J Gastroenterol 2003; 38:751–758 DOI 10.1007/s00535-003-1141-8

Role of a novel oncogenic protein, gankyrin, in hepatocyte proliferation Akio Iwai1, Hiroyuki Marusawa1, Tetsuya Kiuchi2, Hiroaki Higashitsuji3, Koichi Tanaka2, Jun Fujita3, and Tsutomu Chiba1 1

Division of Gastroenterology and Hepatology, Department of Medicine, Graduate School of Medicine, Kyoto University, 54 Kawara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan Department of Transplantation and Immunology, Faculty of Medicine, Kyoto University, Kyoto, Japan 3 Department of Clinical Molecular Biology, Faculty of Medicine, Kyoto University, Kyoto, Japan 2

Background. Gankyrin is a novel oncogenic protein ubiquitously overexpressed in hepatocellular carcinoma. However, little is known about the physiological role of gankyrin in the cell-cycle progression of normal noncancerous hepatocytes at present. We investigated the possible involvement of gankyrin in hepatocyte proliferation and examined the expression of gankyrin in the liver of patients with fulminant hepatic failure (FHF). Methods. We examined the potential cell-cycle dependence of gankyrin expression in primary hepatocytes after mitogenic stimulation. Gankyrin expression level was also measured in the liver tissue of patients with FHF, as a model of active proliferation of human hepatocytes associated with liver regeneration. Results. In primary hepatocytes, gankyrin was expressed in the G1/S phase of the cell cycle at 48 h after mitogenic stimulation, which coincided with the phase of cyclin D1 RNA upregulation and RB1 protein downregulation. Using a quantitative reverse transcription polymerase chain reaction assay, we showed that gankyrin mRNA in the liver tissue of patients with FHF was expressed abundantly compared with that in healthy individuals (median, 109.7 [interquartile range; IQR, 49.0–186.4] vs median, 22.4 [IQR, 1.4–77.3] copies per sample, respectively). Conclusions. The upregulation of gankyrin correlates with cell-cycle progression in normal hepatocyte proliferation. Thus, gankyrin may play a role in cell-cycle progression in normal hepatocytes. Moreover, gankyrin may have a role in the pathophysiology of FHF. Key words: gankyrin, primary hepatocyte, liver regeneration, fulminant hepatic failure (FHF)

Received: October 9, 2002 / Accepted: February 21, 2003 Reprint requests to: T. Chiba

Introduction The liver has a great regenerative capacity; hepatocyte loss caused by various types of injuries or surgical resection triggers liver regeneration.1 Many investigators have attempted to elucidate the mechanisms of liver regeneration using animal models subjected to partial hepatectomy or chemical injuries.1–3 These studies have demonstrated that many genes are upregulated during liver regeneration. For example, the initial phase of liver regeneration is characterized by the expression of immediate early genes, such as c-fos, c-jun, and c-myc, followed by the activation of delayed genes, including Bcl-x, and still later, the expression of cell-cycle genes such as p53 and p21.1,4,5 Eventually, progression of cell proliferation is associated with the expression of DNA replication and mitosis genes such as cyclin D1, E, B, and C.6 Indeed, Haber et al.5 reported that more than 70 genes are expressed during liver regeneration in the rat partial hepatectomy model, and they further stated that it is a difficult but important challenge to determine which among the enhanced genes play essential roles in liver regeneration. Gankyrin is a protein with six ankyrin repeats, and it is ubiquitously overexpressed in hepatocellular carcinoma tissues.7 This oncogenic protein contains the retinoblastoma (RB1)-binding motif, and accelerates the degradation of RB1 both in vitro and in vivo.7 Thus, gankyrin is purported to promote cancer-cell proliferation through interaction with RB1 protein.7,8 However, at present, the physiological role of gankyrin in the cell-cycle progression of normal hepatocytes remains unknown. In the present study, therefore, to address gankyrin function in the cell-cycle progression of normal hepatocytes, we investigated the potential cell-cycledependence of gankyrin expression in rat primary hepatocytes after mitogenic stimulation. In addition, to obtain better understanding of the physiological role

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of gankyrin in human hepatocyte proliferation, we examined the expression of this molecule in the regenerating liver of patients with fulminant hepatic failure (FHF).

Patients, materials, and methods Primary rat hepatocyte culture Adult rat hepatocytes were prepared from 10-week old Fisher rats by the two-step collagenase perfusion method,9 and were centrifuged three times at 50g for 1min. The pellet was used for the preparation of parenchymal hepatocytes. A fraction containing parenchymal hepatocytes was isolated as a pellet obtained by centrifuging the former pellet through 90% Percoll (Amersham Pharmacia Biotech, Uppsala, Sweden) at 50g for 10 min. This fraction was washed twice with Dulbecco’s modified Eagle’s medium (Nissui, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS). Viability of the separated cells was assessed by trypan blue exclusion. Only cell preparations with more than 90% viability were used for further analyses. Fresh rat hepatocytes were seeded in 35-mm diameter type-I collagen-coated plates (Iwaki, Chiba, Japan), at a density of 5 ⫻ 105 cells per well, in 2ml of medium consisting of a 1: 1 mixture of Ham F-12 (Gibco-BRL, Gaithersburg, MD, USA) and Williams’ medium E (Sigma, St. Louis, MO, USA), supplemented with 0.6mg/l insulin, 32.3 mg/l l-proline, 10⫺7 mol/l dexamethasone (Sigma), 100IU/ml penicillin G, and 100µg/ml streptomycin. Preparation of RNA samples from rat hepatocyte and liver tissue Rat hepatocyte samples in a 35-mm diameter type-I collagen-coated plate were homogenized in a total volume of 1ml Trizol Reagent (Life Technologies, Vienna, Austria) for 3min at room temperature. Total RNA was isolated according to the manufacturer’s protocol, followed by ethanol precipitation. The extracted RNA was resuspended in RNase-free water at a final concentration of 1µg/µl, and the quality of these samples was assessed on agarose gels before further use. Liver samples from the patients with FHF and healthy donors were obtained at the time of operation and frozen immediately in liquid nitrogen. Subsequently, tissue samples weighing 50–100mg were homogenized in a total volume of 1 ml Trizol Reagent for 3min at room temperature. RNAs from the patients and donors were prepared using a similar method. These samples were stored at ⫺80°C until use.

Reverse transcription-polymerase chain reaction (RT-PCR) of rat gankyrin mRNA Total RNA (1µg) from rat primary hepatocytes was preincubated for 3min at 70°C with 50µM oligo-dTn primer (Perkin Elmer [PE] Applied Biosystems, Forster City, CA, USA), 0.1M dithiothreitol (DTT; Gibco-BRL), 10mM deoxynucleoside triphosphate (dNTP) mix, and RT Buffer (Gibco-BRL). Samples were then reverse transcribed for 60min at 42°C with 200U Superscript II (Gibco-BRL) in a total volume of 20µl, and then denatured for 30min at 100°C. PCR was performed using TaqMan Gold RT-PCR Reagents, according to the manufacturer’s protocol (PE Applied Biosystems). In brief, each cDNA that was synthesized by RT was added to 1.25U of Taq DNA Polymerase (AmpliTaq Gold; Roche Molecular Systems, Branchburg, NJ, USA), 0.2µM sense primers, 0.2 µM antisense primers, 2 mM MgCl2, 0.2 mM dNTP mix, and PCR reaction buffer (10mM Tris-HCl, pH 8.3, 50 mM KCl) in a total volume of 50µl. Amplification was performed using a thermal cycler (Perkin Elmer 9600). The PCR primers used were: gankyrin sense primer, 5⬘ATTGCTGCTTCCGCTGGC-3⬘ and the corresponding antisense primer, 5⬘-GGATGTTTGTGGATGC TTTG-3⬘; cyclin D1 sense primer, 5⬘-GTGCAGAGGG AGATTGTGCC-3⬘ and the corresponding antisense primer, 5⬘-GCGGCCAGGTTCCATTTGAG-3⬘; cyclin A2 sense primer, 5⬘-CCTGCATTTGGCTGTGAA CTAC-3⬘, and the corresponding antisense, primer 5⬘CACAAACTCTGCTACTTCTGGG-3⬘; porphobilinogen deaminase (PBGD) sense primer, 5⬘-ATGTCC GGTAACGGCGGC-3⬘, and the corresponding antisense primer, 5⬘-CAGCATCGCTACCACAGT GTC-3⬘. Rat PBGD was used as an internal control. Each PCR cycle consisted of a denaturation step for 1min at 94°C, an annealing step for 1min each at 58°C (gankyrin), 66°C (cyclin D1), 63°C (cyclin A2), and 58°C (PBGD), and an extension step for 2min at 72°C. PCR products were separated on 2.0% agarose gels.

Patients Between July 1998 and October 2000, 211 patients underwent living-related liver transplantation at Kyoto University Hospital. Of the 211 patients, 28 were diagnosed as having FHF, and 13 of these 28 patients were randomly selected to be enrolled in this study (Table 1). All 13 patients had had normal liver function before the onset of liver dysfunction; presented with hepatic coma above grade II, as defined at the Inuyama Symposium (1981); and had prolonged prothrombin time (PT), equivalent to less than 40% of control values.10 Of the 13 recipients, 8 were male and 5 were female, with a mean age of 32.9 years (range, 9 months to 59 years).

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Table 1. Characteristics of patients Case no. 1 2 3 4 5 6 7 8 9 10 11 12 13

Age (years)

Sex

Etiology

Clinical course

Coma grade

49 54 18 48 29 47 23 0.9 29 41 28 59 2

M M M M M F F F F M M F M

HBV HBV HBV HBV Unknown Unknown HBV Unknown Unknown HBV Unknown Unknown Unknown

A A A A A A A SA SA SA SA SA SA

IV III IV IV IV IV IV IV IV IV III IV IV

ALT T. Bil (IU/l) (mg/dl)

D/T ratio

PT AFP (%) (ng/ml)

6178 2972 5000 1913 1500 170 6680 2730 1511 339 1480 1102 1039

0.63 0.34 0.28 0.30 0.28 0.48 0.49 0.43 0.48 0.17 0.39 0.52 0.28

10.0 32.0 17.0 21.0 23.0 17.6 4.8 21.9 11.0 5.0 20.0 25.0 28.7

14.0 9.4 21.6 19.4 21.6 16.2 4.1 11.3 28.8 0.6 39.0 13.4 30.6

46.5 9.2 44.0 NT 28.8 5.8 184.0 NT 21.7 19.0 1.8 45.0 8.1

HGF (ng/ml) NT 5.45 1.01 NT 4.67 2.14 NT NT 3.11 NT 2.56 NT NT

LW/BW Gankyrin (%) (copies) 0.95 1.01 0.93 1.21 0.83 1.10 1.14 3.14 1.43 0.88 0.98 0.86 1.59

130.44 106.71 135.55 186.43 155.58 184.21 109.70 112.22 102.38 49.02 93.70 64.63 77.09

All patients underwent liver transplantation due to having fulminant hepatic failure (FHF) The maximum levels of ALT and T. Bil during the clinical course are shown. The minimum levels of PT and the D/T ratio during the clinical course are shown. The levels of AFP and HGF just before operation are shown HBV, hepatitis B virus; A and SA, acute and subacute form of fulminant hepatitis, respectively, as defined in “patients, material, and methods”; ALT, alanine aminotransferase; T. Bil, total bilirubin; D/T ratio, direct bilirubin-to-total bilirubin ratio; PT, prothrombin time; AFP, alphafetoprotein; HGF, hepatocyte growth factor; LW/BW, total liver weight/body weight at liver transplantation; NT, not tested

Before transplantation, liver function in all recipients was evaluated by blood chemistry, ultrasonography, and computed tomography (CT). IgM antibody to hepatitis B core antigen was positive in 6 patients, and the remaining 7 were negative for any serological marker for hepatitis A, B, and C. None of these FHF patients had a history of exposure to any toxin or drug, nor did any have a history of alcohol misuse. For further evaluation, these recipients were classified into two groups based on the criteria of Takahashi and Shimizu:10 recipients having an acute form of FHF, and those with a subacute form of FHF. Patients with the acute-form FHF were defined as those in whom encephalopathy above grade II occurred within 10 days after the onset of liver dysfunction. Patients with the subacute form were defined as those who developed the encephalopathy from 11 days to 8 weeks after the onset of liver dysfunction. Normal liver tissues were obtained from healthy donors (four men and five women; mean age, 35.3 years; range, 29–55 years) by fine needle biopsy at the time of liver transplantation. These donors showed normal liver function and none had a previous history of any liver disease, ongoing liver dysfunction, or any marker for hepatitis B and C. Written informed consent was obtained from all patients and donors, according to the guidelines of the Ethics Committee of Kyoto University. Real-time RT-PCR analysis of human gankyrin mRNA Real-time RT-PCR amplification and data analysis were performed using ABI Prism 7700 Sequence Detec-

tor System (PE Applied Biosystems). The sequences of the oligonucleotide primers and probes were designed according to the consensus sequence of human gankyrin cDNA, as follows: for Gank-148S, 5⬘-GGAAGCTGG AAGAGTTGAAGGA-3⬘; for Gank-245AS, 5⬘TGAGCATGCCCAGTGCAAT-3⬘; and for the GankTaqMan probe, 5⬘-TCTGGCCGATAAATCCCTGGC TACTAGAAC-3⬘. The TaqMan probe was labelled with a 5⬘ FAM reporter and a 3⬘ TAMRA quencher group. RT was performed on 1µg of total RNA from liver tissues, using primers Gank-245AS. First, RNA samples were incubated for 30 min at 55°C with RT buffer (10 mM Tris-HCl at pH 8.3, 50mM KCl), 5.5mM MgCl2, 6.5 units (U) Multiscribe RTase (PE Applied Biosystems), 2 U of RNase Inhibitor (PE Applied Biosystems), 0.2 µM oligonucleotide primer, and 0.5 mM (dNTP) mix, in a final volume of 5µl. This incubation was followed by denaturation of reverse transcriptase for 20 min at 95°C. For amplification of gankyrin sequences, PCR was performed in 40µl of TaqMan Universal PCR Master Mix (PE Applied Biosystems) with 0.3 µM primer sets and 0.3 M TaqMan probe, synthesized by JBioS (Saitama, Japan), using 5 µl of RT product as a template. The PCR conditions were 2min at 50°C, and 10min at 95°C, followed by 45 cycles of 15s at 95°C and 1 min at 60°C. The expression plasmid, hGankyrin CDS pBluescript SK(⫺), was constructed by inserting the EcoRI–XhoI fragment of full-length human gankyrin cDNA into pBluescript SK(⫺) (Stratagene, La Jolla, CA, USA). The sensitivity of our PCR amplification was evaluated by serial dilutions of the plasmid, hGankyrin CDS pBluescript SK(⫺) as a template, and this was deter-

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mined to be higher than 100 copies per sample. Then, a standard curve was constructed by the simultaneous amplification of serial dilutions of hGankyrin CDS pBluescript SK(⫺) plasmid as templates. The standard curve of gankyrin mRNA expression showed linearity over the entire quantification range from 102 to 106 copies, with reliable correlation factors of 0.998, indicating a precise log–linear relationship. All samples were measured at least three times independently, and each PCR run included five gankyrin standards. To rule out possible cross-contamination of genomic DNA, several negative control experiments were carried out, using the following reaction mixtures: reaction mixture without reverse transcriptase, RNA samples pretreated with RNase-A, or PCR reaction without RNA samples. To correct for variations due to differences in the amount of RAN taken for the reaction or due to different levels of inhibition during the RT reaction or PCR, we simultaneously evaluated “housekeeping genes” as internal controls. To date, the commonly used housekeeping genes, such as β-actin, glyceraldehyde-2-phosphate dehydrogenase (GAPDH), and hypoxanthine guanine phosphoribosyl transferase, are purported to lack precision as internal controls, especially in a real-time RT-PCR analysis, because of the existence of pseudogenes or retroposons in human and other mammalian genomes with high sequence homologies.11–13 Recently, Fink et al.14 reported that housekeeping variation of PBGD genes was a suitable internal control, especially in a real-time RT-PCR analysis, because it was pseudogene-free. Based on their findings, we selected the PBGD gene as an internal control in this study. For the PBGD standard, total RNA from the liver tissue of one patient with FHF (patient no. 5 in Table 1) was diluted in ten fold steps from 1:1 to 1 :10 000, and used for internal control of the reference gene in each amplification. Subsequently, the normalized copy number of gankyrin mRNA was defined as follows: Relative gankyrin expression (copy) = (gankyrin sample/gankyrin plasmid [1 copy])/(PBGD sample/ PBGD sample [patient no. 5])

Immunoblotting analysis of human gankyrin and RB1 Homogenates of liver samples from rat primary hepatocytes and FHF patients were diluted in 2⫻ sodium dodecyl sulfate (SDS) sample buffer (62.5mM TrisHCl, pH 6.8; 2% SDS; 5% β-mercaptoethanol; 10% glycerol; and 0.002% bromophenol blue), and boiled for 3min. All protein samples from liver tissues were adjusted to the same concentration with Bradford reagent (Bradford Bio-Rad protein assay; Bio-Rad, Hercules, CA, USA). Protein samples were then separated by

SDS-polyacrylamide gel electrophoresis (PAGE) on 12% (w/v), 12%, and 7.5% polyacrylamide gels for gankyrin, α-tubulin, and RB1, respectively. Protein bands were transferred to nitrocellulose membranes and the blots were individually hybridized with a rabbit polyclonal anti-human gankyrin antibody,7 mouse monoclonal anti-α-tubulin antibody (Calbiochem, San Diego, CA, USA), and mouse monoclonal anti-RB1 antibody (New England Biolabs, Beverly, MA, USA). Immunocomplexes on the filters were detected by an enhanced chemiluminescence assay (Renaissance; NEN Life Science, Boston, MA, USA). Statistical analysis All statistical analyses were performed with Excel computer software (Microsoft). Values of gankyrin for the nonparametric Mann-Whitney U-test were expressed as medians (interquartile ranges [IQRs]). The correlations between gankyrin values and other clinical factors (age, sex, etiology of FHF, serum level of alanine aminotransferase [ALT], total bilirubin [T. Bil], direct bilirubin-tototal bilirubin ratio [D/T ratio], prothrombin time [PT], alpha-fetoprotein [AFP], serum level of hepatocyte growth factor [HGF], and total liver weight/body weight [LW/BW]) were calculated and expressed as correlation coefficients (rs), using Spearman’s correlation coefficient by rank test. Differences were designated as significant at P ⬍ 0.01.

Results Cell-cycle-dependence of gankyrin expression in primary hepatocytes Gankyrin was found to be expressed abundantly in cancer cells of hepatocellular carcinoma.7 To elucidate whether gankyrin upregulation was correlated with cellcycle progression in normal hepatocytes, we examined the potential cell-cycle-dependence of gankyrin expression in rat primary hepatocytes after mitogenic stimulation. It has been shown that rat primary hepatocytes are in G0/G1 phase of the cell cycle at the resting state, but could promptly enter S phase of the cell cycle after stimulation with growth factors, such as epidermal growth factor (EGF) or HGF.15–17 Nagaki et al.18 reported that rat hepatocytes enter G1-S phase at 48h after stimulation by EGF. First, we confirmed that G1S entry was induced in rat primary hepatocytes 24 to 48h after the treatment with EGF or HGF, as determined by cell-cycle analysis by flow cytometry (data not shown). Then, primary rat hepatocytes were plated at an initial density of 5 ⫻ 105 cells per well on type-I collagen-coated dishes, and total RNA was extracted

A. Iwai et al.: Gankyrin in hepatocyte proliferation and liver regeneration

Fig. 1. Time course change of gankyrin mRNA expression in rat primary hepatocytes after mitogenic stimulation. Rat primary hepatocytes were incubated at 37°C for the first 6 h, and then the medium was exchanged for fetal bovine serum (FBS)-free medium. After 24 h, epidermal growth factor (EGF) was added to the FBS-free medium (final concentration, 20 ng/ml). The hepatocytes were harvested and subjected to total RNA isolation immediately before (0 h) and 24, 48, and 72 h after stimulation by EGF (20 ng/ml). Reverse transcription-polymerase chain reaction (RT-PCR) was performed, using 1 µg of each RNA sample as a template, and oligonucleotides specific for gankyrin (upper panel), cyclin D1 (middle panel), and cyclin A2 (lower panel) as primer sets. Representative data from three independent experiments are shown. Porphobilinogen deaminase (PBGD) mRNA was used as an internal control

from these primary hepatocytes at 0, 24, 48, and 72 h after the stimulation with either EGF or HGF. As shown in Fig. 1, endogenous gankyrin mRNA was undetectable by RT-PCR analyses in rat primary hepatocytes in the resting state. However, gankyrin expression was strongly induced at 24h, and its peak level was observed at 48 h after treatment with EGF, whereas the expression of PBGD and GAPDH transcripts was unchanged (Fig. 1, and data not shown). Cyclin D1 expression, which is a marker of cell-cycle progression through late G1 phase, was upregulated, and reached a peak at 48 h after the treatment with EGF. Cyclin A2, which is required for cell-cycle progression through G1/ S transition and for maintenance of the S phase, was upregulated at 24 h and elevated to the highest level at 48 h after EGF stimulation, followed by a decrease at 72 h. Similar results were obtained after HGF stimulation. Namely, endogenous gankyrin mRNA was undetectable in the quiescent cells, increased through G1-S transition, and its expression was maintained in S-phase (data not shown). It has been reported that gankyrin overexpression might contribute to hepatocarcinogenesis by the phosphorylation and degradation of RB1.7 To investigate the relationship between gankyrin and RB1 in cell-cycle progression in hepatocytes, we further examined the expression level of RB1 protein in rat primary hepatocytes after mitogenic stimulation. As shown in Fig. 2,

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Fig. 2. Retinoblastoma 1 (RB1) protein expression in rat primary hepatocytes after mitogenic stimulation. Total protein was isolated from rat hepatocytes immediately before (0 h) and 24, 48, and 72 h after stimulation by EGF (20 ng/ml). A total of 10 µg of each of the protein samples was electrophoresed, blotted, and reacted with antibodies to anti-RB1 (upper panel) or α-tubulin (lower panel). Representative data from three independent experiments are shown. Tubulin protein was used as an internal control

RB1 protein was detectable in the resting state. However, the level of endogenous RB1 protein started to decrease at 48 h, and maintained its low expression levels until 72 h after stimulation with EGF. Taken together, upregulation of gankyrin expression coincided with the decline of RB1 protein level at G1 to S phase in primary hepatocytes after mitogenic stimulation. Increased gankyrin expression in the regenerating liver tissue of patients with FHF The above results suggested the possibility that gankyrin upregulation was induced in proliferating hepatocytes in a cell-cycle-dependent manner. To explore the physiological role of gankyrin in noncancerous human hepatocytes, we analyzed the expression of this protein in the liver tissues of patients with FHF, as a model of liver regeneration. As a control, we also examined normal liver tissues of healthy liver transplant donors. Gankyrin mRNA expression, as determined by quantitive RT-PCR analyses in the liver tissue of healthy individuals was estimated to be a median of 22.4 (IQR, 1.4–77.3) copies. In healthy donors, there were no significant associations between the gankyrin expression level in the liver tissue and age or sex (data not shown). Expression of gankyrin mRNA was then examined in the liver tissue of patients with FHF. As shown in Fig. 3A, the median value of gankyrin mRNA expression in the FHF liver was found to be 109.7 (IQR, 49.0– 186.4) copies, showing that levels of gankyrin transcript were significantly elevated in the liver of FHF patients, compared with normal liver tissues (P ⬍ 0.01). To further confirm the upregulation of gankyrin in the liver of FHF, we examined the expression of gankyrin protein in four FHF patients and two healthy donors by immunoblotting analysis, using anti-gankyrin antibody.

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Fig. 3A,B. Gankyrin mRNA expression in the liver tissue of patients with fulminant hepatic failure (FHF). Relative gankyrin mRNA levels in liver tissue were measured by a realtime RT-PCR method, using the PBGD gene as a reference. The normalized copy number of gankyrin mRNA was calculated as described in “patients, materials, and methods. “Realtime RT-PCR quantification for the gankyrin transcript was performed using Gank-148S and Gank-245AS primers and the Gank-Taqman probe. Each sample was measured on three different occasions, and the mean of the three values is shown. A Patients with FHF (FHF) vs healthy donors (control). B Patients with FHF, acute form (acute-FHF) vs subacute form (sub-FHF). n, number of patients

Consistent with the results of the quantitative RT-PCR analyses, the expression level of gankyrin protein was also abundantly upregulated in all FHF liver samples examined, compared with samples from healthy individuals (Fig. 4). By normalization with α-tubulin levels, gankyrin protein levels were determined to be three- to ten-fold higher in the liver of FHF patients compared with the normal liver, as determined by scanning densitometry analyses of immunoblots. Based on the clinical features, especially focused on the timing of encephalopathy progression, FHF can be further divided into two subgroups, as stated earlier:10 an acute and a subacute form. As shown in Fig. 3B, quantitive RT-PCR revealed significantly higher levels of gankyrin transcripts in the liver of acute FHF compared with that of subacute, FHF (135.6 [IQR, 106.7– 186.4] and 85.4 [IQR, 49.0–112.2] copies, respectively; P ⬍ 0.01). In contrast, there was no significant correlation between the expression level of gankyrin and age (rs ⫽ 0.012; P ⫽ 0.966), sex (rs ⫽ 0.107; P ⫽ 0.711), etiology of FHF (rs ⫽ 0.018; P ⫽ 0.951), ALT (rs ⫽ 0.344; P ⫽ 0.232), T. Bil (rs ⫽ 0.126; P ⫽ 0.662), D/T ratio (rs ⫽ 0.081; P ⫽ 0.779), PT (rs ⫽ ⫺0.061; P ⫽ 0.827), AFP (rs ⫽ 0.143; P ⫽ 0.651), or LW/BW (rs ⫽ ⫺0.262; P ⫽ 0.363). Although the numbers were small, HGF (rs ⫽ ⫺0.3; P ⫽ 0.502) also showed no correlation with the expression level of gankyrin mRNA. Moreover,

Fig. 4. Gankyrin protein expression in the liver of patients with FHF. Protein samples (5µg) from liver tissue of patients with FHF and healthy liver transplant donors were electrophoresed, blotted, and reacted with antibodies to human gankyrin (upper panel) or α-tubulin (lower panel). Lanes 1– 4, liver tissues of FHF patients 1, 3, 6, and 7 (in Table 1); lanes 5 and 6, liver tissues of healthy liver transplant donors. Tubulin protein was used as an internal control

gankyrin expression was not significantly correlated with serum levels of ammonia or creatinine, or with arterial pH (data not shown).

Discussion This is the first report to reveal evidence that a new oncogenic protein, gankyrin, is expressed in proliferating hepatocytes in a cell-cycle-regulated manner. We found that gankyrin expression in rat primary hepatocytes was induced and reached a peak level at 48h after mitogenic stimulation, which coincided with the expression patterns of cyclin D1 and ⫺A2. These results are in agreement with previous findings that gankyrin might lead to cell-cycle progression by increasing the phosphorylation and degradation of RB1 protein in cancer cells.7 In fact, immunoblotting analyses revealed that RB1 expression in rat primary hepatocytes after stimulation by growth factor had started to decline in the phase coinciding with gankyrin upregulation. Interestingly, Fan et al.19 reported that the expression of RB1 protein in a hyperphosphorylated state and shortening of its halflife are important in accelerating the cell cycle in the regenerating rat liver or in Huh7 human hepatoma cells. Taken together, data from previous reports and the present study showing the characteristic expression pattern of gankyrin in rat primary hepatocytes may suggest a role of gankyrin in the regulation of cell-cycle progression in hepatocytes by controlling the expression level of RB1 protein. In this study, we also demonstrated that gankyrin was overexpressed at both the gene and protein levels in the regenerating liver of patients with FHF. Our findings suggested the potent physiological role of gankyrin in the proliferation of hepatocytes in the injured liver, as

A. Iwai et al.: Gankyrin in hepatocyte proliferation and liver regeneration

well as in cancer cells. The liver of patients with FHF has been reported to be a typical example of liver regeneration after functional deficiency.6 Thus, gankyrin expression may be associated with liver regeneration during severe damage in patients with FHF. Notably, the expression of gankyrin in the liver tissue of patients with the acute form of FHF was significantly higher than the expression in those with the subacute form of FHF. It was shown that the acute form of FHF has a better prognosis than the subacute form,9 and the survival rates of patients with the acute and subacute forms of FHF have been reported to be approximately 70% and 30%, respectively.20,21 Therefore, one possibility is that elevated gankyrin expression, particularly in the acute form of FHF, might reflect the active proliferation of hepatocytes in the regenerating liver. On the other hand, various factors, including the grade of encephalopathy, T. Bil, the D/T ratio, PT, AFP, liver volume, blood ammonia levels, and HGF, are reported to predict the prognosis of FHF;22,23 however, we found that gankyrin expression was not associated with any of these factors. In this study, we could not directly evaluate the relationship between gankyrin expression levels and the prognosis of FHF, because all of our patients received liver transplantation. Thus, further studies are required to clarify whether gankyrin expression reflects the actual prognosis of patients with FHF. In addition, because FHF is associated with severe inflammation and hepatocyte necrosis, whether or not inflammation and/or the following hepatocyte necrosis is involved in gankyrin overexpression is an interesting question to be clarified in future. We also attempted to examine the role of gankyrin in human liver cell lines. In contrast to rat primary hepatocytes, however, all the established hepatocyte-derived cell lines tested, including HepG2 and Huh7, had abundant expression of gankyrin protein even in the resting state (data not shown). Therefore, we could not confirm the cell-cycle-dependent expression of gankyrin in human cell lines. Further analysis of gankyrin will be required to understand the overall importance of this oncogenic molecule in the regulation of human hepatocyte proliferation and the development of hepatocellular carcinoma. Acknowledgments. We thank Drs. Shinji Uemoto and Motoshige Nabeshima for their useful advice. Supported by Grants-in-Aid for Scientific and Cancer Research from the Ministry of Education, Culture, Sports, Science, and Technology; a Grant from the Ministry of Health, Labour and Welfare, Japan; and a Grant from the Japan Society for the Promotion of Science (No. JSPS-RFTF97I00201).

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