Study On The Expression And Mutation Of The Tumor Suppressor Gene Mxi1 In Leukemia Cells

Objective: Nowadays, there have been many studies on theexpression and mutation analysis of the tumor suppressor geneMxi1 in solid tumors. And these studies indicat that there is aclose correlation between Mxi1 mutation and the pathogenesisof solid tumors such as neurofibrosarcoma, prostatic carcinoma.But there is no study on the expression and mutation analysis ofMxi1 in leukemic cells,neither domestic nor abroad. OnlyShapiro et al reported that the Mxi1 located-region was ofteninvolved in translocations and deletions in acute and chroniclymphocytic leukemias. However, they didn't mention whetherthere was a mutation of Mxi1. The objective of the present studyis, therefor, to investigate the expression and mutation of Mxi1in leukemic cells and the relationship between thegene-mutation and the patients'prognosis.Methods: We chose 26 patients with acute leukemia (AL),30 healthy volunteers, as well as two myeloid leukemia celllines KG1 and K562 as study subject. All patients were referredto us shortly after the first symptoms occurred and untreated.Total RNA was extracted from bone marrow mononuclear cells(BMMNC) from leukemic patients and peripheral bloodmononuclear cells(PBMNC) from 30 healthy volunteers, as wellas logarithmically growing KG1 and K562 cells. Firstly, wedetected the expression level of Mxi1 mRNA with reversetranscription-polymerase chain reaction(RT-PCR) in all samples.We also compared the relative expression of Mxi1 mRNA ineach group with statistic method. Secondly, we analyzed Mxi1cDNA in SID and bHLHzip encoding domains with singlestrand conformation polymorphism(SSCP) in all samples tosearch for abnormal band mobility. Compared with the normalband mobility in the control group, the abnormal band mobilitywas involved in mutations or polymorphism of Mxi1. Thirdly,the band mobility with abnormal were excised from theacrylamide gels, and DNA fragments were eluted from it. Theeluted DNA fragment was subjected to PCR again with the sameprimers for 35 cycles.The fresh PCR product was cloned intopCR 2.1 vector, sequenced by employing an ABI PRISM DyeTerminator Cycle Sequencing Ready Reaction Kit and an ABIPRISM 310 automated DNA Sequencer. A consistent nucleotidealteration in more than three clones of the same sample wasdefined as mutation. Finally, we analysed the relationshipbetween the mutation of Mxi1 and the clinical prognosis ofleukemic patients.Result:1. All samples expressed Mxi1 mRNA . The averageexpression levels of Mxi1 mRNA(ratio of Mxi1 mRNA toβ-actin mRNA) in control and de novo AL patients were 0.923(range:0.752-1.562) and 0.531(range:0.113-0.835) respectively.The expression levels of Mxi1 mRNA decreased significantly inde novo AL patients compared with control groups (p<0.05).The average expression levels of Mxi1 mRNA in AML and ALLgroups were and 0.503(range: 0.113-0.792) and 0.623(range:0.206-0.835) respectively. Compared with ALL groups, theaverage expression level of Mxi1 mRNA in AML groupsdeclined slightly (p>0.05). The average expression levels ofMxi1 mRNA in AL patients in complete remission were0.812(range: 0.436-1.238). The expression levels of Mxi1mRNA decreased slightly in AL patients with completeremission compared with control groups (p>0.05). Theexpression levels of Mxi1 mRNA increased significantly in ALpatients in complete remission compared with de novo ALpatients (p<0.05). The relative and average expression levels ofMxi1 mRNA in K562 and KG1 cells were 0.583-0.635 and0.613;0.579-0.641 and 0.628 respectively. The expressionlevels of Mxi1 mRNA in K562 and KG1 cells wereremarkablely lower than those in control groups (p<0.05), buthad no statistical significance compared with AL groups(p>0.05).2. We analysed the Mxi1 cDNA with SSCP in all samples.The SID and bHLHzip domains were essential for the functionsof Mxil protein. We therefore focused on searching formutations in SID and bHLHzip encoding domains. SSCPanalysis showed only one pattern of band mobility of SIDdomain in Mxil gene from all samples. No abnormal SSCPband was detected in the SID domain of all samples, suggestingthat neither mutations nor polymorphism existed in this region.However, there were different patterns of band mobility inbHLHzip domain of Mxi1. Same pattens of abnormal bandmobility were shown in KG-1 cell, BMMNC from patients 1, 5,17, 18 and 23, and PBMNC from healthy volunteers 2 and 14.Different pattens of abnormal band mobility presented inpatients 1, 5 and 23. And they appeared between SID and basicdomains in patients 1 and 23, while at bHL domain in patient 5.3. We detected gene sequence of all band with abnormalmobility. We found a GC base order at 390-391 in Mxil, thatcaused an amino acid substitution from Thr to Ser at position 61in all sequenced samples including KG-1 cells, BMMNC frompatients 1, 5, 17, 18 and 23, and PBMNC from healthyvolunteers 2 and 14. This GC base order was consistent with thesequence reported by Shimizu (Genebank accession No.D63940). But Zervos et al reported a CG base order at 390-391in Mxi1 and Thr at position 61. This demonstrated that thetransformation of base order at 390-391 was sequencepolymorphisms. We detected missense mutations in 3 patientsand KG-1cell: 3 in the region between SID and basic motif ofMxil (A362G, G384A and A375G), one in helix I of Mxil(C452T). These four missense mutations were not detectable innormal PBMNC, indicating that these mutations did notrepresent sequence polymorphisms.4.The comparison analysis between the mutation of Mxi1...