DNA Methylation In Chronic Myeloid Leukemia-Blastic Crisis(CML-BC) And A Preliminary Study On The Establishment Of Transplanted Human Chronic Myeloid Leukemia Model In Nude Mice

1. DNA methylation in Chronic Myeloid Leukemia-Blastic Crisis(CML-BC)Objective Investigate the overall methylation status of chronic myeloid leukemiablast crisis(CML-BC) and the correlation with DNA methylatransferase. Observe theinfluence of growth state after decitabine acting on K562cell line. Thus, it could providethe basis for further study methylation status on chronic myeloid leukemia blast crisis invitro. Method1) Collect four paired patients’ bone marrow specimens(That is the samepatient with chronic and acute phase specimens) in the First Affiliated Hospital ofSoochow University, extract of genomic DNA, dectect the methylation status byMspⅠand HapⅡ double enzyme digestion. In addtion, collect newly diagnose CMLchronic phase (CML-CP,15cases) and CML-BC(13cases), extract of total mRNA, anddetect the DNA methyltransferases expression level by quantitive real-timePCR(QRT-PCR), with iron deficiency anemia (10cases) as the control.2) A total of81patients with CML-BC were diagnosed at the Jiangsu Institute of Hematology betweenJanuary2003and December2010and enrolled into squencing of DNMT3A coding exons.3) Put dicitabine into logarithmic phase K562cells, and make sure that the finaloncentration was2μmol/L. And then, we could observe the cell proliferation by0.4%Taiwan phenol blue staining, colony-forming ability by methyl cellulose semi-solidmedium, and determine the cell cycle and apoptosis by flow cytometry, detect theexpression level of DNA methyltransferase enzyme(DNMTs, including DNMT1,DNMT3Aand DNMT3B) by QRT-PCR and western bloting. The K562cells that didn’t be treatedwith dicitabine was used as the control. Result1) MspⅠand HapⅡdouble enzyme digestion suggested that it was at high methylation status both in CML-CP and CML-BC,but there was no obvious difference between CP and BC. Compared with the CML-CP andCML-BC, the expression of DNA methyltranses(DNMTs) in CML-CP was lower than inCML-BC(P＜0.005), and both of them were significiantly higher than Iron DeficiencyAnemia(IDA)(P＜0.05).2) We found none of DNMT3A mutations in CML-BC.3)Compared to the control group, the proferation of exprimental group was degraded, cellgrowth inhibition was increased, colony forming ability decresed, cell cycle was arrested inG2/M phase, apototic rate increased significantly. Whether it was at mRNA or protein level,the expression of DNMT1,DNMT3A,DNMT3B did not decrease. On the contrary, thelevel of DNMTs incresed in exprimental group(P＜0.005). Conclusion1)Hypermethylation invovled in the occurence and progress of CML, but there was notcorrelated with mutation of DNMT3A.2) Although decitabine could influence the cellproliferation, cell cycle and cause cell apototic, it might not play a role by reducing thelevel of DNMTs. Therefore,the mechasim needs to further study.2. A preliminary study on the establishment of transplanted human chronic myeloidleukemia model in nude miceObjective Chronic myeloid leukemia (CML) is a malignant clonal disease derivedfrom hematopoietic stem cells. CML stem cells was thought to be the root which couldlead disease development and ultimately rapid change. However, a stable animal model forstudy the characteristics of CML stem cells is currently lacking. In our present study, wehave tried to establish a transplanted human CML nude-mice model，for further exploringthe biological behavior of CML stem cells in vivo. This model may also be used to enrichCML stem cells in vivo in nude mice by series transplantation. Methods The4-6weeksold BALB/c nu/nu mice pretreated by splenectomy(S), cytoxan intraperitonealinjection(C)and sublethal irradiation(I), were transplanted intravenously with （5-7）×10~7of human CML bone marrow mononuclear cells.2）、Alternatively,4-6weeks old BALB/c nu/nu mice pretreated by lethal irradiation, were transplanted intravenously with5×10~6homologous bone marrow cells of BALB/c nu/nu mice together with（5-7）×10~7of human CML borrow mononuclear cells simultaneously. The leukemic cells engrafted andinfiltrated in organs and bone marrow of the mice were tracked by reversetranscription-polymerase chain reaction (RT-PCR), plastic-embedded biopsy and flowcytometry. Results The result showed that human CML cells engrafted and infiltrating inthe bone marrow of two nude mice which were pretreated with SCI. In spite of the lowsuccessful rate, our preliminary results suggested the feasibility of this method by usingBALB/c nu/nu mice as a human CML animal model. Conclusion It is concluded thathuman CML bone marrow cells could develop leukemia in null mice which werepretreated by SCI. We thus provide a new strategy for establishment of CML animalmodels which deserves further elaboration.