Department of Clinical and Experimental Medicine, University of Perugia, Policlinico Monteluce, 06122 Perugia, Italy
* Author for correspondence (e-mail: labiomol{at}unipg.it)
Accepted 21 April 2005
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: shuttling protein, proliferation, liver, eEF-1A, hepatoma cells, liver regeneration
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Hepatocytes are resting cells in G0 phase that after PH go through G1 phase and progress to the cell cycle. The process that allows hepatocytes to pass from G0 to G1 phase has been described as priming. Growth factors such as hepatocyte growth factor (HGF), transforming growth factor- (TGF-
) and epidermal growth factor (EGF) act primarily on hepatocytes in liver regeneration (Fausto, 2000
). Many researchers have shown that liver regeneration is a multistep process and that priming is necessary for hepatocyte proliferation. HGF, TGF-
and EGF alter gene expression in residual hepatocytes and in the subsequent proliferative steps. Following priming, two major events occur in residual hepatocytes after PH, activation of several genes in response to proliferation and restoration of the quiescent state of hepatocytes by activated genes.
To describe the mechanisms involved in the proliferative response, we focussed on the molecular changes occurring during liver regeneration following PH through characterization of novel genes activated in residual hepatocytes. A cDNA library was constructed with mRNAs derived from residual hepatocytes at different times following PH. We performed a rat regenerating liver cDNA library screening with cDNA-subtracted probes derived from rat regenerating liver cDNAs (2-18 hours after PH) and rat normal liver mRNAs. Screening allowed us to isolate up to 40 genes. All isolated genes were upregulated in the liver after PH and in hepatoma cells. One of these novel genes expressed in liver regeneration has been characterized previously (Della Fazia et al., 2002). This gene Lal-1 is involved during liver regeneration and in the proliferative process. At present our attention has been drawn to another of these genes that we named hepatocyte odd protein shuttling (Hops).
In this paper, we demonstrate that HOPS is a novel shuttling protein, which via CRM-1 (Fornerod et al., 1997), actively directs proliferation of cells controlling protein synthesis. Evidence is provided that cAMP governs HOPS export in hepatocytes of normal and regenerating liver. Following PH, HOPS is rapidly exported from the nucleus and is overexpressed during liver regeneration. It has been established that HOPS binds to elongation factor EF-1A and interferes with protein synthesis. Overexpression of HOPS in H-35 hepatoma cells and 3T3-NIH cells strongly reduces cell proliferation.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animals
Experiments were performed on 3-month-old male animals: Sprague-Dawley rats and SVJ-129 mice. The animals were purchased from Harlan-Nossan and received human care according to NIH guidelines. Animals were maintained in a 12 hours:12 hours light:dark cycle with food and water ad libitum. Liver resection was performed between 8 a.m. and 12 a.m. removing about 70% of the liver mass (Higgins and Anderson, 1931). As control, sham operation by transverse abdominal incision followed by digital manipulation of the liver was performed. Rats were sacrificed at 2, 5, 8, 12, 18, 24, 48 and 72 hours after PH. Mice were sacrificed at 15, 30 minutes, 1, 2, 5, 8, 12, 18, 36, 38, 48, 60 and 72 hours after PH. Four animals per experimental group were used for each time point and livers were pooled prior to analysis.
RNA analysis
Total RNA extraction was performed and analyzed by northern blot analysis as described previously (Sambrook et al., 1989; Della Fazia et al., 1992
).
Cell cycle synchronization and stable clones cells
H-35 cell cycle was synchronized at the G0/G1 boundary in serum-free medium and in G1/S phases by the double thymidine method. Briefly, H-35 were cultured in the absence of serum, arrested for 72 hours and released in fresh culture medium in the presence of serum. The time point at which H-35 received serum after starvation was considered time 0. The double thymidine method to arrest the cells at the G1/S boundary was performed as described previously (Crosio et al., 2002). The time point, corresponding to the G1/S transition, was considered time 0. The percentage of H-35 cells, labeled with propidium iodide, was determined at different phases of the cell cycle by flow cytometry (FACS analysis; Becton Dickinson FACStar Plus flow cytometer).
H-35 stable cell clones overexpressing HOPS were generated by transfection with pcDNA3 vector containing full-length Hops and by selection in culture medium with geneticin (0.4 mg/ml). H-35 stable cell clones generated by transfection with empty pcDNA3 vector were used as control. Thymidine incorporation in H-35 cells and H-35 stable cell clones was performed as described previously (Della Fazia et al., 2001).
Retroviral vector production and cell infection
pBabe-puro, MuLV-based retroviral vector, was used to transduce the Hops gene (Morgenstern et al., 1990). Wild-type Hops cDNA was inserted into pBabe-puro vector to produce pBabe-Hops.
Phoenix cells were plated at 1.5 x106 per plate 2 days before transfection. For each transfection, 2 µg of pBabe-puro or pBabe-Hops plasmid were used. Viral supernatants were concentrated and collected 48 hours after transfection according to Nolan laboratory protocol (www.stanford.edu/group/nolan/tutorials). Culture supernatants containing retroviral vectors were then added to NIH-3T3 with polybrene (8 µg/ml) (Sigma). Cells were cultured for 24 hours and selected in the same medium containing puromycin (2 µg/ml) for 4 days. Resistant cells (1 x104) were plated and counted every 2 days for 10 days after selection.
Hops gene isolation
Hops gene isolation from a regenerating liver library was performed using a subtracted probes procedure. A number of positive clones were isolated and tested by northern blot analysis of the time course of RNA extracted at different times following PH. Selected cDNAs overexpressed during liver regeneration were isolated and sequenced. Hops DNA sequence and putative protein prediction procedures have been previously described (Della Fazia et al., 2002).
Antibody production
Polyclonal anti-HOPS was generated in our laboratory by immunizing rabbits with KHL-coupled peptide (H T T E S T D P L P Q S S G T T T P A Q P S E) corresponding to N-terminal sequence (aa 32-54) of the mouse HOPS protein. The serum of two rabbits was collected and immunopurified on a column Sulfo-Link coupling gel (Pierce) where the specific peptide had been immobilized. To test the specificity of the antibody, a quenching test was performed with different amounts of specific peptide in western blot and in immunohistochemistry analyses (Della Fazia et al., 2002).
Western analyses
Protein extracts were resolved by standard SDS-polyacrylamide gel electrophoresis from total liver and H-35 hepatoma rat cells. Liver and cells were minced immediately in RIPA buffer. Each sample (50 µg) was separated by gel electrophoresis and blotted onto a nitrocellulose membrane (Schleicher and Schuell). The blots were incubated with rabbit anti-HOPS polyclonal antibody and with rabbit anti-CREB polyclonal antibody (Cell Signaling Technology) the signals were detected using an ECL kit (Amersham Pharmacia Biotech).
Histological and immunofluorescence analyses
The livers were embedded into OCT compound for cryosectioning. Sections (7 µm) were cut and mounted on slides. For histological analysis, sections were stained with Hematoxylin-Eosin. Slides were blocked with 3% BSA and then incubated with specific primary polyclonal anti-HOPS antibody. After three washes in PBS, slides were incubated with anti-rabbit Cy-3-conjugated secondary antibody in 3% BSA. DAPI was added at the final concentration of 10 ng/ml. Images were captured with a Zeiss Axioplan fluorescence microscope controlled by a Spot-2 cooled camera (Diagnostic Instruments) with a 40 x objective lens.
H-35 leptomycin B treatment
H-35 hepatoma cells were pretreated for 30 minutes with 10 ng/ml of cycloheximide (CHX; Sigma). The cells were then treated for an additional 6 hours with 20 ng/ml LMB. After CHX treatment PBS was added to the control cell population. At the end of treatment the cells were fixed in 4% paraformaldehyde and hybridized with specific anti-HOPS antibody overnight. The cells were examined using a fluorescence microscope with a standard filter for red fluorescence. Six random fields of cells stained for HOPS in the presence or absence of LMB were counted. The image was captured with a Zeiss Axioplan fluorescence microscope controlled by Spot-2 cooled camera (Diagnostic Instruments). Images were saved as TIFF-files.
Two-hybrid screening and analysis
Hops full-length cDNA was cloned into yeast expression vector pGBKT7. The pGBKT7-Hops plasmid was transformed into yeast strain AH109. Two-hybrid screening was carried out according to the manufacturer's protocol (Clontech) using a VP-16 DNA activation domain fusion library in an E9.5-12.5 mouse embryo cDNA library in the vector pASV3 (Le Douarin et al., 1995). The transformants were plated onto appropriate selective medium supplemented with 25 mM 3-amino-triazole. ß-gal assays were performed on isolated clones and carried out in Y190 yeast strain. The results reported are in Miller units and are the means of triplicate measurements performed using three distinct transformations. The plasmids extracted by lysing cells with acid-washed beads were electroporated in E. coli bacterial strain HB101 and then plated onto M9 (Leu) plates (Vojtek et al., 1993
). The isolated clones were sequenced using the Sanger method.
Immunoprecipitation
H-35 cells were harvested, washed and resuspended in lysis buffer (20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 2% Triton X-100) supplemented with protease inhibitor cocktail and 1 mM phenylmethanesulfonyl fluoride (PMSF). Immunoprecipitation was carried out as described (Bardoni et al., 1999) using the anti-eEF-1A (Upstate Biotech). The proteins bound to the beads were separated by electrophoresis on 10-12% SDS-PAGE and visualized by immunoblot using the anti-HOPS antibody. At the same time, the cell lysate was co-immunoprecipitated using anti-HOPS and detected with anti-eEF-1A.
HOPS recombinant protein: production and purification in bacteria
The HOPS gene was cloned in pET-14Tb expression vector (Novagen). The resulting gene was expressed in BL21 (DE3) cells (Novagen) and after induction with 0.5 mM isopropyl ß-D-1-thiogalactopyranoside (IPTG) for 24 hours at 25°C the recombinant protein was purified under denaturing conditions using His-Bind affinity chromatography. Expression and purification were carried out according to the manufacturer's protocol (Novagen).
In vitro translation inhibition assay
An aliquot (1 µg) of luciferase cDNA (Promega) was added to 100 µl of a rabbit reticulocyte lysate in vitro translation reaction (Promega) in the presence of HOPS or GST purified recombinant proteins at different concentrations: 60, 120, 240, 360 and 420 nM. The reaction mixture was incubated at 30°C for 2 hours. Newly synthesized 35S-labeled luciferase protein was analyzed by 10% SDS-PAGE and results were quantified using densitometry analysis with the Scion Image 4.0 program. Western blot analysis was performed using anti-eEF-1A as control.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
Different HOPS expression and intracellular localization in proliferating cells
Following the results obtained in vivo in liver regeneration, the role of HOPS in H-35 rat hepatoma cells was examined. HOPS western blot analysis in H-35 proliferating cells showed two distinct bands, at 27 and 24 kDa (Fig. 3A). To investigate HOPS expression at different stages of the cell cycle, H-35 cells were synchronized using the double thymidine arrest technique. As demonstrated by flow cytometry, highly synchronized cells were obtained. Thymidine inhibited DNA synthesis and arrested cells on the G1/S border. At the end of the double thymidine arrest (time 0), 90-92% of hepatoma cells arrested in G1/S phase (Fig. 3A).
Protein extracts from H-35 synchronized cells were collected at different times after release from the double thymidine block. HOPS was strongly expressed at time 0 in relation to the arrest of the cell cycle of hepatoma cells and down-regulated rapidly after release (Fig. 3A). Similar results were obtained by blocking the cell cycle of H-35 cells in G0/G1 by serum deprivation. During cell starvation the progressive increase of HOPS expression was analyzed. When the cell cycle was arrested, at the end of 72 hours of serum deprivation, HOPS was overexpressed in the starved cells. Addition of serum to the culture medium down-regulated HOPS expression in the cells (Fig. 3A).
In light of these results, we investigated a possible change of HOPS localization in relation to the cell cycle. The progressive starvation of H-35 cells, following serum deprivation, allowed us to study the redistribution of HOPS localization during cell cycle arrest. HOPS was diffused in the nucleus and cytoplasm of wild-type hepatoma cells. The protein progressively migrated to the nucleus 12 hours after serum deprivation. In the following hours, cells progressively stopped proliferating and HOPS accumulated in the nuclei. At 72 hours after serum deprivation, when almost 90% of the cells were in G0/G1 phase (Fig. 3B) and HOPS was overexpressed, the protein was localized mainly in the nucleus. Addition of serum to the culture medium caused a rapid increase in cell proliferation, as demonstrated by flow cytometry, and redistribution of HOPS in the cells (Fig. 3C).
HOPS is a shuttling protein
Rapid HOPS export from the nucleus to the cytoplasm in proliferating hepatocytes raised the question of whether there are agents that could affect shuttling. During liver regeneration different factors act on residual hepatocytes to modify expression and induce proliferation. In particular, our attention focused on two factors that act rapidly on residual hepatocytes following PH, EGF and cAMP. The effects of EGF and cAMP on HOPS shuttling in the liver were analyzed. In EGF-treated mice a small amount of HOPS migrated into the cytoplasm of hepatocytes at 30 minutes after treatment and almost all protein returned to the nucleus after 60 minutes. At 90 minutes HOPS was detected again in the nucleus (data not shown). In cAMP-treated mice the shuttling protein was detected in the nucleus at 15 and 30 minutes after treatment and migrated in part into the cytoplasm. At 60 minutes HOPS was exported completely into the cytoplasm. At 90 and 120 minutes HOPS returned progressively to the nucleus, showing a distribution pattern similar to normal hepatocytes (Fig. 4).
|
Analysis of the HOPS amino acid sequence revealed the motif LACLLVLALA in the N-terminal region, a typical nuclear export signal (NES) region, present in proteins that are exported from the nucleus to the cytoplasm via CRM-1 (Fig. 1B and Fig. 5A) (Fornerod et al., 1997; Fukuda et al., 1997
; Macara, 2001
).
|
Binding specificity between HOPS and eEF-1A
To gain further insight into the role of HOPS and its shuttling function, screenings were performed using the two-hybrid system in yeast; HOPS was used as bait to identify proteins that specifically bind it. Positive yeast clones were isolated from a cDNA library of total E9.5-12.5 embryos in selected medium during the two-hybrid system screening and all were positive for ß-galactosidase activity. In addition, there was no ß-galactosidase activity in the yeast strain transformed with the empty vector (pASV3; Fig. 6A).
|
High HOPS levels inhibit in vitro translation
Following immunoprecipitation studies on binding specificity between HOPS and eEF-1A, the possibility of a significant role for this interaction was analyzed. Because eEF-1A is essential in protein synthesis in peptide chain elongation, in vitro protein synthesis levels were evaluated in the presence of different HOPS recombinant protein concentrations. In vitro transcription and translation experiments were performed using luciferase cDNA as the reporter gene. The amount of luciferase protein was evaluated in the presence and absence of HOPS recombinant protein. HOPS recombinant protein was added to the in vitro translation (Fig. 7A) at different concentrations (60, 120, 240, 360 and 420 nM). There was a slight increase in protein synthesis when 60 nM of recombinant HOPS was added while 120 nM had no effect. Surprisingly, recombinant HOPS at 240 nM reduced protein synthesis to almost 50% (Fig. 7A). No synthesis of luciferase was detected using 360 and 420 nM (Fig. 7A,B). Analogous experiments performed with the same concentration of GST, used as protein control, had no significant effects on the synthesis of luciferase (Fig. 7B). In reticulate lysates with different HOPS concentrations the expression of eEF-1A was tested by western blot analysis. No differences in eEF-1A expression were detected in all samples examined (Fig. 7A).
|
HOPS and cell proliferation
The increased level of HOPS expression detected in H-35 cells induced to arrest proliferation or in residual hepatocytes following PH suggest a possible involvement of HOPS in proliferation. To test this hypothesis, H-35 stable cells overexpressing HOPS were generated. Stable cell cultures were analyzed by cytofluorimetric analysis and [3H]thymidine incorporation was evaluated. The study was performed on four different stable clones. The results showed that HOPS overexpression blocked H-35 cell growth. Colony growth assay showed that [3H]thymidine incorporation was drastically reduced in H-35 stable clones overexpressing HOPS with respect to H-35 stable clones used as control. In H-35 stable cells overexpressing HOPS the percentage of [3H]thymidine incorporation showed a reduction of about of 65% (Fig. 7C) with respect to controls. In H-35 stable clones HOPS is localized in the nucleus and cytoplasm (Fig. S2 in supplementary material). These results implicate the involvement of HOPS during cell proliferation. To further verify this hypothesis, experiments were performed in NIH-3T3 proliferating cells and the HOPS anti-proliferative effect was quantified. NIH-3T3 cells were infected with pBabe-puro or pBabe-Hops. A growth cell selection by puromycin was carried out for 5 days in both types of infected cells. The results indicate that HOPS inhibits proliferation in NIH-3T3 cells infected with pBabe-Hops. After puromycin selection the number of cells at day 2 decreased in both types of infected cells. In the following days, a stronger proliferation of cells infected with pBabe-puro was observed than in cells infected with pBabe-Hops. The number of the cells switched from approximately 58 x104 with pBabe-puro to about 22 x104 with pBabe-Hops, showing a reduction of almost 65% (Fig. 7D).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In liver regeneration many factors act on residual hepatocytes to organize the reconstitution of the original liver mass. After PH, change occur in stable hepatocytes at many levels (protein synthesis, energy requirement, proliferation) arranging their cell program on the basis of actual needs. It has been demonstrated that the growth factors EGF, TGF- and HGF work as priming factors on the residual hepatocytes in the first hours after PH, and increased levels in cAMP concentration have been detected in the first hours after PH. We observed in vivo that the increased level of cAMP allows HOPS to export in proliferating hepatocytes following PH or in normal hepatocytes after cAMP intraperitoneal injection in mice. Our results show that the rapid export of HOPS from the nucleus to cytoplasm is ascribed to high levels of cAMP in the cells. The return of HOPS to the hepatocyte nucleus, 90 minutes after cAMP injection compared with regenerating hepatocytes where HOPS returns 12 hours after PH, would suggest that HOPS stimulated by cAMP migrates into the cytoplasm, but proliferation of regenerating liver retains HOPS in the cytoplasm.
The compartmentalization of proteins and their regulation in export and import mechanisms from the nucleus to cytoplasm is an important system of control in cell functions. The presence of a NES domain in the HOPS sequence and specific protein accumulation in hepatoma cell nucleus after treatment with LMB indicates an involvement of CRM-1 in nuclear export of HOPS protein. Based on these data we speculate that CRM-1 is responsible for the nuclear export of HOPS and in turn regulates HOPS cytoplasmic functions.
The identification of eEF-1A as a molecular partner binding HOPS in liver and in hepatoma cells shows a specific functional interaction between the two proteins. eEF-1A plays a key role in protein synthesis and controls the first step of elongation of the growing peptide. In the cells, eEF-1A is located predominantly in the cytoplasm. Recent studies showed that eEF-1A is actively exported from the nucleus to keep the nuclear eEF-1A concentration down to 1/100 with respect to the cytoplasmic concentration, preventing eventual nuclear translation (Bohnsack et al., 2002; Calado et al., 2002
). During cell proliferation, translation machinery of protein synthesis rapidly increases and protein synthesis factors, ribosomes and regulating factors guarantee a rapid processing of transcripts (Thomas, 2000
; Ruggero and Pandolfi, 2003
).
In the first hours following PH, in the period preceding cell mitosis, the residual hepatocytes are hypertrophic and protein synthesis is strongly activated. Our findings suggest a molecular mechanism in which, during residual hepatocyte proliferation after PH, HOPS shuttles from the nucleus to cytoplasm playing a pivotal role in the control of protein synthesis by eEF-1A activity regulation (Fig. 8). These assumptions are supported by results of in vitro translation assay in which the addition of recombinant HOPS regulates protein synthesis.
|
Recently it has been suggested that a key role is played by translation factors and protein synthesis in the transformation and regulation of cell proliferation (Caraglia et al., 2000; Ruggero and Pandolfi, 2003
). Alterations in eEF-1A expression correlate with cancer and potential metastatic activity of mammary adenocarcinoma (Edmonds et al., 1996
). Furthermore, the oncogene PTI1 (prostate tumor inducing gene), a hybrid molecule containing a truncated form eEF-1A, appears to play an important role in prostate cancer (Gopalkrishnan et al., 1999
).
The implication of translation factors in protein synthesis in cancer cells may facilitate identification of novel therapeutic agents that act on protein synthesis.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bardoni, B., Schenck, A. and Mandel, J. L. (1999). A novel RNA-binding nuclear protein that interacts with the fragile X mental retardation (FMR1) protein. Hum. Mol. Genet. 8, 2557-2566.
Bohnsack, M. T., Regener, K., Schwappach, B., Saffrich, R., Paraskeva, E., Hartmann, E. and Gorlich, D. (2002). Exp5 exports eEF1A via tRNA from nuclei and synergizes with other transport pathways to confine translation to the cytoplasm. EMBO J. 21, 6205-6215.
Bucher, N. L. R. (1963). Regeneration of the liver. In International Review of Cytology (ed. G. H. Bourne and J. F. Danielli), pp. 245-300. New York: Academic Press.
Calado, A., Treichel, N., Muller, E. C., Otto, A. and Kutay, U. (2002). Exportin-5-mediated nuclear export of eukaryotic elongation factor 1A and tRNA. EMBO J. 21, 6216-6224.
Caraglia, M., Budillon, A., Vitale, G., Lupoli, G., Tagliaferri, P. and Abbruzzese, A. (2000). Modulation of molecular mechanisms involved in protein synthesis machinery as a new tool for the control of cell proliferation. Eur. J. Biochem. 267, 3919-3936.
Cressman, D. E., Greenbaum, L. E., DeAngelis, R. A., Ciliberto, G., Furth, E. E., Poli, V. and Taub, R. (1996). Liver failure and defective hepatocyte regeneration in interleukin-6-deficient mice. Science 274, 1379-1383.
Crosio, C., Fimia, G. M., Loury, R., Kimura, M., Okano, Y., Zhou, H., Sen, S., Allis, C. D. and Sassone-Corsi, P. (2002). Mitotic phosphorylation of histone H3, spatio-temporal regulation by mammalian Aurora kinases. Mol. Cell. Biol. 22, 874-885.
Della Fazia, M. A., Servillo, G. and Viola-Magni, M. (1992). Different expression of tyrosine aminotransferase and serine deydratase in rat livers after partial hepatectomy. Biochem Biophys. Res. Commun. 182, 753-759.[CrossRef][Medline]
Della Fazia, M. A., Servillo, G. and Sassone-Corsi, P. (1997). Cyclic AMP signalling and cellular proliferation: regulation of CREB and CREM. FEBS Lett. 410, 22-24.[CrossRef][Medline]
Della Fazia, M. A., Pettirossi, V., Ayroldi, E., Riccardi, C., Viola Magni, M. and Servillo, G. (2001). Differential expression of CD44 isoforms during liver regeneration in rats. J. Hepatol. 34, 555-561.[CrossRef][Medline]
Della Fazia, M. A., Piobbico, D., Bartoli, D., Castelli, M., Brancorsini, S., Viola Magni, M. and Servillo, G. (2002). lal-1: a differentially expressed novel gene during proliferation in liver regeneration and in hepatoma cells. Genes Cells 7, 1183-1190.
Diehl, A. M. and Rai, R. M. (1996). Liver regeneration 3. Regulation of signal transduction during liver regeneration. FASEB J. 10, 215-227.
Edmonds, B. T., Wyckoff, J., Yeung, Y. G., Wang, Y., Stanley, E. R., Jones, J., Segall, J. and Condeelis, J. (1996). Elongation factor-1 alpha is an overexpressed actin binding protein in metastatic rat mammary adenocarcinoma. J. Cell Sci. 109, 2705-2714.
Ekanger, R., Vintermyr, O. K., Houge, G., Sand, T. E., Scott, J. D., Krebs, E. G., Eikhom, T. S., Christoffersen, T., Ogreid, D. and Doskeland, S. O. (1989). The expression of cAMP-dependent protein kinase subunits is differentially regulated during liver regeneration. J. Biol. Chem. 264, 4374-4382.
Fausto, N. (2000). Liver regeneration J. Hepatol. 32, 19-31.[CrossRef][Medline]
Fausto, N. (2001). Liver regeneration. In The Liver: Biology and Pathobiology. 4th edn (ed. I. M. Arias, J. L. Boyer, F. V. Chisari, N. Fausto, D. Schachter and D. A. Shafritz), pp. 591-610. Philadelphia: Lippincott Williams & Wilkins.
Fausto, N., Laird, A. D. and Webber, E. M. (1995). Liver regeneration. 2. Role of growth factors and cytokines in hepatic regeneration. FASEB J. 9, 1527-1536.
Fornerod, M., Ohno, M., Yoshida, M. and Mattaj, I. W. (1997). CRM1 is an export receptor for leucine-rich nuclear export signals. Cell 90, 1051-1060.[CrossRef][Medline]
Fukuda, M., Asano, S., Nakamura, T., Adachi, M., Yoshida, M., Yanagida, M. and Nishida, E. (1997). CRM1 is responsible for intracellular transport mediated by the nuclear export signal. Nature 390, 308-311.[CrossRef][Medline]
Gopalkrishnan, R. V., Su, Z. Z., Goldstein, N. I. and Fisher, P. B. (1999). Translational infidelity and human cancer: role of the PTI-1 oncogene. Int. J. Biochem. Cell Biol. 31, 151-162.[CrossRef][Medline]
Higgins, G. M. and Anderson, R. M. (1931). Experimental pathology of liver: restoration of liver in white rat following partial surgical removal. Arch. Pathol. 12, 186-202.
Le Douarin, B., Pierrat, B., vom Baur, E., Chambon, P. and Losson, R. (1995). A new version of the two-hybrid assay for detection of protein-protein interactions. Nucleic Acids Res. 23, 876-878.[Medline]
Macara, I. G. (2001). Transport into and out of the nucleus. Microbiol. Mol. Biol. Rev. 65, 570-594.
Malumbres, M. and Barbacid, M. (2001). To cycle or not to cycle: a critical decision in cancer. Nat. Rev. Cancer 1, 222-231.[CrossRef][Medline]
Michalopoulos, G. K. and DeFrances, M. C. (1997). Liver regeneration. Science 276, 60-66.
Morgenstern, J. P. and Land, H. (1990). Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res. 18, 3587-3596.[Abstract]
Ruggero, D. and Pandolfi, P. P. (2003). Does the ribosome translate cancer? Nat. Rev. Cancer 3, 179-192.[CrossRef][Medline]
Sambrook, J., Fritsch, E. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.
Servillo, G., Penna, L., Foulkes, N. S., Viola Magni, M., Della Fazia, M. A. and Sassone-Corsi, P. (1997). Cyclic AMP signalling pathway and cellular proliferation: induction of CREM during liver regeneration. Oncogene 14, 1601-1606.[CrossRef][Medline]
Servillo, G., Della Fazia, M. A. and Sassone-Corsi, P. (1998). Transcription factor CREM coordinates the timing of hepatocyte proliferation in the regenerating liver. Genes Dev. 12, 3639-3643.
Servillo, G., Della Fazia, M. A. and Sassone-Corsi, P. (2001). Cyclic AMP signaling in the liver coupling transcription to physiology and proliferation. In The Liver: Biology and Pathobiology, 4th edn (ed. I. M. Arias, J. L. Boyer, F. V. Chisari, N. Fausto, D. Schachter and D. A. Shafritz), pp. 525-536. Philadelphia: Lippincott Williams & Wilkins.
Servillo, G., Della Fazia, M. A. and Sassone-Corsi, P. (2002). Coupling cAMP signaling to transcription in the liver: pivotal role of CREB and CREM. Exp. Cell. Res. 275, 143-154.[CrossRef][Medline]
Taub, R., Roy, A., Dieter, R. and Koontz, J. (1987). Insulin as a growth factor in rat hepatoma cells. Stimulation of proto-oncogene expression. J. Biol. Chem. 262, 10893-10897.
Thomas, G. (2000). An encore for ribosome biogenesis in the control of cell proliferation. Nat. Cell. Biol. 2, E71-E72.[CrossRef][Medline]
Vojtek, A. B., Hollenberg, S. M. and Cooper, J. A. (1993). Mammalian Ras interacts directly with the serine/threonine kinase Raf. Cell 74, 205-214.[CrossRef][Medline]
Wolff, B., Sanglier, J. J. and Wang, Y. (1997). Leptomycin B is an inhibitor of nuclear export: inhibition of nucleo-cytoplasmic translocation of the human immunodeficiency virus type 1 (HIV-1) Rev protein and Rev-dependent mRNA. Chem. Biol. 4, 139-147.[CrossRef][Medline]
Yamada, Y., Kirillova, I., Peschon, J. J. and Fausto, N. (1997). Initiation of liver growth by tumor necrosis factor: deficient liver regeneration in mice lacking type I tumor necrosis factor receptor. Proc. Natl. Acad. Sci. USA 94, 1441-1446.