Int. J. Cancer: 115, 711–716 (2005) ' 2005 Wiley-Liss, Inc.
Enzymatic detection of precursor cell populations of preneoplastic foci positive for c-Glutamyltranspeptidase in rat liver Kimihiko Satoh1*, Gen Takahashi2, Tomisato Miura1, Makoto Hayakari3 and Ichiro Hatayama4 1 Department of Organic Function, Hirosaki University, School of Health Science, Hirosaki, Japan 2 Department of Radiologic Technology, Hirosaki University, School of Health Science, Hirosaki, Japan 3 Second Department of Biochemistry, Hirosaki University, School of Medicine, Hirosaki, Japan 4 Division of Microbiology, Aomori Prefectural Institute of Health and Environment, Aomori, Japan An improved staining method for c-glutamyltranspeptidase (GGT) was developed using Vibratome-prepared microslices. Microscopic precursor cell populations of preneoplastic foci positive for the marker enzyme were detectable sequentially in rat liver by tracing back from 5 to 1 week after carcinogen injection in a hepatocarcinogenesis model. Mirror-image comparisons of serial sections stained for GGT activity and immunocytochemically stained for GST-P (glutathione S-transferase P-form) revealed that GGT expression was confined within GST-P+ cell populations (GST-P+ minifoci), which are induced in the periportal area (zone 1) of the liver. GGT expression level differed from one minifocus to another, and the larger the GST-P+ focus, the stronger was the GGT expression in it, indicating that GST-P+/GGT– phenotypes are convertible into proliferating GST-P+/GGT+ ones. Our results suggest that there are at least 2 closely related precursors, GST-P+/GGT– and GST-P+/GGT+ phenotypes, of preneoplastic foci in rat chemical hepatocarcinogenesis. ' 2005 Wiley-Liss, Inc. Key words: hepatocarcinogenesis; preneoplastic marker enzyme; g-glutamyltranspeptidase; glutathione S-transferase; activity staining; diethylnitrosamine
Only little information is available on the initiation stage of experimental chemical carcinogenesis compared to compiled data on promotion and progression stages, as superbly reviewed by Gibbs.1 Pitot2 pointed out that the molecular and cellular events in the initiation stage might be minor and latent ones, being ‘‘unknowable’’ in principle. As a matter of course, there is still a dispute whether gene mutations are ‘‘cause or effect’’ in malignant neoplasia.3 To gain insight into the knotty problems of initiation, however, preneoplastic marker enzymes, such as GST-P and GGT, might be of use rather than genetic means.4–6 In a series of studies, we previously identified single cells and minifoci heavily positive for GST-P that were inducible as early as 2–3 days after administration of DEN in the Solt-Farber protocol of rat chemical hepatocarcinogenesis.7–9 Numerous GST-Pþ single cells and minifoci were, however, induced transiently with a maximum peak after 1 week, though whether the microscopic cells are initiated cell precursors or not remains to be established.9,10 GGT has also been extensively used as a marker enzyme for preneoplastic and neoplastic cells.11–14 While the 2 markers are mostly coexpressed in foci and nodules, some of which are negative for either enzyme or both, the distinct relation between the 2 remains to be defined. In addition, the molecular and cellular mechanisms underlying the phenotypic changes of foci and nodules, such as reversion or remodeling, also remain to be identified.15,16 Taking into account the intricate phenomenon of initiation,1,2,6 it must be significant to detect precursor cell species positive for the GGT marker enzyme, similar to the importance of GST-Pþ single cells and minifoci.8–10 We thus improved the GGT activity staining method of Rutenburg et al.17 and show here using the enzymatic approach the presence of GST-Pþ/GGTþ precursor species of preneoplastic foci in rat liver induced in the early stage of chemical hepatocarcinogenesis.
Material and methods Chemicals GMNA was purchased from Sigma-Aldrich (Tokyo, Japan). Anti-GST-P antibody (rabbit) was obtained from Medical and Biologic Laboratories (Nagoya, Japan). Peroxidase-conjugated antirabbit IgG (goat; Envision, labeled polymer, HRP, antirabbit) was purchased from Dako (Kyoto, Japan). Animals Sprague-Dawley male rats (5 weeks old) were purchased from Clea (Tokyo, Japan) and maintained in the Institute for Animal Experiments of Hirosaki University. All animal experiments were conducted according to the guidelines for animal experimentation of Hirosaki University. GGT enzyme activity staining method Rats were killed at appropriate time points, and the right liver lobes were excised and cut into 3–4 mm thick slices, followed by fixation with cold acetone overnight or longer. Liver slices were sectioned in PBS in 25 mm thick sections using a microslicer (Vibratome 1500 sectioning system; Vibratome, New York, NY). GGT activity staining was performed according to the method of Rutenburg et al.17 Microslices were incubated in substrate solution containing 0.57 mM GMNA, 3.3 mM glycylglycine, 93 mM NaCl and 0.52 mM fast blue BB salt in 22 mM Tris-HCl (pH 7.6) for 15–20 min at room temperature under occasional swirling. The azo dye coupling reaction was completed by incubation with 0.1 M CuSO4 for 2 min, followed by washing with saline. Immunocytochemical staining of GST-P Immunocytochemical staining of liver microslices was performed with polyclonal GST-P antibody (rabbit) and peroxidaseconjugated antirabbit IgG (goat) as the first and second antibodies, respectively, with 3,30 -diaminobenzidine as substrate.10 Quantitation of foci and minifoci Color images of microslices were incorporated into Photoshop Elements 2.0 by a scanner (Epson ES-2000; Seiko-Epson, Tokyo, Japan) at a resolution of 600 dpi. Microphotographs were taken Abbreviations: AAF, N-2-acetylaminofluorene; BD, bile duct; DEN, N,N0 -diethylnitrosamine; GGT, g-glutamyltranspeptidase; GMNA, L-glutamic acid g-(4-methoxy-2-naphthylamide); GPE1, GST-P enhancer 1; GSH, reduced glutathione; GST-P, glutathione S-transferase P-form; HRP, horseradish peroxidase; Nrf2, NF-E2 p45-related factor 2; PH, partial hepatectomy. Grant sponsor: Ministry of Education, Culture, Sports, Science and Technology of Japan; Grant sponsor: Intelligent Cosmos Academic Foundation; Grant sponsor: Nippon Boehringer-Ingelheim. *Correspondence to: Department of Organic Function, Hirosaki University, School of Health Science, Hon-Cho 66-1, Hirosaki 036-8564, Japan. Fax: þ81-172-39-5921. E-mail:
[email protected] Received 1 September 2004; Accepted after revision 3 December 2004 DOI 10.1002/ijc.20979 Published online 23 February 2005 in Wiley InterScience (www.interscience. wiley.com).
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FIGURE 1 – (a) Solt-Farber protocol for induction of preneoplastic foci and nodules in rat liver. B.D., basal diet; AAF, basal diet containing AAF (0.02%); PH, two-thirds partial hepatectomy; W, weeks; d, days. Arrows indicate time of death. (b) Areas (mm2/cm2) of GST-Pþ and GGTþ foci induced after PH.18 Data are means of duplicate samples.
within 10–20 sec of exposure to strong light, to avoid drying up and deformation of specimens, by a microscope (Axioskop, Carl Zeiss, Oberkochen, Germany) equipped with a digital camera. The number, area and density of GST-Pþ and GGTþ cell populations were measured with the NIH (Bethesda, MD) image analysis software (version 1.63). Measurement of cell composition as well as size classification were also performed using the software, as reported.10 The correction factor for extension of microslices after GST-P staining was 0.94 6 0.04 (mean 6 SD). Results Macroscopic search for GGTþ precursor cell populations in rat liver Preneoplastic foci and nodules are inducible in rat liver within a short period of 5 weeks according to the Solt-Farber protocol,7 shown schematically in Figure 1a. DEN was administered i.p. in a single dose of 200 mg/kg body weight, followed by application of selection pressure feeding of a basal diet containing 0.02% AAF 2 weeks later combined with PH performed at 3 weeks. Based on the marked growth of GST-Pþ and GGTþ foci after PH (Fig. 1b), the induction process was tentatively divided into latent and logarithmic growth phases. In search of GGTþ precursor cell populations, we traced back GGTþ cell populations sequentially from 5 weeks to 1 week after DEN administration. Daily changes after PH were examined. A number of GST-Pþ and GGTþ foci and nodules were detected at 5 weeks (Fig. 2a), which rapidly and progressively diminished in size and density from 5 weeks to 3 or 2 days after PH (Fig. 2d,e). GST-Pþ minifoci remained visible by the naked eye even at 2 weeks after DEN administration (Fig. 2h), while GGTþ minifoci were discernible only at 2 or 3 days after PH. Microscopic cell populations detected at logarithmic growth phase GST-Pþ and GGTþ cell populations were detectable sequentially after PH (Fig. 3). While mirror images of GST-Pþ and
FIGURE 2 – Macroscopic observation of GST-Pþ and GGTþ cell populations induced in rat liver. Liver microslices were stained for GGT activity (GGT stain) and immunocytochemically for GST-P (GST-P stain).
GGTþ foci appeared to correspond fairly well with each other (Fig. 3a–c), microscopic GGTþ precursor species such as l0 , m0 , u0 and x0 (Fig. 3d,e) were much smaller than the corresponding GSTPþ ones, l, m, u and x; i.e., the degree of GGT expression was different from one minifocus to another, in both activity and area. These precursors were inducible in the periportal area of liver; e.g., u0 /u is directly linked to the BD (Fig. 3e). The relative areas (%) [(GGTþ focus area/GST-Pþ focus area) 100] of larger foci were a0 /a (96.5%), b0 /b (99.5%), g0 /g (105%), d0 /d (101%), e0 /e (74.5%), z0 /z (68.5%), Z0 /Z (90.0%), y0 /y (78.6%) and those of smaller foci were l0 /l (47.0%), m0 /m (15.9%), u0 /u (51.9%) and x0 /x (51.6%). Single cells and minifoci were GGT– (0%). Thus, GGT expression was dependent on the size of the GST-Pþ focus. Microscopic cell populations detected at latent phase Microscopically, GGTþ precursor species were also detected together with GST-Pþ foci at weeks 3, 2 and 1 after DEN administration. The pattern of GGT (and GGTþ cell) expression within GST-Pþ minifoci was much more prominent at the latent phase
GGTþ PRECURSOR CELLS OF PRENEOPLASTIC FOCI
FIGURE 3 – Mirror-image comparison of GST-Pþ and GGTþ precursor cells induced in rat liver at logarithmic phase. Corresponding mirror images were inverted horizontally, where mirror images are the inner sides of 2 serial sections. Values in parentheses denote cell composition. CV, central vein; BD, bile duct or bile ductules. Greek letters (non- and primed ones) represent corresponding GST-Pþ and GGTþ (mini-)foci (arrows).
than at the logarithmic phase. For example, 34% cells were strongly positive for GGT (o0 ) within a GST-Pþ minifocus (o) composed of 35.9 cells (Fig. 4a). In this example, GGT was expressed in the bile canaliculi-like spaces of particular cells. Furthermore, 35% of cells were also strongly positive for GGT (p0 /p), whereby p0 was also directly linked to the BD (Fig. 4b). Both areas showed heterogeneous rather than focal GGT expression. Similarly, 43% of cells were positive for GGT among 8.4 cells (r0 /r) (Fig. 4c). Approximately one cell was GGTþ among 22.9 cells
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FIGURE 4 – Expression of GGT within GST-Pþ minifoci at latent phase. Values in parentheses denote cell composition and area (%) of GGTþ cells against GST-Pþ ones. CV, central vein; BD, bile duct or bile ductules.
(s0 /s) (Fig. 4d). t0 /t is located at the end of bile ductules (Fig. 4e). Despite the nonspecific induction of GGT in the bile ductules at 1 week, GGTþ precursor species were identifiable since GST-P was almost negative in the corresponding BDs and ductules (Fig. 4e). u0 /u is a typical GGT– minifocus (Fig. 4f). While GGT was weakly positive in the BD epithelium of control animal liver, it was markedly inducible in BDs and ductules at the portal area in response to administration of DEN after 1 week. Staining of GGT cells at 1 or 2 weeks was so weak that the developed color faded out within 1–2 days, while staining of large foci was so strong that the color was stable even after 1 month when stored in PBS at room temperature. These findings indicate that
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the amount of GGT enzyme expressed in precursor cell populations at the latent phase is
