Effects of heptachlor epoxide on components of various signal transduction pathways important in tumor promotion in mouse hepatoma cells

Toxicology 160 (2001) 139– 153 www.elsevier.com/locate/toxicol

Effects of heptachlor epoxide on components of various signal transduction pathways important in tumor promotion in mouse hepatoma cells Determination of the most sensitive tumor promoter related effect induced by heptachlor epoxide Mark E. Hansen a, Fumio Matsumura a,b,* a

Institute of Toxicology and En6ironmental Health, Uni6ersity of California, Da6is, CA 95616, USA b Department of En6ironmental Toxicology, Uni6ersity of California, Da6is, CA 95616, USA

Abstract The effects of the organochlorine (OC) liver tumor promoter heptachlor epoxide (HE; 0, 0.1, 1, 10, and 50 mM) on several cellular tumor promoter-sensitive parameters were studied in mouse 1c1c7 hepatoma cells in an effort to identify the most sensitive biomarker for OC promoter exposure and the critical pathway and target of OC promoters. The levels of Ca2 + in the endoplasmic reticulum (ER) store, connexin43 (Cx43), PLCk1, nPKCm, and AP-1 DNA binding in nucleus were studied to screen for effects induced by submicromolar HE levels. While all the parameters tested elicited effects, particulate PLCk1 and AP-1 DNA binding were found to be the most sensitive parameters affected by HE on both dose and temporal bases. Their levels were increased with 10- to 100-fold lower HE concentrations than were required to affect nPKCm or Cx43. Further, with the lower HE dosages, particulate PLCk1 and nuclear AP-1 were positively modulated by HE after 1 h versus 3 or 72 h for nPKCm and Cx43. Ca2 + store depletion was probably the third most sensitive parameter, after AP-1 and PLCk1. These results suggest the tyrosine kinase growth factor receptor pathway is the probable critical pathway for HE-induce tumor promotion with the critical target most likely being upstream of PLCk1 and AP-1. This work also demonstates that upon exposure to a tumor promoter such as HE, many hepatocellular effects or changes result, suggesting that a cellular-program shift occurs similar to that described by the resistant hepatocyte model after exposure to a carcinogen or enzyme inducer. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: AP-1 transcription factors; Connexin-43; Intracellular calcium stores; Liver cancer; Mouse hepatoma cells; Phospholipase C-gamma1; Protein kinase C-epsilon; Tumor promotion

Abbre6iations: Cx43, connexin43; EGMS, electrophoretic gel mobility shift; HE, heptachlor epoxide; NCI, National Cancer Institute; nPKCm, novel protein kinase C-epsilon; PLCk1, phospholipase C-gamma; SDS– PAGE, sodium dodecylsulfate-polyacrylamide gel electrophoresis. * Corresponding author. Tel.: +1-530-7522725; fax: + 1-530-7525330. E-mail address: [email protected] (F. Matsumura). 0300-483X/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S0300-483X(00)00445-5

140

M.E. Hansen, F. Matsumura / Toxicology 160 (2001) 139–153

1. Introduction Heptachlor is a cyclodiene insecticide that was used extensively from the 1950s until the late 1970s to early 1980s when its use was phased out by the US EPA due to its persistence, adverse environmental effects, and suspected carcinogenicity (National Cancer Institute (NCI), 1977; Williams, 1980; WHO, 1984). Heptachlor continued to be applied to pineapples in Hawaii until 1996 to indirectly control the mealy bug wilt which adversely affected this crop (Rick Scudder, 1978 personal communication, Hawaii Heptachlor Research and Education Foundation). Upon absorption by animals, heptachlor is metabolically transformed into its primary, stable metabolite heptachlor epoxide (HE). The biochemical mechanism responsible for HEs chronic effects has not been clearly elucidated, but a number of organochlorine compounds (OCs) have been shown to affect components of the phosphoinositide/protein kinase C (PKC)/activator protein 1 (AP-1) pathways (Suzaki et al., 1988; Moser and Smart, 1989; Rotenberg and Weinstein, 1991; Criswell et al., 1995; Hansen and Matsumura, 2000). Part-per-million levels of dietary HE have been previously shown by this laboratory to down-regulate particulate nPKCo in B6C3F1 male mouse liver tissue, while persistently upregulating AP-1 (activator protein 1) DNA binding activity (Hansen and Matsumura, 2000). Although the dosage levels found to induce these effects are revelant in terms of the etiology of heptocellular carcinoma in this strain of mice, a primary objective of this work was to determine if this or some other parameter was the critical event associated with HE’s tumor promotional effect in the liver. Several factors suspected to be important in tumor promotion were utilized as biomarkers for the effects of HE in mouse liver hepatoma cells. These include: (1) intracellular free Ca2 + ([Ca2 + ]i), an important component of the phosphoinositide cascade (Eto et al., 1995) found to

be increased by a number of tumor promoters (Yamaguchi et al., 1980; Madhukar et al., 1983; Thastrup et al., 1987; Suzaki et al., 1988; Perchellet et al., 1990). (2) connexin43 (Cx43), a gap junction component down-regulated by OCs (bands P1 and P2 containing phosphorylations at one and two critical sites, respectively), known to be important for regulating gap junctional intercellular communication (GJIC) and thought to play a triggering role in tumor promotion (Rose et al., 1977; Peracchia and Peracchia, 1980; Telang et al., 1982; Ruch et al., 1990; Matesic et al., 1994; Criswell and Loch-Caruso, 1995). (3) phospholipase C k1 (PLCk1), since it is activated by receptor-linked tyrosine kinases (RTKs), and up-regulated PLCk1 is associated with malignancy and various cancers (Noh et al., 1994, 1998; Chang et al., 1997; Yang et al., 1998). (4) nPKCm, because it is a key component of the phosphoinositide cascade activated by phorbol esters, it has oncogenic activity (Mischak et al., 1991, 1993; Cacace et al., 1993; Perletti et al., 1996), and its levels are modulated in certain cancer tissues (Hanania et al., 1992; Pongracz et al., 1995), and (5) nuclear activity levels of AP-1, since its transactivation is a downstream effect resulting from modulation of some of the parameters studied here (Nishizuka, 1984, 1992; Ran et al., 1986; Roche and Prentki, 1994; Janknecht et al., 1995; Whitmarsh and Davis, 1996), and it is believed to be a critical component of tumor promotion (Bernstein and Colburn, 1989; Ben-Ari et al., 1992; Dong et al., 1994, 1995; Li et al., 1996). In view of the availability of information on these key hepatic tumorigenic parameters, an effort was made to examine those parameters affected by HE to determine their comparative dose and time-course responses. The ultimate goal of this work was to identify the most sensitive and the earliest responding biochemical target in order to gain insight into the molecular mechanism and critical signal transduction target affected by this type of cancer promoter in hepatocytes. For this purpose, the mouse 1c1c7 hepatoma cell in vitro model was utilized.

M.E. Hansen, F. Matsumura / Toxicology 160 (2001) 139–153

2. Materials and methods

2.1. Chemicals and reagents HE was obtained from Dow Chemical (Midland, MI). Gamma-[32P]-adenosine triphosphate ( = 5000 Ci/mmol) was purchased from Amersham Life Sciences Inc. (Arlington Heights, IL). AP-1 responsive element oligonucleotides were purchased from Promega Corp. (Cat. c : E3300; Madison, WI). Poly (dI-dC) was purchased from Boehringer Mannheim Corp. (Indianapolis, IN). Acetylated bovine serum albumin (BSA) was purchased from Stratagene (La Jolla, CA). AntinPKCm and PLCg1antibodies were purchased from Santa Cruz Biotechnology Inc. (sc c 214, sc c 81; Santa Cruz, CA). Anti-Cx43 antibodies were purchased from Zymed Laboratories Inc. (cat. no. 13-8300; South San Francisco, CA). Fura-2/AM and pluronic F-127 were obtained from Molecular Probes (Eugene, OR). Ionomycin (free acid) was purchased from Calbiochem-Novabiochem Corporation (La Jolla, CA). h-Minimum Essential Media (h-MEM) and antibiotic/antimycotic solution were purchased from Life Technologies Inc. (Gibco BRL, Grand Island, NY). Piperonyl butoxide was purchased from Aldrich Chemical (Milwaukee, WI). All other chemicals and reagents (including, adenosine triphosphate (ATP), aprotinin, benzamidine HCl, calf serum, charcoal-treated dextran, chymostatin, dialyzed calf serum (10 kD maximum pore size), dithiothreitol, E-64, fetal bovine serum (FBS), leupeptin, i-mercaptoethanol, okadaic acid, pepstatin A, 1, 10-phenanthroline, phenylmethylsulfonyl fluoride, sodium bicarbonate, soybean trypsin inhibitor I, sodium vanadate, spermine, spermidine, (9)-sulfinpyrazone, thapsigargin, Triton X-100, 1X trypsin/EDTA medium) were purchased from Sigma Chemical Co. (St Louis, MO).

2.2. Cell culture and treatment protocol Mouse 1c1c7 hepatoma cells (Hankinson et al., 1985; Watson and Hankinson, 1992) were maintained on ‘Culture Medium’ (h-MEM supplemented with 26.2 mM sodium bicarbonate, 10%

141

FBS, and 1% antibiotic-antimycotic solution, pH 7.4, 100 units/ml penicillin G, 100 mg/ml streptomycin sulfate, and 0.25 mg/ml amphotericin B in 0.85% saline); medium was changed every 2 or 3 days and cells were subcultured following trypsinization about every 10 days. Following hepatoma cell growth to confluence or near confluence ( 70% for longer duration treatments), the normal Culture Medium containing 10% FBS was replaced with Culture Medium containing 10% heat-inactivated, charcoal-treated calf-serum (1 h at 55°C then rotated overnight at 4°C with 0.5% charcoal-coated dextran). Cells were treated with the appropriate HE concentration (or vehicle volume: 1 ml ethanol/ml medium [0.1%]; had no effect on the cell parameters assessed here) for the prescribed treatment time. For ER Ca2 + depletion experiments, Western blotting, and EGMS experiments, cells were treated for 1, 3, or 72 h with 0, 0.1, 1, 10, or 50 mM HE. For Western blotting and EGMS experiments, Culture Medium was removed and cells were rinsed and, harvested, in PBS. Cells were then sedimented at 50xg for 5 min at 4°C, and cell pellets were flash frozen in liquid nitrogen and stored at − 80°C until analyzed.

2.3. Cell 6iability experiments Cells plated and grown to confluence on 22× 40 mm glass coverslips were treated for 1, 3, and 72 h with 0, 0.1, 1, 10, or 50 mM HE. Following treatment, 2–4 drops of 0.4% Trypan blue solution (Sigma Chemical Co., cat. no.: T 8154) was applied for 2 min. Excess dye was drained, and total and nonviable (Trypan blue stained) cell numbers were estimated via light microscopy.

2.4. ER Ca 2 + store experiments Hepatoma cells were loaded for 30 min at 37°C with 5 mM fura-2/AM (20% (w/v) pluronic F-127 in DMSO, 5 ml/ml media) in the presence of 1% antibiotic –antimycotic solution, 250 mM sulfinpyrazone (in DMSO) and 100 mM piperonyl butoxide (in DMSO). Cells were then sedimented at 4°C for 3 min. 1156xgmax, rinsed with ‘Isotonic Medium’ (140 mM NaCl, 10 mM HEPES, 10 mM

142

M.E. Hansen, F. Matsumura / Toxicology 160 (2001) 139–153

glucose, 5 mM KCl, 1 mM CaCl2, 1 mM MgSO4, pH 7.4), and re-sedimented. Cells were then resuspended in 3 ml of Isotonic Medium, containing 250 mM sulfinpyrazone and 1% dialyzed FBS (10 kD maximum pore size), 1% antibiotic – antimycotic solution and placed into a 4 ml methacrylate fluorometry cuvette.

2.5. Intracellular Ca 2 + measurements Intracellular Ca2 + was measured using a computer controlled PTI (Photon Technology International Inc., South Brunswick, NJ) fluorimeter with a lens-based sample compartment, single excitation and emission monochrometers, LPS-220 arc lamp supply, SC-550 Shutter Control, and MD-5020 Motor Driver. The excitation wavelengths of 340 and 380 nm were used to monitor the Ca2 + bound and unbound forms of fura-2, respectively, and the emission wavelength utilized was 510 nm. At the end of each experiment, the Ca2 + ionophore ionomycin (20 mM in DMSO) was added to each batch of cells to obtain a fluorescence maximum (Rmax). A fluorescence minimum (Rmin) was obtained by suspending cells in a Ca2 + free Isotonic Medium, treating the cells with 20 mM ionomycin, and then adding 20 mM EGTA. [Ca2 + ]i was determined using the following equation: [Ca2 + ]= Kd ×(R-Rmin)/(Rmax-R) × Sf2/Sb2 according to the method of Grynkiewicz et al. (1985).

2.6. Western blotting analyses Treated cell pellets (see Cell Culture and Treatment Protocol section) were homogenized in 200 ml/dish (100 mm) chilled ‘Homogenization Buffer’ (250 mM sucrose, 20 mM HEPES, 5 mM EGTA, 2 mM EDTA, 50 mM i-mercaptoethanol, 2 mM DTT, and protease and phosphatase inhibitors [2 mg/ml aprotinin, 1 mM benzamidine HCl, 10 – 20 mg/ml chymostatin, 10 mg/ml leupeptin, 10 mM pepstatin A, 2 mM phenylmethylsulfonyl fluoride (PMSF), 10 mg/ml soybean trypsin inhibitor, type I-S, 10 nM okadaic acid], pH 7.5) and homogenized using a glass – teflon homogenizer (Potter –

Elvehjem) with 5 up-and-down strokes. Homogenates were fractionated by centrifugation at 100 000xgavg for 60 min at 4°C. Supernatants were saved as cytosolic fractions for nPKCm Western blots. Pellets were resuspended in the same volume of Homogenization Buffer plus 0.3% Triton X-100; 200 ml/dish), rehomogenized as before, and incubated with rotation at 4°C for 30 min. Protein was determined by the method of Bradford (1976), and samples were flash frozen in liquid nitrogen and stored at − 80°C. Sample protein (100 mg cytosolic and particulate nPKCm [large gels] or 40 mg particulate for Cx43 and [minigels]) was heated for 3 min at 95°C in the appropriate volume of 4X SDS treatment buffer (250 mM Tris –Cl, 8% SDS, 40% glycerol, 20% i-mercaptoethanol, pH 6.8). Proteins were separated on a 10% (nPKCm and PLCg1) or 12% (Cx43) SDS –PAGE gel, transferred to a 0.45 mm PVDF (polyvinylidene difluoride) membrane using a Bio-Rad Trans-Blot apparatus at 100 mA for 24 h at 4°C in Transfer Buffer (25 mM Tris –Cl, 192 mM glycine, 15% methanol, pH 8.3 to 8.0). Membranes were then blocked for 1 h with 5% nonfat dry milk (NFDM; w/v) in TBST (150 mM NaCl, 10 mM Tris –HCl, 0.06% Tween 20, pH 8.3 to 8.0) at room temperature. PVDF membranes were then incubated with 1 mg/ml anti-nPKCm or anti PLCg1 (Santa Cruz Biotechnology, Santa Cruz, CA; cat. no.: sc-214, sc-81) or anti-Cx43 (Zymed Laboratories Inc., South San Francisco, CA; cat. no.: 13-8300) antibody in 15 ml of TBST for 2 h. After a quick rinse (20 ml TBST), membranes were washed three times with 20 ml of TBST for 5 min each at room temperature. A 0.16 mg/ml horseradish peroxidase conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA; antirabbit sc2004, antimouse sc-2005) in 15 ml of TBST was incubated with the membrane for 1 h at room temperature. Membranes were rinsed as described before. ECL™ Western blotting detection kit from Amersham Life Sciences (Arlington Heights, IL) was utilized to detect bands of interest, and bands were quantitated via densitometer (Ambis Systems Inc., San Diego, CA).

M.E. Hansen, F. Matsumura / Toxicology 160 (2001) 139–153

2.7. AP-1 electrophoretic gel mobilization shift experiments Cells were treated as described above. Nuclei were isolated via a modification of the method of Dignam et al. (1983). Briefly, treated cells stored at −80°C were thawed and suspended in 3 ml of ‘Hypotonic Buffer’ (10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol [DTT], 0.74 mM spermidine, 0.15 mM spermine, pH 7.9, plus protease inhibitors) per 0.5 ml cell pellet to remove PBS. Cells were then quickly centrifuged at 1850xg for 5 min, 4°C. The packed cell pellet was resuspended in chilled 1.5 ml of Hypotonic Buffer, allowed to swell for 10 min, before homogenization with a Dounce homogenizer (type B pestle) with 10 up-and-down strokes. Nuclei were then centrifuged at 3300xg for 15 min at 4°C, and the supernatant was frozen and saved. The nuclear pellet was resuspended in one-half nuclear pellet vol of ‘Low Salt Buffer’ (20 mM HEPES, 25% glycerol, 10 mM KCl, 1.5 mM MgCl2, 0.5 mM DTT, pH 7.9), and an equivalent vol of ‘‘High Salt Nuclei Extraction Buffer’’ (20 mM HEPES, 25% glycerol, 800 mM KCl, 1.5 mM MgCl2, 0.5 mM DTT, pH 7.9) was added dropwise, followed by vortexing, to yield a final KCl concentration of 400 mM. The nuclei were extracted for 30 min at 4°C with rotation. Following extraction, nuclei were sedimented at 16 000xg for 30 min at 4°C. Nuclear extracts ( 1 ml) were dialyzed in 500 ml of ‘Dialysis Buffer’ (100 mM KCl, 20 mM HEPES, 20% glycerol, 0.2 mM EDTA, 0.5 mM DTT, plus protease and phosphatase inhibitors, pH 7.9) at 4°C with continuous mixing, and the buffer was changed after 1 h. After 2 h, samples were flash frozen in liquid nitrogen and stored at − 80°C. AP-1 transcription factor binding experiments were performed as described by Liu et al. (1996) with slight modifications. Protein extracts (10 mg) were incubated for 20 min at 25°C in ‘Gel Mobility Shift Incubation Buffer’ (80 mM KCl, 10 mM HEPES, 4% Ficoll, 1 mM EDTA, pH 7.9) with 0.2 to 0.6 ng/lane k-32P-ATP labelled AP-1 responsive element oligonucleotide. Poly (dI-dC) [1 mg/0.1 ng oligonucleotide] and acetylated BSA (5

143

mg/lane) were incubated to inhibit nonspecific binding. Specificity was determined by preincubating selected samples with a 250-fold molar excess of cold AP-1 responsive element. Samples were separated on a 6% nondenaturing polyacrylamide gel for 2–4 h at 30 mA/gel. Gels were dried and incubated with X-ray film, and bands were quantified using Ambis densitometry.

2.8. Statistical analyses Statistical significance was determined via a two-tailed Student’s t-tests, with differences judged significant at P00.05. Each data point was expressed as a mean9standard error of the mean (SE) or percent mean9percent SE.

3. Results

3.1. Cell 6iability experiments The Trypan blue exclusion methodology was employed to assess mouse hepatoma cell viability after exposure to 0, 0.1, 1, 10, or 50 mM HE for 1, 3, and 72 h HE. For all treatment levels tested, HE had a small or no effect on cell viablility. A 0.1 mM HE concentration had no effect on cell

Fig. 1. Effect of HE on cell viability. Hepatoma cells were grown to confluence on glass coverslips as described in Section 2. Cells were treated with 0, 0.1, 1, 10, or 50 mM HE for 1, 3, or 72 h. At the appropriate time, cells were stained with one or two drops of Trypan blue solution then examined under light microscope. Each bar and error bar represents a mean 9 standard error of the mean three individual coverslips per treatment. An asterisk, if present, indicates statistical significance compared to controls at P00.05. (Student’s t-test).

M.E. Hansen, F. Matsumura / Toxicology 160 (2001) 139–153

144

Table 1 Effect of HE on the ER Ca2+ store Treatment time 1h

3h

72 h

Treatment

[Ca2+]i, nMa

% depletion

[Ca2+]i, nMa

% depletionb

[Ca2+]i, nMa

% depletion

Control 0.1 mM HE 1 mM HE 10 mM HE 50 mM HE

82.8 925 54.2 914 55.9 96.1 28.2 97.3c 17.294.1c

0.0 34.6 32.5 65.9 79.2

55.8 9 3.9 56.2 9 7.3 50.0 9 14 51.1 9 3.2 47.0 9 8.0

0.0 −0.7 −10.4 −8.5 −15.8

84.7 97.2 67.5 94.3 66.1 9 17 55.6 9 20c 66.1 915

0.0 20.3 22.0 34.4 22.0

Represents mean 9 standard error of the mean; n = 3 for 1 and 3 h and n = 4 for 72 h. A negative % depletion indicates that calcium stores were increased in treated vs controls. c Represents statistical significance at P00.05 via unpaired Student’s t-test.

a

b

viability up to 72 h (Fig. 1). Viability was slightly decreased (less than 4%) by 50 mM HE for 1 h. Viabilities were slightly increased (less than 4%) at 3 h and slightly decreased (less than 6%) at 72 h with HE treatments of 1 mM and above. Total cell numbers were decreased by 10 and 50 mM HE after 72 h treatment (data not shown). Thus, the effects of HE on cell viability and cell numbers were small and were likely only a concern at 72 h exposure to 10 and 50 mM HE.

3.2. ER Ca 2 + store experiments The effects of prolonged HE exposures on the ER Ca2 + store were studied next, based on the information that a known tumor promoter, thapsigargin induces ER Ca2 + store depletion following binding to and inhibition of its critical target, the ER Ca2 + ATPase or SERCA (smooth endoplasmic reticular Ca2 + ) pump. To do this, the ER Ca2 + store of 1c1c7 cells treated with 0, 0.1, 1, 10, and 50 mM HE for 1, 3, and 72 h was maximally depleted with thapsigargin to assess the remaining store of Ca2 + out of the total store capacity. Although the ER Ca2 + store was found to be depleted by at least 32% with 1h treatments of 0.1 and 1 mM HE, only the 10 and 50 mM HE treatments produced significant levels of ER Ca2 + store depletion, 65.9* and 79.2%* (mean percent; *: statistically significant, P 00.05), respectively (Table 1).

At 3 h, the ER Ca2 + store appeared to be refractory to the effects of HE. While HE treatments for 72 h inhibited ER store capacity by at least 20%, only the 34.4% inhibition induced by 10 mM HE resulted in statistically significant depletion. To summarize, the ER Ca2 + store was depleted by about 32% after 0.1 –1 mM HE for 1 h (although this was not statistically significant), was refractory to HE at 3 h, and was depleted by an average of 20–34% at 72 h. The significance of store depletions of 20–30% with low concentrations of HE is unclear, but at higher concentrations HE-induced over 65% of ER Ca2 + mobilization which is likely to play a role in the cellular changes this compound is known to induce.

3.3. Cx43 experiments Cx43 bands P1 and P2 are known to be specifically down-regulated by organochlorine (OC) pesticide tumor promoters (Matesic et al., 1994; Guan and Ruch, 1996; Hofer et al., 1996; Nomata et al., 1996), and were utilized here for comparative purposes and as a marker for the effect of HE on 1c1c7 hepatoma cells. After 1 h treatment, 10 and 50 mM HE decreased the Cx43 P2 titer by 4.99 13.5 and 9.49 0.6%* (mean9SE; *: statistically significant, P00.05), respectively (Fig. 2). The P1 titer also elicited a 14.891.0%* decrease after 50 mM HE for 1 h. Following 3 h exposure to 1, 10, and 50 mM HE, the P2 titers were decreased by 14.39 12.3, 58.99 19.3*, and 77.29 13.6%*, respectively,

M.E. Hansen, F. Matsumura / Toxicology 160 (2001) 139–153

while the P1 titers were decreased by 24.1912.2 and 39.99 5.9%* after 10 and 50 mM HE, respectively. Treatments with 0.1, 1, 10, and 50 mM HE for 72 h decreased P2 titers by 11.09 8.3, 28.59 7.3*, 43.09 15.8*, and 48.1912.9%*, respectively. Cx43 P1 titers were decreased by 17.09 9.1, 31.9 912.2, 3.4911.0, and 11.19 11.4% after exposure to 0.1, 1, 10, and 50 mM HE, respectively (Fig. 2).

3.4. nPKCm Western blotting experiments Using HE-treated mouse hepatoma cells, nPKCm Western blotting experiments were per-

145

formed to determine the sensitivity of this PKC isoform to HE, since we have previously observed significant changes in nPKCm after dietary HE exposures in mouse liver (Hansen and Matsumura, 2000). The results indicated that cytosolic nPKCm titers were slightly increased after 1 h in a statistically insignificant manner and increased after 3 and 72 h exposure to HE concentrations as low as 1 mM (Fig. 3A–I) in a statistical significance manner. At 3 h cytosolic nPKCm was increased by 30.39 23.6, 44.39 7.9*, 40.4927.9, and 101.19 20.0%* over controls ((mean9 SE); *: statistically significant, P0 0.05) with 0.1, 1, 10, and 50 mM HE, respectively (Fig. 3H). After 72 h

Fig. 2. (A – E) Effect of HE on connexin43. Cx43 Western blots were performed following treatments for 1 h (A), 3 h (B), and (C) 72 h with 0, 0.1, 1, 10, and 50 mM HE. Cx43 proteins were detected using specific antibodies followed by a chemiluminescence detection method. P0, P1, and P2 indicate Cx43 bands phosphorylated at 0, 1, and 2 critical sites. Each lane represents a single sample consisting of five pooled 100 mm culture dishes. Densitometrically quantitated bands, P1 (D) and P2 (E), from a given treatment and treatment time were averaged from three separate experiments, and plotted with error bars (standard errors of the mean; SE). An asterisk, if present, indicates statistical significance compared to controls at P 00.05. (Student’s t-test).

146

M.E. Hansen, F. Matsumura / Toxicology 160 (2001) 139–153

Fig. 3. (A – I) Effect of HE on nPKCm. Immunoblots of nPKCm were performed following treatments for 1 h (A. cytosolic (c); B: particulate (p)), 3 h (C: cytosolic; D: particulate), and 72 h (E: cytosolic; F: particulate) with 0, 0.1, 1, 10, and 50 mM HE. nPKCm was detected using specific antibodies followed by a chemiluminescence detection method. In each representative blot pictured, a given treatment band is derived from five pooled culture dishes. Densitometrically quantitated bands from a given treatment and treatment time were averaged from three separate experiments, and plotted with error bars (standard errors of the mean; SE): 1 h (G), 3 h (H), and 72 h (I). An asterisk, if present, indicates statistical significance compared to controls at P 00.05. (Student’s t-test).

treatment with 0.1, 1, 10, and 50 mM HE, cytosolic nPKCm was increased by 15.69 16.4, 108.6922.4*, 120.99 3.4*, and 273.8965.0%*, respectively, over the control titer (Fig. 3I). Particulate nPKCm titers were variably affected or unchanged by HE after 1 h exposure (Fig. 3G), decreased after 3 h (Fig. 3H), but increased after 72 h (Fig. 3I). With HE concentrations of 1, 10, and 50 mM for 3 h, particulate nPKCm titers were decreased by 24.592.7*, 20.89 7.6*, and 40.69 14.3%*, respectively, as compared to controls. In contrast, particulate nPKCm was increased by 20.6%95.1*, 36.0910.8,* and 36.1 914.3% over the control mean following 72

h exposure to 1, 10, and 50 mM HE, respectively. The 72 h data are suggestive of nPKCm overexpression, which has been correlated to cell proliferation in certain cell lines (Mischak et al., 1991, 1993; Cacace et al., 1993; Perletti et al., 1996).

3.5. PLCk1 Western blotting experiments PLCk1 is known to be activated by receptorlinked tyrosine kinases (RTK) such as the ligand-activated epidermal growth factor receptor kinase and c-Neu receptor kinase and participates in enzymatic activation of PKC.

M.E. Hansen, F. Matsumura / Toxicology 160 (2001) 139–153

147

With all HE treatments employed, the particulate PLCk1 titer was significantly increased except for the 1 h exposure to 50 mM HE (Fig. 4). At 1 h, PLCk1 was increased by 214.7, 58.9, and 37.3% over the control value after 0.1, 1, and 10 mM HE, respectively. The PLCk1 titer was increased by 107.4, 102.8, 93.4, and 233.6% over the control titer after 0.1, 1, 10, and 50 mM HE, respectively, for 3 h. After 72 h exposure, the PLCk1 titer was increased by 195.4, 122.2, 122.2, and 127.6% after respective HE exposures of 0.1, 1, 10, and 50 mM. The significance of these results is that they suggest that HE is activating a RTK, thereby causing PLCk1 to move from the cytosol to the plasma membrane fraction.

Fig. 5. (A – D) Effect of HE on AP-1 transcription factor binding. Gel electrophoretic mobility shift experiments were performed with nuclear extracts isolated following treatments for 1 h (A), 3 h (B), or 72 h (C) with 0, 0.1, 1, 10, and 50 mM HE. Individual bands represent AP-1 nuclear transcription factor dimer/AP-1 DNA concensus sequence complex. Densitometrically quantitated bands (D) from a given treatment and treatment time were averaged from three separate experiments, and plotted with error bars (standard errors of the mean; SE). An asterisk, if present, indicates statistical significance compared to controls at P00.05. (Student’s t-test).

3.6. AP-1 electrophoretic gel mobility shift experiments

Fig. 4. (A – D) Effect of HE on PLCk1. Immunoblots of particulate PLCk1 were performed following treatments for 1 h (A), 3 h (B), or 72 h (C) with 0, 0.1, 1, 10, and 50 mM HE. PLCk1 was detected using specific antibodies followed by a chemiluminescence detection method. In each representative blot pictured, a given treatment band is derived from five pooled culture dishes. Each band was quantitated via densitometer and plotted in (D).

The early-response transcription factor AP-1 is activated and its DNA binding activity is increased after stimulation of a number of upstream signal transduction pathways, including the RTK pathway and the phosphoinositide pathways and by elevated [Ca2 + ]i. Since it has been found to be a critical event in tumor promotion, it was of interest to determine if HE affected AP-1 binding to its DNA responsive element. AP-1 was determined to be one of the most sensitive parameters examined. AP-1 DNA binding was increased after 1 and 3 h HE exposures, while data were variable

148

M.E. Hansen, F. Matsumura / Toxicology 160 (2001) 139–153

after 72 h, with binding significantly decreased at this late time point by HE concentrations of 10 and 50 mM (Fig. 5). Binding of AP-1 to its responsive element was increased by 49.492.8*, 55.7914.4*, 96.99 26.8*, and 93.4910%* over the control mean ([mean9 SE]; *: statistically significant, P 0 0.05) following respective 1 h treatments of 0.1, 1, 10, and 50 mM HE (Fig. 5A, D). At 3 h, treatments of 0.1, 1, 10, and 50 mM HE resulted in increased binding of 81.49 31.4, 164.49 31.1*, 189.59 14.4*, and 110.49 62.8% over the control mean, respectively (Fig. 5B, D). AP-1 binding was increased at 72 h by 47.89 55.9 and 38.79 56.9% over the control mean (100%) at respective HE concentrations of 0.1 and 1 mM, while binding was decreased to only 40.6919.2* and 56.89 53.3% of the control mean following 10 and 50 mM HE, respectively (Fig. 5C, D). These data indicate that HE affects the cell at the level of the nucleus, and therefore, is very likely affecting transcription and translation of various proteins important in cell division and proliferation.

4. Discussion The main objective of this study has been to identify the biochemical parameter, among a number of well-established biomarkers for hepatocyte cancer promotion, most sensitive and rapidly affected by HE. To this end, PLCk1 and AP-1 were found to be the most sensitive parameters affected by HE, with effects being elicited in both parameters at the lowest concentration and at the earliest time point tested. The PLCk1 titer was increased by as much as 300% over the control level following HE exposures of 0.1 mM or more at all time points tested — 1, 3, and 72 h (Fig. 4). In agreement with this observation, elevated PLC activities have been documented by other scientists in several cell lines transformed by oncogenes or carcinogenic stimuli (Alonso et al., 1988; Berggren et al., 1989; Cantley et al., 1991; Punnonen et al., 1994). Further, PLCk1 titers have been found to be elevated in colon tumors (Noh et al., 1994; Park et al., 1994), neoplastic mammary tissue (Arteaga et al., 1991), and the mem-

brane fraction of neoplastic mammary cells (Soderquist et al., 1992). Parallel to the changes in PLCk1, AP-1 DNA binding was increased within 1 h exposure to 0.1 mM HE with up-regulation even more significant after 3 h (Fig. 5). In this work, it was further shown that the sensitivity of PLCk1 and AP-1 to HE was in the range reported for OC-induced decreases in GJIC and Cx43 P2 titers, both markers of OC tumor promoter exposure (Telang et al., 1982; 0.1 and 1 mM heptachlor 72 h; 5 mM chlordane 72 h). In the 1c1c7 mouse hepatoma cell line tested here, PLCk1 and AP-1 were more sensitive than Cx43 to HE. For instance, both PLCk1 and AP-1 responded to HE within 1 h while the Cx43 P2 band, a marker for OC tumor promotion exposure, required 3 h and a 100-fold greater HE concentration to elicit effects. With prolonged exposure (72 h) to higher HE concentrations (10 and 50 mM), AP-1 DNA binding was decreased below that of controls, as has been reported in JB6 mouse epidermal cells after 48 h exposure to 10 ng/ml TPA (12-O-tetradecanoylphorbol-13acetate; Li et al., 1996) and may be related to the negative feedback of cells to compensate for the excessive mitogenic signalling. Under normal physiological conditions, AP-1 activation is a very rapid, transient event that does not require de novo protein synthesis (Herschman, 1991). Up-regulation of AP-1 DNA binding has been shown to be a critically important component of tumor promotion (Bernstein and Colburn, 1989; Ben-Ari et al., 1992; Dong et al., 1994, 1995; Li et al., 1996), for example, as occurs following phorbol ester-induced PKC activation (Sato et al., 1997). The importance of AP-1 in tumor promotion is further supported by the fact that a number of compounds that up-regulate AP-1 DNA binding activity are tumor promoters, including phorbol esters, UV light, deoxycholate, and arsenic (As3 + ; Angel and Karin, 1991; Devary et al., 1991, 1992; Hirano et al., 1996; Matheson et al., 1996), while compounds that inhibit AP-1 DNA binding activity tend to be antitumorigenic such as phenolic antioxidants (Yoshioka et al., 1995), potato proteinase inhibitors I and II (Huang et al., 1997), curcumin (Chen and Tan, 1998), retinoic acid (Dong et al.,

M.E. Hansen, F. Matsumura / Toxicology 160 (2001) 139–153

1994), and fluocinolone acetonide (Dong et al., 1994). AP-1 DNA-binding and transactivating activity is induced in cancer cells following stimulation by peptide growth factors, TPA (Chen et al., 1996), and elevated [Ca2 + ]i (Roche and Prentki, 1994), as well as other agents. Additionally, oncogenic conversion of AP-1, or its individual components c-Jun or c-Fos, has been found to induce various cancers in vivo (Miller et al., 1984; Jenuwein et al., 1985; Ru¨ther et al., 1989; Schu¨tte et al., 1989; Lewin, 1991). Since AP-1 transactivation is a critical event in tumor promotion (Bernstein and Colburn, 1989; Ben-Ari et al., 1992; Dong et al., 1994, 1995; Li et al., 1996), and it is downstream of a number of mitogenic signal transduction pathways, it was reasoned that some upstream, mitogenic signal triggering component(s) such as changes in Ca2 + homeostasis, the phosphoinositide cascade, or an RTK signal transduction pathway might be affected by HE first. Although the HE-induced effects were found to be exhibited with all of the parameters tested, PLCk1 was found to be more sensitive to HE among all other parameters tested. Moreover, the HE dose and time-course of effect for PLCk1 were very similar to those observed for AP-1. The fact that PLCk1 was activated at such a low HE concentration suggests that this upstream component could be very closely connected to the triggering event for HEinduced mitogen signalling. PLCk activation is a key step in activation of the growth factor signal transduction pathway. Following RTK binding by an agonist, receptor dimerization and transphosphorylation can promote coupling of PLCk directly to an autophosphorylated RTK (Heldin, 1996). The key points in understanding the significance of the PLCk activation are as follows: (a) PLCk is the only member of the PLC family activated by tyrosine phosphorylation, (b) PLCk is unique in that it contains both SH2 and SH3 domains, indicating that its recruitment to the growth factor receptor is mediated by tyrosine phosphorylation on the internal domain of the growth factor receptor, (c) PLCk catalyzes the production of diacylglycerol (DAG) and inositol phosphate (IP3), following activation and attachment to the growth factor

149

receptor; DAG can then activate PKC enzymatic activity and IP3 induce ER Ca2 + mobilization after binding to the IP3 receptor, and (d) PLCk activation is, therefore, intimately associated with the activation of growth factor receptors and their tyrosine kinases (RTK). Other proteins that can be turned on by RTK activation include ras and c-Raf, which can lead to signal transduction through the MAP kinase segment of the pathway and induction of AP-1 transactivation in the nucleus. In addition, AP-1 helps regulate several other primary response genes (Herschman, 1991) important in cell proliferation. Further, it can modulate various cellular programs and hormone responses, via transcription factor cross talk, for instance. Thus, the results of the current study are crucial for providing a logical starting point for future in-depth studies into the cellular mechanism and studies of cellular program shifts induced by this group of pesticides. If one accepts the above interpretation, other events become easier to explain, at least from a theoretical viewpoint. First, like PLCk, Cx43 is known to be regulated by phosphorylation by tyrosine kinases (Loo et al., 1995), as well as by the level of internal Ca2 + (Peracchia and Peracchia, 1980). The early effect of HE observed in this study on this gap-junction protein thus appears to be secondary to the modulation of tyrosine kinase by HE, occurring parallel to activation of PLCk1. The fact that Cx43 was less sensitive to HE supports the view that it is not a primary target of HE. Nevertheless, the effect of HE on Cx43 appears to be sustained at high doses. Considering the data reported by others showing pronounced effects on GJIC and gapjunction protein levels following low-level OC treatements (Telang et al., 1982), this aspect of the work presented here requires future attention. Regarding Ca2 + release from ER, although the effects of 0.1 and 1 mM HE were not significant at the earliest point tested, there is a dose-dependent increase in the percent ER Ca2 + store depletion (Table 1). The expected increase in IP3 production resulting from the activation of PLCk1 at the plasma membrane would be expected to induce rapid ER Ca2 + release that could explain the ER Ca2 + store depletion observed here. The apparent

150

M.E. Hansen, F. Matsumura / Toxicology 160 (2001) 139–153

recovery at later time points might be due to cellular compensatory feedback mechanisms designed to maintain Ca2 + homeostasis, following the completion of the initial signal delivery. nPKCm was not affected by HE until 3 h posttreatment. With a 1 mM or greater HE concentration, cytosolic nPKCm tended to be increased, while it was decreased in the particulate fraction, in agreement with previous work in vivo (Hansen and Matsumura, 2000). At 72 h both the cytosolic and particulate nPKCm titers were up-regulated by exposure to 1 mM HE or greater. Therefore, mouse 1c1c7 cells appear to overexpress this isoform in the cytosolic and particulate fraction with longer-term treatments. The role of nPKCm is not well known. Its role in thyrotropin-releasing hormone stimulated pituitary GH4C1 cells appears to be as a messenger to increase prolactin secretion (Akita et al., 1990). This PKC isoform has also been shown to be co-induced by activation of the EGF receptor along with PLCk (Jiang et al., 1996). Activation of PLD to catalyze the hydrolysis of phosphatidylcholine leading to ultimate formation of diacylglycerol has been suggested to lead to selective activation of Ca2 + -independent PKCs like nPKCm. Whatever the true role of this Ca2 + -independent PKC isoform is, a great deal of evidence shows that carcinogenic transformation is frequently associated with changes in PKCm levels. For instance, a number of studies have shown that nPKCm is oncogenic when overexpressed in certain cells (Mischak et al., 1991, 1993; Cacace et al., 1993; Perletti et al., 1996). Other chronic cancer inducing treatments such as a choline-deficient diet have been found to up-regulate particulate PKC levels in liver cells over a period of months (Zeisel et al., 1995). A possibility to consider is that the effects of HE on PKC may be mediated through a RTK, as has been proposed by Blobe et al. (1996). In addition, since other Ca2 + modulating agents such as thapsigargin and ionomycin are tumor promoters, it is probable that HE’s effect on [Ca2 + ]i may play a role in tumor promotion at higher HE concentrations. This study suggests that exposure to the tumor promoter HE induces a cellular-program shift,

whereby a series of effects or changes are induced in liver cells similar to that described by the resistant hepatocyte model of induction/tumor promotion (Schulte-Hermann, 1985). From the work conducted here and listed in decreasing order of sensitivity, HE-induced changes in AP-1, PLCk1, the ER Ca2 + store, nPKCm, and Cx43, all biochemical parameters that have been shown to be important in tumor promotion. These data further suggest that HE is inducing AP-1 up-regulation via activation of a growth factor signalling pathway, with HE possibly targeting growth factor receptor associated tyrosine kinase(s) immediately upstream of PLCk1. In the future, studies will be conducted to further characterize the molecular target of HE with focus concentrated on tyrosine kinases capable of directly modulating PLCk.

Acknowledgements This research was supported by the Hawaii Heptachlor Research and Education Foundation grant c HHHERP 94-04, with additional support from the University of California Toxic Substances Teaching and Research Program’s Ecotoxicology Training Grant Program. Support was also received from the National Institute of Environmental Health Sciences research grants c ES05233 and c ES05707. References Akita, Y., Ohno, S., Yajima, Y., Suzuki, K., 1990. Possible role of Ca2 + -independent protein kinase C isozyme, nPKC-o, in thyrotropin-releasing hormone-stimulated signal transduction: differential down-regulation of nPKC-o in GH4C1 cells. Biochem. Biophys. Res. Commun. 172, 184– 189. Alonso, T., Morga, R.O., Marvizon, J.C., Zarbl, H., Santos, E., 1988. Malignant transformation by ras and other oncogenes produces common alterations in inositol phospholipid signaling pathways. Proc. Natl Acad. Sci. USA 85, 4271– 4275. Angel, P., Karin, M., 1991. The role of Jun, Fos and the AP-1 complex in cell proliferation and transformation. Biochim. Biophys. Acta 1072, 129– 157.

M.E. Hansen, F. Matsumura / Toxicology 160 (2001) 139–153 Arteaga, C.L., Johnson, M.D., Todderud, G., Coffey, R.J., Carpenter, G., Page, D.L., 1991. Elevated content of the tyrosine kinase substrate phospholipase C-g1 in primary human breast carcinomas. Proc. Natl Acad. Sci. USA 88, 10435– 10439. Ben-Ari, E.T., Bernstein, L.R., Colburn, N.H., 1992. Differential c-jun expression in response to tumor promoters in JB6 cells sensitive or resistant to neoplastic transformation. Molec. Carcinogen. 5, 62–74. Berggren, P.O., Hallberg, A., Welsh, N., Arkahammar, P., Nilsson, T., Welsh, M., 1989. Transfection of insulin-producing cells with a transforming c-Ha-ras oncogene stimulates phospholipase C activity. Biochem. J. 259, 701–707. Bernstein, L.R., Colburn, N.H., 1989. AP1/jun function is differentially induced in promotion-sensitive and resistant JB6 cells. Science 244, 566–569. Blobe, G.C., Stribling, S., Obeid, L.M., Hannun, Y.A., 1996. Protein kinase C isoenzymes: regulation and function. Cancer Sur. 27, 213–248. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248– 254. Cacace, A.M., Guadagno, S.N., Krauss, R.S., Fabbro, D., Weinstein, I.B., 1993. The epsilon isoform of protein kinase C is an oncogene when overexpressed in rat fibroblasts. Oncogene 8, 2095–2104. Cantley, L.C., Auger, K.R., Carpenter, C., Duckworth, B., Graziani, A., Kapeller, R., Soltoff, S., 1991. Oncogenes and signal transduction. Cell 64, 281–302. Chang, J.S., Noh, D.Y., Park, I.A., Kim, M.J., Song, H., Ryu, S.H., Suh, P.G., 1997. Overexpression of phospholipase C-gamma1 in rat 3Y1 fibroblast cells leads to malignant transformation. Cancer Res. 57, 5465–5468. Chen, T.K., Smith, L.M., Gebhardt, D.K., Birrer, M.J., Brown, P.H., 1996. Activation and inhibition of the AP-1 complex in human breast cancer cells. Molec. Carcinogen. 15, 215– 226. Chen, Y.-R., Tan, T.-H., 1998. Inhibition of the c-Jun N-terminal kinase (JNK) signalling pathway by curcumin. Oncogene 17, 173– 178. Criswell, K.A., Loch-Caruso, R., 1995. Lindane-induced elimination of gap junctional communication in rat uterine myocytes is mediated by an arachidonic acid-sensitive cAMP-independent mechanism. Toxicol. Appl. Pharmacol. 135, 127– 138. Criswell, K.A., Loch-Caruso, R., Stuenkel, E.L., 1995. Lindane inhibition of gap junctional communication in myometrial myocytes is partially dependent on phosphoinositide-generated second messengers. Toxicol. Appl. Pharmacol. 130, 280–293. Devary, Y., Gottlieb, R.A., Lau, L.F., Karin, M., 1991. Rapid and preferential activation of the c-jun gene during the mammalian UV response. Molec. Cell. Biol. 11, 2804– 2811. Devary, Y., Gottlieb, R.A., Smeal, T., Karin, M., 1992. The mammalian ultraviolet response is triggered by activation of Src tyrosine kinases. Cell 71, 1081–1091.

151

Dignam, J.D., Lebovitz, R.M., Roeder, R.G., 1983. Accurate transcription initiation by RNA polymeraseII in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11, 1475– 1489. Dong, Z., Birrer, M.J., Watts, R.G., Matrisian, L.M., Colburn, N.H., 1994. Blocking of tumor promoter-induced AP-1 activity inhibits induced transformation in JB6 mouse epidermal cells. Proc. Natl Acad. Sci. USA 91, 609– 613. Dong, Z., Lavrovsky, V., Colburn, N.H., 1995. Transformation reversion induced in JB6 RT101 cells by AP-1 inhibitors. Carcinogenesis 16, 749– 756. Eto, A., Akita, Y., Saido, T.C., Suzuki, K., Kawashima, S., 1995. The role of the calpain-calpastatin system in thyrotropin-releasing hormone-induced selective down-regulation of a protein kinase C isozyme, nPKC-o in rat pituitary GH4C1 cells. J. Biol. Chem. 270, 25115– 25120. Grynkiewicz, G., Poenie, M., Tsien, R.Y., 1985. A new generation of Ca2 + indicators with greatly improved fluorescence properties. J. Biol. Chem. 260, 3440– 3450. Guan, X., Ruch, R.J., 1996. Gap junction endocytosis and lysosomal degradation of connexin43-P2 in WB-F344 rat liver epithelial cells treated with DDT and lindane. Carcinogenesis 17, 1791– 1798. Hanania, N., Lezenes, J.R., Castagna, M., 1992. Tumorigenicity-associated expression of protein kinase C isoforms in rhabdomyosarcoma-derived cells. FEBS Lett. 303, 15 – 18. Hankinson, O., Andersen, R.D., Birren, B.W., Sander, F., Negishi, M., Nebert, D.W., 1985. Mutations affecting the regulation of transcription of the cytochrome P1-450 gene in the mouse Hepa-1 cell line. J. Biol. Chem. 260, 1790– 1795. Hansen M.E., Matsumura F., 2000. Down-regulation of particulate protein kinase Cm and up-regulation of nuclear AP-1 DNA binding in liver following in vivo exposure of B6C3F1 male mice to heptachlor epoxide, J. Biochem. Molec. Toxicol., in press Heldin, C.-H., 1996. Protein tyrosine kinase receptors. Cancer Sur. 27, 7 – 24. Herschman, H.R., 1991. Primary response genes induced by growth factors and tumor promoters. Annu. Rev. Biochem. 60, 281– 319. Hirano, F., Tanaka, H., Makino, Y., Okamoto, K., Hiramoto, M., Handa, H., Makino, I., 1996. Induction of the transcription factor AP-1 in cultured human colon adenocarcinoma cells following exposure to bile acids. Carcinogenesis 17, 427– 433. Hofer, A., Sa´ez, J.C., Chang, C.C., Trosko, J.E., Spray, D.C., Dermietzel, R., 1996. C-erbB2/neu transfection induces gap junctional communication incompetence in glial cells. J. Neurosci. 16, 4311– 4321. Huang, C., Ma, W.-Y., Ryan, C.A., Dong, Z., 1997. Proteinase inhibitors I and II from potatoes specifically block UV-induced activator protein-1 activation through a pathway that is independent of extracellular signal-regulated kinases, c-Jun N-terminal kinases, and P38 kinase. Proc. Natl Acad. Sci. USA 94, 11957– 11962.

152

M.E. Hansen, F. Matsumura / Toxicology 160 (2001) 139–153

Janknecht, R., Cahill, M.A., Nordheim, A., 1995. Signal integration at the c-fos promoter. Carcinogenesis 16, 443–450. Jenuwein, T., Mu¨ller, D., Curran, T., Mu¨ller, R., 1985. Extended life span and tumorigenicity of nonestablished mouse connective tissue cells transformed by the fos oncogene of FBR-MuSV. Cell 41, 629–637. Jiang, Y., Lu, Z., Zang, Q., Foster, D.A., 1996. Regulation of phosphatidic acid phosphohydrolase by epidermal growth factor. Reduced association with the EGF receptor followed by increased association with protein kinase C epsilon. J. Biol. Chem. 271, 29529–29532. Lewin, B., 1991. Oncogenic conversion by regulatory changes in transcription factors. Cell 64, 303–312. Li, J.-J., Dong, Z., Dawson, M.I., Colburn, N.H., 1996. Inhibition of tumor promoter-induced transformation by retinoids that transrepress AP-1 without transactivating retinoic acid response element. Cancer Res. 56, 483–489. Liu, P.C., Phillips, M.A., Matsumura, F., 1996. Alteration by 2,3,7,8-Tetrachlorodibenzo-p-dioxin of CCAAT/enhancer binding protein correlates with suppression of adipocyte differentiation in 3T3-L1 cells. Molec. Pharmacol. 49, 989– 997. Loo, L.W., Berestecky, J.M., Kanemitsu, M.Y., Lau, A. F., 1995. pp60src-mediated phosphorylation of connexin 43, a gap junction protein. J. Biol. Chem. 270, 12751–12761. Madhukar, B.V., Yoneyama, M., Matsumura, F., Trosko, J.E., Tsushimoto, G., 1983. Alteration of calcium transport by tumor promoters 12-O-tetradecanoyl phorbol-13-acetate and p, p’-dichlorodiphenyltrichloroethane, in the Chinese hamster V79 fibroblast cell line. Cancer Lett. 18, 251– 259. Matesic, D.F., Rupp, H.L., Bonney, W.J., Ruch, R.J., Trosko, J.E., 1994. Changes in gap-junction permeability, phosphorylation, and number mediated by phorbol ester and nonphorbol-ester tumor promoters in rat liver epithelial cells. Molec. Carcinogen. 10, 226–236. Matheson, H., Branting, C., Rafter, I., Okret, S., Rafter, J., 1996. Increased c-fos mRNA and binding to the AP-1 recognition sequence accompanies the proliferative response to deoxycholate of HT29 cells. Carcinogenesis 17, 421– 426. Miller, A.D., Curran, T., Verma, I.M., 1984. c-fos protein can induce cellular transformation: a novel mechanism of activation of a cellular oncogene. Cell 36, 51–60. Mischak, H., Kolch, W., Goodnight, J., Davidson, W.F., Rapp, U., Rose-John, S., Mushinski, J.F., 1991. Expression of protein kinase C genes in hemopoietic cells is cell-type-and B cell-differentiation stage specific. J. Immunol. 147, 3981– 3987. Mischak, H., Goodnight, J., Kolch, W., Martiny-Baron, G., Schaechtle, C., Kazanietz, M.G., Blumberg, P.M., Pierce, J.H., Mushinski, J.F., 1993. Overexpression of protein kinase C-d and -o in NIH 3T3 cells induces opposite effects on growth, morphology, anchorage dependence, and tumorigenicity. J. Biol. Chem. 268, 6090–6096. Moser, G.J., Smart, R.C., 1989. Hepatic tumor-promoting chlorinated hydrocarbons stimulate protein kinase C activity. Carcinogenesis 10, 851–856.

National Cancer Institute (NCI), 1977. Bioassay of heptachlor for possible carcinogenicity, Technical Report Series No. 9, Washington, DC, Department of Health, Education and Welfare. Publication No. (DHEW; NIH); 77-809: pp. 1111. Nishizuka, Y., 1984. The role of protein kinase C in cell surface signal transduction and tumour promotion. Nature 308, 693– 698. Nishizuka, Y., 1992. Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science 258, 607– 614. Noh, D.Y., Kang, H.S., Kim, Y.C., Youn, Y.K., Oh, S.K., Choe, K.J., Park, I.A., Ryu, S.H., Suh, P.G., 1998. Expression of phospholipase C-gamma 1 and its transcriptional regulators in breast cancer tissues. Anticancer Res. 18, 2643– 2648. Noh, D.Y., Lee, Y.H., Kim, S.S., Kim, Y.I., Ryu, S.H., Suh, P.G., Park, J.G., 1994. Elevated content of phospholipase C-gamma 1 in colorectal cancer tissues. Cancer 73, 36 – 41. Nomata, K., Kang, K.-S., Hayashi, T., Matesic, D., Lockwood, L., Chang, C.C., Trosko, J.E., 1996. Inhibition of gap junctional intercellular communication in heptachlorand heptachlor epoxide-treated normal human breast epithelial cells. Cell Biol. Toxicol. 12, 69 – 78. Park, J.-G., Lee, Y.H., Kim, S.S., Park, K.J., Noh, D.-Y., Ryu, S.H., Suh, P.-G., 1994. Overexpression of phospholipase C-gamma 1 in familial adenomatous polyposis. Cancer Res. 54, 2240– 2244. Peracchia, C., Peracchia, L.L., 1980. Gap junction dynamics: reversible effects of divalent cations. J. Cell Biol. 87, 708– 718. Perchellet, E.M., Jones, D., Perchellet, J.P., 1990. Ability of the Ca2 + ionophores A23187 and ionomycin to mimic some of the effects of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate on hydroperoxide production, ornithine decarboxylase activity, and DNA synthesis in mouse epidermis in vivo. Cancer Res. 50, 5806– 5812. Perletti, G.P., Folini, M., Lin, H.-C., Mischak, H., Piccinini, F., Tashjian, A.H., 1996. Overexpression of protein kinase C epsilon is oncogenic in rat colonic epithelial cells. Oncogene 12, 847– 854. Pongracz, J., Clark, P., Neoptolemos, J.P., Lord, J.M., 1995. Expression of protein kinase C isoezymes in colorectal cancer tissue and their differential activation by different bile acids. Int. J. Cancer 61, 35 – 39. Punnonen, K., Denning, M.F., Rhee, S.G., Yuspa, S.H., 1994. Differences in the regulation of phosphatidylinositol-specific phospholipase C in normal and neoplastic keratinocytes. Molec. Carcinogen. 10, 216– 225. Ran, W., Dan, M., Levine, R.A., Henkle, C., Campisi, J., 1986. Induction of c-fos and c-myc mRNA by epidermal growth factor or calcium ionophore is cAMP dependent. Proc. Natl Acad. Sci. USA 83, 8216– 8220. Roche, E., Prentki, M., 1994. Calcium regulation of immediate-early response genes. Cell Calcium 16, 331– 338. Rose, B., Simpson, I., Loewenstein, W.R., 1977. Calcium ion produces graded changes in permeability of membrane channels in cell junction. Nature 267, 625– 627.

M.E. Hansen, F. Matsumura / Toxicology 160 (2001) 139–153 Rotenberg, S.A., Weinstein, I.B., 1991. Two polychlorinated hydrocarbons cause phospholipid-dependent protein kinase C activation in vitro in the absence of calcium. Molec. Carcinogen. 4, 477– 481. Ruch, R.J., Fransson, R., Flodstrom, S., Warngard, L., Klaunig, J.E., 1990. Inhibition of hepatocyte gap junctional intercellular communication by endosulfan, chlordane and heptachlor. Carcinogenesis 11, 1097–1101. Ru¨ther, U., Komitowski, D., Schubert, F.R., Wagner, E.F., 1989. c-fos expression induces bone tumors in transgenic mice. Oncogene 4, 861–865. Sato, N., Sadar, M.D., Bruchovsky, N., Saatcioglu, G., Rennie, P.S., Sato, S., Lange, P.H., Gleave, M.E., 1997. Androgenic induction of prostate-specific antigen gene is repressed by protein-protein interaction between the androgen receptor and AP-1/c-Jun in the human prostate cancer cell line LNCaP. J. Biol. Chem. 272, 17485–17494. Schulte-Hermann, R., 1985. Tumor promotion in the liver. Arch. Toxicol. 57, 147–158. Schu¨tte, J., Minna, J.D., Birrer, M.J., 1989. Deregulated expression of human c-jun transforms primary rat embryo cells in cooperation with an activated c-Ha-ras gene and transforms rat-1a cells as a single gene. Proc. Natl Acad. Sci. USA 86, 2257– 2261. Soderquist, A.M., Todderud, G., Carpenter, G., 1992. Elevated membrane association of phospholipase C-g1 in MDA-468 mammary tumor cells. Cancer Res. 52, 4526– 4529. Suzaki, E., Inoue, B., Okimasu, E., Ogata, M., Utsumi, K., 1988. Stimulative effect of chlordane on the various functions of the guinea pig leukocytes. Toxicol. Appl. Pharmacol. 93, 137– 145. Telang, S., Tong, C., Williams, G.M., 1982. Epigenetic membrane effects of a possible tumor promoting type on cultured liver cells by the non-genotoxic organochlorine

.

153

pesticides chlordane and heptachlor. Carcinogenesis 3, 1175– 1178. Thastrup, O., Foder, F., Scharff, O., 1987. The calcium mobilizing and tumor promoting agent, thapsigargin elevates the platelet cytoplasmic free calcium concentration to a higher steady state level. A possible mechanism of action for the tumor promotion. Biochem. Biophys. Res. Commun. 142, 654– 660. Watson, A.J., Hankinson, O., 1992. Dioxin- and Ah receptordependent protein binding to xenobiotic responsive elements and G-rich DNA studied by in vivo footprinting. J. Biol. Chem. 267, 6874– 6878. Whitmarsh, A.J., Davis, R.J., 1996. Transcription factor AP-1 regulation by mitogen-activated protein kinase signal transduction pathways. J. Molec. Med. 74, 589– 607. WHO, 1984, Environmental Health Criteria 38: Heptachlor. World Health Organization, Geneva, Switzerland, pp. 181. Williams, G.M., 1980. Classification of genotoxic and epigenetic hepatocarcinogens using liver culture assays. Ann. N.Y. Acad. Sci. 349, 273– 282. Yamaguchi, I., Matsumura, F., Kadous, A.A., 1980. Heptachlor epoxide: effects on calcium mediated transmitter release from brain synaptosomes in rat. Biochem. Pharmacol. 29, 1815– 1823. Yang, H., Shen, F., Herenyiova, M., Weber, G., 1998. Phospholipase C (EC 3.1.4.11): a malignancy linked signal transduction enzyme. Anticancer Res. 18, 1399– 1404. Yoshioka, K., Deng, T., Cavigelli, M., Karin, M., 1995. Antitumor promotion by phenolic antioxidants: inhibition of AP-1 activity through induction of Fra expression. Proc. Natl Acad. Sci. USA 92, 4972– 4976. Zeisel, S.H., da Costa, K.A., Albright, C.D., Shin, O.H., 1995. Choline and hepatocarcinogenesis in the rat. Advan. Exper. Med. Biol. 375, 65 – 74.