|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Prepublished online as a Blood First Edition Paper on June 14, 2002; DOI 10.1182/blood-2001-11-0089.
NEOPLASIA
From the European Institute of Oncology, Department of
Experimental Oncology, Milan, Italy; Department of Physiology and
General Biochemistry, and Department of Veterinary Pathology, Hygiene
and Public Health, School of Veterinary Medicine, University of Milan,
Italy; IFOM-FIRC Institute of Molecular Oncology, Milan, Italy; and
Department of Human Biotechnology and Hematology, University of Rome
"La Sapienza," Rome, Italy.
Acute promyelocytic leukemia (APL) is associated with chromosomal
translocations resulting in fusion proteins of the retinoic acid
receptor (RAR). Here, we report a novel murine model system for APL,
based on the transduction of purified murine hematopoietic progenitors
(lin Expression of the promyelocytic leukemia-retinoic
acid receptor (PML-RAR) fusion protein Studies conducted in cell lines showed that PML-RAR is able to block
differentiation on several differentiating stimuli and that this effect
is reverted by RA treatment.4 These results suggested that
PML-RAR is responsible for both the pathogenesis of the disease and for
its RA sensitivity. In vivo studies using transgenic chicken and mice
confirmed this hypothesis.5-13 Expression of PML-RAR in
mice under different myeloid-specific promoters gave 2 kinds of
results: (1) in the case of promoters for genes expressed during early
myeloid-promyelocytic stages of differentiation, such as cathepsin-G
and MRP8, PML-RAR induced slight alterations in myelopoiesis
("preleukemic syndrome") in 100% of mice, and an APL-like leukemia
at low penetrance (15%-20%) and long latency (6-18 months)6,8-10; (2) in the case of promoters for late
stages of myeloid differentiation, such as CD11b, expression of PML-RAR provoked a modest impairment in hematopoiesis, without induction of an overt leukemic syndrome.7 These results underscore
the relevance of the proper targeting of PML-RAR expression and suggest a "2-hit" model, where expression of the fusion protein is not sufficient for leukemogenesis, but creates a cellular environment favorable for the accumulation of additional genetic
lesions.12,13
We have developed an alternative strategy to express PML-RAR in the
hematopoietic compartment, by retroviral-based transduction of murine
hematopoietic progenitors and subsequent reinoculation of syngeneic
mice. Our results confirm and extend the observations obtained by the
conventional transgenic models, and offer a new, more versatile model
system to study APL pathogenesis.
Plasmids
Purification of lin Transduction and sorting of GFP+ cells Lin cells were grown for 36 hours in medium
containing interleukin 3 (IL-3), IL-6, and stem cell factor (SCF). The
cells were then plated onto retronectin-coated (Takara-Shuzo, Shiga,
Japan), non-tissue culture-treated plates and exposed to the
supernatant of packaging, ecotropic Phoenix cells transiently
transfected with the indicated retroviral vectors. Transduced cells
were sorted using a Becton Dickinson (Franklin Lakes, NJ) FACS Vantage
instrument. Purity of sorted cells was more than 98% in all experiments.
Differentiation and survival assays To analyze differentiation in vitro, sorted cells or uninfected control lin cells were plated (5000 cells/plate) in methylcellulose medium containing fetal bovine serum
and the following cytokines: IL-3 (20 ng/mL), IL-6 (20 ng/mL), SCF (100 ng/mL), granulocyte colony-stimulating factor (G-CSF; 60 ng/mL), and
granulocyte-macrophage colony-stimulating factor (GM-CSF; 20 ng/mL).
Seven to 10 days after plating, cells were harvested and characterized
by analysis of surface myeloid markers (MAC1 and GR1). For the in vivo
studies, cells from peripheral blood, bone marrow, or spleen of healthy
and leukemic mice were analyzed for the presence of surface progenitor
markers (C-KIT, CD34, and SCA1) and myeloid markers (GR1 and MAC1).
Murine antibodies for C-KIT, CD34, SCA1, GR1, MAC1, and their matched
isotype controls were purchased from Pharmingen (San Diego, CA)
and used according to the manufacturer's instruction. Cells were
analyzed by a Becton Dickinson FACScan, using Cell Quest software.
In survival assays in vitro, cells were plated in methylcellulose medium as described for the differentiation assays. After 10 days, pooled colonies were reseeded (10 000 cells/plate) in semisolid medium. The assay was repeated until no colonies developed in control plates (usually, 2-3 platings), due to terminal differentiation of the cells.16 Repopulation of lethally irradiated recipients 129SvEv wild-type mice were lethally irradiated (9 Gy) and reinjected intravenously with 300 000 sorted cells plus 500 000 spleen cells (obtained from an untreated mouse); after 7 days, mice were reinjected intravenously with 500 000 spleen cells. Mice reinjected with spleen cells only survived slightly longer than untreated, irradiated mice (approximately 4 weeks versus 1-2 weeks), but eventually they all died due to impaired hematopoiesis.The animals were checked periodically for clinical signs of disease and for presence of blast cells by May-Grünwald-Giemsa staining of blood smears. Morphologic analysis Mice were humanely killed by CO2 inhalation and underwent necropsy. Main organs were fixed in 10% buffered formalin and processed for histopathologic examination. Samples were embedded in paraffin, sectioned at 4 µm and stained with hematoxylin and eosin. Blood smears, bone marrow smears, and spleen cell imprints were stained with May-Grünwald-Giemsa and Sudan black B.DNA, RNA, and protein analysis DNA polymerase chain reaction (PCR) and reverse transcription-PCR (RT-PCR) from colonies derived from methylcellulose plating, or from leukemic animals, were performed according to standard techniques, using primers specific for the human PML-RAR cDNA.17 As a routine control for DNA quality of all of our genomic samples, we have used primers to amplify a genomic subregion of the murine p21 gene. In all of the samples shown in the figures, that test result was positive. For Southern blot analysis, genomic DNAs from spleen, liver, or bone marrow of leukemic mice (primary or secondary recipients) were digested with HindIII, run on an agarose gel, and then blotted to nylon membranes (Amersham, Piscataway, NJ). The filters were hybridized using an HindIII-EcoRI 3-kb fragment containing the entire PML-RAR cDNA. Eight primary leukemic samples and 12 secondary/tertiary leukemic samples were analyzed in this study; 4 representative primary cases/2 secondary cases are shown in Figure 6. For protein analysis, immunoblotting and immunofluorescence experiments were performed using antibodies specific for the human and mouse PML.18 Immunoprecipitations were performed using antibodies against human PML, followed by analysis of the immunoprecipitates by immunoblotting using anti-RAR antibodies.FISH analysis Metaphase spleen cells were obtained from leukemic mice as described.19 PINCO PML/RAR was labeled as described.20 Fluorescent in situ hybridization (FISH) analysis was performed using Applied Spectral Imaging (ASI, Carlsbad, CA) and the software SKY View 1.6.1.RA treatment of secondary recipients Mice with overt leukemias were humanely killed, and leukemic cells were harvested from the spleen. Leukemic cells were reinjected intravenously (1 × 107 cells/mouse) in nonirradiated, syngeneic recipient mice. When the secondary recipient mice developed overt leukemia (in about 2-3 weeks), a 21-day release pellet containing 5 mg RA or placebo (Innovative Research of America [IRA], Sarasota, FL) was implanted subcutaneously. Absence/presence of blast cells, differentiation, and recovery were evaluated on peripheral blood smears stained with May-Grünwald-Giemsa or Sudan black B.All procedures involving animals were done in accordance with national and international laws and policies.
Ectopic expression of PML-RAR blocks myeloid differentiation and
increases proliferative potential of lin cells); (2) infection of lin
cells with high-titer retroviruses expressing PML-RAR, or wild-type RAR, from the 5' viral long terminal repeat (LTR), and GFP from an internal promoter (cytomegalovirus [CMV]); (3) separation of GFP+ cells by fluorescence-activated cell sorter (FACS);
and (4) use of GFP+ cells for in vitro differentiation
assays (colony formation in methylcellulose) or in vivo reconstitution
of lethally irradiated, syngeneic mice (Figure
1A). Western blot analysis showed
expression of PML-RAR in the sorted lin cell population
(Figure 1F). Anti-PML immunofluorescence analysis of GFP+
cells showed the typical punctuated or microspeckled patterns in cells
infected with the empty or PML-RAR retroviruses, respectively (Figure 1G).
Uninfected or GFP+ lin The capacity of PML-RAR to block myeloid differentiation (at the promyelocytic stage), and to prolong cell survival in a RA-reversible fashion, indicates that our model system recapitulates the biologic properties of the human disease in vitro, thus prompting us to examine the effect of fusion protein expression in vivo. PML-RAR-expressing cells cause an APL-like disease in mice Immediately after sorting, GFP+ cells from control, PML-RAR, or RAR-transduced cells were reinoculated into lethally irradiated recipient mice. More than 95% of the mice reconstituted with control, GFP-expressing cells survived for more than 6 months, whereas the nonreinoculated mice died within 1 to 2 weeks after irradiation (Table 1 and Figure 2).
Mice reconstituted with lin Massive splenomegaly and hepatomegaly were present in all PML-RAR mice,
with frequent lymphoadenomegaly (Figure 5A and data not shown).
Histopathologic examination showed leukemic cells diffusely invading
the bone marrow (Figure 3B), the splenic
red pulp, and the liver (not shown and Figure 5C). Moderate to severe infiltration of leukemic cells was also frequently detectable in the
lymph nodes, lungs, meninges, and less frequently in the kidneys.
Cytologically, the disease was characterized by extensive prevalence of
promyelocytes, and, to a lesser degree, metamyelocytes (Figures 3C and
5E). Promyelocytes were intensely positive for Sudan black B (Figure
3F), had fewer intracytoplasmic granules, and did not show Auer rods,
as compared to the human APL cells, thereby resembling the hypogranular
variant of human APL.22 Analysis of differentiation
surface markers revealed, unlike normal cells, simultaneous expression
of the myeloid lineage markers GR1 and MAC1, and of the progenitor
markers CD34 and C-KIT, but not of SCA1 (Figure
4A, and data not shown). Notably,
CD34+ human APL cells are also more frequently associated
with the hypogranular variant of disease.23 In all the
leukemic mice analyzed, the leukemic cells expressed PML-RAR, as shown
by immunoprecipitation, RNA, and immunofluorescence analyses (Figure
4B-C). In contrast, leukemic cells were uniformly negative for GFP,
reproducing in vivo the phenotype observed in vitro (see Figure 7).
A hallmark of APL is the dramatic differentiation response observed on treatment with pharmacologic doses of RA.2,3 To examine the RA response of leukemic mice in vivo, we obtained secondary leukemias by reinoculating spleen cell suspensions of leukemic mice (with 30%-50% infiltration of leukemic cells) into syngeneic, nonirradiated recipients. The reinoculated mice developed (100% cases) secondary leukemias with
minimal latency (2 weeks), with characteristics identical to those
observed in the primary recipient mice (including transplantability to
further recipients; data not shown). Because the leukemic phenotype was
observed synchronously in the secondary recipients, we could treat a
cohort of mice that were at the same stage of disease, and carrying the
same leukemic clone(s). RA was administered by subcutaneous
implantation of a 21-day release pellet containing 5 mg RA or placebo.
Mice were killed at time intervals to monitor the response in the
various tissues or maintained until death to monitor survival. Starting
from the third day of therapy, cells within the leukemic population
revealed clear signs of granulocytic differentiation, which was
extensive by the seventh day of treatment (Figures 3D-E and
5E). Immunofluorescence studies revealed
that RA, as previously shown, reverted almost completely the
delocalized pattern of mouse PML to the normal "nuclear bodies"
appearance (Figure 4C: from 30% of cells showing a microspeckled
pattern in spleen cells, to < 2% on RA treatment). The leukemic
infiltrates in the various organs appeared reduced, as shown by their
parallel reduction to normal size (Figure 5A,C,D). All of the mice
implanted with a subcutaneous placebo died by progressive multifocal
invasion of parenchymal tissues by leukemic blasts, whereas RA
treatment significantly prolonged survival (P < .0001),
with no apparent signs of disease during treatment, in more than 90%
of mice (Figure 5B). Identical results were obtained in 5 independent
experiments, using as donors 3 mice that developed APL in 3 independent
series of experiments (data not shown).
In human APL, treatment with RA alone results in disease remission and subsequent relapse in virtually all patients.2,3 Likewise, suspension of RA treatment in the leukemic mice resulted in relapse in 100% of mice (Figure 5B). Taken together, these results show that our mouse model faithfully reproduced the main biologic and clinical features of human APL. APL in mice is a monoclonal or oligoclonal disease, which follows a preleukemic state To evaluate the polyclonal or clonal origin of the mouse APL leukemias, we performed Southern blotting analysis of integrated proviruses. A polyclonal origin is consistent with the capacity of PML-RAR to directly transform target cells, whereas a monoclonal or oligoclonal origin suggests a 2-hit model of leukemogenesis, that is, the requirement for additional genetic lesions.6,13 The genomic DNAs from spleen/bone marrow of 4 primary and 2 secondary leukemic mice were digested with the HindIII restriction enzyme (which cuts once in the integrated retroviral sequence, allowing recognition of individual integration events) and hybridized with a probe representative of the human PML-RAR cDNA.As observed in Figure 6A, all the primary
leukemias showed a discrete number of hybridizing DNA fragments (2-4 in
the cases shown). Primary and secondary leukemia samples revealed the
same patterns of multiple hybridizing fragments (compare lanes 1 and 2, 3 and 4 in Figure 6A). The presence of multiple hybridizing fragments
can be the consequence of a single, yet complex, integration site, or
of several integration sites in the leukemia initiating cell. To
clarify this issue, we performed FISH analysis of leukemic metaphases
using a probe encompassing the entire integrated sequence. Strikingly,
in the case where 2 hybridizing fragments were observed, we detected 2 hybridizing regions, confirming the monoclonality of the leukemia, and
the presence of 2 integration events (Figure 6B). Overall, these
results support the monoclonal or oligoclonal origin of the leukemic
blasts and the hypothesis that leukemia arose from one PML-RAR
expressing cell undergoing additional genetic lesions.
We next investigated whether expression of PML-RAR is sufficient to
induce a detectable phenotype, prior to the onset of an overt leukemia.
In the transgenic mice carrying PML-RAR under the MRP-8 or cathepsin G
promoters, a mild defect in myeloid maturation was consistently seen in
100% of the mice, defined as a "preleukemic" state and
characterized by a slight decrease of MAC1 or GR1 expression in the
whole bone marrow.6,13 We did not, however, detect a
similar phenotype in our PML-RAR mice, as shown by the immunophenotypic analysis of bone marrow cells at 30, 60, and 90 days after
reinoculation (Figure 7A and data not
shown). Because we could not follow directly the fate of
PML-RAR-expressing cells, due to loss of GFP expression, we performed
DNA-PCR analysis on several colonies derived from methylcellulose
platings of lin
These results suggest that, as in the transgenic mice, a "preleukemic" state may be observed in our experimental system, and corresponds to an alteration in the differentiation potential induced by the fusion protein in hematopoietic progenitors.
We have presented here a novel model system to study APL in mice,
based on transplantation of transduced murine hematopoietic progenitors. This system fully recapitulates the most relevant features
of human APLs, that is, a differentiation block at the promyelocyte
stage and RA sensitivity. Leukemia is obtained by the expression of
PML-RAR (but not RAR) in lin Expression of PML-RAR is not sufficient to transform cells; in all
model systems, additional genetic/epigenetic events are required, which
are favored by PML-RAR expression.25 The fusion protein
provokes a so-called "preleukemic" state, characterized, in vivo,
by a mild impairment in myeloid differentiation. Lin The possibility to study the same cell populations in vitro and in vivo
is, indeed, one of the greatest advantages of our experimental system.
In vitro, we have shown that PML-RAR blocks differentiation and extend
survival of lin Finally, RA sensitivity of primary and secondary leukemias, as well as of the preleukemic state observed in vitro, shows that this system is also available for pharmacologic studies. Aberrant recruitment of histone deacetylases (HDACs) is considered critical for the leukemogenic potential of PML-RAR and other AML fusion proteins, and treatment with HDAC inhibitors is able to induce differentiation in vitro.26 Treatment of the leukemic mice with HDAC inhibitors, or other pharmacologically active compounds, may lead to new proposals for the therapy of APL, and possibly other types of cancers.
We thank Tim Ley, Silvia Soddu, Jeff Medin, Mirco Fanelli, and Francesco Bertolini for discussions and help with the set up of some experimental techniques. S. Monestiroli and S. Giavara contributed equally to this work.
Submitted November 28, 2001; accepted June 6, 2002.
Prepublished online as Blood First Edition Paper, June 14, 2002; DOI 10.1182/blood-2001-11-0089.
Supported by grants from European Community (EEC), Italian Foundation for Cancer Research (FIRC), Italian Association for Cancer Research (AIRC), and Ministero della Sanitá to S.M. and P.G.P.
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: Pier Giuseppe Pelicci, European Institute of Oncology, Department of Experimental Oncology, Via Ripamonti 435 Milan, 20141 Italy; e-mail: pgpelicci{at}ieo.it; and Saverio Minucci, European Institute of Oncology, Department of Experimental Oncology, Via Ripamonti 435 Milan, 20141 Italy; e-mail: sminucci{at}ieo.it.
1.
Melnick A, Licht JD.
Deconstructing a disease: RARalpha, its fusion partners, and their roles in the pathogenesis of acute promyelocytic leukemia.
Blood.
1999;93:3167-3215 2. Fenaux P, Chomienne C, Degos L. All-trans retinoic acid and chemotherapy in the treatment of acute promyelocytic leukemia. Semin Hematol. 2001;38:13-25[Medline] [Order article via Infotrieve].
3.
Warrell RP Jr, de The H, Wang ZY, Degos L.
Acute promyelocytic leukemia.
N Engl J Med.
1993;329:177-189 4. Grignani F, Ferrucci PF, Testa U, et al. The acute promyelocytic leukemia-specific PML-RAR alpha fusion protein inhibits differentiation and promotes survival of myeloid precursor cells. Cell. 1993;74:423-431[CrossRef][Medline] [Order article via Infotrieve]. 5. Altabef M, Garcia M, Lavau C, Bae SC, Dejean A, Samarut J. A retrovirus carrying the promyelocyte-retinoic acid receptor PML- RARalpha fusion gene transforms haematopoietic progenitors in vitro and induces acute leukaemias. EMBO J. 1996;15:2707-2716[Medline] [Order article via Infotrieve].
6.
Brown D, Kogan S, Lagasse E, et al.
A PMLRARalpha transgene initiates murine acute promyelocytic leukemia.
Proc Natl Acad Sci U S A.
1997;94:2551-2556
7.
Early E, Moore MA, Kakizuka A, et al.
Transgenic expression of PML/RARalpha impairs myelopoiesis.
Proc Natl Acad Sci U S A.
1996;93:7900-7904
8.
Grisolano JL, Wesselschmidt RL, Pelicci PG, Ley TJ.
Altered myeloid development and acute leukemia in transgenic mice expressing PML-RAR alpha under control of cathepsin G regulatory sequences.
Blood.
1997;89:376-387
9.
He LZ, Tribioli C, Rivi R, et al.
Acute leukemia with promyelocytic features in PML/RARalpha transgenic mice.
Proc Natl Acad Sci U S A.
1997;94:5302-5307
10.
Kogan SC, Hong SH, Shultz DB, Privalsky ML, Bishop JM.
Leukemia initiated by PMLRARalpha: the PML domain plays a critical role while retinoic acid-mediated transactivation is dispensable.
Blood.
2000;95:1541-1550 11. Lavau C, Heard JM, Danos O, Dejean A. Retroviral vectors for the transduction of the PML-RARalpha fusion product of acute promyelocytic leukemia. Exp Hematol. 1996;24:544-551[Medline] [Order article via Infotrieve]. 12. Pandolfi PP. In vivo analysis of the molecular genetics of acute promyelocytic leukemia. Oncogene. 2001;20:5726-5735[CrossRef][Medline] [Order article via Infotrieve].
13.
Westervelt P, Ley TJ.
Seed versus soil: the importance of the target cell for transgenic models of human leukemias [see comments].
Blood.
1999;93:2143-2148
14.
Grignani F, Kinsella T, Mencarelli A, et al.
High-efficiency gene transfer and selection of human hematopoietic progenitor cells with a hybrid EBV/retroviral vector expressing the green fluorescence protein.
Cancer Res.
1998;58:14-19 15. Minucci S, Maccarana M, Cioce M, et al. Oligomerization of RAR and AML1 transcription factors as a novel mechanism of oncogenic activation. Mol Cell. 2000;5:811-820[CrossRef][Medline] [Order article via Infotrieve].
16.
Du C, Redner RL, Cooke MP, Lavau C.
Overexpression of wild-type retinoic acid receptor alpha (RARalpha) recapitulates retinoic acid-sensitive transformation of primary myeloid progenitors by acute promyelocytic leukemia RARalpha-fusion genes.
Blood.
1999;94:793-802
17.
Lo Coco F, Diverio D, Falini B, Biondi A, Nervi C, Pelicci PG.
Genetic diagnosis and molecular monitoring in the management of acute promyelocytic leukemia.
Blood.
1999;94:12-22
18.
Flenghi L, Fagioli M, Tomassoni L, et al.
Characterization of a new monoclonal antibody (PG-M3) directed against the aminoterminal portion of the PML gene product: immunocytochemical evidence for high expression of PML proteins on activated macrophages, endothelial cells, and epithelia.
Blood.
1995;85:1871-1880
19.
Le Beau MM, Bitts S, Davis EM, Kogan SC.
Recurring chromosomal abnormalities in leukemia in PML-RARA transgenic mice parallel human acute promyelocytic leukemia.
Blood.
2002;99:2985-2991 20. Specchia G, Cuneo A, Liso V, et al. A novel translocation t(1;7)(p36;q34) in three patients with acute myeloid leukaemia. Br J Haematol. 1999;105:208-214[CrossRef][Medline] [Order article via Infotrieve].
21.
Angulo A, Suto C, Heyman RA, Ghazal P.
Characterization of the sequences of the human cytomegalovirus enhancer that mediate differential regulation by natural and synthetic retinoids.
Mol Endocrinol.
1996;10:781-793 22. Neame PB, Soamboonsrup P, Leber B, et al. Morphology of acute promyelocytic leukemia with cytogenetic or molecular evidence for the diagnosis: characterization of additional microgranular variants. Am J Hematol. 1997;56:131-142[CrossRef][Medline] [Order article via Infotrieve]. 23. Foley R, Soamboonsrup P, Carter RF, et al. CD34-positive acute promyelocytic leukemia is associated with leukocytosis, microgranular/hypogranular morphology, expression of CD2 and bcr3 isoform. Am J Hematol. 2001;67:34-41[CrossRef][Medline] [Order article via Infotrieve].
24.
Pollock JL, Westervelt P, Kurichety AK, Pelicci PG, Grisolano JL, Ley TJ.
A bcr-3 isoform of RARalpha-PML potentiates the development of PML-RARalpha-driven acute promyelocytic leukemia.
Proc Natl Acad Sci U S A.
1999;96:15103-15108
25.
Zimonjic DB, Pollock JL, Westervelt P, Popescu NC, Ley TJ.
Acquired, nonrandom chromosomal abnormalities associated with the development of acute promyelocytic leukemia in transgenic mice.
Proc Natl Acad Sci U S A.
2000;97:13306-13311 26. Minucci S, Nervi C, Lo Coco F, Pelicci PG. Histone deacetylases: a common molecular target for differentiation treatment of acute myeloid leukemias? Oncogene. 2001;20:3110-3115[CrossRef][Medline] [Order article via Infotrieve].
© 2002 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  E. Radaelli, F. Marchesi, V. Patton, and E. Scanziani Diagnostic Exercise: Sudden Death in a Mouse with Experimentally Induced Acute Myeloid Leukemia Vet. Pathol., November 1, 2009; 46(6): 1301 - 1305. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Morey, C. Brenner, F. Fazi, R. Villa, A. Gutierrez, M. Buschbeck, C. Nervi, S. Minucci, F. Fuks, and L. Di Croce MBD3, a Component of the NuRD Complex, Facilitates Chromatin Alteration and Deposition of Epigenetic Marks Mol. Cell. Biol., October 1, 2008; 28(19): 5912 - 5923. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Hoemme, A. Peerzada, G. Behre, Y. Wang, M. McClelland, K. Nieselt, M. Zschunke, C. Disselhoff, S. Agrawal, F. Isken, et al. Chromatin modifications induced by PML-RAR{alpha} repress critical targets in leukemogenesis as analyzed by ChIP-Chip Blood, March 1, 2008; 111(5): 2887 - 2895. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. van Wageningen, M. C. Breems-de Ridder, J. Nigten, G. Nikoloski, C. A. J. Erpelinck-Verschueren, B. Lowenberg, T. de Witte, D. G. Tenen, B. A. van der Reijden, and J. H. Jansen Gene transactivation without direct DNA binding defines a novel gain-of-function for PML-RAR{alpha} Blood, February 1, 2008; 111(3): 1634 - 1643. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. T. Chan, J. L. Kutok, I. R. Williams, S. Cohen, S. Moore, H. Shigematsu, T. J. Ley, K. Akashi, M. M. Le Beau, and D. G. Gilliland Oncogenic K-ras cooperates with PML-RAR{alpha} to induce an acute promyelocytic leukemia-like disease Blood, September 1, 2006; 108(5): 1708 - 1715. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. B. Kindle, P. J. F. Troke, H. M. Collins, S. Matsuda, D. Bossi, C. Bellodi, E. Kalkhoven, P. Salomoni, P. G. Pelicci, S. Minucci, et al. MOZ-TIF2 Inhibits Transcription by Nuclear Receptors and p53 by Impairment of CBP Function Mol. Cell. Biol., February 1, 2005; 25(3): 988 - 1002. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. A. Lane and T. J. Ley Neutrophil Elastase Is Important for PML-Retinoic Acid Receptor {alpha} Activities in Early Myeloid Cells Mol. Cell. Biol., January 1, 2005; 25(1): 23 - 33. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
P. Westervelt, A. A. Lane, J. L. Pollock, K. Oldfather, M. S. Holt, D. B. Zimonjic, N. C. Popescu, J. F. DiPersio, and T. J. Ley High-penetrance mouse model of acute promyelocytic leukemia with very low levels of PML-RAR{alpha} expression Blood, September 1, 2003; 102(5): 1857 - 1865. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2002 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
originated by the
t(15;17) chromosomal translocation
) and GR1
(
). (C) Control, empty, or PML-RAR cells were plated as described.
Colonies were pooled and 10 000 cells were reseeded in methylcellulose
plates. This procedure was repeated twice, and in the third plating RA
was also included in the medium. (D) Typical morphology of the colonies
observed in control (× 50), or PML-RAR expressing cells (× 100).
The "clusters" of PML-RAR-expressing cells are
particularly evident from the second plating. Control colonies derive
from cells infected with the empty vector. Identical results were
obtained from uninfected cells. (E) Wright-Giemsa staining of PML-RAR
pooled colonies (third plating), after cytospins. (Fi) Western
blot analysis using an anti-RAR antibody of sorted cells from control
and PML-RAR infections. The arrow indicates the PML-RAR protein. (Fii)
DNA-PCR analysis of several individual colonies (lanes 3-8) obtained
from growing PML-RAR expressing cells in semisolid medium. Lane 1:
negative control, from one control colony; lane 2: positive control,
from plasmid DNA. The arrow indicates the specific, amplified PCR
product. (Gi) PML expression pattern in lin
