Correlation between Bcl-2 Expression and

Transcription

1 American Journal of Pathology, Vol. 151, No. 4, October 1997 Copyright American Societyfor Investigative Pathology Correlation between Bcl-2 Expression and Histopathology in Diethylnitrosamine-Induced Mouse Hepatocellular Tumors Gang-Hong Lee From the Department of Pathology, Asahikawa Medical College, Asahikawa, Hokkaido, Japan It is generally accepted that suppression of apoptosis in chemically initiated hepatocytes results in promotion of rodent hepatocarcinogenesis. Using immunohistochemical methods, I studied the expression of Bcl-2, an anti-apoptotic protein, in hepatocellular tumors of B6C3F1 mice. Although normal mouse hepatocytes did not express detectable amounts of Bcl-2, most diethylnitrosamine-induced tumors were positive for this protein. Virtually all of the Bcl-2-positive tumors were composed of small basophilic hepatocytes, whereas the rare cases of Bcl-2-negative tumors demonstrated an eosinophilic appearance. To confirm this difference, tumors initiated with diethylnitrosamine and promoted by phenobarbital were also studied, as this initiation-promotion protocol has been shown to selectively produce eosinophilic lesions. All such tumors were immunohistochemically negative for Bcl-2. The relatively infrequent basophilic tumors found with phenobarbital treatment, however, did express Bcl-2. Thus, the concordance with basophilia was observed regardless of the nature of the promotion agent. These results indicate that the two types of tumors are qualitatively distinct and may develop through independent mechanisms. (Am J Pathol 1997, 151: ) The bcl-2 oncogene was first isolated as a gene showing constitutive overexpression in human B cell lymphocytic malignancies, including follicular lymphomas and chronic lymphocytic leukemias.1' 2 It is well documented that bcl-2 becomes deregulated in such tumor cells as a result of translocation into the immunoglobulin heavychain locus.1-4 Recent evidence indicates that this oncogene plays a role in tumorigenesis by inhibiting apoptotic death rather than promoting cell growth.5`a The Bcl-2 oncoprotein product is a putative membrane-associated 26-kd protein, containing a hydrophobic carboxy terminus that may locate it to intracellular membranes, leaving the remainder of the molecule in the cytosol.9 In mice, Bcl-2 is abundantly expressed in the thymus, spleen, and cerebrum but not in the liver.10 Rodent hepatocarcinogenesis has been extensively studied in terms of the multistage concept. The earliest stage, designated initiation, is considered to occur in hepatocytes through mutational events, by which the cells become initiated or preneoplastic The second stage, promotion, is characterized by gain in volume of initiated lesions, this being positively modified by nongenotoxic compounds called hepatopromoters represented by phenobarbital (PB) At least two possible cellular mechanisms regulating this promotion stage have been implicated, one being enhancement of growth and the other being inhibition of apoptosis of preneoplastic hepatocytes. Recent studies by Schulte-Hermann and his co-workers have suggested that the latter is the major mechanism for PB promotion of chemically initiated rat hepatocarcinogenesis.14 They found that preneoplastic rat hepatocytes initiated with a chemical carcinogen and promoted chronically with PB showed enhanced apoptosis after withdrawal of the promoter. There was, in contrast, no influence of PB on their [3H]thymidine-labeling index. 15 In the present study, the expression of Bcl-2 in mouse hepatocellular tumors was immunohistochemically investigated to clarify its possible involvement in their genesis. Attention was focused on the link between expression of Bcl-2 and the histopathological type, with demonstration of a clear difference between basophilic and eosinophilic lesions. Materials and Methods Animals B6C3F1 mice were produced in our laboratory by mating female C57BL/6J with male C3H/HeJ mice. The parental strains were purchased from Japan CLEA, Tokyo, Japan. Except for the PB promotion case, the mice were maintained on a basal diet (CE-Il, Japan CLEA) and allowed free access to tap water throughout. Only male B6C3F1 mice were used in this study. Supported in part by grants from the Ministry of Health and Welfare, the Ministry of Education, Culture, and Science, and the Akiyama Foundation, Japan. Accepted for publication July 23, Address reprint requests to Dr. Gang-Hong Lee, Department of Pathology, Asahikawa Medical College, Nishikagura, Asahikawa, Hokkaido 078, Japan. 957

2 958 Lee Induction of Liver Tumors Following the protocol introduced by Vesselinovitch and Mihailovich,'6 eight 12-day-old male infant B6C3F1 mice were injected intraperitoneally with diethyinitrosamine (DEN), dissolved in physiological saline, at the dose level of 5,ug/g body weight, and, after weaning, fed the basal diet. At 25 weeks of age, they were sacrificed by venesection under ether anesthesia, and tumors on the liver surface larger than 2 mm in diameter were enumerated. Portions of liver tissues were fixed overnight in 10% neutral formalin and used as materials for histological studies. For each relatively large tumor, a part of the tissue was freshly frozen on dry ice and used for Western blot studies. To induce eosinophilic lesions, the protocol of Pitot et a11 was used. Eighteen 5-week-old male B6C3F1 mice underwent twothirds partial hepatectomy under ether anesthesia, and after 36 hours were injected intraperitoneally with DEN dissolved in physiological saline at the dose level of 20,ug/g body weight. Two weeks later, six of the mice were maintained on a diet containing 500 ppm PB, whereas the remainder received the basal diet. At 55 weeks of age, all of the mice were sacrificed and treated as described above except that fresh-frozen tissues were not collected. Histology The formalin-fixed liver tissues were embedded in paraffin, and serial sections were cut. For each sample, one section was routinely stained with hematoxylin and eosin (H&E), and a serial section was used for immunohistochemistry. Hepatocellular tumors were classified into two major types by histological examination of the H&E-stained sections in line with the literature.17'18 The first type features enhanced cytoplasmic basophilia and usually smaller cells relative to normal hepatocytes. Tumors of the second, eosinophilic, type were distinguished from the basophilic lesions on the basis of their cytoplasmic eosinophilia, similar to that of normal hepatocytes, and large size. A few unclassifiable tumors composed of mixtures of basophilic and eosinophilic tumor cells were also encountered. Immunohistochemistry To immunohistochemically detect the Bcl-2 oncoprotein, paraffin sections were deparaffinized in xylene and microwaved four times in 0.01 mol/l citrate buffer (ph 6.0), each for 5 minutes. Staining was performed with the avidin-biotin complex method using rabbit anti-mouse Bcl-2 serum (13456E, PharMingen, San Diego, CA; 1: 1000 dilution) and a HISTOFINE kit (Nichirei, Tokyo, Japan). The sections were lightly counterstained with hematoxylin to aid orientation. Western Blot Analysis For Western blot analysis of Bcl-2 protein, total tissue lysates containing 50,ug of denatured proteins were run on 12.5% polyacrylamide gels containing sodium dodecyl sulfate and electroblotted onto nitrocellulose filters. The filters were then blocked with 5% nonfat dry milk in phosphate-buffered saline with 0.5% Tween 20 (PBST) and incubated with rabbit anti-mouse Bcl-2 serum diluted in PBST (1:1000 dilution). After extensive washing in PBST, the filters were reacted with peroxidase-labeled anti-rabbit secondary antibodies (Amersham, Little Chal- Table 1. Expression of Bcl-2 in Mouse Hepatocellular Tumors Age at DEN Promotion Number of Number of Bcl-2-positive injection by PB Tumor type tumors tumors (%) 12 days Basophilic Adenoma (100%) Adenoma 0 Adenoma 1 1 (100%) 5 weeks Basophilic Adenoma (100%) Carcinoma 4 4 (100%) Adenoma 4 0(0%) Adenoma 2 2 (100%) Carcinoma 1 1 (100%) 5 weeks + Basophilic Adenoma (100%) Adenoma 88 0 (0%) Carcinoma 7 0 (0%) Adenoma 0 Carcinoma 1 1 (100%)

3 Bcl-2 Expression in Mouse Liver Tumors 959 Normal Livers Liver Tumors Bcl-2 - (26 kda) Figure 2. Western blot study of Bcl-2. Bands of Bcl-2 at 26 kd are visible for the five liver tumors but not for three normal liver samples.. "_a a.. E^P s S~~~~~~~~, 1 Figure 1. a and b: Semiserial sections through a basophilic adenoma induced by DEN treatment at 12 days of age. a: H&E staining. Ad, basophilic adenoma; N, normal liver; BD, bile duct. Note the cytoplasmic basophilia of the adenoma cells. b: Bcl-2 staining. Adenoma cells and bile ductular cells are positively stained, whereas normal hepatocytes are negative. c and d: Serial sections through a small basophilic focus induced by DEN treatment at 12 days of age. c: H&E staining. The focus area is indicated by arrowheads. d: Bcl-2 staining. Focus cells are positive. font, UK) in PBST and again washed. Finally, the immunoreaction was visualized using the enhanced chemiluminescence system (Amersham). Results Development of Liver Tumors In mice treated with DEN at 12 days of age, 4.6 ± 4.6 (mean ± SD) tumors were observed per mouse at 25 weeks of age. Mice treated with DEN at 5 weeks and sacrificed at 55 weeks of age developed 8.2 ± 4.4 tu- Figure 3. a and b: Serial sections through an eosinophilic adenoma induced by DEN treatment at 5 weeks of age alone. a: H&E staining. Ad, adenoma; N, normal liver. Note cytoplasmic eosinophilia of adenoma cells. b: Bcl-2 staining. Both adenoma cells and normal hepatocytes are negative. c and d: Serial sections through juxtaposed eosinophilic (lower left) and basophilic (upper right ) adenomas induced by DEN treatment at 5 weeks of age followed by PB promotion. C: H&E staining. The two different types of adenomas appear to be colliding. Ad, adenoma. d: Bcl-2 staining. Only the basophilic adenoma cells are positive. mors per mouse without and 46 ± 11 with PB promotion (P < 0.01 by the Mann-Whitney U test). Expression of BcI-2 in Mouse Hepatocellular Tumors In total, 226 mouse hepatocellular tumors were examined. The results are summarized in Table 1.

4 960 Lee Among 87 tumors taken from mice treated with DEN at 12 days of age, 86 were basophilic adenomas and only 1 was an unclassifiable adenoma (Table 1). Immunohistochemically, all of the tumors were positive for Bcl-2, whereas the surrounding normal hepatocytes were negative (Table 1 and Figure 1, a and b). The staining was specifically seen in the cytoplasm, and the tumor cells did not exhibit significant heterogeneity in staining degree (Figure lb). In nontumorous portions of the livers, positive Bcl-2 staining of intrahepatic bile ductular epithelium was also noted (Figure 1b). Occasionally, small altered foci composed of basophilic hepatocytes were also found to be positive (Figure 1, c and d). To confirm the specificity of the immunohistochemical detection of Bcl-2, specimens of five liver tumors and three normallooking areas of liver tissue in their vicinity, taken from different animals, were examined by Western blot analysis. As shown in Figure 2, the Bcl-2 protein, 26 kd, was detectable only in the tumor tissues. A total of 31 tumors developing in mice given DEN at 5 weeks of age and then maintained without further treatment were examined. The 24 basophilic lesions, of which 20 were diagnosed as adenomas and 4 as carcinomas, were again consistently positive for Bcl-2 (Table 1). In contrast, the four eosinophilic adenomas in this group were all negative for Bcl-2 (Table 1 and Figure 3, a and b). Three tumors judged unclassifiable were all positive for Bcl-2. Of a total of 108 tumors examined in the DEN-PB group, 95 were eosinophilic, of which 88 were adenomas and 7 were carcinomas (Table 1). Only 12 were basophilic, with the remaining one being an unclassifiable carcinoma. All of the eosinophilic tumors were negative for Bcl-2, irrespective of their malignancy, whereas all of the basophilic tumors were again positive (Table 1 and Figure 3, c and d). Discussion The present study, to our knowledge, is the first to demonstrate that the Bcl-2 oncoprotein, which is known to suppress apoptosis,68 is expressed in mouse hepatocellular tumors. The finding that Bcl-2 is differentially expressed in basophilic and eosinophilic tumors, provides further evidence that these two types of lesions are qualitatively distinct. As the eosinophilic tumors were almost exclusively observed under PB promotion conditions, PB may selectively confer a growth advantage on this type of lesion. Interestingly, Rumsby et al reported that DEN-induced basophilic liver tumors in C3H/He mice often contain activating point mutations in the H-ras oncogene, whereas the eosinophilic tumors associated with PB promotion do not.19 This, together with the present findings, suggests that those two types of tumors develop through independent molecular mechanisms. Mouse skin papillomas initiated with a carcinogen and promoted by phorbol esters have also been shown to be qualitatively different from those induced by a carcinogen alone.20 The positive staining observed here for Bc1-2 of normal mouse bile ductular epithelium is in line with that reported for human bile ductular cells.21 In the rat liver, it is a common phenomenon that altered hepatocytes show some bile-duct-like features,22'23 and thus, the Bcl-2 expression in basophilic mouse tumors might suggest a metaplastic shift to a less differentiated phenotype. Alternatively, the basophilic tumors might originate from Bcl- 2-positive bile ductular cells rather than the negative hepatocytes, as some investigators have proposed that bile ductular cells can serve as precursors for hepatocellular tumors.22'23 It is tempting to assume that the Bcl-2 expression causes some resistance to apoptosis and contributes to promotion of basophilic tumors in B6C3F1 mice, because both cell death and cell division are important parameters with regard to the growth of tumors. During rodent hepatocarcinogenesis, apoptosis occurs as programed cell death in both preneoplastic and neoplastic hepatocellular lesions.14'24 Its inhibition by hepatopromoters may result in enhancement of hepatocarcinogenesis, whereas agents that selectively induce apoptosis in altered hepatocytes may prevent tumor formation.14'2527 However, this study provided no evidence of a relationship between Bcl-2 expression and resistance to apoptosis, and elucidation of the role of Bcl-2 in mouse hepatocarcinogenesis therefore awaits future investigations. Finally, it should be pointed out that both Bcl-2 expression and ras gene mutations are very rare phenomena in human hepatocellular carcinomas Consequently, the basophilic mouse liver tumors cannot be simply regarded as murine counterparts of human hepatocellular carcinomas. This issue deserves serious consideration, as the mouse liver model has been extensively used for experimental risk assessment of suspected human carcinogens.29 Clearly, additional studies are needed to elucidate the molecular mechanisms of mouse hepatocarcinogenesis and their relevance to the human situation. References 1. Tsujimoto Y, Finger LR, Yunis J, Nowell PC, Croce CM: Cloning of the chromosome break point of neoplastic B cells with the t(14;18) chromosome translocation. Science 1984, 226: Tsujimoto Y, Gorham J, Cossman J, Jaffe E, Croce CM: The t(14;18) chromosome translocation involved in B-cell neoplasms results from mistakes in VDJ joining. Science 1985, 229: Bakhshi A, Jensen JP, Goldman P: Cloning the chromosomal break point of t(14;18) human lymphomas: clustering around JH on chromosome 14 and near a transcriptional unit on 18. 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