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Expressions of transforming growth factor (TGF)-β1 and TGF-β type II receptor and their relationship with apoptosis during chemical hepatocarcinogenesis in rats
Hepatology Research 27 (2003) 205–213
Expressions of transforming growth factor (TGF)-1 and TGF- type II receptor and their relationship with apoptosis during chemical hepatocarcinogenesis in rats Do Youn Park a , Mee Young Sol a,∗ , Kang Suek Suh a , Eui-Cheol Shin b , Chul Hoon Kim c a
Department of Pathology, College of Medicine Pusan National University, Ami-1 Ga, Seo-Gu, Busan 602-739, South Korea b Department of Microbiology, Yonsei University College of Medicine, Seoul, South Korea c Department of Pharmacology, Yonsei University College of Medicine, Seoul, South Korea Received 14 January 2003; received in revised form 2 June 2003; accepted 17 June 2003
Abstract The expression of transforming growth factor (TGF)-1, which regulates cell proliferation, is tightly associated with that of TGF- type II receptor (TGR2), and has been regarded as an important change during hepatocarcinogenesis. Our aim in this study was to investigate the expression and localization of TGF-1 and TGR2 and to determine their relationships with apoptosis in chemical hepatocarcinogenesis of the rat produced by Solt and Farber’s method. Northern blot analysis showed that a slight increase of TGF-1 transcripts and a decrease of TGR2 transcripts during hepatocarcinogenesis. Immunohistochemistry revealed that TGF-1-positive preneoplastic hepatocytes increased with time, and that this correlated with a reduction TGR2 expressing preneoplastic lesions. Hepatocellular carcinoma (HCC) tissues showed higher levels of TGF-1 transcripts and protein and lower levels of TGR2 transcripts and protein compared to the paired adjacent liver parenchyme. TUNEL revealed that apoptotic cells increased with time and were more numerous in the adjacent liver parenchyme than in preneoplastic lesions and HCC tissues. Our data suggest that the down regulation of TGR2 in preneoplastic lesions and HCC tissues might contribute to resistance to the growth inhibitory effects of TGF-1, and to the roles of TGF-1 in the development and progression of preneoplastic lesions and HCC in a chemically induced rat hepatocarcinogensis model. © 2003 Elsevier B.V. All rights reserved. Keywords: TGF-1; TGF- type II receptor; Northern blot; Immunohistochemistry; Apoptosis; Chemical hepatocarcinogenesis; Rat
1. Introduction Transforming growth factor (TGF)-1 is a multipotent polypeptide, which inhibits the growth of epithelial cells, including hepatoma cell lines, hepatocytes and oval cells by inducing apoptosis [1–3]. Many reports have suggested that the ability of TGF-1 to control hepatocyte proliferation depends, in part, on the cellular concentration of various TGF- receptors [4,5], and to date, five such receptors have been identified. The type II serine/threonine kinase receptor forms a heterodimeric complex upon binding to TGF-, and generates the first step in the signal transduction pathway leading to growth inhibition in coordination ∗ Corresponding author. Tel.: +82-51-240-7717; fax: +82-51-253-7319. E-mail address: [email protected] (M.Y. Sol).
1386-6346/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S1386-6346(03)00264-X
with type I receptor [6,7]. In humans, elevated levels of TGF-1 mRNA have been reported in hepatocellular carcinoma (HCC), and TGF-1 was also found to be increased in the plasma of HCC patients [8,9]. Furthermore, the loss of responsiveness of hepatocellular carcinoma cells to TGF-1-mediated growth inhibition has been implicated in hepatocarcinogenesis [10]. Experimental hepatocarcinogenesis in rats occurs in distinctly defined stages, that is, initiation, promotion, and progression. The resistant hepatocyte model that was developed by Solt et al. [11] and Farber and Sarma [12] has been widely used in the study of multistep chemical hepatocarcinogenesis, in which the balance between cell death (apoptosis) and cell proliferation is a major determinant [13]. In chemical hepatocarcinogenesis, apoptosis plays a major role in each of these three stages. However, the temporal expressions and cellular localizations of TGF-1 and
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the TGF- type II receptor (TGR2) and their relationships with apoptosis in chemical hepatocarcinogenesis have not been clearly elucidated. The purpose of this study was to investigate the temporal expression and cellular localization of TGF-1 and TGR2 and their relationships with apoptosis during chemical hepatocarcinogenesis, especially during the stages of progression and HCC.
2. Materials and methods 2.1. Induction of chemical hepatocarcinogenesis in rats Diethylnitrosamine (DEN), 2-actetylaminofluorene (2AAF), carboxymethylcellulose and dimethylsulfoxide were obtained the Sigma Chemical Co. (St. Louis, MO, USA). 2-AAF was dissolved in a small volume of dimethylsulfoxide and suspended in 1% aqueous solution of carboxymethylcellulose to a final concentration of 1.5 mg/ml. Male Sprague–Dawley rats, weighing 200 gm, were maintained on standard pelleted chow and had access to water ad libitum. Solt and Farber’s method was used to induce hepatocarcinogenesis. Briefly, rats were initiated with a single dose (200 mg/kg) of DEN administered intraperitoneally by injection. Two weeks after initiation, all rats received a daily oral gavage of 10 mg/kg of 2-AAF for a period of up to 14 days. A partial hepatectomy (PH) was performed 1 week after starting the 2-AAF treatment. Five animals were sacrificed as indicated at 1 and 2 months after PH, that is during the promotion stage, at 6 and 8 months after PH, as progression stages, and 10 animals at 12 months after PH, as HCC stage. Two animals were sacrificed as an age-matched sham-operated control, which did not receive AAF or DEN treatment. After gross examination of each liver sample, a portion liver was fixed in 10% neutral buffered formalin solution and routinely processed for hematoxylin and eosin and immunohistochemical staining. The remainder of each sample was frozen in liquid nitrogen and kept at −70 ◦ C until required for RNA isolation. All experiments were conducted in accord with the institutional review committee’s guidelines. 2.2. RNA isolation, probe synthesis and Northern blot analysis Hybridization probes for Northern blot analysis were created by reverse transcription polymerase chain reaction (RT-PCR) amplification. Total RNA was isolated from each liver sample using an RNeasy kit (Qiagen, Santa Clarita, CA) under manufacturer’s instructions, which uses a silica-gel based membrane and guanidine isothiocyanate. cDNA was synthesized from 5 g of total RNA using 2 g of random hexamer (Pharmacia, Uppsala, Sweden), 10 mM dNTP (Boehringer Mannheim, Mannheim, Germany) and 200 U of M-MLV reverse transcriptase (GIBCO BRL) in a final volume of 25 l. PCR was performed using 1.25 mM
dNTP, 0.25 U of Taq polymerase (Perkin-Elmer, Branchburg, NJ), 10 pmol of the primer pairs compatible with rat TGF-1, TGR2, glutathione S-transferase placental form (GST-P), or glyceraldehydes-3-phosphate dehydrogenase (GAPDH), and cDNA using a thermal cycler (Perkin-Elmer, Branchburg, NJ). The following primer pairs were used: TGF-1
TGR2 GST-P GAPDH
5 -GAGAGCCCTGGATACCAACTACTG-3 and 5 -GTGTGTCCAGGCTCCAAATGTAG-3 5 GCTTCACTCTGGAAGATGCC-3 and 5 -AAGGAGTGTGGTCACTGTGC-3 5 -AAGTTTGAAGATGGAGACCT-3 and 5 -GATAGTTAGTGTAGATGAGGG-3 5 -ACCACAGTCCATGCCATCAC-3 and 5 CCACCACCCTGTTGCTGTA-3
The PCR cycling conditions used for TGF-1 were, 25 cycles of; denaturation at 94 ◦ C for 50 s, annealing at 55 ◦ C for 30 s, and extension at 72 ◦ C for 2 min, with a final extension at 72 ◦ C for 10 min, with a predicted product size of 173 bp. For TGR2 these were, 28 cycles of; denaturation at 94 ◦ C for 1 min, annealing at 60 ◦ C for 1 min, and extension at 72 ◦ C for 1 min, with a final extension at 72 ◦ C for 10 min, with a predicted product size of 774 bp. For GST-P were, 25 cycles of; denaturation at 94 ◦ C for 1 min, annealing at 57 ◦ C for 1 min, and extension at 72 ◦ C for 1 min, with a final extension at 72 ◦ C for 10 min, with a predicted product size of 167 bp, and finally for GAPDH were, 25 cycles of; denaturation at 94 ◦ C for 1 min, annealing at 57 ◦ C for 1 min, and extension at 72 ◦ C for 2 min, with a final extension at 72 ◦ C for 10 min, with predicted product size of 452 bp. Amplified PCR products were analyzed on 1.5% agarose gel and extracted using a QIAquick gel extraction kit (Qiagen, Santa Clarita, CA). PCR products were sequenced and labeled with [32 P]-dCTP by the random priming method using a High Prime random labeling kit (Boehringer Mannheim, Mannheim, Germany). Labeled probes were purified using a QIAquick nucleotide removal kit (Qiagen, Santa Clarita, CA). Total RNA was separated on denaturating formaldehyde 1.0% agarose gels (20 g total RNA per lane), and transferred to a Hybond (+) nylon membrane (Boehringer Mannheim, Mannheim, Germany) using the capillary transfer method. The RNAs were then cross-linked to the membrane by using a UV cross-linker and baked in an oven at 80 ◦ C for 1 h. The RNAs were hybridized with specific probes in an ExpressHyb hybridization solution (Clontech, Palo Alto, CA) for 1 h at 68 ◦ C, rinsed twice with 2× SSC (containing 0.1% SDS) at room temperature, a finally rinsed with 0.1× SSC (containing 0.1% SDS) at 50 ◦ C. Autoradiography was performed using Fuji RX film and an intensifying screen at −80 ◦ C for 24 h. Densitometric analysis of Northern blots was used to obtain the ratio of optical densities for TGF-1 and TGR2 and the corresponding GAPDH signals using a color image processor (Image Pro-plus, Mediacybernetics, Maryland, USA).
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in each chosen area were used to generate the results shown in Table 1 and Fig. 5.
2.3. Detection of apoptotic cells Cells undergoing programmed cell death (apoptosis) were detected in situ by specifically labeling nuclear DNA fragmentation (TUNEL method), using an ApopTag in situ detection kit/peroxidase (Intergen, Purchase, NY, USA), in accord with the manufacturer’s instructions. The number of TUNEL-positive apoptotic cells under the light microscope at 400× was counted and expressed as a percentage of the total nuclei counted; this involved 1000 nuclei per view in a randomly selected preneoplastic lesion (altered cellular foci and hyperplastic nodules), the HCC tissues, and the adjacent liver parenchyme.
2.6. Statistical analysis The results are expressed as means ± standard deviations (S.D.). The Kruskal–Wallis and the Mann–Whitney U-tests were used for the statistical analysis. Significance was defined as P < 0.05.
3. Results 3.1. Histopathologic findings and the sequential analysis of GST-P expression
2.4. Immunohistochemistry Sections were dewaxed and rehydrated according to a standard procedure, and washed with PBS. For the immunohistochemical staining of TGF-1 and TGR2, sections were heated in a microwave oven at 600 W for 2 × 7 min and for 5 min in 0.01 M citrate buffer, pH 6.0. Sections were then immersed in 3% H2 O2 to quench endogenous peroxidase activity, and unspecified binding was blocked with 5% normal goat serum (0.1% BSA in PBS). Immunohistochemical staining was performed by the avidin–biotin peroxidase complex method with aminoethylcarbazole as a chromogen using a Vectastain ABC elite kit (Vector Laboratories, Burlingame, CA, USA), according to the manufacturer’s instructions. Sections were counterstained with Mayer’s hematoxylin solution. To detect TGF-1, TGR2 and GST-P, rabbit polyclonal antibodies against TGF-1 (sc-146, Santa-Cruz, CA, USA), TGR2 (sc-400, Santa-Cruz, CA, USA.), and GST-P (311, MBL, Nagoya, Japan) were used at a dilution of 1:200, 1:500 and 1:1000, respectively.
In brief, 1 and 2 months after PH, variable numbers of preneoplastic lesions (clear cell foci, basophilic foci, eosinophilic foci and hyperplastic nodules) were apparent. An increase in the number of hyperplastic nodules was observed at 6 and 8 months after PH. Of the 10 animals examined at 12 months after PH, six cases had HCC. Sequential quantitation of the GST-P-positive lesions, including altered cellular foci (Fig. 1A), hyperplastic nodules (Fig. 1B) and HCC (Fig. 1C), is summarized in Fig. 1D. The areas of GST-P-positive lesions per unit area (mm2 /cm2 ) rapidly increased with time (P < 0.05). Northern blot analysis showed a linear increase of GST-P transcripts with time and a higher level of GST-P transcripts in HCC tissues (Fig. 2A) (P < 0.05). 3.2. Expressions of TGF-β1 and of the TGF-β type II receptor transcript To verify the expressions of the TGF-1 and of TGF- type II receptor transcripts in chemical hepatocarcinogenesis in the rat, Northern blot analysis was performed in each liver sample 1, 2, 4, 6, and 12 months after PH. A slight increase in the level of TGF-1 transcripts was observed during the promotion and progression stage versus the sham-operated control liver, but no statistically significant difference was observed for TGF-1 expression throughout the experiment (Fig. 2) (P > 0.05). In HCC, the level of TGF-1 transcripts in the HCC had a tendency to be higher than in the corresponding adjacent liver parenchyma
2.5. Sequential quantification of GST-P-positive lesions The expression of glutathione S-transferase placental form in focal areas of hepatocytes has been widely used as a marker to identify preneoplastic lesions in the rat liver [17]. Areas of GST-P-positive lesions were measured using a color image processor (Image Pro-plus, Mediacybernetics, Maryland, USA) and results were expressed in mm2 of stained area per cm2 . The means and the standard deviations of the areas of GST-P-positive lesions in each animal and
Table 1 Distribution of TUNEL-positive apoptotic cells in chemical hepatocarcinogenesis in the rat Months after partial hepatectomy
Preneoplastic lesion and HCC∗ Adjacent liver parenchyme∗
S
1
2
6
8
12
0.0 ± 0.0 0.0 ± 0.0
5.6 ± 2.8 24.6 ± 9.1
12.6 ± 6.8 39.7 ± 5.8
15.7 ± 11.4 43.4 ± 12.9
21.5 ± 12.8 64.6 ± 19.1
25.6 ± 21.8 79.7 ± 15.8
Values presented are the percent of TUNEL-positive nuclei to total nuclei and means ± S.D. S: sham-operated control liver. ∗ Significance of value difference between preneoplastic lesion and hepatocellular carcinoma and the adjacent liver parenchyme at each experimental point (P < 0.05).
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Fig. 1. Sequential changes in the GST-P-positive altered cellular foci (A) at 1 month after partial hepatectomy (PH), hyperplastic nodule (B) at 6 months after PH, and hepatocellular carcinoma (C) at 12 months after PH. Magnification 40× (A–C). The areas of GST-P-positive lesions per unit area (mm2 /cm2 ) rapidly increased with time (D). The values presented are means ± S.D. S indicates sham-operated control liver. (∗) Significance of value difference among each experimental point are seen (P < 0.05).
(Fig. 3) (P < 0.05). The transcripts of TGR2 were strongly expressed at 1 month after PH and in the sham-operated control liver. However, TGR2 significantly decreased with time, especially during the progression stage and HCC (Fig. 2) (P < 0.05). Also HCC tissues showed statistically significant lower levels of TGR2 transcripts compared to the paired adjacent liver parenchyma (Fig. 3) (P < 0.05). 3.3. Detection of apoptotic cell death Fig. 4 shows that the numbers of apoptotic cells increased with time and were significantly more numerous in the adja-
cent liver parenchyme than in preneoplastic lesions (foci and hyperplastic nodules (Fig. 4A) and in HCC tissues (Fig. 4B) (P < 0.05). 3.4. Localization and temporal expressions of TGF-β1 and TGR2 protein To confirm and localize TGF-1 expression at the protein level, immunohistochemical staining was performed for TGF-1. One month after PH, some TGF-1-positive cells were noted in preneoplastic lesions and the adjacent liver parenchyme was either weakly positive or negative
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Fig. 2. Northern blot analysis showing a slight increase of TGF-1 transcripts in the experimental groups vs. the sham-operated control group (S), but no statistically significant difference during the course of the experiment and a significant decrease of TGR2 transcripts during the progression stage (6 months after partial hepatectomy). A significant increase of GST-P transcripts was also noted (A). Densitometric analysis of Northern blots was used to obtain the ratio of optical densities of TGF-1 (open bar) and TGR2 (filled bar) with the corresponding GAPDH signals (B). The figures show a representative example of three independent Northern blots. The values are means ± S.D. S indicates sham-operated control liver. (∗) Significance of TGR2 difference is seen between progression stage (6 and 8 months after partial hepatectomy), HCC (12 months after hepatectomy) and other experimental point with sham operative control liver (P < 0.05).
(Fig. 5A). With the lapse of time, the number of positive cells increased in the preneoplastic lesions, which presented as clear and basophilic foci, and hyperplastic liver nodules as well as adjacent normal liver parenchyma (Fig. 5C). Scattered TGF-1-positive clusters of hepatocytes were also observed around preneoplastic lesions. The HCC showed strong TGF-1 positivity, and the staining intensity of HCC was stronger than that of the adjacent normal liver parenchyma (Fig. 5E). These results are in accord with the findings of Northern blot analysis. TGF-1 expression was mainly located in cytoplasm of the preneoplastic and neoplastic hepatocytes. TGR2 expression at the protein level was confirmed and localized immunohistochemically. At 2 months after PH, there were few TGR2 negative preneoplastic lesions (Fig. 5B). With the passage of time, the number of preneoplastic lesions that were negative for TGR2 and had
weaker positivity for the TGR2 compared with the adjacent liver parenchyma increased (Fig. 5D). HCC showed no or weak TGR2 positivity as compared to the adjacent liver parenchyma (Fig. 5F). TGR2 was mainly expressed in the cytoplasm of the hepatocytes without linear reinforcement.
4. Discussion The ability of TGF-1 to induce apoptosis depends, in part, upon the cellular concentration of various TGF- receptors [4,5]. In terms of chemical hepatocarcinogenesis, the down regulation of the TGF- receptors in hepatic tumors might provide a selective growth advantage to the neoplastic hepatocytes by reducing the ability of TGF-1 to inhibit their growth [14,15]. The present study shows that TGF- type II receptor transcript and protein expression are reduced
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Fig. 3. Northern blot analysis showed that there was an increase in TGF-1 transcripts and a decrease of TGR2 transcripts in hepatocellular carcinoma (HCC) tissues (T) compared to the corresponding adjacent liver parenchyma (P) (A). Densitometric analysis of Northern blots was used to obtain optical density ratios for TGF-1 (open bar) and TGR2 (filled bar) vs. the corresponding GAPDH signals (B). The figures show two representative examples of six independent HCCs and of the corresponding adjacent liver parenchyma. The values are means ± S.D. Significance of value of TGF-1 (∗) and TGR2 (∗∗) transcripts is noted between HCC and corresponding adjacent liver parenchyma (P < 0.05).
with time. This result suggests that a decrease in TGR2 contributes to the abrogation of the growth inhibitory effect of TGF-1 and indirectly to the clonal expansion of preneoplastic lesions and the ultimate progression to hepatocellular carcinoma. This contention is in agreement with studies that found reduced TGF- receptor expression, mainly of TGR2, in preneoplastic and malignant hepatocytes in mice, rats and humans [16–21]. In our study, there was no considerable expressional difference of TGF-1 transcripts during chemical hepatocarcinogenesis of rats. But, immunohistochemistry revealed that TGF-1-positive preneoplastic and neoplastic hepatocytes were increased with time. We suggest some possible explanations of this discrepancy. There are possibilities of post-transcriptional modifications of TGF-1 expression. So, result of Northern blot might be blurred. But, more precise in vitro and in vivo investigations are needed for confirmation of this discrepancy. One striking observation made in this study was that TGF-1-positive preneoplastic and neoplastic hepatocytes increased with time. Because of the loss of TGR2 in preneoplastic lesions and HCC, increased TGF-1 in preneoplastic and neoplastic hepatocytes has little or no effect on the
growth of preneoplastic lesions or upon disease progression. So, we are perplexed about the functions of the TGF-1 expressed by preneoplastic and neoplastic hepatocytes. Generally, TGF-1 has a local immunosuppressive effect [22] and inhibits the growth of epithelial cells by inducing apoptosis [1–3]. Many reports have revealed that many malignant tumors, including HCC, show increased TGF-1 expression [23–25]. However, other investigators have reported that the role of TGF-1 may vary to the extent that it may either inhibit or stimulate carcinogenesis. It was further suggested that this wide range of action depends on factors, such as the stage of carcinogenesis or the status of other oncogenes and growth factors [26–28]. So, it is tempting to speculate that the TGF-1 produced by preneoplastic and neoplastic hepatocytes might induce apoptosis of the adjacent TGR2 positive liver parenchyme in a paracrine fashion but not induce apoptosis in the preneoplastic and neoplastic hepatocytes. Moreover, this mechanism might have some role in growth and expansion of preneoplastic lesions and HCC tissues into the adjacent hepatic parenchyme. Our study supports these hypotheses, which explain why apoptotic cells were more numerous in the adjacent liver parenchyme than in the preneoplastic lesions and HCC tissues. This contention
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Fig. 4. Sequential changes in TUNEL-positive apoptotic cells. TUNEL-positive apoptotic cells increased with time and were more numerous in the adjacent hepatic parenchyma than in the hyperplastic nodules (A) and hepatocellular carcinoma (B). The values shown are means ± S.D. from measurements made on randomly selected separate preneoplastic lesions and hepatocellular carcinoma (HCC) (open bar) and surrounding hepatic parenchyme (filled bar) (C). S indicates sham-operated control liver. N, H, and L indicate hyperplastic nodule, hepatocellular carcinoma and adjacent liver parenchyma, respectively. (∗) Significant difference between preneoplastic lesions, HCC and the adjacent liver parenchyma is noted at each experimental point (P < 0.05).
is in agreement with a study which found that hepatoma cells might generate TGF--mediated peritumoral apoptosis of hepatocytes in a paracrine manner, which could facilitate their expansion in situ [29]. Also, in the promotion stage, the clonal expansion of initiated, i.e. genetically altered, hepatocytes was evident [30]. We suggest that TGF-1 in preneoplastic hepatocytes might also induce the apoptotic cell death of genetically unaltered hepatocytes, and thus accelerate the clonal expansion of initiated hepatocytes. However, more in vitro and in vivo studies are needed to confirm these findings. TGF-1 in HCC tissues might have several roles in chemical hepatocarcinogenesis of rats. Many investigators have reported upon the roles of TGF-1 in malignant neoplasms. TGF-1 has been found to stimulate angiogenesis and to increase tumor vascularity [31–33], and in human pancreatic cancer cells, TGF-1 was found to cause a paradoxical increase in c-myc mRNA expression [34]. As increased c-myc overexpression is associated with enhanced cell proliferation, these observations raise the possibility that TGF-1 may contribute to HCC cell growth in
the same way. Furthermore, it has been shown that TGF-1 potentiates the invasive capacity of malignant tumor cells [35,36]. Taken together, we suggest that after the progression stage of chemical hepatocarcinogenesis, TGF-1 may induce paradoxical effects on neoplastic hepatocytes by increasing their growth and invasiveness. We suggest that TGF-1 acts in the development and progression of preneoplastic lesions and HCC tissues by inducing local immunosuppressive effects on cytotoxic T cells and NK cells [22]. By this mechanism preneoplastic lesions and HCC tissues might have the capacity to escape from effector cells. In summary, we propose that the down regulation of TGR2 observed after the promotion stage might contribute to an attenuation of the growth inhibitory effects of TGF-1. Further, we suggest that TGF-1 has an important role in the development and progression of preneoplastic lesions and of HCC in chemical hepatocarcinogenesis in the rat. More in vivo and in vitro investigations are needed on the role of TGF-1 in preneoplastic lesions and HCC tissues during hepatocarcinogenesis.
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Fig. 5. Immunohistochemical staining for TGF-1 (A, C, and E) and TGR2 (B, D, and F). Some TGF-1-positive cells showed in clear cell foci (A) 1 month after partial hepatectomy (PH), and an increase in the number of TGF-1-positive cells in hyperplastic nodules was observed 6 months after PH (C), and in hepatocellular carcinoma tissues at 12 months after PH (E). Loss of TGR2 expression in clear cell foci (B) at 2 months after PH, in hyperplastic nodules at 6 months after PH (D), and in hepatocellular carcinoma tissue at 12 months after PH (F). Note the increased expression of TGR2 in hepatocytes surrounding the preneoplastic lesions and hepatocellular carcinoma. Magnification 200×. F, N, H, and L indicate altered cellular foci, hyperplastic nodule, hepatocellular carcinoma and adjacent liver parenchyma, respectively.
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Expressions of transforming growth factor (TGF)-1 and TGF- type II receptor and their relationship with apoptosis during chemical hepatocarcinogenesis in rats Do Youn Park a , Mee Young Sol a,∗ , Kang Suek Suh a , Eui-Cheol Shin b , Chul Hoon Kim c a
Department of Pathology, College of Medicine Pusan National University, Ami-1 Ga, Seo-Gu, Busan 602-739, South Korea b Department of Microbiology, Yonsei University College of Medicine, Seoul, South Korea c Department of Pharmacology, Yonsei University College of Medicine, Seoul, South Korea Received 14 January 2003; received in revised form 2 June 2003; accepted 17 June 2003
Abstract The expression of transforming growth factor (TGF)-1, which regulates cell proliferation, is tightly associated with that of TGF- type II receptor (TGR2), and has been regarded as an important change during hepatocarcinogenesis. Our aim in this study was to investigate the expression and localization of TGF-1 and TGR2 and to determine their relationships with apoptosis in chemical hepatocarcinogenesis of the rat produced by Solt and Farber’s method. Northern blot analysis showed that a slight increase of TGF-1 transcripts and a decrease of TGR2 transcripts during hepatocarcinogenesis. Immunohistochemistry revealed that TGF-1-positive preneoplastic hepatocytes increased with time, and that this correlated with a reduction TGR2 expressing preneoplastic lesions. Hepatocellular carcinoma (HCC) tissues showed higher levels of TGF-1 transcripts and protein and lower levels of TGR2 transcripts and protein compared to the paired adjacent liver parenchyme. TUNEL revealed that apoptotic cells increased with time and were more numerous in the adjacent liver parenchyme than in preneoplastic lesions and HCC tissues. Our data suggest that the down regulation of TGR2 in preneoplastic lesions and HCC tissues might contribute to resistance to the growth inhibitory effects of TGF-1, and to the roles of TGF-1 in the development and progression of preneoplastic lesions and HCC in a chemically induced rat hepatocarcinogensis model. © 2003 Elsevier B.V. All rights reserved. Keywords: TGF-1; TGF- type II receptor; Northern blot; Immunohistochemistry; Apoptosis; Chemical hepatocarcinogenesis; Rat
1. Introduction Transforming growth factor (TGF)-1 is a multipotent polypeptide, which inhibits the growth of epithelial cells, including hepatoma cell lines, hepatocytes and oval cells by inducing apoptosis [1–3]. Many reports have suggested that the ability of TGF-1 to control hepatocyte proliferation depends, in part, on the cellular concentration of various TGF- receptors [4,5], and to date, five such receptors have been identified. The type II serine/threonine kinase receptor forms a heterodimeric complex upon binding to TGF-, and generates the first step in the signal transduction pathway leading to growth inhibition in coordination ∗ Corresponding author. Tel.: +82-51-240-7717; fax: +82-51-253-7319. E-mail address: [email protected] (M.Y. Sol).
1386-6346/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S1386-6346(03)00264-X
with type I receptor [6,7]. In humans, elevated levels of TGF-1 mRNA have been reported in hepatocellular carcinoma (HCC), and TGF-1 was also found to be increased in the plasma of HCC patients [8,9]. Furthermore, the loss of responsiveness of hepatocellular carcinoma cells to TGF-1-mediated growth inhibition has been implicated in hepatocarcinogenesis [10]. Experimental hepatocarcinogenesis in rats occurs in distinctly defined stages, that is, initiation, promotion, and progression. The resistant hepatocyte model that was developed by Solt et al. [11] and Farber and Sarma [12] has been widely used in the study of multistep chemical hepatocarcinogenesis, in which the balance between cell death (apoptosis) and cell proliferation is a major determinant [13]. In chemical hepatocarcinogenesis, apoptosis plays a major role in each of these three stages. However, the temporal expressions and cellular localizations of TGF-1 and
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the TGF- type II receptor (TGR2) and their relationships with apoptosis in chemical hepatocarcinogenesis have not been clearly elucidated. The purpose of this study was to investigate the temporal expression and cellular localization of TGF-1 and TGR2 and their relationships with apoptosis during chemical hepatocarcinogenesis, especially during the stages of progression and HCC.
2. Materials and methods 2.1. Induction of chemical hepatocarcinogenesis in rats Diethylnitrosamine (DEN), 2-actetylaminofluorene (2AAF), carboxymethylcellulose and dimethylsulfoxide were obtained the Sigma Chemical Co. (St. Louis, MO, USA). 2-AAF was dissolved in a small volume of dimethylsulfoxide and suspended in 1% aqueous solution of carboxymethylcellulose to a final concentration of 1.5 mg/ml. Male Sprague–Dawley rats, weighing 200 gm, were maintained on standard pelleted chow and had access to water ad libitum. Solt and Farber’s method was used to induce hepatocarcinogenesis. Briefly, rats were initiated with a single dose (200 mg/kg) of DEN administered intraperitoneally by injection. Two weeks after initiation, all rats received a daily oral gavage of 10 mg/kg of 2-AAF for a period of up to 14 days. A partial hepatectomy (PH) was performed 1 week after starting the 2-AAF treatment. Five animals were sacrificed as indicated at 1 and 2 months after PH, that is during the promotion stage, at 6 and 8 months after PH, as progression stages, and 10 animals at 12 months after PH, as HCC stage. Two animals were sacrificed as an age-matched sham-operated control, which did not receive AAF or DEN treatment. After gross examination of each liver sample, a portion liver was fixed in 10% neutral buffered formalin solution and routinely processed for hematoxylin and eosin and immunohistochemical staining. The remainder of each sample was frozen in liquid nitrogen and kept at −70 ◦ C until required for RNA isolation. All experiments were conducted in accord with the institutional review committee’s guidelines. 2.2. RNA isolation, probe synthesis and Northern blot analysis Hybridization probes for Northern blot analysis were created by reverse transcription polymerase chain reaction (RT-PCR) amplification. Total RNA was isolated from each liver sample using an RNeasy kit (Qiagen, Santa Clarita, CA) under manufacturer’s instructions, which uses a silica-gel based membrane and guanidine isothiocyanate. cDNA was synthesized from 5 g of total RNA using 2 g of random hexamer (Pharmacia, Uppsala, Sweden), 10 mM dNTP (Boehringer Mannheim, Mannheim, Germany) and 200 U of M-MLV reverse transcriptase (GIBCO BRL) in a final volume of 25 l. PCR was performed using 1.25 mM
dNTP, 0.25 U of Taq polymerase (Perkin-Elmer, Branchburg, NJ), 10 pmol of the primer pairs compatible with rat TGF-1, TGR2, glutathione S-transferase placental form (GST-P), or glyceraldehydes-3-phosphate dehydrogenase (GAPDH), and cDNA using a thermal cycler (Perkin-Elmer, Branchburg, NJ). The following primer pairs were used: TGF-1
TGR2 GST-P GAPDH
5 -GAGAGCCCTGGATACCAACTACTG-3 and 5 -GTGTGTCCAGGCTCCAAATGTAG-3 5 GCTTCACTCTGGAAGATGCC-3 and 5 -AAGGAGTGTGGTCACTGTGC-3 5 -AAGTTTGAAGATGGAGACCT-3 and 5 -GATAGTTAGTGTAGATGAGGG-3 5 -ACCACAGTCCATGCCATCAC-3 and 5 CCACCACCCTGTTGCTGTA-3
The PCR cycling conditions used for TGF-1 were, 25 cycles of; denaturation at 94 ◦ C for 50 s, annealing at 55 ◦ C for 30 s, and extension at 72 ◦ C for 2 min, with a final extension at 72 ◦ C for 10 min, with a predicted product size of 173 bp. For TGR2 these were, 28 cycles of; denaturation at 94 ◦ C for 1 min, annealing at 60 ◦ C for 1 min, and extension at 72 ◦ C for 1 min, with a final extension at 72 ◦ C for 10 min, with a predicted product size of 774 bp. For GST-P were, 25 cycles of; denaturation at 94 ◦ C for 1 min, annealing at 57 ◦ C for 1 min, and extension at 72 ◦ C for 1 min, with a final extension at 72 ◦ C for 10 min, with a predicted product size of 167 bp, and finally for GAPDH were, 25 cycles of; denaturation at 94 ◦ C for 1 min, annealing at 57 ◦ C for 1 min, and extension at 72 ◦ C for 2 min, with a final extension at 72 ◦ C for 10 min, with predicted product size of 452 bp. Amplified PCR products were analyzed on 1.5% agarose gel and extracted using a QIAquick gel extraction kit (Qiagen, Santa Clarita, CA). PCR products were sequenced and labeled with [32 P]-dCTP by the random priming method using a High Prime random labeling kit (Boehringer Mannheim, Mannheim, Germany). Labeled probes were purified using a QIAquick nucleotide removal kit (Qiagen, Santa Clarita, CA). Total RNA was separated on denaturating formaldehyde 1.0% agarose gels (20 g total RNA per lane), and transferred to a Hybond (+) nylon membrane (Boehringer Mannheim, Mannheim, Germany) using the capillary transfer method. The RNAs were then cross-linked to the membrane by using a UV cross-linker and baked in an oven at 80 ◦ C for 1 h. The RNAs were hybridized with specific probes in an ExpressHyb hybridization solution (Clontech, Palo Alto, CA) for 1 h at 68 ◦ C, rinsed twice with 2× SSC (containing 0.1% SDS) at room temperature, a finally rinsed with 0.1× SSC (containing 0.1% SDS) at 50 ◦ C. Autoradiography was performed using Fuji RX film and an intensifying screen at −80 ◦ C for 24 h. Densitometric analysis of Northern blots was used to obtain the ratio of optical densities for TGF-1 and TGR2 and the corresponding GAPDH signals using a color image processor (Image Pro-plus, Mediacybernetics, Maryland, USA).
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in each chosen area were used to generate the results shown in Table 1 and Fig. 5.
2.3. Detection of apoptotic cells Cells undergoing programmed cell death (apoptosis) were detected in situ by specifically labeling nuclear DNA fragmentation (TUNEL method), using an ApopTag in situ detection kit/peroxidase (Intergen, Purchase, NY, USA), in accord with the manufacturer’s instructions. The number of TUNEL-positive apoptotic cells under the light microscope at 400× was counted and expressed as a percentage of the total nuclei counted; this involved 1000 nuclei per view in a randomly selected preneoplastic lesion (altered cellular foci and hyperplastic nodules), the HCC tissues, and the adjacent liver parenchyme.
2.6. Statistical analysis The results are expressed as means ± standard deviations (S.D.). The Kruskal–Wallis and the Mann–Whitney U-tests were used for the statistical analysis. Significance was defined as P < 0.05.
3. Results 3.1. Histopathologic findings and the sequential analysis of GST-P expression
2.4. Immunohistochemistry Sections were dewaxed and rehydrated according to a standard procedure, and washed with PBS. For the immunohistochemical staining of TGF-1 and TGR2, sections were heated in a microwave oven at 600 W for 2 × 7 min and for 5 min in 0.01 M citrate buffer, pH 6.0. Sections were then immersed in 3% H2 O2 to quench endogenous peroxidase activity, and unspecified binding was blocked with 5% normal goat serum (0.1% BSA in PBS). Immunohistochemical staining was performed by the avidin–biotin peroxidase complex method with aminoethylcarbazole as a chromogen using a Vectastain ABC elite kit (Vector Laboratories, Burlingame, CA, USA), according to the manufacturer’s instructions. Sections were counterstained with Mayer’s hematoxylin solution. To detect TGF-1, TGR2 and GST-P, rabbit polyclonal antibodies against TGF-1 (sc-146, Santa-Cruz, CA, USA), TGR2 (sc-400, Santa-Cruz, CA, USA.), and GST-P (311, MBL, Nagoya, Japan) were used at a dilution of 1:200, 1:500 and 1:1000, respectively.
In brief, 1 and 2 months after PH, variable numbers of preneoplastic lesions (clear cell foci, basophilic foci, eosinophilic foci and hyperplastic nodules) were apparent. An increase in the number of hyperplastic nodules was observed at 6 and 8 months after PH. Of the 10 animals examined at 12 months after PH, six cases had HCC. Sequential quantitation of the GST-P-positive lesions, including altered cellular foci (Fig. 1A), hyperplastic nodules (Fig. 1B) and HCC (Fig. 1C), is summarized in Fig. 1D. The areas of GST-P-positive lesions per unit area (mm2 /cm2 ) rapidly increased with time (P < 0.05). Northern blot analysis showed a linear increase of GST-P transcripts with time and a higher level of GST-P transcripts in HCC tissues (Fig. 2A) (P < 0.05). 3.2. Expressions of TGF-β1 and of the TGF-β type II receptor transcript To verify the expressions of the TGF-1 and of TGF- type II receptor transcripts in chemical hepatocarcinogenesis in the rat, Northern blot analysis was performed in each liver sample 1, 2, 4, 6, and 12 months after PH. A slight increase in the level of TGF-1 transcripts was observed during the promotion and progression stage versus the sham-operated control liver, but no statistically significant difference was observed for TGF-1 expression throughout the experiment (Fig. 2) (P > 0.05). In HCC, the level of TGF-1 transcripts in the HCC had a tendency to be higher than in the corresponding adjacent liver parenchyma
2.5. Sequential quantification of GST-P-positive lesions The expression of glutathione S-transferase placental form in focal areas of hepatocytes has been widely used as a marker to identify preneoplastic lesions in the rat liver [17]. Areas of GST-P-positive lesions were measured using a color image processor (Image Pro-plus, Mediacybernetics, Maryland, USA) and results were expressed in mm2 of stained area per cm2 . The means and the standard deviations of the areas of GST-P-positive lesions in each animal and
Table 1 Distribution of TUNEL-positive apoptotic cells in chemical hepatocarcinogenesis in the rat Months after partial hepatectomy
Preneoplastic lesion and HCC∗ Adjacent liver parenchyme∗
S
1
2
6
8
12
0.0 ± 0.0 0.0 ± 0.0
5.6 ± 2.8 24.6 ± 9.1
12.6 ± 6.8 39.7 ± 5.8
15.7 ± 11.4 43.4 ± 12.9
21.5 ± 12.8 64.6 ± 19.1
25.6 ± 21.8 79.7 ± 15.8
Values presented are the percent of TUNEL-positive nuclei to total nuclei and means ± S.D. S: sham-operated control liver. ∗ Significance of value difference between preneoplastic lesion and hepatocellular carcinoma and the adjacent liver parenchyme at each experimental point (P < 0.05).
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Fig. 1. Sequential changes in the GST-P-positive altered cellular foci (A) at 1 month after partial hepatectomy (PH), hyperplastic nodule (B) at 6 months after PH, and hepatocellular carcinoma (C) at 12 months after PH. Magnification 40× (A–C). The areas of GST-P-positive lesions per unit area (mm2 /cm2 ) rapidly increased with time (D). The values presented are means ± S.D. S indicates sham-operated control liver. (∗) Significance of value difference among each experimental point are seen (P < 0.05).
(Fig. 3) (P < 0.05). The transcripts of TGR2 were strongly expressed at 1 month after PH and in the sham-operated control liver. However, TGR2 significantly decreased with time, especially during the progression stage and HCC (Fig. 2) (P < 0.05). Also HCC tissues showed statistically significant lower levels of TGR2 transcripts compared to the paired adjacent liver parenchyma (Fig. 3) (P < 0.05). 3.3. Detection of apoptotic cell death Fig. 4 shows that the numbers of apoptotic cells increased with time and were significantly more numerous in the adja-
cent liver parenchyme than in preneoplastic lesions (foci and hyperplastic nodules (Fig. 4A) and in HCC tissues (Fig. 4B) (P < 0.05). 3.4. Localization and temporal expressions of TGF-β1 and TGR2 protein To confirm and localize TGF-1 expression at the protein level, immunohistochemical staining was performed for TGF-1. One month after PH, some TGF-1-positive cells were noted in preneoplastic lesions and the adjacent liver parenchyme was either weakly positive or negative
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Fig. 2. Northern blot analysis showing a slight increase of TGF-1 transcripts in the experimental groups vs. the sham-operated control group (S), but no statistically significant difference during the course of the experiment and a significant decrease of TGR2 transcripts during the progression stage (6 months after partial hepatectomy). A significant increase of GST-P transcripts was also noted (A). Densitometric analysis of Northern blots was used to obtain the ratio of optical densities of TGF-1 (open bar) and TGR2 (filled bar) with the corresponding GAPDH signals (B). The figures show a representative example of three independent Northern blots. The values are means ± S.D. S indicates sham-operated control liver. (∗) Significance of TGR2 difference is seen between progression stage (6 and 8 months after partial hepatectomy), HCC (12 months after hepatectomy) and other experimental point with sham operative control liver (P < 0.05).
(Fig. 5A). With the lapse of time, the number of positive cells increased in the preneoplastic lesions, which presented as clear and basophilic foci, and hyperplastic liver nodules as well as adjacent normal liver parenchyma (Fig. 5C). Scattered TGF-1-positive clusters of hepatocytes were also observed around preneoplastic lesions. The HCC showed strong TGF-1 positivity, and the staining intensity of HCC was stronger than that of the adjacent normal liver parenchyma (Fig. 5E). These results are in accord with the findings of Northern blot analysis. TGF-1 expression was mainly located in cytoplasm of the preneoplastic and neoplastic hepatocytes. TGR2 expression at the protein level was confirmed and localized immunohistochemically. At 2 months after PH, there were few TGR2 negative preneoplastic lesions (Fig. 5B). With the passage of time, the number of preneoplastic lesions that were negative for TGR2 and had
weaker positivity for the TGR2 compared with the adjacent liver parenchyma increased (Fig. 5D). HCC showed no or weak TGR2 positivity as compared to the adjacent liver parenchyma (Fig. 5F). TGR2 was mainly expressed in the cytoplasm of the hepatocytes without linear reinforcement.
4. Discussion The ability of TGF-1 to induce apoptosis depends, in part, upon the cellular concentration of various TGF- receptors [4,5]. In terms of chemical hepatocarcinogenesis, the down regulation of the TGF- receptors in hepatic tumors might provide a selective growth advantage to the neoplastic hepatocytes by reducing the ability of TGF-1 to inhibit their growth [14,15]. The present study shows that TGF- type II receptor transcript and protein expression are reduced
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Fig. 3. Northern blot analysis showed that there was an increase in TGF-1 transcripts and a decrease of TGR2 transcripts in hepatocellular carcinoma (HCC) tissues (T) compared to the corresponding adjacent liver parenchyma (P) (A). Densitometric analysis of Northern blots was used to obtain optical density ratios for TGF-1 (open bar) and TGR2 (filled bar) vs. the corresponding GAPDH signals (B). The figures show two representative examples of six independent HCCs and of the corresponding adjacent liver parenchyma. The values are means ± S.D. Significance of value of TGF-1 (∗) and TGR2 (∗∗) transcripts is noted between HCC and corresponding adjacent liver parenchyma (P < 0.05).
with time. This result suggests that a decrease in TGR2 contributes to the abrogation of the growth inhibitory effect of TGF-1 and indirectly to the clonal expansion of preneoplastic lesions and the ultimate progression to hepatocellular carcinoma. This contention is in agreement with studies that found reduced TGF- receptor expression, mainly of TGR2, in preneoplastic and malignant hepatocytes in mice, rats and humans [16–21]. In our study, there was no considerable expressional difference of TGF-1 transcripts during chemical hepatocarcinogenesis of rats. But, immunohistochemistry revealed that TGF-1-positive preneoplastic and neoplastic hepatocytes were increased with time. We suggest some possible explanations of this discrepancy. There are possibilities of post-transcriptional modifications of TGF-1 expression. So, result of Northern blot might be blurred. But, more precise in vitro and in vivo investigations are needed for confirmation of this discrepancy. One striking observation made in this study was that TGF-1-positive preneoplastic and neoplastic hepatocytes increased with time. Because of the loss of TGR2 in preneoplastic lesions and HCC, increased TGF-1 in preneoplastic and neoplastic hepatocytes has little or no effect on the
growth of preneoplastic lesions or upon disease progression. So, we are perplexed about the functions of the TGF-1 expressed by preneoplastic and neoplastic hepatocytes. Generally, TGF-1 has a local immunosuppressive effect [22] and inhibits the growth of epithelial cells by inducing apoptosis [1–3]. Many reports have revealed that many malignant tumors, including HCC, show increased TGF-1 expression [23–25]. However, other investigators have reported that the role of TGF-1 may vary to the extent that it may either inhibit or stimulate carcinogenesis. It was further suggested that this wide range of action depends on factors, such as the stage of carcinogenesis or the status of other oncogenes and growth factors [26–28]. So, it is tempting to speculate that the TGF-1 produced by preneoplastic and neoplastic hepatocytes might induce apoptosis of the adjacent TGR2 positive liver parenchyme in a paracrine fashion but not induce apoptosis in the preneoplastic and neoplastic hepatocytes. Moreover, this mechanism might have some role in growth and expansion of preneoplastic lesions and HCC tissues into the adjacent hepatic parenchyme. Our study supports these hypotheses, which explain why apoptotic cells were more numerous in the adjacent liver parenchyme than in the preneoplastic lesions and HCC tissues. This contention
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Fig. 4. Sequential changes in TUNEL-positive apoptotic cells. TUNEL-positive apoptotic cells increased with time and were more numerous in the adjacent hepatic parenchyma than in the hyperplastic nodules (A) and hepatocellular carcinoma (B). The values shown are means ± S.D. from measurements made on randomly selected separate preneoplastic lesions and hepatocellular carcinoma (HCC) (open bar) and surrounding hepatic parenchyme (filled bar) (C). S indicates sham-operated control liver. N, H, and L indicate hyperplastic nodule, hepatocellular carcinoma and adjacent liver parenchyma, respectively. (∗) Significant difference between preneoplastic lesions, HCC and the adjacent liver parenchyma is noted at each experimental point (P < 0.05).
is in agreement with a study which found that hepatoma cells might generate TGF--mediated peritumoral apoptosis of hepatocytes in a paracrine manner, which could facilitate their expansion in situ [29]. Also, in the promotion stage, the clonal expansion of initiated, i.e. genetically altered, hepatocytes was evident [30]. We suggest that TGF-1 in preneoplastic hepatocytes might also induce the apoptotic cell death of genetically unaltered hepatocytes, and thus accelerate the clonal expansion of initiated hepatocytes. However, more in vitro and in vivo studies are needed to confirm these findings. TGF-1 in HCC tissues might have several roles in chemical hepatocarcinogenesis of rats. Many investigators have reported upon the roles of TGF-1 in malignant neoplasms. TGF-1 has been found to stimulate angiogenesis and to increase tumor vascularity [31–33], and in human pancreatic cancer cells, TGF-1 was found to cause a paradoxical increase in c-myc mRNA expression [34]. As increased c-myc overexpression is associated with enhanced cell proliferation, these observations raise the possibility that TGF-1 may contribute to HCC cell growth in
the same way. Furthermore, it has been shown that TGF-1 potentiates the invasive capacity of malignant tumor cells [35,36]. Taken together, we suggest that after the progression stage of chemical hepatocarcinogenesis, TGF-1 may induce paradoxical effects on neoplastic hepatocytes by increasing their growth and invasiveness. We suggest that TGF-1 acts in the development and progression of preneoplastic lesions and HCC tissues by inducing local immunosuppressive effects on cytotoxic T cells and NK cells [22]. By this mechanism preneoplastic lesions and HCC tissues might have the capacity to escape from effector cells. In summary, we propose that the down regulation of TGR2 observed after the promotion stage might contribute to an attenuation of the growth inhibitory effects of TGF-1. Further, we suggest that TGF-1 has an important role in the development and progression of preneoplastic lesions and of HCC in chemical hepatocarcinogenesis in the rat. More in vivo and in vitro investigations are needed on the role of TGF-1 in preneoplastic lesions and HCC tissues during hepatocarcinogenesis.
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Fig. 5. Immunohistochemical staining for TGF-1 (A, C, and E) and TGR2 (B, D, and F). Some TGF-1-positive cells showed in clear cell foci (A) 1 month after partial hepatectomy (PH), and an increase in the number of TGF-1-positive cells in hyperplastic nodules was observed 6 months after PH (C), and in hepatocellular carcinoma tissues at 12 months after PH (E). Loss of TGR2 expression in clear cell foci (B) at 2 months after PH, in hyperplastic nodules at 6 months after PH (D), and in hepatocellular carcinoma tissue at 12 months after PH (F). Note the increased expression of TGR2 in hepatocytes surrounding the preneoplastic lesions and hepatocellular carcinoma. Magnification 200×. F, N, H, and L indicate altered cellular foci, hyperplastic nodule, hepatocellular carcinoma and adjacent liver parenchyma, respectively.
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