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Decreased cytosolic levels of the heme binding Z protein in rat hepatocyte nodules and hepatocellular carcinomas
Cancer Letters, 43 (1988) 55-58 Usevier Scientific Publishers Ireland Ltd.
55
Decreased cytosolic levels of the heme binding Z protein in rat hepatocyte nodules and hepatocellular carcinomas M.W. Roomi, Departments Pharmacology
S.H. Vincent,
ojPothology,
E. Farber and U. Muller-Eberhard
Uniuersity of Toronto,
and Biochemistry,
Toronto, Ontario (Canada) and the Departments Cornell University Medical College, New York, NY 10021 (U.S.A.)
ofPediatrics,
(Received 20 February 1988) (Revision received 8 August 1988) (Accepted 9 August 1988)
Summary
Introduction
Hepatocyte nodules and hepatocellular carcinomas were induced in male Fischer rats using the resistant hepatocyte model. The immunoreactive cytosolic levels of the hemebinding Z protein (HBP) were reduced by 56 %i (P < 0.001; Z-tailed t-test) in early hepatocyte nodules (25 weeks) and hepatoceNular carcinomas (lo-12 months). This finding is in accordance with the previously reported reduced heme content of hepatocyte nodules and is consistent with the postulated role for HBP in intracellular heme transport and distribution. The immunoreactive levels of the glutathione S-transferase isozymes (GST) which like HBP bind heme, were elevated Z-fold (P < 0.01) in early and late hepatocyte nodules and were unchanged in hepatocellular carcinomas.
Rat hepatocyte nodules, a precursor population of liver cancer, exhibit in several different models a biochemical pattern of enzyme induction which is characterized by decreased activity of phase I enzymes of the microsomal drug metabolizing system including cytochrome P-450 and several cytochrome P-450mediated dealkylating enzymes [ 101, The decrease in phase I enzyme activity is associated with an increase in phase II components such as glutathione and GST, including a placental form [4,5,11]. This biochemical pattern has been considered to be responsible for the resistance of the nodules to the cytotoxic effect of several hepatotoxic agents. Our current hypothesis is that the rat hepatocyte nodules exhibit a basic interference in heme synthesis. This hypothesis is based on several observations. In addition to a generalized decrease in several cytochrome P-450 isozymes, with no apparent preference for any particular P-450, the nodules exhibit decreases in another microsomal cytochrome, cytochrome b,, and in two other heme containing proteins, catalase and tryptophan 2,3-dioxygenase as well as in the total microsomal heme content [9]. Decreases in total cytochrome P-450, cytochrome b, tryptophan 2,3-
Keywords: heme binding 2 protein; hepatocyte nodules and carcinomas; glutathione Stransferase; male Fischer rats. Correspondence to: Ursula Muller-Eberhard. Department of Pediatric Hem/One., Cornell University Medical College, 525 East 68th Street, Room 804, New York, New York 10021, U.S.A.
0304-3835/88/$03.50 0 1988 Elsevier Scientific Publishers Ireland Ltd Published and Printed in Ireland
56
dioxygenase, catalase and aminolevulinic acid synthase as well as an increase in microsomal heme oxygenase have also been reported for mouse liver tumors [16]. The hepatocyte nodules in the rat also showed low levels of total iron and delta-aminolevulinic acid activity
PI. To further test this hypothesis, we measured the level of the cytosolic HBP [15] in hepatocyte nodules and hepatocellular carcinomas generated when using the resistant hepatocyte model [13]. HBP, which comprises 3-5% of total liver cytosolic protein, has been suggested to be involved in the intracellular transport and distribution of heme [7]. It is also known as fatty acid-binding protein, L-FABP [ 1,171 as it binds fatty acids although with a lower affinity than heme [l, 151. For comparison, we also measured the levels of GST whose enzymatic activity is known to be increased in hepatocyte nodules [5,10] and which bind heme with a similar affinity as HBP. Materials and methods Hepatocyte nodules and hepatocellular carcinomas were induced according to the resistant hepatocyte model [13]. Male Fischer rats, initially weighing 150-170 g, were given diethylnitrosamine (Eastman Kodak Co., Rochester, NY) 200 mg/kg i.p. After a Z-week recovery period, the rats were placed on 0.02% 2-acetylaminofluorene for 1 week, subjected to a standard partial hepatectomy and continued for an additional week on the 2acetylaminofluorene diet. The rats were then placed on normal chow diet until the termination of the experiment. At various time periods rats were killed to give early nodules (25 weeks) and hepatocellular carcinomas (lo12 months). Control rats were fed normal chow diet for 12 months. The normal liver tissue, hepatocyte nodules and carcinomas were homogenized in Tris buffer containing 10m4 M PMSF (phenylethylsulfonyl fluoride), 1 mM EDTA and 1 mg aprotinin/lOO ml buffer. Cytosolic fractions were obtained by centrifuging the homogenates at
100,000 x g and protein concentration determined by the method of Bradford [3]. SDSpolyacrylamide gel electrophoresis (SDSPAGE) of cytosolic fractions were performed according to Laemmli [6]. Cytosolic HBP concentration was measured by rocket immunoelectrophoresis [8] and a solid-phase radioimmunoassay (RIA) [16,17] using an antibody raised in rabbits against HBP purified by the 4-step procedure described [15]. Results by the two assays agreed within f 12%. GST concentration was measured by RIA using an antibody raised in rabbits against a mixture of GST isozymes obtained by affinity chromatography on glutathione-agarose 1121. The antibody recognized by 3 major subunits as judged by Western blotting after SDS-PAGE of purified GST and liver cytosol. The data were analyzed by the 2-tailed Student’s t-test. Results and discussion Electrophoretic analysis of cytosolic protein fractions revealed a substantial decrease in the intensity of the M, 14,000 band in cytosol from early hepatocyte nodules as compared to cytosol from control liver tissue (Fig. 1). That the decrease in band intensity is accounted for by a decrease in the concentration of HBP, an abundant i’$l4,000 cytosolic protein, was confirmed by determining that the immuno-reactive levels of this protein are significantly reduced in cytosolic fractions from early and late hepatocyte nodules and hepatocellular carcinomas. As shown in Table I, HBP cytosolic levels were reduced by 56% (P < 0.001) in early hepatocyte nodules and hepatocellular carcinomas. As shown in Table 1, HBP cytoThe reduced levels of this abundant hemebinding protein are in accord with the reduced heme content of hepatocyte nodules [l] and are consistent with a postulated role for this protein in intracellular heme transport and distribution [7]. Given these observations and the general finding of reduced levels of total cytochrome P-450, cytochrome b,, heme content and decreased delta-aminolevulinic acid synthase activity in the nodules it becomes
57
Table 1. Cytosolic concentrations of HBP and GST isozymes in hepatocyte nodules and hepatocellular carcinomas in male Fischer rats. Determined by (combined homogenates of 4 nodules) rocket immunoelectrophoresis and/or solid-phase radioimmunoassay in samples from 4 rats in each group. Data expressed as % (mean f S.D.) of control (normal rat liver cytosol) Cytosolic concentration % of control
Early nodules Late nodules Hepatocellular carcinomas “P< 0.001; bP< 0.01.
Sodium dodecyl sulfate-polyacrylamide gel Fig. 1. electrophoresis of cytosolic proteins from normal rat liver tissue (lane 1, 30 pg) and early hepatocyte nodules (lane 2, 30 pg). The developing gel was 15% acrylamide. Lane 3 contains molecular weight markers.
reasonable to entertain the hypothesis that one or more changes relating to iron metabolism, including intracellular iron transport, may be fundamental to the characteristic phenotype of hepatocyte nodules. Recently, L-FABP equivalent with HBP as the cytoplasmic [17], was identified polypeptide p14 the mitosis-associated target of activated metabolites of the carcinogen 2acetylaminofluorene [2]. The concentration
HBP
GST
44&P 10 f & 11 + 9
213 f 38b 210 + 45b 131 + 36
2-tailed t-test.
of ~14 was found to be increased in proliferating hepatocytes of hyperplastic foci and hepatocellular carcinomas in rats that were fed 0.02% acetylaminofluorene continuously for 4-32 weeks without prior initiation with diethylnitrosamine or partial hepatectomy [ 141. It is not known whether heme synthesis is affected under these conditions. In contrast to the decrease in HBP levels, the immunoreactive levels of GST isozymes, which like HBP also bind heme, were elevated by 2-fold (P < 0.01) in both early and late hepatocyte nodules and were essentially in hepatocellular carcinomas unchanged (Table 1). The observed increase in GST levels is in agreement with the previously reported increase in GST enzymatic activity in hepatocyte nodules [lo, 111 and suggests that the primary role of GST isozymes is to function as Phase II conjugating enzymes rather than as transporters of heme whose synthesis in hepatocyte nodules is greatly reduced [9]. In conclusion, we have found that in hepatocyte nodules and hepatocellular carcinomas the cytosolic concentration of the abundant HBP is significantly reduced in accord with the previously reported decrease in heme synthesis. In contrast, the cytosolic concentration of the GST isozymes, which also bind heme, was elevated in hepatocyte nodules.
58
References Bass, N.M. (1985) Function and regulation of hepatic and intestinal fatty acid-binding proteins. Chem. Phys. Lipids, 38.95-114. Bassuk, J.A., Tsichlis. P.N. and Sorof, S. (1987) Liver fatty acid binding protein is the mitosis-associated polypeptide target of a carcinogen in rat hepatocytes. Proc. Natl. Acad. Sci. USA, 84,7547-7551. 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. Farber, E. (1984) The biochemistry of preneoplastic liver: a common metabolic pattern in hepatocyte nodules. Cancer J. Biochem. Cell Biol.. 62, 486-494. Kitahara, A., Satoh, K., Nishimuta, K., Ishikawa, T., Ruike. K., Sato, K., Tsuda, H. and Ito, N. (1984) Changes in molecular forms of rat hepatic glutathione S-transferase during chemical hepatocarcinogenesis. Cancer Res., 44, 2698-2703. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227,680-685. Muller-Eberhard, U. and Vincent, S.H. (1985) Concepts of heme distribution within hepatocytes. Biochem. Pharmacol., 34.719-725. Plumley, F.G. and Schmidt, G.W. (1983) Rocket and crossed immunoelectrophoresis of proteins solubilized with sodium dodecyl sulfate. Anal. Biochem., 134, 86-95. Roomi, M.W., Cameron, R.G. and Farber, E. (1984) Is the reduced cytochrome P-450 content in hepatocyte
10
11
12
13 14
15
16
17
nodules a reflection of decreased heme synthesis? Cancer Fed. Biol. Sot., 27, 161. Roomi, M.W., Ho, R.K., Sarma, D.S.R. and Farber, E. (1985) A common biochemical pattern in preneoplastic hepatocyte nodules generated in four different models in the rat. Cancer Res., 45,564-571. Rushmore, R.H., Sharma, R.N.S., Roomi, M.W.. Harris, L., Satoh, K., Sato, K., Murray, R.K. and Farber, E. (1987) Identification of a characteristic cytosolic polypeptide of rat preneoplastic hepatocyte nodules as placental glutathione S-transferase. Biochem. Biophys. Res. Commun., 143,98-103. Simons, P.C. and Vander Jagt, D.L. (1981) Purification of glutathione S-transferases by glutathione-affinity chromatography. Methods Enzymol., 77,235-237. Solt, D. and Farber, E. (1976) A new principle for the analysis of chemical carcinogenesis. Nature, 263.701-703. Sorof, S. and Custer, R.P. (1987) Elevated expression and cell cycle deregulation of a mitosis-associated target polypeptide of a carcinogen in hyperplastic and malignant rat hepatocytes. Cancer Res., 47, 210-220. Vincent, S.H. and Muller-Eberhard. U. (1985) A protein of the Z class of liver cytosolic proteins in the rat that preferentially binds heme. J. Biol. Chem., 260, 14521-14528. Stout, D.L. and Becker, F.F. (1986) Heme enzymes patterns in genetically and chemically induced mouse tumors. Cancer Res., 46.2756-2759. Vincent, S.H., Bass, N.M., Snider, J.M. and Muller-Eberhard, U. (1987) Are the rat liver cytosolic fatty acid-binding (L-FABP) and heme-binding (HBP) proteins identical? Biochem. Arch., 3.443-451.
55
Decreased cytosolic levels of the heme binding Z protein in rat hepatocyte nodules and hepatocellular carcinomas M.W. Roomi, Departments Pharmacology
S.H. Vincent,
ojPothology,
E. Farber and U. Muller-Eberhard
Uniuersity of Toronto,
and Biochemistry,
Toronto, Ontario (Canada) and the Departments Cornell University Medical College, New York, NY 10021 (U.S.A.)
ofPediatrics,
(Received 20 February 1988) (Revision received 8 August 1988) (Accepted 9 August 1988)
Summary
Introduction
Hepatocyte nodules and hepatocellular carcinomas were induced in male Fischer rats using the resistant hepatocyte model. The immunoreactive cytosolic levels of the hemebinding Z protein (HBP) were reduced by 56 %i (P < 0.001; Z-tailed t-test) in early hepatocyte nodules (25 weeks) and hepatoceNular carcinomas (lo-12 months). This finding is in accordance with the previously reported reduced heme content of hepatocyte nodules and is consistent with the postulated role for HBP in intracellular heme transport and distribution. The immunoreactive levels of the glutathione S-transferase isozymes (GST) which like HBP bind heme, were elevated Z-fold (P < 0.01) in early and late hepatocyte nodules and were unchanged in hepatocellular carcinomas.
Rat hepatocyte nodules, a precursor population of liver cancer, exhibit in several different models a biochemical pattern of enzyme induction which is characterized by decreased activity of phase I enzymes of the microsomal drug metabolizing system including cytochrome P-450 and several cytochrome P-450mediated dealkylating enzymes [ 101, The decrease in phase I enzyme activity is associated with an increase in phase II components such as glutathione and GST, including a placental form [4,5,11]. This biochemical pattern has been considered to be responsible for the resistance of the nodules to the cytotoxic effect of several hepatotoxic agents. Our current hypothesis is that the rat hepatocyte nodules exhibit a basic interference in heme synthesis. This hypothesis is based on several observations. In addition to a generalized decrease in several cytochrome P-450 isozymes, with no apparent preference for any particular P-450, the nodules exhibit decreases in another microsomal cytochrome, cytochrome b,, and in two other heme containing proteins, catalase and tryptophan 2,3-dioxygenase as well as in the total microsomal heme content [9]. Decreases in total cytochrome P-450, cytochrome b, tryptophan 2,3-
Keywords: heme binding 2 protein; hepatocyte nodules and carcinomas; glutathione Stransferase; male Fischer rats. Correspondence to: Ursula Muller-Eberhard. Department of Pediatric Hem/One., Cornell University Medical College, 525 East 68th Street, Room 804, New York, New York 10021, U.S.A.
0304-3835/88/$03.50 0 1988 Elsevier Scientific Publishers Ireland Ltd Published and Printed in Ireland
56
dioxygenase, catalase and aminolevulinic acid synthase as well as an increase in microsomal heme oxygenase have also been reported for mouse liver tumors [16]. The hepatocyte nodules in the rat also showed low levels of total iron and delta-aminolevulinic acid activity
PI. To further test this hypothesis, we measured the level of the cytosolic HBP [15] in hepatocyte nodules and hepatocellular carcinomas generated when using the resistant hepatocyte model [13]. HBP, which comprises 3-5% of total liver cytosolic protein, has been suggested to be involved in the intracellular transport and distribution of heme [7]. It is also known as fatty acid-binding protein, L-FABP [ 1,171 as it binds fatty acids although with a lower affinity than heme [l, 151. For comparison, we also measured the levels of GST whose enzymatic activity is known to be increased in hepatocyte nodules [5,10] and which bind heme with a similar affinity as HBP. Materials and methods Hepatocyte nodules and hepatocellular carcinomas were induced according to the resistant hepatocyte model [13]. Male Fischer rats, initially weighing 150-170 g, were given diethylnitrosamine (Eastman Kodak Co., Rochester, NY) 200 mg/kg i.p. After a Z-week recovery period, the rats were placed on 0.02% 2-acetylaminofluorene for 1 week, subjected to a standard partial hepatectomy and continued for an additional week on the 2acetylaminofluorene diet. The rats were then placed on normal chow diet until the termination of the experiment. At various time periods rats were killed to give early nodules (25 weeks) and hepatocellular carcinomas (lo12 months). Control rats were fed normal chow diet for 12 months. The normal liver tissue, hepatocyte nodules and carcinomas were homogenized in Tris buffer containing 10m4 M PMSF (phenylethylsulfonyl fluoride), 1 mM EDTA and 1 mg aprotinin/lOO ml buffer. Cytosolic fractions were obtained by centrifuging the homogenates at
100,000 x g and protein concentration determined by the method of Bradford [3]. SDSpolyacrylamide gel electrophoresis (SDSPAGE) of cytosolic fractions were performed according to Laemmli [6]. Cytosolic HBP concentration was measured by rocket immunoelectrophoresis [8] and a solid-phase radioimmunoassay (RIA) [16,17] using an antibody raised in rabbits against HBP purified by the 4-step procedure described [15]. Results by the two assays agreed within f 12%. GST concentration was measured by RIA using an antibody raised in rabbits against a mixture of GST isozymes obtained by affinity chromatography on glutathione-agarose 1121. The antibody recognized by 3 major subunits as judged by Western blotting after SDS-PAGE of purified GST and liver cytosol. The data were analyzed by the 2-tailed Student’s t-test. Results and discussion Electrophoretic analysis of cytosolic protein fractions revealed a substantial decrease in the intensity of the M, 14,000 band in cytosol from early hepatocyte nodules as compared to cytosol from control liver tissue (Fig. 1). That the decrease in band intensity is accounted for by a decrease in the concentration of HBP, an abundant i’$l4,000 cytosolic protein, was confirmed by determining that the immuno-reactive levels of this protein are significantly reduced in cytosolic fractions from early and late hepatocyte nodules and hepatocellular carcinomas. As shown in Table I, HBP cytosolic levels were reduced by 56% (P < 0.001) in early hepatocyte nodules and hepatocellular carcinomas. As shown in Table 1, HBP cytoThe reduced levels of this abundant hemebinding protein are in accord with the reduced heme content of hepatocyte nodules [l] and are consistent with a postulated role for this protein in intracellular heme transport and distribution [7]. Given these observations and the general finding of reduced levels of total cytochrome P-450, cytochrome b,, heme content and decreased delta-aminolevulinic acid synthase activity in the nodules it becomes
57
Table 1. Cytosolic concentrations of HBP and GST isozymes in hepatocyte nodules and hepatocellular carcinomas in male Fischer rats. Determined by (combined homogenates of 4 nodules) rocket immunoelectrophoresis and/or solid-phase radioimmunoassay in samples from 4 rats in each group. Data expressed as % (mean f S.D.) of control (normal rat liver cytosol) Cytosolic concentration % of control
Early nodules Late nodules Hepatocellular carcinomas “P< 0.001; bP< 0.01.
Sodium dodecyl sulfate-polyacrylamide gel Fig. 1. electrophoresis of cytosolic proteins from normal rat liver tissue (lane 1, 30 pg) and early hepatocyte nodules (lane 2, 30 pg). The developing gel was 15% acrylamide. Lane 3 contains molecular weight markers.
reasonable to entertain the hypothesis that one or more changes relating to iron metabolism, including intracellular iron transport, may be fundamental to the characteristic phenotype of hepatocyte nodules. Recently, L-FABP equivalent with HBP as the cytoplasmic [17], was identified polypeptide p14 the mitosis-associated target of activated metabolites of the carcinogen 2acetylaminofluorene [2]. The concentration
HBP
GST
44&P 10 f & 11 + 9
213 f 38b 210 + 45b 131 + 36
2-tailed t-test.
of ~14 was found to be increased in proliferating hepatocytes of hyperplastic foci and hepatocellular carcinomas in rats that were fed 0.02% acetylaminofluorene continuously for 4-32 weeks without prior initiation with diethylnitrosamine or partial hepatectomy [ 141. It is not known whether heme synthesis is affected under these conditions. In contrast to the decrease in HBP levels, the immunoreactive levels of GST isozymes, which like HBP also bind heme, were elevated by 2-fold (P < 0.01) in both early and late hepatocyte nodules and were essentially in hepatocellular carcinomas unchanged (Table 1). The observed increase in GST levels is in agreement with the previously reported increase in GST enzymatic activity in hepatocyte nodules [lo, 111 and suggests that the primary role of GST isozymes is to function as Phase II conjugating enzymes rather than as transporters of heme whose synthesis in hepatocyte nodules is greatly reduced [9]. In conclusion, we have found that in hepatocyte nodules and hepatocellular carcinomas the cytosolic concentration of the abundant HBP is significantly reduced in accord with the previously reported decrease in heme synthesis. In contrast, the cytosolic concentration of the GST isozymes, which also bind heme, was elevated in hepatocyte nodules.
58
References Bass, N.M. (1985) Function and regulation of hepatic and intestinal fatty acid-binding proteins. Chem. Phys. Lipids, 38.95-114. Bassuk, J.A., Tsichlis. P.N. and Sorof, S. (1987) Liver fatty acid binding protein is the mitosis-associated polypeptide target of a carcinogen in rat hepatocytes. Proc. Natl. Acad. Sci. USA, 84,7547-7551. 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. Farber, E. (1984) The biochemistry of preneoplastic liver: a common metabolic pattern in hepatocyte nodules. Cancer J. Biochem. Cell Biol.. 62, 486-494. Kitahara, A., Satoh, K., Nishimuta, K., Ishikawa, T., Ruike. K., Sato, K., Tsuda, H. and Ito, N. (1984) Changes in molecular forms of rat hepatic glutathione S-transferase during chemical hepatocarcinogenesis. Cancer Res., 44, 2698-2703. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227,680-685. Muller-Eberhard, U. and Vincent, S.H. (1985) Concepts of heme distribution within hepatocytes. Biochem. Pharmacol., 34.719-725. Plumley, F.G. and Schmidt, G.W. (1983) Rocket and crossed immunoelectrophoresis of proteins solubilized with sodium dodecyl sulfate. Anal. Biochem., 134, 86-95. Roomi, M.W., Cameron, R.G. and Farber, E. (1984) Is the reduced cytochrome P-450 content in hepatocyte
10
11
12
13 14
15
16
17
nodules a reflection of decreased heme synthesis? Cancer Fed. Biol. Sot., 27, 161. Roomi, M.W., Ho, R.K., Sarma, D.S.R. and Farber, E. (1985) A common biochemical pattern in preneoplastic hepatocyte nodules generated in four different models in the rat. Cancer Res., 45,564-571. Rushmore, R.H., Sharma, R.N.S., Roomi, M.W.. Harris, L., Satoh, K., Sato, K., Murray, R.K. and Farber, E. (1987) Identification of a characteristic cytosolic polypeptide of rat preneoplastic hepatocyte nodules as placental glutathione S-transferase. Biochem. Biophys. Res. Commun., 143,98-103. Simons, P.C. and Vander Jagt, D.L. (1981) Purification of glutathione S-transferases by glutathione-affinity chromatography. Methods Enzymol., 77,235-237. Solt, D. and Farber, E. (1976) A new principle for the analysis of chemical carcinogenesis. Nature, 263.701-703. Sorof, S. and Custer, R.P. (1987) Elevated expression and cell cycle deregulation of a mitosis-associated target polypeptide of a carcinogen in hyperplastic and malignant rat hepatocytes. Cancer Res., 47, 210-220. Vincent, S.H. and Muller-Eberhard. U. (1985) A protein of the Z class of liver cytosolic proteins in the rat that preferentially binds heme. J. Biol. Chem., 260, 14521-14528. Stout, D.L. and Becker, F.F. (1986) Heme enzymes patterns in genetically and chemically induced mouse tumors. Cancer Res., 46.2756-2759. Vincent, S.H., Bass, N.M., Snider, J.M. and Muller-Eberhard, U. (1987) Are the rat liver cytosolic fatty acid-binding (L-FABP) and heme-binding (HBP) proteins identical? Biochem. Arch., 3.443-451.