A new myeloblastic leukemia cell line with double minute chromosomes. Induction of methotrexate resistance and dihydrofolate reductase gene amplification

Department of Hygiene and Oncology, Kyoto Prefectural University of Medicine, Kyoto, Japan
Leukemia Research (Impact Factor: 2.35). 02/1992; 16(3):217-226. DOI: 10.1016/0145-2126(92)90059-G


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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    • "Cytogenetic evidence of gene amplification can be manifested as either HSR or dmin, although low-level amplification may be present without visible cytogenetic alterations (Stark et al. 1989). Although the KY821 parent cell line constitutionally exhibits dmin, previous studies using Southern and in-situ hybridization techniques yielded no evidence for amplification of either MYC or DHFR (Saito et al. 1992). It remains possible that a novel DNA target(s), containing genes associated with cell growth and differentiation, is amplified in KY821. "
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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 · 2.46 Impact Factor
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    ABSTRACT: The p53 gene is currently considered to function as a tumor-suppressor gene in various human malignancies. In hematologic malignancies, alterations in the p53 gene have been shown in some human leukemias and lymphomas. Although mutations in the p53 gene are infrequent in acute myelogenous leukemia (AML) patients, we show in this report that alterations in the p53 gene are frequent in myeloid leukemia cell lines. We studied alterations of the p53 gene in nine human myeloid leukemia cell lines by reverse transcriptase-polymerase chain reaction (RT-PCR), single-strand conformation polymorphism (SSCP) analysis, and direct sequencing. Expression of the p53 gene was not detected at all by RT-PCR in two of the nine cell lines. In these two cell lines, Southern blot analysis showed gross rearrangements and deletions in both of the p53 alleles. Six of the nine cell lines were found to express only mutant p53 mRNA by RT-PCR/SSCP analysis and direct sequencing, and wild-type p53 mRNA was not detected. Two of the mutant p53 mRNAs were shown to be products of abnormal splicing events induced by intronic point mutations. Taken together, eight of nine human myeloid leukemia cell lines expressed no or an undetectable amount of wild-type p53 mRNA. Three of the eight cell lines were growth factor-dependent. Our results suggest that inactivation of the p53 gene may be a common feature in myeloid leukemia cell lines and may play an important role in the establishment of these cell lines.
    Blood 06/1992; 79(9):2378-83. · 10.45 Impact Factor
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    ABSTRACT: Human myelomonocytic leukemic cell line, designated as KY-821, and its sublines KY-Ra, KY-VCR, and KY-MTX, which were resistant to cytosine arabinoside, vincristine, and methotrexate, respectively, were compared for response to various hematopoietic growth factors. Cells of KY-Ra and KY-VCR proliferated in response to natural interleukin-1 (nIL-1), whereas the proliferation of KY-821 and KY-MTX was inhibited. Unexpectedly, recombinant IL-1 alpha and IL-1 beta had no effect on the proliferation of each cell line. The effect of nIL-1 was partially deleted by an addition of optimal anti-IL-1. Supernatants of each cell line had no IL-1 activity. Interferon gamma (IFN gamma) and tumor necrosis factor alpha (TNF alpha) also had an inhibitory effect for KY-821 and KY-MTX, but lacked such effect in KY-RA and KY-VCR. nIL-1, IFN gamma and TNF alpha could not differentiate between any of the cell lines but IFN gamma and TNF alpha induced monocytic surface antigens. In addition, there was no difference in the number of IL-1 and TNF alpha receptors in each cell line. These results indicate that there is a difference in biological effects between nIL-1 and recombinant IL-1 species and acquirement of resistance for some types of drugs may associate closely with different responses to hematopoietic growth factors, probably through altered postmembranous transduction.
    Leukemia Research 05/1993; 17(4):325-32. DOI:10.1016/0145-2126(93)90019-H · 2.35 Impact Factor
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