Establishment of the acute myeloid leukemia cell line Kasumi-6 from a patient with a dominant-negative mutation in the DNA-binding region of the C/EBPalpha gene.
A myeloid leukemia cell line designated Kasumi-6 was established from the bone marrow cells of an individual with acute myeloid leukemia, subtype M2. Both the original leukemic cells and the Kasumi-6 cell line harbor a hemizygous point mutation in the gene encoding the CCAAT/enhancer binding protein alpha (C/EBPalpha), a critical myeloid transcriptional factor. The C to G transition at nucleotide 1063 of the C/EBPalpha gene results in amino acid transition R305P in the fork or hinge region between the DNA-binding basic region and the leucine zipper dimerization domain of the C/EBPalpha protein. The Kasumi-6 cells expressed both the p42 and p30 isoforms of the C/EBPalpha protein endogenously, but electrophoretic mobility shift assays demonstrated an absence of C/EBPalpha binding to its respective site. Exogenous expression of the mutant form of C/EBPalpha demonstrated that it was unable to bind DNA and activate transcription from a G-CSF receptor-luciferase reporter construct. Furthermore, coexpression of the wild-type and mutant forms revealed that the mutant form repressed reporter gene activation by the wild type in a dose-responsive manner. This was concomitant with a dose-responsive decrease in wild-type protein binding to the G-CSF receptor C/EBP site. The data suggest that the R305P alteration confers a dominant-negative property on the mutant C/EBPalpha protein whereby the mutant polypeptide heterodimerizes with the wild-type polypeptide and prevents it from binding to DNA, thus blocking transcriptional activation. The Kasumi-6 cell line can serve as a model to study the cellular and molecular biology of the non-t(8;21) M2 type of myeloid leukemia and can elucidate the role of mutated C/EBPalpha in leukemogenesis.
Asou
Hiroya
H
Department of Molecular Oncology, Research Institute for Radiation Biology & Medicine, Hiroshima University, Hiroshima, Japan. asobu@hiroshima-u.ac.jp
Gombart
Adrian F
AF
Takeuchi
Seisho
S
Tanaka
Hideo
H
Tanioka
Maki
M
Matsui
Hirotaka
H
Kimura
Akiro
A
Inaba
Toshiya
T
Koeffler
H Phillip
HP
D016475:3T3 Cells / Q000378:metabolism; D000818:Animals; D001854:Bone Marrow Cells / Q000378:metabolism; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000187:drug effects; D005786:Gene Expression Regulation / Q000235:genetics; D005799:Genes, Dominant / Q000235:genetics*; D018922:HL-60 Cells / Q000378:metabolism; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000235:genetics* / Q000378:metabolism; D008297:Male; D051379:Mice; D008875:Middle Aged; D009364:Neoplasm Recurrence, Local; D017354:Point Mutation / Q000235:genetics*; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured; D020298:U937 Cells / Q000378:metabolism
D022763:CCAAT-Enhancer-Binding Protein-alpha