Establishment And Characterization Of A Human Acute Myelocytic Leukemic Cell Line, SH-2, Carrying Non-balanced T(16;17) (q24;q12) Chromosomal Translocation

Objective To establish a novel human acute myelocyte leukemia cell line SH-2 with non-balanced t(16;17)(q24;q12) translocation and analysed its biologic characteristics.Methods1.The cell line SH-2 was established from an acute myelocyte leukemia (AML-M2a) patient.The patient was male,32 years old.He was refractory to multiple chemotherapentants and died of leukemia relapsed one month after haploidentical bone marrow transplantation.2.Mononuclear cells were isolated from the bone marrow,inoculated by IMDM medium containing 20%FBS,and cultured in flask at 37C in humidified air with 5%CO₂.2/3 or 1/2 medium was changed every 3～4 days,stromal cells were found under the botton and co-cultured with flooting cells,in the culture system rh-IL-3 10ug/ml was added when the cells growth slowly one month later.The cells was cultured without rh-IL-3 three months later when its speed of proliferation was acceleeated.To date the cells was cultured 22 months in vitro,named as SH-2 which can resistant to cryopreserved and thawing.3.The morphology of cell line was analyzed by Wright-Gimesa-staining and cytochemical staining.4.Electronic microscope was used to analysis the ultrastructure of cells5.The immunoprofile was studied by flow cytometry(FCM).6.RHG banding was employed for karyotypic analysis.7.FISH was used to study the PML/RARa and RARa gene,p53 gene and the translocation between chromosomes 16 and 17. 8.The clonality was assayed by semi-liquid methylcellulose clonogeneic culture.9.Quantitative fluorescent polymerase chain reaction was used to detect EBV DNA,mycoplasma contamination was determined by nested RT- polymerase chain reaction.10.Effect of cytokines on cell line was analysed.11.Cell line authentication was performed by short tandem repeating sequences (STR).12.P53 mutation was checked by analysis of the sequence of its extron 5,6,7 and 8.13.expression of multiple multidrug resistant related proteins MDR1 was analysed by RT-PCR and FCM.14.Transplantation into immunodeficient mice.15.A series of BAC/PAC clones were mapped to 16q and 17q to identify the breakpoints of t(16;17).DNA was isolated and nick translated,FISH mapping was performed using one probe from a set of BAC/PAC clones along with the centromeric probe for chromosome 16.ResultsThe cell line has proliferated continuously in vitro for 22 months.Its morphology showed typical features of myelocyte.The cell line's immunoprofile was accordant with the origin of myelocyte:positive for CD13,CD33,and MPO,CD16/56 and CD56 expression were found on SH-2 cells.Karyotypic analysis revealed non-balanced translocation t(16;17)(q24;q12),loss of chromosome 17 and trisomy 19.The PML-RARa fusion transcription was not detected by FISH.The loss of p53 gene and translocation between chromosomes 16 and 17 were conformed by FISH method.SH-2 cells can form leukemia clones on methylcellulose semisolid culture.Infection of the EBV and the mycoplasma were also excluded.Cytokines of GM-CSF,IL-3,IL-6,SCF,IL-4 and IL-5 can improve the proliferation of SH-2.Cell line authentication by STR showed that the SH-2 cell line originate from the primary cells of the patient.P53 mutation of extron 5 was found by analysis of its sequence of extron 5:a point mutation of CAG→CAT.MDR1 expression was not found by RT-PCR and FCM.The tumorigenicity of the SH-2 cell line was also confirmed.The breakpoints of chromosomes 16 and 17 were localized RP11-356C4(16q24.3) and between BAC clone RP1-161P9 and RP11-47L3 corresponding to 17q12.ConclusionWe established a novel human myelocyte leukemic cell line SH-2 with non-balanced t(16;17)(q24;q12) which was a novel rare translocation in acute myelocyte leukemia and may predict a poor prognosis.SH-2 cells expressed mylocytic and NK related antigens.The karyotype of SH-2 was 45,X,-Y,der(16) t(16;17),-17,+19 at first which evolved into the polyploidy near tetraploidy with two non-balanced 16;17 translocation 22 months after passage cultures.Through a series of FISH,the precise localization of breakpoints for derivation chromosome 16 was mapped to 16q24.3(in or below RP11-356C4) and 17q12 between BAC clone RP1-161P9 and RP11-47L3,respectively.Cytokines of GM-CSF,IL-3,IL-6,SCF, IL-4 and IL-5 can improve the proliferation of SH-2.The clonality and the tumorigenicity of the SH-2 cell line were confirmed.Infection of the EBV and the mycoplasma were also excluded,extron 5 of p53 existed mutation,cell line originated from the primary cells of the patient.Our study provides SH-2,a useful tool with a clear biology background for the study of leukemia origin and also settled the base for cloning t(16;17)(q24;q12) fusion gene.