Establishment And Characterization Of A Human Acute Monocytic Leukemic Cell Line, SHI-1, Carrying T(6;11)(q27;q23) And Having High Tumorigenicity In Nude Mice

For the last 20 years human leukemic cell lines have represented extremely useful tools for studying the mechanisms of tumorigenesis and the strategy of treatment of human leukemia and other tumors. However, the efficiency of cell line establishment is rather low, and the deliberate establishment of new leukemic cell line remains by and large an unpredictable random process. To date a few human leukemic cell lines have been reported in China. However, some of them cannot be maintained in long-term culture in vitro. And most of them are not sufficiently characterized in the cytogenetic, molecular genetic, and other biologic features. Actually some of them may be the misidentified Epstein-Barr virus-transformed B-lymphoblastoid cell lines (EBV+ B-LCL). Today almost all the available leukemic cell lines in China are derived from foreign laboratories. We wish to establish a novel human acute myeloid leukemia (AML) cell line carrying specific chromosomal abnormality by culturing the primary leukemic cells from numerous AML patients in vitro and to provide a novel well-characterized AML cell line for the research of leukemia. MethodsThe primary leukemic cells from over 300 acute leukemic patients were cultured in vitro in last two years. And the first acute monocytic leukemic cell line of China, SHI-1, was established from the bone marrow specimen of an acute monocytic leukemia (AML-M5b) patient. In the first part, we performed comprehensive genetic characterization on the AML-M5b patient. Conventional RHG banding was employed for karyotypic analysis. Chromosome painting (CP) was performed using whole chromosome paint probes (WCPs) for chromosomes 6 and 11. Fluorescence in situ hybridization (FISH) was used to identify the rearrangement of MLL gene and thedeletion of p53 gene. The MLL-AF6 fusion transcript was studied by reverse transcriptase polymerase chain reaction (RT-PCR). The exon 5, 6, 7, and 8 of p53 gene were amplified by PCR respectively and sequenced to exclude the mutation.In the second part, the process of establishment of the SHI-1 cell line was described. And the basic biologic features of SHI-1 were characterized. The growth feature of the SHI-1 cell line in flask was studied under the inverted microscope. The ultrastructure was assayed by the transmitted electronic microscope. The cell line was routinely stained with the Wright' s staining method. Cytochemical studies were performed by a -naphthyl acetate esterase, a -naphthyl butyrate esterase, and peroxidase staining. The growth curve of the SHI-1 was obtained by incubating the cells in 24-well plate. And the doubling time was calculated from the curve. The immunoprofile and the cell cycle distribution of SHI-1 were studied by flow cytometry (FCM). RHG banding was employed for dynamic karyotypic analysis in the process of establishment of the cell line. The MLL-AF6 fusion transcript was studied by RT-PCR. The clonality was assayed by semi-solid methylcellulose clonogenic culture. Quantitative fluorescent polymerase chain reaction was used to detect EBV genomic DNA. DNA fluorochrome staining and culture of mycoplasma were performed to determine the contamination of mycoplasma. Gelatin Zymography method was used to study the expression of MMP-9 and MMP-2 in the supernatant of the SHI-1 cell line. Matrigel invasion assay was employed for the study of migration of the SHI-1 cell in vitro. CP was performed using WCPs for chromosomes 6 and 11. FISH was used to identify the rearrangement of MLL gene and deletion of p53 gene. The exon 5, 6, 7, and 8 of p53 gene were amplified by PCR and sequenced respectively. Cell line authentication was performed by short tandem repeating sequences-PCR (STR-PCR). Multiplex-FISH (M-FISH) was used to exclude cryptic chromosomal abnormalities. CP was performed to verify the results of M-FISH. The proliferative responses of the cell line to various cytokines were examined by standard 3H-thymidine incorporation assay.The multidrug resistance of the SHI-1 cell and its mechanisms were studied in the third part.