Mechanism Of P53Proliferation Promoting In Rat Primary Hepatocyte

1. Objects:P53gene, a tumor suppress gene, is closely related to tumorigenesis. There is P53function loss or mutant in50%of all cancers. Although P53generaly function as an inhibitor of proliferation, it is discovered that inhibition of P53reduce proliferation of primary hepatocytes. This suggests that P53has a positive function in cell proliferation. However it was reported that P53may act as one of the upstream regulators of erk activation. And P53may positively regulate hepatocyte proliferation through endogenous TGFa. In this study, we will go further in the mechanism of P53positive regulate proliferation.2. Methods.2.1Cell isolation and cultureYoung adult male Wistar rats, weighing200-220g, were kept on a12-h light: dark cycle and were fed ad libitum. Hepatocytes were isolated in a two-step in vitro version of the collagenase perfusion technique with modification. Cell death was measured using the trypan blue exclusion technique. Cells were seeded at density of20000cells/cm2. Serum-free culture medium consisted of a1:1combination of Williams Medium E and Dulbecco’s modified Eagle’s medium, with a final glucose concentration of8.4mm. Medium was supplemented with penicillin (67mg/L), streptomycin (100mg/L), collagen (3mg/L), dexamethasone (25nmol/L) and insulin (100nmol/L). The cells were plated with5%serum during the first three hours. Then cultures were maintained in medium without serum in a5%CO2atmosphere at37℃. Hepatocytes were exposed to lOnmol/L EGF.2.2WesternCultured hepatocytes were lysed in Tris-lysis buffer, pH7.4, with60mmol/L Tris-HCl,10%glycerol,3%sodium dodecyl sulfate (SDS),1mol/L EDTA,0.2mmol/L AEBSF,20μmol/L leupeptin,200units/mL aprotinin,65μmol/L sodium orthovanadate and10mmol/L β-glycerophosphate. Lysates were sonicated. Protein concentrations of all samples were measured by DC protein assay (Bio-Rad, Hercules, CA, USA), and were adjusted to equal concentrations in the lysis buffer containing5%P-mercaptoethanol and0.0025%bromophenol blue. Protein samples were boiled for4min and were stored at-20℃. Proteins were separated by SDS-PAGE and were electrotransferred to nitrocellulose membranes for subsequent protein detection as described (Laemmli1970; Towbin et al.1979). Filters were blocked in Tris-buffered saline containing5%fat-free dry milk and were incubated with primary antibodies diluted in1%fat-free dry milk in Tris-buffered saline over night at4℃. The following primary antibodies were used:anti-Thr202/204phosphorylated Erk and CDK4from Cell Signalling Technology, mouse anti-Cyclin E and anti-P53from Cell Signalling Technology, rabbit anti-Cyclin Dl and mouse anti-P21from Upstate Biotechnology, mouse anti-β-tubulin from Sigma-Aldrich, rabbit anti-pY1173-EGF receptor as previously described.After washing, filters were incubated with horse radish peroxidase (HRP)-conjugated goat anti-rabbit IgG, donkey anti-mouse IgG and donkey anti-sheep IgG from Sigma Aldrich at room-temperature for90min. Immunobinding was detected by the enhanced chemiluminescence (ECL) method plus exposure to Hyperfilm-ECL. 2.3Measurement of DNA synthesisFor thymidine incorporation cells were cultured in six-well plates and grown as described above.[3H]-thymidine(1nCi/L) was added to the cultures48h after plating.DNA synthesis was assessed by determining the amount of radioactivity incorporated into DNA. The cellular material was precipitated with2ml5%trichloroacetic acid for2×10min. The acid-precipitated material was dissolved in0.3ml KOH (1mol/L), followed by liquid scintillation counting. Protein was measured as earlier described.2.4ELISAMedium was collected and assayed with a TGFa quantitative ELISA (Calbiochem; published sensitivity of5pg/ml) according to the manufacturer’s protocol. Briefly,polyclonal antibodies corresponding to epitopes from the50-amino acid extracellular region as well as epitopes corresponding to the full-length molecule were immobilized onto the surface of the microtiter plate wells (to capture antigen), the wells were rinsed, and then uterine lavage samples that had been mixed with biotinylated goat antihumanTGFa antibodies were added to the wells. After the3-h incubation, the wells were washed (×3) and rinsed (×1) to remove any unbound material. Streptavidin-horseradish peroxidase was then incubated with the samples for30min. After washing (×3)and rinsing (×1), the chromogenic substrate O-phenylenediamine was incubated with the samples in the dark, and then4N sulfuric acid was added to stop the reaction. Spectro-photometric absorbency of light at490nm for the intraluminal samples was quantified against a standard curve using known amounts of lyophilized control TGFa peptide reconstituted in dH2O and then serially diluted in assay buffer to concentrations of250,125, and63pg/ml. In addition, control samples were set up at concentrations of63,125, and250pg/ml reconstituted with a1:1mix of intraluminal fluid and assay buffer to rule out the possibility of a factor being present in the intraluminal fluid that was interfering with the ability of the assay to detect TGFa. All assays were carried out in duplicate. 2.5Immunocytochemistry and confocal immunofluorescence microscopyThe primary hepatocytes were fixed in4%buffered paraformaldehyde for10min. Then the cells were washed with PBS for2×10min and dried in room temperature for30min. The first antibody was added after there is no liquid over the cells surface. Then the cells were incubated over night in room temperature. PBS washed the sample for2×10min and then the immunofluorescence labeled second antibody was added. The cells were incubated for1hour in room temperature. Then the sample was washed with PBS for2×10min and dried for30min. Then the sample was examed with confocal immunofluorescence microsopy(60X oil objective).3. Results:3.1P53and cell proliferation1) Stimulation cells with EGF induced P53activationWhen24and30hours after the medium was supplemented with EGF, increased P21expression was seen. Pifithrin-a, the inhibitor of P53, can completely reduce P21expression induced by EGF. We draw the conclusion that EGF can activate P53and thus induce P21expression.2) Induction of Cyclin D1dependond on P53functionWhen24and30hours after the medium was supplemented with EGF, increased Cyclin D1expression was seen whereas Pifithrin-a can inhibit its induction. The results showed that the induction of Cyclin D1deponded on P53activation.3) Dose-Reponse relationshiop of P53function and cell proliferationHepatocytes were incubated with or without EGF for30hours.1hour prior to growth factor addition, increasing concentrations of pifithrin-a was added. Measurements of thymidine incorporation showed that proliferation was effectively inhibited, whith a close to100%total inhibition at (30μmol/L) concentration. There is a dose response relationship between P53function and proliferation.4) Time-Response relationship of P53function and cell proliferation Primary cultures of hepatocytes were exposed to pifithrin-a at different time point. Following thymidine incorporation, the inhibitor of P53totally inhibited EGF-stimulated hepatocytes proliferation when added1hour before or12hours after the growth factor. A partial effect was found when the antibody was added24hours after EGF. Thus, it appeared as P53activation12hours after EGF stimulation was necessary for proliferation.3.2The mechanism of cell proliferation induced by P531) TGFa secretion depended on P53activationHepatocytes were incubated with EGF alone for24hours, and TGFa concentrations in the medium assayed with an ELISA kit (expressed with average values of there expreriments). Results showed that TGFa levels in medium from control hepatocytes were below8pg/ml, medium from hepatocytes exposed to EGF for24hours contained38.24pg/ml TGFa. If cells were exposed to30μmol/L pifithrin-a1hour before EGF stimulation, the medium contained12.3pg/ml TGFa. Thus we can get conclusion that TGFa secretion depended on P53activation.2) Dose-reponse relationshiop of TGFa and cell proliferationHepatocytes were incubated with or without EGF for30hours.1hour prior to growth factor addition, increasing concentrations of a neutralizing antibody to TGFa was added. Measurements of thymidine incorporation showed that proliferation was effectively inhibited, whith a close to100%total inhibition at (0.5mg/L) concentration. There is a dose response relationship between autocrine TGFa and proliferation.The antibody or a control antibody was incubated for1hour with the100ul medium containing EGF. This medium was applied to hepatocyte cultures, which were harvested for Western blotting10minutes thereafter. Western blotting for EGF receptor phosphorylation and Erkl/2phosphorylations showed that preincubation of EGF with the neutralizing antibody did not reduce the initial, EGF-induced phosphorylations. Result showed that the effect of the neutralizing antibody was not an effect of cross-reactivity with EGF. 3) Time-response relationship of TGFa and cell proliferationPrimary cultures of hepatocytes were exposed to a neutralizing mouse monoclonal tgfα antibody or control antibody of the same isoclass and subclass at different time point. Following thymidine incorporation, the antibody to TGFa totally inhibited EGF-stimulated hepatocytes proliferation when added1hour before or12hours after the growth factor. A partial effect was found when the antibody was added24hours after EGF. Thus, it appeared as autocrine TGFa stimulation12hours after EGF stimulation was necessary for proliferation. The similar time-respose relationship like P53suggests that P53positive regulate cell proliferation through TGFa pathway.3.3The mechanism of cell proliferation induced by TGFa1) Autocrine TGFa induced Cyclin D expressionBecause autocrine TGFa stimulation12hours after EGF stimulation was necessary for proliferation, primary cultures of hepatocytes were exposed to a neutralizing mouse monoclonal tgfa antibody12hours after EGF stimulation and harvested24and30hours after EGF thereafter. Results showed that autocrine TGFa was necessary for Cyclin D induction. It proved that P53induced Cyclin D expression through autocrine TGFa.4) Autocrine TGFa induced Cyclin D nuclear accumulationThe function of Cyclin D depends on its nuclear accumulation. Cyclin D and CDK4was translocated into the nuclear30hours after EGF stimulation. But if primary cultures of hepatocytes were exposed to a neutralizing mouse monoclonal tgfa antibody1hour before EGF, there is no Cyclin D and CDK4accumulated in the nuclear30hours after EGF stimulation. Results showed that autocrine TGFa induced Cyclin D and CDK4nuclear accumulation.4. Conclusions:P53was activated and traslocated into nuclear to induce TGFa gene translation after the medium of primary hepatocytes was supplement with EGF. TGFa can bind to EGF receptor, finally Cyclin D was induced and nuclear accumulated. Thus primary hepatocytes continue its cell cycle and proliferate.