SPECIAL DEPARTMENTS
Authors: Fumihiko Ishimaru, Fatih M. Uckun
Journal: Journal of Clinical Oncology (2000)

Article Tools SPECIAL DEPARTMENTS Article Tools OPTIONS & TOOLS Export Citation Track Citation Add To Favorites Rights & Permissions COMPANION ARTICLES No companion articles ARTICLE CITATION DOI: 10.1200/JCO.2000.18.6.1395 Journal of Clinical Oncology - published online before print September 21, 2016 PMID: 10715313 Fumihiko IshimaruxFumihiko IshimaruSearch for articles by this author Fatih M. UckunxFatih M. UckunSearch for articles by this author Show More University of Okayama OkayamaJapanCCG Biology Reference Laboratory Parker Hughes Institute St PaulMN https://doi.org/10.1200/JCO.2000.18.6.1395 First Page Full Text PDF Figures and Tables © 2000 by American Society of Clinical OncologyjcoJ Clin OncolJournal of Clinical OncologyJCO0732-183X1527-7755American Society of Clinical OncologyResponse13032000In Reply:Nakayama et al1 recently published an article entitled “Decreases in Ikaros Activity Correlate With Blast Crisis in Patients With Chronic Myelogenous Leukemia.” Contrary to what the title implies, the authors did not show any DNA binding or other functional data to indicate that Ikaros activity is indeed altered in leukemic cells from their patients. Furthermore, the authors reported that they found in nuclear extracts of leukemic cells from chronic myelogenous leukemia (CML) patients in blast crisis Ikaros-6,1 a small Ikaros isoform that should localize to the cytoplasm.2 The authors claimed that the identity of the isoform was confirmed by sequence analysis, but no sequence data were shown in the article.1 We could not replicate their reported findings regarding Ikaros-6 expression in leukemic cells from adult CML patients. In three patients examined, none of the 21 polymerase chain reaction (PCR) clones had the Ikaros-6 coding sequence. Recently, two abstracts claiming similar findings in adult acute lymphoblastic leukemia (ALL) patients were published by the same authors, but these abstracts were not presented at the American Society of Hematology meeting.3,4 Furthermore, these abstracts incorrectly claimed that the results we recently published in the Proceedings of the National Academy of Sciences of the United States of America5 demonstrated Ikaros gene inactivation by the dominant-negative isoforms in leukemia cells. First, it is against our policy to cite abstracts or publications that have not undergone peer review. In addition, these abstracts appeared after the publication of our articles in Clinical Cancer Research6 and the Journal of Clinical Oncology.7 We found Ikaros-6 in a single case of ALL among 59 ALL patients studied.7 We have no explanation as to why Nakayama et al apparently detected Ikaros-6 in 13 of 42 patients studied,3 but look forward to the formal presentation of their data at a scientific meeting or in a peer-reviewed publication.In his letter, Ishimaru requests that we show the PCR evidence of Ikaros-4 expression and lack of Ikaros-1 expression in NALM-6 as well as Jurkat leukemia cells. The PCR data on Jurkat cells were recently published elsewhere.6 Of 11 clones sequenced, eight had one wild-type Ikaros-4 coding sequence, one had the Ikaros-8 coding sequence, and only two had the Ikaros-2 coding sequence.6 Our PCR data for NALM-6 cells are presented in Figure 1. In contrast to what was reported by Ishimaru, NALM-6 cells maintained in our laboratory did not express a 1.5-kb PCR product corresponding to Ikaros-1, whereas normal fetal thymocytes did (Fig 1B.1). The 1.2- to 1.3-kb PCR band(s) in NALM-6 cells hybridized to both exon 4– and exon 7–specific Ikaros probes, confirming their identity as Ikaros mRNA as well as ruling out that they correspond to Ikaros-6, which does not have exon 4 (Fig 1B.2 and 1B.3). The PCR clones from NALM-6 cells with the Ikaros-4 coding sequence lacked exon 3 (Fig 1C) as well as exon 5 (Fig 1D), confirming their identity. While PCR studies are important for molecular characterization of Ikaros isoforms, we disagree with Ishimaru regarding their relevance in the absence of Western blot and confocal microscopy data. mRNA for dominant-negative Ikaros isoforms could have a potential biologic significance only if they are expressed and their expression is confirmed at the protein level. High-level mRNA expression of course does not ensure high-level protein expression. Furthermore, it is highly unlikely that the Ikaros bands we observed in Western blots of whole cell lysates from B-lineage ALL cell lines are nonspecific bands, since they were not observed in the normal bone marrow or thymocyte lysate samples analyzed side by side.7 Finally, the PCR methods used by Nakayama et al. are not quantitative and therefore cannot be used for comparing the relative expression levels of Ikaros mRNAs.1 Fig 1. Detection of Ikaros 4 transcripts in NALM-6 human pre-B leukemia cells. (A) Schematic representation of Ikaros isoforms 1, 2, 4, and 6 with specific composition of domains encoded by exons (E) 1 through 7 and the PCR primers. (B.1) Representative ethidium bromide–stained gels of the PCR products from fetal thymocytes (FT) and NALM-6 leukemia cells. (B.2) Southern blot hybridization of the PCR products shown in B.1. with an exon 7-specific probe. (B.3) Southern blot hybridization of the PCR products shown in B.1. with an exon 4-specific probe. (C & D) Cloned PCR products were subjected to automated sequencing, as described.5,6 Sequence traces spanning the junctions between exon 2 and exon 4 as well as between exon 4 and exon 6 are shown for representative PCR clones from fetal thymocytes and NALM-6 leukemia cells.6. Nakayama H, Ishimaru F, Avitahl N, et al: Decreases in Ikaros activity correlate with blast crisis in patients with chronic myelogenous leukemia. Cancer Res 59::3931,1999-3934, Medline, Google Scholar6. Sun L, Lue A, Georgopoulos K: Zinc finger-mediated protein interactions modulate Ikaros activity: A molecular control of lymphocyte development. EMBO J 15::5358,1996-5369, Crossref, Medline, Google Scholar6. Sezaki N, Ishimaru F, Nakase K, et al: The dominant-negative isoform of the Ikaros gene confers resistance to glucocorticoid-induced apoptosis in human pre-B acute lymphoblastic leukemia cell line. Blood 94::190b,,1999 (abstr) Google Scholar6. Nakase K, Ishimaru F, Sezaki N, et al: Ikaros gene inactivation in patients with adult acute lymphoblastic leukemia. Blood 94::189b,,1999 (abstr) Google Scholar6. Sun L, Heerema N, Crotty L, et al: Expression of dominant-negative and mutant isoforms of the antileukemic transcription factor Ikaros in infant acute lymphoblastic leukemia. Proc Natl Acad Sci U S A 96::680,1999-685, Crossref, Medline, Google Scholar6. Sun L, Crotty ML, Sensel M, et al: Expression of dominant-negative Ikaros isoforms in T-cell acute lymphoblastic leukemia. Clin Cancer Res 8::2112,1999-2120, Google Scholar6. Sun L, Goodman PA, Wood CM, et al: Expression of aberrantly spliced oncogenic Ikaros isoforms in childhood acute lymphoblastic leukemia. J Clin Oncol 17::3753,1999-3766, Link, Google Scholar