The Study Of Palladin Gene Fuction

This research paper mainly includes the following two aspects:(1)The study of Palladin gene function during all-trans retinoic acid(ATRA)induced differentiation of acute promyelocytic leukemia(APL)cells;(2)The in-depth analysis of the processes where Palladin participates inphagocytosis and the research on related cell and molecular mechanisms.PART? The function of Palladin in ATRA induced differentiation of APL cellsAcute promyelocytic leukemia(APL)accounts for about 10-15%of acute myelogenous leukemia(AML).However,it is one of the few human cancers which can be cured by targeting therapy.Clinically,APL is a very dangerous disease especially during chemotherapy induction.For example,disseminated intravascular coagulation(DIC)is one of the fatal complications.The induction of all-trans retinoic acid(ATRA)and arsenic trioxide(ATO)into APL treatment has greatly improved the outcome of the patients,representing milestones in the therapy of this disease~1.Pilot studies of combination treatment with arsenic trioxide and ATRA have shown high efficacy and reduced hematologic toxicity,and the 5-year overall survival rate is now about 85-90%~2.Palladin gene,also known as RIG-K(Retinoid acid inducible gene-K),was upregulated during ATRA induced APL cells differentiation.Our laboratory was one of the three earliest groups who found this gene,and our interests on Palladin gene function have being focused on on its role played in the differentiation of APL cells.Previous work found that Palladin was upregulated during APL cell line NB4 cell differentiation.The first part of this thesis mainly addresses the function of Palladin during the process of NB4 cell differentiation.Our findings are:1.Cell morphological studies and flow cytometer features suggest that,knockdown of Palladin has minor effect on NB4 cell differentiation both at cell both morphology and expression level of differentiation maker CD11b;2.Palladin exerts effect on proliferation and apoptosis of APL cells,and its knockdown can be partly relieved this proliferation inhibitory effect by ATRA and can also remarkably reduce apoptosis rate at relatively late time periods;3.Palladin is an actin related protein.In differentiated NB4 cells Palladin takes part in the formation of lamellipodia,podosomes and focal adhesions,Palladin knockdown can destroy these structures,result in the inhibition of neutrophil associated function such as adhesion,migration,spreading,phagocytosis and so on;4.Differentiated NB4cell can secrete many chemokines.Results from quantitative RT-PCR and chemokine array show that,Palladin knockdown reduces a number of chemokines such as IL-8,CCL3,CCL4,CCL20 and CCL22,suggesting that Palladin may a play role in gene transcriptional regulation;5.In vitro model shown knockdown of Palladin can reduce the infiltration degree of differentiated NB4 cells into mouse lung tissues,indicating that Palladin maybe participate in the ATRA-induced Retinoic Acid Syndrome(RAS).PART? The study of Palladin functions involved in phagocytosisNow that Palladin has no significant effcet on morphological maturation and expression of differentiation antigens of APL cells,but plays important roles in neutrophil associated function such as migration,spreading,phagocytosis,and chemtaxis,in this part of the thesis,our research interests have being mainly focused on on the roles of Paladin in phagocytosis,including concrete cellular processes in which Palladin is involved and the related molecular mechanisms.Our results show that:1.Palladin exerts distinct functions in different receptors-mediated phagocytosis.Palladin plays important roles in Fc?R and complement receptors-mediated phagocytosis,but not in scavenger receptor-mediated phagocytosis;2.Palladin acts on the adhesion,phagocytic cup formation and its internalization,but has no significant effcet on phagosome maturation;3.Palladin takes part in the receptor activation such as Fc?RIIA clustering,and this effect maybe related with Palladin's role in regulating microfilament cytoskeleton assembly and the interactation between Palladin and c-SRC;4.OCRL is a new parter of Palladin because Palladin and OCRL can colocalize at phagocytic cup whereas Palladin knockdown reduces the recruitment of OCRL in phagocytic cup.Since OCRL is a 5-phosphatase enzyme involved in phosphoinositide remodeling and actin polymerization,Palladin may recruit OCRL protein to phagocytic cup to regulated the phosphatidylinositol turnover and then actin depolymerization.