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Blood, 15 January 2001, Vol. 97, No. 2, pp. 578-579
CORRESPONDENCE
To the editor:
Human A1 expression in acute myeloid leukemia and
its relationship to Bcl-2 expression
Konopleva et al1 describe the
expression of Bcl-2 in 2 acute myeloid leukemia (AML) cell
lines, HL-60 and HL-60-doxorubicin-resistant, and in fresh AML cells
obtained from patients with newly diagnosed or recurrent AML. The focus
of their study was to investigate the effect of liposomal Bcl-2
antisense oligonucleotides (Bcl-2-ASs) on proliferation and
chemotherapy sensitivity of these AML cells. Indeed, the authors report
convincingly that Bcl-2-ASs reduce Bcl-2 protein levels and increase
cytosine-arabinoside cytotoxicity in both HL-60 cell lines and in 11 of
19 primary AML samples. On the other hand, the data concerning the levels of human A1 protein
are less convincing for at least 2 reasons. First, in "Materials and methods" under "Western blot analysis,"
the authors cite references related to the use of polyclonal antibodies
to A1 which in fact do not have any data on A1. Second, the authors show results on the protein expression for Bcl-XL, Bag-1,
Mcl-1, Bax, Bad, Bak, and A1 (in Table 4), but an example of Western blot analysis for A1 protein was strikingly missing (in Figure 12). We have been studying the role of human A1 in acute leukemia for the
last 3 years.2-4 We have used mainly Northern blot
analysis because of the unavailability of a human A1 antibody. We
tested several batches of a polyclonal antibody marketed by Santa Cruz Biotechnology (Santa Cruz, CA) but without success. Thus it would be
important for Konopleva et al to provide the proper source for the A1
antibody that they used and the Western blot evidence for their ability
to detect the human A1 protein. Using Northern blot analysis, we have shown that A1 mRNA expression is
upregulated with myelomonocytic differentiation.2 Exposure
to bleomycin at 1 µg/mL induces myeloid differentiation in the U937
leukemic cell line (increased granularity and CD11b expression)5 and upregulates A1 mRNA expression
(Figure 1) in wild-type (WT) cells and in control
cells transfected with a pLXSN retroviral vector (lanes 2-4 and 8-10, respectively). But in U937 cells transduced with pLXSN containing the
murine Bcl-2 cDNA, the untreated and the bleomycin-treated
cells lack A1 mRNA expression (lanes 5-7). These results
suggest a regulatory relationship between Bcl-2
overexpression and A1 downregulation.
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| Figure 1.
Northern analysis for human A1 mRNA
expression.
WT U937 cells or U937 cells transfected with pLSXN retroviral
vector or pLXSN containing Bcl-2 in the sense orientation were exposed
to 1 µg/mL bleomycin, which induces myeloid differentiation in these
cells (increased granularity and CD11b expression), and assayed for
the expression of A1 mRNA. Approximately 20 µg total RNA
was loaded in each lane. IL-1-treated A549 lung carcinoma cell line
was included (lane 1) for positive control. Other experimental groups
included WT U937 cells that were either untreated or treated with
bleomycin for 2 and 5 days (lanes 2, 3, and 4, respectively); U937
cells expressing Bcl-2 that were either untreated or treated
with bleomycin for 2 and 5 days (lanes 5, 6, and 7, respectively); and
vector-transfected U937 cells that were either untreated or treated
with bleomycin for 2 and 5 days (lanes 8, 9, and 10, respectively).
Northern blot was hybridized to a randomly primed
32P-labeled A1 cDNA. Autoradiograph was developed after 6 days exposure to x-ray film using 2 intensifying screens at  80°.
Photograph of the corresponding ethidium bromide-stained gel is shown
below the autoradiograph for loading control.
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In summary, although Konopleva et al show that Bcl-2 plays a central
role in the proliferation and drug resistance of AML, the nature of its
interaction with other antiapoptotic proteins, including A1, remains
largely unknown.
Jan S. Moreb and James Zucali
Division of Hematology/Oncology University of Florida
College of Medicine Gainesville, FL
References
1.
Konopleva M, Tan AM, Estrov Z, et al.
Liposomal Bcl-2 antisense oligonucleotides enhance proliferation, sensitize acute meyloid leukemia to cytosine-arabinoside, and induce apoptosis independent of other antiapoptotic proteins.
Blood.
2000;95:3929-3938[Abstract/Free Full Text].
2.
Moreb JS, Schweder M.
Human A1, a Bcl-2 related gene, is induced in leukemic cells by cytokines as well as differentiating factors.
Leukemia.
1997;11:998-1004[CrossRef][Medline]
[Order article via Infotrieve].
3.
Moreb JS, Schweder M.
Human A1, a Bcl-2 related gene, protects against 4-hydroperoxycyclophosphamide (4-HC) toxicity [abstract].
Blood.
1997;90(suppl 1, part 2):169b.
4.
Moreb JS, Perry NAD, Pollock B, et al.
Lack of expression of human A1 mRNA in acute leukemic patients correlates with good prognostic features [abstract].
Proc Amer Assoc Cancer Res.
2000;41:757.
5.
Guedez L, Zucali J.
Bleomycin-induced differentiation of Bcl-2-transfected U937 leukemia cells.
Cell Growth Differ.
1996;7:1625-1631[Abstract].
Response:
Human A1 expression in acute myeloid leukemia
We thank Drs Moreb and Zucali for their comments on our
report concerning the effects of Bcl-2 antisense
oligonucleotides (Bcl-2-ASs) on proliferation, apoptosis, and
sensitivity to Ara-C in primary acute myeloid leukemia (AML) and
myeloid leukemia cell lines.1 We further stated that
apoptosis was induced by Bcl-2-ASs although other antiapoptotic
proteins were expressed, thus establishing Bcl-2-ASs as a critical
target for AS strategies in AML. While Drs Moreb and Zucali agree with these findings, they find the
data on one of the antiapoptotic proteins, A1, "less convincing." The Bcl-2 family member A1/Bfl-1 was cloned by Choi et al2 and by Karsan et al.3 It was found to be expressed in bone marrow cells and is induced by cytokines and differentiation inducers in leukemic cells.4 Drs Moreb and Zucali state that they "used mainly Northern blot
analysis because of the unavailability of a human A1 antibody." The
A1 antibody used in our study was kindly provided by Dr John C. Reed.
It generated immunoblots of better quality than those obtained with the
polyclonal antibody marketed by Santa Cruz. But a paper has recently
been published utilizing the Santa Cruz antibody
successfully.5 We here provide our own method of A1 detection: Cells were washed twice with phosphate buffered saline (PBS) buffer and
lysed at 4 × 104 cells/µL in cell lysis buffer (20 mM
Hepes, pH 7.4, 0.25% NP-40 containing protease inhibitor cocktail;
Boehringer Mannheim, Indianapolis, IN) for 10 minutes on ice. Lysates
were spun at 25 000g for 10 minutes, and an equal amount of
2 × gel-loading buffer (0.25 M Tris-HCl, 2% SDS, 4%
 -mercaptoethanol, 10% glycerol, 0.02% bromophenol blue) was added
to the supernatants. Equal amounts of lysate (equivalent to
5 × 105 cells) were subjected to sodium-dodecyl-sulfate
polyacrylamide-gel electrophoresis (SDS-PAGE) onto 12% polyacrylamide
gels. Afterward, proteins were transferred to Hybond-P membranes
(Amersham Pharmacia Biotech, Buckinghamshire, England). The membranes
were blocked overnight at 4°C with 7% milk in PBS buffer containing
0.3% Tween-20, followed by incubation with polyclonal antibody against
A1 (1:1000 dilution) (kindly provided by Dr J. C. Reed) for 1 hour
at room temperature. Membranes were washed 3 times with PBS buffer
containing 0.3% Tween-20, probed with a horseradish peroxydase
(HRP)-conjugated secondary antibody and then reacted with enhanced
chemiluminescence (ECL) reagent (Amersham Pharmacia Biotech). Signals
were detected by a phosphoimager (Storm 860 Version 4.0; Molecular
Dynamics, Sunnyvale, CA). Using this method, the A1 bands are clearly discernible at the correct
location, and these results are supported by the detection of A1 mRNA
by reverse-transcriptase polymerase chain reaction (RT-PCR) in
primary AML samples (Figure 1).
Oligonucleotide primers (F, forward; R, reverse) used for expression
analysis by RT-PCR were as follows: human A1- F
5'-CGGCATCATTAACTGGGGAAG-3' and R 5'-GATCTTTCCTGTAACTTCTAG-3'; the
expected PCR product is 250 base pairs (bp). Other pro- and
antiapoptotic genes are also expressed. In a series of 135 AML analyzed
for expression of A1 by semiquantitative RT-PCR, no correlation with
white blood cell count or bone marrow blast count was observed.
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| Figure 1.
A1 detection by Western blot analysis and
reverse-transcriptase polymerase chain reaction.
(A) Detection of A1 by Western blot analysis. The A1 bands are at the
correct location of 20 kd. (B) Detection of A1 mRNA by
reverse-transcriptase polymerase chain reaction (RT-PCR) in primary
AML samples.
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In conclusion, we have demonstrated the presence of the antiapoptosis
protein A1 in primary AML samples by RT-PCR and immunoblot analyses. We
agree with Drs Moreb and Zucali that the nature of the interaction
between Bcl-2 and other antiapoptotic proteins remains unknown.
Marina Konopleva, Zhong Xie, Shourong Zhao, and Michael Andreeff
University of Texas M. D. Anderson Cancer Center
Houston, TX
References
1.
Konopleva M, Tari AM, Estrov Z, et al.
Liposomal Bcl-2 antisense oligonucleotides enhance proliferation, sensitize acute myeloid leukemia to cytosine-arabinoside, and induce apoptosis independent of other antiapoptotic proteins.
Blood.
2000;95:3929-3938.
2.
Choi SS, Park IC, Yun JW, Sung YC, Hong SI, Shin HS.
A novel Bcl-2 related gene, Bfl-1, is overexpressed in stomach cancer and preferentially expressed in bone marrow.
Oncogene.
1995;11:1693-1698[Medline]
[Order article via Infotrieve].
3.
Karsan A, Yee E, Kaushansky K, Harlan JM.
Cloning of human Bcl-2 homologue: inflammatory cytokines induce human A1 in cultured endothelial cells.
Blood.
1996;87:3089-3096[Abstract/Free Full Text].
4.
Moreb JS, Schweder M.
Human A1, a Bcl-2-related gene, is induced in leukemic cells by cytokines as well as differentiating factors.
Leukemia.
1997;11:998-1004.
5.
Bellavia D, Campese AF, Alesse E, et al.
Constitutive activation of NF- B and T-cell leukemia/lymphoma in Notch3 transgenic mice.
EMBO J.
2000;19:3337-3348[CrossRef][Medline]
[Order article via Infotrieve].
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Liposomal Bcl-2 antisense oligonucleotides enhance proliferation, sensitize acute myeloid leukemia to cytosine-arabinoside, and induce apoptosis independent of other antiapoptotic proteins
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