TOXICOLOGIC PATHOLOGY, vol 29, no 4, pp 479 482, 2001 Copyright C 2001 by the Society of Toxicologic Pathology

Greater Expression of Transforming Growth Factor and Proliferating Cell Nuclear Antigen Staining in Mouse Hepatoblastomas Than Hepatocellular Carcinomas Induced by a Diethylnitrosamine-Sodium Phenobarbital Regimen
TETSUYA SAKAIRI, KIYOSHI KOBAYASHI, KAZUHIRO GOTO, MIYOKO OKADA, MANAMI KUSAKABE, TAKAYUKI TSUCHIYA, JIRO SUGIMOTO, FUMIKO SANO, AND MAMORU MUTAI
Toxicology Laboratory, Research Center, Mitsubishi-Tokyo Pharmaceuticals, Inc., 100-5, Yana, Kisarazu, Chiba 292-0812, Japan
ABSTRACT Transforming growth factor a (TGF-a ) is a potent stimulator of normal hepatocyte proliferation, considered to have relationship to the liver regeneration or carcinogenesis . In this study, we investigated immunohistochemicall y the association between expression of TGF-a and cell proliferation activity in mouse hepatoblastoma s (HBs) and hepatocellular carcinomas (HCCs) induced in B6C3F1 mice by diethylnitrosamine and sodium phenobarbital . The TGF-a -positive rate in HBs (29.2%) was signi cantly higher than that in HCCs (12.7%). Likewise, the proliferating cell nuclear antigen-positive rate (22.2%) was higher than the HCC value (14.5%). On the individual data for both TGF-a and PCNA, most of the HBs showed higher positive rates than HCCs. In HBs, TGF-a was localized only in the nuclei, whereas some HCC cells stained positive both in their nuclei and cytoplasm (0.6%). These results suggest expression of TGF-a and its localization might be linked to cell proliferation and play a role in malignant progression of mouse HBs. Keywords. B6C3F1 mice; cell proliferation; hepatoblastoma ; immunohistochemistry ; liver; transforming growth factor a .

INTRODUCTION Mouse hepatoblastomas (HBs) occur spontaneously in aged animals and can be induced by chemical treatment in some strains of mice (6, 8, 17, 24, 30). Mouse HBs are histologically similar to the embryonal or small cell types of human HBs (5, 7, 30). Their histogenesis is still unclear, but there is evidence that the mouse HB is a highly malignant tumor of hepatocyte origin (2, 5). Transforming growth factor a (TGF-a ), a member of epidermal growth factor (EGF) family, is a 50-amino acid polypeptide (29). It shares about 30% structural similarity with EGF and can bind to the EGF receptor (3). Several reports have suggested TGF-a to be a potent stimulator of normal hepatocyte proliferation and its overexpression may play an important role in hepatocarcinogenesis (13, 22, 23). Thus, TGF-a has been detected in many malignant cell lines including human and rodent hepatocellular tumors (4, 19, 20), and overexpression in the liver results in hepatocarcinogenesis in transgenic mice (18, 26). In the present study, we investigated the expression and immunohistochemical localization of TGF-a in HBs and HCCs induced in B6C3F1 mice with diethylnitrosamine (DEN) and sodium phenobarbital (PB), and analyzed the relationship between expression of TGF-a and cell proliferation activity in these tumors.

Address correspondenc e to: Tetsuya Sakairi, Mitsubishi-Toky o Pharmaceuticals 100-5, Yana, Kisarazu, Chiba 292-0812 , Japan; e-mail: 4307434@ mitsubishi-pharm.co.jp.

MATERIALS AND METHODS A total of 21 male 6-week-old B6C3F1 mice were purchased from Charles River Japan (Kanagawa, Japan) and acclimatized for 1 week in an air-conditioned animal room at 22 C with a 12-hour light/12-hour dark cycle. They were fed basal powdered diet (MF, Oriental Yeast Co. Ltd., Tokyo, Japan) and tap water ad libitum. All mice were intraperitoneally administered 80 mg/kg of DEN (Tokyo Kasei Co. Ltd., Tokyo, Japan), then fed a diet containing PB at a concentration of 500 ppm or basal diet alone until the end of experiment at week 50, when all animals were sacri ced under ether anesthesia. Livers were xed in 10% neutral-buffered formalin, embedded in paraf n, cut in 3 4-l m sections, and stained with hematoxylin eosin (HE). Hepatocellular tumors were histopathologically classi ed into hepatocellular adenomas (HCAs), hepatocellular carcinomas (HCCs), and HBs. They were also examined immunohistochemically using antiTGF-a (Oncogene Research Products, Cambridge, MA, USA) and antiproliferating cell nuclear antigen (PCNA; Coulter Immunology, Hialeah, FL, USA) antibodies. Tissue sections were exposed to these primary antibodies, and then subjected to the avidin-biotin-peroxidas e complex (ABC) method. For quantitative comparisons, 1,000 nuclei were counted for each lesion, and the percentages of PCNApositive nuclei for the HB and HCC cells were calculated. Similarly, 1,000 cells were counted for each lesion and percentages with TGF-a -positive nuclei or cytoplasm were calculated. TGF-a positively stained cells were classi ed as nuclear or with extranuclear staining, and ratios were calculated for 1,000 tumor cells.

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Statistical analyses to compare the PCNA- and TGF-a positive rates between HBs and HCCs were carried out using the Students t -test. RESULTS HCAs, HCCs, and HBs were induced in 17, 13, and 13 of the 21 mice, respectively. Seven of 13 HBs (53.8% ) were observed in and/or adjacent to HCAs or HCCs. Representative immunohistochemical staining patterns for TGF-a and PCNA are illustrated in Figure 1. Data for TGF-a expression and its localization are summarized in Figure 2, and relationship between TGF-a and PCNA positive rates for individual HBs and HCCs is illustrated in Figure 3. Seven HBs (Figure 1A ) and 10 HCCs (Figure 1D) were microscopically examined. The TGF-a -positive rates were 29.2%

and 12.7%, respectively. All TGF-a positive HB cells were positively stained only in their nuclei, whereas some HCC cells demonstrated both nuclear and cytoplasmic staining. The TGF-a positive rate of HB was signi cantly higher than that of HCC ( p < 0.01). PCNA-positive rates for HBs and HCCs were 22.2% and 14.5% respectively, the difference being also signi cant ( p < 0.05, Figure 2). In almost every HB, the individual data for the TGF-a -positive rate was greater than the HCC values, and most of the HBs also showed higher positive rates as to PCNA (Figure 3). DISCUSSION In line with earlier reports (2, 5), HBs were observed within and/or adjacent to hepatocellular tumors in the present study. The mouse HB is thought to be a highly malignant neoplasm

FIGURE 1.Histological features of mouse hepatoblastoma s (A, B, C) and hepatocellular carcinomas (D, E, F). A and D) hematoxylin-eosi n staining. B and E) transforming growth factor a (TGF-a ) immunohistochemica l staining. C and F) proliferating cell nuclear antigen (PCNA) immunostaining. 145. Note the nuclear staining for TGF-a in the HB. Some HCC cells are also stained positively in their cytoplasm.

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FIGURE 2.A) TGF-a -positive rates. ; Nuclear and j ; cytoplasmic staining. B) PCNA-positive rates. All data are means SD. TGF-a -positive and PCNApositive rates in hepatoblastoma s are signi cantly higher than in hepatocellular carcinomas ( p < 0.01 and p < 0.05, respectively).

arising in the late stages of hepatocarcinogenesis, because it may metastasize to the lungs (6, 7). The present results for cell proliferation rate are in line with this conclusion. In the present study, we observed an association between TGF-a expression and cell proliferating activity in chemically induced mouse HBs. Mead et al (22) reported that TGF-a might have a function as a physiological inducer of hepatocyte DNA synthesis during liver regeneration. Thus, the higher TGF-a positive rates in HBs may be partly responsible for the increased cell proliferating activity. Steinmetz

FIGURE 3.Relationship between TGF-a and PCNA positive rates for individual HBs and HCCs.

et al (28) and Imai et al (10) described expression of TGF-a and PCNA to be increased in foci of cellular alteration. Several studies have pointed to TGF-a overexpression playing an important role in hepatocarcinogenesis (12, 25, 29). Recently, we reported linkages to cell proliferation in chemically induced rat HCCs (16). From the literature, as well as the present study, TGF-a expression may be associated with cell proliferation activity of hepatocellular tumor cells. The human HB is a distinctive tumor of the liver in infants and children that consists of cells resembling hepatocytes with varying degrees of maturation (15). In contrast, mouse HBs occur only in aged animals and exhibit histologically a poorly differentiated status resembling the embryonal or small cell type of human HB (5, 7, 30). Kiss et al (15) reported that well-differentiated human HBs show high expression of TGF-a in cytoplasm, but this is lacking in other forms, for example embryonal or small cell type. Although the reason for this discrepancy with the present ndings is unclear, less differentiated human HBs may not depend on growth stimulation of TGF-a . It is noteworthy that all mouse HBs and most HCCs showed nuclear staining for TGF-a in this study in line with the previous reports (9, 14) of nuclear TGF-a staining in human HCCs. We recently demonstrated less differentiated types of rat HCC to be positive for TGF-a , with a clear tendency for exhibition of nuclear staining pattern with increased cell proliferation (16). Moreover, the cytoplasm of hepatocytes is positive for TGF-a 6 12 hours from partial hepatectomy but becomes localized to the nuclei after 24 36 hours when cell division is at its peak (paper in preparation). Reportedly, EGF is handled very differently in the regenerating rat liver than the normal adult rat liver (21). EGF is normally internalized on plasma membrane receptors and destroyed in lysosomes. In regenerating hepatocytes, however, EGF accumulates in the nuclei, suggesting the presence of

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nuclear EGF receptors. Thus, this might also be the case for TGF-a . Brown et al (1) demonstrated a speci c nuclear reaction for TGF-a in mammary tissues of rats. Nuclear localization has been also reported for broblast growth factor and nerve growth factor in BALB/3T3 cells and rat pheochromocytoma cells, respectively (11, 27, 31). Growth factors conceivably act directly on nuclear events such as DNA replication and transcription, and nuclear TGF-a expression may be related to a shift in localization associated with advances in the cell cycle. Further investigations are now needed to clarify the relationship between TGF-a and progression of hepatocellular neoplasms. In conclusion, mouse HBs exhibit higher cell proliferating activity than HCCs, and this might be linked to abundant TGF-a expression and nuclear localization. ACKNOWLEDGMENTS We thank Yasuko Ogawa and Yoshie Kawazu for expert technical assistance. REFERENCES
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