* Biosafety Research Center, Foods, Drugs and Pesticides, 5822 Arahama, Shioshinden, Fukude-cho, Iwata-gun, Shizuoka 437-1213, Japan; and
Graduate School of Nutritional and Environmental Sciences, University of Shizuoka, 521 Yada, Shizuoka-shi, Shizuoka 422-8526, Japan
Received January 3, 2000; accepted April 6, 2000
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ABSTRACT |
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Key Words: Bcl-2; H-ras; B6C3F1 mouse; liver tumor.
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INTRODUCTION |
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Numerous reports on the inhibitory role of the bcl-2 oncogene in apoptosis have been published recently (Goldsworthy et al., 1996a,b
; Reed, 1998
; Williams, 1991
; Wyllie, 1997
). This gene was identified by cloning breakpoints of 1418 translocations, t(14;18), a characteristic of human B-cell lymphomas (Tsujimoto et al., 1985
). This translocation determines the juxtaposition of the bcl-2 gene on the immunoglobulin heavy chain locus (Bakhshi et al., 1985
) resulting in deregulation of overexpression of the bcl-2 gene. The bcl-2 gene inhibits the release of cytochrome c and apoptosis-inducing factor (AIF) from the mitochondria, thereby preventing apoptotic death (Kluck et al., 1997
; Yang et al., 1997
). Prevention of induced or spontaneous apoptosis via bcl-2 may result in the selective survival of preneoplastic cells and ultimate neoplastic transformation. Lee (1997) reported that the Bcl-2 protein is overexpressed in diethylnitrosamine-induced hepatocellular tumors of B6C3F1 mice, and demonstrated the difference in Bcl-2 expression among histological phenotypes of hepatocellular tumors induced by the combined effects of diethylnitrosamine and phenobarbital using a 2-step initiator-promoter model. However, the role of the Bcl-2 protein on naturally occurring hepatocellular tumors in B6C3F1 mice has not been demonstrated. The present study was conducted to examine the role of Bcl-2 expression in the development of the naturally occurring hepatocellular tumors in B6C3F1 mice and to demonstrate the correlation between Bcl-2 expression and H-ras activation in carcinogenesis.
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MATERIALS AND METHODS |
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Immunohistochemistry
Immunohistochemical staining for Bcl-2 or PCNA was performed with an antigen retrieval system. Sections were autoclaved at 120°C for 5 min in distilled water for Bcl-2 or with a microwave oven in 10 mM citrate buffer (pH 6.0) for PCNA. Following cooling, the sections were incubated with primary antibodies overnight at 4°C. Rabbit anti-mouse Bcl-2 serum (PharMingen, San Diego, CA) was employed as the Bcl-2 antibody and was diluted l:500 in 1% bovine serum albumin/phosphate-buffered saline (PBS). Sections were stained by the LSAB method (Labeled Streptavidin Biotin Kit, DAKO, Japan). Mouse monoclonal anti-PCNA (PC10; DAKO, Japan) was used as a PCNA antibody at a dilution of 1:200 and stained by the LSAB method. Finally, the color indicative of immune-reactivity was developed using diaminobenzidine (DAB) for 7 min. Slides were rinsed with distilled water, lightly counterstained with hematoxylin, and mounted. The primary antibody was omitted in negative control samples. The PCNA labeling index (LI) was calculated as the percentage of positive cells per 1000 cells in randomly selected fields.
TUNEL Method
The Apop Tag in situ Apoptosis Detection Kit-Peroxidase (Oncor, Gaithersburg, MD) was used to identify apoptosis by the method described by Gavrieli et al. (1992). Sections were deparaffinized with xylene, rinsed in ethanol, and digested with 20 µg/ml Proteinase K (Boeringer-Mannheim, Germany) for 15 min at room temperature and rinsed with distilled water. Endogenous peroxidase activity was blocked with 2% hydrogen peroxide in PBS for 5 min and rinsed with PBS. Sections were incubated in equilibration buffer for 15 min at room temperature and incubated in working strength TdT enzyme at 37°C for 1 h. Slides were washed in stop/wash buffer for 10 min, rinsed with PBS, incubated with anti-digoxigenin-peroxidase for 30 min at room temperature, and washed with PBS. Slides were developed with DAB, washed with distilled water, counterstained with methyl green, and mounted. Distilled water was used in place of the TdT enzyme, in negative controls. The apoptotic index (AI) was calculated as the percentage of positive cells per 1000 cells in randomly selected fields.
Western Blot Analysis
We used Western blotting to analyze Bcl-2 expression at the protein level. Total tissue lysates containing 25 µg of denatured protein/lane were run on 12% Ready Gel J (Bio-Rad, California) and electroblotted onto a nitrocellulose membrane (Hybond-ECL, Amersham Pharmacia, Uppsala, Sweden). The membrane was blocked with 0.5% non-fat dry milk in PBS with 0.1% Tween 20 (PBST) and incubated with rabbit anti-mouse Bcl-2 serum (PharMingen) diluted in PBST with 5% non-fat dry milk (1:1000 dilution) at 4°C overnight. After washing in PBST, the membrane was incubated with horseradish peroxidase-labeled anti-rabbit secondary antibodies (Amersham Pharmacia) diluted in PBST with 5% non-fat dry milk (1:2000 dilution) at 4°C overnight and washed again. Finally, the immunoreaction was visualized using enhanced chemiluminescence (Amersham Pharmacia).
Extraction and Amplification of DNA from Paraffin-Embedded Hepatocellular Tissue
DNA was obtained for oncogenic analysis from paraffin-embedded hepatocellular tissues. Tissue samples obtained from normal liver tissues and hepatocellular proliferative lesions, 12 FCA (3 Bcl-2-positive and 9 Bcl-2-negative), 34 HCA (18 Bcl-2-positive and 16 Bcl-2-negative) and 14 Bcl-2-positive HCC were deparaffinized with xylene and rinsed with ethanol. Sections from these specimens were collected with a sterile disposable scalpel and placed in 1.5 ml microcentrifuge tubes. DNA was extracted from each sample by the same method previously reported (Iida et al., 1999). The polymerase chain reaction (PCR) was carried out as described previously (Iida et al., 1997
) to amplify a 116 bp fragment of exon 2 of the H-ras gene, which includes codon 61. An aliquot (10 µl) of DNA extracts was used for PCR in a PCR reaction mixture containing 50 µl reaction buffer (10 mM Tris-HCl/pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.001% (w/v) gelatin, 200 µM dNTPs (dATT, dCTP, dGTP, and dTTP), 0.2 µM of each primer and 5 units of AmpliTaq (PE Biosystems, California). The sequences of primers 1 and 2 were 5'-GAGACATGTCTACTGGACATCTT-3' (sense) and 5'-GTGTTGTTGATGGCAAATACACAGAGG-3' (antisense), respectively. The mixture was covered with a layer of mineral oil (Sigma, St. Louis, MO) to prevent evaporation and transferred to the thermal cycler (TR-100; Taitec, Japan) for 38 cycles. Each cycle consisted of 30 seconds at 94°C, one min at 60°C, and 30 s at 72°C. Following this reaction, the mixture was incubated at 72°C for 8 min. Each amplification reaction containing DNA from normal liver tissue, positive controls for H-ras mutation, and controls without DNA template was run with all sets of reactions. DNA amplification was confirmed by electrophoresis on a 1.5% agarose gel containing (0.5 µg/ml) of ethidium bromide.
Non-RI SSCP
Non-radioisotope single strand conformation polymorphism (non-RI SSCP) technique, in combination with the silver staining method, was chosen to detect point mutations, since it is highly sensitive and simple. Aliquots of 10 µl of the PCR products were added to 45 µl of loading buffer (95% formamide, 20 mM EDTA, 0.05% BPB, 0.05% xylene cyanol), denatured by heating for 3 min at 80°C, and then chilled on ice. Aliquots of 3 µl of this mixture were electrophoresed by the Phast System (Amersham Pharmacia) using PhastGel Homogeneous 12.5 (Amersham Pharmacia) and PhastGel Native Buffer strips (Amersham Pharmacia). The Phast System was run under the following conditions: (i) pre-run, 15 mA, 300 V, 2.5 W, 4°C, 100 Vh; (ii) Sample application, 15 mA, 300 V, 2.5 W, 4°C, 2 Vh; and (iii) Run, 15 mA, 300 V, 2.5 W, 4°C, 200 Vh. The relationship between immunohistochemical staining for Bcl-2 and the mutation at codon 61 of the H-ras gene in HCA was analyzed using Fisher's exact test, following classification of the patterns of H-ras mutations.
Direct DNA Sequencing
Representative samples were selected for direct sequencing to determine the nucleotide substitution detected by non-RI SSCP. Aliquots of 30 µl were purified following amplification using Ultrafree DA (Millipore, Bedford, MA). Samples were sequenced using an ABI PRISM Big dye Terminator Cycle Sequencing Ready Reaction Kit (PE Biosystems) according to the manufacturer's instructions. The sequencing primer used was a nested primer, 5'-GATGGCAAATACACAGAG-3'. An ABI PRISM 310 genetic analyzer (PE Biosystems) analyzed reaction products. All the mutations were confirmed by sequencing at least twice.
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RESULTS |
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The extent of apoptosis in the Bcl-2-positive and Bcl-2-negative sections that were used to determine PCNA LI was also examined by the TUNEL method. Since the AI was very low (0.10.5%) in all hepatocellular proliferative lesions examined, it was concluded that an adequate incidence for comparison did not exist (data not shown).
Nine FCA/Bcl-2-negative, 3 FCA/Bcl-2-positive, 16 HCA/Bcl-2-negative, 18 HCA/Bcl-2-positive, and 14 HCC/Bcl-2-positive DNA samples were analyzed by SSCP as having a mutation at codon 61 in the H-ras gene. The H-ras gene with mutated codon 61 was detected in 3 cases of HCA/Bcl-2-negative, 7 cases of HCA/Bcl-2-positive, and 8 cases of HCC. However, no mutations of the H-ras gene were detected in any of 9 FCA/Bcl-2-negative or 3 FCA/Bcl-2-positive samples (Table 3 and Fig. 3
). Mutations in hepatocellular tumors were confirmed by DNA sequencing. Three HCA/Bcl-2-negative, 4 HCA/Bcl-2-positive and 4HCC/Bcl-2-positive, which showed the shift-positive results in non-RI PCR-SSCP, were found to contain C to A transversion at the first base of the codon 61 in the H-ras gene (see lanes 2, 3, and 4 in Fig. 3
). In addition, 3 HCA/Bcl-2-positive and 4 HCC/Bcl-2-positive were found to contain A to G transition at the second base (see lane 5 in Fig. 3
). Mutations at codon 61 in the H-ras gene were found in 7 (38.9%) of 18 HCA specimens that stained positive for Bcl-2, and in 3 (18.8%) of 16 HCA specimens stained negative for Bcl-2 (Table 3
). The incidence of mutations at codon 61 in the H-ras gene was higher in Bcl-2-positive specimens than in Bcl-2-negative specimens.
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DISCUSSION |
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The AI values in the majority of the hepatocellular proliferative lesions were low in this study. Goldsworthy et al. (1996a,b) also reported that apoptosis occurs infrequently in hepatocellular tumors in B6C3F1 mice (0.010.1% in females). Although the specificity of Bcl-2 expression and a definitive correlation of Bcl-2 protein with PCNA (LI) were not demonstrated in HCA, marked and increased expression of Bcl-2 in HCC demonstrates its usefulness in the evaluation of the malignant behavior of the naturally occurring hepatocellular tumors in B6C3F1 mice.
Mutation at codon 61 of the H-ras gene was detected in 7 (38.9%) of 18 HCA that stained positive for Bcl-2 immunohistochemically, but in only 3 (18.8%) of 16 HCA stained negative for Bcl-2, suggesting a higher expression of Bcl-2 in HCA with H-ras mutation. These results suggest a cooperative synergistic effect between ras oncogenes and cell growth-regulatory factors, including Bcl-2, in the development of naturally occurring hepatocellular tumors in B6C3F1 mice. Kinoshita et al. (1995) proposed that H-ras gene mutations cause overexpression of the Bcl-2 protein and homologous proteins such as Bcl-xL, resulting in suppression of cell death in hematopoietic cells. Weinberg (1989) reported that ras-oncogene activation occurs during the cellular growth phase of benign tumors or preneoplastic lesions, resulting in the formation of large adenomas that may progress to malignant tumors. Overexpression of Bcl-2 was observed in cell lines that contain the human c-H-ras oncogene with an activating point mutation on codon 61 (Osanai et al., 1997). These observations suggest that activating point mutations in H-ras gene may induce Bcl-2 expression, which might. result in the selective survival of the initiated hepatocytes via inhibition of apoptosis and the accumulation of alterations in other tumor-related genes.
A mutation at codon 61 in the H-ras gene was not detected by non-RI SSCP or DNA sequencing in 11 of 18 HCA that had stained positive for Bcl-2 immunohistochemically. Furthermore, although all the HCC stained positive for Bcl-2, 6 (47.1%) of 14 HCC contained no mutation at codon 61. These results suggest that in naturally occurring hepatocellular proliferative lesions factors other than the mutation at codon 61 in the H-ras gene lead to the enhanced expression of the Bcl-2 protein, and that the overexpression of Bcl-2 is more important for the development of malignancy in the naturally occurring hepatocellular carcinoma in the B6C3F1 mouse than H-ras activation by the mutation at codon 61.
Data supporting the correlation between Bcl-2 overexpression and the H-ras gene mutation will shed light on the molecular basis of cooperation between endogenous mutational effects of oncogenes and the regulatory factors of cellular growth as interactive and inter-related steps in the neoplastic turnover in hepatocytes. Further studies using molecular and morphologically specific markers will be required to elucidate the pathogenesis of naturally occurring hepatocellular tumors commonly found in the aged B6C3F1 mice.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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Enomoto, M., Kobayashi, K., and Inoue, H. (1990). Significance of naturally occurring tumors in evaluating the carcinogenicity of a test compound: A review and an improved carcinogenicity bioassay for chemicals. J. Toxicol. Pathol. 3, 117.
Gavrieli, Y., Sherman, Y., and Ben-Sasson, S. A. (1992). Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell Biol. 119, 493501.[Abstract]
Goldsworthy, T. L., Conolly, R. B., and Fransson-Steen, R. (1996a). Apoptosis and cancer risk assessment. Mutat. Res. 365, 7190.[ISI][Medline]
Goldsworthy, T. L., Fransson-Steen, R., and Maronpot, R. R. (1996b). Importance of and approaches to quantification of hepatocyte apoptosis. Toxicol. Pathol. 24, 2435.[ISI][Medline]
Goldsworthy, T. L., Romach, E. H., and Fox, T. R. (1998). Mouse liver carcinogenesis. In Carcinogenicity Testing, Predicting, and Interpreting Chemical Effects (K. T. Kitchin, Ed.), pp. 145195. Marcel Dekker, New York.
Iida, M, Iwata, H., Yamakawa, S., and Enomoto, M. (1997). Analysis of Ha-ras gene in the B6C3F1 mouse liver by non-RI PCR-SSCP. J. Toxicol. Pathol. 10, 1317.
Iida, M., Iwata, H., Enomoto, M., Horie, N., and Takeishi, K. (1999). Analysis of ras gene mutations in main spontaneously occurring non-epithelial tumors of B6C3F1 mouse. J. Toxicol. Pathol. 12, 6570.
Kinoshita, T., Yokota, T., Arai, K., and Miyajima, A. (1995). Regulation of Bcl-2 expression by oncogenic Ras protein in hematopoietic cells. Oncogene 10, 22072212.[ISI][Medline]
Kluck, R. M., Bossy-Wetzel, E., Green, D. R., and Newmeyer, D. D. (1997). The release of cytochrome c from mitochondria: A primary site for Bcl-2 regulation of apoptosis. Science 275, 11321136.
Korsmeyer, S. J. (1999). Bcl-2 gene family and the regulation of programmed cell death. Cancer Res. 59(Suppl. 7), 16931700.
Lee, G. H. (1997). Correlation between Bcl-2 expression and histopathology in diethylnitrosamine-induced mouse hepatocellular tumors. Am. J. Pathol. 151, 957961.[Abstract]
Maronpot, R. R., Haseman, J. K., Boorman, G. A., Eustis, S. E., Rao, G. N., and Huff, J. E. (1987). Liver lesions in B6C3F1 mice: The National Toxicology Program, experience and position. In: Archives of Toxicology. (P. L. Chambers, D. Henschler, and F. Oesch, Eds.), pp. 1026. Springer-Verlag, Berlin, Heidelberg.
Maronpot, R. R., Fox, T., Malarkey, D. E., and Goldsworthy, T. L. (1995). Mutations in the ras proto-oncogene: Clues to etiology and molecular pathogenesis of mouse liver tumors. Toxicology 101, 125156.[ISI][Medline]
Osanai, M., Ogawa, K., and Lee, G. H. (1997). Phenobarbital causes apoptosis in conditionally immortalized mouse hepatocytes depending on deregulated c-myc expression: Characterization of an unexpected effect. Cancer Res. 57, 28962903.[Abstract]
Reed, J. C. (1998). Bcl-2 family proteins. Oncogene 17, 32253236.[ISI][Medline]
Tsujimoto, Y., Cossman, J., Jaffe, E., and Croce, C. M. (1985). Involvement of the bcl-2 gene in human follicular lymphoma. Science 228, 14401443.[ISI][Medline]
Weinberg, R. A. (1989). Oncogenes and multistep carcinogenesis. In Oncogenes and the Molecular Origins of Cancer. (R. A. Weinberg, Ed.), pp. 307326. Cold Spring Harbor Laboratory Press, Plainview, New York.
Williams, G. T. (1991). Programmed cell death: Apoptosis and oncogenesis. Cell 65, 10971098.[ISI][Medline]
Wyllie, A. H. (1997). Apoptosis and carcinogenesis. Eur. J. Cell Biol. 73, 189197.[ISI][Medline]
Yang, J., Liu, X., Bhalla, K., Kim, C. N., Ibrado, A. M., Cai, J., Peng, T.-I., Jones, D. P., and Wang, X. (1997). Prevention of apoptosis by Bcl-2: Release of cytochrome c from mitochondria blocked. Science 275, 11291132.
Zhao, M., Zhang, N.-X., Economou, M., Blaha, I., Laissue, J. A., and Zimmermann, A. (1994). Immunohistochemical detection of Bcl-2 protein in liver lesions: Bcl-2 protein is expressed in hepatocellular carcinomas but not in liver cell dysplasia. Histopathology 25, 237245.[ISI][Medline]