Histone H4 histidine kinase displays the expression pattern of a liver oncodevelopmental marker
Eiling Tan1,
Paul G. Besant1,2,
Xin Lin Zu1,
Christoph W. Turck3,
Marie A. Bogoyevitch1,
Seng Gee Lim4,
Paul V. Attwood1 and
George C. Yeoh1,2,5
1 Biochemistry and Molecular Biology, School of Biomedical and Chemical Sciences, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia, 2 Laboratory for Cancer Medicine, The UWA Centre for Medical Research, West Australian Institute for Medical Research, Level 5, MRF Building, 50 Murray Street, Perth, WA 6000, Australia, 3 Max-Planck Institute of Psychiatry, Molecular, Cellular, Clinical Proteomics, Kraepelinstr., 2, D-80804 Munich, Germany and 4 Division of Gastroenterology, Department of Medicine, National University Hospital, 5 Lower Kent Ridge Rd, Singapore 119074
5 To whom correspondence should be addressed Email: yeoh{at}cyllene.uwa.edu.au
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Abstract
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Protein phosphorylation is a vital process in the regulation of mammalian cell division and the protein kinases that catalyze the phosphorylation of proteins on serine, threonine and tyrosine residues have been well characterized. In contrast, little is known about the kinases involved in protein histidine phosphorylation, which have been described in various mammalian cells that are highly proliferative. Histone H4 histidine kinase (HHK) activity is highly active in regenerating rat liver. Using a novel and specific assay, we demonstrate that it is active in human fetal liver, essentially absent in adult liver and highly expressed in liver tumours. Normal liver surrounding the HCC contains low to undetectable levels of HHK. In a rodent model of chronic liver injury that leads to HCC, its activity is induced. Two lines of evidence suggest that liver progenitor (oval) cells, which populate the liver at early stages following induction of liver damage are responsible for the increased activity. Purified oval cells, as well as cell lines established from primary cultures of oval cells express high levels of HHK. We propose that the pattern of expression of histone H4 histidine kinase activity justifies its classification as an oncodevelopmental marker and suggest it may be useful as a diagnostic marker for hepatocellular carcinoma as well for identifying preneoplastic lesions.
Abbreviations: HHK, histone H4 histidine kinase; PCNA, proliferating cell nuclear antigen.
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Introduction
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The link between phosphorylation and the regulation of cell growth has focused predominantly on tyrosine, serine and threonine kinases. Interestingly, their substrates collectively account for only 50% of the total phosphoamino acids in mammalian cells (1). Phosphohistidine constitutes 6% of all phosphoamino acids of basic nuclear proteins in lower eukaryotes (2), exceeding by at least two orders of magnitude, the cellular abundance of phosphotyrosine (3). Whilst the importance of histidine kinases in lower eukaryotic and prokaryotic cellular function is well recognized (4,5), little is known about their role in mammalian cells.
Histidine phosphorylation of histone H4 occurs during liver regeneration (68). Its up-regulation in nuclei of regenerating liver prior to DNA synthesis suggests it may positively regulate cell proliferation. The exact role of histidine-phosphorylated histone H4 in this process has not been elucidated. It may prevent the premature formation of nucleosome complexes during DNA replication (1). Protein histidine phosphorylation is observed in highly proliferative transformed cells such as the Walker 256 carcinosarcoma cell line (9), rat hepatoma Fao cells (10) and HL-60 leukaemia cells (11,12), consistent with its postulated role in cell growth. It has also been observed in the liver following administration of clofibrate, a hepatocellular carcinogen (13), which damages hepatocytes and promotes their proliferation.
Mammalian histidine kinases have been partially isolated and characterized from a variety of sources. These include the nuclear extract from porcine thymus (14), the Walker 256-carcinomasarcoma cell line (9) and the HIT-T15 pancreatic cell line (14). A histidine kinase has also been purified from Saccharomyces cerevisiae (15). All possess the ability to phosphorylate histone H4 in vitro at one of its two histidine residues. However, these kinases differ with respect to molecular size and regulation. For example, the yeast and thymic histidine kinases have a molecular mass in the range of 3241 kDa (15,16), whereas the pancreatic histidine kinase has a molecular mass of 6070 kDa (14). The histidine kinase from the Walker 256-carcinomasarcoma cell line has a requirement for Mg2+, and is susceptible to nucleotide inhibition (9), whereas the pancreatic histidine kinase is dependent on G-protein activation (14). Studies of regenerating rat liver following acute damage suggest that histone H4 histidine kinase (HHK) activity is mechanistically associated with liver growth (1). In this instance, restoration of the liver occurs by the proliferation of hepatocytes (17), which are normally quiescent. In contrast, chronic liver injury-induced regeneration is facilitated by the proliferation of liver progenitor (oval) cells (18,19). In several experimental animal models, this form of liver injury is repaired by a process that is independent of growth from pre-existing hepatocytes (2022). The status of HHK activity in these conditions has not been addressed.
The preponderance of reports describing increased HHK activity in transformed cell lines suggests it may be an oncoprotein. This study was undertaken to define the developmental pattern of HHK activity and to determine its level of expression in pre-cancerous liver and in liver tumours. We show that HHK activity is elevated in fetal liver but is low in postnatal, weaning and adult liver. It is rapidly up-regulated when rats are placed on a carcinogenic diet, which suggests that it is expressed in progenitor oval cells, which appear in pre-cancerous liver. Elevated levels of HHK activity in a liver progenitor oval cell line also support this view. Finally, we establish that human hepatocarcinoma biopsies have elevated HHK activity, whereas normal surrounding tissue, as well as biopsies from normal liver, has very low levels of activity. Both the developmental pattern of HHK activity and its relative levels of expression in normal and transformed liver justifies its classification as an oncodevelopmental marker and further investigations into its role in tumorigenesis.
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Materials and methods
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Purification of histone H4 substrate
Histone H4 was prepared as described previously by Wei and Matthews (23) with some modifications. Briefly, Type II-S histone (Sigma) (33 mg ml1 in 8 M urea, 10 mM HCl) was loaded onto a Bio-Gel P-10 (200400 mesh) (Bio-Rad), which was equilibrated for 24 h with 10 mM HCl prior to loading. The column was eluted with 10 mM HCl at a flow rate of 1.25 ml min1. Ten-milliliter fractions were collected, and peak fractions corresponding to histone H4 were pooled. The partially purified H4 was then re-loaded onto the same column under conditions described above. Fractions corresponding to the peak of histone H4 were pooled, dialysed against H2O lyophilized and stored at 4°C.
Detection of histidine phosphorylation of histone H4 by a filter-based kinase assay and phosphoamino acid analysis
A filter-based kinase assay to detect alkali-stable phosphorylation was performed as described by Wei and Matthews (23) with some modifications (24). Histone H4 (25 µg) was phosphorylated in 20 µl of a reaction mixture [15 mM TrisHCl pH 7.5, 15 mM MgCl2, 50 mM NaF, 100 µM ATP and 1 µCi [
-32P]ATP (6000 Ci/mmol) (Amersham-Pharmacia)] with 30 µl of crude nuclear protein (1 mg ml1) extracted from liver. The reaction was performed at 30°C for 1 h (unless otherwise stated) and terminated by adding 6 µl of stop solution (3 M NaOH, 2 mM ATP) then incubated at 60°C for 30 min to deplete phosphoserine and phosphothreonine. Half of the reaction mixture was used to estimate acid-labile phosphorylation of histone H4 by addition of 10 µl HCl (3.6 M), and the other half received 10 µl of water. Following incubation at 60°C for 30 min, samples were returned to their original pH by addition of 10 µl of NaOH (3.6 M) to the acid-treated samples, and 10 µl of NaCl (3.6 M) to the non-acid-treated samples. Samples were adsorbed onto NytranTM membranes (Schleicher and Schuell), washed in 10 mM sodium pyrophosphate, dried and 32P incorporation into histone H4 was determined by Cherenkov counting (23). Activity is expressed as U mg1 of total nuclear protein ± SEM, where 1 U = 1 pmol Pi incorporated into histone H4 per min. Assays were performed in triplicate on groups of three rats (n = 3). HHK activity was determined by subtracting the Pi incorporated into histone H4 in the acid-treated sample from the corresponding non-acid treated sample (24).
Phosphoamino acid analysis was performed as described previously (16,24,25) with modifications. Histone H4 (25 µg) was immunoprecipitated with 10 µg of the monoclonal anti-histone pan antibody (Roche) for 1 h at room temperature. Immunoprecipitates were adsorbed to protein ASepharose (Pharmacia) [30 µl of a 50%(w/v) suspension] for 1 h at room temperature. Samples were centrifuged briefly and the supernatants discarded, before addition of the phosphorylation reaction mixture. Phosphorylation and reaction termination were performed as described above. IPs were washed three times (3 x 1 ml of 1 mM ATP, 10 mM sodium pyrophosphate), then digested overnight using 10 µg of pronase E (Streptomyces griseus protease) (Sigma) in 40 µl of 20 mM ammonium bicarbonate. Samples were centrifuged briefly, the supernatants dried by vacuum, the residue re-suspended in 1 µl of water and spotted onto a RP-18 F254s pre-coated silica reverse-phase thin layer chromatography (RP-TLC) plate (Merck). In addition, 2 µg each of phosphohistidine, phosphotyrosine (Sigma), phosphoserine (Sigma), phosphothreonine (Sigma) and phosphoarginine (Fluka-Biochemika) standards dissolved in water were spotted. The samples were resolved using 72 ml redistilled ethanol, 10 ml ammonium solution (28%v/v) (Chem-Supply) and 9 ml water. When the solvent front was
1 cm from the top of the plate, it was removed, air-dried, exposed to a BAS IIIs phosphorimaging plate (Fuji), then stained with ninhydrin (BDH). Radioactive spots corresponding to the migration of the phosphohistidine standard were eluted as described by Besant and Attwood (16) and re-run on another RP-TLC plate. In some instances, phosphoamino acid standards were mixed with the samples.
Animals, oval cell induction and isolation
Wistar rats were cared for in accordance with the guidelines stipulated by the National Health and Medical Research Council of Australia. Male rats (200 g) were anaesthetized with 1 µl of nembutal (Rhone Merieux)/g body wt, and subjected to a 70% liver resection as described by Higgins and Anderson (26). Animals were killed at the indicated time points by cervical dislocation. Portions of remnant liver were removed for nuclear preparation (90%), or fixed in Carnoys fixative for 2 h prior to paraffin embedding (10%). To induce oval cell proliferation, male rats (120 g, 4-week-old) were placed on a choline-deficient chow (ICN) supplemented with 0.165% ethionine (Sigma) in the drinking water. Three rats were killed at each of the indicated time points by cervical dislocation. Control rats were fed standard rat chow and water ad libitum. Liver progenitor oval cells were isolated and purified as described previously (27,28). Portions of individual rat liver from 19-day gestation rat embryos (E19), newborns (1 day), juveniles (3 week) and adults (6 week) were collected for nuclear preparation (
90%) and paraffin embedding (
10%).
Human tissue acquisition
Normal human fetal and adult liver tissue, as well as hepatocellular carcinoma tissue, were obtained after informed consent, approved by the Ethics Committee of Gleneagles Hospital and National University Hospital, Singapore. Samples were placed immediately in Williams' E solution on ice and transferred to the laboratory. A sterile scalpel was used to divide the sample into
5 mm blocks, which were snap-frozen in liquid nitrogen and stored at 80°C.
Cell culture conditions
PIL-2 cells were plated in Williams' E medium (Sigma) with 10% FCS as described previously (18). Cells were grown in an atmosphere of 5% CO2, 90% humidity at 37°C. The medium was replaced 24 h after plating in Williams' E medium (Sigma) minus FCS and grown to 80% confluency, then harvested for nuclear isolation or fixation for imunohistochemical staining.
Isolation of nuclei
All steps were performed at 4°C. Cells were washed twice with balanced salts solution (BSS) then scraped from the dish with a teflon policeman in 10 ml of BSS and centrifuged at 600 g for 5 min. Cells were suspended in 5 ml buffer containing 15 mM Tris, pH 7.5, 60 mM KCl, 15 mM NaCl, 2 mM EDTA, 0.5 mM EGTA, 0.3 M sucrose, 0.5 mM spermidine, 0.15 mM spermine and 14 mM 2-mercaptoethanol, pelleted, and the supernatant discarded. Cells were lysed in the above buffer containing 0.1% Triton-X-100. Nuclei released were pelleted (600 g for 5 min) and washed twice with 5 ml of buffer containing 50 mM HEPES, pH 8.0, 5 mM MgCl2, 0.5 mM dithiothreitol (DTT), 1 µg ml1 BSA and 25% glycerol. Nuclei (107 ml1) were stored in the above buffer at 80°C, or immediately used for nuclear protein extraction as described below.
Nuclear protein extraction
Cellular nuclei were pelleted by centrifugation (500 g for 5 min), then lysed in ice cold buffer containing 0.5 mM HEPES, pH 8.0, 1.5 mM MgCl2, 420 mM KCl, 0.2 mM EDTA, 0.5 M DTT, 2 µg/ml leupeptin. Sixty microlitres of this buffer was added to 100 µl of nuclear pellet. After 15 min, an equal volume of buffer containing 20 mM HEPES, pH 8.0, 0.5 mM DTT, 0.2 mM EDTA, 0.5 mM PMSF, 20% glycerol and 2 µg ml1 leupeptin was added. The solution was centrifuged at 4000 g for 1 h and the supernatant containing the soluble nuclear proteins was stored at 80°C.
Immunohistochemical staining
Five micrometer sections of paraffin-embedded liver were de-waxed in histoclear (National Diagnostics), followed by decreasing, graded ethanol washes, then finally water. Cells were washed three times with PBS, then fixed for 5 min with 4% paraformaldehyde/0.1% glutaraldehyde in 0.1 M cacodylate buffer at pH 7.45, then stored in PBS containing 0.01% Thimerosal at 4°C. Proliferating cell nuclear antigen (PCNA) staining was performed on areas on the culture dish delineated with a wax pen. Immunohistochemical staining was performed using reagents supplied by DakoCytomation (CA). Briefly, endogenous peroxidases were inactivated with 2.5% periodic acid for 5 min, prior to neutralizing free aldehyde groups with 0.02% sodium borohydride for 2 min. All sections were blocked with 10% FCS for 1 h prior to detection of PCNA with mouse monoclonal anti-PCNA antibody (Santa Cruz). The primary antibody was detected with a secondary anti-mouse immunoglobulin G coupled to biotin (DakoCytomation). The signal was amplified using a streptavidinbiotin complex coupled to horseradish peroxidase (DakoCytomation), and cells positive for PCNA were visualized using a DAB peroxidase substrate kit (DakoCytomation). Cells positive for the PCNA antigen were counted over 10 fields at 40x magnification, and were expressed as a percentage of total cells in each field. Scores for all samples were expressed as the mean ± SEM.
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Results
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HHK activity is up-regulated by growth induced by partial hepatectomy, and increases prior to DNA synthesis
HHK activity peaked 18 h post-partial hepatectomy, with a specific activity of 115 ± 4 U mg1 of nuclear protein (Figure 1a). Hepatocyte proliferation, judged by PCNA staining of cells with large round nuclei and hepatocyte morphology continued to increase beyond 18 h (Figure 1a). Liver from sham-operated rats exhibited negligible histidine kinase activity (5.4 ± 5.4 U mg1) and few PCNA-positive cells (0.04%). These values are similar to those observed for untreated rats. Phosphorylation of a histidine residue is confirmed by phosphoamino acid analysis of the phosphorylated histone H4 (Figure 1b).

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Fig. 1. HHK activity and PCNA-positive hepatocytes increase following acute liver injury induced by partial hepatectomy (PHx). (a) HHK activity is expressed as U mg1 nuclear protein extract and cell proliferation is expressed as PCNA-positive cells as a percentage of the total cells. Results are means (n = 3) ± SE. (b) Phosphoamino acid analysis of phosphorylated histone H4 showing specific histidine phosphorylation. The right panel is the phosphorimage of the TLC plate, showing 32P-labelled phosphohistidine. The left panel is the same plate showing the position of ninhydrin-stained free histidine (H), phosphohistidine (PH), phosphotyrosine (PY), phosphoserine (PS), phosphothreonine (PT) and phosphoarginine (PR) standards.
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HHK activity increases in chronically damaged liver
To ascertain whether HHK activity was induced by chronic liver damage we subjected rats to a hepatotoxic CDE diet. They displayed elevated levels of HHK activity after 3 weeks (54 ± 1 U mg1). Little activity was detected in rats fed the diet for 1 or 2 weeks, or in rats fed a normal diet for 1, 2 or 3 weeks (Figure 2a). PCNA staining reveals that damaged hepatocytes do not proliferate in contrast to liver progenitor oval cells, as only oval cells with PCNA-stained ovoid-shaped nuclei, which are clearly different to round large nuclei of hepatocytes were observed in the liver of CDE treated rats. Phosphoamino acid analysis confirmed that only nuclear extracts from 3-week CDE-treated liver phosphorylate histidine in histone H4 and not liver nuclear extracts from rats that received the control diet (Figure 2b).

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Fig. 2. HHK activity is upregulated by chronic liver injury induced by a CDE diet. (a) HHK activity in the liver of control diet and CDE fed-rats after 1, 2 and 3 weeks. Activity is expressed as U mg1 nuclear protein extract. Cell proliferation is expressed as PCNA-positive cells as a percentage of total cells. Results are presented as means (n = 3) ± SE. (b) Phosphoamino acid analysis of histone H4 phosphorylated with nuclear protein derived from the liver rat placed on a CDE or control (CON) diet for 3 weeks. The (+) lanes contained the substrate (histone H4), while the () lanes did not. The left panel shows the migration of free histidine (H), phosphohistidine (PH), phosphotyrosine (PY), phosphoarginine (PR), phosphothreonine (PT), phosphoserine (PS) on the ninhydrin-stained plate. The right panel is the phosphorimage of the RP-TLC plate (left) showing 32P-labelled phosphohistidine only in the CDE (+) lane.
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HHK activity is confined to the liver progenitor oval cell population in chronically injured liver
To confirm that liver progenitor oval cells were responsible for the increased HHK in chronically damaged liver we isolated cells from the liver and separated them by centrifugal elutriation. This method separates cells according to size and provides a fraction that contains small oval cells devoid of large hepatocytes, and a fraction that contains the converse. HHK activity was present only in small liver progenitor oval cells (712 ± 67 U mg1) and not in hepatocytes. PCNA-staining of cytocentrifuged preparations of the respective fractions showed the majority of oval cells were positive whereas none of the hepatocytes were labelled.
HHK activity is high in fetal liver, very low in postnatal liver and just detectable in adult liver
To ascertain changes in HHK activity in liver during development, we determined its activity in fetal, newborn, 2-week, 4-week and adult liver. Fetal liver nuclear extracts prepared from 19-day gestation rats displayed HHK activity (110 ± 22 U mg1). PCNA-staining of sections confirms that the proliferating cellular population comprises small hepatocytes and hepatoblasts (Figure 3i). In comparison, HHK activity is very low in postnatal (2.3 ± 0.6 U mg1), 2-week (3.4 ± 2.0 U mg1) and 4-week rat liver (6.5 ± 4.4 U mg1), and it is not detected in normal adult rat liver. Phosphoamino acid analysis of histone H4 phosphorylated by the nuclear extract of fetal rat liver confirms that the site of phosphorylation was a histidine residue (data not shown).

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Fig. 3. There is an abundance of proliferating cells in fetal liver and in cultures of PIL2 oval cell line. (i) The majority of E19 fetal liver cells are PCNA positive. These are mostly hepatoblasts and small hepatocytes. (ii) The majority of PIL2 cells in culture are PCNA positive. Magnification bar = 100 µm.
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HHK activity is elevated in the human hepatocellular carcinoma cell line HepG2 and in the mouse liver progenitor cell line PIL-2
HHK activity of the human hepatocellular carcinoma cell line HepG2 (7.5 ± 3.0 U mg1), is 12.5-fold higher than that of normal adult human liver (0.6 ± 0.2 U mg1). This is in agreement with findings in other cell types which link HHK activity with cellular proliferation. PIL2 cells, a tumorigenic liver progenitor cell line derived from a p53 knockout mouse, also display elevated levels of HHK activity (912 ± 1 U mg1). PCNA-staining of a representative PIL2 culture shows that 97% of cells are proliferating (Figure 3ii).
HHK activity is elevated in human fetal liver and hepatocellular carcinomas
To ascertain the HKK status of human liver, we determined its activity in normal adult and fetal liver, the human hepatoma cell line HepG2 and pathological specimens taken from patients with HCC as well as the normal surrounding tissue which was excised with the tumour. Normal liver is material excess to the requirement for tranplants. The HCCs are reported to contain pleomorphic malignant epithelial cells and classified as between grade III and IV using the Edmondson Grading System. The normal surrounding tissue is described as being free of malignancy, containing mild infiltrate of inflammatory cells and in one sample, mild focal fatty change. HHK is just detectable (0.6 ± 0.2 U mg1) in normal adult human liver (Figure 4). In contrast, its activity is considerably higher in three human HCC (262 ± 10 U mg1). Normal liver tissue, which lies adjacent to the same tumour, displays negligible levels of enzyme activity (1.4 ± 2.6 U mg1). HHK activity in human fetal liver is elevated (56 ± 8 U mg1). This transition in HHK activity from a high level in fetal liver though to negligible levels in normal adult liver and back to elevated levels in hepatocellular carcinoma tissue, suggests that HHK is an oncodevelopmental marker.

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Fig. 4. HHK activity is elevated in human fetal liver and hepatocellular carcinomas. HKK activity is high in fetal liver (FE) and hepatocellular carcinoma (HCC). It is barely detected in normal tissue adjacent to HCC (N-HCC) and in normal adult (AD) liver. Activity is expressed as U mg1 nuclear protein extract and presented as means (n = 3) ± SE.
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Discussion
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The number of cellular proteins reported to undergo histidine phosphorylation is limited when compared with the reported incidence of serine, threonine and tyrosine phosphorylation. This is primarily due to the acid-labile nature of the phosphoramidate bond of phosphohistidine. Most biochemical procedures used in protein purification or biochemical detection of protein phosphorylation do not detect acid-labile protein phosphorylations. Histone H4 histidine phosphorylation is one such phosphorylation that can be measured using an assay specific for detecting acid-labile/alkali-stable phosphorylation (24). A HHK has been characterized in yeast (15) but its mammalian equivalent remains unidentified.
In regenerating rat liver (7), and transformed cells such as Walker 256 carcinosarcoma cell line (9), histone H4 histidine phosphorylation has been reported. This suggests that HHK activity is associated with cell proliferation, and perhaps cancer. HHK activity increases when growth is induced by partial hepatectomy, peaking 18 h after the procedure. This precedes the peak of [3H]thymidine incorporation reported by Smith et al. (6). Smith and co-workers (7) also reported the induction of HHK with similar kinetics based on alkali-stable phosphorylation of histone H4. Since tyrosine phosphorylation is also alkali-stable, HHK activity was not unequivocally demonstrated. In this study, we employ an HHK assay developed by this laboratory (24), which detects acid-labile, alkali-stable phosphorylation and thus distinguishes histidine phosphorylation from tyrosine phosphorylation. Combined with phosphoamino acid analysis, this assay specifically measures histidine phosphorylation of histone H4. We conclusively demonstrate the activation of HHK in regenerating rat liver with kinetics similar to that reported by Chen et al. (7) using our assay and confirm that HHK activity is maximal 18 h following partial hepatectomy while cell proliferation shown by PCNA staining continues to increase from 18 to 24 h.
We have also evaluated changes in HHK activity associated with regeneration following chronic liver injury, liver development and cancerous cell growth. We show high levels of HHK activity in fetal liver, and declining levels during development, which reduce to very low or undetectable levels in the adult liver. In chronically damaged liver, HHK activity is induced, and this can be ascribed to the liver progenitor oval cells, which appear as a consequence of the liver injury. Assays of nuclear extracts purified from oval cells were shown to contain high levels of HHK activity compared with extracts from hepatocytes from the same liver that had negligible HHK activity. To further support this, we also show elevated levels of HHK activity in a liver progenitor cell line (PIL-2) established from p53-null mice subjected to the same CDE diet to induce chronic liver injury. These cells proliferate continuously in culture and the elevated levels of HHK activity are consistent with the hypothesis that HHK activity is elevated in proliferating liver progenitor cells. The PIL-2 cells are also transformed and give rise to tumours in nude mice (29); hence one may speculate that a link exists between HHK activity and transformation. This pattern of expression of HHK activity from fetal liver, through adult, and to tumour is similar to the pattern of other genes such as alpha-fetoprotein (30), isoenzymes of pyruvate kinase, aldolase and lactic dehydrogenase (31), that have been described as oncodevelopmental markers. Its association with cell proliferation and cancer suggests that HHK may be oncogenic. Confirmation will require the establishment of a causal relationship between HHK expression and cell proliferation and its over-expression in transformed cells. Work towards this objective is proceeding in our laboratories.
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Acknowledgments
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Support for this study was received from The Australian Grants Commission (Grant ID DP 0208385), The National Health & Medical Research Council of Australia (Grant ID 254666) and The National University of Singapore (Grant R-172-000-001-731). P.G.B. is the recipient of a fellowship from the Faculty of Medicine & Dentistry, The University of Western Australia.
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Received March 16, 2004;
revised May 31, 2004;
accepted June 22, 2004.