XENOBIOTICA,

1991, VOL. 21,

NO.

7, 945-960

Fast Track Paper Protooncogene expression in rat liver by polychlorinated biphenyls (PCB) H.-S. JENKET, G. MICHEL, S. HORNHARDT and J. B E R N D T Gesellschaft fur Strahlen- und Umweltforschung, Abteilung fur Zellchemie, Ingolstaedter Landstr. 1, D-8042 Neuherberg, Germany

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Received 21 January 1991; accepted 14 March 1991 1. T h e expression of 10 protooncogenes was studied in control rat liver and at various times after exposure to polychlorinated biphenyls (PCB), a known tumour promoter.

2. The expression of protooncogenes in liver is more pronounced in those rats treated with PCB beginning at weaning (‘weanlings’) than in adult rats. 3. T h e RNA levels of c-Ha-ras, c-raf, c-yes, c-erbA and c-erbB are elevated after PCB feeding.

4. Nuclear run-on transcription analysis revealed that the altered expression of the protooncogenes is transcriptionally regulated. 5. In one group the prompt rise of the protooncogene transcription rate is followed by a decline (c-Ha-ras, c-raf, c-yes). In a second group a further increase in transcription at later feeding times (c-erbA, c-erbB) was observed. 6. A correlation between the altered expression of these protooncogenes and the action of PCB as a tumour promoter remains to be determined.

Introduction It is widely accepted that protooncogenes are involved in signal transduction pathways, growth control and differentiation in mammalian cells (Weinstein 1987). There are several ways of protooncogene activation including the alteration in gene expression (Buick and Pollack 1984, Weinstein 1988). Inappropriate expression of protooncogenes may disturb the process of signal transduction (Bishop 1983, Reymond et al. 1986). An altered expression of protooncogenes can be seen in tumour tissues and transformed cells (Land et al. 1983b, Hsieh et al. 1987, Thiele et al. 1988). T h e development of certain tumour types is preceded by preneoplastic lesions (Bannasch 1986, Schulte-Herrmann and Timmermann-Trosiener 1984, Farber 1973, 1980). Whether an altered protooncogene expression is critical for the development of focal areas of preneoplastic cells is not known. Polychlorinated biphenyls (PCB) are highly lipophilic compounds, which are widely distributed and persistent in the environment. These xenobiotics have toxic potency (Kirnbrough 1980, 1987), induce drug metabolizing enzymes (Hinton et al. 1978, Traber et al. 1988) and alter lipid metabolism, including steroid metabolism (Garthoff et al. 1977, Jenke 1985, Jenke et al. 1988). Utilizing the initiation/promotion model, PCB have been shown to be a hepatic tumour promoter (Oesterle and Demel1983, Kobush et al. 1989, Preston et al. 1981). PCB enhances the number of preneoplastic enzyme-altered islands (pre-stages of both benign and malignant neoplasms) in rat liver (Deml and Oesterle 1986, Norback and Weltrnann 1985).

t T o whom correspondence

should be addressed.

0049-8254/91 $3.00 0 1991 Taylor & Francis Ltd.

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Among the pleiotropic effects caused by tumour promoters, changes in gene expression may be one of the most important events (Hiwasa and Sakiyama 1986). Little is known about the mechanism. A wide variety of chlorinated hydrocarbons, which are considered to be hepatic tumour promoters, stimulate protein kinase C (Moser and Smart 1989). A PCB receptor like the Ah-receptor for tetrachlorodibenzodioxin (TCDD) has not yet been isolated. For TCDD, dioxinresponsive elements (DRE) and transcription factors have been identified (Denison et al. 1988), but not yet for PCB. A PCB binding activity to the thyroxin nuclear receptor (McKinney et al. 1987) and to the soluble uterine oestrogen receptor (Korach et al. 1988) has been described, the relative binding affinity depending on the various congeners of PCB (Kannan et al. 1988). In the present paper the results of investigations are described, which were aimed at identifying altered expression of protooncogenes caused by the tumour promoter PCB. Where appropriate, the mechanism involved was studied further in more detail.

Materials and methods Chemicals Deoxycytidine 5’-(a-32P)-triphosphate,triethylammonium salt, ca. 3000 Ci/nmol; uridine-5’-(a32P)-triphosphate, triethylammonium salt, ca. 400 Cilmmol and (meth~l-~H)thyrnidine, ca. 90 Ci/mmol were purchased from Amersham Buchler GmbH, D-3300 Braunschweig, Germany. Nitrocellulose paper was obtained from Schleicher & Schuell, D-3354 Dassel, Germany. Restriction endonucleases and E. coli DNA polymerase (Klenow fragment) and all other enzymes were obtained from Boehringer Mannheim, Germany. All other chemicals and molecular biological reagents were purchased from E. Merck, D-6100 Darmstadt 1, Germany or from Sigma GmbH, D-8024 Deisenhofen, Germany. Animals and diet Beginning at weaning (3 weeks old) one group of female Sprague-Dawley rats were kept on a standard diet o r on a 0.05% PCB supplemented diet for 0 2 5 , 0.5, 1, 2, 4, 10, 21, and 42 days (‘weanling rats’). In a second group all animals were 10 weeks of age at the end of these different feeding periods (‘adult rats’). The standard diet, Altromin 1320 (Altromin International GmbH, D-4937 Lage, Germany) contains carbohydrates (SO, 5%), cellulose (673, proteins (19%), fat (4%) and appropriate amounts of salts (including trace elements) and vitamins. The metabolizing energy is 14235 kJ/kg. This standard diet was supplemented with 0.05% Clophen A 50 (a kind gift from Dr Wrabetz, Bayer AG, Leverkusen, Germany), a commercial mixture of polychlorinated biphenyls with a mean chlorine content of about 54%, the main components being trichlorobiphenyls (973, tetrachlorobiphenyls (28%), pentachlorobiphenyls (44%), hexachlorobiphenyls (16%) and heptachlorobiphenyls (2%). Food and water were provided ad libitum. For the initiation/promotion bioassay diethylnitrosamine (DEN) was dissolved in water immediately before use. One does of 30mg/kg body weight was given i.p. to the weanling rats. The animals were then kept on a 0.05% PCB-containing diet for the indicated periods until 6 weeks. Autoradiography and histology [methyl-3H]Thymidine (200pCi) was injected i.p. into the control- and PCB-fed rats 6 h prior to slaughter. Formaldehyde-fixed, paraffin-embedded, haematoxylin and eosin-stained 3 pm liver sections were obtained with standard procedures and autoradiographed by exposing to Kodak emulsion type NTB 2 for 6 days. The labelled nuclei per area were determined. The histology examinations were performed by A. Lutz and W. Schmahl, Pathological Institute, GSF. Plasmids and D N A probes The following DNA fragments were used: v-abl, 1.5 kilobase (kb) BglII fragment from the A-MuLV genome (Reddy et al. 1983, Wang et al. 1984); v-Ha-ras, 0.62 kb HindIII to BamHI fragment from the Balb-MuSV-DNA (Andersen et al. 1981a, b); v-erbA, 0 5 kb PstI fragment of the AEV genome (Vennestrom et a f . 1980); v-erbB, 0.5 kb BamHI fragment of the AEV genome (Vennestrom et at. 1980); v-fms, 0.5 kb PstI fragment excised from the Kpnl to BglII fragment of a SM-FeSV genome cloned into the PstI site of pBR322 (Donner et al. 1982); v-mos; 1.2 kb XbaI to HindIII fragment excised from the EcoRI to HindIII fragment of a MuSV genome (Van Beveren et al. 1981); c-myc, 4 8 kb XbaI to BamHI fragmentofthemousecellularmycgene(Landetal.1983 a)clonedinpSV2gpt(Mulliganand Berg 1981); v-raf, 1.38kb XhoI to BglII v-raf specific fragment of the 3611 MSV genome (Rapp et al. 1983); c-sis, 2.1 kb SstII to Sstl fragment of a c-human sis specific c-DNA clone (Ratner et al. 1985); v-yes, 1.1 kb

Protooncogene expression in rat liver by PCBs

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EcoRI to HindIII fragment of avian sarcoma virus Y73 genome (Kitamura et al. 1982); b-actin, 0 9 k b HindIII fragment of HAc-69A clone (Moos and Gallwitz 1983). Glyceraldehyde phosphate dehydrogenase (GAPDH) 1.1 kb PstI fragment of the GAPDH gene cloned in pBR322.

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R N A extraction, slot-blot and Northern blot analysis Livers were removed immediately at the end of feeding times, placed in liquid nitrogen and stored at -80°C. Total RNA was isolated by the guanidinium isothiocyanate method according to Chirgwin et al. (1979). For slot-blot hybridization total RNA (10 or 20pgglslot) was denatured with formaldehyde and applied on nitrocellulose filter using the Minifold I 1 system. For Northern blot hybridizations total cellular RNA (20 pg/lane) was denatured with glyoxal and electrophoresed on 1%agarose gels according to Maniatis et al. (1982). Gels were stained with acridine orange prior to the transfer of the RNA onto nitrocellulose membranes. RNA was subsequently fixed to the membrane by baking at 80°C for 2 h. RNA blots were prehybridized for about 24 h at 42°C in hybridization mixture containing 40% formamide, 5 x Denhardt's (50 x Denhardt's is 1% bovine serum albumin, 1% Ficoll, 1% polyvinylpyrollidone), 1% sodium dodecylsulphate (SDS), 5 x standard saline citrate (SSC) (20 x SSC is 3 M NaC1, 0 3 M Nacitrate), 0 2 % Na,P,O,, 10H,O, 0 2 5 mg/ml denatured DNA, and 0 5 mg/ml denatured RNA. Hybridizations were performed in the same buffer containing approx. 2.5 x lo6 cpm/ml of 32P-labelled DNA probes for 1-3 days at 42°C. T h e DNA probes were 32Plabelled by nick translation (Rigby et al. 1977). After hybridization the filters were washed under stringent conditions at 55°C in 0.5 x SSC, 1% S D S and exposed to Kodak X-Omat S film or Konica X-ray film A2 for desired periods. T h e relative abundance of transcripts was determined by densitometric analysis of the autoradiographs using an Elscript 400, Hirschmann, D-8025 Unterhaching, Germany. It was confirmed that the experiments were done in the linear range of the film. Run-on transcription assay Nuclei from rat liver were isolated by a modification of the procedure described by Osborne et al. (1987). Liver tissue was powdered under liquid nitrogen and homogenized at W " C in buffer A (0.25 M sucrose, lOmM Tris-HCI, p H 7 5 , 5 mM MgCI,, 1 g tissue/lOml buffer A) by 20 strokes with a motordriven Teflon pestle in a Potter-Elvehjem-type glass homogenizer. T h e lysis was monitored by phase contrast microscopy. T h e homogenate was centrifuged for 10 min at LO00 x g.The crude nuclei sediment was resuspended in 6 ml buffer A, mixed with 6 ~ 0 1of . buffer B (2.4 M sucrose, lOmM Tris-HCI, p H 7.5, 5 mM MgCI,), put on a cushion of buffer C (2.2 M sucrose, 1OmM Tris-HCI, p H 7.5, 5 mM MgCI,) and centrifuged for 80min at 90000g in SW25.1 tubes. T h e nuclei were washed with buffer A and stored in 50mM Tris-acetate, pH8.3, 5 m MgCI,, ~ 0 1 M EDTA, 40% glycerol in liquid nitrogen. In wztro transcription assays were performed in principle as described (McKnight and Palmiter 1979): RNA was synthesized from 1 x l o 7 nuclei for 30min at 26°C on a total vol. of 200pl containing 25 mM Tris-acetate, 7.5 m M MgCI,, 2.5 m M dithiothreitol (DDT), 0.15 mM EDTA, 0 . 4 m each ~ of ATP, pH 8 , 3 , 7 0 m KCI, ~ G'I'P, C T P , 1 . 6 UTP ~ ~ and 65pCi of ( C X - ~ UTP, ~ P ) 20% glycerol and 60 units RNAsin. After the addition of another 6 units RNAsin and 20p1 DNAse I (1 unitlml) the incubation was continued for 15min at 26°C. S E T buffer (200pl) (1 x SET is 2% SDS, 1OmM EDTA, 20mM Tris-HCI, p H 7.4) and 40p1 proteinase K (1 mg/ml) were added followed by incubation for 30min at 42°C. Subsequently, 40pg of carrier RNA from Saccharomyces cereviseae and 1 vol. of 10% trichloroacetic acid (TCA) containing 6 0 m ~Na,P,O,, 10H,O were added and after 30min at 0°C the precipitate was collected on nitrocellulose filter and washed with 3% TCA containing 3 0 m ~Na,P,O,, 10H,O. The RNA was ~ 1 mM extracted from the precipitate by adding 0.9ml DI-buffer ( 2 0 m Hepes, ~ pH7.5, 5 m MgCI,, CaCI,), 120 units RNAsin and 11 pI DNAse I (1 unit/ml) to the nitrocellulose filter and by incubation at 37°C for 30min. The reaction was stopped by adding E D T A to 15 mM and SDS to 1%, followed by an incubation at 60°C for 1Omin. T h e filters were extracted with 5OOp1 of 1 x S E T buffer at 60°C for IOmin. Both extracts were combined and treated with proteinase K (25 pg/ml extract) for 30min at 37°C. From this solution the RNA was extracted with phenol and chloroform (1 vol. phenol-chloroform 1 : 1 v/v) and ethanol precipitated in the presence of 0.3 M acetate and 1OOpg of carrier RNA at -20°C. Typically, 1-2 x 10' cpm of radiolabelled RNA were obtained per reaction. Equal amounts of radiolabelled transcripts were hybridized to nitrocellulose-bound single-stranded probes. Hybridization were performed in 10 ml hybridization mix (see above) for 3 days at 42°C. Washing and autoradiography was performed as described above.

Immunoblot analysis Immunoblot analysis was performed as described by Towbin et of. (1 979). Liver tissue of control- and PCB-fed weanlings, stored in liquid N,, was powdered and homogenized in lysis buffer (1OmM sodium phosphate, pH 7.5, 0.5% sodium deoxycholate, 0.1% SDS, 0.1 M NaC1, 5 mM EDTA, 0.01% sodium azide). Proteins (200pg) were separated on a S D S polyacrylamide (10%) gel according to Laemmli (1970) and the proteins were electrophoretically transferred to nitrocellulose membranes (0.9 mA/cm2, 2.5 h, 20% methanol). T h e membrane was washed in transfer buffer being Tris-HC1, p H 8.3, 1 9 0 m glycine, ~ 20 mM phosphate buffer, pH 7.4, 200mM NaC1, 3% bovine serum albumin for 4 h at 37°C and incubated with the appropriate dilution of the first antibody (mouse monoclonal anti protein kinase C, IgG,,

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antibody class, provided by Amersham Buchler, GmbH, Braunschweig, Germany) for 2 h at 37°C in washing buffer (20mM phosphate buffer, p H 7 , 4 , 2 0 O m ~NaCl, 1 mM EDTA, 0 3 %Triton X100). After treatment with washing buffer the membrane was incubated with the second antibody (goat anti-mouse IgG, conjugated with horseradish peroxidase, provided by Dianova, Hamburg, Germany) in washing buffer. Antigens were visualized by a peroxidase-based detection method. With a monoclonal antibody for protein kinase C (provided by Amersham Buchler GmbH, Braunschweig, Germany) a 79 kDa protein was identified. This protein band was determined densitometrically.

Results Feeding schedule, liver histology, liver toxicity and D N A synthesis rate In our approach to evaluate the role of PCB in altering the expression of protooncogenes two feeding designs were used. In one group of rats all animals received PCB for 025,0*5,1,2,4,10,21 and 42 days beginning at weaning (3 weeks old). The liver of these weanlings is still growing. At the end of the indicated feeding periods the rats were from 3 to 10 weeks old (‘weanling rats’). In a second group, all animals were at the same age at the end of these different feeding times, i.e. almost full-grown at 10 weeks, normal weight 240g (adult rats). Chronic administration of 005% PCB in the diet produced a gradual decline in body weight gain which became more pronounced after longer feeding periods although the food consumption was not reduced; this has already been described in detail by others (Garthoff e t a l . 1977, Baumann et al. 1983). It is also known that PCB results in hepatomegaly (Garthoff et al. 1977, Jenke 1985, Norback and Weltmann 1985). We have observed that after the 6-week feeding period the livers of weanling rats reached about 10% of body weight compared to 4% in controls. T h e pathological-anatomical evaluation of the livers of weanling rats revealed hepatocellular lesions only after long-term exposure. After 42 days some hypertrophic hepatocytes were observed (figure 1). T o examine whether PCB has an overall growth-promoting effect on rat liver we studied the DNA synthesis in the liver of weanling rats. DNA synthesis in PCBtreated rat liver (120 and 500ppm PCB in the diet) exhibited only negligible differences from control rats. Furthermore, the autoradiographs show that DNA synthesis occurred in the periphery of the hepatic lobuli of PCB-treated rats rather than in the centre (data not shown here). From this it can be concluded that the toxicity of PCB is obviously not high enough to cause a general liver regeneration. Analysis of protooncogene expression T o screen the expression of a broad spectrum of protooncogenes we performed slot-blot analyses. Increasing amounts of rat liver RNA were slot-blotted and hybridized with a 32P-labelled probe-DNA. There was a linear increase in the intensity of the slot bands on the range of 1-20pg (data not shown). Rats were treated with PCB for different times and the expression of 10 protooncogenes was examined. For the hybridization experiments, DNA probes of protooncogenes were chosen whose protein products are localized in the nucleus, in the cytoplasm or in the plasma membrane. Figure 2 shows the expression of c-Ha-ras, c-raf, c-erbA and c-myc in control- and PCB-treated weanling rats examined by slot-blot analysis. These autoradiographs were scanned by a densitometer and the m-RNA levels of the protooncogenes in PCB-fed rats were compared with those of the controls (figure 3 (A)-(D)). As can be seen from the slot-blots the expression of protooncogenes even in controls change during the investigation period of 6 weeks. This is probably due to the growth of the weanlings during this

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Protooncogene expression in rat liver by PCBs

Figure 1. Photomicrographs of representative areas of ( n ) normal liver, and (b) liver removed from a rat fed 0.05% PCB for 42 days beginning at weaning (3 weeks old). Magnification: x 875.

H.-S. Jenke et al.

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Figure 2.

Autoradiograms from slot blots of total cellular RNA (1Opg) from livers of control- and PCBfed weanling rats. Five weanling ratseach were fed a control- or a PCB-containing diet for the indicated time periods. Total liver RNA was isolated and slot-blotted on nitrocellulose. The slot-blots were divided into halves. One half was hybridized to 32P-labelled onc probe of interest, the other to 32P-labelled GAPDII probe as a normalizing control.

time. Therefore, to show the PCB-specific effect, results are presented as percentage of controls. T o ensure that equal amounts of RNA were slot-blotted, the densitometric signals were normalized to the constitutively expressed GAPDH gene. PCB affects the expression of c-Ha-ras, c-raf, c-erbA and c-myc to different extents, and it can be seen that the m-RNA level of c-Ha-ras in weanlings is stimulated to about 3.5-fold within 24 h, drops back to normal and remains normal despite further PCB feeding. For c-raf a maximum of the m-RNA level is reached after 7 days of PCB feeding. T h e time-course of the m-RNA level for c-erbA is similar to that of c-raf, while that of c-myc does not change significantly. Beside these oncogenes there are others which were not influenced by PCB. I n table 1 the results of the slot-blot analyses of the 10 protooncogenes investigated are summarized. In general, the PCB effects on the expression of protooncogenes are similar in weanlings and in adult rats, but they are must more pronounced in the liver of weanlings. T h e protooncogenes can be grouped: those which are stimulated very early, those which are stimulated at later feeding times, and those which show no effect. T h e specificity of the probe fragments used in the slot-blot experiments was proved for c-raf, c-Ha-ras and c-erbA. T h e probes recognize the correct transcript sizes: 3.4 kb for c-raf (Miilders et al. 1985), 1.2 kb for c-Ha-ras (Hsieh et al. 1987, 1989) and ra. 5 k b for c-erbA (van de Vijver et al. 1987). Figure 4 shows the expression of c-raf in PCR-fed weanling liver determined by densitometric

Protooncogene expression in rat liver by PCBs

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Expression of (A) c-Ha-ras, (B) c-raf, (C) c-erbA, and (D) c-myc in weanling rats fed a 0.05% PCB diet. Five weanling rats each were fed a control- or a PCB-containing diet for the indicated time periods. RNA was isolated from the combined livers, slot-blotted and hybridized as described in Materials and methods. Autoradiographic signals depicted in figure 2 were quantified densitometrically, normalized to the GAPDH mRNA values obtained from the same sample and presented as percentage of the control.

Table 1 .

Expression of 10 protooncogenes in adult and weanling rat livers after feeding a 0.05%PCBcontaining diet compared to controls. Protooncogene

Adult rat liver

Weanling rat liver

c-abl c-Ha-ras c-erbA c-erbB c-fms c-mos c-myc c-raf c-sis c-yes

N.E.

N.E.

9

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9 N.E. N.E. N.E.

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N.E., no effect; 4 , mRNA levels are affected after very short-term PCB treatment (< 1 day); 9 , mRNA levels are affected after short-term PCB treatment ( < 10 days); +, mRNA levels are affected after long-term PCB treatment ( 221 days).

H . - S . Jenke et al.

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10 21 (DAYS) FEEDING TIME

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Northern hybridizationanalysis of c-raf RNA in weanling rat liver fed 0 0 5 % PCB containing diet compared to controls. RNA (20pg) of treated and control rats were used for analysis as described in Materials and methods. The autoradiographic signals (B) were quantified and normalized to the GAPDH mRNA signals and presented as percent of control (A). Changes in GAPDH are due to different amounts of RNA, not to changes in GAPDH expression.

Figure 4.

quantification after Northern blotting. In principle, examinations by the slot-blot (figure 3 (€3)) or the Northern blot method (figure 4) yielded qualitatively similar results.

Nuclear run-on transcription analysis T o ascertain whether the changes in expression of these protooncogenes are due to transcriptional control we isolated the liver cell nuclei from control- and PCB-fed weanlings and performed nuclear run-on experiments. T h e transcription rates of c-Ha-ras, c-raf, c-yes, c-erbA and c-erbB (figure 5) and their densitometric analysis (figure 6) show a 2-%fold transient increase after PCB treatment, those of c-Ha-ras, c-raf and c-yes then decline but they are higher than controls even after 42 days. T h e transcription rate of c-erbA and c-erbB show an increase again after long-time feeding. C-fms is a homologue of the CSF1-receptor which is mainly localized in macrophages, i.e. the ‘Kupffer’ cells of the liver. PCB, however, attacks mainly the parenchymic cells, and the unchanged expression of c-fms (figure 6) is therefore expected; c-fms is thus valid as an internal control. In figure 7, the slot-blot, Northern blot and nuclear run-on transcription analyses of the expression of c-raf are placed side by side. With regard to the m-RNA level the results obtained by these methods are quite consistent. From the nuclear run-on transcription analyses it can be concluded that PCBdependent changes in the expression of these protooncogenes are the consequences of alterations in the transcriptional control.

PCR as tumour promoter It is known that the tumour promoter 12-O-tetradecanoyl-phorbol-l3-acetate (TPA) acts by binding to protein kinase C and thus activating this calcium- and

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Protooncogene expression in rat liver by P C B s

Figure 5.

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Autoradiograms of nuclear run-on transcription analysis of protooncogenes in livers of control and 0.05% PCB-treated weanling rats.

The indicated protooncogene DNAs (2 pg) was immobilized on nitrocellulose filters. Isolation of the nuclei, preparation of 32P-labelled nuclear RNA transcripts and hybridization to nitrocellulose bound single stranded DNA probes was performed as described in Materials and methods. The nuclei were prepared and pooled from five weanling rat livers for each feeding time point.

1

c-erb B

c-erb A c-Ha-ras c-raf c-yes

c-fms

1

42

10

PCB

FEEDING

TIME [ D A Y S )

Figure 6. Transcription rate of protooncogenes in livers of weanling rats fed 0.05% PCB for different time periods. The autoradiographic signals depicted in figure 5 were quantified densitometrically, normalized to the actin mRNA signals and presented as percentage of controls.

H.-S. Jenke et al.

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Figure 7. Comparison of the expression of c-raf examined by (A) slot blot, (9) Northern blot, and (C) nuclear run on transcription analysis in weanling rat livers fed PCB for different time periods. Experimental procedure as in figures 3, 4 and 6

phospholipid-dependent enzyme (Nishizuka 1984). Since PCB is considered also to be a tumour promoter, we measured the expression of protein kinase C in PCBtreated weanlings. Figure 8 shows that the amount of protein kinase C changes in weanlings during the investigation period. PCB feeding induces the enzyme very rapidly (within 1 day) but then the level of protein kinase C falls and after 42 days the amount detectable by Western blot analysis is far below control. In particular, the disappearance of protein kinase C after PCB treatment is remarkably similar to the effects of TPA on protein kinase C carried out with rat glioma cells (Young et al. 1987). The promoting effect of PCB on diethylnitrosamine (DEN)-initiated preneoplastic foci has been shown in a rat liver foci bioassay, using ATPase deficiency as a marker (Oesterle and Deml 1983, Deml and Oesterle 1986). In DEN-initiated weanling rats, enzyme-altered islands develop frequently after 12 weeks of a promoting PCB treatment. These hyperplastic liver nodules are a well-studied model system of chemical carcinogenesis (Norback and Weltmann 1985, Farber and Cameron 1980). In contrast to this rather long-term model system, we studied whether the expression of cellular oncogenes is affected by PCB in a short-term cancer initiation/promotion bioassay. Figure 9 shows the results: D E N alone has a stimulating effect neither on c-Ha-ras nor on c-raf oncogene expression. DEN in combination with PCB results in a stimulation of protooncogene expression which is similar to that of PCB alone (figure 3). Thus, the effect seems to be PCR-specific. During the observation period (6 weeks) of this in vitro short-term initiation/promotion bioassay, hyperplastic nodules did not develop; this is a later event as shown by others (Deml and Oesterle 1986, Norback and Weltmann 1985). Therefore, a correlation between the altered expression of protooncogenes and the action of PCB as a tumour promoter (based on this bioassay) remains to be determined. However, it cannot be excluded that the altered PCB-induced protooncogene expression is an early event of tumour promotion.

Protooncogene expression in rat liver by PCBs

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0.01 21 42 FEEDING TIME (DAYS)

1 4 10 Figure 8.

Relative amount of protein kinase C in control and 0.05%PCB-treated weanling rat livers.

Western blotting procedure was performed as described in Materials and methods. The protein kinase C band (79 kDa) was quantified densitometrically. For each time-point the liver lysate from PCB-treated rats five treated rat livers was used for the Western blot analysis. Control rats (0); (0).

Discussion PCB have been shown to result in the development of preneoplastic lesions (Oesterle and Deml 1983), Norback and Weltmann 1985) which are considered as pre-stages of both benign and malignant neoplasms (Bannasch 1986, Farber 1980). Alterations in protooncogene expression may change cellular differentiation and proliferation (Weinstein 1988, Reymond et al. 1986, Thiele et al. 1988, Goyette et al. 1984) and are common in tumour cells and in transformed cell lines (Bishop 1983, Land et al. 1983 b, Sistonen et al. 1989). Of particular interest to us has been whether the tumour promoter PCB interacts with protooncogene expression. T h e experiments reported here demonstrate qualitatively altered mRNA transcription of multiple protooncogenes in weanling and adult rats. T h e five cellular protooncogenes which are most affected by PCB in v i v o are c-Ha-ras, c-raf, c-yes, c-erbA and c-erbB, the changes of each being transcriptionally regulated. Weanlings are more susceptible than adult rats. PCB feeding to weanlings results in a very short-term and transient rise of the rat liver c-Ha-ras mRNA, exhibiting a maximum at 24 h. DEN-initiated/PCBpromoted animals show a similar time-course of the c-Ha-ras expression, whereas DEN alone has no effect. A comparable time-course and an increased expression of c-Ha-ras has been shown by others in rats treated according to the resistant hepatocyte model (Porsch-Hallstrom et al. 1989). With respect to our results it has to be considered that the ras gene can be malignantly activated also through quantitative mechanisms (Bos 1988). For instance, an increased expression of c-Haras is observed in rat hepatocellular carcinomas (Ishikawa et al. 1986, Beer et al. 1986). Moreover, increased c-Ha-ras expression is an early and stable event in liver lesions associated with hepatocarcinogenesis (Galand et al. 1988). T h e plasma membrane bound c-Ha-ras oncogene product, p21, is expressed in many human

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1 10 42 FEEDING TIME (DAYS)

A B C

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TREATED

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Figure 9.

Expression of (A) c-raf, and (B) c-Ha-ras in livers of DEN-initiated and DEN-initiated/PCBpromoted weanling rats.

The initiation/promotion bioassay protocol1 was performed as described in Materials and methods. The autoradiographic signals of the Northern blots were quantified densitometrically and presented as percentage of control. The RNA of five weanling rat liver for each feeding time was used for Northern blots.

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cancers (Tanaka et al. 1986), and in rat-1 cells p21 is assumed to be involved in signal transduction (Burgering et al. 1989). PCB is known to be incorporated into cell membranes and to result in a protein-lipid interaction (Jenke 1985); it may also interact with c-Ha-ras. T h e c-raf expression is elevated transiently until about 10 days of PCB feeding to weanlings. In the initiation/promotion bioassay, again DENinitiated/PCB-promoted animals show a similar time-course of c-raf expression; DEN alone has no effect. Additionally, both the results obtained by Northern blotting experiments, and those obtained by nuclear run-on transcription assays, indicate a slightly elevated c-raf expression even after longer PCB feeding times. Indeed, in neoplastic nodules and hepatocellular carcinomas the endpoint of the rat liver carcinogenesis in the resistant hepatocyte model, elevated levels of mRNA for c-raf oncogene has been shown (Beer et al. 1988). By preliminary results we can also confirm that in isolated rat liver neoplastic nodules generated by DENinitiation/PCB-promotion treatment, the c-raf mRNA is elevated more than 15fold. In these neoplastic nodules the elevated c-raf expression is linked with known markers of preneoplastic foci, as reactivation of y-glutamyltranspeptidase and ATPase deficiency (data not shown). Usually high levels of normal c-raf 1 mRNA are found in mouse lung carcinoma cell lines and in human tumour cell lines (Rapp et al. 1988). Their contribution to tumour induction has still to be determined, but it is assumed that the c-raf oncogene product has basic regulatory functions in a platelet-derived growth factor (PDGF)-receptor-mediated signal transduction (Morrison et al. 1989). T h e c-erbA expression was elevated transiently between 2 and 7 days of PCB feeding to weanlings. In contrast with the other protooncogenes there is an obvious difference between the time-course of the mRNA level of c-erbA (figure 3 (C)) and that of the transcription rate (figure 6). T h e transcription rate is further increased at the end of the PCB feeding period. This might be due to a posttranscriptional regulation. On the one hand, the c-erbA gene encodes a thyroid hormone receptor (Weinberger et al. 1986), and on the other, PCB interacts with specific binding sites for thyroid hormone in rat liver nuclear extracts (McKinney et al. 1987). Therefore, PCB might interfere with the c-erbA oncogene product and modulate the expression. In this context it is interesting that the thyroid hormone plays a critical role in induction of neoplastic transformation (Borek et al. 1983). Of special interest to us is a possible correlation between the protooncogene expression shown here and the effects of the tumour promoter PCB creating preneoplastic lesions. T h e search for PCB-binding proteins, transcription factors and (corresponding to the dioxin responsive elements (DRE)) in the cytochrome P-450 genes (Denison et al. 1988, Whitlock 1987) for xenobiotic-responsive elements, in the 5'-upstream sequences of the protooncogenes are of importance. Furthermore, investigations are necessary to elucidate the mechanism and a possible contribution of PCB to the development of preneoplastic lesions.

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