Sequential Alteration of Apoptosis, p53 Expression, and Cell

Toxicologic Pathology, 31:625–631, 2003 C by the Society of Toxicologic Pathology Copyright ISSN: 0192-6233 print / 1533-1601 online DOI: 10.1080/01926230390241855

Sequential Alteration of Apoptosis, p53 Expression, and Cell Proliferation in the Rat Pancreas Treated with 4-Hydroxyaminoquinoline 1-Oxide TAKAYOSHI IMAZAWA,1 AKIYOSHI NISHIKAWA,1 KAZUHIRO TOYODA,2 FUMIO FURUKAWA,2 MASAYUKI MITSUI,1 AND MASAO HIROSE1 1

Division of Pathology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan and 2 Present address: Japan Tobacco Co, Ltd, 2-2-1 Toranomon, Minato-ku, Tokyo 105-8422, Japan ABSTRACT

Changes in p53 expression, apoptosis and cell proliferation after treatment with 4-hydroxyaminoquinoline 1-oxide (4HAQO) were investigated in the rat pancreas and liver, target and nontarget organs for tumorigenesis, respectively. Male rats were given a single intravenous injection of 4HAQO at a dose of 20 mg/kg body weight and control rats received vehicle alone and were euthanized after 2–72 hours. Pancreata and livers were removed for histopathological examination, immunohistochemistry for p53 protein, PCNA and Ki-67, and TUNEL labeling and electron microscopic observation for detecting apoptosis. In the pancreas, p53 expression and apoptosis were significantly increased first at 4 and 6 hours, respectively, while no change was evident in the liver. The rates peaked at 24 hours, consistent with the peak for PCNA-labeling, while Ki-67-labeling rates peaked at 72 hours. Electron microscopically, apoptotic changes in pancreatic acinar cells were observed after 2 hours. No significant apoptosis, p53 expression or cell proliferation were noted in the pancreatic tissues of the control rats nor in liver cells regardless of 4HAQO treatment. Taken together with our previous data, the results suggest that apoptosis, p53 expression, and enhanced cell replication are closely related phenomena involved in the carcinogenesis of 4HAQO following DNA adduct formation. Keywords. p53 protein; apoptosis; cell proliferation; 4-hydroxyaminoquinoline 1-oxide; rat; pancreas.

INTRODUCTION It is generally accepted that covalent binding between an ultimate carcinogen and DNA is of prime importance for early steps of tumor initiation (26). 4-Nitroquinoline 1-oxide (4NQO) is a heterocyclic aromatic compound of which mutagenicity and carcinogenicity have been extensively studied (36). 4-Hydroxyaminoquinoline 1-oxide (4HAQO), a reactive oxidative metabolite of 4NQO, is known as a potent carcinogen with clear organotrophism and species specificity (21, 26, 30). It has been well documented that a single intravenous administration of 4HAQO preferentially induces pancreatic acinar cell tumors in rats (14, 15, 23, 31) and its repeated injection results in the development of islet cell tumors (18). Extensive studies on the positive interaction of 4HAQO or 4NQO with DNA have been carried out using bacteria, transplantable ascites tumor cells, cultured mammalian cells and DNA itself (16, 28, 33), and it has been shown that guanine and adenine adducts are produced (6, 37). In our previous study (14), DNA adduct formation was consistently demonstrated in the target tissue in rats receiving a single injection of 20 mg/kg body weight of 4HAQO. The immunohistochemical staining pattern of 4HAQO-DNA adducts differed according to anatomic location and cell type. The pancreatic acinar cells were clearly positive, but not cells of the liver, which is a nontarget organ. Almost simultaneously or a little later an ultrastructural change, nucleolar segregation, was noted in pancreatic acinar cells.

The tumor suppressor gene p53 plays an important role in the control of cell proliferation, induction of programmed cell death (apoptosis), and cell differentiation (4). Mutations in the p53 gene, which are often of missense type, result in a single amino acid change, and the resultant abnormal protein may exhibit a prolonged half-life (13, 24). The accumulated abnormal protein is easily detectable by immunohistochemistry and there appears to be a good correlation between overexpression of p53 protein and p53 gene mutation (2). p53 has been shown to induce apoptosis when overexpressed (1, 27). Apoptosis is an important mechanism of negative selection that removes damaged cells deleterious to the host, serving as an important defense against cancer development (40). Proliferating cell nuclear antigen (PCNA) is closely involved in DNA replication and repair, in addition to increasing the efficiency of DNA synthesis. PCNA is also readily detected simultaneously with p53 expression after genotoxic insult (3, 11). p53 may directly control DNA replication and repair by modulating the levels of PCNA. In addition to the essential roles of PCNA in DNA metabolism, its interactions with cellular regulatory proteins might establish a pathway through which p53 influences multiple cellular activities. The present study was conducted to concomitantly examine the induction of p53 protein, apoptosis and PCNA in the pancreatic tissue of rats given a single injection of 4HAQO. Furthermore, to clarify changes in cell cycle/DNA replication of tissues the expression of proliferation-associated nuclear antigen Ki-67 was also evaluated because Ki-67 reacts with a nuclear antigen present throughout the proliferative phases of the cell cycle (G1, G2, and M phases) but is absent in quiescent (G0) cells (5, 8). For comparison, the liver was selected as a no-ntarget organ for 4HAQO carcinogenicity. In order to confirm ultrastructural changes such as apoptosis and

Address correspondence to: Dr. Takayoshi Imazawa, Division of Pathology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan; e-mail: [email protected]

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nucleolar segregation, electron microscopical examination was also applied. MATERIALS AND METHODS Animals The protocol for this study was approved by the Animal Care and Utilization Committee of the National Institute Health Sciences. Male 6-week-old Sprague-Dawley rats (Sankyo Laboratory, Tokyo, Japan), initially weighing approximately 160 g, were used in the experiment. Rats were housed, 4 per wire cage, in an air-conditioned room maintained at 24 ± 2◦ C and 55 ± 5% relative humidity under a daily cycle of alternating 12-hour periods of light and darkness. The animals were given commercial standard diet (CRF-1: Charles River Inc, Tokyo) and tap water ad libitum. Experimental Design After a 1-week acclimatization period, 42 rats received a single injection of 4HAQO (Iwai Chemicals, Tokyo) dissolved in 0.005 N hydrochloric acid into the saphenous vein at a dose of 20 mg/kg body weight in 0.3 ml of solution. Groups of 6 rats each were sacrificed under ether anesthesia at 2, 4, 6, 8, 24, 48, and 72 hours after injection. Thirty control rats received a single intravenous injection of acidic aqueous solution alone, and 6 each were sacrificed at 2, 6, 24, 48, and 72 hours thereafter. At necropsy, samples of the pancreas and liver were immediately excised from each rat. Immunohistochemistry for p53 Protein, PCNA, and Ki-67 Tissue specimens were fixed in a 10% buffered formalin solution for 24 hours at room temperature, routinely processed, embedded in paraffin, and then cut at 4 µm. For immunohistochemical staining, all tissues were heated by microwave in distilled water or autoclave in citrate buffer for antigen retrieval (8, 34), and endogenous peroxidase was blocked by exposure to an aqueous solution of 1% peroxidic acid for 15 minutes. Sections were then rinsed in phosphated-buffered saline (PBS; pH 7.2), and examined with antibodies against each antigen using the ABC method (Vectastain ABC Elite kit; Vector Lab, Burlingame, CA). Wild-type and mutant reactive anti-p53 rabbit polyclonal antibody (CM-5; Novocastra Lab, Newcastle, UK) was used at a dilution of 1:500; and anti-PCNA and anti-Ki-67 mouse monoclonal antibodies (DAKO Japan, Kyoto, Japan) at dilution of 1:400 and 1:50, respectively. Incubation with the primary antibody was performed at 4◦ C, overnight. Binding was visualized by reaction of the enzyme complex with 0.05% 3,3 -diaminobenzidinetetrachloride (DAB; Dojindo Lab, Kumamoto, Japan) plus 0.01% hydrogen peroxide in Tris-buffered saline containing 10 mM sodium azide. The sections were subsequently counterstained with hematoxylin, dehydrated through graded alcohol and xylene, and mounted for microscopic examination. Terminal Deoxynucleotidyl Transferase-Mediated X-dUTP Nick-End Labeling (TUNEL) for Apoptosis The TUNEL method was performed as described previously (7). Tissue sections were mounted on coated slides, deparaffinized, hydrated, and treated for 20 minutes at 37◦ C with proteinase-K (pH 7.4; Boehringer Mannheim Co, Mannheim, Germany) followed by 3 washes with distilled

TOXICOLOGIC PATHOLOGY

water. The sections were treated with 3% hydrogen peroxide for 5 minutes to suppress endogenous peroxidase activity, and washed in distilled water for 10 minutes. After incubation for 10 minutes in TdT buffer (140 mM sodium cacodylate, 5 mM CoCl2 , and 30 mM Tris-HCl buffer, pH 7.2) at room temperature, terminal transferase (Boehringer Mannheim Co) and biotinylated dUTP (Boehringer Mannheim Co) were added in fresh TdT buffer followed by incubation at 37◦ C in a humidified chamber for 60 minutes. The sections were then subjected to 3 washes with PBS and incubated with TB buffer (300 mM NaCl and 30 mM sodium citrate) for 30 minutes at room temperature. Nonspecific background staining was reduced by incubation for 10 minutes at room temperature with PBS containing 1% bovine serum albuminutes (PBS-BSA), and the sections were then incubated with the peroxidase-conjugated avidin-biotin complex for 5 minutes at room temperature in a humidified chamber. The sections were washed twice for 5 minutes in PBS and incubated with DAB solution for 5 minutes at room temperature, then washed for 10 minutes in distilled water. They were subsequently counterstained with hematoxylin, dehydrated through graded alcohol and xylene, and mounted for microscopic examination. Electron Microscopic Examination Small specimens of tissues were fixed in phosphatebuffered 2.5% glutaraldehyde for 2 hours at 4◦ C, postfixed in phosphate-buffered 1.5% osmium tetroxide for 60 minutes, dehydrated through a series of alcohols, and embedded in Epon 812. Ultrathin sections were stained with uranyl acetate and lead citrate, and examined under a transmission electron microscope (JEM 100CXS, JEOL, Tokyo). Quantitative Analysis and Statistics For counting the numbers of cells positive for p53, TUNEL, PCNA, and Ki-67, approximately 300 cells were randomly selected from 4 different areas in each section and examined under a light microscope at a magnification of X400. Data were statistically evaluated using the Student’s t-test. RESULTS Representative histological, immunohistochemical and TUNEL findings are shown in Figure 1 (A–H). Except for single cell death frequently found in pancreatic acinar cells, no histopathological lesions were seen in the 4HAQO-treated rats (Figure 1B). Using the CM-5 polyclonal antibody, p53 protein was clearly detectable in nuclei of pancreatic acinar cells after 4HAQO-treatment (Figure 1D), whereas in hepatocytes, or pancreatic acinar cells without carcinogen treatment, no nuclear p53 expression was observed (Figure 1C). In the 4HAQO-treated rats, nuclei of apoptotic cells in the pancreatic acini showed strong TUNEL staining (Figure 1F) but few TUNEL-positive hepatocytes were found. Similarly, the pancreas without carcinogen treatment demonstrated only few apoptotic cells (Figure 1E). The results of sequential quantitation of cells expressing p53 and apoptosis in response to 4HAQO are summarized in Figures 2 and 3, respectively. Stainings of p53 and apoptotic cells were already noted in acinar cells 2 hours after injection of 4HAQO and significantly ( p < 0.05) increased after 4 and 6 hours, respectively. Both indices showed the highest frequency 24 hours after the

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FIGURE 1.—Histopathology of the pancreas. (A) Normal pancreatic acinar cells in a control rat. (H&E), ×240. (B) Pancreatic acinar cells showing frequent apoptotic cell death and nuclear condensation, 24 hours after treatment with 4HAQO. (H&E), ×240. (C) No evidence of p53 immunoreactivity in pancreatic acinar cells of a control rat, ×240. (D) p53 immunoreactivity is evident in pancreatic acinar cells 24 hours after the treatment with 4HAQO, ×240. (E) A few acinar cells positive for TUNEL staining in a control rat, ×240. (F) 24 hours after the treatment with 4HAQO, many apoptotic cells show strong TUNEL staining, ×240. (G) Infrequent PCNA immunoreactivity in pancreatic acinar cells of a control rat, ×180. (H) 24 hours after the treatment with 4HAQO, acinar cells show frequent PCNA immunoreactivity, ×240. (I) Infrequent Ki-67 immunoreactivity in pancreatic acinar cells of a control rat, ×240. (J) 72 hours after the treatment with 4HAQO, acinar cells show frequent Ki-67 immunoreactivity, ×240.

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FIGURE 2.—Summary of sequential changes in p53 expression in the exocrine pancreas of rats treated with 4HAQO and control rats. The values for the treatment group at the 4- and 8-hour time points were compared with the average control values throughout 2-, 6-, 24-, 48-, and 72-hour time points. Data are mean ± standard deviation values. Significant at ∗ p < 0.05, ∗∗ p < 0.01 by Student’s t-test; n = 6.

FIGURE 4.—PCNA indices for pancreatic acinar cells of rats treated with 4HAQO and controls. Data are mean ± standard deviation values. Significant at ∗ p < 0.05, ∗∗ p < 0.01 by Student’s t-test.

carcinogen injection with values of about 12% and 11% , respectively, but they were only about 0.1% in the respective controls. Appreciable numbers of pancreatic acinar cells exhibited strong staining for PCNA and Ki-67 in the 4HAQO-treated rats (Figure 1H and 1J), whereas in control rats the positive rate was constantly very low (Figure 1G and 1I). As compared with the respective control groups, PCNA labeling was significantly ( p < 0.05) elevated in pancreatic acinar cells 6 hours after carcinogen treatment (Figure 4). The highest rate (about 8%) in pancreatic acinar cells was noted at 24 hours. PCNA levels persisted at the higher levels through 6–72 hours after the treatment with 4HAQO. On the other hand, Ki-67 levels showed a tendency to decrease at 6 and 24 hours, but significantly ( p < 0.05) increased after 72 hours, the value being

about 4% (Figure 5). No increase in cell proliferation activity was seen in the hepatocytes of 4HAQO-treated rats (Figures 6 and 7). Electron microscopically, the numbers of zymogen granules in the affected cells were markedly decreased at 24 hours and degenerative and apoptotic changes of acinar cells were evident in addition to necrosis at 48 hours. Starting at 2 hours after injection of 4HAQO, acinar cells showing ultrastructural features of apoptosis characterized by condensation and margination of chromatin against the nuclear membrane were consistently increased, some demonstrating a possible conglomeration of interchromatin particles (Figure 8). Such nuclear alterations were not seen in hepatocytes at any time after 4HAQO-treatment. Furthermore, nucleolar alteration, with segregation of nucleolar components into granular and fibrillar compartments, was observed in cells of the target organ exocrine pancreas at 2 hours after injection of 4HAQO, but not in the non-target liver parenchyma (data not shown).

FIGURE 3.—Summary of sequential changes in numbers of apoptotic positive cells by TUNEL method in the exocrine pancreas of rats treated with 4HAQO and in controls. The values in the treatment group at 4- and 8-hour time points were compared with the average control values throughout the 2-, 6-, 24-, 48-, and 72-hour time points. Data are mean ± standard deviation values. Significant at ∗ p < 0.05, ∗∗ p < 0.01 by Student’s t-test.

FIGURE 5.—Ki-67 indices for pancreatic acinar cells of rats treated with 4HAQO and controls. Data are mean ± standard deviation values. Significant at ∗ p < 0.05 by Student’s t-test.

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FIGURE 6.—PCNA indices for liver cells of rats treated with 4HAQO and controls. Data are mean ± standard deviation values.

DISCUSSION In the present study, expression of p53 protein was slightly increased within 2 hours after 4HAQO treatment. An important physiological function of p53 seems to be regulation of cell cycle by blockage in the G1 phase after induction of DNA damage, which allows the cell to repair its DNA damage or trigger apoptosis (21, 25, 29). Under physiological circumstances, the wild-type p53 protein has a very short half-life and is present in such small quantities as to be immunohistochemically undetectable (9, 12). There are different mechanisms that lead to accumulation of the p53 protein up to immunohistochemically detectable levels. DNA damage gives rise to a temporary accumulation of wild-type p53 protein resulting in an arrest of cell cycle assumed to prevent replication of damaged DNA (19). Missense mutation in the p53 gene in general leads to a dramatic increase in the half-life of the p53 protein (12). In contrast to transient accumulation of wild-type p53, mutation can lead to constitutively high p53 levels in the cell. Although CM-5 detects both wildtype and mutated p53, the nuclear staining seen in the acute phase probably indicates an increased amount of wild-type rather than mutated p53, since the peak of apoptosis was accompanied by positive immunohistochemistry for p53. Such

FIGURE 7.—Ki-67 indices for liver cells of rats treated with 4HAQO and controls. Data are mean ± standard deviation values.

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detectable and putatively wild-type p53 may be either due to increased mRNA levels as a response to chemical damage or through induction of a cellular protein which binds to p53 and consequently increases its half-life. Hainaut and Milner (10) have found that oxidizing agents can convert p53 from a wild-type to a mutant conformation and increase its stability at the same time. It is possible that certain carcinogenic agents may exert some of their transforming effects by inducing a transient conformation shift in p53 from a suppressing to a mutant phenotype. Carcinogenesis involves an imbalance between regulations of cell proliferation and apoptotic cell death. We therefore quantitated the sequential changes occurring in both parameters after 4HAQO-treatment in the rat pancreas. Apoptosis is an essential and important part of normal tissue development (32), as well as a defense against the development of cancer (40). Accumulation of wild-type p53 protein levels is associated with increased levels of apoptosis, which may arise from conflicting growth signals (41). The results of the present study show that 4HAQO induces p53 expression and almost simultaneously brings about apoptosis and increased PCNA expression in the pancreatic acinar cells. Most nongrowing cells contain little PCNA mRNA and protein (20). Thus, activated synthesis is necessary for the purpose of DNA repair after genotoxic insult in populations of nongrowing cells. PCNA is well established as a useful marker for replication of DNA (39). In the present study, PCNA rates were markedly elevated in pancreatic acini of 4HAQO-treated rats as compared with control rats, the peak being coincident with positive immunohistochemistry for p53 at 24 hours. It has been reported that p53 may induce PCNA expression (35). In contrast, no significant alterations of apoptosis, p53 expression or cell proliferation were noted in the tissues of control rats. The fact that Ki-67 index was significantly increased 72 hours after the carcinogen treatment, indicates that cell proliferation was elevated in pancreatic acinar cells. Since PCNA expression is associated with cell proliferation as well as DNA synthesis and repair (20, 38), increased PCNA-positive before 48 hours may have resulted from DNA damage. Thus, the immunohistochemical approach allowed comparative morphological analysis. Electron microscopically, apoptotic changes including nucleolar alteration in acinar cells were already evident at 2 hours after injection of 4HAQO in the present study. Segregation of nucleolar components into granular and fibrillar compartments was evident in cells of the target organ (exocrine pancreas) but not of the nontarget hepatocytes. As we have previously reported, DNA adduct formation and nucleolar alteration can be consistently demonstrated by immunohistochemical methods using antibodies directed against 4HAQO-DNA adducts and electron microscopy for segregation of nucleolar components in the target organs of rats given a single injection of 4HAQO (17). In addition, sequential observation clarified that such alterations were highest in frequency 6 hours after 4HAQO administration in pancreatic acinar cells (17). In conclusion, the results of the present study suggest that p53 overexpression, apoptosis, and enhanced cell replication are closely related phenomena involved in the early phase of 4HAQO carcinogenesis following DNA damage.

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FIGURE 8.—Condensation of chromatin in a pancreatic acinar cell nucleus, indicative of the first stage of apoptosis, in a rat 8 hours after 4HAQO administration. Note sharply delineated dense and lucent areas. Bar = 2 µm.

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