Little expression of proto-oncogene Bcl-2 in tumourous cells of hepatocellular carcinoma with chronic hepatitis C virus infection

International

COIllIIlUIli~tiOIlS

Hepatology Communications 4 (1996) 316-325

Little expression of proto-oncogene Bcl-2 in tumourous cells of hepatocellular carcinoma with chronic hepatitis C virus infection Yoshiki Ito”, Norio Hayash?“, Yutaka Sasaki”, Masayoshi Horimoto”, Shigeo Wada”, Yuji Tanaka”, Taizo Hijioka”, Kunio Suzukib, Hideyuki Fusamoto”, Jiro Fujimotoc, Eizo Okamoto”, Takenobu Kamada” “First

‘First

Department

of Medicine,

Unicersity School 565, Japan bOsuka Rousai Hospital, I1 79-3 Nakasone, Department of Surgery. Hvogo College of’ Medicine, Japan Received

Osaka

27 November

of

Medicine,

2-2

Sakai, Osaka I-l Mukogawu,

1995: accepted

Yamadaoka, 565, Japan Nishinomiya,

28 November

Suita,

Hyogo

Osaka

663,

1995

Abstract The proto-oncogene bcl-2, which was first found overexpressed in human follicular lymphoma, is now considered to inhibit apoptotic cell death, resulting in greater cell susceptibility to genetic alteration. Thus, this gene might cause hyperplasia or occasionally lead to a more malignant phase, i.e. carcinoma. Overexpression of this gene has been reported in several tumors. However, little has been clarified about its contribution to hepatocarcinogenesis. In this study, using immunoblotting, immunohistochemical and in situ hybridization techniques, we examined the expression of the bcl-2 protein and messenger RNA in patients having hepatocellular carcinoma with chronic hepatitis C virus infection. Immunoblot analysis indicated no significant differences in the amounts of bcl-2 protein between tumorous and nontumorous lesions. Furthermore, immunohistochemistry as well as in situ hybridization technique demonstrated that only lymphocytes infiltrating the tissues and tumors were bcl-2 positive. These findings suggest that the proto-oncogene bcl-2 may make no or little contribution, if any, to hepatocarcinogenesis. Keyworcls: Hepatocarcinogenesis; Tumor-infiltrating ting; Immunohistochemistry; In-situ hybridization

lymphocyte

(TIL);

* Corresponding author. @medone.med.osaka-uacjp.

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1. Introduction The proto-oncogene bcl-2, whose name is derived from its association with B-cell lymphoma, was first found overexpressed due to the t(14;18) chromosoma1 translocation in human follicular lymphoma [l]. Later, this gene was revealed to inhibit programmed cell death, so-called ‘apoptosis’ [2,3]. This inhibitory effect promotes the maintenance and regulation of stem cells, longlived post mitotic cells hematopoietic cells, and hormone- or growth factor-regulatory glandular epithelia [4]. Since the bcl-2-expressing cell is not only susceptible to further genetic alteration due to the extension of its life but also evades elimination by apoptosis, this gene might play an important role in tumorigenesis, neoplastic growth and resistance to therapy [5]. For instance, co-expression of bcl-2 with inappropriate c-myc expression prevents apoptosis, resulting in hyperplasia or occasionally leading to a more malignant phase, i.e. carcinoma [6,7]. Up to now, several neoplastic cells have been reported to express bcl-2, including lymphoma, leukemia, lung cancer and neuroblastoma [& 101. However, little has been clarified about its contribution to hepatocarcinogenesis. Previous reports have described that hepatitis C viruses (HCV) were detected in tumorous cells of hepatocellular carcinoma (HCC) [l l] and that apoptosis mediated by the Fas antigen is one of the pathways leading to elimination of HCV-infected hepatocytes in chronic HCV infection [12]. These observations suggested that there might be some mechanism whereby tumorous cells bearing HCV could escape apoptosis. As bcl-2 was reported to interfere with the apoptotic process mediated by the Fas antigen [13], this proto-oncogene might be involved in hepatocarcinogenesis. To clarify this issue: we examined the expression of the bcl-2 protein and messenger RNA (mRNA) in tumorous and adjacent nontumorous liver lesions of patients with chronic HCV infection and HCC, using immunoblot immunohistochemical and in situ hybridization techniques.

2. Materials

and methods

2.1. Subjects The five patients of this study were all males, aged from 58 to 71 years old, suffering from chronic HCV infection and HCC. Their clinical characteristics are shown in Table 1. All patients had to undergo surgery. None had current infection of hepatitis B virus at the time of operation. Tumorous lesions were histologically classified to be moderately differentiated adenocarcinoma in four patients and a mixture of moderately and well differentiated adenocarcinoma in one. In each case, the removed liver tissues were histologically divided into tumorous and nontumorous lesions soon after the surgical resection, and in each lesion, one part was homogenized for immunoblot analysis, and the remaining portion was snap-

This

1 2 3 4 5

Patient Male Male Male Male Male

Sex

is according

61 61 66 58 71

Age

of the patients

stage grouping

No.

Table 1 Clinical features

to the International

I II IV-A 11 IV-A

Stage

Union

20 30 105 45 15

Tumor

Against

size (mm)

Cancer

TNM

classification

Well-moderate Moderate Moderate Modcratc Moderate

Llifferentiation Cirrhosis Cirrhosis Cirrhosis Cirrhosis Cirrhosis

Adjacent

tissue

+ + + + +

HCV

Ab

-/+ -I+ -it -I-/+

HBsAg/Ab

2 G 2.

6

II 6’ c E p:

Y. Ito et al. : International

frozen in powdered studies. 2.2. Immunoblot

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and in-situ

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hybridization

analysis

Preparation of the tissue homogenate and immunoblotting were performed as reported previously [14] with slight modification. In brief, tissues were homogenized in a glass-Teflon homogenizer containing Triton-lysis buffer. Protein concentrations of the homogenates were determined using BCA protein assay (Pierce, Rockford, IL). The homogenates containing 100 pg of protein were boiled with 6 x concentrated Quench buffer, fractionated by 15% sodium dodesyl sulfate polyacrylamide gel electrophoresis, and transferred to PVDF filters (Millipore, Bedford, MA). Filters were blocked in Tris-buffered saline (TBS) (pH 7.6) containing 4% non-fat dry milk overnight at 4°C and incubated with mouse anti-human bcl-2 monoclonal IgG (DAK0 A/S, Denmark) 1:80 diluted in TBS at 4°C for 24 h, then with biotinylated horse anti-mouse IgG antibody 1:lOOO diluted in TBS overnight at 4°C. Finally, conjugated biotin was calorimetrically detected using Vectastain ABC kit (Vector Laboratories, Burlingame, CA) with diaminobenzidine tetrahydrochloride (DAB) as oxidizable peroxidase substrate. The homogenate containing an equal amount of protein obtained from surgically resected human spleen was used as the positive control for the bcl-2 protein expression [15]. The homogenate from human normal liver was also used as the normal control for that. 2.3. Tissue section preparation Eight-micrometer sections were cut on a cryostat, thaw-mounted on glass slides coated with a subbed of 3-aminopropyltriethoxysilane (Aldrich Chemical Co., Milwaukee, WI) at - 20°C and then stored at - 80°C until processing for immunohistochemistry and in situ hybridization. 2.4. Immunohistochemical procedures Immunohistochemistry was performed as previously reported [12] with several modifications. Briefly after fixation with ice-cold acetone for 10 min, sections were blocked with 2% normal horse serum and 1 “/o bovine serum albumin in TBS for 10 min, and incubated with 1:40 diluted mouse anti- human bcl-2 monoclonal IgG (DAKO) or 1:50 diluted mouse anti-human CD3 monoclonal IgG UCHTl (DAKO) overnight at 4°C. Next, sections were incubated with biotinylated horse anti-mouse IgG antibody 1:1000 diluted in TBS for 1 h at ambient temperature. After treatment with 0.03% hydrogen peroxide in methanol, immunostaining was carried out using the Vectastain ABC kit and developed with DAB, followed by counterstaining with methylgreen. As negative controls, sequential sections of both tumorous and nontumorous lesions were immunostained with mouse preimmune IgG as the first antibody instead of immune-specific antibody.

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2.5. In situ hybridization pBIISK( + )hbcl-2 was established by insertion of pYTlS-4H, the plasmid coding partial human hcl-2 gene [16], into the Sac1 and Sac11 sites of pBluescriptIISK( + ) phagemid vector. Digoxigenin(DIG)-labeled antisense RNA probe was made using in vitro transcription with DIG-labeled UTP (Boehringer Mannheim GmbH, Germany) by T7 RNA polymerase (Promega, Madison, WI) with BssHII-digested pBIISK( + )hbcl-2 as a template. DIG-labeled sense RNA probe was also made by T3 RNA polymerase (Stratagene, La Jolla, CA) with HincII-digested pBIISK( + )hbcl-2 as a template. In situ hybridization was performed as previously described [17]. In brief, sections were fixed with 4% paraformaldehyde (PFA) in phosphate buffer (pH 7.4) and treated with proteinase K. After another PFA fixation, sections were treated subsequently with 0.2 N HCl, 0.25%(V/V) acetic anhydride in 0.1 M triethanolamine, followed by dehydration with ethanol series and chloroform treatment. After preincubation with the hybridization buffer, sections were incubated with the same buffer containing DIG-labeled bcl-2 antisense RNA probe overnight at 50°C. Subsequently, they were rinsed in 5 x SSC, 50% formamide in 2 x SSC, treated with RNase A, and then rinsed again in 50% formamide in 2 x SSC. Hybridization products were detected according to the manufacturer’s instructions for the DIG Nucleic Acid Detection Kit (Boehringer Mannheim). The sequential sections of both lesions hybridized with bcl-2 sense probe were studied as negative controls.

3. Results and discussion 3.1. Expression tissues

of bcl-2 protein und mRNA

in tumorous und nontumorous

liver

We first explored expression of the bcl-2 protein in HCC using immunoblot analysis with anti-bcl-2 IgG. Bcl-2 protein was detected as a protein with molecular weight of 26 kDa. In cases with HCC, there seemed no tendency regarding amount of the bcl-2 protein between tumorous and nontumorous lesions; the amount in tumorous lesion was higher in two cases and lower in three cases than that in the corresponding nontumorous lesion (Fig. 1). A trace of bcl-2 expression was observed even in normal liver tissue, that might originate slightly infiltrated or remaining peripheral blood cells in the liver. To reveal how bcl-2 is expressed in the tissues and which population of hepatic cells participates in bcl-2 expression, an immunohistochemical study with anti-bcl-2 IgG was performed. In tumorous tissues small round-shaped cells were bcl-2 positive and scattered in tumorous lesions and capsules. No positive staining was detected in tumorous cells (Fig. 2A and B). In nontumorous tissues, bcl-2 positive cells were also small and round-shaped, which were detected mainly in portal areas and interlobular fibrous lesions, gathered in lymphfollicles and also scattered in

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lobules. These immuno-positive cells are different from hepatocytes, Kuppfer cells, endothelial cells, Ito cells and epithelial cells of bile ducts or vessels (Fig. 2C and D). No immununopositive cell was found on incubation with preimmune IgG (Fig. 2E and F). Tha data obtained from immununohistochemistry were compatible with those from immunoblotting. We next investigated the expression of the bcl-2 mRNA using in situ hybridization technique to confirm the above findings. With bcl-2 antisense RNA probe, cells expressing bcl-2 mRNA were scattered among tumorous cells in HCC, but no tumorous cell was positively labeled for bcl-2 (Fig. 3A and B). In nontumorous tissue, labeled cells were similarly detected in portal areas, interlobular fibrous lesions, and lymphfollicles as a result of immunostaining with anti bcl-2 IgG, but intralobular distribution of the labeled cells was equivocal. Neither parenchymal nor nonparenchymal cells were distribution of the labeled cells was equivocal. Neither parenchymal nor nonparenchymal cells were positively labeled for bcl-2 mRNA (Fig. 3C and D). Degrees of bcl-2 mRNA expression were less than those of bcl-2 protein, that might be caused by translational enhancement of bcl-2 protein expression. No labeled cell was detected in either tumorous or nontumorous lesions with bcl-2 sense RNA probe (Fig. 3E and F). Zhao et al. previously demonstrated an immnuno-positive structure for bcl-2 protein in lymphocytes and rarely in HCCs [18], however, there has been no report on the contribution of this proto-oncogene to hepatocarcinogenesis nor characteristic assessment of the backgrounds of nontuniorous lesions. This is the first study verifying with both immunohistochemical and in situ hybridization techniques that neither bcl-2 protein nor mRNA is expressed in tumorous cells of HCC with chronic HCV infection. 3.2. Expression

of CD3 protein

in tumorous and non tumor0 us liver tissues

In view of the distribution and appearance of bcl-2 positive cells, these cells might be tumor- or tissue-infiltrating lymphocytes, considering that lymphocytes have

N

WW 46

case1 NT T

case2 NT T

case3 NT T

case4 NT T

case5 NT T

S

-

Fig. 1. Immunoblot analysis of bcl-2 expression in cases. Liver samples (100 pg each) were subjected Prestained protein marker is shown on the left side bcl-2 protein of around 26 kDa that migrated. N. nontumorous lesion; S, spleen as positive control.

tumorous and adjacent nontumorous lesions in 5 to Western blotting with anti-human bcl-2 IgG. with Arabic numerals. Open arrowheads indicates normal liver; T. tumorous lesion: NT, adjacent

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Fig. 2. Representative presentation of bcl-2 immunopositive structures in tumorous and adjacent nontumorous lesions. Liver sections from case 2 were immunostained with anti-human bcl-2 IgG as described in Materials and methods. (A and C) Sections from tumorous and nontumorous lesions immunostained with anti-bcl-2 IgG, respectively. (B and D) Same sections as (A and C) at higher magnification. (E and F) Sequential secdons from tumorous and nontumorous lesions immunostained with preimmune IgG, respectively. Arrowheads in the panel indicate positively stained cells. Original magnifications: x 20 in A, C, E and F: x 50 in B and D. p, portal vein.

been reported to express the bcl-2 protein [4] and might infiltrate into tissues with inflammation. To identify the bcl-2 positive cells in both tumorous and nontumorous lesions, we performed an immunohistochemical study of CD3, a specific surface antigen of T cell lymphocytes [19]. With HCC, CD3 positive cells were distributed in the capsules and among tumorous cells (Fig. 4A and C). In nontumorous lesion, CD3 positive cells were distributed in portal areas, interlobular fibrous lesions and some lymphfollicles, and slightly in lobules (Fig. 4B and D). The localization and appearance of CD3 positive cells were almost accordant with those of bcl-2 positive

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cells, suggesting that T lymphocytes gathering at an inflammation site may account for the bcl-2 positive structures in such tissue. In conclusion, these findings demonstrate that the contribution of bcl-2 expression to hepatocarcinogenesis may be negligible. Other causes of hepatocarcinogenesis should he further investigated.

Fig. 3. Detection of bcl-2 mRNA in tumorous and adjacent nontumorous lesions using in situ hybridization analysis. Sequential liver sections used in Fig. 2A and C were hybridized with DIGlabeled human bcl-2 RNA probes as described in Materials and methods. Representative results are shown. (A and C) Sections from tumorous and nontumorous lesions hybridized with bcl-2 antisense RNA probe. respectively. (B and D) Same sections as (A and C) at higher magnification. (E and F) Sequential sections from tumorous and nontumorous lesions hybridized with bcl-2 sense RNA probe, respectively. Arrowheads in the panel indicate positively laheled cells. Original magnifications: x 20 in A, C, E and F; x 50 in B and D. p , portal vein.

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Fig. 4. Immunohistochemical localization of CD3 in tumorous and adjacent nontumorous lesions. Sections were immunostained with anti-human CD3 IgG as described in Materials and methods. Representatives are presented. (A and C) Immunostaining of sequential sections to those used in Fig. 2A and C from tumorous and nontumorous lesions, respectively, with anti-CD3 IgG. (B and D) Same sections as (A and C) at higher magnifications. Arrowheads indicate positively stained cells. Original magnifications: x 20 in A and C; x 50 in B and D. p, portal vein.

Acknowledgements pYTl8-4H was kindly provided from Dr. Yoshihide Tsujimoto, Department Medical Genetics, Biomedical Research Center, Osaka University School Medicine, Osaka, Japan.

[l] [2] [3] [4]

[5]

of of

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