Overexpression of tumour suppressor retinoblastoma 2 protein (pRb2/p130) in hepatocellular carcinoma
Hung Huynh
Laboratory of Molecular Endocrinology, Division of Cellular and Molecular Research, National Cancer Centre of Singapore, Singapore 169610
Tel: +65 436 8347; Fax: +65 226 5694; Email: cmrhth{at}nccs.com.sg
 |
Abstract
|
---|
Hepatocellular carcinoma (HCC) is one of the most common malignancies in Southeast Asia. Although inactivation of pRb2/p130 has been reported in a variety of human cancers, its function in HCC has not been established. In this study we report that loss of expression of pRb2/p130 was detected by immunohistochemistry and western blotting in 15.2% (7 of 46) HCCs examined. High levels of pRb2/p130 expression were found in 84.8% (39 of 46) HCCs studied. Western blot analysis revealed that HCC had 3.5-fold higher pRb2/p130 than adjacent benign liver (ABL) tissues. 71.7% (33 of 46) of HCCs examined exhibited both nuclear and cytoplasmic staining for pRb2/p130. Cytoplasmic staining was found in 93.5% (43 of 46) of ABL tissues. Overproduction of pRb2/p130 in HepG2 cells led to growth suppression, cell cycle arrest in G0/G1, altered cell morphology, inhibition of in vitro colony formation and reduction in tumourigenicity in SCID mice. This demonstration suggests a role of pRb2/p130 as a tumour suppressor protein in HCC and the loss of this protein may lead to the development or progression of HCC. Overexpression of pRb2/p130 in HCC was, therefore, suggested to be a programmed protective response of the organism to uncontrolled proliferation.
Abbreviations: ABL, adjacent benign liver; FBS, foetal bovine serum; HCC, hepatocellular carcinoma; MEM, modified Eagle's medium; PMSF, phenylmethylsulphonyl fluoride
 |
Introduction
|
---|
Hepatocellular carcinoma (HCC) accounts for >90% of all primary liver cancers. The annual incidence of HCC varies from 250 000 to 1 200 000 cases (1,2). The disease is 25 times more prevalent in men than in women. It is frequently seen as both a unifocal as well as a multifocal disease. The disease is associated with environmental exposure to hepatitis B virus, hepatitis C virus and aflatoxin B1 (1,2). Treatment outcomes for HCC have remained generally poor. The majority of patients with HCC have inoperable disease with very poor prognosis (3). The five year survival rate is limited to 2539% after surgery (4) and much lower elsewhere (5,6). Long-term survival is uncommon because of the frequent presence of recurrence, metastasis or the development of new primaries (7,8). Current adjuvant or palliative treatment modalities have not been conclusively shown to prolong survival in HCC (9).
Although the molecular mechanisms of hepatocarcinogenesis remain unclear, a number of genetic lesions have been identified (10). More than 20 cellular genes have been found to be either down- or up-regulated or mutated in HCC. They include Ras, c-myc, c-fos and c-jun, rho, TGF-
, HGF and c-met, c-ErbB-2, u-PA, MXR7, MDM2, MAGE, matrix metalloproteinase, Smads, p21WAF1/CIP1, p27Kip1, PTEN, E-cadherin, ß-catenin, AXIN1 and HCCA1 (reviewed in 11). Different spectra of p53 and pRB alterations have been found in HCCs (12,13). Hypermethylation of p16 (14,15) and p15 (16), a cyclin-dependent kinase inhibitor gene that regulates cell cycling, has been detected in HCC. p15 has been postulated to be a tumour suppressor gene modulating pRb phosphorylation (17).
Proteins of the pRB family are highly homologous in the pocket region, which is targeted by viral oncoproteins and is responsible for many functional interactions (1823). Functionally, all the pRB family members show cell type-specific growth inhibition properties unique to each member (24,25). Interplay between the pRB family and the E2F family is hypothesized to regulate transcription and progression of the cell cycle. Although the different members of the pRB gene family may complement each other, they are not fully functionally redundant (24).
The human pRb2/p130 gene is localized on chromosome 16q12.2, an area frequently altered in some human cancers, including hepatic carcinoma (26). pRB2/p130 displays a cell cycle-regulated phosphorylation pattern (27) and forms complexes with different members of the E2F family of transcription factors (2830). It is localized mainly in the nuclear compartment of the cell. However, a recent study showed that point mutations in the nuclear localization signal peptide of pRb2/p130 disrupted its nuclear localization (31).
A role of pRb2/p130 in the development and/or progression of lung cancer has been reported (32,33). pRb2/p130 expression is a strong predictor of overall survival in lung cancer patients (34). Loss of pRb2/p130 expression significantly correlated with a negative prognosis in endometrial (35) and oral squamous cell carcinomas (36), choroidal melanoma (37) and malignant lymphomas (38). Genomic mutations in the pRb2/p130 gene has been reported for nasopharyngeal carcinomas (39) and lung tumours (40). Introduction of the pRb2/p130 gene into HONE-2 human nasopharyngeal carcinoma cells caused a significant reduction in cell proliferation and changes in cellular morphology (24). pRb2/p130 also mediates the G0/G1 phase cell cycle arrest in the T98G glioblastoma cell line (reviewed in 40).
In this study we demonstrate that elevation and loss of pRb2/p130 protein was detected in 84.8% (39 of 46) and 15.2% (7 of 46) HCCs examined, respectively. Overproduction of pRb2/p130 in HepG2 cells resulted in cell cycle arrest at G0/G1, growth inhibition in vitro and a reduction in tumour formation in vivo. These observations lend support to the speculation that pRb2/p130 may play a vital role in growth regulation and differentiation of liver epithelial cells and its elevation in HCCs may serve as a protective mechanism to limit the uncontrolled growth of cancer cells.
 |
Materials and methods
|
---|
Patients and tissue samples
Prior written informed consent was obtained from all patients and the study received ethics board approval at the National Cancer Centre of Singapore as well as the Singapore General Hospital. Tissue samples were obtained intraoperatively from tumours and adjacent benign liver (ABL) tissue during liver resection for HCC in 46 patients at the Singapore General Hospital. Fourteen of 46 HCC samples had a single tumour and 32 of 46 had two tumours. The samples were snap frozen in liquid nitrogen and stored at 80°C until analysis. A similar set of samples was fixed in neutral buffer containing 10% formalin and paraffin embedded. The diagnosis of HCC was confirmed histologically in all cases.
Staging of tumours was performed using the TNM system (41). In addition, every tumour was examined macroscopically and microscopically for the presence of capsule formation, satellites, multiplicity and necrosis. Dysplasia and cirrhosis in the surrounding liver tissue were noted. Fifteen of 46 tumours were associated with cirrhosis. Of 15 cirrhotic HCCs, 12 had dysplastic tissue. Multifocality was defined as multiple small uniformly sized tumours that likely represented independent primary tumours (42). This was distinguished from satellites, which were defined as tumour nodules, smaller than the main tumour mass, located within a maximum distance of 2 cm. The term multiplicity was used for both multifocal tumours and for multiple intrahepatic metastasis from a single primary tumour that were situated >2 cm from the edge of the main tumour mass.
Immunohistochemical analysis and assessment
For immunohistochemical analysis of pRb2/p130, 5 µm thick sections were cut, dewaxed in xylene and then rehydrated as described (43). The sections were incubated with mouse anti-human pRb2/p130 and mouse anti-Ki-67 (NeoMarker, Fremont, CA) overnight at 4°C. Immunohistochemistry was performed using the streptavidinbiotinperoxidase complex method according to the manufacturer's instructions (Lab Vision, Fremont, CA) using aminoethyl carbazole as the chromogen. Sections known to stain positively were incubated in each batch and negative controls were also prepared by replacing primary antibody with preimmune sera. The slides were examined and pictures taken using an Olympus BX60.
Tumour sections were considered negative if staining was absent or present in <5% of tumour cells. Immunostainning was scored using the formula IS = (i + 1) x PI as described (44), where i is the intensity of staining, varying between 1+ and 3+, and PI is the percentage of positive cells. At least 20 high power fields were chosen randomly and 2000 cells were counted. Weak, average and strong staining of pRb2/p130 expression in carcinoma cells was considered when IS was between 20 and 100, 104 and 240 and 244 and 400, respectively.
Western blot analysis
To determine changes in expression of the indicated proteins, snap frozen HCC tumours and ABL tissues were thawed and homogenized in lysis buffer as described (43). Tissue or cell lysate was subjected to western blot analysis as described (43). Blots were incubated with the indicated primary antibody and 1:7500 diluted horseradish peroxidase-conjugated donkey anti-mouse or anti-rabbit secondary antibody (Pierce, Rockford, IL). The blots were then visualized with a chemiluminescent detection system as described by the manufacturer (Amersham Pharmacia Biotech, Arlington Heights, IL).
Expression of pRb2/p130 in HCC and ABL tissues via reverse transcription PCR
Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA). An aliquot of 1 µg of total RNA was reversed transcribed using a One Step RTPCR Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. Each RTPCR reaction tube contained 1x Qiagen OneStep RTPCR buffer, 400 µM each dNTP, 0.6 µM each primer, 1 µl Qiagen OneStep RTPCR Enzyme Mix and 4 U RNase inhibitor (Promega) in a total volume of 25 µl. The primers for amplifying pRb2/p130 were 5'-CCGCCATGCCGTCGGGAGGTGACCAG-3' and 5'-CCGTTCAGACACCTTGAGAGAG-3'. The primers for amplifying S16 rRNA were 5'-TCCAAGGGTCCGCTGCAGT-3' and 5'-TCACGATGGGCTTATCGGTA-3'. The One Step RTPCR was performed for the following cycles: reverse transcription at 50°C for 30 min; activating Taq polymerase at 96°C for 1.5 min; 30 cycles of 95°C for 30 s, 58°C for 30 s, 72°C for 2 min; a final extension at 72°C for 10 min. The reagents used for internal control S16 rRNA RTPCR were similar to those stated above except that the amount of RNA template and concentration of each primer used were 500 ng and 0.4 µM, respectively. The PCR cycle for S16 rRNA was 30 cycles of 94°C for 30 s, 56°C for 30 s, 72°C for 1 min and a final extension at 72°C for 10 min. The amplified products were separated on a 1% agarose gel, stained with ethidium bromide and photographs taken.
Immunoprecipitations
HCC and ABL tissues were lysed in lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerolphosphate, 2 mM Na3VO4, 1 µg/ml leupeptin) containing 1 mM phenylmethylsulphonyl fluoride (PMSF) and lysates were centrifuged at 12 000 g for 20 min at 4°C. A total of 500 µg protein from the supernatants was diluted with washing buffer (without PMSF) and precleared with protein G-coupled Sepharose. An aliquot of 50 µl of 50% (v/v) agarose-conjugated rabbit anti-human pRb2/p130 antibody (raised against a peptide mapping at the C-terminus of pRb2/p130 of human origin) (Santa Cruz Biotechnology, Santa Cruz, CA) were added to the precleared lysate and incubated with gentle rocking overnight at 4°C. Immunoprecipitates were resuspended in 30 µl of 2x Laemmli buffer and analysed by western blot analysis as described above. Blots were incubated with either rabbit anti-pRb2/p130 or mouse anti E2F-5 (an epitope corresponding to amino acids 89346 of human origin) or mouse anti-E2F-4 antibodies (Santa Cruz Biotechnology).
Transfection of pRb2/p130 cDNA into HepG2 cells
Human hepatoma HepG2 cells were obtained from the American Type Culture Collection (Rockville, MD) and maintained as monolayer cultures in modified Eagle's medium (MEM) (Life Technology Inc.) supplemented with 10% foetal bovine serum (FBS). The pCMV130 plasmid containing the entire coding region of human pRb2/p130 cDNA was kindly provided by Prof. Peter Whyte (McMaster University, Ontario, Canada). For transfection, HepG2 cells were seeded at 2 x 105 in 100 mm culture dishes in 90% MEM containing 10% FBS with Garamycin 24 h prior to transfection. Cells were transfected with 5 µg of pCMV130 or pDNA3.0 control plasmid DNA and 28 µl of Lipofectamine reagent (Life Technologies Inc.) following the manufacturer's recommendations. Forty-eight hours post-transfection, cells were subcultured at a ratio of 1:10 and replaced with selective growth medium containing 800 µg/ml G418 (Calbiochem, La Jolla, CA). Four weeks post-transfection, individual clones were isolated, expanded and assayed for pRb2/p130 expression.
For cell proliferation studies, parental HepG2, pcDNA-transfected (pcDNA-1 and -2) and pRb2/p130-transfected (pRb2/p130-43, pRb2/p130-44 and pRb2/p130-16) cells were seeded at a density of 2.5 x 104 cells/well in 24-well plates in MEM supplemented with 10% FBS. Cell number was determined by counting the cells using a haemocytometer for 5 days. Means were determined from quadruplicate wells and in no case did the standard deviation exceed 15% of the mean value.
Soft agar colony formation assay
Cells (1 x 103 cells/well) were plated in 6-well plates in semi-solid medium containing 0.35% Bacto Agar (Difco, Detroit, MI) supplemented with 20% FBS and 200 µg/ml G418 over a 0.8% agarose layer. Colonies were scored after 21 days incubation at 37°C in 5% CO2 in air.
Colony formation assay
HepG2 cells stably transfected with either plasmid pCMV130 or pDNA-3 were plated (1 x 106 cells in 100 mm dishes) in triplicate. After 3 weeks selection in 800 µg/ml G418 cells were fixed and stained with methylene blue in 50% ethanol (Difco) for 20 min, photographed and scored.
Cell cycle analysis
Parental HepG2, pcDNA-1 and pcDNA-2, pRb2/p130-43, pRb2/p130-44 and pRb2/p130-16 cells were seeded at a density of 2 x 106 cells/dish in MEM supplemented with 2.5% FBS for 2 days. Cell cycle analysis was performed using a CycleTESTTMPLUS DNA Reagent Kit as described by the manufacturer (Becton Dickinson, San Jose, CA). Fluorescence-activated cell sorting (FACS) analysis was performed three separate times in triplicate on a FACSCalibur system (Becton Dickinson). The experiments were repeated at least three times and data are expressed as means ± SD.
Tumorigenicity in SCID mice
Parental HepG2, pcDNA-1 and pcDNA-2, pRb2/p130-43, pRb2/p130-44 and pRb2/p130-16 cells were suspended in calcium-free phosphate-buffered saline and 5 x 106 cells s.c. injected into both sides of male SCID mice. Tumour growth was monitored at least twice weekly by Vernier caliper measurement of the length (a) and width (b) of tumours. Tumour volume was calculated as (a x b2)/2. Six animals per group were tested in three sets of independent experiments. Differences in tumour incidence and tumour volume among treatment groups were analyzed by ANOVA.
Statistical analysis
The pRB2/p130, E2F-5,
-tubulin and E2F-4 bands were scanned using a PhosphorImager (Molecular Imager Fx; Bio-Rad, Hercules, CA). For quantitative analysis, the sum of the density of bands corresponding to protein blotting with the antibody under study was calculated and normalized to the amount of
-tubulin. Differences in cell number, colony formation and the levels of proteins under study were analysed by ANOVA. Linear-by-linear and KruskalWallis association tests were used to examine any possible correlation between pRb2/p130 expression and clinico-pathological parameters (TNM status and tumour stage) or patient survival time. Statistical significance was established at P < 0.01.
 |
Results
|
---|
The pathologic data are summarized as follows. Concomitant liver cirrhosis occurred in 15 (33%) of 46 patients and dysplasia within the cirrhotic liver was diagnosed in 12 (26%) of 46 patients. Thirty-two of 46 patients (70%) had multiple HCC nodules. Satellite formation occurred in 26 (56%) of 46 patients. The overall observed 1 year disease-related survival rate of all patients was 74% (34 of 46).
Because loss of pRb2/p130 expression was found in a variety of human cancers (3540) and pRb2/p130 played an important role in regulating cell growth (24,40), the abundance of pRb2/p130 was investigated in ABL tissues and HCC. Figure 1 shows that a doublet of
130 and 140 kDa was detected by pRb2/p130 antibody. pRb2/p130 from HCC lysate migrated more heterogeneously, suggesting the presence of both hyper- and hypophosphorylated pRb2/p130 species. Loss of pRb2/p130 expression was observed in seven of 46 (15.2%) HCCs examined. The levels of pRB2/p130 were much lower in ABL tissues compared with cancerous tissues (Figure 1). When the blots were stripped and reprobed with
-tubulin antibody, roughly equal amounts of protein were observed. After correcting for the amount of protein loaded per lane, the levels of pRb2/p130 were significantly higher in HCCs as compared with ABL tissues (P < 0.01).
It has been demonstrated that growth suppression by pRb2/p130 was, at least in part, a consequence of its ability to bind to E2Fs and to repress activity of the E2F target genes required for cell cycle progression (45,46). Since pRb2/p130 preferentially binds to E2F-4 and E2F-5, pRb2/p130E2F-4 and pRb2/p130E2F-5 complexes in vivo were determined. Lysate from HCCs and ABL tissues were immunoprecipitated using anti-human pRb2/p130 antibody. The immunoprecipitates were subjected to western blot analysis. Blots were incubated with either mouse anti-E2F-5 or mouse anti-E2F-4 or rabbit anti-pRb2/p130 antibodies. As shown in Figure 2, pRb2/p130E2F-4 and pRb-2/p130E2F-5 complexes were found in both ABL and HCC samples. Only a slight increase in the amount of E2F-4 co-precipitated with pRb2/p130 was observed in HCC samples, even though they had approximately 4-fold higher levels of pRb2/p130 than ABL. The E2F-5 antibody recognized three bands of
45 kDa. A similar pattern was observed for E2F-5 when U2-OS cells were transfected with E2F-5 expression vector and lysates from transfected cells were immunoprecipitated with both polyclonal and monoclonal anti-E2F-5 antibodies (29). The differences in the migration of E2F-5 might be due to differences in the levels of phosphorylation or glycosylation.

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 2. pRb2/p130 complexes with E2F-4 and E2F-5 in vivo. Human HCC and ABL tissues were collected and lysed in lysis buffer as described in Materials and methods. Lysates were immunoprecipitated using agarose-conjugated rabbit anti-human pRb2/p130 antibody. Immunoprecipitates were separated by SDSPAGE and western blot analysis was performed as described in Materials and methods. Blots were incubated with either mouse anti-pRb2/p130 (A), mouse anti-E2F-5 (B) or mouse anti-E2F-4 (C) antibodies. Lysate from MCF-7 cells was used as an internal control of co-immunoprecipitation. The experiment was repeated twice.
|
|
Since HCC tumours are heterogeneous with respect to cell type, it is critical to identify the cell type responsible for the elevation or loss of pRb2/p130. Forty-six surgical HCC specimens were examined by immunohistochemistry. For each specimen, an adjacent paraffin section was processed simultaneously for immunohistochemistry. Antibody specific for pRb2/p130 was used to stain the tissues. Figure 3 shows that a strong staining for pRb2/p130 (IS = 280360) was seen in HCC nodules. Thirty-eight of 46 (82.6%) HCCs examined exhibited intense nuclear staining (i = 3+). Both strong nuclear (IS = 250300) and cytoplasmic staining for pRb2/p130 was observed in 33 of 46 (71.7%) HCC (Table I). Loss of pRb2/p130 expression was detected in seven of 46 (15.2%) HCCs examined. pRb2/p130 was not detected by either immunohistochemistry or western blot analysis in this subset of HCC. Both weak nuclear (IS = 4060) and cytoplasmic staining were observed in 100% (46 of 46) of adjacent normal and cirrhotic hepatocytes (Figure 3 and Table I). No distinct staining pattern for pRb2/p130 was observed in cirrhotic and normal hepatocytes. Nuclei of bile duct epithelial cells were strongly stained for pRb2/p130 (IS = 280380), as were the nuclei of the fibrovascular stroma within cirrhotic liver tissue. While intense nuclear and cytoplasmic staining for pRb2/p130 was often observed in stage 3 or 4 tumours, weak nuclear and cytoplasmic staining for pRb2/p130 was found more frequently in stage 1 and stage 2 tumours. Loss of pRb2/p130 was often found in stage 1 and 2 tumours (Table I). Because each of the subsets of HCCs consists of relatively few cases, the association between immunohistochemical expression of pRb2/p130 and tumour stage could not be established. When tissues were stained with Ki-67 antibody, the percentage of Ki-67-positive cells was <0.01% for benign hepatocytes and from 8 to 25% for cancerous cells. Tumours that had high nuclear staining for pRb2/p130 also had a high Ki-67 index (P = 0.003), as determined by the KruskalWallis association test. For 15.2% (7 of 46) of HCC samples where loss of expression of pRb2/p130 was detected by immunohistochemistry and western blotting, the Ki-67 index ranged from 8 to 20%. The results indicate that Ki-67 immunostaining in the tumours was independent of pRb2/p130 status. When the sections were stained with hepatitis B and C antibodies, positive staining signal was observed only in benign liver tissues but not in the HCC (data not shown).

View larger version (109K):
[in this window]
[in a new window]
|
Fig. 3. Immunolocalization of pRb2/p130 in HCC and ABL tissues. Human HCC and ABL tissues were collected and paraffin blocks were prepared as described in Materials and methods. Sections (5 µm) were subjected to immunohistochemical analysis as described in Materials and methods. The sections were stained with mouse anti-pRb2/p130 antibody. Benign adjacent liver (N) and HCC (T) tissues. Original x400.
|
|
To determine whether the loss of pRb2/p130 expression in a subset of HCCs occurred at the transcriptional or post-transcriptional level, RTPCR was performed on four normal and 16 HCCs. Figure 4 shows that a mild elevation in pRb2/p130 mRNA was observed in 44% (7 of 16) of HCCs examined, while 44% (7 of 16) of HCCs had very low or undetectable levels of pRb2/p130 mRNA. The loss of pRb2/p130 immunostaining was correlated with very low levels or the absence of pRb2/p130 mRNA (P = 0.0001).

View larger version (46K):
[in this window]
[in a new window]
|
Fig. 4. Agarose gel electrophoresis of the RTPCR product of pRb2/p130 in ABL and HCC. One-step RTPCR was performed using total RNA as described in Materials and methods. The amplified products were separated on a 1% agarose gel, stained with ethidium bromide and photographs taken. The RTPCR products of pRb2/p130 and control S16 rRNA are shown. Samples are ABL (N) and HCC (T).
|
|
Although the results obtained by immunohistochemistry and western blot analysis suggest a link between HCC and expression of pRb2/p130, its role in hepatoma cell proliferation and hepatocarcinogenesis was not well understood. It has been reported that the pRb2/p130 protein can, at least when overproduced, negatively regulate the G1/S transition of the proliferative cell cycle (47) and suppress the tumorigenicity of cancer cells (24,48). To determine the possible role of pRb2/p130 in HCC, HepG2 cells were stably transfected with a plasmid containing the coding sequence for wild-type human pRb2/p130 or with pcDNA-3 vector. As shown in Figure 5, HepG2 cells transfected with full-length pRb2/p130 and pcDNA gave rise to 126 ± 27 and 423 ± 45 colonies in a colony formation assay. The differences in colony growth of cells transfected with pRb2/p130 cDNA as compared with pcDNA vector were significant (P < 0.01). After 4 weeks selection, individual clones were isolated, expanded and assayed for pRb2/p130 expression by western blot analysis. As shown in Figure 5B, HepG2 cells expressed a normal sized pRb2/p130 protein of
130140 kDa. The slower migrating band corresponded to the putative hyperphosphorylated form while the faster migrating one corresponded to the hypophosphorylated form of pRb2/p130. pRb2/p130-tranfected clones (pRb2/p130-43, -44 and -16) expressed 6- to 8-fold higher pRb2/p130 than pcDNA-transfected cells (pcDNA-1 and -2). The three pRb2/p130-transfected clones and two pc-DNA-transfected clones were selected for further analysis.

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 5. Colony formation and pRb2/p130 expression in HepG2 cells transfected with pCMVp130 cDNA. For colony formation, HepG2 cells were transfected with pcDNA-3 or pCMV130 plasmid containing full-length pRb2/p130 cDNA as described in Materials and methods. After 3 weeks selection in 800 µg/ml G-418, cells were fixed and stained with methylene blue and counted. The number of colonies is shown in (A). Bars with different letters are statistically significant at P < 0.01, as determined by ANOVA. For detection of pRb2/p130, pcDNA-transfected (pc-DNA-1 and -2) and pRb2/p130-transfected (pRb2/p130-43, -44 and -16) clones were grown and cell lysate was prepared for western blot analysis as described in Materials and methods. Total protein extracts (100 µg) underwent 8% SDSPAGE under reducing conditions as described in Materials and methods. Blots were incubated with mouse anti- -tubulin and anti-human pRb2/p130 antibodies (B). Experiments were repeated three times with similar results.
|
|
To determine whether overexpression of pRb2/p130 would alter HepG2 cell morphology, pcDNA-transfected and pRb2/p130-transfected HepG2 cells were seeded at low density, allowed to grow for 5 days and photographed. Figure 6A shows that the morphology of pcDNA-1 cells, like parental HepG2 cells grown as a clump in monolayer, was essentially unchanged after vector transfection, but was markedly altered by pRb2/p130 transfection. The pRb2/p130-transfected cells became more flattened and greatly enlarged in average diameter compared with pcDNA-transfected cells. We evaluated the proliferative behaviour of two vector-transfected and three pRb2/p130-expressing clones in vitro by determining cell number on plastic dishes after 5 days culture. As shown in Figure 6(B), pRb2/p130-transfected cells grew much more slowly than colonies of pcDNA-transfected cells or parental HepG2 cells. The number of cells was significantly less (P < 0.01) in pRb2/p130-43, -44 and -16 clones than in parental HepG2, pcDNA-1 and -2 cells. The results suggest that overproduction of pRb2/p130 in HepG2 cells influenced cell morphology and significantly inhibited growth rate.

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 6. Soft agar colony assay for parental HepG2, pcDNA-transfected and pRb2/p130 transfected cells. Parental HepG2 cells, two pcDNA-transfected clones (pcDNA-1 and -2) and three pRb2/p130-transfected clones (pRb2/p130-43, -44 and -16) were plated in semi-solid medium containing 0.35% Bacto agar over a 0.8% agarose layer for the clones. After 21 days, colonies were fixed and stained with methylene blue and scored. Values represent the means ± SD of three replicates in two independent experiments. Bars with different letters were statistically significant at P < 0.01, as analysed by ANOVA.
|
|
To determine whether overproduction of pRb2/p130 in HepG2 cells would affect their ability to form colonies in soft agar, a soft agar colony assay was performed. Figure 7 shows that soft agar formation was significantly inhibited in each of the pRb2/p130-transfected cells compared with that of pcDNA-transfected cells (P < 0.01).

View larger version (44K):
[in this window]
[in a new window]
|
Fig. 7. Morphology and growth characteristics of pcDNA-transfected and pRb2/p130-transfected clones. For morphological study, pcDNA-1 and pRb2/p130-43, -44 and -16 clones were grown in growth medium for 5 days and photographed using an inverse microscope (A). Magnification x800. For the proliferation study, parental HepG2 cells and two pcDNA-transfected and three pRb2/p130-transfected clones were grown on plastic dishes for 5 days. Cell number was determined daily for 5 days and plotted (B). The number of cells was significantly less (P < 0.01, ANOVA) in pRb2/p130-transfected than in pcDNA-transfected cells. Experiments were repeated three times with similar results (<10% variation).
|
|
Because pRb2/p130 has been implicated in mediating the G0/G1 phase cell cycle arrest (reviewed in 40), we wished to determine whether the slow growth of pRb2/p130-transfected cells was due to cell cycle alteration. pcDNA-transfected and pRb2/p130-transfected cells were grown in MEM supplemented with 2.5% FBS for 24 h and then harvested. Their cell cycle profiles were examined by flow cytometry analysis. As shown in Table II, 7078% of the cells were in the G0/G1 stage. The results are in agreement with previous studies (48,49) showing that the growth suppressive action of pRb2/p130 is specific to the G0/G1 phase of the cell cycle.
View this table:
[in this window]
[in a new window]
|
Table II. Flow cytometry analysis (fluorescence-activated cell sorting (FACS) showing the cell cycle profile of pcDNA-transfected and pRb2/p130-transfected HepG2 cells
|
|
To test the neoplastic behaviour of pRb2/p130-transfected cells, parental HepG2, pcDNA-transfected and pRb2/p130-transfected cells were injected into both flanks of SCID mice. Parental HepG2 and pcDNA-transfected cells formed tumours in SCID mice 3 weeks after injection of 5 x 106 cells. The rate of tumor formation following injection of HepG2 cells was 100% (8 of 8), that of pcDNA-1 and -2 clones 100 (8 of 8) and 100% (8 of 8), respectively, and that of pRb2/p130-42, -43 and -16 clones 87.5 (7 of 8), 87.5 (7 of 8) and 50% (7 of 14), respectively. The growth of parental HepG2 and pcDNA-transfected clones in SCID mice was significantly faster than that of pRb2/p130-transfected clones (P < 0.01) (Figure 8A). The tumour volumes evaluated at 9 weeks after injection for parental HepG2, pcDNA-1, pcDNA-2, pRb2/p130-43, pRb2/p130-44 and pRb2/p130-16 were 0.543 ± 0.09, 0.613 ± 0.08, 0.621 ± 0.09, 0.162 ± 0.07, 0.194 ± 0.05 and 0.116 ± 0.05, respectively (Figure 8B). The differences in tumour growth and tumour volume between pcDNA-transfected and pRb2/p130-transfected clones were statistically significant (P < 0.01). The results suggest that overproduction of pRb2/p130 in HepG2 cells affected cellular morphology, growth rate and tumorigenicity in SCID mice.

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 8. Neoplastic behaviour of pRb2/p130-transfected cells in SCID mice. Parental HepG2, pcDNA-transfected and pRb2/p130-transfected cells were injected (5 x 106 cells/injection) into both flanks of SCID mice as described in Materials and methods. Parental HepG2 and pcDNA-transfected cells formed tumours in SCID mice within 4 weeks. Tumour growth curves of HepG2, pcDNA-1, pcDNA-2 and three pRb2/p130-transfected clones (pRb2/p130-43, -44 and -16) are shown in (A). Parental HepG2 cells and pcDNA-transfected clones grew significantly faster than pRb2/p130-transfected ones (P < 0.01). At the end of week 9 the mice were killed and tumours were harvested weighed and plotted (B). Experiments were repeated twice with similar results. Differences in final tumour weights between the control group and pRb2/p130-transfected one were statistically significant at P < 0.01, as analysed by ANOVA.
|
|
 |
Discussion
|
---|
Deranged expression of cell cycle-related proteins is one of the major factors contributing to HCC development (5052). Loss or inactivation of pRB family proteins allows the E2F family of transcription factors to induce the expression of genes necessary for S phase entry, such as cyclin A and PCNA (reviewed in 53). We have demonstrated that expression of pRb2/p130 is detected by immunohistochemistry in 84.8% (39 of 46) of HCCs examined. A complete absence of pRb2/p130 was found in 15.2% (7 of 46) of cases. This observation was similar to previous studies showing that a reduction in or loss of pRB2/p130 is found in lung and endometrial cancers (32,33,54). A positive pRb2/p130 signal is localized both in the cell nucleus and cytoplasm of hepatocarcinoma cells. Weak nuclear and cytoplasmic staining is found more often in dysplastic or benign hepatocytes. Transfection of HepG2 cells with a plasmid carrying the pRb2/p130 ORF leads to overproduction of pRb2/p130 protein, growth suppression, cell cycle arrest at G0/G1, a reduction in colony formation and a decrease in tumour formation in SCID mice compared with cells transfected with a plasmid containing only the neomycin gene. In the present study, pRb2/p130 function can be seen even in the presence of a functional wild-type pRb2/p130 product in HepG2 cells. A TUNEL assay showed that overproduction of pRb2/p130 is growth suppressive in HepG2 cells without any toxic effect, such as an increase in apoptosis (data not shown). These observations are in agreement with previous reports (48,49,55). These results also demonstrate that overproduction of pRb2/p130 alters cellular morphology. This observation is consistent with previous reports showing that overproduction of pRb2/p130 in HONE-1 cells causes a significant reduction in cell proliferation and a change in cell morphology (24).
Although the aetiology and pathogenesis of HCC remain unclear, our observations suggest a role for the pRb2/p130 regulatory network. Genetic alterations in the pRb2/p130 gene in a variety of cancer cell lines and in primary tumours has been reported (31,40). The remarkable homology between pRB and pRb2/p130, together with their ability of pRb2/p130 to induce cell cycle arrest, suggests that pRb2/p130 can act as a tumour suppressor gene in HCC. The observations are in agreement with previous studies (24,40,49). The most strongly conserved regions of p107, pRb2/p130 and pRB correspond to the pocket region. Like pRB, pRb2/p130 and p107 display a cell cycle-regulated phosphorylation pattern (27) and form complexes with different members of the E2F family of transcription factors. Both pRb2/p130 and p107, like pRB, display growth suppressive properties, although the growth arrest mediated by these proteins is not identical (20,24,25). Interplay between the RB family and the E2F family is hypothesized to regulate transcription and progression of the cell cycle. It has been demonstrated that pRb2/p130 is not evenly distributed within the nucleus and that cell cycle-dependent binding with E2F-4 changes as a function of its subnuclear localization. In the nucleoplasm pRB2/p130E2F-4 complexes are more numerous during G0/G1, while in the nucleolus it increases in S phase. In G0/G1 pRb2/p130 exists predominantly in a hypophosphorylated form and becomes hyperphosphorylated in S phase. pRb2/p130 inactivation by hyperphosphorylation corresponded to weaker binding to the nuclear matrix. This suggests that pRb2/p130 could exert fine repressive control by modulating its binding with the nuclear matrix as the cell moves from G1 to S phase.
In the present study, the amount of E2F-4 co-precipitating with pRb2/p130 was not significantly different between ABL and HCC, even though HCC had more pRb2/p130 than ABL. It remains to be determined whether pRb2/p130 in HCC has a lower affinity for E2Fs than that found in ABL. Since pRb2/p130 in HCC retains its ability to form complexes with E2F-5 and E2F-4, it is likely that pRb2/p130 is still functional. At the present time the mechanism(s) responsible for elevation of pRb2/p130 in a subset of HCC is still not known. It is possible that overexpression of pRb2/p130 in HCC is a defensive mechanism against aberrant proliferation. This hypothesis is supported by a similar finding where overexpression of p73 is detected in a majority of HCCs (56). It is also possible that pRb2/p130, like p53 and p73, can increase its expression in response to oncogenic activation or hypoxia or growth factor depletion. In response to these stimuli, pRb2/p130 expression increases to induce cell cycle arrest.
Our data, identifying high expression levels of pRb2/p130 in HCC, provide, to our knowledge, the first analysis of this protein in patients with HCC. Previous studies showed a negative correlation between histological grading and pRb2/p130 staining in specimens of human lung cancer (34) and endometrial carcinoma (35) and patients with lack of pRb2/p130 expression have at least a 5-fold increased risk of dying from the disease (35). Our present study indicates that while absent or low pRb2/p130 expression is often detected in stage 1 and stage 2 HCC, many stage 3 and 4 HCC express high levels of pRb2/p130. The molecular basis of this finding remains to be elucidated. Because the number of HCC cases examined in the present study is small, the association between pRb2/p130 expression and histological staging or survival time cannot be established.
In conclusion, we show that elevation and loss of pRb2/p130 protein is detected by both immunohistochemistry and western blot analysis in 84.8 (39 of 46) and 15.2% (7 of 46) of HCCs examined, respectively. Introduction of pRb2/p130 cDNA into HepG2 cells causes cell cycle arrest in G0/G1, growth inhibition in vitro and a reduction in tumor formation in vivo. These observations suggest that pRb2/p130 plays a vital role in the growth regulation of liver cancer cells and its elevation in HCCs may serve as a protective mechanism to limit the uncontrolled growth of cancer cells. In consideration of these data, the impact of HCC in terms of mortality in Southeast Asia and the results presented in this report and others (24,40), Rb2/p130 gene therapy may serve as a therapeutic alternative or adjuvant in combating HCC and be worth further investigation.
 |
Acknowledgments
|
---|
We would like to thank Professor Peter Whyte (McMaster University, Ontario, Canada) for the gift of the pCMV130 plasmid. This work was supported by the National Cancer Centre Tissue Repository and grants from the National Medical Research Council of Singapore (NMRC/0541/2001), SingHealth Cluster Research Fund (EX 008/2001), A*STAR-BMRC (LS/00/017) and A*STAR-BMRC (LS/00/019) to Huynh Hung.
 |
References
|
---|
- Schafer,D.F. and Sorrell,M.F. (1999) Hepatocellular carcinoma. Lancet, 353, 12531257.[CrossRef][ISI][Medline]
- Ince,N. and Wands,J.R. (1999) The increasing incidence of hepatocellular carcinoma. N. Engl. J. Med., 340, 798799.[Free Full Text]
- Okuda,K., Ohtsuki,T., Obata,H., Tomimatsu,M., Okazaki,N., Hasegawa,H., Nakajima,Y. and Ohnishi,K. (1985) Natural history of hepatocellular carcinoma and prognosis in relation to treatment. Study of 850 patients. Cancer, 56, 918928.[ISI][Medline]
- Colombo,M. (1992) Hepatocellular carcinoma. J. Hepatol., 15, 225236.[ISI][Medline]
- Lai,E.C., Fan,S.T., Lo,C.M., Chu,K.M., Liu,C.L. and Wong,J. (1995) Hepatic resection for hepatocellular carcinoma. An audit of 343 patients. Ann. Surg., 221, 291298.[ISI][Medline]
- Takenaka,K., Kawahara,N., Yamamoto,K., Kajiyama,K., Maeda,T., Itasaka,H., Shirabe,K., Nishizaki,T., Yanaga,K. and Sugimachi,K. (1996) Results of 280 liver resections for hepatocellular carcinoma. Arch. Surg., 131, 7176.[Abstract]
- Huguet,C., Stipa,F. and Gavelli,A. (2000) Primary hepatocellular cancer: Western experience. In Blumgart,L. (ed.) Surgery of the Liver and Biliary Tract. Churchill Livingstone, London, pp. 13651369.
- Lai,E. and Wong,J. (1994) Hepatocellular carcinoma: the Asian experience. In Blumgart,L. (ed.) Surgery of the Liver and the Biliary Tract. Churchill Livingstone, London, pp. 13491363.
- Chan,E.S., Chow,P.K., Tai,B., Machin,D. and Soo,K. (2000) Neoadjuvant and adjuvant therapy for operable hepatocellular carcinoma. Cochrane Database. Syst. Rev., CD001199.
- Montesano,R., Hainaut,P. and Wild,C.P. (1997) Hepatocellular carcinoma: from gene to public health. J. Natl Cancer Inst., 89, 18441851.[Abstract/Free Full Text]
- Zeng,J.Z., Wang,H.Y., Chen,Z.J., Ullrich,A. and Wu,M.C. (2002) Molecular cloning and characterization of a novel gene which is highly expressed in hepatocellular carcinoma. Oncogene, 21, 49324943.[CrossRef][ISI][Medline]
- Murakami,Y., Hayashi,K., Hirohashi,S. and Sekiya,T. (1991) Aberrations of the tumor suppressor p53 and retinoblastoma genes in human hepatocellular carcinomas. Cancer Res., 51, 55205525.[Abstract]
- Lee,A.V. and Yee,D. (1995) Insulin-like growth factors and breast cancer. Biomed. Pharmacother., 49, 415421.[CrossRef][ISI][Medline]
- Wong,I.H., Johnson,P.J., Lai,P.B., Lau,W.Y. and Lo,Y.M. (2000) Tumor-derived epigenetic changes in the plasma and serum of liver cancer patients. Implications for cancer detection and monitoring. Ann. N. Y. Acad. Sci., 906, 102105.[Free Full Text]
- Kamb,A., Gruis,N.A., Weaver-Feldhaus,J., Liu,Q., Harshman,K., Tavtigian,S.V., Stockert,E., Day,R.S.,III, Johnson,B.E. and Skolnick,M.H. (1994) A cell cycle regulator potentially involved in genesis of many tumor types (see comments). Science, 264, 436440.[ISI][Medline]
- Wong,I.H., Lo,Y.M., Yeo,W., Lau,W.Y. and Johnson,P.J. (2000) Frequent p15 promoter methylation in tumor and peripheral blood from hepatocellular carcinoma patients (In Process Citation). Clin. Cancer Res., 6, 35163521.[Abstract/Free Full Text]
- Stone,S., Dayananth,P., Jiang,P., Weaver-Feldhaus,J.M., Tavtigian,S.V., Cannon-Albright,L. and Kamb,A. (1995) Genomic structure, expression and mutational analysis of the P15 (MTS2) gene. Oncogene, 11, 987991.[ISI][Medline]
- Whyte,P., Buchkovich,K.J., Horowitz,J.M., Friend,S.H., Raybuck,M., Weinberg,R.A. and Harlow,E. (1988) Association between an oncogene and an anti-oncogene: the adenovirus E1A proteins bind to the retinoblastoma gene product. Nature, 334, 124129.[CrossRef][ISI][Medline]
- Ewen,M.E., Xing,Y.G., Lawrence,J.B. and Livingston,D.M. (1991) Molecular cloning, chromosomal mapping and expression of the cDNA for p107, a retinoblastoma gene product-related protein. Cell, 66, 11551164.[ISI][Medline]
- Zhu,L., van deneuvel,H.S., Helin,K., Fattaey,A., Ewen,M., Livingston,D., Dyson,N. and Harlow,E. (1993) Inhibition of cell proliferation by p107, a relative of the retinoblastoma protein. Genes Dev., 7, 11111125.[Abstract]
- Mayol,X., Grana,X., Baldi,A., Sang,N., Hu,Q. and Giordano,A. (1993) Cloning of a new member of the retinoblastoma gene family (pRb2) which binds to the E1A transforming domain. Oncogene, 8, 25612566.[ISI][Medline]
- Li,Y., Graham,C., Lacy,S., Duncan,A.M. and Whyte,P. (1993) The adenovirus E1A-associated 130-kD protein is encoded by a member of the retinoblastoma gene family and physically interacts with cyclins A and E. Genes Dev., 7, 23662377.[Abstract]
- Hannon,G.J., Demetrick,D. and Beach,D. (1993) Isolation of the Rb-related p130 through its interaction with CDK2 and cyclins. Genes Dev., 7, 23782391.[Abstract]
- Claudio,P.P., Howard,C.M., Baldi,A., De Luca,A., Fu,Y., Condorelli,G., Sun,Y., Colburn,N., Calabretta,B. and Giordano,A. (1994) p130/pRb2 has growth suppressive properties similar to yet distinctive from those of retinoblastoma family members pRb and p107. Cancer Res., 54, 55565560.[Abstract]
- Zhu,L., Enders,G., Lees,J.A., Beijersbergen,R.L., Bernards,R. and Harlow,E. (1995) The pRB-related protein p107 contains two growth suppression domains: independent interactions with E2F and cyclin/cdk complexes. EMBO J., 14, 19041913.[Abstract]
- Yeung,R.S., Bell,D.W., Testa,J.R., Mayol,X., Baldi,A., Grana,X., Klinga-Levan,K., Knudson,A.G. and Giordano,A. (1993) The retinoblastoma-related gene, RB2, maps to human chromosome 16q12 and rat chromosome 19. Oncogene, 8, 34653468.[ISI][Medline]
- Baldi,A., De Luca,A., Claudio,P.P., Baldi,F., Giordano,G.G., Tommasino,M., Paggi,M.G. and Giordano,A. (1995) The RB2/p130 gene product is a nuclear protein whose phosphorylation is cell cycle regulated. J. Cell Biochem., 59, 402408.[ISI][Medline]
- Cobrinik,D., Whyte,P., Peeper,D.S., Jacks,T. and Weinberg,R.A. (1993) Cell cycle-specific association of E2F with the p130 E1A-binding protein. Genes Dev., 7, 23922404.[Abstract]
- Hijmans,E.M., Voorhoeve,P.M., Beijersbergen,R.L., van't Veer,L.J. and Bernards,R. (1995) E2F-5, a new E2F family member that interacts with p130 in vivo. Mol. Cell. Biol., 15, 30823089.[Abstract]
- Vairo,G., Livingston,D.M. and Ginsberg,D. (1995) Functional interaction between E2F-4 and p130: evidence for distinct mechanisms underlying growth suppression by different retinoblastoma protein family members. Genes Dev., 9, 869881.[Abstract]
- Cinti,C., Claudio,P.P., Howard,C.M., Neri,L.M., Fu,Y., Leoncini,L., Tosi,G.M., Maraldi,N.M. and Giordano,A. (2000) Genetic alterations disrupting the nuclear localization of the retinoblastoma-related gene RB2/p130 in human tumor cell lines and primary tumors. Cancer Res., 60, 383389.[Abstract/Free Full Text]
- Baldi,A., Esposito,V., De Luca,A., Howard,C.M., Mazzarella,G., Baldi,F., Caputi,M. and Giordano,A. (1996) Differential expression of the retinoblastoma gene family members pRb/p105, p107 and pRb2/p130 in lung cancer. Clin. Cancer Res., 2, 12391245.[Abstract]
- Baldi,A., Esposito,V., De Luca,A., Fu,Y., Meoli,I., Giordano,G.G., Caputi,M., Baldi,F. and Giordano,A. (1997) Differential expression of Rb2/p130 and p107 in normal human tissues and in primary lung cancer. Clin. Cancer Res., 3, 16911697.[Abstract]
- Caputi,M., Groeger,A.M., Esposito,V., De Luca,A., Masciullo,V., Mancini,A., Baldi,F., Wolner,E. and Giordano,A. (2002) Loss of pRb2/p130 expression is associated with unfavorable clinical outcome in lung cancer. Clin. Cancer Res., 8, 38503856.[Abstract/Free Full Text]
- Susini,T., Baldi,F., Howard,C.M., Baldi,A., Taddei,G., Massi,D., Rapi,S., Savino,L., Massi,G. and Giordano,A. (1998) Expression of the retinoblastoma-related gene Rb2/p130 correlates with clinical outcome in endometrial cancer. J. Clin. Oncol., 16, 10851093.[Abstract]
- Tanaka,N., Ogi,K., Odajima,T., Dehari,H., Yamada,S., Sonoda,T. and Kohama,G. (2001) pRb2/p130 protein expression is correlated with clinicopathologic findings in patients with oral squamous cell carcinoma. Cancer, 92, 21172125.[CrossRef][ISI][Medline]
- Massaro-Giordano,M., Baldi,G., De Luca,A., Baldi,A. and Giordano,A. (1999) Differential expression of the retinoblastoma gene family members in choroidal melanoma: prognostic significance. Clin. Cancer Res., 5, 14551458.[Abstract/Free Full Text]
- Leoncini,L., Bellan,C., Cossu,A. et al. (1999) Retinoblastoma-related p107 and pRb2/p130 proteins in malignant lymphomas: distinct mechanisms of cell growth control. Clin. Cancer Res., 5, 40654072.[Abstract/Free Full Text]
- Claudio,P.P., Howard,C.M., Fu,Y., Cinti,C., Califano,L., Micheli,P., Mercer,E.W., Caputi,M. and Giordano,A. (2000) Mutations in the retinoblastoma-related gene RB2/p130 in primary nasopharyngeal carcinoma. Cancer Res., 60, 812.[Abstract/Free Full Text]
- Claudio,P.P., Howard,C.M., Pacilio,C. et al. (2000) Mutations in the retinoblastoma-related gene RB2/p130 in lung tumors and suppression of tumor growth in vivo by retrovirus-mediated gene transfer. Cancer Res., 60, 372382.[Abstract/Free Full Text]
- Spiessl,B., Beahrs,O.H., Hermanek,P., Hutter,R.V.P., Scheibe,O., Sobin,L.H. and Wagner,G. (1992) TNM Atlas. Illustrated Guide to the TNM/pTNM Classification of Malignant Tumours. Springer Verlag, Berlin, pp. 104111.
- Ishak,K.G., Goodman,Z.D. and Stocker,J.T. (2001) Tumors of the liver and intrahepatic bile. Atlas of Tumor Pathology, Third Series. Armed Forces Institute of Pathology, Washington, DC, pp. 199230.
- Huynh,H., Chow,P.K., Ooi,L.L. and Soo,K.C. (2002) A possible role for insulin-like growth factor-binding protein-3 autocrine/paracrine loops in controlling hepatocellular carcinoma cell proliferation. Cell Growth Differ., 13, 115122.[Abstract/Free Full Text]
- Claudio,P.P., Zamparelli,A., Garcia,F.U. et al. (2002) Expression of cell-cycle-regulated proteins pRb2/p130, p107, p27(kip1), p53, mdm-2 and Ki-67 (MIB-1) in prostatic gland adenocarcinoma. Clin. Cancer Res., 8, 18081815.[Abstract/Free Full Text]
- Taya,Y. (1997) RB kinases and RB-binding proteins: new points of view. Trends Biochem. Sci., 22, 1417.[CrossRef][ISI][Medline]
- Dyson,N. (1998) The regulation of E2F by pRB-family proteins. Genes Dev., 12, 22452262.[Free Full Text]
- Adams,P.D. (2001) Regulation of the retinoblastoma tumor suppressor protein by cyclin/cdks. Biochim. Biophys. Acta, 1471, M123M133.[CrossRef][ISI][Medline]
- Pupa,S.M., Howard,C.M., Invernizzi,A.M., De Vecchi,R., Giani,C., Claudio,P.P., Colnaghi,M.I., Giordano,A. and Menard,S. (1999) Ectopic expression of pRb2/p130 suppresses the tumorigenicity of the c-erbB-2-overexpressing SKOV3 tumor cell line. Oncogene, 18, 651656.[CrossRef][ISI][Medline]
- Howard,C.M., Claudio,P.P., Gallia,G.L., Gordon,J., Giordano,G.G., Hauck,W.W., Khalili,K. and Giordano,A. (1998) Retinoblastoma-related protein pRb2/p130 and suppression of tumor growth in vivo. J. Natl Cancer Inst., 90, 14511460.[Abstract/Free Full Text]
- Santoni-Rugiu,E., Jensen,M.R. and Thorgeirsson,S.S. (1998) Disruption of the pRb/E2F pathway and inhibition of apoptosis are major oncogenic events in liver constitutively expressing c-myc and transforming growth factor alpha. Cancer Res., 58, 123134.[Abstract]
- Deane,N.G., Parker,M.A., Aramandla,R., Diehl,L., Lee,W.J., Washington,M.K., Nanney,L.B., Shyr,Y. and Beauchamp,R.D. (2001) Hepatocellular carcinoma results from chronic cyclin D1 overexpression in transgenic mice. Cancer Res., 61, 53895395.[Abstract/Free Full Text]
- Masaki,T., Shiratori,Y., Rengifo,W. et al. (2003) Cyclins and cyclin-dependent kinases: comparative study of hepatocellular carcinoma versus cirrhosis. Hepatology, 37, 534543.[CrossRef][ISI][Medline]
- Dyson,N. (1998) The regulation of E2F by pRB-family proteins. Genes Dev., 12, 22452262.[Free Full Text]
- Susini,T., Baldi,F., Howard,C.M., Baldi,A., Taddei,G., Massi,D., Rapi,S., Savino,L., Massi,G. and Giordano,A. (1998) Expression of the retinoblastoma-related gene Rb2/p130 correlates with clinical outcome in endometrial cancer. J. Clin. Oncol., 16, 10851093.[Abstract]
- Claudio,P.P., De Luca,A., Howard,C.M., Baldi,A., Firpo,E.J., Koff,A., Paggi,M.G. and Giordano,A. (1996) Functional analysis of pRb2/p130 interaction with cyclins. Cancer Res., 56, 20032008.[Abstract]
- Tannapfel,A., Wasner,M., Krause,K., Geissler,F., Katalinic,A., Hauss,J., Mossner,J., Engeland,K. and Wittekind,C. (1999) Expression of p73 and its relation to histopathology and prognosis in hepatocellular carcinoma. J. Natl Cancer Inst., 91, 11541158.[Abstract/Free Full Text]
Received October 21, 2003;
revised March 10, 2004;
accepted March 22, 2004.