.1. Acute Lymphoblastic Leukemia, Flt3 Gene Mutation Discovery And Its Clinical Significance 2.iaspp Gene And Genome Instability Relationship. Recombinant Lentiviral Pcdh-iaspp And Its Packaging Infection Lcl Cells

Objective Deregulatedtyrosinekinaseactivity(RTK) haslong been implicated in the molecular pathogenesisof cancer,including leukemia,and mutant forms of FLT3,c-KIT, ABL,and platelet-derived growth factor receptor(PDGFR)。FMS-liketyrosinekinase-3 (FLT3),anew member of RTKⅢsubfamily,was originally identified by its expression in hematopoietic stem/progenitor cells,and its importance in normal lymphohematopoietic stem cell function is now well established.It has been reported that FLT3 receptor appears to play an important role in the pathogenesisof acute myeloid leukemia(AML),but it's rarely reported in acute Iymphoblastic leukemia(ALL) patient.We investigate theALL patients with FLT3 mutation-internal tandem duplication(FLT3-ITD) or mutation in the tyrosinekinasedomain(FLT3-TKD) and discuss its clinical significance.Methods The genomic DNA from sixty-one ALL patients was screened by polymerase chain reaction(PCR) and gel electrophoresis for FLT3/ITD and point mutation.The FLT3/ITD amplification yielded a higher molecular weight product on a 3%agarose gel stained with ethidium bromide.The point mutation amplification was digested with EcoRV.The digested products were separated on a 3%agarose gel,and undigested PCR product indicated the presence of a mutant.One of the mutants was sequenced to confirm mutation.The PCR products of those patients who were detected with FLT3-ITD or FLT3-TKD would be sent to sequence analysis.Results In these 61 ALL patients,two FLT3-ITD and oneFLT3-TKD cases were found. Sequence analysis of these three cases showed the internal tandem duplications and the tandem duplication.The sequence analysis results of the PCR products confirmed the FLT-3 mutations really exist.The sequences of two patients with FLT3/ITD~+ both are involved in the -589YFYVDFREYEY599-sequence with tyrosine residue rich.One FLT3-TKD patient is proved with a D835 mutation which is incidenced in AML patients most commonly.Analysising the clinical data of those three cases patients,we find FLT3 mutations with acute Iymphoblastic leukemia incidenced in early adolescence,the total number of leukocytes can be high and also may be lower.Except of one case who gived up the treatment,the other two patients were complete remissed after the first induction of remission.Conclusion FLT3-ITD and FLT3-TKD also could be found in ALL patients.Due to the small patients number,a common clinical feature of ALL patients with FLT3 mutation has not yet found.But untill now,it seemed the appearance of FLT3 mutation has nothing to do with the ease of treatment and prognosis. Objective:To investigate the relationship between iASPP gene and genomic instabilityMethods:iASPP-FL(iASPP-full length) and iASPP-SV(iASPP-splice variant) plasmid were transfected into MCF-7 cells.After Geneticin(G418) oppression growth,limit dilution method was used to cultrue these transfected cells to get several monoclonal cell line.By the application of reverse transcriptase PCR(RT-PCR) and western Blotting methods,we identified and picked out the stable monoclonal cell lines with empty vector,iASPP-FL and iASPP-SV gene cells,these iASPP-FL and iASPP-SV monoclonal cell line express iASPP proten stably.Respectively,radiation and Etoposide(VP16) were used to cause DNA damage to these cell lines.Flow cytometry to detect the various cell apoptosis with DNA damage.By immunohistochemistry,alkaline single cell gel electrophoresis(comet assay)and western Blotting,we observe the existence of genomic instability and the difference between these cell lines.Results:1.By flow cytometer assay,afer some strike to DNA such as radiation and Etopside,compared with the cell line which was transfected with empty plasmid,less number cells would apoptosis in these cell lines with iASPP-FL and iASPP-SV transfected.2.Afer the DNA damage,compared with the control group,immunohistochemistry and western Blotting show us a significant up-regulated H2AX protein in iASPP-FL and iASPP-SV monoclonal cell lines which means a serious DNA double strains breakage. Alkaline single cell gel electrophoresis(comet assay) also display a clearer DNA damage in iASPP-FL and iASPP-SV cell lines.Conclusion:iASPP gene could inhibit the cell apoptosis after DNA damage,compared with the wild type group,more cells with iASPP gene overexpressed could even survived in spite of these DNA damages,therefor to keep a more serious phenomenon of genomic instability.