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HEMATOPOIESIS
From the Department of Molecular and Experimental
Medicine, The Scripps Research Institute, La Jolla, CA; and Department
of Medicine, Harvard Medical School, Boston, MA.
As reported previously, AML1-ETO knock-in mice were generated to
investigate the role of AML1-ETO in leukemogenesis and to mimic the
progression of t(8;21) leukemia. These knock-in mice died in
midgestation because of hemorrhaging in the central nervous system and
a block of definitive hematopoiesis during embryogenesis. Therefore,
they are not a good model system for the development of acute myeloid
leukemia. Therefore, mice were generated in which the expression of
AML1-ETO is under the control of a tetracycline-inducible system.
Multiple lines of transgenic mice have been produced with the AML1-ETO
complementary DNA controlled by a tetracycline-responsive element. In
the absence of the antibiotic tetracycline, AML1-ETO is strongly
expressed in the bone marrow of AML1-ETO and tet-controlled transcriptional activator double-positive transgenic mice. Furthermore, the addition of tetracycline reduces AML1-ETO expression in
double-positive mice to nondetectable levels. Throughout the normal
murine lifespan of 24 months, mice expressing AML1-ETO have not
developed leukemia. In spite of this, abnormal maturation and
proliferation of progenitor cells have been observed from these
animals. These results demonstrate that AML1-ETO has a very restricted
capacity to transform cells. Either the introduction of additional
genetic changes or the expression of AML1-ETO at a particular stage of
hematopoietic cell differentiation will be necessary to develop a model
for studying the pathogenesis of t(8;21).
(Blood. 2000;96:2108-2115) Acute myeloid leukemia (AML) is a major
hematopoietic malignancy characterized by the proliferation of a
malignant clone of myeloid progenitor cells. One of the most common
targets of translocations that have been implicated in AML is the
AML1 gene.1-3 The AML1 gene, also
known as RUNX1, encodes a novel transcription factor that
forms a complex with core binding factor To study the effect of AML1-ETO on hematopoiesis, we previously
generated mice in which wild-type AML1 was replaced by
AML1-ETO by using a knock-in strategy.12 These
mice recapitulate human t(8;21) by substituting the fusion gene for the
wild-type allele. In these studies, a block in definitive hematopoiesis
was seen in the heterozygous AML1+/AML1-ETO+
embryos. These embryos died in midgestation because of severe hemorrhaging in the central nervous system. The resulting phenotype in
these mice was similar to the phenotypes seen in AML1 and
CBF We report here the development of unique lines of transgenic mice that
express AML1-ETO under the control of a tetracycline-responsive element Plasmid construction and generation of transgenic mice
pUHD-AML1/ETO was digested with DraI and EcoRV. A
3-kilobase (kb) band containing the AML1-ETO cDNA and the
tet-responsive promoter was isolated and used for microinjection.
AML1-ETO transgenic mice were produced in the transgenic facility of
Beth Israel Deaconess Medical Center (Boston, MA) by using inbred FVB zygotes.
Transgenic mice containing the murine mammary tumor
virus-tet-controlled transcriptional activator (MMTV-tTA) construct
were obtained from Dr L. Hennighausen (National Institutes of Health, Bethesda, MD).22 These mice were bred with our
pUHD-AML1/ETO mice to generate double-positive mice with inducible
AML1-ETO expression.
Administration of tetracycline to transgenic mice
Southern blot analysis Tail DNA samples prepared as previously described23 were digested with EcoRI, electrophoresed in a 0.8% agarose gel, and transferred to positively charged nylon membrane (Biotrans plus, ICN, Costa Mesa, CA). ETO cDNA (1.7-kb KpnI/XbaI DNA fragment from pKS-ETO) and the tTA-VP16 DNA (1-kb BamHI/EcoRI DNA fragment from pUHD15-1) were radiolabeled with 32P-dCTP and used as probes to hybridize with the genomic tail DNA samples to test for the presence of the 2 transgene constructs.Isolation of RNA and Northern blot analysis Peritoneal macrophages were harvested from the abdominal cavities of mice 48 to 72 hours after the intraperitoneal injection of thioglycollate (0.1 g/mL, 1.5 mL/mouse). Total RNA was extracted from these macrophages, various tissues from the injected mice, and cell lines by guanidine isothiocyanate extraction were followed by purification on a cesium chloride gradient. Total RNA (10 µg) for each sample was electrophoresed in a 1% agarose gel containing 0.22 mol/L formaldehyde. The RNA was transferred to positively charged nylon membrane (Biotrans plus), using capillary transfer with 20 × sodium chloride-sodium citrate (SSC). The blots were then UV cross-linked using a Stratalinker (Stratagene). The blots were subsequently hybridized with 32P-dCTP radiolabeled probes: ETO cDNA or tTA-VP16 DNA. RNA from bulk colonies was isolated by RNAzol extraction (Tel-Test Inc, Friendswood, TX). Total RNA (5 µg) for each sample was electrophoresed and blotted as described above.Isolation of protein and Western blot analysis One mouse from each of the 5 founder lines as well as a mouse that was positive for tTA but not AML1-ETO (negative control) was killed, and the bone marrow was flushed from the femurs with phosphate-buffered saline (PBS). The cells were centrifuged for 5 minutes at 1000 rpm in a GPKR centrifuge (Beckman, Fullerton, CA) and resuspended in a resuspension buffer (100 mmol/L NaCl, 10 mmol/L Tris pH 7.6, 1 mmol/L EDTA, 1 µg/mL aprotinin, 100 µg/mL PMSF) to which an equal volume of 2 × SDS loading buffer (4% SDS, 20% glycerol, 1.43 mol/L 2-mercaptoethanol, 125 mmol/L Tris) was then added. The samples were boiled for 10 minutes, followed by microcentrifugation for 10 minutes. Aliquots of the supernatant equivalent to 2 × 106 cells were stored at 80°C. The samples were
boiled and spun as above immediately prior to loading in an 8%
resolving SDS-PAGE gel (bis:acrylamide = 1:19). Nuclear extracts from
wild-type Ba/F3 cells and Ba/F3 cells transfected with an AML1-ETO
expression construct were also loaded as controls (35 µg each). The
protein was transferred to PVDF membrane (Immobilon P, Millipore,
Bedford, MA) by using a Mini Trans-Blot cell (BioRad, Hercules, CA).
The blot was then blocked in 5% dry milk in TBS + 0.2% NP-40.
The blot was incubated with a primary monoclonal antibody against the
ETO portion of the human fusion protein (a gift from Dr P. Erickson,
University of Colorado).24 The blot was then incubated with a secondary antibody conjugated to horseradish peroxidase. The
blot was developed by using a chemiluminescent substrate (ECL, Amersham, Buckinghamshire, UK) and exposed on Hyperfilm (Amersham).
CFU and replating assay Double-positive MMTV-tTA/AML1-ETO transgenic mice and wild-type control mice were killed, and the bone marrow was flushed from the femurs and the tibias. The cells were washed in PBS and resuspended in MethoCult (M3434) containing 10 ng/mL recombinant mouse (rm) interleukin (IL)-3, 10 ng/mL recombinant human (rh) IL-6, 50 ng/mL rm stem cell factor (SCF), 3 U/mL rh erythropoietin, 15% fetal bovine serum (Stem Cell Technologies, Vancouver, British Columbia, Canada), and Pen-Strep (2.5 mL/35-mm plate). The cells were then plated out at densities of 1 × 104, 2 × 104, 3 × 104, or 6 × 104 cells/plate and incubated at 37°C with 5% CO2. The colonies on the plates were counted and classified 7 to 10 days after plating. To analyze the replating efficiency of bone marrow cells from double-positive tTA/AML1-ETO mice and MMTV-tTA mice, bulk populations of colonies were harvested 7 to 10 days after plating as previously described.16,25 Cells (1 × 104) were then replated in MethoCult and 1-2 × 104 cells were cytocentrifuged onto slides for Wright-Giemsa staining. As before, colonies were counted and harvested 7 to 10 days following plating.
Strong inducible expression of AML1-ETO is seen in the bone marrow of 3 of the 5 transgenic founder lines To study AML1-ETO expression in mice, we generated transgenic mice containing the AML1-ETO cDNA driven by a tetracycline-responsive promoter using pUHD-AML1/ETO. We obtained 5 unique founder lines (#2, #6, #7, #8, and #13). The mice from these 5 lines were bred with mice containing the MMTV-tTA DNA to produce double-positive mice for further study (Figure 1). As seen in Figure 2, tTA is ubiquitously expressed in various tissues of the tTA-positive transgenic mice. The level of tTA expression was variable in different tissues. Founder line #2 did not show any inducible AML1-ETO expression, which could be due to the disruption or the mutation of the pUHD-AML1/ETO DNA construct during the generation of the transgenic mice (data not shown). Three (#6, #8, and #13) of the 5 founder lines had a very similar pattern of AML1-ETO expression (Figure 2). AML1-ETO was expressed in a tissue-specific manner in the bone marrow of the double-positive mice, and it was not expressed in the AML1-ETO-positive mice without the MMTV-tTA construct. A basal level of endogenous ETO expression can be seen in the brain RNA of both single- and double-positive mice (data not shown and Figure 3). AML1-ETO is also weakly expressed in the spleen, thymus, and peritoneal macrophages of the various founder lines (Figure 2 and data not shown). The level varies from founder line to founder line and is probably caused by different integration sites of the transgenic construct.
Strong inducible expression of AML1-ETO is seen in the bone marrow and macrophages of founder line #7 In 1 of the 5 unique founder lines that we obtained, a slightly different pattern of expression was observed (Figure 3). Founder line #7 expressed AML1-ETO RNA in peritoneal macrophages as well as in the bone marrow. The expression level in the macrophages was relatively higher than the level in the bone marrow. This pattern is possibly related to the integration site of the AML1-ETO construct in this particular line.Functional AML1-ETO fusion protein is detectable in the inducible founder lines Expression of AML1-ETO was observed by Northern blot analysis of tissues from the inducible mice. To check whether there was any detectable translation of the AML1-ETO fusion protein, we performed a Western blot analysis of bone marrow samples from the double-positive transgenic mice (Figure 4A). We used protein extracts from transfected Ba/F3 cells as a control. In 4 (#6, #7, #8, and #13) of our 5 founder lines, we observed detectable levels of the AML1-ETO fusion protein. No protein expression was seen in founder line #2, the wild-type mice, or untransfected cell controls. Furthermore, the different founder lines expressed the AML1-ETO fusion protein at varying levels. This finding indicates that under tTA regulation, the transgenic construct is expressed not only at the transcriptional level but also at the protein level. To demonstrate that the expressed AML1-ETO protein is functional, we tested whether AML1-ETO expression had an effect on UBP43 gene expression. UBP43 is a novel ubiquitin-specific protease. We cloned this gene by comparing gene expression in the yolk sacs of AML1-ETO knock-in mice and wild-type mice.26 UBP43 is expressed at a relatively higher level in the knock-in mice, indicating its up-regulation by AML1-ETO. In adult mice, UBP43 is expressed at the highest level in the thymus and at a slightly lower level in the peritoneal macrophages. It is also expressed at a detectable level in the bone marrow. As shown in Figure 4B, total RNA was prepared from the bone marrow and peritoneal macrophages of either AML1-ETO transgenic mice or AML1-ETO and tTA double-positive transgenic mice from founder line #7 and subjected to Northern blot analysis. In the single-positive mice, which do not express AML1-ETO, a basal level of UBP43 expression is observed. In the mice with AML1-ETO expression, UBP43 is strongly up-regulated in the bone marrow and macrophages. This finding indicates that inducibly expressed AML1-ETO is a functional protein.
Tetracycline can control the expression of AML1-ETO in transgenic mice To test whether AML1-ETO expression was inducible in our double-positive transgenic mice, we bred mice in which the mother had a subcutaneous time-release tetracycline pellet to keep the AML1-ETO gene off in the developing embryos. Double-positive offspring of founder line #6 were identified by Southern blot analysis, and these mice were given tetracycline in their drinking water. When these offspring were 58 days old, the tetracycline was removed from some of these mice. After 3 weeks in the presence or absence of tetracycline, the bone marrow was harvested from these mice, and total RNA was analyzed by Northern blot analysis (Figure 5). The tet-off system of controllable gene expression was functional in these mice. The level of MMTV-tTA expression was equivalent in both the mice that continued to receive tetracycline and the mice from which tetracycline was removed. The mice from which tetracycline was removed showed a high level of AML1-ETO expression, whereas the mice that continued to receive tetracycline showed a trace level of AML1-ETO expression. This finding demonstrates that transgenic animals can be generated in which AML1-ETO expression can be controlled by administration of tetracycline.
Transgenic mice that express AML1-ETO have normal hematopoiesis Our goal was to investigate the effect of AML1-ETO on hematopoiesis and its potential role in leukemogenesis. Because of the lethal effect of AML1-ETO on embryogenesis, we used the tet-off inducible system of gene expression. We observed adult double-positive mice that express AML1-ETO to study their hematopoiesis. These mice exhibited no outward signs of illness. Their coat appeared normal, and their level of activity was consistent with that of wild-type mice. We performed blood smears and differential counts of the blood from these mice. In all of the founder lines, the differential blood counts were normal (Table 1). We then used bone marrow cells from founder lines #7 and #8 to perform in vitro CFU assays. The numbers of different colonies observed for both double-positive and wild-type mice were approximately the same (Table 1). These mice showed no abnormal hematopoiesis. CFU assays were also performed in which cells isolated from the same MMTV-tTA/AML1-ETO double-positive mice were plated in the presence or absence of tetracycline. No difference was observed in number or type of colonies generated (data not shown). Total RNA was then harvested from colonies from the CFU assay for founder line #8 and analyzed by Northern blot analysis. A similar level of AML1-ETO expression is seen in both the bone marrow of double-positive mice and colonies derived from the bone marrow of double-positive mice (Figure 6). Bone marrow cells were also analyzed for their morphology (Figure 7). No significant difference can be observed between bone marrow samples from the control and AML1-ETO-expressing mice.
Alteration of the differentiation and proliferation ability of progenitor cells expressing AML1-ETO To examine the self-renewal capacity of progenitor cells expressing AML1-ETO, we performed serial replatings of bulk populations of bone marrow-derived colonies in methocellulose cultures. Seven to 10 days after plating, entire methocellulose cultures from MMTV-tTA mice and MMTV-tTA/AML1-ETO mice were harvested, and 1 × 104 cells were replated in methocellulose under conditions optimal for the differentiation of multipotential progenitors. As described earlier, primary cultures from bone marrow of MMTV-tTA/AML1-ETO mice exhibited no difference in the number or types of colonies generated as compared with MMTV-tTA mice in the presence or absence of tetracycline. However, on serial replating of the colonies, several differences became apparent. After 3 to 4 generations in methocellulose, the cultures from MMTV-tTA mice failed to form colonies (Figure 8A). Instead, large diffusely distributed cells populated the plates. On examination by cytospin and Wright-Giemsa staining, these cells were easily identifiable as fully differentiated macrophages. In contrast, colonies derived from mice expressing AML1-ETO continued to replate and to generate colonies. On these plates, we observed very few migrating, fully differentiated macrophages, but rather we saw small, compact colonies containing 50 to 100 cells. Cytospin and Wright-Giemsa staining of these colonies revealed a polymorphic population of cells, including very early progenitors as well as early myeloid cells. To be certain that these colonies were expressing AML1-ETO, we isolated RNA from colonies derived from MMTV-tTA mice and MMTV-tTA/AML1-ETO mice and performed a Northern analysis. This analysis (Figure 8B) clearly demonstrates AML1-ETO expression in the colonies from MMTV-tTA/AML1-ETO mice after culture in methocellulose.
To further examine the role played by the expression of AML1-ETO in
these cells, we replated colonies expressing AML1-ETO, which had gone
through 9 generations in culture without tetracycline, in the presence
or absence of tetracycline. Interestingly, when AML1-ETO expression was
turned off in these cells by the addition of tetracycline to the media,
we saw 3-fold more colony formation as well as an increase in average
colony size (150 to 300 cells). When we analyzed these colonies by
cytocentrifugation and Wright-Giemsa staining, we observed an increased
percentage of fully differentiated cells in both the macrophage and
granulocyte lineages and a significant decrease in the percentage of
blasts and immature cells (Table 2 and
Figure 8C). We also observed that, following one passage in the absence
of AML1-ETO expression, these cells did not replate. These data
indicate that AML1-ETO expression reduces myeloid differentiation, causing the cells to pause in an immature stage. On suppression of
AML1-ETO expression, these cells are fully capable of complete differentiation.
The t(8;21) translocation is a frequent chromosomal aberration found in AML.7,10,11,27 We have previously shown that when AML1-ETO is knocked into the AML1 locus in mice, an embryonic lethal phenotype is observed.12 To further investigate the role of AML1-ETO in hematopoiesis and leukemogenesis, we generated mice that have tetracycline-inducible expression of AML1-ETO. This allowed us to control the expression of AML1-ETO by removing tetracycline to activate expression of the gene. In our mice, we have found that functional AML1-ETO is expressed in a highly inducible manner in bone marrow cells. We generated 5 unique founder lines containing the AML1-ETO construct. We compared mice that were positive for the AML1-ETO construct with mice that were positive for both AML1-ETO and tTA. Although the expression of tTA in the double-positive mice was seen in all tissues tested, AML1-ETO was only highly expressed in the bone marrow of 4 (#6, #7, #8, and #13) of the 5 founder lines (#2, #6, #7, #8, and #13). In addition, founder line #7 showed a higher level of expression in the macrophages. The variation in the expression pattern may be associated with the different integration sites for the AML1-ETO construct in different founder lines. It is unclear why AML1-ETO is not expressed in all tissues that express tTA. One possibility is that AML1-ETO messenger RNA (mRNA) is transcribed but is not stable in some of the tissues that we have analyzed. To test this possibility, we performed nuclear run-on experiments with nuclei prepared from livers and kidneys of mice expressing AML1-ETO in the bone marrow and also from mice lacking such expression. We did not detect any AML1-ETO transcription in the livers and kidneys of mice that do express AML1-ETO in their bone marrow (data not shown). This result did not favor the RNA stability theory. Therefore, the MMTV-tTA tet-off system may be valuable for researchers interested in targeting inducible gene expression specifically to hematopoietic cells. The purpose of this project was to create a model system in which leukemia could be brought on by the induction of AML1-ETO expression at a time after its expression would normally cause embryonic lethality. In tetracycline-inducible mice, however, we did not observe onset of leukemia. This is a clear example to support the concept proposed by Westervelt and Ley28 in their recent review that target cells are a critical component in the development of transgenic animal leukemia models. A good example of this is seen in the analysis of PML/RARA fusion protein transgenic mice. Because of the different expression patterns of cathepsin G, MRP8, and CD11b, the mice produced with the regulatory elements of these genes target different cell populations and show different leukemic phenotypes.29-32 Therefore, it may be necessary for AML1-ETO to be expressed at a specific time during cell differentiation to exhibit a phenotype. Unfortunately, although the specific antibody used in this report is good for Western blot analysis, it still cannot detect AML1-ETO expression in an immunostaining assay. This difficulty makes it impossible to analyze the exact hematopoietic stage of AML1-ETO expression in t(8;21) leukemia patients or in these transgenic mice. Also, Western analysis is not practical because the number of cells necessary to produce enough protein to analyze would be difficult to acquire. Furthermore, there is no clear data correlating AML1-ETO expression during a particular stage of hematopoiesis with the development of leukemia in humans. Although our double-positive AML1-ETO-expressing transgenic mice
exhibit no disease and appear to have normal hematopoiesis as
determined by analysis of differential counts of blood and bone marrow
smears, we have made an important observation concerning the effect of
AML1-ETO expression on the development and proliferation of a
subpopulation of hematopoietic progenitors. Furthermore, we have used
CFU assays to study in vitro proliferation and differentiation of bone
marrow hematopoietic progenitor cells. Although we did not detect a
difference between AML1-ETO-expressing bone marrow cells and
nonexpressing cells in primary cultures, on serial replatings we have
observed a difference in the ability of AML1-ETO-expressing cells to
differentiate and proliferate. In their analysis of hematopoietic progenitors from AML1-ETO knock-in mice, Okuda et al.16
also observed an increase in the self-renewal capacity of these cells. Our replating experiments with tetracycline-inducible expression of
AML1-ETO indicate that hematopoietic progenitor cells from mice
expressing AML1-ETO may be partially blocked from differentiation into
mature myeloid cells. On addition of tetracycline to suppress the
expression of AML1-ETO, these cells differentiate more efficiently into
mature myeloid cells. These cells also proliferate in response to the
removal of AML1-ETO expression. Taken together, these data indicate
that, although differentiation is partially blocked or slowed very
early in response to AML1-ETO, proliferation is not enhanced. This
failure to proliferate may be the reason why we do not observe disease
in these animals. To date, the AML1-ETO-expressing mice have exhibited
normal hematopoiesis. Besides the concern of the time window of
AML1-ETO expression as discussed above, the presence of normal
hematopoiesis in these mice suggests the possibility that AML1-ETO
alone is not sufficient to cause leukemia. It has been reported that
the AML1-ETO transcript is detectable in t(8;21) patients with
long-term remission.33-35 Therefore, it is possible to
express AML1-ETO and not exhibit disease. Additional mutation or
abnormal expression of another gene(s) may be necessary to promote
leukemogenesis, perhaps through increased proliferation. It has been
reported that retrovirus-mediated BCR/ABL or HRX-ENL expression in bone
marrow cells causes leukemia.25,36,37 This demonstrates
that expression of either BCR/ABL or HRX-ENL alone is sufficient to
generate leukemia. When the same tetracycline-inducible system is used
to direct BCR/ABL expression in the transgenic mice, MMTV-tTA/BCR/ABL
double-positive mice developed leukemia.38 Conversely,
when CBF
We wish to thank Lothar Hennighausen for the MMTV-tTA transgenic mice and Joel Lawitts, Pu Zhang, and Claudia Huettner for valuable discussion and technical assistance.
Submitted February 15, 2000; accepted May 18, 2000.
Supported by National Institutes of Health grant CA72009, and American Cancer Society grant DHP-166. D.E.Z. is a Leukemia and Lymphoma Society Scholar.
K.L.R., C.J.H., and N.H. contributed equally to this work.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Dong-Er Zhang, MEM-L51, The Scripps Research Institute, 10550 North Torrey Pines Rd, La Jolla, CA 92037; e-mail: dzhang{at}scripps.edu.
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M. Yan, E.-Y. Ahn, S. W. Hiebert, and D.-E. Zhang RUNX1/AML1 DNA-binding domain and ETO/MTG8 NHR2-dimerization domain are critical to AML1-ETO9a leukemogenesis Blood, January 22, 2009; 113(4): 883 - 886. [Abstract] [Full Text] [PDF]
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E.-Y. Ahn, M. Yan, O. A. Malakhova, M.-C. Lo, A. Boyapati, H. B. Ommen, R. Hines, P. Hokland, and D.-E. Zhang Disruption of the NHR4 domain structure in AML1-ETO abrogates SON binding and promotes leukemogenesis PNAS, November 4, 2008; 105(44): 17103 - 17108. [Abstract] [Full Text] [PDF]
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N. Watanabe-Okochi, J. Kitaura, R. Ono, H. Harada, Y. Harada, Y. Komeno, H. Nakajima, T. Nosaka, T. Inaba, and T. Kitamura AML1 mutations induced MDS and MDS/AML in a mouse BMT model Blood, April 15, 2008; 111(8): 4297 - 4308. [Abstract] [Full Text] [PDF]
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F. Dayyani, J. Wang, J.-R. J. Yeh, E.-Y. Ahn, E. Tobey, D.-E. Zhang, I. D. Bernstein, R. T. Peterson, and D. A. Sweetser Loss of TLE1 and TLE4 from the del(9q) commonly deleted region in AML cooperates with AML1-ETO to affect myeloid cell proliferation and survival Blood, April 15, 2008; 111(8): 4338 - 4347. [Abstract] [Full Text] [PDF]
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L. F. Peterson, A. Boyapati, E.-Y. Ahn, J. R. Biggs, A. J. Okumura, M.-C. Lo, M. Yan, and D.-E. Zhang Acute myeloid leukemia with the 8q22;21q22 translocation: secondary mutational events and alternative t(8;21) transcripts Blood, August 1, 2007; 110(3): 799 - 805. [Abstract] [Full Text] [PDF]
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L. F. Peterson, M. Yan, and D.-E. Zhang The p21Waf1 pathway is involved in blocking leukemogenesis by the t(8;21) fusion protein AML1-ETO Blood, May 15, 2007; 109(10): 4392 - 4398. [Abstract] [Full Text] [PDF]
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E. Bockamp, C. Antunes, M. Maringer, R. Heck, K. Presser, S. Beilke, S. Ohngemach, R. Alt, M. Cross, R. Sprengel, et al. Tetracycline-controlled transgenic targeting from the SCL locus directs conditional expression to erythrocytes, megakaryocytes, granulocytes, and c-kit-expressing lineage-negative hematopoietic cells Blood, September 1, 2006; 108(5): 1533 - 1541. [Abstract] [Full Text] [PDF]
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S. Nishida, N. Hosen, T. Shirakata, K. Kanato, M. Yanagihara, S.-i. Nakatsuka, Y. Hoshida, T. Nakazawa, Y. Harada, N. Tatsumi, et al. AML1-ETO rapidly induces acute myeloblastic leukemia in cooperation with the Wilms tumor gene, WT1 Blood, April 15, 2006; 107(8): 3303 - 3312. [Abstract] [Full Text] [PDF]
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K. Maki, T. Yamagata, T. Asai, I. Yamazaki, H. Oda, H. Hirai, and K. Mitani Dysplastic definitive hematopoiesis in AML1/EVI1 knock-in embryos Blood, September 15, 2005; 106(6): 2147 - 2155. [Abstract] [Full Text] [PDF]
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H. G. Nguyen, G. Yu, M. Makitalo, D. Yang, H.-X. Xie, M. R. Jones, and K. Ravid Conditional overexpression of transgenes in megakaryocytes and platelets in vivo Blood, September 1, 2005; 106(5): 1559 - 1564. [Abstract] [Full Text] [PDF]
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J. C. Mulloy, V. Jankovic, M. Wunderlich, R. Delwel, J. Cammenga, O. Krejci, H. Zhao, P. J. M. Valk, B. Lowenberg, and S. D. Nimer AML1-ETO fusion protein up-regulates TRKA mRNA expression in human CD34+ cells, allowing nerve growth factor-induced expansion PNAS, March 15, 2005; 102(11): 4016 - 4021. [Abstract] [Full Text] [PDF]
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Y.-Y. Wang, G.-B. Zhou, T. Yin, B. Chen, J.-Y. Shi, W.-X. Liang, X.-L. Jin, J.-H. You, G. Yang, Z.-X. Shen, et al. AML1-ETO and C-KIT mutation/overexpression in t(8;21) leukemia: Implication in stepwise leukemogenesis and response to Gleevec PNAS, January 25, 2005; 102(4): 1104 - 1109. [Abstract] [Full Text] [PDF]
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J. L. Hess and B. A. Hug Fusion-protein truncation provides new insights into leukemogenesis PNAS, December 7, 2004; 101(49): 16985 - 16986. [Full Text] [PDF]
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M. Yan, S. A. Burel, L. F. Peterson, E. Kanbe, H. Iwasaki, A. Boyapati, R. Hines, K. Akashi, and D.-E. Zhang From the Cover: Deletion of an AML1-ETO C-terminal NcoR/SMRT-interacting region strongly induces leukemia development PNAS, December 7, 2004; 101(49): 17186 - 17191. [Abstract] [Full Text] [PDF]
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T. S. Fenske, G. Pengue, V. Mathews, P. T. Hanson, S. E. Hamm, N. Riaz, and T. A. Graubert Stem cell expression of the AML1/ETO fusion protein induces a myeloproliferative disorder in mice PNAS, October 19, 2004; 101(42): 15184 - 15189. [Abstract] [Full Text] [PDF]
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S. Tsuzuki, M. Seto, M. Greaves, and T. Enver Modeling first-hit functions of the t(12;21) TEL-AML1 translocation in mice PNAS, June 1, 2004; 101(22): 8443 - 8448. [Abstract] [Full Text] [PDF]
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X. Zheng, T. Beissert, N. Kukoc-Zivojnov, E. Puccetti, J. Altschmied, C. Strolz, S. Boehrer, H. Gul, O. Schneider, O. G. Ottmann, et al. {gamma}-Catenin contributes to leukemogenesis induced by AML-associated translocation products by increasing the self-renewal of very primitive progenitor cells Blood, May 1, 2004; 103(9): 3535 - 3543. [Abstract] [Full Text] [PDF]
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M. Schwieger, J. Lohler, M. Fischer, U. Herwig, D. G. Tenen, and C. Stocking A dominant-negative mutant of C/EBP{alpha}, associated with acute myeloid leukemias, inhibits differentiation of myeloid and erythroid progenitors of man but not mouse Blood, April 1, 2004; 103(7): 2744 - 2752. [Abstract] [Full Text] [PDF]
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J. C. Mulloy, J. Cammenga, F. J. Berguido, K. Wu, P. Zhou, R. L. Comenzo, S. Jhanwar, M. A. S. Moore, and S. D. Nimer Maintaining the self-renewal and differentiation potential of human CD34+ hematopoietic cells using a single genetic element Blood, December 15, 2003; 102(13): 4369 - 4376. [Abstract] [Full Text] [PDF]
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J. L. Grisolano, J. O'Neal, J. Cain, and M. H. Tomasson An activated receptor tyrosine kinase, TEL/PDGF{beta}R, cooperates with AML1/ETO to induce acute myeloid leukemia in mice PNAS, August 5, 2003; 100(16): 9506 - 9511. [Abstract] [Full Text] [PDF]
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Q. Xu, S.-E. Simpson, T. J. Scialla, A. Bagg, and M. Carroll Survival of acute myeloid leukemia cells requires PI3 kinase activation Blood, August 1, 2003; 102(3): 972 - 980. [Abstract] [Full Text] [PDF]
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O. Heidenreich, J. Krauter, H. Riehle, P. Hadwiger, M. John, G. Heil, H.-P. Vornlocher, and A. Nordheim AML1/MTG8 oncogene suppression by small interfering RNAs supports myeloid differentiation of t(8;21)-positive leukemic cells Blood, April 15, 2003; 101(8): 3157 - 3163. [Abstract] [Full Text] [PDF]
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J. Cammenga, J. C. Mulloy, F. J. Berguido, D. MacGrogan, A. Viale, and S. D. Nimer Induction of C/EBPalpha activity alters gene expression and differentiation of human CD34+ cells Blood, March 15, 2003; 101(6): 2206 - 2214. [Abstract] [Full Text] [PDF]
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M. L. Kalev-Zylinska, J. A. Horsfield, M. V. C. Flores, J. H. Postlethwait, M. R. Vitas, A. M. Baas, P. S. Crosier, and K. E. Crosier Runx1 is required for zebrafish blood and vessel development and expression of a human RUNX1-CBF2T1 transgene advances a model for studies of leukemogenesis Development, March 6, 2003; 129(8): 2015 - 2030. [Abstract] [Full Text] [PDF]
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A. Tonks, L. Pearn, A. J. Tonks, L. Pearce, T. Hoy, S. Phillips, J. Fisher, J. R. Downing, A. K. Burnett, and R. L. Darley The AML1-ETO fusion gene promotes extensive self-renewal of human primary erythroid cells Blood, January 15, 2003; 101(2): 624 - 632. [Abstract] [Full Text] [PDF]
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R. K. Vangala, M. S. Heiss-Neumann, J. S. Rangatia, S. M. Singh, C. Schoch, D. G. Tenen, W. Hiddemann, and G. Behre The myeloid master regulator transcription factor PU.1 is inactivated by AML1-ETO in t(8;21) myeloid leukemia Blood, January 1, 2003; 101(1): 270 - 277. [Abstract] [Full Text] [PDF]
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K. Barseguian, B. Lutterbach, S. W. Hiebert, J. Nickerson, J. B. Lian, J. L. Stein, A. J. van Wijnen, and G. S. Stein Multiple subnuclear targeting signals of the leukemia-related AML1/ETO and ETO repressor proteins PNAS, November 26, 2002; 99(24): 15434 - 15439. [Abstract] [Full Text] [PDF]
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J. Basecke, L. Cepek, C. Mannhalter, J. Krauter, S. Hildenhagen, G. Brittinger, L. Trumper, and F. Griesinger Transcription of AML1/ETO in bone marrow and cord blood of individuals without acute myelogenous leukemia Blood, August 28, 2002; 100(6): 2267 - 2267. [Full Text] [PDF]
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H. Mori, S. M. Colman, Z. Xiao, A. M. Ford, L. E. Healy, C. Donaldson, J. M. Hows, C. Navarrete, and M. Greaves Chromosome translocations and covert leukemic clones are generated during normal fetal development PNAS, June 11, 2002; 99(12): 8242 - 8247. [Abstract] [Full Text] [PDF]
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D. A. Sweetser, C.-S. Chen, A. A. Blomberg, D. A. Flowers, P. C. Galipeau, M. T. Barrett, N. A. Heerema, J. Buckley, W. G. Woods, I. D. Bernstein, et al. Loss of heterozygosity in childhood de novo acute myelogenous leukemia Blood, August 15, 2001; 98(4): 1188 - 1194. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
(CBF
the tet-off system. The tet-off system has been successfully utilized to control gene expression in transgenic
mice.
) alone or MMTV-tTA/AML1-ETO (
), and 1 × 104
cells were plated in MethoCult in the absence of tetracycline. Bulk
cultures were harvested after 7 to 10 days in culture, and
1 × 104 cells were replated for each sample. Each point
represents the number of colonies generated per 104 cells
seeded. After 9 (G9, 
) and 11 (G11, 
) generations, cells derived
from MMTV-tTA/AML1-ETO mice were replated in MethoCult in the presence
or absence of tetracycline. (B) Northern analysis of AML1-ETO
expression in colonies harvested at various generations in
methocellulose. RNA was harvested from bulk populations of colonies
grown for 7 to 10 days in culture. Total RNA (5 µg) was loaded on
each lane. The RNA was then transferred to a nylon membrane and
hybridized with ETO cDNA. The ethidium bromide staining of the 18S
ribosomal RNA is presented to show the loading of the RNA samples.
tTA+ indicates RNA prepared from MMTV-tTA bone marrow cell
culture; +/+, RNA prepared from
MMTV-tTA/AML1-ETO bone marrow cell culture. (C) Morphology of cells
obtained from colonies grown in the presence or absence of
tetracycline. After 9 generations, cells were cultured in the presence
or absence of tetracycline. Cytospins of colonies replated in the
presence or absence of tetracycline after culture for 9 generations
without tetracycline were stained with Wright-Giemsa solution.