Effects Of Arsenic On P53 Function In Keratinocyte And Mechanism Of Action

It has been proved by epidemiology that exposure of low concentration arsenic is associated with risk of cancers such as skin cancer.Although arsenic carcinogenesis is well documented,the mechanism of action is still debated.Some scientists intsist that arsenic can induce carcinogenesis by itself.Others hold that arsenic is only a co-carcinogen.P53 gene is one of the well-known antioncogenes.The signal trasnduction pathway mediated by p53 play the most importmant role in the cellular proliferation,differentiation,stress and carcinogenesis.P53 gene is named as "guardian of genome".MDM2(murine double minute 2, MDM2)gene is one of the downstream genes of p53.p53 can stimulate the transcription of MDM2 gene while MDM2 can inhibit the function of p53.So p53 and MDM2 form an autoregulated feed-back loop which keep p53 at low activity under normal condition.Accurate sub-cellular location is very important to p53 function. After activated,p53 go to nucleus and activate the transcription of target genes.Then p53 is degradated at cytoplasm.MDM2 protein can shuttle back and forth beween nucleus and cytoplasm due to its NES and NLS. NES can pull MDM2 protein from nucleus to cytoplasm while NLS can pull MDM2 protein from cytoplasm to nucleus.MDM2 protein can transport p53 /MDM2 complex between nucleus and cytoplasm.The ability of shuttling back and forth is very important for MDM2 to inhibit the function of p53.In our experment,the hTERT immortalized human keratinocytes were used to explore the effects of arsenic on p53 and MDM2.It will provide novel research findings to clarify arsenic carcinogenesis in the skin.The first chapter Low concentration arsenic functional inactivated p53 by cytoplasmic distribution through upregulation of MDM2Objective:To explore the effects of low concentration arsenic on MDM2 protein and association with p53 subcellular location and function in human keratinocytes.Methods:hTERT immmortalized human keratinocyte were used as model.Western blotting was used to explore the protein levels of MDM2, p53 and p53 phosphorylation at serine 15 site.After treatment of 0.5μmol/L,1μmol/L and 2μmol/L sodium arsenite for 12 hours or 24 hours.Immunofluorescence staining was used to observe the subcellular location of p53 protein before and after arsenic treatment and the effects of LMB and Nutlin-3.Results:The protein level of MDM2 was upregulated after low concentration arsenic administration while p53 protein has no obvious change.MDM2 protein increased steadily with the increase of dose and duration of arsenic treatment.Most of p53 protein located at cytoplasm in the keratinocyte after arsenic treatment.LMB and Nutlin-3 can block arsenic-induced p53 cytoplasmic distribution.Pretreatment of arsenic on keratinocyte can interfere with 5Fu-induced p53 activation.Conclusion:Low concentration arsenic upregulate MDM2 protein in a dose and time dependent manner.Low concentration arsenic induce p53 cytoplasmic distribution through upregulation of MDM2.Low dose arsenic-induced p53 cytoplasmic distribution functionally inactivated p53.The second chapter Mechanism of low concentration arsenic-induced upregulation of MDM2Objective:To explore the mechanism of action of upregulation of MDM2 protein after low dose arsenic treatment.Methods:The luciferase report gene system was used to observe the effects of sodium arsenite on MDM2 gene P₁,P₂ promoter.RT-PCR was used to explore the mRNA level of MDM2 after 1μmol/L and 2μmol/L sodium arsenite treatment in keratinocytes for 24 hours.Pretreatment of PD98059,SB203580 and LY294002 was used to interfere with the effects of sodium arsenite on MDM2 protein expression in keratinocytes.Results-The luciferase activity of MDM2 P₁ promoter was induced obviously after low concentration arsenic treatment(p＜0.05).MDM2 mRNA level increased steadily with the increase of sodium arsenic. Pretreatment of PD98059 can totally block arsenic-induced MDM2 upregulation while SB203580 and LY294002 have no effect.Conclusion:Through MAPK/ERK pathway,low dose aresnic can induce the transciptional activity of P₁ promoter of MDM2 gene promoter, and then upregulate the expression of MDM2.The third chapter low concentration arsenic acts as a co-carcinogen by functional inactivation of p53Objective:To explore the effect of low dose arsenic on UV-induced apoptosis of keratinocyte and association with arsenic carcinogenesis in the skin.Methods:Flow cytometry was used to measure UV-induced apoptosis after 0.5μmol/L and 1μmol/L sodium arsenite treatment for 24 hours in keratinocytes.Results:The apoptotic rate of 0.5μmol/L and 1μmol/L sodium arsenite was 1.2%and 1.5%.There is no difference compared to 1.5%of control(P＞0.05).After pretreated by 0.5μmol/L and 1μmol/L sodium arsenite,UV-induced apoptotic rate was 48.8%and 39.9%.There is significant difference compared to 64.7%of control(P＜0.05).The results of Hoechst nuclei staining is the same as flow cytometry.Conclusion:Low dose arsenic exposure interfered with UVinduced apoptosis in keratinocytes.Low concentration of arsenic acts as a co-carcinogen by functional inactivation of p53.