Purpose and Experimental Design: CDC25 genes are cell cycle-activating phosphatases that positively regulate the activity of cyclin-dependent kinase. CDC25A and CDC25B, being oncogenes, are overexpressed in a variety of human malignancies. To investigate the potential roles of CDC25s in hepatocellular carcinoma (HCC), expression of CDC25A and CDC25B was examined in human HCC samples.

Results: Reverse transcription-PCR showed that overexpression of CDC25A and CDC25B mRNAs was found in 9 of 13 (69%) and 4 of 13 (31%) HCCs, respectively. Immunohistochemistry of 59 HCCs showed marked increase in CDC25A expression, but not CDC25B, in HCC compared with noncancer tissues, and high expression of CDC25A in 33 of 59 (56%) HCCs. Overexpression of CDC25A in HCC was confirmed by Western blot analysis. High expression of CDC25A was associated with dedifferentiated phenotype and portal vein invasion (P = 0.001 and 0.031, respectively), and expression of CDC25A correlated well with proliferating cell nuclear antigen labeling index (P = 0.005). Univariate analysis indicated that high expression of CDC25A and proliferating cell nuclear antigen were both significant predictive factors for shorter disease-free survival (P = 0.004 and 0.039, respectively). Multivariate analysis indicated that CDC25A was an independent prognostic marker for disease-free survival (risk ratio for cancer relapse, 2.98; P = 0.029), even when analyzed with several clinicopathologic factors. On the other hand, expression of CDC25B did not correlate with any clinicopathological features.

Conclusion: Our findings suggest that CDC25A, but not CDC25B, could be used as an independent prognostic marker for HCC. Our data would also contribute to forward understanding of tumor biology of HCC that is associated with cell cycle regulation.

Primary HCC3 is one of the most common tumors in Southeast Asia and Africa with an estimated incidence of ∼30/100,000 men per year. The prognosis of HCC is generally poor, and the 5-year survival rate is limited to 25–49% after surgery (1, 2). In an effort to understand the biological features of this aggressive type of carcinoma, several studies have shown alterations of several key molecules including β-catenin, Rb, p53, and c-myc (3, 4, 5), but much remains to be clarified.

Mammalian cell cycle is controlled by sequential activation and inactivation of a family of CDKs. CDKs interact with a specific subset of cyclins during different phases of the cell cycle to determine the proper timing and coordination of cell cycle progression (6). The negative control of CDK is exerted by various CDK inhibitors (7). An alternative negative regulation of CDK action is modulated by phosphorylation of threonine 14 and tyrosine 15 residues by Wee-1 or Mik1 (8). On the other hand, positive regulation of CDK activity is exerted by CDK activating kinase or cell cycle-activating phosphatase CDC25 genes. The latter molecules activate CDK by removal of the inhibitory phosphates of threonine and tyrosine residues in the ATP-binding sites of the CDK. Three CDC25-related genes, CDC25A, CDC25B, and CDC25C, which share approximately 40–50% amino acid identity, have been identified in humans (9, 10, 11). CDC25 phosphatases act at different points of the cell cycle, including G1-S and G2-M transition (9, 10, 12).

Dysregulation of cell cycle progression has been characterized in a variety of human malignancies (13). HCC tissues overexpress cyclins D1 and E (14, 15). The CDK inhibitor p21waf1/cip1 is reduced, methylation of the p16INK4 gene occurs in the promoter region, and p27Kip1 appears to be decreased in a subset of HCCs (15, 16, 17). Recent studies point out the relevance of CDC25 phosphatases in human neoplasms. In the CDC25 family, A and B types, but not CDC25C, appear to be potential oncogenes as they have been found to transform primary murine fibroblasts in cooperation with either mutated Ha-ras or loss of Rb1(18). Transgenic mice that overexpress the CDC25B gene display enhanced sensitivity to carcinogen 9,10-dimethyl-1,2-benzanthracene (19) or develop mammary gland hyperplasia (20). Concordant to in vitro and in vivo findings, overexpression of CDC25A and CDC25B has been demonstrated in many types of human malignancies (21, 22, 23, 24, 25, 26, 27, 28).

Although dysfunction of the cell cycle machinery occurs infrequently in human HCC, the expression and clinical significance of CDC25A and CDC25B proteins have not yet been elucidated. Therefore, in the present study, we examined 59 primary HCCs immunohistochemically and determined the correlation between the levels of these proteins, and various clinical and pathological features including prognosis. In addition, in an effort to assess proliferation of HCC cells, we examined the expression of a nonhistone nuclear protein, PCNA, which accumulates from late G1 to S-phase and acts as an adjunct to DNA polymerase (29).

Cell Culture.

HEK 293 cells were obtained from the American Type Culture Collection (ATCC CRL-1573) and cultured in DMEM supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin in 5% CO2 at 37°C.

Patients and Tissue Specimens.

Tissue samples were obtained from 59 patients with HCC (49 males and 10 females; 44–76 years of age; mean, 61 ± 6 years), who underwent hepatectomy at the Department of Surgery and Clinical Oncology, Osaka University. Before hepatectomy for HCC, 31 patients were treated with transarterial embolization. All of the patients had hepatitis C virus infection but none had hepatitis B viral infection. The mean follow-up period in the prognosis study was 3.4 ± 1.8 years. The resected surgical specimens were fixed in 10% neutral buffered formalin, processed through graded ethanol solution, and embedded in paraffin. A piece of each tissue sample was immediately frozen in liquid nitrogen and stored at −80°C for analysis by RT-PCR and Western blotting.

Pathological Examination.

Tissue sections (4-μm thick) were deparaffinized in xylene, rehydrated with graded concentrations of ethanol, and stained with H&E solution. Pathological diagnosis of tissues into nontumor and tumor tissues was determined by a pathologist (N. M.) who was blinded to the clinical background. For 59 nontumor tissues, the presence of active inflammation and cirrhotic nodule was examined and 23 cases with CH and 36 with liver cirrhosis were identified. Tumor tissues were examined for the following characteristics: tumor site, cell differentiation (well, moderately, poorly differentiated, or undifferentiated), hepatic vein invasion, portal vein invasion, number of tumors, septal formation, capsular formation, and capsular invasion.

Antibodies.

The rabbit polyclonal antibodies for CDC25A and CDC25B phosphatases and their blocking peptides that were used as immunogen for generation of the antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA; Refs. 25, 26). The mouse monoclonal antihuman PCNA antibody was purchased from DAKO (Carpinteria, CA; Ref. 29).

Immunohistochemistry.

Immunostaining was performed on the TechMate Horizon automated staining system (DAKO, Glostrup, Denmark) using the Vectastain ABC-peroxidase kit (Vector Laboratories, Burlingame, CA), as described previously (25, 26). The primary antibodies were applied to the sections at the following dilutions: anti-CDC25A antibody, 1:100; anti-CDC25B antibody, 1:50; and anti-PCNA antibody, 1:400. Counter nuclear staining was performed briefly with hematoxylin solution. For the positive controls of CDC25 phosphatase, sections of colon cancer expressing intense CDC25A or CDC25B protein (25) were included in each staining procedure. For negative controls, nonimmunized rabbit or mouse IgG (Vector Labs), or PBS was used as a substitute for the primary antibody. In addition, absorption tests were performed to verify the specificity of the CDC25A and CDC25B antibodies.

Immunohistochemical Evaluation.

All of the immunostained sections were evaluated in a blinded manner without knowledge of the clinical and pathological parameters of the patients. For assessment of CDC25A and PCNA, four high-power fields in each specimen were selected randomly, and nuclear staining was examined under high power magnification. More than 500 cells were counted to determine the LI, which represented the percentage of immunostained cells relative to the total number of cells. Because CDC25B expression was found mainly in the cytoplasm and its level was generally less than strong, the intensity of staining was scored on the two scales where “positive” represented moderate staining and “negative” indicated none or only faint staining. In half of the samples, staining was repeated twice to avoid possible technical errors, but similar results were obtained in these samples. The above procedures of evaluation were performed by X. X. The obtained results were confirmed by two investigators (H. Y. and N. M.) using a multihead microscope, and a consensus was achieved.

Semiquantitative RT-PCR Analysis for CDC25A and CDC25B mRNAs.

Semiquantitative analysis for expression of CDC25B or CDC25A mRNA was performed by the multiplex RT-PCR technique, using PBGD as the internal standard, as described previously (25, 26). PCRs were performed in a total volume of 25 μl reaction mixture containing 1 μl of cDNA template, 1× Perkin Elmer PCR buffer, 1.5 mm MgCl2, 0.8 mm deoxynucleotide triphosphates, 20 pmol of each primer for CDC25A or CDC25B, 4 pmol each for PBGD, and 1 unit of TaqDNA Polymerase (AmpliTaq Gold; Roche Molecular Systems, Inc., Belleville, NJ). The sequences of these PCR primers and PBGD were described previously (25, 26, 30). The sizes of the amplicons for CDC25A, CDC25B, and PBGD were 272, 416, and 127 bp, respectively.

Western Blot Analysis.

Western blotting was performed as described previously (25). Briefly, 50 μg of the total protein were subjected to 10% PAGE, followed by electroblotting onto a polyvinylidene difluoride membrane. After blocking in 5% skim milk, the membrane was incubated with 1 μg/ml CDC25A antibody, followed by incubation with the secondary antibody at a dilution of 1:3000. For detection of the immunocomplex, the enhanced chemiluminescence Western blot detection system (Amersham, Aylesbury, United Kingdom) was used.

Transfection and β-Gal Staining

The human CDC25A cDNA (length 1.6 Kb) was obtained from D. Beach (Wolfson Institute for Biomedical Research, University College, London) (18) and subcloned into the NotI site of the pcDNA3 vector (Invitrogen, Carlsbad, CA) in the sense orientation. HEK 293 cells were seeded 24 h before transfection at a density of 1 × 105/well in 6-cm dishes. To monitor the transfection efficiency, either the CDC25A plasmid or pcDNA3 vector was cotransfected with a cytomegalovirus β-gal plasmid at a ratio of 2:1 (1 μg:0.5 μg), using the lipofectin reagent (Life Technologies, Inc., Gaithersburg, MD), as recommended by the supplier. Forty-eight h after transfection the cells were collected. For immunostaining, cells were fixed on the glass slides in 10% buffered formalin for 10 min and in 70% ethanol solution for 30 min. For β-gal staining, β-gal staining kit (ACTIVE MOTIF, Carlsbad, CA) was used. Briefly, cells were fixed with 0.7% formaldehyde and 0.05% glutaraldehyde solution, and washed twice with PBS.

β-Gal-introduced cells were visualized by incubation with β-gal staining solution (4 mm potassium ferrocyanide, 4 mm potassium ferricyanide, 2 mm MgCl2, and 0.02% 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) at 37°C for 60 min.

Statistical Analysis.

Statistical analysis was performed using the Statview J-5.0 program (Abacus Concepts, Inc., Berkeley, CA). The postoperative period extended from the date of surgery to the date of the last follow-up or death. Survival was censored if the patient was still alive or died from other causes. The Kaplan-Meier method was used to estimate recurrence or death from HCC, and the log-rank test was used to examine statistical significance. A Cox proportional hazards model was used to assess the risk ratio under simultaneous contribution from several covariates. The associations between the discrete variables were assessed using the χ2 test or Fisher’s exact probability test. Mean values were compared using the Mann-Whitney test. Data are expressed as mean ± SD. A P < 0.05 denoted the presence of a statistically significant difference.

Specificity of CDC25A and CDC25B Antibodies.

Immunohistochemical assays were performed on a series of 59 paired HCCs and their matched nontumor tissues. Immunohistochemical staining of sections of colon cancer expressing CDC25A and CDC25B served as positive controls (Fig. 1, A and B; Ref. 25). In contrast, no staining was observed when the primary antibody was substituted with nonimmunized rabbit IgG or PBS (data not shown). The use of antibody preabsorbed with excess amount of immunogens abolished staining (data not shown), indicating that these antibodies were highly specific to CDC25A and CDC25B proteins on tissue sections. The CDC25A protein was mainly found in the nucleus, whereas the CDC25B protein was localized in the cytoplasm, consistent with the results of previous studies showing CDC25A expression in the nuclear fraction and abundant CDC25B expression in the cytoplasm (31, 32).

Immunohistochemistry for CDC25A.

CDC25A protein was expressed in all of the nontumor and carcinoma tissues tested, mainly in the nucleus. The percentage of cells positive for nuclear CDC25A was relatively low in noncancerous tissues (Fig. 1,C); the LI was 13.5 ± 4.9% in CH-based hepatocytes (n = 23) and 15.7 ± 4.3% in cirrhotic livers (n = 36). In carcinoma tissues, 25 cases (42.4%) expressed nuclear CDC25A with moderate to strong intensity (Fig. 1,D), whereas the remaining 34 cases (57.6%) displayed a weak CDC25A expression. LI varied widely in carcinoma tissues with a mean value of 42.3 ± 12.7%. The difference in LI for CDC25A between HCC and CH-based hepatocytes or cirrhotic livers was significant (P < 0.001 for both; Fig. 2), and overexpression rate of the CDC25A protein was 78% (46 of 59).

Transfection Assay and Western Blot Analysis.

The above immunohistochemical assay showed that most of the HCCs expressed increased levels of CDC25A protein when compared with noncancerous tissues. To additionally rule out a possibility of nonspecific staining with the CDC25A antibody, we performed additional experiments for confirmation of the results. Transfection assay in duplicate cultures showed that transfection efficiency using β-gal construct was ∼12% in pcDNA3-transfected cultures and 10% in CDC25A-transfected cultures, respectively (Fig. 3, A and B). By staining with the CDC25A antibody, pcDNA3-transfected cultures did not express nuclear CDC25A, whereas CDC25A-transfected cultures did display an intense nuclear CDC25A staining in ∼5% of the cells (Fig. 3, C and D). The findings indicate that the CDC25A antibody specifically reacts with CDC25A protein. Western blot analysis for CDC25A in six pairs of cancer and its noncancer counterpart revealed that majority of HCCs indeed exhibited overexpression, and a subset of HCCs expressed considerable levels of CDC25A (Fig. 4). There was a significant correlation between CDC25A level on immunoblot and LI determined by immunohistochemistry (γ2) = 0.672; P < 0.001).

Immunohistochemistry for CDC25B.

Approximately half of the nontumor tissues (28 of 59: 47.5%; 11 of 23 CH and 17 of 36 cirrhosis) expressed the CDC25B protein in moderate intensity (Fig. 1,E) and the other half (31 of 59: 52.5%) displayed none or only faint CDC25B expression. On the other hand, over half of the HCCs expressed the CDC25B protein with none or only faint intensity (39 of 59: 66.1%; Fig. 1,F) and the remaining 20 HCCs (33.9%) expressed it with moderate intensity. Among the HCC samples, none expressed as strong a CDC25B expression as the positive control sample of colon cancer shown in Fig. 1 B. As a result, 12 of 59 HCCs (20.3%) expressed increased levels of the CDC25B protein, compared with their noncancer counterparts.

RT-PCR Analysis for CDC25A and CDC25B mRNAs.

RT-PCR analysis was performed using 13 paired nontumor and tumor mRNA extracts. The relative value of CDC25A and CDC25B band to PBGD band was calculated, and the T:N ratio was determined in each case. Three representative cases are shown in Fig. 5. The T:N ratios of cases 1, 2, and 3 were 2.5, 3.1, and 2.2 for CDC25A, and 1.3, 1.6, and 0.4 for CDC25B, respectively. When the T:N ratio of >2.0 was defined as overexpression, CDC25A was overexpressed in 9 of 13 (69%) cases tested, whereas CDC25B was overexpressed in 4 of 13 (31%) cases. When mRNA levels of CDC25A or CDC25B were compared with expression levels detected by immunohistochemistry, no discrepant results were found (data not shown).

PCNA Expression.

To examine the possible involvement of CDC25 phosphatases in growth of carcinoma cells, we examined PCNA expression and compared it with that of CDC25A and CDC25B. Carcinoma tissues displayed a wide variety of PCNA expression ranging from 16 to 92%, with a mean value of 49.4 ± 14.8%, which was significantly higher than 20.5 ± 7.2% in CH-based hepatocytes, and 15.5 ± 6.0% in cirrhotic hepatocytes (P < 0.001 for both; Fig. 1,G). When the patients were divided into two groups according to a cutoff of the mean value of PCNA expression, high PCNA expression was associated with dedifferentiated carcinoma (P = 0.012) but not with other clinicopathological parameters (data not shown). Moreover, the expression of CDC25A and PCNA displayed a clear correlation (Fig. 6; P = 0.005; γ2 = 0.361), whereas no correlation was found between CDC25B expression and PCNA expression (data not shown).

Relationship between Clinicopathological Parameters and CDC25A or CDC25B Expression.

For statistical analysis of the expression of CDC25A, the carcinoma specimens were divided into two groups: 33 (56.0%) high expressors and 26 (44.0%) low expressors, according to the percentage of nuclear CDC25A-positive cells, using a cutoff level of 42.3%, representing the mean value of CDC25A expression. We then analyzed the relationship between CDC25A expression and various clinicopathological parameters listed in Table 1. There was a significant correlation between high expression of CDC25A and dedifferentiated phenotype (poorly differentiated carcinoma and undifferentiated carcinoma), and portal vein invasion (P = 0.001 and 0.031, respectively). No correlation was found between CDC25A expression and other variables including age, gender, tumor size, hepatic vein invasion, number of tumor, septal formation, capsular formation, and capsular invasion.

With regard to CDC25B expression, comparison between the moderate expression group (n = 20) and none or faint expression group (n = 39) showed no significant differences in the correlations between the expression and the clinicopathological features listed in Table 1 (data not shown).

Analysis of Survival Rates.

We then analyzed the DFS rates based on CDC25A and CDC25B expression, as well as clinicopathological features of HCCs. Univariate analysis showed that high expression of CDC25A was a significant predictor of disease relapse, when assessed by Kaplan-Meier curves (Fig. 7; P < 0.01). Among the various parameters listed in Table 1, the histological grade and portal vein invasion were also indicative of disease relapse (P = 0.003 and P = 0.002, respectively). Hepatic vein invasion showed a significant value, but the positive cases were too small (only 3 of 59) for adequate statistical analysis. We also found that PCNA expression was also indicative of disease relapse (Fig. 8; P = 0.039). In contrast, expression of CDC25B and other clinicopathological parameters were not significant prognostic factors.

Multivariate analysis using the Cox regression model showed that CDC25A expression was a significant covariate (P = 0.029; Table 2), as well as portal vein invasion (P = 0.017) and PCNA expression (P = 0.022). The relative risk of cancer relapse in patients with carcinomas expressing high levels of CDC25A was 2.98 that of patients with tumors expressing low levels of CDC25A.

CDC25A and CDC25B are overexpressed in various types of human carcinomas (21, 22, 23, 24, 25, 26, 27, 28). Importantly, overexpression of CDC25A and CDC25B is often associated with malignant features of human neoplasms, which include poor prognosis of carcinomas of the esophagus, breast, colon, and ovary, advanced stage gastric cancer, poorly differentiated non-small cell lung cancer, and aggressive types of lymphoma (22, 23, 24, 25, 26, 27, 28). These findings suggest that CDC25 phosphatases are useful markers for the malignant potential of various carcinomas, and they may function to enhance cancer growth and expansion, although information regarding their biological functions in malignant cells is limited.

Our results showed a preferential expression of CDC25A over CDC25B in HCC. The overexpression rate for CDC25A was 69% and 78%, at RNA level and protein level, respectively, whereas those for CDC25B were only 31% and 20%. These findings are in contrast with our previous reports on concurrent overexpression of CDC25A and CDC25B in carcinomas of the colon and esophagus (25, 26). The mechanism for CDC25A overexpression in HCC is unknown at present, although a few molecules such as c-myc, E2F, transforming growth factor β could be considered as a key candidates to affect the synthesis of CDC25A (33, 34, 35). Our data indicated that CDC25A was not only up-regulated but also associated with aggressive cancer phenotypes including portal vein invasion and dedifferentiated histology (Table 1). Moreover, univariate and multivariate analyses showed that high expression of CDC25A was associated significantly with disease relapse of HCC. In contrast, CDC25B expression was not associated with conventional clinicopathological parameters, and it was not helpful in predicting prognosis. These data again highlight the relevance of CDC25A in HCC rather than CDC25B. Then what is the functional significance of CDC25A molecule in HCC?

One of the mechanisms related to malignant potential could be that CDC25A may function to inhibit cellular apoptosis. The survival promoting activity by CDC25A was demonstrated in the serum-starved condition (36). It is postulated that increased expression of CDC25A may contribute to reduced cellular responsiveness to oxidative stress (37). However, at present, only limited evidence is provided in relation to apoptosis. On the other hand, there is cumulative evidence concerning its contribution to cell cycle control. Previous studies have shown that CDC25A is a rate-limiting, positive regulator of the cell cycle. A gain in CDC25A activity can result in defects in transforming growth factor β antiproliferative responsiveness (35). Ablation of CDC25A function by microinjection of a specific antibody blocks entry into S phase (12). Conversely, inducible overexpression of CDC25A leading to activation of cyclin E-Cdk2 and cyclin A-Cdk2 revealed that these complexes act as critical targets for CDC25A (38, 39). This evidence emphasizes the relevance of CDC25A in G1-S transition and, accordingly, it is suggested that disturbance of G1-S transition may be important in the progression of human HCC.

Human HCC takes a unique natural course of tumor progression. Early HCC usually shows well-differentiated phenotype and grows relatively slowly. As the tumor stage advances, HCC gradually grows faster, associated with dedifferentiation (40, 41). In accordance with this model, we found that high expression of PCNA was significantly associated with poorly differentiated tumors. Furthermore, PCNA expression was associated significantly with the disease relapse, consistent with reports from other laboratories (42, 43). Likewise, active cell growth is an important aspect that defines the prognosis of patients with carcinomas of the breast and lung (44, 45), whereas cell proliferation alone does not always result in fatal events in certain types of human malignancies, including carcinomas of the colon (46, 47). Probably other features apart from cell growth, such as invasive capability and metastasis potential, are more important in this cancer type. Therefore, in the case of HCC, it is postulated that uncontrolled cell growth may particularly play a crucial role in disease progression. Finally, we found a clear correlation between PCNA index and CDC25A expression. Taken together, the present work suggests the following scenario. Increased level of CDC25A in HCC facilitates progression of the cell cycle from G1 phase to S phase, leading to active DNA synthesis. Cancer cells then acquire a high proliferative activity and cause disease recurrence.

In conclusion, we have demonstrated in the present study that CDC25A, but not CDC25B, is a novel independent prognostic marker for patients with HCC. Our findings also suggest that CDC25A might be a therapeutic target for an aggressive form of HCC by using a CDC25A-specific inhibitor or antisense technology. Indeed, several inhibitors are being developed, including novel arylating K vitamin analogues, vitamin D3 analogues, and steroidal derived inhibitors, and some of them showed antiproliferative activity in cancer cell lines including HCC (48, 49).

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.

1

Supported by a grant-in-aid for Cancer Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (to H. Y.; No. 12213078) and by an award from the Osaka Medical Research Foundation for Incurable Diseases (to H. Y.).

3

The abbreviations used are: HCC, hepatocellular carcinoma; CDK, cyclin-dependent kinase; PCNA, proliferating cell nuclear antigen; RT-PCR, reverse transcription-PCR; PBGD, porphobilinogen deaminase; LI, labeling index; CH, chronic hepatitis; β-gal, β-galactosidase; T:N, tumor:nontumor; DFS, disease-free survival.

Fig. 1.

Immunostaining with antihuman CDC25A and CDC25B antibodies in colorectal carcinoma tissues. A, a representative colorectal carcinoma tissue with low expression level of CDC25A. B, a representative colorectal carcinoma with high expression level of CDC25B. Immunostaining with antihuman CDC25A antibody in noncancerous tissue of the liver (C) and HCC tissue (D). C, note the low expression level of CDC25A in noncancerous tissue of the liver. D, a representative HCC expressing a high level of CDC25A (LI = 42%). Immunostaining with antihuman CDC25B antibody in normal and noncancerous tissue of the liver (E) and HCC tissue (F). E, moderate expression of CDC25B was noted in the noncancerous tissue. F, weak expression of CDC25B was noted in the cytoplasm of hepatoma cells. Immunostaining with antihuman PCNA antibody in HCC tissue (G). G, a representative HCC that expressed a high level of PCNA (LI = 49%). Magnification: A–F, ×100.

Fig. 1.

Immunostaining with antihuman CDC25A and CDC25B antibodies in colorectal carcinoma tissues. A, a representative colorectal carcinoma tissue with low expression level of CDC25A. B, a representative colorectal carcinoma with high expression level of CDC25B. Immunostaining with antihuman CDC25A antibody in noncancerous tissue of the liver (C) and HCC tissue (D). C, note the low expression level of CDC25A in noncancerous tissue of the liver. D, a representative HCC expressing a high level of CDC25A (LI = 42%). Immunostaining with antihuman CDC25B antibody in normal and noncancerous tissue of the liver (E) and HCC tissue (F). E, moderate expression of CDC25B was noted in the noncancerous tissue. F, weak expression of CDC25B was noted in the cytoplasm of hepatoma cells. Immunostaining with antihuman PCNA antibody in HCC tissue (G). G, a representative HCC that expressed a high level of PCNA (LI = 49%). Magnification: A–F, ×100.

Close modal
Fig. 2.

LI of CDC25A in CH, liver cirrhosis, and HCC. HCC displays significantly higher LI than CH or liver cirrhosis (both for P < 0.001); bars, ±SD.

Fig. 2.

LI of CDC25A in CH, liver cirrhosis, and HCC. HCC displays significantly higher LI than CH or liver cirrhosis (both for P < 0.001); bars, ±SD.

Close modal
Fig. 3.

Transfection assays. Transfection efficiency was ∼12% in pcDNA3-transfected cultures (A) and 10% in CDC25A-transfected cultures (B). β-Gal-positive HEK 293 cells were in blue. pcDNA3 transfected cultures did not express nuclear CDC25A (C), whereas CDC25A-transfected cultures did display an intense nuclear CDC25A staining in ∼5% cells (D). The arrows indicate positive staining.

Fig. 3.

Transfection assays. Transfection efficiency was ∼12% in pcDNA3-transfected cultures (A) and 10% in CDC25A-transfected cultures (B). β-Gal-positive HEK 293 cells were in blue. pcDNA3 transfected cultures did not express nuclear CDC25A (C), whereas CDC25A-transfected cultures did display an intense nuclear CDC25A staining in ∼5% cells (D). The arrows indicate positive staining.

Close modal
Fig. 4.

Western blot analysis for CDC25A. Western blotting was performed on six pairs of HCC and its noncancer counterpart. There was a significant correlation between CDC25A level on immunoblot and LI determined by immunohistochemistry (γ2) = 0.672; P < 0.001).

Fig. 4.

Western blot analysis for CDC25A. Western blotting was performed on six pairs of HCC and its noncancer counterpart. There was a significant correlation between CDC25A level on immunoblot and LI determined by immunohistochemistry (γ2) = 0.672; P < 0.001).

Close modal
Fig. 5.

RT-PCR analysis for CDC25A and CDC25B mRNAs. RT-PCR analysis was performed for CDC25A (top panel) or CDC25B (bottom panel) mRNA in paired normal and carcinoma tissues. The value of CDC25A or CDC25B band relative to PBGD band was calculated for each sample, and the T:N ratio was determined in each case.

Fig. 5.

RT-PCR analysis for CDC25A and CDC25B mRNAs. RT-PCR analysis was performed for CDC25A (top panel) or CDC25B (bottom panel) mRNA in paired normal and carcinoma tissues. The value of CDC25A or CDC25B band relative to PBGD band was calculated for each sample, and the T:N ratio was determined in each case.

Close modal
Fig. 6.

Relationship between CDC25A and PCNA expression, significant correlation was found (γ2 = 0.361; P < 0.01).

Fig. 6.

Relationship between CDC25A and PCNA expression, significant correlation was found (γ2 = 0.361; P < 0.01).

Close modal
Fig. 7.

Kaplan-Meier curves for DFS according to CDC25A expression in 59 patients with HCC. High expression of CDC25A was significantly associated with worse prognosis (P = 0.004).

Fig. 7.

Kaplan-Meier curves for DFS according to CDC25A expression in 59 patients with HCC. High expression of CDC25A was significantly associated with worse prognosis (P = 0.004).

Close modal
Fig. 8.

Kaplan-Meier curves for DFS according to PCNA expression in 59 patients with HCC. High expression of PCNA was significantly associated with worse prognosis (P = 0.039).

Fig. 8.

Kaplan-Meier curves for DFS according to PCNA expression in 59 patients with HCC. High expression of PCNA was significantly associated with worse prognosis (P = 0.039).

Close modal
Table 1

Relationship between CDC25A expression and clinicopathological parameters in cases with HCC

Number of casesCDC25AP
LowHigh
Age ≧60 49 23 26 N.S.a 
 <60 10  
Gender Male 49 23 26 N.S. 
 Female 10  
Tumor size <2 cm 18 13 N.S. 
 ≧2 cm 41 21 20  
Histological gradeb Well/mod. 38 23 15 0.001c 
 Poor./undiff. 21 18  
Hepatic vein invasion Yes N.S. 
 No 56 26 30  
Portal vein invasion Yes 0.031c 
      
No 50 25 25   
Number of tumord Solitary 42 20 22 N.S. 
 Multiple 17 11  
Septal formation Yes 41 19 22 N.S. 
 No 18 11  
Capsular formation Yes 46 22 24 N.S. 
 No 13  
Capsular invasione Yes 21 14 N.S. 
 No 25 15 10  
Number of casesCDC25AP
LowHigh
Age ≧60 49 23 26 N.S.a 
 <60 10  
Gender Male 49 23 26 N.S. 
 Female 10  
Tumor size <2 cm 18 13 N.S. 
 ≧2 cm 41 21 20  
Histological gradeb Well/mod. 38 23 15 0.001c 
 Poor./undiff. 21 18  
Hepatic vein invasion Yes N.S. 
 No 56 26 30  
Portal vein invasion Yes 0.031c 
      
No 50 25 25   
Number of tumord Solitary 42 20 22 N.S. 
 Multiple 17 11  
Septal formation Yes 41 19 22 N.S. 
 No 18 11  
Capsular formation Yes 46 22 24 N.S. 
 No 13  
Capsular invasione Yes 21 14 N.S. 
 No 25 15 10  
a

N.S., not significant.

b

Mod., moderately; poor., poorly; undiff., undifferentiated.

c

Statistically significant.

d

This category includes both intrahepatic metastasis and tumors generated by multicentric carcinogenesis.

e

The capsular invasion is composed in the category of capsular formation.

Table 2

Multivariate analysis (Cox proportional hazards model)

CategoriesaPRisk ratio95% Confidence interval
CDC25A (H:L) 0.029 2.98 0.126–0.894 
Portal vein invasion (yes:no) 0.017 4.13 0.075–0.774 
PCNA (H:L) 0.022 2.66 1.154–6.107 
Differentiation (well/mod.:poor./undiff.) 0.541 1.31 0.319–1.822 
Number of tumor (solitary:multiple) 0.406 1.49 0.580–3.844 
Capsular formation (yes:no) 0.133 2.16 1.170–1.263 
CategoriesaPRisk ratio95% Confidence interval
CDC25A (H:L) 0.029 2.98 0.126–0.894 
Portal vein invasion (yes:no) 0.017 4.13 0.075–0.774 
PCNA (H:L) 0.022 2.66 1.154–6.107 
Differentiation (well/mod.:poor./undiff.) 0.541 1.31 0.319–1.822 
Number of tumor (solitary:multiple) 0.406 1.49 0.580–3.844 
Capsular formation (yes:no) 0.133 2.16 1.170–1.263 
a

H:L, high:low; well/mod.:poor./undiff., well/moderately differentiated:poorly differentiated/undifferentiated.

1
Grazi G. L., Ercolani G., Pierangeli F., Del Gaudio M., Cescon M., Cavallari A., Mazziotti A. Improved results of liver resection for hepatocellular carcinoma on cirrhosis give the procedure added value.
Ann. Surg.
,
234
:
71
-78,  
2001
.
2
Nagasue N., Ono T., Yamanoi A., Kohno H., El-Assal O. N., Taniura H., Uchida M. Prognostic factors and survival after hepatic resection for hepatocellular carcinoma without cirrhosis.
Br. J. Surg.
,
88
:
515
-522,  
2001
.
3
Murakami Y., Hayashi K., Hirohashi S., Sekiya T. Aberrations of the tumor suppressor p53 and retinoblastoma genes in human hepatocellular carcinomas.
Cancer Res.
,
51
:
5520
-5525,  
1991
.
4
Nishida N., Fukuda Y., Ishizaki K. Molecular aspects of hepatocarcinogenesis and their clinical implications.
Int. J. Oncol.
,
4
:
615
-622,  
1994
.
5
De La Coste A., Romagnolo B., Billuart P., Renard C. A., Buendia M. A., Soubrane O., Fabre M., Chelly J., Beldjord C., Kahn A., Perret C. Somatic mutations of the β-catenin gene are frequent in mouse and human hepatocellular carcinomas.
Proc. Natl. Acad. Sci. USA
,
95
:
8847
-8851,  
1998
.
6
Sherr C. J. G1 phase progression: cycling on cue.
Cell
,
79
:
551
-555,  
1994
.
7
Sherr C. J., Roberts J. M. Inhibitors of mammalian G1 cyclin-dependent kinases.
Genes Dev.
,
9
:
1149
-1163,  
1995
.
8
Parker L. L., Atherton-Fessler S., Piwnica-Worms H. p107wee1 is a dual-specificity kinase that phosphorylates p34cdc2 on tyrosine 15.
Proc. Natl. Acad. Sci. USA
,
89
:
2917
-2921,  
1992
.
9
Galaktionov K., Beach D. Specific activation of cdc25 tyrosine phosphatases by B-type cyclins: evidence for multiple roles of mitotic cyclins.
Cell
,
67
:
1181
-1194,  
1991
.
10
Nagata A., Igarashi M., Jinno S., Suto K., Okayama H. An additional homolog of the fission yeast cdc25+ gene occurs in humans and is highly expressed in some cancer cells.
New Biol.
,
3
:
959
-968,  
1991
.
11
Sadhu K., Reed S. I., Richardson H., Russell P. Human homolog of fission yeast cdc25 mitotic inducer is predominantly expressed in G2.
Proc. Natl. Acad. Sci. USA
,
87
:
5139
-5143,  
1990
.
12
Jinno S., Suto K., Nagata A., Igarashi M., Kanaoka Y., Nojima H., Okayama H. Cdc25A is a novel phosphatase functioning early in the cell cycle.
EMBO J.
,
13
:
1549
-1556,  
1994
.
13
Hall M., Peters G. Genetic alterations of cyclins, cyclin-dependent kinases, and Cdk inhibitors in human cancer.
Adv. Cancer Res.
,
68
:
67
-108,  
1996
.
14
Zhang Y. J., Jiang W., Chen C. J., Lee C. S., Kahn S. M., Santella R. M., Weinstein I. B. Amplification and overexpression of cyclin D1 in human hepatocellular carcinoma.
Biochem. Biophys. Res. Commun.
,
196
:
1010
-1016,  
1993
.
15
Ito Y., Matsuura N., Sakon M., Miyoshi E., Noda K., Takeda T., Umeshita K., Nagano H., Nakamori S., Dono K., Tsujimoto M., Nakahara M., Nakao K., Taniguchi N., Monden M. Expression and prognostic roles of the G1-S modulators in hepatocellular carcinoma: p27 independently predicts the recurrence.
Hepatology
,
30
:
90
-99,  
1999
.
16
Hui A. M., Sakamoto M., Kanai Y., Ino Y., Gotoh M., Yokota J., Hirohashi S. Inactivation of p16INK4 in hepatocellular carcinoma.
Hepatology
,
24
:
575
-579,  
1996
.
17
Hui A. M., Kanai Y., Sakamoto M., Tsuda H., Hirohashi S. Reduced p21(WAF1/CIP1) expression and p53 mutation in hepatocellular carcinomas.
Hepatology
,
25
:
575
-579,  
1997
.
18
Galaktionov K., Lee A. K., Eckstein J., Draetta G., Meckler J., Loda M., Beach D. CDC25 phosphatases as potential human oncogenes.
Science (Wash. DC)
,
269
:
1575
-1577,  
1995
.
19
Yao Y., Slosberg E. D., Wang L., Hibshoosh H., Zhang Y. J., Xing W. Q., Santella R. M., Weinstein I. B. Increased susceptibility to carcinogen-induced mammary tumors in MMTV-Cdc25B transgenic mice.
Oncogene
,
18
:
5159
-5166,  
1999
.
20
Ma Z. Q., Chua S. S., DeMayo F. J., Tsai S. Y. Induction of mammary gland hyperplasia in transgenic mice over-expressing human Cdc25B.
Oncogene
,
18
:
4564
-4576,  
1999
.
21
Gasparotto D., Maestro R., Piccinin S., Vukosavljevic T., Barzan L., Sulfaro S., Boiocchi M. Overexpression of CDC25A and CDC25B in head and neck cancers.
Cancer Res.
,
57
:
2366
-2368,  
1997
.
22
Wu W., Fan Y. H., Kemp B. L., Walsh G., Mao L. Overexpression of cdc25A and cdc25B is frequent in primary non-small cell lung cancer but is not associated with overexpression of c-myc.
Cancer Res.
,
58
:
4082
-4085,  
1998
.
23
Kudo Y., Yasui W., Ue T., Yamamoto S., Yokozaki H., Nikai H., Tahara E. Overexpression of cyclin-dependent kinase-activating CDC25B phosphatase in human gastric carcinomas.
Jpn. J. Cancer Res.
,
88
:
947
-952,  
1997
.
24
Hernandez S., Hernandez L., Bea S., Cazorla M., Fernandez P. L., Nadal A., Muntane J., Mallofre C., Montserrat E., Cardesa A., Campo E. cdc25 cell cycle-activating phosphatases and c-myc expression in human non-Hodgkin’s lymphomas.
Cancer Res.
,
58
:
1762
-1767,  
1998
.
25
Takemasa I., Yamamoto H., Sekimoto M., Ohue M., Noura S., Miyake Y., Matsumoto T., Aihara T., Tomita N., Tamaki Y., Sakita I., Kikkawa N., Matsuura N., Shiozaki H., Monden M. Overexpression of CDC25B phosphatase as a novel marker of poor prognosis of human colorectal carcinoma.
Cancer Res.
,
60
:
3043
-3050,  
2000
.
26
Nishioka K., Doki Y., Shiozaki H., Yamamoto H., Tamura S., Yasuda T., Fujiwara Y., Yano M., Miyata H., Kishi K., Nakagawa H., Shamma A., Monden M. Clinical significance of CDC25A and CDC25B expression in squamous cell carcinomas of the oesophagus.
Br. J. Cancer
,
85
:
412
-421,  
2001
.
27
Cangi M. G., Cukor B., Soung P., Signoretti S., Moreira G., Jr, Ranashinge M., Cady B., Pagano M., Loda M. Role of the Cdc25A phosphatase in human breast cancer.
J. Clin. Investig.
,
106
:
753
-761,  
2000
.
28
Broggini M., Buraggi G., Brenna A., Riva L., Codegoni A. M., Torri V., Lissoni A. A., Mangioni C., D’Incalci M. Cell cycle-related phosphatases CDC25A and B expression correlates with survival in ovarian cancer patients.
Anticancer Res.
,
20
:
4835
-4840,  
2000
.
29
Adachi E., Hashimoto H., Tsuneyoshi M. Proliferating cell nuclear antigen in hepatocellular carcinoma and small cell liver dysplasia.
Cancer (Phila.)
,
72
:
2902
-2909,  
1993
.
30
Nagel S., Schmidt M., Thiede C., Huhn D., Neubauer A. Quantification of Bcr-Abl transcripts in chronic myelogenous leukemia (CML) using standardized, internally controlled, competitive differential PCR (CD-PCR).
Nucleic Acids Res.
,
24
:
4102
-4103,  
1996
.
31
Dixon D., Moyana T., King M. J. Elevated expression of the cdc25A protein phosphatase in colon cancer.
Exp. Cell Res.
,
240
:
236
-243,  
1998
.
32
Gabrielli B. G., De Souza C. P., Tonks I. D., Clark J. M., Hayward N. K., Ellem K. A. Cytoplasmic accumulation of cdc25B phosphatase in mitosis triggers centrosomal microtubule nucleation in HeLa cells.
J. Cell Sci.
,
109
:
1081
-1093,  
1996
.
33
Galaktionov K., Chen X., Beach D. Cdc25 cell-cycle phosphatase as a target of c-myc.
Nature (Lond.)
,
382
:
511
-517,  
1996
.
34
Chen X., Prywes R. Serum-induced expression of the cdc25A gene by relief of E2F-mediated repression.
Mol. Cell. Biol.
,
19
:
4695
-4702,  
1999
.
35
Iavarone A., Massague J. Repression of the CDK activator Cdc25A and cell-cycle arrest by cytokine TGF-β in cells lacking the CDK inhibitor p15.
Nature (Lond.)
,
387
:
417
-422,  
1997
.
36
Fuhrmann G., Leisser C., Rosenberger G., Grusch M., Huettenbrenner S., Halama T., Mosberger I., Sasgary S., Cerni C., Krupitza G. Cdc25A phosphatase suppresses apoptosis induced by serum deprivation.
Oncogene
,
20
:
4542
-4553,  
2001
.
37
Zou X., Tsutsui T., Ray D., Blomquist J. F., Ichijo H., Ucker D. S., Kiyokawa H. The cell cycle-regulatory CDC25A phosphatase inhibits apoptosis signal-regulating kinase 1.
Mol. Cell. Biol.
,
21
:
4818
-4828,  
2001
.
38
Blomberg I., Hoffmann I. Ectopic expression of Cdc25A accelerates the G(1)/S transition and leads to premature activation of cyclin E- and cyclin A-dependent kinases.
Mol. Cell. Biol.
,
19
:
6183
-6194,  
1999
.
39
Sandhu C., Donovan J., Bhattacharya N., Stampfer M., Worland P., Slingerland J. Reduction of Cdc25A contributes to cyclin E1-Cdk2 inhibition at senescence in human mammary epithelial cells.
Oncogene
,
19
:
5314
-5323,  
2000
.
40
Kenmochi K., Sugihara S., Kojiro M. Relationship of histologic grade of hepatocellular carcinoma (HCC) to tumor size, and demonstration of tumor cells of multiple different grades in single small HCC.
Liver
,
7
:
18
-26,  
1987
.
41
Kojiro M. Pathology of early hepatocellular carcinoma: progression from early to advanced.
Hepatogastroenterology
,
45
:
1203
-1205,  
1998
.
42
Ng I. O., Lai E. C., Fan S. T., Ng M., Chan A. S., So M. K. Prognostic significance of proliferating cell nuclear antigen expression in hepatocellular carcinoma.
Cancer (Phila.)
,
73
:
2268
-2274,  
1994
.
43
King K. L., Hwang J. J., Chau G. Y., Tsay S. H., Chi C. W., Lee T. G., Wu L. H., Wu C. W., Lui W. Y. Ki-67 expression as a prognostic marker in patients with hepatocellular carcinoma.
J. Gastroenterol. Hepatol.
,
13
:
273
-279,  
1998
.
44
Aaltomaa S., Lipponen P., Syrjanen K. Prognostic value of cell proliferation in breast cancer as determined by proliferating cell nuclear antigen (PCNA) immunostaining.
Anticancer Res.
,
12
:
1281
-1286,  
1992
.
45
Fukuse T., Hirata T., Naiki H., Hitomi S., Wada H. Prognostic significance of proliferative activity in pN2 non-small-cell lung carcinomas and their mediastinal lymph node metastases.
Ann. Surg.
,
232
:
112
-118,  
2000
.
46
Jansson A., Sun X. F. Ki-67 expression in relation to clinicopathological variables and prognosis in colorectal adenocarcinomas.
APMIS
,
105
:
730
-734,  
1997
.
47
Kubota Y., Petras R. E., Easley K. A., Bauer T. W., Tubbs R. R., Fazio V. W. Ki-67-determined growth fraction versus standard staging and grading parameters in colorectal carcinoma. A multivariate analysis.
Cancer (Phila.)
,
70
:
2602
-2609,  
1992
.
48
Peng H., Xie W., Otterness D. M., Cogswell J. P., McConnell R. T., Carter H. L., Powis G., Abraham R. T., Zalkow L. H. Syntheses and biological activities of a novel group of steroidal derived inhibitors for human Cdc25A protein phosphatase.
J. Med. Chem.
,
44
:
834
-848,  
2001
.
49
Wang Z., Southwick E. C., Wang M., Kar S., Rosi K. S., Wilcox C. S., Lazo J. S., Carr B. I. Involvement of Cdc25A phosphatase in Hep3B hepatoma cell growth inhibition induced by novel K vitamin analogs.
Cancer Res.
,
61
:
7211
-7226,  
2001
.