DNA Synthesis In Hepatocytes Research Articles

Comparison of the effects of the synthetic pyrethroid Metofluthrin and phenobarbital on CYP2B form induction and replicative DNA synthesis in cultured rat and human hepatocytes

High doses of Metofluthrin (MTF) have been shown to produce liver tumours in rats by a mode of action (MOA) involving activation of the constitutive androstane receptor leading to liver hypertrophy, induction of cytochrome P450 (CYP) forms and increased cell proliferation. The aim of this study was to compare the effects of MTF with those of the known rodent liver tumour promoter phenobarbital (PB) on the induction CYP2B forms and replicative DNA synthesis in cultured rat and human hepatocytes. Treatment with 50 μM MTF and 50 μM PB for 72 h increased CYP2B1 mRNA levels in male Wistar rat hepatocytes and CYP2B6 mRNA levels in human hepatocytes. Replicative DNA synthesis was determined by incorporation of 5-bromo-2′-deoxyuridine over the last 24 h of a 48 h treatment period. Treatment with 10–1000 μM MTF and 100–500 μM PB resulted in significant increases in replicative DNA synthesis in rat hepatocytes. While replicative DNA synthesis was increased in human hepatocytes treated with 5–50 ng/ml epidermal growth factor or 5–100 ng/ml hepatocyte growth factor, treatment with MTF and PB had no effect. These results demonstrate that while both MTF and PB induce CYP2B forms in both species, MTF and PB only induced replicative DNA synthesis in rat and not in human hepatocytes. These results provide further evidence that the MOA for MTF-induced rat liver tumour formation is similar to that of PB and some other non-genotoxic CYP2B form inducers and that the key event of increased cell proliferation would not occur in human liver.

Characterization of the Enhancing Effect of Protamine on the Proliferative Activity of Hepatocyte Growth Factor in Rat Hepatocytes

The aim of the present study was to characterize the mechanism of the stimulatory effect of protamine on HGF activity. The enhancing effects of protamine on the proliferative activity of HGF were investigated in vivo, in primary cultured rat hepatocytes, and in perfused rat liver. In alpha-naphthylisothiocyanate-intoxicated rats, pretreatment with protamine increased HGF-induced autophosphorylation of the HGF receptor in liver. The maximum enhancing effect of protamine on HGF-induced DNA synthesis of hepatocytes required a 10 min-pretreatment period both in vivo and in vitro, and the stimulatory effect of protamine was not observed when it was administered simultaneously with HGF. Preperfusion of the liver with protamine for 10 min decreased the non-saturable portion of hepatic clearance for (125)I-HGF, which is mainly mediated by cell-surface heparan-sulfate proteoglycan (HSPG). Inhibition of HGF binding to heparin by protamine was confirmed using heparin-coated sepharose. This inhibition also required 10 min of pretreatment, for protamine to bind heparin. The enhancing effect of protamine on the mitogenic activity of HGF on hepatocytes requires pretreatment with protamine for a short period presumably required for its binding to cell-surface heparin, implying possible regulation of c-met autophosphorylation by HSPG.

Tumor necrosis factor (TNF) receptor-2-mediated DNA synthesis and proliferation in primary cultures of adult rat hepatocytes: The involvement of endogenous transforming growth factor-α

We investigated the effects of tumor necrosis factor (TNF)-α on DNA synthesis and proliferation, and its signal transduction pathways in primary cultures of adult rat hepatocytes. TNF-α induced time- and dose-dependent increases in hepatocyte DNA synthesis and proliferation. The hepatocyte proliferation stimulated by 30 ng/ml TNF-α was significantly inhibited by anti-TNF receptor 2 antibody, but not by anti-TNF receptor 1 antibody. TNF-α-induced hepatocyte DNA synthesis and proliferation were blocked by AG1478 (10 − 7 M), PD98059 (10 − 6 M), LY 294002 (10 − 7 M), and rapamycin (100 ng/ml). TNF-α at 30 ng/ml significantly increased phosphorylation of receptor tyrosine kinase (175 kDa) and p42 mitogen-activated protein (MAP) kinase. This data suggests that the proliferative signal for primary cultured hepatocytes induced by TNF-α is mediated by TNF receptor 2 and the receptor tyrosine kinase/MAP kinase pathway. In addition, TNF-α-induced hepatocyte mitogenesis was significantly blocked by somatostatin (10 − 6 M), adenylate cyclase inhibitor dideoxyadenosine (10 − 7 M), protein kinase A inhibitor H-89 (10 − 7 M), and neutralizing antibody to transforming growth factor (TGF)-α in culture. Indeed, 30 ng/ml TNF-α was found to rapidly stimulate secretion of TGF-α, and this secretion was also blocked by anti-TNF receptor 2 antibody. Moreover, TGF-α secretion induced by TNF-α was suppressed by dideoxyadenosine, H-89, and somatostatin. Together, these results indicate that stimulation of TNF receptor 2 by 30 ng/ml TNF-α induces autocrine secretion of TGF-α via the adenylate cyclase/protein kinase A pathway, after which TGF-α induces hepatocyte DNA synthesis and proliferation through the TGF-α receptor-linked tyrosine kinase (175 kDa)/MAP kinase signaling system.

Microarray analysis of mitogenic effects of T3 on the rat liver

A single dose of the thyroid hormone tri-iodothyronine, T3, can enhance both size and function of normal rodent liver, which is potentially of value in the treatment of liver disease. However the mechanism of this has not been fully elucidated, and it cannot be modeled in vitro. We therefore investigated the transcriptome response to T3 in rat liver in vivo. After adult rats were administered 5 microg T3 subcutaneously, a whole rat genome microarray comparing global hepatic gene expression against vehicle-only treated liver after 3 h was performed. Informative transcripts which had identifiable gene ontology biological processes were grouped according to function, broadly reflecting general metabolic effects and those linked to cell-proliferation control. We then compared the transcriptome response after 5-microg T3 initiating hepatocyte DNA synthesis (mitogenic) with that after 0.1 microg T3, a supraphysiological amount not initiating hepatocyte DNA synthesis. We compared the results with published results of the response to other primary mitogens, and identified the Gadd45beta/MyD118 gene as a common early factor upregulated during proliferation.

TNF-α and IL-6 signals from the bone marrow derived cells are necessary for normal murine liver regeneration

In the present study, we used bone marrow transplanted mice and revealed the role of bone marrow derived cells in liver regeneration after partial hepatectomy (PH). Irradiated wild type (WT) mice received a bone marrow transplant from either WT, TNF (tumor necrosis factor)-α knockout (KO), or interleukin (IL)-6 KO donors. Both TNF-α KO- and IL-6 KO-transplanted mice compared with WT-transplanted mice showed decreased hepatocyte DNA synthesis after PH. TNF-α KO-transplanted mice showed no nuclear factor kappa B (NF-κB) and signal transducer and activator of transcription (STAT) 3 binding after PH, while IL-6 KO-transplanted mice showed NF-κB, but not STAT3, binding. Lack of AP-1 or C/EBP binding or expression of c- jun or c-myc mRNA after PH was unrelated to the timing and amount of DNA replication. In conclusion, The TNF-α and IL-6 signals from the blood are necessary for liver regeneration and NF-κB and STAT3 binding are activated via TNF-α and IL-6 signal pathways.

T Cell-Derived Lymphotoxin Regulates Liver Regeneration

The ability of the liver to regenerate hepatic mass is essential to withstanding liver injury. The process of liver regeneration is tightly regulated by distinct signaling cascades involving components of the innate immune system, cytokines, and growth factors. However, the role of the adaptive immune system in regulation of liver regeneration is not well-defined. The role of adaptive immune system in liver regeneration was investigated in lymphocyte-deficient mice and in conditional lymphotoxin-deficient mice. A model of liver regeneration after 70% partial hepatectomy was used, followed by examination of liver pathology, survival, DNA synthesis, and cytokine expression. We found that mice deficient in T cells show a reduced capacity for liver regeneration following partial hepatectomy. Furthermore, surface lymphotoxin, provided by T cells, is critical for liver regeneration. Mice specifically deficient in T-cell lymphotoxin had increased liver damage and a reduced capacity to initiate DNA synthesis after partial hepatectomy. Transfer of splenocytes from wild-type but not lymphotoxin-deficient mice improved liver regeneration in T cell-deficient mice. We found that an agonistic antibody against the lymphotoxin beta receptor was able to facilitate liver regeneration by reducing liver injury, increasing interleukin-6 production, hepatocyte DNA synthesis, and survival of lymphocyte-deficient (Rag) mice after partial hepatectomy. The adaptive immune system directly regulates liver regeneration via a T cell-derived lymphotoxin axis, and pharmacological stimulation of lymphotoxin beta receptor might represent a novel therapeutic approach to improve liver regeneration.

Reciprocal Regulation of Prothrombin Secretion and Tyrosine Aminotransferase Induction in Hepatocytes

Multicellular spheroids of hepatocytes are known to maintain liver functions for a long period. Rat hepatocytes were isolated to form spheroids by rotation culture and immobilized within calcium alginate. Immobilized spheroids had a much higher extent of tyrosine aminotransferase induction, which is one of the liver-specific differentiated functions, than immobilized non-aggregated cells, while the spheroids secreted significantly less prothrombin than non-aggregated cells. Co-culture of hepatocytes and non-parenchymal liver cells in a monolayer enhanced tyrosine aminotransferase induction and suppressed prothrombin secretion, while conditioned medium prepared from non-parenchymal cells greatly stimulated tyrosine aminotransferase induction and suppressed the prothrombin secretion and DNA synthesis in monolayer-cultured hepatocytes. Prothrombin secretion in hepatocytes was subjected to cell-density-dependent regulation. In a similar manner to other growth-related functions, prothrombin secretion was stimulated at low cell density. It has been reported that thrombin activates the zymogen of hepatocyte growth factor activator [Shimomura, T., Kondo, J., Ochiai, M., Naka, D., Miyazawa, K., Morimoto, Y. & Kitamura, N. (1993) J. Biol. Chem. 268, 22,927-22,932]. Therefore, prothrombin secretion could be one of the growth-related functions and involved in wound healing and liver regeneration.

Zinc Finger Protein A20 Promotes Regeneration of Small-for-Size Liver Allograft and Suppresses Rejection and Results in a Longer Survival in Recipient Rats

Small-for-size liver allografts without immunosuppression have decreased survival compared with full-for-size grafts for the concomitant regeneration-induced accelerated rejection. This study was designed to examine the effect of zinc finger protein A20 on liver allograft regeneration and acute rejection using a high responder rat model (DA-->Lewis) of 30% partial liver transplantation. Adenovirus carrying the full length of A20 was introduced into liver grafts by ex vivo perfusion via the portal vein during preservation, physiological saline (PS), and empty Ad vector rAdEasy served as controls; then small-sized liver transplants were performed. Liver graft regeneration was assessed, as well as graft rejection, hepatocyte apoptosis, nuclear factor kappa B activation, and intercellular adhesion molecule-1 mRNA expression in liver graft sinusoidal endothelial cells (LSECs), infiltration of liver graft infiltrating mononuclear cells (LIMCs), and the subproportion of NK and NKT cells, activity of liver graft NK-like cells, interferon gamma (IFN-gamma) production, and animal survival. Ex vivo transfer of the A20 gene resulted in overexpression of A20 protein in LSECs and hepatocytes 24 h after partial liver transplantation. Regeneration of the small-sized liver allograft was augmented by A20 overexpression, the DNA synthesis of hepatocytes on d 4 post-transplant was increased in A20 group compared with PS and rAdEasy groups (P < 0.01). Hepatocyte apoptosis was inhibited by A20 (P < 0.001). On d 4 after transplantation, histological examination revealed a more exiguous cellular infiltration and mild rejection in A20 group but a more vigorous cellular infiltration in the sinusoidal area and more severe rejection in PS and rAdEasy group. Nuclear factor kappa B activation and intercellular adhesion molecule-1 mRNA expression in LSECs were suppressed by A20 overexpression. Flow cytometry analysis showed a marked down-regulation of LIMCs number by A20, including more prominent decrease in the subproportion of NK and NKT cells. Activity of liver graft NK-like cells, IFN-gamma mRNA expression in LIMCs, and serum IFN-gamma protein level were also suppressed by A20 overexpression (P < 0.05), respectively. Survival days of A20 rats were longer than those of PS rats and rAdEasy rats (P < 0.01), whereas survival days of rAdEasy rats were shorter than those of PS rats (P < 0.01). These data suggest that A20 overexpression could effectively promote small-sized liver allograft regeneration, suppress rejection, and prolong survival of recipient rat. These effects of A20 could be related to an inhibition of LSECs activation, suppression of infiltration of LIMCs, and the subpopulations such as NK and NKT cells into liver graft, and inhibition of hepatocyte apoptosis.

Cultured rat hepatocytes upregulate Akt and ERK in an ErbB-2-dependent manner

Epidermal growth factor (EGF) stimulates freshly plated adult hepatocytes to synthesize DNA, but only after they pass through a lag phase of 40 h following EGF exposure. The longer the cells are maintained, they become more responsive to EGF and the lag phase shortens. Maximal EGF-mediated stimulation of DNA synthesis requires the induction of ErbB2, which is not normally expressed in adult hepatocytes. We used immunological methods to demonstrate increased expression during culture of two gene families required for EGF to stimulate hepatocyte DNA synthesis: Akt and ERK 1/2. Both families showed hyperexpression in culture particularly when cells were exposed to insulin and EGF. Unlike CDK-2 and cyclin D1, integral mediators of the G1/S phase transition, ERK 1/2 and Akt appeared in the absence of EGF, particularly when insulin was present. This hyperexpression, which high concentrations of dexamethasone reversed, increased basal and growth factor-stimulated phosphorylation of Akt and ERK 1/2. Pharmacological blockade of phosphatidylinositol kinase suppressed the Akt increase whereas pharmacological blockade or small interfering RNA downregulation of ErbB2 inhibited both Akt and ERK 1/2 expression. All three Akt isoforms contributed to the increase in total Akt. EGF but not insulin specifically upregulated Akt 2 and 3. Since Akt and ERK 1/2 are also hyperexpressed in poorly differentiated hepatomas, their dysregulation in cancer may involve transcriptional mechanisms normally operative in cultured hepatocytes. We hypothesize that the induction and activation of ErbB2 increases the expression of these kinases, enhancing the responsiveness of hepatocytes to EGF as they adapt to culture.

A new technique for primary hepatocyte expansion in vitro

The current application for many potential cell-based treatments for liver failure is limited by the low availability of mature functional hepatocytes. Although adult hepatocytes have a remarkable ability to proliferate in vivo, attempts to proliferate adult hepatocytes in vitro have been less successful. In this study, we investigated the effect of coculture cell type on the proliferative response and the functional activities of hepatocytes. We show, for the first time, a robust proliferative response of primary adult rat hepatocytes when cocultured with mouse 3T3-J2 fibroblasts. Hepatocytes cultured at low density on growth-arrested 3T3-J2 fibroblast feeder layers underwent significantly higher proliferation rates than when cultured on feeder layers made of four other cell types. Increasing colony size correlated with an increase in hepatocellular functions. The proliferating hepatocytes retained their morphologic, phenotypic, and functional characteristics. Using a cell patterning technique, we found that 3T3-J2 fibroblasts stimulate DNA synthesis in hepatocytes by short-range heterotypic cell-cell interactions. When hepatocytes that proliferated in cocultures were harvested and further subcultured either on 3T3-J2 fibroblast feeders or in the collagen sandwich configuration, their behavior was similar to that of freshly isolated hepatocytes. We conclude that adult rat hepatocytes can proliferate in vitro in a coculture cell type-dependent manner, and can be serially propagated by coculturing with 3T3-J2 fibroblasts while maintaining their differentiated characteristics. Our results also suggest that one of the major reasons for the functional differences in hepatocyte cocultures may be due to the different proliferative responses of hepatocytes as a function of coculture cell type. This study provides new insights in the roles of coculture cell types and cell-cell interactions in the modulation of hepatic proliferation and function.

Species-Specific Kinetics and Zonation of Hepatic DNA Synthesis Induced by Ligands of PPARα

Peroxisome proliferator-activated receptor alpha (PPARalpha) ligands evoke a profound mitogenic response in rodent liver, and the aim of this study was to characterize the kinetics of induction of DNA synthesis. The CAR ligand, 1,4-bis[2-(3,5-dichoropyridyloxy)]benzene, caused induction of hepatocyte DNA synthesis within 48 h in 129S4/SvJae mice, but the potent PPARalpha ligand, ciprofibrate, induced hepatocyte DNA synthesis only after 3 or 4 days dosing; higher or lower doses did not hasten the DNA synthesis response. This contrasted with the rapid induction (24 h) reported by Styles et al., 1988, Carcinogenesis 9, 1647-1655. C57BL/6 and DBA/2J mice showed significant induction of DNA synthesis after 4, but not 2, days ciprofibrate treatment. Alderley Park and 129S4/SvJae mice dosed with methylclofenapate induced hepatocyte DNA synthesis at 4, but not 2, days after dosing and proved that inconsistency with prior work was not due to a difference in mouse strain or PPARalpha ligand. Ciprofibrate-induced liver DNA synthesis and growth was absent in PPARalpha-null mice and are PPARalpha dependent. In the Fisher344 rat, hepatocyte DNA synthesis was induced at 24 h after dosing, with a second peak at 48 h. Lobular localization of hepatocyte DNA synthesis showed preferential periportal induction of DNA synthesis in rat but panlobular zonation of hepatocyte DNA synthesis in mouse. These results characterize a markedly later hepatic induction of panlobular DNA synthesis by PPARalpha ligands in mouse, compared to rapid induction of periportal DNA synthesis in rat.

Integrin α5‐induced EGFR activation by prothrombin triggers hepatocyte apoptosis via the JNK signaling pathway

We have previously shown that prothrombin, a blood coagulation factor, can cause an inhibition of DNA synthesis in normal rat hepatocytes. To explore the mechanisms of this prothrombin action, we examined its effects on the activation of fibronectin receptor integrin alpha5, since fibronectin was found to be degraded by prothrombin actions in primary hepatocyte cultures. We found that prothrombin treatment of rat hepatocytes without addition of any growth factor induced tyrosine phosphorylation of integrin alpha5 and interaction of integrin alpha5 with epidermal growth factor receptor (EGFR), leading to EGFR tyrosine phosphorylation at tyrosine residues Tyr-845 and Tyr-1173. EGFR tyrosine phosphorylation triggered phosphorylation of its down-stream target Shc and the activation of the c-Jun N-terminal kinase (JNK) pathway. Prothrombin also induced hepatocyte apoptosis, a change in cell shape and activation of caspase 3 pathway. The JNK pathway is most likely involved in prothrombin-induced hepatocyte apoptosis, because pre-treatment of hepatocytes with JNK kinase inhibitor II (SP600125) antagonized these prothrombin actions. The data suggest that integrin-related EGFR activation by prothrombin can induce cell growth inhibition and apoptosis via an EGFR-JNK signaling pathway.

A mesoscale model of G1/S phase transition in liver regeneration

The liver regenerates and maintains its function and size after injury by counterbalancing cell death with compensatory cell division. During liver regeneration, injured sites release cytokines, which stimulate normally quiescent hepatocytes to re-enter cell division cycle. Using a mesoscale approach, we have implemented the first mathematical model that describes cytokine-induced dedifferentiation of hepatocytes and the subsequent initiation of DNA synthesis (G0/G1 and G1/S phase transitions of the cell cycle). The model accurately reproduces experimentally measured kinetics of various signaling intermediates and DNA synthesis in hepatocytes for varying degrees of liver damage, in both wild type and knockout backgrounds. Liver regeneration is known to be a robust process, as liver mass reconstitution still occurs in various knockout mice (albeit with different kinetics). We analyze the robustness of the model using methods of control analysis. Moreover, we discuss the system's bandpass filtering properties and delays, which arise from feedbacks and nested feed-forward loops.

Cr (VI) inhibits DNA, RNA and protein syntheses in hepatocytes: Involvement of glutathione reductase, reduced glutathione and DT-diaphorase

In patients with orthopaedic implants, metallic particles have been shown to be disseminated widely throughout the body, particularly in the liver, spleen and lymph nodes. Levels of metal particles and ions in distant organs were highest in patients with loose, corroded prostheses, and when stainless steel and cobalt chrome alloy corrode, chromium is released predominantly as Cr (VI), a toxic ion. This manuscript investigates the interaction of Cr (VI) with liver cells in terms of inhibition of macromolecular synthesis, and the contribution of reduced glutathione (GSH), DT-diaphorase and glutathione reductase (GRd) to the toxicity of Cr (VI). Cr (VI) caused concentration dependent inhibition of protein, DNA and RNA synthesis in hepatocytes. GRd and to a lesser extent DT-diaphorase activities were involved in the generation of toxic intermediates. GRd activity was markedly inhibited during the reduction of Cr (VI), and GSH levels decreased. The concentrations of Cr (VI) found to inhibit macromolecular syntheses in this study are clinically relevant: it is therefore important to develop implants with minimum wear potential.

Role of Interleukin-6 in the Control of DNA Synthesis of Hepatocytes: Involvement of PKC, p44/42 MAPKs, and PPARδ

Interleukin-6 (IL-6) is a pleiotropic cytokine with a pivotal role in normal hepatic growth and liver regeneration. Therefore, in the present study, we examined the effect of IL-6 on cell proliferation and the related signaling pathways in primary cultured chicken hepatocytes. IL-6 increased the level of [³H]thymidine incorporation in a time (≥ 6 hr)- and a dose (≥ 0.1 ng/ml)-dependent manner. Indeed, IL-6 increased the number of BrdU-positive cells and the total number of cells. IL-6 (10 ng/ml) increased the level of IL-6Rα and glycoprotein (gp) 130 (IL-6Rβ) protein expression, Janus Kinase (JAK) 2, signal transducer and activator of transcription (STAT) 3, PKC, p44/42 MAPKs phosphorylation, and PPARδ protein expression. Inhibition of each pathways blocked IL-6-induced [³H]thymidine incorporation increase. IL-6 increased c-fos, c-jun, and c-myc proto-oncogene mRNA levels and the percentage of cells in the S phase according to fluorescence-activated cell sorter (FACS) analysis. IL-6-induced G1/S phase progression was inhibited by AG 490 (2x10^-5 M, JAK2 inhibitor), a STAT3 inhibitor peptide (10^-5 M), bisindolylmaleimide I (10^-6 M, PKC inhibitor), PD 98059 (10^-5 M, p44/42 MAPKs blocker), or PPARδ-specific small interfering RNAs (siRNAs). In conclusion, IL-6 stimulates the proliferation of primary cultured chicken hepatocytes through PKC, p44/42 MAPKs, and PPARδ pathways.

Temporal treatment with interferon-β prevents hepatocellular carcinoma in hepatitis B virus X gene transgenic mice

The preventive effect of interferon (IFN) against hepatocellular carcinoma (HCC) has been confirmed clinically. We sought to determine whether the temporal administration of IFN-beta prevents hepatocarcinogenesis in a mouse model where HCC develops without necroinflammation. Hepatocarcinogenic mice that are transgenic for the hepatitis B virus X gene (HBx-Tg) were treated with IFN-beta or saline (control) for three months, from 3 to 6 months of age, and the incidence of HCC was determined at 18 months of age. The effects of IFN-beta on DNA synthesis and apoptosis were tested. The incidence of HCC was significantly lower in the IFN-beta-treated mice than the controls (0 vs. 50%, P<0.01). Inhibition of DNA synthesis in hepatocytes by IFN-beta was observed in the livers of HBx-Tg, without any significant induction of apoptosis. Although the treatment of IFN-beta was temporal, the number of hepatocytes with DNA synthesis remained lower 3 and 12 months later in life. Temporal administration of IFN-beta has a significant preventive effect on the occurrence of HCC in a mouse model where HCC develops without inflammation. The mechanisms are the inhibition of DNA synthesis and cell cycle progression of hepatocytes.

Activation of the p53–p21Cip1 pathway is required for CDK2 activation and S-phase entry in primary rat hepatocytes

p53 plays a major role in the prevention of tumor development. It responds to a range of potentially oncogenic stresses by activating protective mechanisms, most notably cell-cycle arrest and apoptosis. The p53 gene is also induced during normal liver regeneration, and it has been hypothesized that p53 serve as a proliferative 'brake' to control excessive proliferation. However, it has lately been shown that p53 inhibition reduces hepatocyte growth factor-induced DNA synthesis of primary hepatocytes. Here we show that epidermal growth factor (EGF) activated p53 in a phosphatidylinositol-3 kinase-dependent way, and thus induced the cyclin-dependent kinase inhibitor p21(Cip1) in primary rat hepatocytes. p53 inactivation with a dominant-negative mutant (p53(V143A)) attenuated EGF-induced DNA synthesis and was associated with reduced CDK2 phosphorylation and retinoblastoma protein hyperphosphorylation. When p21(Cip1) was ectopically expressed in p53-inactivated cells, these effects were neutralized. In conclusion, our results demonstrate that in normal hepatocytes, EGF-induced expression of p53 is involved in regulating CDK2- and CDK4 activity, through p21(Cip1) expression.

Role of STAT3 in liver regeneration: survival, DNA synthesis, inflammatory reaction and liver mass recovery

The hepatoprotective effect of interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) has been well documented. However, reports on the role of IL-6/STAT3 in liver regeneration are conflicting probably due to the fact that the model of Stat3 knockout mice were complicated with obesity and fatty liver, which may cause some secondary effects on liver regeneration. To study the direct role of STAT3 and to circumvent the problems of obesity and fatty liver in liver regeneration, we generated conditional STAT3 knockout in the liver (L-Stat3−/−) using a transthyretin-driven Cre-lox method. The L-Stat3−/− mice were born with the expected Mendelian frequency and showed no obesity or other obvious phenotype. After partial hepatectomy, mortality in the L-Stat3−/− mice was significantly higher than the littermate Stat3f/+ controls in the early time points (<24 h). Hepatocyte DNA synthesis in the survived L-Stat3−/− mice slightly decreased as compared with Stat3f/+ mice at 40 h after partial hepatectomy, whereas similar hepatocyte DNA synthesis was found at other time points and liver mass could be completely recovered in the L-Stat3−/− mice. In another model of liver regeneration induced by subcutaneous injection of carbon tetrachloride (CCl4), hepatocyte DNA synthesis in the CCl4-treated L-Stat3−/− mice also decreased as compared with Stat3f/+ mice at 40 h after injection but not at other time points. In addition, infiltration of neutrophils and monocyte increased in the liver of CCl4-treated L-Stat3−/− mice compared to wild-type mice. In conclusion, STAT3 is required for survival in the acute stage after 70% hepatectomy and plays a role in inflammatory reaction after hepatocyte necrosis. However, the hepatocytic STAT3 may have limited role in liver mass recovery although DNA synthesis may be impaired.

Growth inhibitory actions of prothrombin on normal hepatocytes: Influence of matrix

Most hepatomas have a defect in prothrombin carboxylation, and can secrete under-carboxylated prothrombin or des-gamma-carboxy-prothrombin (DCP), the function of which is unknown. We considered that the prothrombin-DCP axis might also be involved in growth control. Hepatocytes and hepatoma cells were treated with prothrombin and DNA synthesis and cytoskeletal changes were studied. Prothrombin inhibited DNA synthesis in hepatocytes on fibronectin, but not collagen matrix. Hepatoma cell lines were not inhibited. We found that hepatoma cell matrix conferred resistance to hepatocytes. Prothrombin decreased fibronectin but not collagen amounts, but only in the presence of hepatocytes and not hepatoma cells, indicating that it has a differential action on matrix proteins. It also caused changes in cell shape and actin depolymerization. In vivo, there was a decrease in plasma prothrombin activity after a partial hepatectomy (PH), concomitant with the peak of DNA synthesis in the hepatocytes at 24h after PH. Injection of warfarin at the time of PH, further inhibited PT activity and enhanced this 24h peak of DNA synthesis. Furthermore, repeated injection of prothrombin lowered the peak DNA synthesis after PH. The data support the hypothesis that prothrombin can act as a hepatocyte growth inhibitor, likely at the level of fibronectin loss and result in cytoskeletal changes. Hepatomas resist this action, possibly due to their different matrix proteins. This represents a novel mechanism for growth regulation and provides a possible biological significance for the tumor marker DCP.

Decreased Hepatic 5-HT1A Receptors During Liver Regeneration and Neoplasia in Rats

In the present study we investigated the role of 5-hydroxytryptamine (5-HT) and 5-HT1A receptor during liver regeneration after partial hepatectomy (PH) and N-nitrosodiethylamine (NDEA) induced hepatocellular carcinoma in male Wistar rats. 5-HT content was significantly increased during liver regeneration after PH and NDEA induced hepatocellular carcinoma. Scatchard analysis using 8-OH-DPAT, a 5-HT1A specific agonist showed a decreased receptor during liver regeneration after PH and NDEA induced hepatocellular carcinoma. 5-HT when added alone to primary hepatocyte culture did not increase DNA synthesis but was able to increase the EGF mediated DNA synthesis and inhibit the TGF beta 1 mediated DNA synthesis suppression in vitro. This confirmed the co-mitogenic activity of 5-HT. 8-OH-DPAT at a concentration of 10(-4) M inhibited the basal and EGF-mediated DNA synthesis in primary hepatocyte cultures. It also suppressed the TGF beta 1-mediated DNA synthesis suppression. This clearly showed that activated 5-HT1A receptor inhibited hepatocyte DNA synthesis. Our results suggest that decreased hepatic 5-HT1A receptor function during hepatocyte regeneration and neoplasia has clinical significance in the control of cell proliferation.