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CORRESPONDENCE Recently, Yagi et al1 reported on expression of
Ikaros isoforms in patients with childhood acute myeloid leukemia
(AML). Ikaros expression was assessed by nested polymerase chain
reaction (PCR) and immunoblotting. The authors found that
Ikaros isoform 6 (Ik-6) was detected in 7 of 10 cases of M4 and M5, but
in none of the remaining FAB (French-American-British)
subtypes. They conclude that the pathogenesis of
myelomonocytic/monocytic AML may involve aberrant regulation of
apoptosis by Bcl-XL up-regulation due to unscheduled expression of
Ik-6. Over the past several years, there has been a controversy regarding the
expression of Ikaros isoforms in human leukemia. Sun et al reported
that leukemic cells from infants with B-cell acute lymphoblastic
leukemia (ALL) expressed dominant-negative Ikaros isoforms Ik-4, Ik-7,
Ik-8, and their deletion mutants.2 They also reported
similar observations with childhood T-cell ALL3 and
childhood ALL4 using reverse transcriptase (RT)
PCR and immunoblotting. Contrary to their reports, we demonstrated
overexpression of dominant-negative Ikaros isoform Ik-6 in patients
with blast crisis of chronic myelogenous leukemia (CML)5
and adult B-cell ALL6 using similar methods. Recently,
Payne et al reported that Ik-4, Ik-7, Ik-8, and their deletion mutants,
previously linked to leukemia by Sun et al,2-4 are
expressed in normal human cells.7 As I have already raised
a technical concern over immunoblotting of the articles of Sun et
al,8 I would like to ask a question about RT-PCR
in the article of Yagi et al. We extensively examined expression of Ikaros isoforms in patients with
human hematologic malignancies and could not find any overexpression of
dominant-negative isoforms in patients with AML, including 6 cases of
M4 and 5 cases of M5 (T. Tabayashi, F. I., unpublished data,
February 2002). Is the discrepancy between our observation and
the interpretation of Yagi et al coming from the difference in
patients' age (adult vs children)? I realized that Yagi et al were
using nested RT-PCR for detection of Ikaros isoforms, although we are
using just single RT-PCR. In general, PCR amplifies shorter products
more efficiently than longer ones. I am afraid that with nested RT-PCR,
by amplifying Ikaros isoforms not proportionately, we might get more
shorter PCR products than expected. In case we could detect Ikaros
expression by immunoblotting, we should easily amplify the products by
single PCR. As shown in Table 1, Sun et
al also used nested RT-PCR. The authors need to show the results of the
first-round PCR to avoid skewed amplification of shorter PCR products,
since the relative amount of dominant-negative isoform to full-length
isoform is important to discuss the pathogenesis.
Fumihiko Ishimaru
References
1.
Yagi T, Hibi S, Takanashi M, et al.
High frequency of Ikaros isoform 6 expression in acute myelomonocytic and monocytic leukemias: implications for up-regulation of the antiapoptotic protein Bcl-XL in leukemogenesis.
Blood.
2002;99:1350-1355
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.
Proc Natl Acad Sci U S A.
1999;96:680-685
3.
Sun L, Crotty ML, Sensel M, et al.
Expression of dominant-negative Ikaros isoforms in T-cell acute lymphoblastic leukemia.
Clin Cancer Res.
1999;5:2112-2120
4.
Sun L, Goodman PA, Wood CM, et al.
Expression of aberrantly spliced oncogenic Ikaros isoforms in childhood acute lymphoblastic leukemia.
J Clin Oncol.
1999;17:3753-3766
5.
Nakayama H, Ishimaru F, Avitahl N, et al.
Decreases in Ikaros activity correlate with blast crisis in patients with chronic myelogenous leukemia.
Cancer Res.
1999;59:3931-3934
6.
Nakase K, Ishimaru F, Avitahl N, et al.
Dominant negative isoform of the Ikaros gene in patients with adult B-cell acute lymphoblastic leukemia.
Cancer Res.
2000;60:4062-4065
7.
Payne KJ, Nicolas JH, Zhu JY, et al.
Predominant expression of a novel Ikaros isoform in normal human hemopoiesis.
J Immunol.
2001;167:1867-1870
8.
Ishimaru F.
Expression of Ikaros isoforms in acute lymphoblastic leukemia cell lines.
J Clin Oncol.
2000;18:1395-1397
Response:The expression of Ikaros in childhood AMLWe thank Dr Ishimaru for his remarks about our
article.1 As described, we determined the expression of
Ikaros dominant-negative isoform 6 (Ik6) with use of first-round as
well as nested reverse transcriptase polymerase chain reaction
(RT-PCR). As a result, we detected Ik6 expression in 7 of 10 cases of
M4 and M5 leukemia, but in none of the remaining
French-American-British subtypes. Although only nested RT-PCR
results have been shown in the article,1 in 5 of these 7 samples, first-round RT-PCR clearly detected Ik6 (4 of these 5 shown in
Figure 1, upper panel). Accordingly, our observation was not limited to nested RT-PCR. In Figure 1, bands of Ik6
appeared to be denser than that of Ik1 or Ik2 in the Ik6-positive cases. However, for precisely quantifying the isoforms in these leukemic specimens, more meticulous procedures may be necessary. Because Dr Ishimaru and his colleagues could not detect Ik6 by single
RT-PCR in adult acute myeloid leukemia (AML) cases, they raised the
question whether there is a difference between adult and pediatric AML.
Unfortunately, we do not have any data on adult de novo AML for
comparison. Contrary to the report by Payne et al,2 we
detected none of the dominant-negative Ik isoforms including Ik6 with
either first-round or nested RT-PCR in healthy human controls.
Since details of the healthy controls were not shown in the
article,1 we present here the data, where we tested fractionated CD4+, CD8+, CD14+,
CD19+ subsets in peripheral blood and placenta cDNA
(CLONTECH Laboratories, Palo Alto, CA) (Figure 1, lower
panel).
Another point Dr Ishimaru raised was the report of Sun et al.3 In cases of infant leukemia, Sun et al3 reported a high incidence of detectable mutant Ik isoforms by nested RT-PCR. We found, however, that none of the dominant-negative Ik isoforms including Ik6 could be detected even with nested RT-PCR (M.T. et al, unpublished observation, 2000), indicating that there are yet-to-be-clarified differences aside from PCR technical issues that Dr Ishimaru is most concerned with. In accord with other studies in Japan,4,5 we demonstrated that among childhood lymphoid leukemia, Ik 6 was detectable in 26.3% of B-precursor acute lymphoblastic leukemia (ALL) with first-round RT-PCR and Western blot.6 Finally, in our article,1 we clearly showed that the pathogenesis of myelomonocytic/monocytic AML may involve aberrant regulation of apoptosis due to unscheduled expression of the Ik6 isoform.
Tomohito Yagi, Shigeyoshi Hibi, Mami Takanashi, Gen Kano, Yasuhiro Tabata, Toshihiko Imamura, Tohru Inaba, Akira Morimoto, Shinjiro Todo, and Shinsaku Imashuku
References
1.
Yagi T, Hibi S, Takanashi M, et al.
High frequency of Ikaros isoform 6 expression in acute myelomonocytic and monocytic leukemias: implications for up-regulation of the antiapoptotic protein Bcl-XL in leukemogenesis.
Blood.
2002;99:1350-1355
2.
Payne KJ, Nicolas JH, Zhu JY, et al.
Cutting edge: predominant expression of a novel Ikaros isoform in normal human hemopoiesis.
J Immunol.
2001;167:1867-1870
3.
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.
1999;96:680-685
4.
Nakase K, Ishimaru F, Avitahl N, et al.
Dominant negative isoform of the Ikaros gene in patients with adult B-cell acute lymphoblastic leukemia.
Cancer Res.
2000;60:4062-4065 5. Nishii K, Katayama N, Usui E, et al. Expression of non-functional Ikaros gene in acute lymphocytic leukemia [abstract]. Jpn J Clin Hematol. 2000;41:1020. 6. Takanashi M, Yagi T, Imamura T, et al. Expression of the Ikaros gene family in childhood acute lymphoblastic leukemia. Br J Haematol. 2002;17:525-530.   CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
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