| | Expression of DNA methyltransferases in multistep hepatocarcinogenesis☆☆☆Accepted 30 September 2002. Abstract Hypermethylation of cell cycle regulators and increased DNA methyltransferase 1 (Dnmt1) mRNA level have been reported in hepatocarcinogenesis. However, the expression of Dnmts has not yet been examined in hepatocellular carcinomas (HCCs). We examined 13 cases of HCCs in dysplastic nodules (DNs) and 28 cases of advanced HCCs for Dnmt1 and Dnmt3a, and compared the results with those of 9 cases of low-grade DNs, 24 cases of high-grade DNs, and 59 cases of nonneoplastic liver tissues from 59 cases of surgically resected livers by immunohistochemical staining. Nuclear expression of Dnmt1 was increased significantly in all HCCs in DNs and advanced HCCs compared with those of nonneoplastic livers, low-grade DNs, and high-grade DNs (P <0.05). Nuclear expression of Dnmt3a was not detectable in nonneoplastic liver and low-grade DN, whereas it was observed in high-grade DNs (7 of 24, 29.2%), HCCs in DNs (7 of 13, 53.8%), and advanced HCCs (11 of 28, 39.3%). Different from Dnmt1 immunostaining, cytoplasmic immunoreactivity for Dmnt3a was significantly decreased or absent in 13 of 24 cases of high-grade DNs (54.1%), 12 of 13 cases of HCCs in DNs (92.3%), and 22 of 28 cases of advanced HCCs (78.6%), compared with nonneoplastic livers and low-grade DNs (P <0.05). Our data suggest that Dnmt1 and Dnmt3a play a role in the early stage of hepatocarcinogenesis and that dysregulation of Dnmt3a may be involved in the progression of HCC. Furthermore, the significantly decreased cytoplasmic immunoreactivity for Dnmt3a in high-grade DNs and HCCs can be used as a diagnostic adjunct. HUM PATHOL 34:11-17. Copyright 2003, Elsevier Science (USA). All rights reserved.
Abbreviations:
CN
, cirrhotic nodule,
DN
, dysplastic nodule,
Dnmt
, DNA methyltransferase,
HBV
, hepatitis B virus,
HCC
, hepatocellular carcinoma,
HCV
, hepatitis C virus
The methylation of DNA is an epigenetic modification involved in the control of gene expression of mammalian cells.1 In mammalian cells, approximately 3% to 5% of the cytosine residues in genomic DNA are present as 5-methylcytosine, 70% to 80% of which are found in CpG dinucleotide-rich regions termed “CpG islands.”2, 3 In normal cells, CpG methylation patterns in genomes can be maintained precisely through DNA replication and mitosis via the action of a DNA methyltransferase (Dnmt).4 In cancer cells, however, genome-wide hypomethylation is a general phenomenon,5 and specific CpG island sequences are hypermethylated.6, 7 Such aberrant methylation of CpG islands is one of the most consistent epigenetic mechanisms of gene expression in human cancers.7 Hypermethylation of tumor-suppressor genes causes gene inactivation, leading to functional impairment.8 Furthermore, aberrant DNA methylation facilitates gene mutation9 or is closely associated with allelic loss.10, 11, 12
The enzymatic methylation machinery is composed of 3 catalytically active Dnmts: Dnmt1, Dnmt3a, and Dnmt3b. Dnmt1 is the most abundant Dnmt targeted to replication foci and has a 10- to 40-fold preference for hemimethylated DNA substrates.13, 14 It seems to be the main enzyme responsible for copying the methylation pattern after each round of DNA replication.15 However, studies of Dnmt1-deficient embryonic stem cells have revealed that other enzymes must exist for de novo methylation to take place after the wave of global demethylation that occurs during early embryonic development.16, 17 Independently encoded new Dnmts, such as Dnmt2,18 Dnmt3a, and Dnmt3b,19 have been identified recently. Dnmt3a and Dnmt3b are essential for embryonic development and are responsible for the de novo methylation during embryogenesis and tumorigenesis.20, 21
Dnmts have been found to be overexpressed in tumorigenic cells22 and in a few types of human tumors.23, 24 An increased level of Dnmt1 mRNA is known to be dependent on cell proliferation25, 26, 27, 28 and seems to be regulated by a posttranscriptional mechanism, such as differential mRNA stability in normal somatic cells.26 Thus the increased Dnmt1 enzymatic activity might not always be correlated with the Dnmt1 mRNA levels in cancer cells. Dnmt3a and 3b are differentially expressed in normal and tumor cells. Dnmt3b mRNA levels were nearly undetectable, whereas Dnmt3a mRNA levels were less sensitive to cell cycle alterations and maintained at a slightly higher level in tumor cell lines than in normal cell strains.29 However, contrasting results on the expression of Dnmts and decreased methylation of CpG dinucleotides have been reported in human colon cancers.30, 31 In the development of human hepatocellular carcinomas (HCCs), the average levels of Dnmt1 and Dnmt3a mRNA were significantly higher in noncancerous liver tissues showing chronic hepatitis or cirrhosis and much higher in HCCs than in histologically normal liver tissues.32 However, data on Dnmt expression in human HCCs are not available. In the present study, we investigated the expression of Dnmt1 and Dnmt3a in nonneoplastic liver, including cirrhotic nodules (CNs), low-grade and high-grade dysplastic nodules (DNs), HCCs arising in DNs, and advanced HCCs by immunohistochemical analysis to characterize the different degrees of expression of Dnmt1 and Dnmt3 during multistep human hepatocarcinogenesis.
Materials and methods  Tissue samples We selected 59 cases of surgically resected livers from the surgical pathology files of the Department of Pathology, Asan Medical Center from May 1996 to April 2000. These cases included 9 low-grade DNs, 24 high-grade DNs, and 41 HCCs. Diagnosis of DNs and HCCs was made according to previously described criteria.33 Thirteen cases of HCCs arose within the high-grade DNs, and 28 cases were advanced HCCs. Nonneoplastic livers displayed cirrhotic nodules in 47 cases and chronic hepatitis in 9 cases, and were unremarkable in 3 cases. Fifty cases were associated with hepatitis B virus (HBV) infection, and 6 cases were positive for hepatitis C virus (HCV) RNA or an antibody for HCV. Three cases were negative for both HBV and HCV. Cells HepG2 (human hepatoblastoma cell line) and Hep3B (human hepatocellular carcinoma cell line) were cultured in Dulbeco's modified essential medium with 10% fetal calf serum in 5% CO2 incubation at 37°C. Cells of 2×105/mL were plated on round cover slips measuring 12 mm in diameter and cultured in 24-well culture plates. Immunohistochemical and immunofluorescent staining Sections (4 to 6 μm thick) were cut from 10% buffered formalin-fixed, paraffin-embedded tissues. The sections were mounted on poly-L-lysine–coated glass slides and baked at 60°C for 15 minutes. Slides were deparaffinized in xylene, rehydrated in graded alcohol, and washed in tap water. Endogeneous peroxidase activity was blocked by incubating sections with 3% H2O2 . Slides were placed in a steam cooker filled with 10 mmol sodium citrate buffer, pH 6.0, for antigen retrieval. After treatment with 10% normal goat serum for 10 minutes to block nonspecific protein binding, mouse monoclonal antibodies for Dnmt1 (IMG-261, 60B1220, dilution 1:125; Imgenex, San Diego, CA) and Dnmt3a (IMG-268, 64B1446, dilution 1:125; Imgenex) were applied for 1 hour. After reaction with a biotinylated anti-mouse antibody for 1 hour, antigen-antibody complexes were visualized using a streptavidin-horseradish peroxidase conjugate (LSAB kit; Dako, Carpenteria, CA) with diaminobenzidine as a chromogen. Slides were counterstained with Harris's hematoxylin for 3 to 5 minutes. Both nuclear and cytoplasmic immunopositivity was evaluated using the scoring system according to the staining intensity (0, negative; 1, mild; 2, moderate; 3, severe) and the proportion of positive cells (0, negative; 1, positive cells in ≤10% of cells; 2, positive cells in >10% and ≤1/3 of cells; 3, positive cells in >1/3 and ≤2/3 of cells; 4, positive cells in >2/3 of cells). Two scores were added in each case, and the expression was graded as negative (0), weakly positive (1 or 2), intermediate positive (3 to 5), and strongly positive (6 or 7). Cultured cells were fixed in 2% paraformaldehyde for 20 minutes when they were in 70% to 80% confluency and further penetrated in 0.2% Triton X for 10 minutes. The cells were incubated in the anti-Dnmt1 and Dnmt3a antibodies for 1 hour and then in fluorescein isothiocyanate–conjugated anti-mouse antibody for 30 minutes. Cells were stained for DNA with 0.5 mg/mL bisbenzimide (Hoechst 33258; Sigma, St. Louis, MO) in phosphate-buffered saline for 5 minutes. Cells and tissue sections were examined with an Olympus light microscope and an Olympus Venox fluorescent microscope (Olympus Corp., Lake Success, NY). Statistical analyses The SAS program (SAS Institute, Cary, NC) was used for all statistical analyses. The grade of immunoreactivity for Dnmt1 and Dnmt3a in the nucleus and cytoplasm in nonneoplastic liver tissues, low-grade DNs, high-grade DNs, HCCs in DNs, and advanced HCCs were compared using Cochran-Mantel-Haenszel tests for correlated ordered categorical outcomes. A P value <0.05 was considered significant, and all P values were 2-sided. Results As a positive control, 2 liver cancer cell lines, Hep3B and HepG2, were analyzed for the expression of Dnm1 and Dnmt3a by immunoflourescent staining. Diffuse and granular staining for Dnmt1 was exclusively observed in the nucleus of cells in interphase, and cells in metaphase showed localization of Dnmt1 in chromosomes (Fig 1A and B).
The expression of Dnmt3a was similar to that of Dnmt1; however, a few cells also displayed granular cytoplasmic stainability ( Fig 1C and D). CNs, low-grade DNs, high-grade DNs, and HCCs were classified on the basis of morphologic characteristics as were previously described (Fig 2A, B, C, and D).33 Immunoreactivity for Dnmt1 in tissue sections was evident only in nuclei of liver cells and sinusoidal lymphocytes (Fig 3).
Immunopositivity for Dnmt1 was sequentially increased through the multistep hepatocarcinogenesis ( Fig 3A, B, C, and D; Table 1).
Forty-two of 59 (71.2%) nonneoplastic liver tissues showed no immunoreactivity ( Fig 3A); 12 of 59 (20.3%) displayed weak immunopositivity and 5 of 59 (8.5%) displayed intermediate immunopositivity. In 9 cases of low-grade DNs, immunoreactivity for Dnmt1 was not detectable in 5 (55.6%) and weak in 4 (44.4%) ( Table 1). In high-grade DNs, 15 of total 24 cases (62.5%) displayed weak immunopositivity ( Fig 3B), and 9 cases (37.5%) showed no detectable immunoreactivity for Dnmt1. In HCCs arising in DNs, the immunoreactivity for Dnmt1 was weak in 8 cases (61.5%), intermediate in 4 cases (30.8%), and strong in 1 case (7.7%) ( Fig 3C). The increase in Dnmt1 immunoreactivity in HCCs in DNs was statistically significant compared with the Dnmt1 immunoreactivity in nonneoplastic livers ( P <0.0001), low grade DNs ( P=0.0028) and high-grade DNs ( P=0.004). All 28 cases of advanced HCCs displayed Dnmt1 nuclear immunoreactivity with 10 cases (35.7%) showing strong immunoreactivity ( Fig 3D), 14 cases (50.0%) showing intermediate immunoreactivity, and 4 cases (14.3%) showing weak immunoreactivity for Dnmt1. There was a statistically significant difference in the Dnmt1 nuclear immunoreactivity between advanced HCCs and HCCs in DNs ( P=0.0031). | | |  | | Nuclear immunoreactivity for Dnmt 1 (%) | | |
|---|
| Pathologic diagnosis | − | + | ++ | +++ | Total cases | |
|---|
| Nonneoplastic liver | 42 (71.2) | 12 (20.3) | 5 (8.5) | 0 (0.0) | 59 | |
| Low-grade DN | 5 (55.6) | 4 (44.4) | 0 (0.0) | 0 (0.0) | 9 | |
| High-grade DN | 9 (37.5) | 15 (62.5) | 0 (0.0) | 0 (0.0) | 24 | |
| HCC in DN* | 0 (0) | 8 (61.5) | 4 (30.8) | 1 (7.7) | 13 | |
| Advanced HCC† | 0 (0) | 4 (14.3) | 14 (50.0) | 10 (35.7) | 28 | |
| *Statistically significant difference of nuclear Dnmt1 immunoreactivity between HCC in DN and high-grade DN using Cochran-Mantel-Haenszel tests (P = 0.004). †Statistically significant difference of nuclear Dnmt1 immunoreactivity between advanced HCC and HCC in DN using Cochran-Mantel-Haenszel tests (P = 0.0031). There was no significant difference of nuclear Dnmt1 immunoreactivity between nonneoplastic liver, low-grade DN, and high-grade DN. | | | | |
Dnmt3a immunopositivity was detected both in the nucleus (Table 2) and in the cytoplasm (Table 3).
Nuclear immunoreactivity was not detected in any case of nonneoplastic liver (Fig 4A and B) or low-grade DN (Fig 4C).
In some high-grade DNs and HCCs, nuclear immunoreactivity for Dnmt3a was observed ( Table 2). Seven of 24 cases (29.2%) of high-grade DNs and 7 of 13 cases (53.8%) of HCCs in DNs displayed weak to intermediate nuclear immunopositivity for Dnmt3a ( Table 2). In advanced HCCs, 11 of 28 cases (39.3%) showed variable degrees of nuclear immunoreactivity for Dnmt3a (Fig 5).
Although fewer cases displayed nuclear immunoreactivity for Dnmt3a in advanced HCCs than in HCCs in DNs (39.7% versus 53.8%), the staining intensity was stronger and the number of positive tumor cells was higher in advanced HCCs than in HCCs in DNs ( Table 2). The difference in nuclear Dnmt3a immunopositivity was not statistically significant between cases of low-grade DN and high-grade DN, between cases of high-grade DN and HCC in DN, and between cases of HCC in DN and advanced HCC. However, there was a statistically significant difference between low-grade DNs and HCCs in DNs ( P=0.0146) or advanced HCCs ( P=0.0494) ( Table 2). | | | | | Nuclear immunoreactivity for Dnmt3a (%) | | |
|---|
| Pathologic diagnosis | − | + | ++ | +++ | Total cases | |
|---|
| Nonneoplastic liver | 59 (100.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 59 | |
| Low-grade DN | 9 (100.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 9 | |
| High-grade DN | 17 (70.8) | 7 (29.2) | 0 (0.0) | 0 (0.0) | 24 | |
| HCC in DN* | 6 (46.2) | 6(46.2) | 1 (7.6) | 0 (0.0) | 13 | |
| Advanced HCC† | 17 (60.7) | 5 (17.9) | 4 (14.3) | 2 (7.1) | 28 | |
| *Statistically significant difference of nuclear Dnmt3a immunoreactivity between HCC in DN and low-grade DN using Cochran-Mantel-Haenszel tests (P = 0.0146) †Statistically significant difference of nuclear Dnmt3a immunoreactivity between advanced HCC and low-grade DN using Cochran-Mantel-Haenszel tests (P = 0.0494). There was no significant difference of nuclear Dnmt3a immunoreactivity between low-grade DN and high-grade DN, between high-grade DN and HCC in DN, and between HCC in DN and advanced HCC. | | | | |
| | | | | Cytoplasmic immunoreactivity of Dnmt3a (%) | | |
|---|
| Pathologic diagnosis | − | + | ++ | +++ | Total cases | |
|---|
| Nonneoplastic liver | 0 (0.0) | 0 (0.0) | 1 (1.7) | 58 (98.3) | 59 | |
| Low-grade DN* | 0 (0.0) | 1 (11.1) | 0 (0.0) | 8 (88.9) | 9 | |
| High-grade DN† | 2 (8.3) | 11 (45.8) | 7 (29.2) | 4 (16.7) | 24 | |
| HCC in DN§ | 5 (38.5) | 7 (53.8) | 0 (0.0) | 1 (7.7) | 13 | |
| Advanced HCC | 3 (10.7) | 19 (67.9) | 5 (17.8) | 1 (3.6) | 28 | |
| *Statistically significant difference of cytoplasmic Dnmt3a immunoreactivity between low grade DN and nonneoplastic liver using Cochran-Mantel-Haenszel tests (P = 0.0351). †Statistically significant difference of cytoplasmic Dnmt3a immunoreactivity between high-grade DN and low-grade DN using Cochran-Mantel-Haenszel tests (P = 0.0023). §indicates statistically significant difference of cytoplasmic Dnmt3a immunoreactivity between HCC in DN and high-grade DN using Cochran-Mantel-Haenszel tests(P = 0.0115). There was no significant difference of cytoplasmic Dnmt3a immunoreactivity between HCC in DN and advanced HCC. | | | | |
In addition to nuclear immunoreactivity, cytoplasmic immunopositivity was also present for Dnmt3a (Fig 4A and B). In all cases of nonneoplastic livers, a diffusely strong (58 of 59, 98.3%) or intermediate (1 of 59, 1.7%) degree of cytoplasmic immunopositivity was observed (Fig 4B and Table 3). In low-grade DNs, 8 of 9 cases (88.9%) displayed strong cytoplasmic immunopositivity and 1 case (11.1%) showed weak cytoplasmic immunopositivity. In high-grade DNs, cytoplasmic immunoreactivity for Dnmt3a was significantly lower than that of nonneoplastic liver and low-grade DNs (Table 3 and Fig 4C). More than 50% of total cases with high-grade DN showed no detectable or only weak cytoplasmic immunoreactivity for Dnmt3a; only 4 cases (16.7%) displayed strong immunopositivity. Of the 13 cases of HCC in DN, 5 (38.5%) displayed no detectable cytoplasmic immunoreactivity for Dnmt3a (Fig 4D), 7 (53.8%) showed weak cytoplasmic immunopositivity, and only 1 (7.7%) showed strong immunopositivity for Dnmt3a. Sequential decreases in cytoplasmic immunoreactivity for Dnmt3a through multistep hepatocarcinogenesis was statistically significant in each type of liver tissue except for advanced HCCs (Table 3). In cases of advanced HCC, cytoplasmic expression of Dnmt3a was generally weak, but quite variable between case to case and area to area in the same case (Fig 5). Thus there was no statistical significance in the cytoplasmic immunoreactivity for Dnm3a between cases of HCC in DN and advanced HCC. Discussion Genome-wide hypomethylation and aberrant methylation of specific gene loci are characteristics of human cancers including HCCs.8, 34 Dnmts are the most important proteins involved in such abnormal methylation processes, and their transcript levels have been analyzed in various types of human cancers.15, 24, 32 However, their protein expression has been rarely examined in primary human cancers, such as colon and prostate cancers, due to the lack of specific antibodies for Dnmts.24, 35 Furthermore, Dnmt expression has never been examined in relation to precancerous lesions of human cancers. The recent commercial availability of monoclonal antibodies for Dnmts led us to examine Dnmt expression in nonneoplastic and neoplastic liver tissues as well as precancerous dysplastic liver tissues. In the present study, we first demonstrated different degrees of Dnmt1 and Dnmt3a immunoreactivity in different stages of multistep hepatocarcinogenesis. HCC is well documented to develop through the stages of dysplasia and early HCC in the background of chronic liver diseases, including chronic hepatitis and liver cirrhosis. Thus HCC with a nodule-in-nodule pattern arising in DN is representative of the early stage of HCC in multistep hepatocarcinogenesis. Regarding Dnmt1 expression, there was no significant difference in immunoreactivity between nonneoplastic liver tissues and DNs, but the immunopositivity for Dnmt1 was significantly increased in HCCs in DNs and in advanced HCCs. These results indicate that Dnmt1 may play an important role in the early stage of HCC development. Furthermore, the degree of Dnmt1 expression was even stronger in advanced HCCs than in HCCs arising in DNs. This result further suggests that Dnmt1 is also involved in the progression of HCCs. In the present study, 28.8% of nonneoplastic liver tissues with either chronic hepatitis or liver cirrhosis showed variable nuclear immunopositivity for Dnmt1. The result was compatible with the previous report showing that the level of Dnmt1 mRNA was significantly higher in chronic hepatitis and cirrhosis than in normal liver tissues adjacent to HCCs.32 Immumolocalization of Dnmts in cancer cells or tissues has been reported only rarely. Using a polyclonal antibody that they developed, De Marzo et al24 detected Dnmt1 expression only in the nucleus of normal human and colon cancer tissues. In the present study, unlike the immunoreactivity for Dnmt1, immunoreactivity for Dnmt3a was detected in the cytoplasm as well as in the nucleus. Furthermore, strong immunopositivity of Dnmt3a was detected consistently in the cytoplasm of nonneoplastic hepatocytes and in low-grade DNs. The cytoplasmic immunoreactivity for Dnmt3a may be attributable to either the presence of Dnmt3a in the cytoplasm or a cross-reactivity to another proteins. The sequential decrease in cytoplasmic immunoreactivity for Dnmt3a and the concurrent increase in nuclear Dnmt3a in high-grade DNs and in HCCs arising in DNs suggest that nucleocytoplasmic shuttling of Dnmt3a may be involved in the development of HCCs, and the nuclear expression of Dnmt3a may be closely related to DNA methylation. From a diagnostic standpoint, the significant decrease in cytoplasmic immunoreactivity for Dnmt3a in HCCs in DNs can be useful in the diagnosis of carcinoma developed from DN. The degree of nuclear Dnmt3a expression was generally lower than that of Dnmt1, and the difference in increased immunoreactivity for Dnmt3a between high-grade DNs and HCCs was not statistically significant. However, nuclear Dnmt3a and Dnmt1 may cooperate to silence genes in the early stage of hepatocarcinogenesis, as suggested by Rhee.36 Furthermore, the heterogeneity of Dnmt3a expression in the cytoplasm as well as in the nucleus of advanced HCCs suggests that a progressive dysregulation of Dnmt3a expression is involved in the late stage of human hepatocarcinogenesis and may contribute much more to the abnormal CpG island methylation patterns of dysplastic hepatocytes and HCC cell DNA as in the case of Dnmt1 in human colon cancers.24 The interaction of Dnmt3a with other nuclear proteins involved in cell cycle or proliferation is a topic for future study. In addition, the significance of the cytoplasmic immunopositivity for Dnmt3a should be further analyzed to elucidate the functional role of Dnmt3a in human hepatocarcinogenesis, In conclusion, our data suggest that nuclear Dnmt1 and Dnmt3a play an important role in the early stage of multistep human hepatocarcinogenesis, and dysregulation of Dnmt3a is involved in the progression of HCC. Furthermore, the decreased cytoplasmic immunoreactivity of Dnmt3a in high-grade DNs and HCCs arising in DNs compared with the surrounding DNs can be useful in the diagnosis of high-grade DNs and well-differentiated HCCs in the needle biopsy samples. Acknowledgements We thank Ms. Yoon Jung Lee and Ms. Se Jin Shon for their technical assistance. References 1.
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Departments of Pathology, Internal Medicine, and Surgery, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Korea and Department of Biological Sciences and Bio/Molecular Informatics Center, Konkuk University, Seoul, Korea ☆ Supported by the Korean Ministry of Science and Technology through the 21C Frontier Project, Functional Genomics of the Human Genome, grant FG-1-4-01 (to Y.K.P.). ☆☆ Address correspondence and reprint requests to Eunsil Yu, MD, PhD, Department of Pathology, University of Ulsan College of Medicine, Asan Medical Center, 388-1 Pungnap-dong, Songpa-gu, Seoul 138-736, Korea. PII: S0046-8177(02)29705-1 doi:10.1053/hupa.2003.5 © 2003 Elsevier Science (USA). All rights reserved. | |
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