 40
o9 20
I
\',
- - i - - - FAO
--~5,--- thSF = Ftf
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0 50 100 150 200 250
EA (gmol/I)
Fig. 5. Killing curves of parental cells and chromosomally reduced hybrids treated with erucic acid. The experimental conditions are similar to those for Fig. 2. The data for only four chromosomally reduced hybrid clones are provided. The test was performed on WlF12-A and WIF12-9 too. The results are similar to those recorded for the other Wifl2 clones shown. Ftf is a pool of hybrids isolated in parallel with the Ftfl2 and 13 clones. This cell population shares with the Ftf series the same karyological and phenotypic characteristics.
Basically, the hybrids exhibit phenotypic traits of the fibroblast parental cells, characterized by reduced peroxisomal activities compared to the hepatoma cells. This involves both the number of organelles and metabolic activities. It is worth noting that most peroxisomal enzymes are inactive when they are not localized in the organelle. This is illustrated, for example, in patients deficient in peroxisome assembly, where failure of enzyme transport into the particle compar tment leads to abnormal molecular forms and the absence of catalytic activities. Yet some enzymes, including catalase, remain active when localized in the cytosol. Thus, the correlation between the number of peroxisomes per cell and the catalase activity illustrates the coordinated expression of the protein and biogenesis of the organelle. Such a correlation is observed in the liver cells, the fibroblasts, and the hybrid ceils too. Therefore, the extinction of peroxisomal functions in these hybrid cell experiments parallels observations of the same well-documented extinction
88 Bioukar et al.
kb A
3.8
B 1 . 1 - " 1 ~
1 2 3 4 5
Fig. 6. Detection of rat acyl-CoA oxidase R N A in chromosomally reduced hybrids. To- tal R N A was prepared, fractionated in an agarose gel, transferred on nylon mem- branes, and probed with radiolabeled probes. Lanes: (1) WIF12-1; (2) WIF12-6; (3) WIF12-E; (4) FHA7; (5) thSF. Probes: (A) rat acyl-CoA oxidase cDNA; (B) G A P D H cDNA. Before exposure to X-ray films, the filters were washed in 2 x SSC, 0.1% NaDodSO4 at 65°C.
of differentiated functions in such products of fusion (Ephrussi, 1972; Ringertz and Savage, 1976; Weiss, 1982). It is worth noting that extinction of specialized functions does not reflect a hierarchy in the specific traits of different cell types. In products of fusion where expression of both parental specific traits could be tested, extinction of the two sets of differentiated functions was observed (Foug6re and Weiss, 1978). This indicates that the high level of expression of peroxisomal activities in hepatoma cells is part of the hepatocyte epigenotype.
The mechanisms responsible for extinction are still poorly understood. Using microcell hybrids, a locus and ultimately the gene encoding the RloL
Table III. Catalase Activities in Parental and Hybrid Cells a
aSee Materials and Methods for the assay. One unit of catalase decomposes 1 Ixmol of H202 per min at pH 7.0 and 25°C, when the H202 concentration falls from 10.3 to 9.2 raM.
bValues are means of data from two enzymatic assays. CThe hepatoma clone parent of this hybrid is Faza967, progenitor of Fao.
Peroxisomal Functions in Hepatoma CelI-Fibroblast Hybrids 89
subunit of cAMP-dependent protein kinase A were identified and shown to be involved in the negative control of tyrosine aminotransferase expression (Killary and Fournier, 1984; Boshart et al., 1991). Also, a correlation was established between the presence in hepatoma-fibroblast hybrid cells of a recombined mouse chromosome and the extinction of serum albumin pro- duction (Petit et al., 1986). Yet whether or not these particular examples reflect general mechanisms for the extinction phenomenon observed in somatic hybrids is unclear. Griffo et al. (1993) characterized a series of hybrid clones between rat hepatoma Fao cells and human fibroblasts, where extensive human chromosomes segregation occurs. The authors revealed a correlation between reexpression of an entire group of liver functions and loss of human chromosome 2. The high levels of acyl-CoA oxidase mRNA and of catalase activity observed here are consistent with this pattern of liver gene expression. Yet cells of this series of hybrid clones exhibit levels of resistance to erucic acid close to that of hepatoma cells. This may be related to relative activities of the three enzymes of [3-oxidation pathway. Both extinction and reexpression of peroxisomal functions were observed, in parallel with tissue-specific regulation of liver functions. Peroxisome prolif- eration triggered by some xenobiotics (i.e., hypolipidemic compounds) has been documented and differences in the response to proliferators were reported, depending on species and cell types. This suggests that the observed extinction may be relevant to the disruption of a chronic prolifera- tion signaling pathway or a direct repression of the genes encoding peroxi- somal proteins.
ACKNOWLEDGMENTS
This work was supported by grants from l'Association Fran~aise contre les Myopathies. Jean Deschatrette is a CNRS Research Fellow. E1 Bachir Bioukar was a recipient of fellowships from la Socidt6 d'Etudes et de Soins pour les Enfants paralys6s et polymalformds, le Conseil Scientifique de l'Universit6 Paris XI, and l'Association pour la recherche sur le cancer. We thank Dr. Hashimoto for providing us with antiserum against acyl-CoA oxidase and cDNAs encoding acyl CoA-oxidase and thiolase. We thank Dr. Mary Weiss and Dr. Doris Cassio for the WIF12 and FHA7 hybrids.
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