| Part I. Genetic polymorphisms of NAT2 and risk of AML Objective:Acute myeloid leukemia (AML) takes the most part of adult acute lekemia, and it is origin from malignant stem cells. Extensive and in-depth researches have deepened our understanding of AML, especially in the area of pathogenesis. Currently , heredity and environment is still seen as the most accepted pattern to describe causes of AML. In the study of genetic factors, correlations between mutations in genes , which may play important roles in physiology, and the onset of AML has been revealed.N-acetyltransferase 2(NAT2) , as a member of acetyl transferase ,takes part in the individual physiological response to various xenobiotic compounds, such as a wide range of exogenous chemicals and several clinically useful drugs. A number of single nucleotide polymorphisms (SNPs) have been detected in NAT2 gene,and some of them, which can result in a slow phenotype of NAT2,including rs1799930 (G590A) and rs1799931 (G857A),have been identified as regulators of human susceptibility to various cancer. in recent years,it has been reported abroad that rsl799930 and rs1799931 may contribute to the risk of leukemia.Howere,there is no report about the condition in Chinese,let alone in Han population. Our purpose was to investigate the possible associations between NAT2 gene polymorphisms and the risk of AML in a Chinese Han population.Methods and results:A case control study was conducted including 98 AML cases and 112 healthy controls.NAT2 gene two polymorphisms rs1799930 and rsl799931 were genotyped in the two groups. Chi-square test was employed to compare the genotype and allele distribution differences between groups. Odds ratio (OR) with 95% confidence interval (CI) was estimated to calculate the association between NAT2 gene polymorphisms and AML onset.A remarkable decrease trend of rs1799931 GA genotype was detected in AML patients compared with controls, while the ancestral GG genotype frequency increased in cases(P<0.05). And the mutant A allele of rs1799931 significantly reduced the risk of AML by 0.585 fold versus the ancestral G allele carriers (OR=0.585, 95%CI=0.361-0.950). But no significant differences of rs1799930 both genotype and allele distribution differences were found between groups (P>0.05).Conclusion:Our findings suggested that the NAT2 gene polymorphism rs1799931 was associated with decreased risk of AML and was likely to be a protective factor against AML development.Part ?. Crosstalking between NPC1 and canonical wnt signalingObjective:Wnt signaling, activated by secreted wnt ligands, is one of the fundamental mechanisms in development and homeostasis. As the most studied pathway, the canonical wnt signaling has been demonstrated to be very critical in many aspects of physiology, and mutations in this pathway are often linked to human cancer, degeneration and other diseases.Abundant studies have shown that the canonical Wnt signaling plays important roles in normal and abnormal hematopoiesis. Searching for new therapies for cancers ,including leukemia,through targeting canonical Wnt signaling,becomes a hot area.Niemann-Pick disease type C1 (NPC1) lies in the membrane of lysosome/late endosome,and functions with NPC2 to transport cholesterol and some sphingolipid out of the lysosome/late endosome system to the other parts of cells.Therefor,mutations either in NPC1 or NPC2 will result in a lethal disease, Niemann-Pick type C.According to some previous studies, several signalings are disregulated in NPC1 mutated cells,and wnt signaling may also be involed. Interestingly,NPC1, as an indispensable glycoprotein in cholesterol transport, may also be regulated by canonical wnt signaling, as the latter can directs efflux of endosomal cholesterol.However,there is no precise report demonstrating that NPC1 can influence the canonical wnt signaling or be regulated by the latter yet. So our research is aimed to explor the relationship between NPC1 and canonical wnt signaling.Methods and results:1. We built NPC1 knocked down (KD) cell lines using lentivirus.Then we tested?-catenin in NPC1 KD or NPC1 inhibitor(U18666A) treated cells and controls with western blotting(WB).A significant decrease of total?-catenin was detected in NPC1 KD cells.Then nuclei extract was test by WB, and decrease of?-catenin in nuclei was deteced as well.Immunofluorescent staining (IF) was used to identify the distribution of ?-catenin in those cells, and the result was consistant with that detected by WB ,both total and nuclear?-catenin was decreased in NPC1 KD cells. We also used quantitative polymerase chain reaction(QPCR)and top-flash reporter system to dectect the change of transcriptive activity mediated by canonical wnt signaling.Decrease was found as our predict.Then we over expressed NPC1 in NPC1 KD cells by transient transfection, and the decrease of?-catenin was rescued.2. No significant change of mRNA of ?-catenin was detected in NPC1 KD cells vs control cells with QPCR.WB found accelerated degradation of?-catenin in NPC1 KD cells,as the synthesis was block by cycloheximide(CHX) previously. The result of immunoprecipitation (IP) showed that(3-catenin was more ubiquitylated in NPC1 KD or U18666A treated cells.3. Decreases in both protein and mRNA of NPC1 was detected in ?-catenin KD cells by WB and QPCR.Using of wnt inhibitor DKK-1 also down regulated the protein level of NPC1, while wnt activators Wnt-3a and LiCl up regulated it. Over expressing ?-catenin through transient transfection can lead to a same result as using wnt activators.4. Down regulation of canonical wnt signaling, by knocking down NPC1 with lentivirus or treatment of U18666A, inhibited the proliferation of K562 cells.Combined utilization of U18666A and imatinib could sensitize imatinib-resisted K562 cells to imatinib again.Conclusion:NPC1 can modulate canonical wnt signaling by influencing the stability of ?-catenin,and canonical wnt signaling can modulate the expression of NPC1 as well, a crosstalking between NPC1 and canonical wnt signaling existing in cells. Leukemia cells ,at least those arosed by wnt signaling, can been depressed by using inhibitor of NPC1 through down regulation of canonical wnt signaling.lt even can sensitize leukemia cells to drugs if the resistance was caused by over activation of wnt signaling. |
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