A new myeloblastic leukemia cell line with double minute chromosomes. Induction of methotrexate resistance and dihydrofolate reductase gene amplification
ABSTRACT To test the relationship between DMs and drug resistance in newly established AML cell lines, KY821, and its clone KY821A3, the latter had lost DMs during cloning, were cultured in increasing concentrations of MTX, KY821 became resistant against 2 × 10−4 M MTX, whereas KY821A3 did against 2 × 10−5 M MTX in a same period. Enhanced enzyme activities of DHFR were correspondent to the increased DMs numbers and DHFR gene amplification in both resistant clones. The amplified DHFR gene was located on DMs by in situ hybridization. These data indicated that the presence of DMs in KY821 would facilitate the acquisition of drug resistance.
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ABSTRACT: Double minute chromosomes (dmin) are cytogenetic hallmarks of amplified genes. Using spectral karyotyping (SKY) and comparative genomic hybridization (CGH), we identified the origin of amplified DNA in a leukemic cell line, KY821, that harbors numerous dmin. The SKY revealed that the DNA sequences of dmin are derived from materials of chromosome 8, and CGH showed a high degree of overrepresentation only at 8q22-24, indicating that in KY821 only chromosomal material of 8q22-24, containing MYC, is amplified in dmin. An approach combining SKY with CGH should facilitate efforts to identify novel chromosomal regions of gene amplification and contribute information about genetic lesions that underly neoplastic tumors.Journal of Human Genetics 02/1998; 43(3):187-90. DOI:10.1007/s100380050067 · 2.53 Impact Factor
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ABSTRACT: We developed an improved technique that permits simultaneous DNA and RNA quantitation by a flow cytofluorometry using 7-amino-actinomycin D (7AAD) and pyronin Y (PY), respectively. Detailed cell cycle analyses based upon the cellular DNA/RNA levels were performed using cells suspended in a buffer containing 0.004% saponin. This method preserved the light scattering properties of human peripheral blood cells, thus lymphocyte, monocyte and granulocyte populations could be evaluated. In addition, since 7AAD and PY exhibit red (> 650 nm) and orange fluorescence (570 nm) respectively, the green fluorescence channel of the flow cytometer was reserved for surface phenotyping using FITC-conjugated antibodies. The 7AAD/PY method is applicable to the simultaneous three-color analysis of the surface phenotype and DNA-RNA quantitation when combined with FITC-conjugated surface markers in heterogeneous samples. To demonstrate the three-color analysis, PHA-activated human peripheral blood lymphocytes were stained for cell surface markers with monoclonal antibodies. The cells were suspended in buffer containing 0.004% saponin, then stained with 7AAD and PY. The DNA and RNA were analyzed in indivisual CD4+, CD8+ and CD20+ cells, and the characteristic cell cycle status was found. Cell activation was further analyzed using antibodies against interleukin-2 (IL-2) receptors (CD25), transferrin receptors (CD71) or HLA-DR molecules. Transferrin receptors were expressed in late G1 phase (G1B) just before the initiation of DNA synthesis, whereas IL-2 receptors and HLA-DR were expressed very early in the G1 phase (G1T). Since this technique preserves both light scatter properties as well as cell surface proteins, it is ideally suited for detailed cell cycle analyses of heterogeneous samples such as peripheral blood or bone marrow cells.Journal of Immunological Methods 07/1995; 182(2):193-207. DOI:10.1016/0022-1759(95)00050-K · 2.01 Impact Factor
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ABSTRACT: New adriamycin (ADR) resistant human leukemic cell lines (KY-ADR1 and KY-ADR2) have been established. KY-ADR1 was selected from a cytosine arabinoside (Ara C) resistant cell line by gradually increasing the concentration of ADR and KY-ADR2 from the parental cell line, KY-821, by the same method. The IC50s of both cell lines were 4.3 x 10(-5) and 3.6 x 10(-5) M ADR, respectively. Both lines revealed a similar cross resistance to various anticancer drugs, but KY-ADR1 was resistant to Ara C, whereas KY-ADR2 was sensitive. MDR1 gene was over-expressed and P-glycoprotein was expressed on the cytoplasmic membrane in both lines. Neither verapamil nor cyclosporin A could completely reverse ADR resistance. In addition, no significant changes in topoisomerase II and glutathione-s-transferase levels were detected. These findings indicate that ADR resistance in both cell lines is mainly mediated by P-glycoprotein and some other mechanism may be present. Interestingly, growth of both cell lines was stimulated by natural IL-1 and not affected by TNF alpha and IFN gamma, whereas growth of parental KY-821 was inhibited by these factors. These cell lines will provide new biological aspects on drug resistant leukemic cells.Leukemia Research 10/1994; 18(9):709-15. DOI:10.1016/0145-2126(94)90071-X · 2.69 Impact Factor