Peak Of DNA Synthesis Research Articles

Lack of Abcc3 expression impairs bile-acid induced liver growth and delays hepatic regeneration after partial hepatectomy in mice

Bile acids (BA) are increasingly recognized as important modulators of liver regeneration. Increased enterohepatic BA flux has been proposed to generate specific signals that activate hepatocyte proliferation after partial hepatectomy (PH). We have investigated the role of the BA membrane transporter Mrp3 (Abcc3), which is expressed in the liver and gut, in the hepatic growth response elicited by BA and in liver regeneration after PH. Liver growth and regeneration, and the expression of growth-related genes, were studied in Mrp3(+/+) and Mrp3(-/-) mice fed a cholic acid (CA) supplemented diet and after 2/3 PH. Activation of the BA receptor FXR was measured in mice after in vivo transduction of the liver with a FXR-Luciferase reporter plasmid. BA levels were measured in portal serum and liver tissue by high performance liquid chromatography-tandem mass spectrometry. Liver growth elicited by CA feeding was significantly reduced in Mrp3(-/-) mice. These animals showed reduced FXR activation in the liver after CA administration and decreased portal serum levels of BA. Liver regeneration after PH was significantly delayed in Mrp3-deficient mice. Proliferation-related gene expression and peak DNA synthesis in Mrp3(-/-) mice occurred later than in wild types, coinciding with a retarded elevation in intra-hepatic BA levels. Lack of Abcc3 expression markedly impairs liver growth in response to BA and after PH. Our data suggest that Mrp3 plays a non-redundant role in the regulation of BA flux during liver regeneration.

Ultraviolet stress delays chromosome replication in light/dark synchronized cells of the marine cyanobacterium Prochlorococcus marinus PCC9511.

BackgroundThe marine cyanobacterium Prochlorococcus is very abundant in warm, nutrient-poor oceanic areas. The upper mixed layer of oceans is populated by high light-adapted Prochlorococcus ecotypes, which despite their tiny genome (~1.7 Mb) seem to have developed efficient strategies to cope with stressful levels of photosynthetically active and ultraviolet (UV) radiation. At a molecular level, little is known yet about how such minimalist microorganisms manage to sustain high growth rates and avoid potentially detrimental, UV-induced mutations to their DNA. To address this question, we studied the cell cycle dynamics of P. marinus PCC9511 cells grown under high fluxes of visible light in the presence or absence of UV radiation. Near natural light-dark cycles of both light sources were obtained using a custom-designed illumination system (cyclostat). Expression patterns of key DNA synthesis and repair, cell division, and clock genes were analyzed in order to decipher molecular mechanisms of adaptation to UV radiation.ResultsThe cell cycle of P. marinus PCC9511 was strongly synchronized by the day-night cycle. The most conspicuous response of cells to UV radiation was a delay in chromosome replication, with a peak of DNA synthesis shifted about 2 h into the dark period. This delay was seemingly linked to a strong downregulation of genes governing DNA replication (dnaA) and cell division (ftsZ, sepF), whereas most genes involved in DNA repair (such as recA, phrA, uvrA, ruvC, umuC) were already activated under high visible light and their expression levels were only slightly affected by additional UV exposure.ConclusionsProchlorococcus cells modified the timing of the S phase in response to UV exposure, therefore reducing the risk that mutations would occur during this particularly sensitive stage of the cell cycle. We identified several possible explanations for the observed timeshift. Among these, the sharp decrease in transcript levels of the dnaA gene, encoding the DNA replication initiator protein, is sufficient by itself to explain this response, since DNA synthesis starts only when the cellular concentration of DnaA reaches a critical threshold. However, the observed response likely results from a more complex combination of UV-altered biological processes.

HDAC1 Cooperates with C/EBPα in the Inhibition of Liver Proliferation in Old Mice

Epigenetic control of liver proliferation involves cooperation between transcription factors and chromatin-remodeling proteins. In this work, we found that the levels of HDAC1 (histone deacetylase 1) are increased in quiescent livers of old mice. The elevation of HDAC1 in liver is mediated by the RNA-binding protein CUGBP1. We found that the age-associated CUGBP1-eIF2 complex binds to the 5' region of HDAC1 mRNA and increases translation of HDAC1 in the liver. Further analyses showed that CUGBP1 also increases expression of HDAC1 in cultured cells, in the livers of CUGBP1 transgenic mice, and in the livers of mice injected with cyclin D3, which enhances the formation of the CUGBP1-eIF2 complex. In livers of old mice, HDAC1 interacts with the transcription factor C/EBPalpha and is recruited by this protein to E2F-dependent promoters as a component of high M(r) C/EBPalpha-Brm complexes. The recruitment of HDAC1 to c-Myc and FoxM1B promoters leads to deacetylation of histone H3 at Lys-9 on these E2F-dependent promoters. We show that HDAC1 is an important mediator of growth-inhibitory activity of C/EBPalpha and that small interfering RNA-mediated inhibition of HDAC1 reduces the ability of C/EBPalpha to inhibit cell proliferation. In addition, we have found that both elevation of HDAC1 and interaction of C/EBPalpha with HDAC1 are controlled by cyclin D3-dependent mechanisms. Treatment of old mice with growth hormone, which reduces cyclin D3 levels, leads to the reduction of the CUGBP1-eIF2 complex, normalization of HDAC1 levels, and inhibition of interactions of HDAC1 with C/EBPalpha-Brm complexes. Thus, our data demonstrate that translational elevation of HDAC1 in livers of old mice is involved in the assembly of high M(r) protein-protein complexes that inhibit liver proliferation.

Soluble HLA class I antigen secretion by normal lymphocytes: relationship with cell activation and effect of interferon-gamma

HLA class I antigens are thought to be integral membrane proteins. However, soluble forms of these molecules have been detected. Our laboratory has recently shown that the predominant form of these soluble proteins present in human serum, spleen tissue and culture supernatant of activated lymphocytes exhibits molecular weight and structure similar to classical HLA class I antigens, but lacks HLA A or B polymorphic determinants. In the present study, the secretion of such soluble proteins by lymphocytes has been further explored. Phytohaemagglutinin-stimulated normal lymphocytes secrete considerable quantities of soluble HLA (sHLA) class I proteins. This secretion seems to be a general property of lymphocytes, since activation of T as well as B cells by appropriate mitogens equally induce sHLA I secretion. Lymphocytes require RNA and protein synthesis, but not DNA synthesis, for the secretion to occur. Kinetic studies reveal that maximal sHLA I secretion precedes the peak of DNA synthesis by 24 h. In vitro stimulation with antigens or alloantigens also provokes sHLA I secretion. Moreover, this phenomenon has also been detected for in vivo-activated lymphocytes, as enhanced spontaneous sHLA I secretion was observed in cultures of low-density blastic B and T cells, and of blood lymphocytes obtained from normal subjects who had received a booster immunization 5 days earlier. Interferon-gamma (IFN-gamma) increases the expression of membrane-bound class I antigens but does not induce any sHLA I secretion, suggesting that both molecules are under different regulatory mechanisms. Our results indicate that human lymphocytes, upon stimulation, actively secrete considerable amounts of a soluble form of these biologically relevant proteins.

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.

Involvement of retinoic acid‐induced transglutaminase activity in zonal differences of hepatocyte proliferation after partial hepatectomy

The authors have recently demonstrated that there is inverse correlation between transglutaminase (TGase) activity and DNA synthesis in periportal hepatocytes (PPH) and perivenous hepatocytes (PVH) at 1 day after partial hepatectomy in rats. In order to ensure the involvement of TGase in the differential growth capacities between periportal and perivenous regions of regenerating liver, the aim of this study was to investigate the effect of retinoic acid, an inducer of TGase expression, on zonal differences of hepatocyte proliferation between PPH and PVH isolated from regenerating rat liver. Regenerating liver was prepared by 70% partial hepatectomy. PPH and PVH subpopulations were isolated by the digitonin/collagenase perfusion technique. Cell cycle was evaluated for incorporation of BrdU into hepatocytes and detected by flow cytometric analysis. TGase activity was determined by incorporation of 14C-putrescine into dimethylcasein. When retinoic acid was injected immediately after partial hepatectomy, TGase activity greatly increased in both PPH and PVH at 1 day after partial hepatectomy, and activity was higher in PPH than in PVH. DNA synthesis in both subpopulations did not increase 1 day after partial hepatectomy, with peaks of DNA synthesis shifting to 2 days, and synthesis was higher in PVH than in PPH. These results suggest that TGase might be involved in differential growth capacities between periportal and perivenous regions of regenerating rat liver after partial hepatectomy.

Enhanced β-adrenergic receptors in the brain and pancreas during pancreatic regeneration in weanling rats

Adrenergic stimulation has an important role in the pancreatic beta-cell proliferation and insulin secretion. In the present study, we have investigated how sympathetic system regulates the pancreatic regeneration by analyzing Epinephrine (EPI), Norepinephrine (NE) and beta-adrenergic receptor changes in the brain as well as in the pancreas. EPI and NE showed a significant decrease in the brain regions, pancreas and plasma at 72 hrs after partial pancreatectomy. We observed an increase in the circulating insulin levels at 72 hrs. Scatchard analysis using [(3)H] propranolol showed a significant increase in the number of both the low affinity and high affinity beta-adrenergic receptors in cerebral cortex and hypothalamus of partially pancreatectomised rats during peak DNA synthesis. The affinity of the receptors decreased significantly in the low and high affinity receptors of cerebral cortex and the high affinity hypothalamic receptors. In the brain stem, low affinity receptors were increased significantly during regeneration whereas there was no change in the high affinity receptors. The pancreatic beta-adrenergic receptors were also up regulated at 72 hrs after partial pancreatectomy. In vitro studies showed that beta-adrenergic receptors are positive regulators of islet cell proliferation and insulin secretion. Thus our results suggest that the beta-adrenergic receptors are functionally enhanced during pancreatic regeneration, which in turn increases pancreatic beta-cell proliferation and insulin secretion in weanling rats.

Identification of a novel gata binding activity of cyclin D1 promoter during Epidermal Growth Factor (EGF)-induced hepatocyte proliferation and after partial hepatectomy

Background: GATA factors contain zinc finger motifs and act by binding to the consensus DNA sequence (A/T) GATA (A/G). GATA factors have been shown to play critical roles in gene regulation of cell proliferation through the interaction with other transcription factors such as serum response factor (SRF). Following 70% partial hepatectomy (PH) in the rat, we have observed increased GATA 4 and SRF nuclear protein expression at 24 hours, the known peak of DNA synthesis. We have previously reported increased SRF binding to the Cyclin D1 promoter in hepatocytes stimulated to proliferate with EGF and in regenerating liver after PH.

028. Cytokine networks and regulation of spermatogenesis - what should we really believe?

Spermatogenesis is a complex yet highly organised process involving intimate interactions between the supporting Sertoli cells and germ cells at various stages of development. The repeating pattern of the cycle of the seminiferous epithelium is due to the fact that spermatogonia enter spermatogenesis at regularly spaced intervals and proceed through the process at a species-specific rate. How this degree of coordination is maintained remains poorly understood, but recent evidence has focussed attention on the role of growth factors produced by the Sertoli cells and germ cells. Several of these growth factors, such as interleukin-1α (IL-1α), IL-6, tumour necrosis factor (TNFα) and activin A, are also inflammatory cytokines. This has led some researchers to question the physiological significance of these data with respect to normal testicular function. For example, in spite of the fact that IL-1α is produced by the Sertoli cell and regulates spermatogonial proliferation and development in vitro, mice lacking the IL-1R, and hence unresponsive to IL-1α, possess relatively normal fertility. So what role, if any, do these cytokines play in the normal testis, or are they only important during inflammation? It is quite evident that these cytokines have stimulatory and/or inhibitory effects on spermatogonial and spermatocyte development. These cytokines also interact at multiple levels within each other’s signalling pathways and have considerable redundancy of action. Moreover, expression of these cytokines varies across the cycle of the seminiferous epithelium, with major changes in production coinciding with two key events within the cycle: the release of sperm from the epithelium, and the major peaks of DNA synthesis by spermatogonia and preleptotene spermatocytes. It is therefore possible to hypothesise that release of sperm and resorption of the residual cytoplasm triggers a self-regulating inflammatory cascade within the epithelium that initiates and then modulates the next round of spermatogenic development, ensuring that spermatogonia enter the process at the appropriately spaced intervals.

Intact signaling by transforming growth factor beta is not required for termination of liver regeneration in mice.

Transforming growth factor beta (TGF-beta) is a potent inhibitor of hepatocyte proliferation in vitro and is suggested to be a key negative regulator of liver growth. To directly address the role of TGF-beta signaling in liver regeneration in vivo, the TGF-beta type II receptor gene (Tgfbr2) was selectively deleted in hepatocytes by crossing "floxed" Tgfbr2 conditional knockout mice with transgenic mice expressing Cre under control of the albumin promoter. Hepatocytes isolated from liver-specific Tgfbr2 knockout (R2LivKO) mice were refractory to the growth inhibitory effects of TGF-beta1. The peak of DNA synthesis after 70% partial hepatectomy occurred earlier (36 vs. 48 hours) and was 1.7-fold higher in R2LivKO mice compared with controls. Accelerated S-phase entry by proliferating R2LivKO hepatocytes coincided with the hyperphosphorylation of Rb protein and the early upregulation of cyclin D1 and cyclin E. However, by 120 hours after partial hepatectomy, hepatocyte proliferation was back to baseline in both control and R2LivKO liver. Regenerating R2LivKO liver showed evidence of increased signaling by activin A and persistent activity of the Smad pathway. Blockage of activin A signaling by the specific inhibitor follistatin resulted in increased hepatocyte proliferation at 120 hours, particularly in R2LivKO livers. In conclusion, TGF-beta regulates G(1) to S phase transition of hepatocytes, but intact signaling by TGF-beta is not required for termination of liver regeneration. Increased signaling by activin A may compensate to regulate liver regeneration when signaling through the TGF-beta pathway is abolished, and may be a principal factor in the termination of liver regeneration.

Bone modeling: biomechanics, molecular mechanisms, and clinical perspectives

Abstract Bone modeling is a mechanically mediated adaptive process for changing a bone's size, shape, or position. Site specific, anabolic and catabolic modeling events are manifestations of overload hypertrophy and disuse atrophy, respectively. Catabolic bone modeling at the periodontal ligament (PDL) surface is the rate-limiting step in tooth movement. In initiating tooth movement, connective tissue growth factor (CTGF) is expressed in osteoblasts and osteocytes, and osteopontin (Opn) is expressed by osteoclasts and osteocytes. Undermining resorption occurs by recruiting osteoclasts to the site of maximal PDL compression and a paravascular osteogenic response is initiated in widened areas of the PDL: (1) preosteoblast formation at ∼10 hours, (2) peak DNA synthesis at ∼20 hours, (3) maximum rate of mitosis at ∼30 hours, and (4) initiation of bone formation at ∼48 hours. At other skeletal sites, additional genes have been linked to mechanical activation of bone: glutamate/aspartate transporter (GLAST), nitric oxide synthetase (NOS), prostaglandin G/H synthetase (PGHS-2), Msx1 , and c-fos . An intricate series of endocrine, paracrine, and autocrine factors has been described. Parathyroid hormone (PTH) enhances expression of insulin-like growth factor I (IGF-I) and the estrogen receptor beta (ER-β), which are both modulators of mechanical loading. Transforming growth factor beta (TGF-β) and the related bone morphogenetic proteins (BMPs) help control anabolic modeling. Catabolic modeling is mediated by PGE2, IL-1β, and other inflammatory cytokines. Osteoclast differentiation and activation is controlled by genes related to tumor necrosis factor (TNF) and its receptor (TNFR): colony stimulating factor 1 (CSF-1), osteoprotegerin (OPG), receptor activator of nuclear factor (RANK), and RANK ligand (RANKL). Orthodontic aspects of bone modeling are reviewed relative to morphology, biomechanics, neurology, molecular control mechanisms, and the periodontal adaptation to loading. Clinical correlations discussed are orthodontic analgesics, inflammatory cytokines, surgical enhancement of tooth movement, distraction osteogenesis, closure of atrophic defects, periodontal compromise, traumatized periodontium, and systemic disease.

Follistatin 315 accelerates proliferation of hepatocytes after peak DNA synthesis in hepatectomized rat

Follistatin 315 accelerates proliferation of hepatocytes after peak DNA synthesis in hepatectomized rat

A new suppressive agent against interleukin-1β and tumor necrosis factor-α enhances liver regeneration after partial hepatectomy in rats

In the present study, the effect of FR167653, a novel suppressive agent against interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), on liver regeneration was investigated in rats after partial hepatectomy (PH). Doses of 1, 3 and 5 mg/kg per h FR167653 (FR-1, FR-3 and FR-5, respectively) were given intravenously 30 min before PH, while the control was given normal saline. Serum chemistries were serially monitored, and liver regeneration was evaluated by remnant liver weight ratio, proliferating cell nuclear antigen (PCNA) labeling index and mitotic index. Accumulation of IL-1β and TNF-α messenger RNA (mRNA) in the remnant liver was also measured. In FR167653-treated groups, the releases of alanine transaminase (ALT) and aspartate transaminase (AST) were lower. The PCNA labeling index was enhanced by FR167653-administration in a dose-dependent manner, FR-3 and FR-5 groups showed significantly higher peak DNA synthesis than the control group at 24 h post-PH ( P<0.01). The mitotic index was also enhanced by FR167653-administration in a dose-dependent manner. In FR-5 group, the remnant liver weight ratio was significantly higher than that in the control group ( P<0.05). The accumulation of IL-1β messenger RNA (mRNA) in the remnant liver was obviously suppressed in FR-3 and FR-5 groups, but the expression of TNF-α mRNA was not apparently reduced. In conclusion, FR167653 ameliorates liver injury and enhances liver regeneration after PH in rats.

Antiproliferative property of sphingosine 1-phosphate in rat hepatocytes involves activation of Rho via Edg-5

Background & Aims: Sphingosine 1-phosphate (S1P), a ligand for G protein-coupled endothelial differentiation gene-1 (Edg-1), Edg-3, Edg-5, Edg-6, and Edg-8, elicits a variety of responses by cells. Prominent among these is cell proliferation. S1P is abundantly stored in platelets and released upon their activation, suggesting that S1P plays a pathophysiologic role in vivo. Because the major part of injected S1P was distributed into the liver in mice, we wondered whether the liver would be one of its targets. The effects of S1P on hepatocytes, the major constituent cells in the liver, were examined. Methods & Results: Northern blot analysis revealed the expression of Edg-1 and Edg-5 messenger RNA (mRNA) in cultured rat hepatocytes, in which S1P decreased DNA synthesis induced by hepatocyte growth factor (HGF) or epidermal growth factor (EGF) without affecting total protein synthesis. This inhibitory effect was attenuated by inactivation of small GTPase Rho with C3 exotoxin but not by inactivation of G i with pertussis toxin. Moreover, in the presence of JTE-013, a newly developed and specific binding antagonist for Edg-5, the inhibitory effect was also cancelled. Finally, the administration of S1P after 70% partial hepatectomy in rats reduced the peak of DNA synthesis in hepatocytes with increased Rho activity. Furthermore, Edg-5 but not Edg-1 mRNA expression was enhanced in hepatocytes 24–72 hours after partial hepatectomy, which coincides with decreasing hepatocyte proliferation. Conclusions: S1P has an antiproliferative property in rat hepatocytes by activating Rho via Edg-5. Our results raise the possibility that S1P is a negative regulator in liver regeneration.

Impaired Ras membrane association and activation in PPARalpha knockout mice after partial hepatectomy.

Liver regeneration after partial hepatectomy (PH) involves several signaling mechanisms including activation of the small GTPases Ras and RhoA in response to mitogens leading to DNA synthesis and cell proliferation. Peroxisome proliferator-activated receptor-alpha (PPARalpha) regulates the expression of several key enzymes in isoprenoid synthesis, which are key events for membrane association of Ras and RhoA. Thus the role of PPARalpha in cell proliferation after PH was tested. After PH, an increase in PPARalpha DNA binding was observed in wild-type mice, correlating with an increase in the PPARalpha-regulated enzyme acyl-CoA oxidase. In addition, the PPARalpha-regulated genes farnesyl pyrophosphate synthase and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase were significantly increased in wild-type mice. However, these increases were not observed in PPARalpha knockout (PPARalpha -/-) mice. The peak in DNA synthesis observed 42 h after PH was reduced by approximately 60% in PPARalpha -/- mice, despite increases in TNF-alpha and IL-1. Also, under these conditions, membrane association of Ras was high in wild-type mice after PH but was impaired in PPARalpha -/- mice. Accordingly, Ras was significantly elevated in the cytosol in PPARalpha -/- mice. This observation correlated with lower levels of active GTP-bound Ras after PH in PPARalpha -/- mice compared with wild-type mice. Similar observations were made for RhoA. Moreover, deletion of PPARalpha blunted the activation of cyclin-dependent kinase (cdk)2/cyclin E and cdk4/cyclin D complexes. Collectively, these results support the hypothesis that PPARalpha is necessary for cell cycle progression in regenerating mouse liver via mechanisms involving prenylation of small GTPases Ras and RhoA.

Cell kinetic studies in the murine ventral tongue epithelium: thymidine metabolism studies and circadian rhythm determination.

The oral mucosa is a rapidly replacing body tissue that has received relatively little attention in terms of defining its cell kinetics and cellular organization. The tissue is sensitive to the effects of cytotoxic agents, the consequence of which can be stem cell death with the subsequent development of ulcers and the symptoms of oral mucositis. There is considerable interest in designing strategies to protect oral stem cells and, hence, reduce the mucositis side-effects in cancer therapy patients. Here we present details of a new histometric approach designed to investigate the changing patterns in cellularity in the ventral tongue mucosa. This initial paper in a series of four papers presents observations on the changing patterns in the labelling index following tritiated thymidine administration, which suggest a delayed uptake of tritiated thymidine from a long-term intracellular thymidine pool, a phenomenon that will complicate cell kinetic interpretations in a variety of experimental situations. We also provide data on the changing pattern of mitotic activity through a 24-h period (circadian rhythms). Using vincristine-induced stathmokinesis, the data indicate that 54% of the basal cells divide each day and that there is a high degree of synchrony in mitotic activity with a mitotic peak occurring around 13.00 h. The mitotic circadian peak occurs 9-12 h after the circadian peak in DNA synthesis. The data presented here and in the subsequent papers could be interpreted to indicate that basal cells of BDF1 mice have an average turnover time of about 26-44 h with some cells cycling once a day and others with a 2- or 3-day cell cycle time.

Enhanced GABA(B) receptor in neoplastic rat liver: induction of DNA synthesis by baclofen in hepatocyte cultures.

In the present study, the involvement of GABA(B) binding parameters were analyzed in partial hepatectomized (PH), lead nitrate (LN) induced hyperplastic and N-nitrosodiethylamine (NDEA) treated neoplastic rat livers at the peak DNA synthesis. The receptor up-regulated significantly in NDEA treated group compared with respective control. The affinity of the receptor decreased in PH while it increased in LN treated rats. In the other groups, the binding parameters remained unaltered. The displacement analysis using GABA(B) receptor agonist, [3H]baclofen, against baclofen showed a shift in affinity of the receptor towards high-affinity in PH rats and towards low-affinity in LN treated rats. Baclofen dose-dependently induced EGF mediated DNA synthesis in primary hepatocyte cultures. Also, it significantly reduced the TGFbeta1 suppression of EGF induced DNA synthesis. The effect of baclofen on hepatocyte DNA synthesis was abolished by the addition of G(i)-protein inhibitor, pertussis toxin, suggesting the involvement of GABA(B) receptor mechanisms in hepatocyte DNA synthesis. Baclofen alone could not elicit any significant change in DNA synthesis. Thus, our results show that GABA(B) receptor enhancement induce hepatic neoplasia. Also, baclofen is seen to act as a potent co-mitogen, triggering DNA synthesis in primary cultures of rat hepatocytes, mediated through the G(i) protein coupled GABA(B) receptors.

Calorie restriction influences cell cycle protein expression and DNA synthesis during liver regeneration.

Calorie restriction without essential nutrient deficiency (calorie restriction, CR) abrogates experimental carcinogenesis and extends healthful life span. To test whether CR influences cell-cycle protein expression during the hepatocellular proliferation induced by 70% partial hepatectomy (PH), BALB/c mice were separated into two groups, fed comparable semi-purified diets for 10 weeks that differed 40% in caloric offering, and were then subjected to PH. When PH was performed, CR mice weighed 36% less than ad libitum (AL)-fed mice (P < 0.01), but liver-to-body weight ratios were similar. During the regenerative hyperplasia, hepatocytes of CR mice demonstrated evidence of accelerated entrance and passage through G1 and S phases, and an earlier exit from the cell cycle. The first peak of DNA synthesis occurred 6 hr earlier, and the second peak was significantly greater among CR mice with 38% +/- 13% bromodeoxyuridine (BrdU)-positive hepatocytes, compared with 14% +/- 4% in AL mice (P < 0.01). More E2F-1 expression was induced at the hepatic G1/S boundary just prior to each peak of DNA synthesis in regenerating livers of CR mice (P < 0.01), and 8 hr earlier among CR mice. More hyperphosphorylated retinoblastoma p110 was detected during hepatic G1 and the G1-S transition among CR mice, coincident with the early hepatocellular proliferative wave. Cyclin A was induced during the first peak of DNA synthesis 4 hr earlier among CR mice, and it continued 4 hr longer in AL mice, indicating an earlier post-replicative exit by hepatocytes in CR mice. p21 was induced during the G1 phase at 4 hr post-PH, and was maximally expressed during and after peak DNA synthesis in both dietary groups. These results indicate that CR influences cell cycle protein expression levels, causing hepatocytes to enter into S phase earlier and exit abruptly from the cell cycle, and they support the premise that CR enhances induced cell responsiveness by influencing cell cycle regulatory controls.

Hepatic GABA A receptor functional regulation during rat liver cell proliferation

Gamma aminobutyric acid (GABA A) receptor functional status was analysed in partial hepatectomised (PH), lead nitrate (LN) induced hyperplastic and N-nitrosodiethylamine (NDEA) treated neoplastic rat livers during peak DNA synthesis. The high-affinity [ 3H]GABA binding significantly decreased in PH and NDEA rats and the receptor affinity decreased in NDEA and increased in LN rats compared with control. In NDEA, displacement analysis of [ 3H]GABA with muscimol showed loss of low-affinity site and a shift of high-affinity site towards low-affinity. The affinity sites shifted towards high-affinity in LN rats. The number of low-affinity [ 3H]bicuculline receptors decreased significantly in NDEA and PH whereas it increased in LN rats. GABA A receptor agonist, muscimol, dose dependently inhibited epidermal growth factor (EGF) induced DNA synthesis and enhanced the transforming growth factor β1 (TGFβ1) mediated DNA synthesis suppression in primary hepatocyte cultures. Our results suggest that GABA A receptor act as an inhibitory signal for hepatic cell proliferation.

Acute glucose-induced downregulation of PKC-betaII accelerates cultured VSMC proliferation.

Accelerated vascular smooth muscle cell (VSMC) proliferation contributes to the formation of atherosclerotic lesions. To investigate protein kinase C (PKC)-betaII functions with regard to glucose-induced VSMC proliferation, human VSMC from aorta (AoSMC), a clonal VSMC line of rat aorta (A10), and A10 cells overexpressing PKC-betaI (betaI-A10) and PKC-betaII (betaII-A10) were studied with the use of three techniques to evaluate glucose effects on aspects affecting proliferation. High glucose (25 mM) increased DNA synthesis and accelerated cell proliferation compared with normal glucose (5.5 mM) in AoSMC and A10 cells, but not in betaI-A10 and betaII-A10 cells. The PKC-betaII specific inhibitor CGP-53353 inhibited glucose-induced cell proliferation and DNA synthesis in AoSMC and A10 cells. In flow cytometry analysis, high glucose increased the percentage of A10 cells at 12 h after cell cycle initiation but did not increase the percentage of betaI-A10 or betaII-A10 cells entering S phase. PKC-betaII protein levels decreased before the peak of DNA synthesis, and high glucose further decreased PKC-betaII mRNA and protein levels in AoSMC and A10 cells. These results suggest that high glucose downregulates endogenous PKC-betaII, which then alters the normal inhibitory role of PKC-betaII in cell cycle progression, resulting in the stimulation of VSMC proliferation through acceleration of the cell cycle.