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/EBPα gene
✍ Scribed by Hiroya Asou; Adrian F. Gombart; Seisho Takeuchi; Hideo Tanaka; Maki Tanioka; Hirotaka Matsui; Akiro Kimura; Toshiya Inaba; H. Phillip Koeffler
- Publisher
- John Wiley and Sons
- Year
- 2002
- Tongue
- English
- Weight
- 229 KB
- Volume
- 36
- Category
- Article
- ISSN
- 1045-2257
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✦ Synopsis
Abstract
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/EBPα), a critical myeloid transcriptional factor. The C to G transition at nucleotide 1063 of the C/EBPα 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/EBPα protein. The Kasumi‐6 cells expressed both the p42 and p30 isoforms of the C/EBPα protein endogenously, but electrophoretic mobility shift assays demonstrated an absence of C/EBPα binding to its respective site. Exogenous expression of the mutant form of C/EBPα 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/EBPα 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/EBPα in leukemogenesis. © 2003 Wiley‐Liss, Inc.