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Expression of Ikaros Isoforms in Acute Lymphoblastic Leukemia Cell Lines

University of Okayama Okayama, Japan

To the Editor:Sun et al¹ reported the expression of aberrantly spliced oncogenic Ikaros isoforms in childhood acute lymphoblastic leukemia (ALL) in the December 1999 issue of the Journal of Clinical Oncology. Inconsistent with their previous publications,^2,3 they report that dominant-negative isoforms or mutants are expressed in ALL cell lines and leukemic cells from patients with ALL. Recently we published a similar analysis⁴; however, our results are quite different from theirs. There may be variabilities with age (child or adult), race (white or Asian), and disease (ALL or blast crisis of chronic myelogenous leukemia [CML]), although our results are consistent with those of ALL patients.⁵ I would like to raise a technical concern over the article.

Inconsistent with the previous report,⁶ our reverse transcription (RT) polymerase chain reaction (PCR) analysis showed no dominant-negative isoforms in Jurkat and Nalm6, although Ik-4 is expressed normally at a low level.⁴ However, Sun et al reported that Jurkat and Nalm6 showed dominant-negative isoforms as a major band by immunoblotting.¹ In general, RT-PCR is more sensitive than immunoblotting. We confirmed the sequence of the dominant-negative isoform (Ik-6), which was overexpressed in patients with CML blast crisis, by subcloning the PCR products.⁴ In their article, Sun et al do not show any data for RT-PCR analysis. They showed no evidence of those bands as dominant-negative isoforms (Ik-4 through -8), since they estimated those bands as dominant-negative isoforms just from their molecular weight. We argue that those bands might be just nonspecific bands, which we frequently encounter in immunoblotting even with monoclonal antibodies. To demonstrate that those bands are dominant-negative isoforms, the authors definitely need to show the RT-PCR analysis data. They should also discuss why their results are so different from those of the previous reports.^4,6

REFERENCES

1. Sun L, Goodman PA, Wood CM, et al: Expression of aberrantly spliced oncogenic Ikaros isoforms in childhood acute lymphoblastic leukemia. J Clin Oncol 17:3753-3766, 1999[Abstract/Free Full Text]

2. 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. A 96:680-685, 1999[Abstract/Free Full Text]

3. Sun L, Crotty ML, Sensel M, et al: Expression of dominant-negative Ikaros isoforms in T-cell acute lymphoblastic leukemia. Clin Cancer Res 8:2112-2120, 1999

4. Nakayama H, Ishimaru F, Avitahl N, et al: Decreases in Ikaros activity correlate with blast crisis in patients with chronic myelogenous leukemia. Cancer Res 59:3931-3934, 1999[Abstract/Free Full Text]

5. Nakase K, Ishimaru F, Sezaki N, et al: Ikaros gene inactivation in patients with adult acute lymphoblastic leukemia. Blood 94:189b, 1999 (abstr)

6. Molnar A, Wu P, Largespada DA, et al: The Ikaros gene encodes a family of lymphocyte-restricted zinc finger DNA binding proteins, highly conserved in human and mouse. J Immunol 156:585-592, 1996[Abstract]

Response

CCG Biology Reference Laboratory Parker Hughes Institute St Paul, MN

In 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

View larger version (45K): [in this window] [in a new window]   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.

1. Nakayama H, Ishimaru F, Avitahl N, et al: Decreases in Ikaros activity correlate with blast crisis in patients with chronic myelogenous leukemia. Cancer Res 59:3931-3934, 1999

2. Sun L, Lue A, Georgopoulos K: Zinc finger-mediated protein interactions modulate Ikaros activity: A molecular control of lymphocyte development. EMBO J 15:5358-5369, 1996[Medline]

3. 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)

4. Nakase K, Ishimaru F, Sezaki N, et al: Ikaros gene inactivation in patients with adult acute lymphoblastic leukemia. Blood 94:189b, 1999 (abstr)

5. 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. A 96:680-685, 1999

6. Sun L, Crotty ML, Sensel M, et al: Expression of dominant-negative Ikaros isoforms in T-cell acute lymphoblastic leukemia. Clin Cancer Res 8:2112-2120, 1999

7. Sun L, Goodman PA, Wood CM, et al: Expression of aberrantly spliced oncogenic Ikaros isoforms in childhood acute lymphoblastic leukemia. J Clin Oncol 17:3753-3766, 1999

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