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Clinical Cancer Research Vol. 11, 7354-7361, October 15, 2005
© 2005 American Association for Cancer Research

Imaging, Diagnosis, Prognosis

The Up-Regulation of Y-Box Binding Proteins (DNA Binding Protein A and Y-Box Binding Protein-1) as Prognostic Markers of Hepatocellular Carcinoma

Mahmut Yasen1,2, Kazunori Kajino1, Sayaka Kano1, Hiroshi Tobita1, Junji Yamamoto3, Takeshi Uchiumi4, Shigeyuki Kon5, Masahiro Maeda5, Gulanbar Obulhasim1, Shigeki Arii2 and Okio Hino1

Authors' Affiliations: 1 Second Department of Pathology, Juntendo University School of Medicine, Tokyo, Japan; 2 Department of Hepato-Biliary-Pancreatic Surgery, Tokyo Medical and Dental University, Tokyo, Japan; 3 Department of Surgery, Japanese Foundation for Cancer Research, Cancer Institute Hospital, Tokyo, Japan; 4 Department of Molecular Biology, University of Occupational and Environmental Health, Fukuoka, Japan; and 5 Immuno-Biological Laboratories Co., Ltd., Gunma, Japan

Requests for reprints: Okio Hino, Second Department of Pathology, Juntendo University School of Medicine, 2-1-1, Bunkyo-ku, Hongo, 113-8421 Tokyo, Japan. Phone: 81-3-5802-1038; Fax: 81-3-5684-1646; E-mail: ohino{at}med.juntendo.ac.jp.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: The development of hepatocellular carcinoma is associated with the chronic inflammation of the liver caused by various factors such as hepatitis B or C virus infection. Previously, we reported DNA binding protein A (dbpA) as a candidate molecule that can accelerate inflammation-induced hepatocarcinogenesis. DbpA belongs to the Y-box binding protein family, and Y-box binding protein-1 (YB-1), the prototype member of this family, is reported to be a prognostic marker of malignant diseases other than hepatocellular carcinoma. The purpose of this study is to examine the significance of the expression of dbpA or of the T-to-G transversion in the dbpA promoter region, which enhances the promoter activity in vitro, for the progression of hepatocellular carcinoma.

Experimental Design: We studied the expression of dbpA (as well as of YB-1) in 82 formalin-fixed hepatocellular carcinoma tissues by immunohistochemistry and determined the sequence of the dbpA promoter region in 42 frozen hepatocellular carcinoma tissues. We examined the relationship between these findings and the clinicopathologic factors of hepatocellular carcinoma patients.

Results: DbpA expression was associated with the advanced stages of hepatocellular carcinoma, and the cases with the nuclear dbpA expression had a poor prognosis. DbpA contributed more significantly to this association than YB-1. Furthermore, the T-to-G transversion in the dbpA promoter region was related to the nuclear localization of dbpA.

Conclusion: DbpA was a more significant prognostic marker of hepatocellular carcinoma than YB-1. The T-to-G transversion in the dbpA promoter region was suggested to be a predisposing factor for the progression of hepatocellular carcinoma.

Hepatocellular carcinoma is the sixth most common cancer in the world (1), but it is the third most common cause of death from cancer because of its poor prognosis. It is particularly common in China, Japan, and African countries. Various screening and treatment protocols have been used for hepatocellular carcinoma during the past 25 years but they have not significantly improved the chances of survival of patients. A major risk factor affecting hepatocellular carcinoma development is the persistent inflammation of the liver caused by chronic hepatitis B or C virus infection. We previously reported that DNA binding protein A (dbpA) was a candidate molecule to accelerate the process of the inflammation-induced hepatocarcinogenesis (2). DbpA belongs to the Y-box binding protein family and its expression is enhanced in the cellular proliferative state, such as the regenerating liver of rat and mouse (3, 4).

Members of Y-box binding protein family all contain a DNA binding domain, called the cold shock domain, of ~80-amino-acid residues, which is highly conserved from prokaryotes to eukaryotes (5). Y-box binding protein-1 (YB-1) is the prototype member of this family and was originally isolated as the DNA binding protein recognizing the Y-box sequence in the MHC class II gene promoter region (6). This box is found in the promoter region of several genes involved in positive regulation of cell cycle, such as proliferating cell nuclear antigen, DNA topoisomerase II{alpha}, and MDR1 (7–10). Consequently, YB-1 is thought to play crucial roles in cell proliferation in carcinogenesis (11–13). Also, another member of this family, dbpA, was identified as the protein binding to the epidermal growth factor receptor enhancer or c-erbB-2 promoter (8). These Y-box binding proteins are reported to have multiple functions, such as the regulations of transcription and translation (14).

Recently, YB-1 has been reported to be a prognostic marker of breast cancer (15), ovarian cancer (16), lung cancer (17), thyroid cancer (18), and synovial sarcoma (19). These findings indicate the significance of YB-1 for the progression of malignant diseases, but thus far there is no report of its expression in hepatocellular carcinoma.

In a previous report, we showed the occurrence of a T-to-G transversion in the dbpA promoter region (20). The promoter sequence having this transversion has a higher promoter activity than the wild-type promoter in vitro, but its significance in vivo is not known. There has been no study regarding the expression of dbpA or about the significance of the T-to-G transversion in human hepatocellular carcinoma.

Here, we studied the relationship between the expression of dbpA as well as of YB-1 and the clinicopathologic factors of hepatocellular carcinoma patients. Our data showed that their expression was associated with the advanced stages of hepatocellular carcinoma and that the involvement of dbpA was more obvious than that of YB-1 for the progression of hepatocellular carcinoma. Moreover, the T-to-G transversion in the dbpA promoter region was related to the nuclear localization of dbpA, and hepatocellular carcinoma cases with the nuclear dbpA expression had a poor prognosis.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Patients. This study included 82 hepatocellular carcinoma patients who had undergone surgical resection of hepatocellular carcinoma between May 2000 and September 2004 in the Department of Hepato-Biliary-Pancreatic Surgery in Tokyo Medical and Dental University. All samples were obtained with the patients' informed consents. A part of the resected sample was fixed in formalin and embedded in paraffin for histologic diagnosis and for immunohistochemical study. In 42 cases, the tissue was also frozen and kept at –80°C for DNA analysis. Histologic diagnosis was made when two pathologists specializing in liver disease reached the same conclusion. The patients consisted of 60 (73.2%) males and 22 (26.8%) females, 28 to 83 (mean 65.1) years old. The clinical and pathologic classifications of hepatocellular carcinoma, including its stages from I to IV, were based on the previously published criteria (21). Fourteen (17.1%) patients were hepatitis B surface antigen–positive and 45 (54.9%) were positive for hepatitis C virus antibody. Other clinicopathologic features are shown in Table 1.


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  		Table 1. Expression of dbpA or YB-1 and clinicopathologic findings

 
Preparation of anti–Y-box binding protein-1 and anti-DNA binding protein A antibodies. Mouse monoclonal anti–YB-1 and rabbit polyclonal anti-dbpA antibodies were prepared as follows. For anti–YB-1 antibody, whole YB-1 expressed in Escherichia coli as a glutathione S-transferase–tagged fusion protein was purified by chromatography on glutathione Sepharose 4B (Amersham Biosciences, Piscataway, NJ). The purified glutathione S-transferase–tagged YB-1 was coupled with thyroglobulin and injected into mice. The splenocytes of the mice were fused with a myeloma cell line X63-Ag8.653. The supernatants of hybridoma cells were screened by their reactivity to YB-1 expressed in E. coli as a His-tagged fusion protein, and several positive clones were selected by the limiting dilution method; one clone, 21A3, was chosen for use in this study. For anti-dbpA, rabbits were immunized with the synthetic peptide TENPAPPTQQSSAE coupled with thyroglobulin. This peptide sequence corresponds to the COOH-terminal end of dbpA. The IgG fraction was obtained from the sera of the immunized rabbits using a column of thiol-Sepharose beads (Amersham Biosciences) coupled with the peptide used for immunization following the method described previously (22). Eluted IgG was designated anti-dbpA (COOH terminal).

Expression of Y-box binding protein-1 or DNA binding protein A in Escherichia coli and Western blotting to confirm the specificity of antibodies. Whole dbpA or YB-1 coding regions were inserted into pQE vector (Qiagen, Chatsworth, CA) so that they could be expressed in E. coli as His-tagged fusion proteins, using QIAexpressionist system (Qiagen). E. coli containing the expressed dbpA or YB-1 and HepG2 cells were lysed in a solution containing 2% SDS, 10% glycerol, 50 mmol/L Tris-HCl (pH 6.8), and 100 mmol/L DTT and were then boiled. These crude lysates were electrophoresed on 10% Laemmli gels (23) and transferred to a nitrocellulose membrane, BA85 (Schleicher & Schuell, Keene, NH). The two identical membranes (Fig. 1, left and right) were blocked in 1% skim milk in PBS with 0.1% Tween 20 for 1 hour at room temperature. Each of the two membranes was incubated with 1 µg/mL anti-dbpA (COOH terminal; Fig. 1, left) or anti–YB-1 (21A3; Fig. 1, right) in 1% skim milk in PBS with 0.1% Tween 20 for 1 hour at room temperature. Goat anti-rabbit immunoglobulin (for dbpA) or goat anti-mouse immunoglobulin (for YB-1) conjugated to peroxidase-labeled dextran polymer (Envision K4002 or K4000; DAKO, Carpinteria, CA) was used as secondary antibody and was diluted 50 times in 1% skim milk in PBS with 0.1% Tween 20. The reaction was done at room temperature for 1 hour. DbpA or YB-1 on the membranes was visualized by the enhanced chemiluminescence detection system (Amersham Biosciences).



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  		Fig. 1. No cross-reactivity between anti-dbpA and anti–YB-1. DbpA (lanes 1) or YB-1 (lanes 2) expressed in E. coli or HepG2 lysate (lanes 3) were electrophoresed on 10% polyacrylamide gels and transferred to nitrocellulose membranes. The immobilized proteins were detected by reaction with anti-dbpA (COOH terminal; left) or by anti–YB-1 (21A3; right).

 
Immunohistochemistry. Four-micrometer-thick tissue sections were prepared from formalin-fixed, paraffin-embedded specimens. After deparaffinization, tissue sections were heated in 10 mmol/L citrate buffer (pH 6.0) for antigen retrieval and treated with 3% hydrogen peroxide. Then, the sections were incubated with primary antibodies, anti-dbpA (COOH terminal) or anti–YB-1 (21A3), 1 µg/mL each, in PBS with 0.1% Tween 20 at room temperature overnight. As secondary antibodies, Envision K4002 or K4000 (for dbpA or YB-1, respectively), without dilution, was applied to the tissue sections. 3,3'-Diaminobenzidine was used as the substrate for peroxidase. Staining was evaluated by two independent observers and interpreted to be positive when >10% of tumor cells showed the positive signal. As a negative control, we stained the normal liver tissue obtained by the surgical resection of metastatic liver cancer (originated from breast cancer).

Determination of the DNA binding protein A promoter sequence. Genomic DNA was extracted from 42 frozen samples by the phenol/chloroform method (24). The dbpA promoter region was amplified by PCR using Advantage-GC Genomic PCR kit (BD Biosciences Clontech, Palo Alto, CA) with 25-mer sense (5'-GACTCCCGAGCCAAGTTTCCCCACCC-3') and antisense (5'-CACGAAGCTCGAGCCGCCTCCGCCA-3') primers. The sequence of the PCR product was directly determined and compared with the sequence in Genbank database (Genbank accession no. L29064). If the result of the direct sequence suggested the T-to-G transversion at the position –6 (six nucleotide upstream from the transcription initiation site) in the dbpA promoter region, the PCR product was cloned and the sequences of 10 clones per one PCR product were determined.

Statistical analysis. Student's t test was used to analyze differences in age of patients. Fisher's exact test or {chi}2 test was used to compare categorical data between groups. The disease-free survival rate was calculated by the Kaplan-Meier method and rates are reported with 95% confidence intervals. Differences were tested for significance using the log-rank test. The disease-free survival rate was measured from the date of resection until the date when the recurrence of hepatocellular carcinoma was detected or when the patient died. P < 0.05 was deemed to be statistically significant.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Specificity of anti-DNA binding protein A and anti–Y-box binding protein-1 antibodies. Western blotting confirmed that anti-dbpA and anti–YB-1 did not cross-react with each other. As shown in Fig. 1, anti-dbpA (COOH terminal) or anti–YB-1 (21A3) detected only the 60 kDa dbpA (lanes 1) or the 50 kDa YB-1 (lanes 2) expressed in E. coli. These antibodies also detected the corresponding antigens in HepG2 lysate (lanes 3). The data proved that the antibodies used in this study specifically recognized the antigens used for immunization. Figure 2 shows the representative data of immunohistochemical staining. The hepatocytes in the normal liver used as a negative control did not show any signal of dbpA nor of YB-1, whereas the tumor part (metastatic liver cancer originated from breast cancer) in the same slice showed the positive signals (Fig. 2, left column; a tumor part is in the left bottom corner). In the normal liver, the bile duct cells showed positivity for dbpA but not for YB-1. In Fig. 2 (middle or right column), the representative hepatocellular carcinoma tissues having their main expression in the cytoplasm or in the nucleus are shown, respectively.



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  		Fig. 2. Representative immunohistochemical data of the expression of dbpA or YB-1. Left column (control), the normal liver adjacent to the metastatic liver cancer. The part of metastatic liver cancer is in the left bottom corner. Middle (C) or right (N) column, hepatocellular carcinoma (HCC) in which dbpA or YB-1 is mainly expressed in the cytoplasm or in the nucleus, respectively. HE, H&E staining.

 
Similar but not identical distribution of DNA binding protein A or Y-box binding protein-1–positive cells in the liver. The distributions of dbpA- or YB-1–positive cells in hepatocellular carcinoma were similar but not the same (Fig. 3, left column). H&E staining of hepatocellular carcinoma (left column, top) showed three cancerous nodules of which only two nodules were positive for dbpA (left column, middle) but all three were positive for YB-1 (left column, bottom). In cirrhotic hepatocytes of nontumorous region (middle column), both proteins were weakly expressed and the cells expressing the proteins were not as numerous as in hepatocellular carcinoma. DbpA was expressed in bile duct cells of both nontumorous and hepatocellular carcinoma but YB-1 was not.



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  		Fig. 3. Nonidentical expression of dbpA and YB-1. Hepatocellular carcinoma, nontumorous liver (NT), or portal vein invasion of hepatocellular carcinoma [(Vp+)] are in left, middle, or right columns, respectively.

 
Expression of DNA binding protein A or Y-box binding protein-1 and clinicopathologic features. Sixty-two percent (51 of 82) or 89% (73 of 82) of hepatocellular carcinoma cases were positive for the expression of dbpA or YB-1, respectively. All positive cases showed the expression of these proteins in the cytoplasm and some of them also showed the expression in the nucleus (9 of 51 or 8 of 73 for dbpA or YB-1, respectively). At first, we studied the relationship between the expression of these proteins regardless of their intracellular localization and clinicopathologic factors of hepatocellular carcinoma. As shown in Table 1, the expression of dbpA and YB-1 showed similar but not identical tendencies. Both were associated with portal vein invasion (P < 0.001 or P = 0.010 for dbpA or YB-1, respectively) and with the advanced stages of disease (III and IV; P = 0.017 or P = 0.024). DbpA was more significantly related to these factors than YB-1. A representative case of portal vein invasion of tissues positive both for dbpA and YB-1 is shown in Fig. 2 [right column; Vp(+)]. The expression of dbpA was furthermore associated with a higher level of serum {alpha}-fetoprotein (P = 0.017), hepatic vein invasion (P = 0.020), and to the undifferentiated state of hepatocellular carcinoma. Well-, moderately, or poorly differentiated hepatocellular carcinoma showed 35% (6 of 17), 66% (31 of 47), or 78% (14 of 18) positive incidence of dbpA (P = 0.025). YB-1 was not related to these factors. The expression of dbpA or YB-1 did not show association with positive hepatitis B surface antigen, anti–hepatitis C virus, or the other factors listed in Table 1.

Nuclear localization of DNA binding protein A or Y-box binding protein-1 and clinicopathologic features. As for the intracellular localization, both dbpA and YB-1 showed the following three patterns of expressions, respectively, for dbpA or YB-1: C(–) and N(–), 31 of 82 or 9 of 82; C(+) and N(–), 42 of 82 or 65 of 82; and C(+) and N(+), 9 of 82 or 8 of 82. There was no C(–) and N(+) case, indicating that all hepatocellular carcinomas positive in the nucleus (N) were positive in the cytoplasm (C) for both YB-1 and dbpA. We compared the clinicopathologic factors between N(+) and N(–) groups among the all C(+) cases (51 or 73 for dbpA or YB-1, respectively; Table 2). The nuclear dbpA was associated with the histologic structures. It was more frequently detected in a compact type (47%, 7 of 15) than trabecular (5%, 1 of 22), pseudoglandular (0 %, 0 of 8), and scirrhous types (17%, 1 of 6; P = 0.005). A compact type hepatocellular carcinoma generally corresponds to poorly differentiated or undifferentiated states (25). Supporting this association, 0% (0 of 6), 16% (5 of 31), and 29% (4 of 14) of well, moderate, and poorly differentiated hepatocellular carcinomas, respectively, were positive for the nuclear dbpA although this tendency was not statistically significant (P = 0.289). The nuclear YB-1 showed association with the hepatic vein invasion (P = 0.008).


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  		Table 2. Nuclear expression of dbpA or YB-1 and clinicopathologic findings

 
Expression of DNA binding protein A or Y-box binding protein-1 and disease-free survival rate. We were able to follow the postoperative course of 37 patients for longer than 3 years after the operation. The follow-up period until death or the end point of this study was 31 to 1,509 days (mean 677 days). Twelve of 37 patients survived for longer than 3 years without recurrence of hepatocellular carcinoma. Figure 4 shows the disease-free survival rate of dbpA(+/–) or YB-1(+/–) patients (A and B, respectively). There were no differences in the survival rate between dbpA(+) and dbpA(–) nor between YB-1(+) and YB-1(–) patients. Then, in the 21 dbpA(+) patients, we compared the survival rate between the 3 N(+) and 18 N(–) cases (Fig. 5A). All three N(+) patients died on the 58th, 67th, and 231st postoperative days, whereas 7 of 18 N(–) patients survived without recurrence of hepatocellular carcinoma for longer than 3 years. There was a significant difference in the disease-free survival rates between the dbpA N(+) and N(–) patients (P = 0.003; Fig. 5A). Similarly, in the 34 YB-1(+) patients, we compared the disease-free survival rate between the 2 N(+) and 32 N(–) cases. The difference of the disease-free survival rate between N(+) and N(–) cases was also statistically significant (P = 0.010; Fig. 5B).



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  		Fig. 4. The disease-free survival curves of dbpA (+)/(–) (A) or YB-1 (+)/(–) (B) patients.

 


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  		Fig. 5. The disease-free survival curve of the nuclear dbpA (+)/(–) (A) or the nuclear YB-1 (+)/(–) (B) patients.

 
T-to-G transversion in the DNA binding protein A promoter region and the nuclear localization of DNA binding protein A. We were able to analyze the T-to-G transversion at position –6 in the dbpA promoter region in 42 of 82 hepatocellular carcinoma cases. These 42 cases were divided into 6 N(+) and 36 N(–) cases and the frequency of the transversion was compared between these two groups. Among them, the transversion was detected in three cases. One case was due to single nucleotide polymorphism and two were the result of somatic mutations, confirmed by the comparison of the sequences amplified from hepatocellular carcinoma and nontumorous liver. Table 3 shows that the transversion was associated with the nuclear localization of dbpA (P = 0.048). We then studied the relationship between the transversion and the clinicopathologic features of hepatocellular carcinoma. Three transversion (+) cases all showed the hepatic vein invasion although it was seen in only 8 of 39 transversion (–) cases, and this difference was statistically significant (P = 0.014). As for the histologic structures, the transversion was seen only in compact-type hepatocellular carcinoma (3 of 14) and not in trabecular (0 of 18), pseudoglandular (0 of 6), or scirrhous (0 of 4) types, although this difference was not statistically significant (P = 0.091).


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  		Table 3. Nuclear dbpA expression and the T-to-G transversion in the promoter region of dbpA

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Previously, we reported dbpA as the candidate molecule to accelerate the inflammation-induced hepatocarcinogenesis. DbpA binds to the ssDNA via its cold shock domain and homodimerizes via the charged zipper domain in its COOH terminus.6 We suspect that homodimerization draws the two ssDNAs closer, thus making it possible for dbpA to accelerate the pairing of the ssDNA and increase the chance of inaccurate homologous recombination. In support of this concept, YB-1, which is the prototype member of this family and contains both the cold shock domain and charged zipper domain, accelerates the pairing of DNA (26). Here, we studied the significance of dbpA in the clinical viewpoint, for the progression of hepatocellular carcinoma, and showed that the expression of dbpA was associated with the advanced stages of hepatocellular carcinoma. The involvement of dbpA was more obvious than that of YB-1 for the progression of hepatocellular carcinoma. No signal was detected in hepatocytes of the normal liver that was used as the negative control of staining. Also, in animal experiments, their expression level in normal hepatocytes is reported to be very low (3, 4, 27). Our data also showed that they were expressed in metastatic liver cancer originated from breast cancer. We have not studied the significance of dbpA in breast cancer, but the expression of YB-1 is already reported to be associated with the progression of breast cancer (15).

YB-1 and dbpA share the structural features characteristic to the Y-box binding protein family. In both dbpA and YB-1, the DNA binding domain, called cold shock domain, is highly conserved at the nucleotide sequence level. Anti–YB-1 antibody used in this study was produced by immunizing mice with whole YB-1 protein expressed in E. coli and it was possible that the antibody recognized the cold shock domain and cross-reacted with dbpA. To rule out this possibility, we checked the reactivity of anti-dbpA or anti–YB-1 to the overexpressed dbpA or YB-1 by Western blotting. Figure 1 showed that these antibodies, anti-dbpA (COOH terminal) and anti–YB-1 (21A3), specifically recognized the immunized antigens. This result confirmed the specificity of the antibodies used in this study.

It has been suggested that YB-1 and dbpA are derived from a common ancestral gene (28). In these two genes, there are similarities of the motif structures in their transcriptional regulatory regions although the nucleotide sequences are poorly conserved. Characteristically, the regulatory regions of the two genes are highly GC-rich, and neither of them has a typical TATA box. Multiple E-boxes, recognized by basic helix-loop-helix proteins, such as c-Myc, and the binding sites for Sp1 family of transcription factors are conserved in these regions. This structural similarity of the promoter region could explain the similar, although not identical, expression pattern of the two genes studied here.

In all dbpA- or YB-1–positive cases, the proteins were detected in cytoplasm. In some of these positive cases, the proteins were also detected in the nucleus. Nuclear localization of YB-1 has been documented as a prognostic marker of breast, ovary, and lung cancers (15–17). Shibahara et al. (17) proposed that the translocation of YB-1 into the nucleus occurs during the process of the tumor progression in non–small cell lung cancer and emphasized the importance of the nuclear localization for the progression of the tumor. On the other hand, Ito et al. (18) argued that the expression of YB-1, regardless of intracellular localization, is important for the progression of thyroid cancer. Our analysis showed that the cytoplasmic expression of dbpA or YB-1 was associated with the progressed state of hepatocellular carcinoma as shown in Table 1 and that their nuclear localization, albeit infrequent, as evidenced by the low number of positive cases, was associated with poor prognosis (Fig. 5). Hence, our data of cytoplasmic expression and that of the nuclear localization did not concur. However, we were able to show that the expression of these proteins was generally associated with the advanced stages of hepatocellular carcinoma. Further studies are needed with a large number of cases to confirm the result obtained in this study. As hepatocellular carcinoma progresses, these proteins start to be expressed and detected, at first in the cytoplasm, and then, as hepatocellular carcinoma progresses to the more advanced stage, in the nucleus as well as in the cytoplasm, as suggested by Shibahara et al. for the expression of YB-1.

DbpA positivity was related to high serum {alpha}-fetoprotein value (Table 1). In the promoter region of {alpha}-fetoprotein (Genbank accession no. L34019), there are two "inverted CCAAT," the core sequence of Y-box; however, we do not have the functional data to prove that dbpA could positively regulate the expression of the {alpha}-fetoprotein gene.

Previously, we reported the existence of T-to-G transversion in the dbpA promoter region (20). It was the result of both single nucleotide polymorphism and of somatic mutation. The sequence containing this transversion has a higher promoter activity than the wild-type sequence in vitro (20). In this study, we examined the clinical significance of this transversion. We determined the sequence of dbpA promoter region in hepatocellular carcinoma and adjacent nontumorous of 42 cases. Among them, three showed the transversion, which was associated with the nuclear localization of dbpA (Table 3). The data suggested that the transversion, which leads to enhanced promoter activity in vitro (20), can also be a prognostic marker of hepatocellular carcinoma.

In conclusion, dbpA was associated with the advanced stages of hepatocellular carcinoma and its nuclear localization was suggested to be a marker of poor prognosis. YB-1 showed the similar tendency but the involvement of dbpA was more significant than YB-1. Furthermore, the T-to-G transversion in the dbpA promoter region was related to the nuclear dbpA expression. YB-1 has been reported to be a prognostic marker of several malignant diseases other than hepatocellular carcinoma and the present study showed that, as for hepatocellular carcinoma, dbpA can be a more significant prognostic marker than YB-1.


   Footnotes
 
Grant support: Grant-in-Aid for Cancer Research and Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, and Science and Technology of Japan and the Ministry of Health, Labor, and Welfare of Japan.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

6 K. Kajino, unpublished data. Back

Received 5/ 9/05; revised 7/12/05; accepted 7/25/05.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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