|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Prepublished online as a Blood First Edition Paper on December 5, 2002; DOI 10.1182/blood-2002-06-1656.
HEMATOPOIESIS
From the Center for Cancer Research, the Department of
Biology, and the Whitehead Institute, Massachusetts Institute of
Technology, Cambridge; the Department of Medicine, Children's
Hospital, Boston; and the Department of Pediatrics, Harvard Medical
School, Boston, MA.
The WT1 tumor-suppressor gene is
expressed by many forms of acute myeloid leukemia. Inhibition of this
expression can lead to the differentiation and reduced growth of
leukemia cells and cell lines, suggesting that WT1 participates in
regulating the proliferation of leukemic cells. However, the role of
WT1 in normal hematopoiesis is not well understood. To investigate this
question, we have used murine cells in which the WT1 gene
has been inactivated by homologous recombination. We have found that
cells lacking WT1 show deficits in hematopoietic stem cell function.
Embryonic stem cells lacking WT1, although contributing
efficiently to other organ systems, make only a minimal contribution to
the hematopoietic system in chimeras, indicating that hematopoietic
stem cells lacking WT1 compete poorly with healthy stem
cells. In addition, fetal liver cells lacking WT1 have an
approximately 75% reduction in erythroid blast-forming unit (BFU-E),
erythroid colony-forming unit (CFU-E), and colony-forming
unit-granulocyte macrophage-erythroid-megakaryocyte (CFU-GEMM).
However, transplantation of fetal liver hematopoietic cells lacking
WT1 will repopulate the hematopoietic system of an
irradiated adult recipient in the absence of competition. We conclude
that the absence of WT1 in hematopoietic cells leads to functional
defects in growth potential that may be of consequence to leukemic
cells that have alterations in the expression of WT1.
(Blood. 2003;101:2570-2574) WT1 was isolated as a tumor-suppressor
gene on the basis of its deletion in a subset of patients with Wilms
tumor, a pediatric cancer of the kidney.1 In addition to
its mutation in 5% to 10% of Wilms tumor patients, mutations of
WT1 have been implicated in a variety of other cancers
including mesotheliomas, desmoplastic round cell tumors, and
leukemias.1 WT1 encodes a protein of 52 to 54 kD, containing 4 zinc fingers of the Cys2 His2 class, that has been
demonstrated to act as transcription factor.1 Potential
transcriptional targets for WT1 include a variety of growth factor
and growth factor receptor genes.1 An additional role for
WT1 in RNA processing has also been proposed.1
Many leukemias, including up to 80% of acute myeloid leukemias (AMLs),
have been demonstrated to express WT1.2-10 Mutations of WT1 have been found in approximately 5% to 10% of AMLs,
similar to the level seen in Wilms tumors, and sporadically in a
variety of other leukemias.11-18 Manipulation of WT1
expression in leukemia cells results in alterations in differentiation
and growth potential.19-28 In the treatment of leukemia,
expression of WT1 in the peripheral blood and bone marrow has been
suggested to be an indicator of clinical relapse. Attempts have been
made to correlate WT1 expression with progression of disease
and prognosis in several types of hematologic
malignancies.3,29-37 The clinical use of anti-WT1 cytotoxic T-lymphocytes (CTLs) for preferential destruction of WT1-expressing malignant cells in leukemia is under
evaluation.38-41
A number of lines of evidence suggest that WT1 may play a role in
regulating hematopoiesis. In the human hematopoietic system, WT1
appears to be normally expressed in bone marrow CD34+
cells.2,20,42-47 WT1 mRNA levels are modulated
during the in vitro differentiation of hematopoietic cells and leukemic
cells lines.23,44,48,49 In vitro manipulation of WT1
expression has been shown to affect the growth potential and
differentiation of hematopoietic cells, leukemia cells, and cell
lines.19-28
Despite the use of WT1 as a therapeutic target and an indicator of
prognosis in leukemia, the role of the WT1 gene in
hematopoiesis is not clearly understood. We have used the targeted
deletion of WT1 gene function in the mouse to directly
investigate the role of WT1 in normal hematopoiesis. Because the
absence of WT1 is lethal at embryonic day 13.5 (E13.5) to E15.5,
we have used a combination of approaches Mice
Chimera production
GPI assays Glucose-phosphate-isomerase (GPI) assays were performed as described.51 In brief, samples were lysed by 4 cycles of freezing and thawing, and GPI isoforms were analyzed by cellulose acetate electrophoresis of the samples followed by detection in an agarose gel containing fructose-6-phosphate, nicotinamide-adenine dinucleotide phosphate (NADP), methyl thiazolyl tetrazolium (MTT), phenazine methylsulfate (PMS), and glucose-6-phosphate dehydrogenase (all reagents were from Sigma [St Louis, MO]) at room temperature. This method permits the discrimination of the isoforms GPI-Iaa and GPI-Ibb present in 129Sv and C57BL strains, respectively.Reconstitution of irradiated mice Mating pairs of mice heterozygous for the absence of WT1 were checked daily for the presence of a vaginal plug. The morning of detection was designated as E0.5. On E13.5, pregnant females were killed and the embryos were harvested. Each fetal liver was dissociated in sterile phosphate-buffered saline (PBS) and quantitated. Trypan blue-excluding nucleated cells (1 × 106) were injected in 200 µL PBS retro-orbitally into female C57/B6 Ly5.2 (8- to 10-week-old) recipients. These mice were lethally irradiated in 2 doses of 8 Gy and 4 Gy separated by 3 hours before receiving the donor cells.Flow cytometry For analysis of the contribution of ES cells to the peripheral blood and bone marrow, a 2-month-old female 90% (by coat color) chimera was anesthetized using tribromoethanol. One milliliter blood was obtained by heart puncture. Red cells were removed using 1% dextran T 500 followed by ammonium chloride lysis. Bone marrow cells were obtained by flushing both hind femurs and tibias into PBS. Peripheral blood and bone marrow cells were fractionated on a FACStar Plus (Becton Dickinson, San Jose, CA) using forward scatter and side scatter gates and then analyzed for ES cell contribution using the GPI assay. For analysis of the hematopoietic system of animals reconstituted with embryonic liver, blood samples were collected from the retro-orbital sinus. Red cells were removed using 1% dextran T 500 followed by ammonium chloride lysis. Cells were washed with PBS and then incubated with saturating quantities of fluorescein isothiocyanate (FITC)-conjugated anti-Ly5.2 (Pharmingen, San Diego, CA) and biotin-conjugated anti-Ly5.1 followed by phycoerythrin (PE)-conjugated streptavidin. Cells were washed and analyzed using FACStar Plus (Becton Dickinson) with forward scatter and side scatter gates. FITC emission was collected using a 530/30 dichroic filter (DF), and PE emission was collected using a 575/26 DF. A 560SP dichroic mirror (DM) was used to split the FITC and PE emissions.In vitro progenitor cell assays Mice heterozygous for the absence of WT1 were mated, and the morning of detection of a vaginal plug was designated as E0.5. On E14.5, pregnant females were killed and the embryos were harvested. Each fetal liver was dissociated in 1 mL Iscove/2% fetal bovine serum (FBS) and was cultured in Methocult GF M3434 (StemCell Technologies, Vancouver, BC, Canada) in triplicate at 1.5 × 104 cells/mL at 37°C and 5% CO2. CFU-Es were counted after 2 days of culture, CFU-GMs after 10 days, and CFU-GEMMs after 14 days. Significance was determined using analysis of variance (ANOVA) in the Statview statistical analysis program (SAS, Cary, NC).
Embryonic stem cells lacking WT1 do not contribute to the hematopoietic system Mice lacking WT1 develop to between day E13.5 and E15.5 before they die in utero, apparently of hemorrhage into the pericardial cavity.50,52 At the time of death, the embryos appear to have a grossly normal hematopoietic system. To further investigate the hematopoietic functions of the WT1 gene, chimeric adult mice were generated from ES cells lacking WT1 and normal C57/Bl6 blastocysts to allow hematopoietic cells lacking WT1 the opportunity to develop past 15.5 days of embryogenesis. Tissues from mice with 90% to 100% agouti coat color were analyzed for ES cell contribution using the GPI assay. ES cells lacking WT1 contributed strongly to a number of organs, including the liver. However, the ES cell contribution to whole blood was at or below the detection threshold of the GPI assay system (approximately 5% ES cell or 2% 129 isoform because both forms of GPI were contained by the ES cells) (Figure 1). Peripheral blood and bone marrow from a 90% agouti coat color mouse were fractionated by size and side scatter into red cell, lymphocyte, and granulocyte populations. GPI assays on these cell fractions also did not detect any significant contribution by ES cells (data not shown). These results demonstrate that ES cells lacking WT1 failed to contribute to these populations significantly in adults.
Cells lacking WT1 can repopulate the mouse hematopoietic system Because no abnormalities in early hematopoiesis had been reported in embryos lacking WT1, the results on the chimeric animals suggested it was possible that WT1 was necessary to effectively populate the hematopoietic system of the adult animal. The ability to distinguish donor from recipient cells by the polymorphism of the Ly5 antigen was used to assess the ability of early hematopoietic cells lacking WT1 to populate the adult hematopoietic lineages. NIH C57 female mice congenic for the Ly5.1 antigen were lethally irradiated and then reconstituted with 1 × 106 fetal liver cells from E13.5 WT1 mutant, WT1 heterozygote, or wild-type embryos. Fourteen days after reconstitution, 4 of 4 of the buffer-injected control mice had died or been killed because of extreme morbidity, whereas 2 of 2 mice injected with cells from wild-type embryos, 7 of 7 injected with WT1 heterozygote cells, and 9 of 11 injected with WT1 mutant cells survived. At 4 weeks these mice all survived and had normal hematocrit levels of approximately 44%. At 2 months only one additional mouse reconstituted with cells lacking WT1 had died of unknown cause (Table 1). When peripheral blood was analyzed for its donor/host composition using staining with anti-Ly5.2 and Ly5.1, more than 90% of the staining cells were donor derived regardless of the genotype of the donor embryo. There was no alteration in this ratio when side and forward scatter were used to subdivide the cells into lymphocyte, granulocyte, and monocyte populations (Figure 2). These results demonstrate that stem cells lacking WT1 are capable of reconstituting and maintaining the adult hematopoietic system for at least 2 months after transplantation.
Cells lacking WT1 have reduced in vitro colony-forming potential The ability of stem cells lacking WT1 to reconstitute the hematopoietic system of lethally irradiated adults, together with the failure of ES cells lacking WT1 to contribute effectively to the hematopoietic system of chimeric embryos in a competitive situation, suggested that WT1 might play a role in regulating the proliferation of differentiating hematopoietic cells. To characterize the growth and differentiation properties of hematopoietic cells lacking WT1 in vitro, fetal liver cells from E14.5 WT1 mutant, heterozygous, and wild-type embryos were placed in culture. Cultures from mutant embryos gave less than 30% the number of erythrocyte, granulocyte macrophage, and granulocyte macrophage erythroidmegakaryocyte colonies as those derived from
WT/WT embryos. Cultures of WT1 heterozygous cells gave less
than 50% of the numbers of all classes of colonies as wild-type
cultures (Figure 3). This suggests that the WT1 effect is quantitative and that upward or downward regulation of the level of WT1 expression can play a significant role in the
number, proliferative potential, or survival of hematopoietic precursors. To determine whether the decrease in colony-forming potential was mirrored by hematologic deficiencies after birth, blood
counts were taken on 31 wild-type and 21 heterozygous offspring. No
statistically significant differences were found (data not shown).
Hematopoietic cells lacking WT1 demonstrate functional deficits. When chimeric mice are formed between normal blastocysts and ES cells lacking WT1, contributions to the blood by the WT1-negative cells are negligible compared with those previously seen with normal 129 ES cells, suggesting a competitive disadvantage for hematopoietic stem cells lacking WT1.53,54 When WT1-negative fetal liver cells are tested by in vitro differentiation assay, colony numbers are significantly reduced, suggesting that the regulation of the formation or function of hematopoietic progenitors is also compromised. Despite these deficits, the observation that murine E13.5 fetal liver cells lacking WT1 can repopulate a lethally irradiated adult host demonstrates that WT1 function is not absolutely essential either for the initial development of the hematopoietic system or for the differentiation of its various lineages in the adult. These defects of the hematopoietic system suggest that, as a transcription factor expressed in early members of the hematopoietic lineage, WT1 may play a role in regulating the expression of one or more genes that, in turn, are significant regulators of function of hematopoietic stem cells, progenitors, or both. WT1 has been shown to be capable of controlling the transcription of members of several classes of genes, which could account for this apparent regulation of hematopoiesis. These include a number of cell-signaling molecules, their receptors, and genes involved in key decision points in regulating the balance between cell cycle progression, differentiation, and apoptosis.1 The phenotypes of hematopoietic cells deficient for a number of such genes show significant resemblance to the phenotypes of WT1-deficient hematopoietic cells. The phenotype of mice with targeted deletion of p21 cyclin-dependent kinase inhibitor may be of particular relevance to the phenotype of WT1-negative hematopoietic cells. The absence of p21 has a clear negative effect on the long-term proliferative capacity of hematopoietic stem cells because of an alteration in the balance between cell cycling and quiescence.55 p21-Deficient hematopoietic stem cells show greater 5-fluorouracil (5-FU) sensitivity than normal cells, presumably as a consequence of an increase in the proportion of actively cycling hematopoietic stem cells. In addition, p21-deficient cells are unable to undergo repeated serial bone marrow transplantation, suggesting that increased cycling leads to early depletion of these cells. WT1 has been shown to transcriptionally activate p21, and it may play a critical role in the regulation of p21 in hematopoietic stem cells.20,56 If so, WT1 absence could lead to a decrease in the competitive efficiency of stem cells through a failure to properly up-regulate p21 expression. Mutations of the tyrosine kinase receptors c-kit and flk2/flt3 lead to hematopoietic phenotypes that show parallels to the phenotype of WT1 mutant mice. In each case, the hematopoietic system of animals with reduced or absent function for these tyrosine kinase receptors have decreased competitive efficiency of hematopoietic stem cells and negative effects on the differentiation of committed progenitors.57,58 Deficiencies in stem cells and committed progenitors resembling the WT1 defect have also been described for mice lacking the thrombopoietin (TPO) receptor c-mpl and for mice lacking the growth factors interleukin-6 (IL-6), kit ligand and Lif, and the HOX cofactor Pbx1.57,59-62 Hematopoietic defects of WT1-deficient mice could be the consequence of the failure of regulation by WT1 of one or more hematopoietic signaling pathways. If receptor expression levels are reduced by the absence of WT1, a hematopoietic phenotype resembling the WT1-negative hematopoietic cell would be expected. Alternatively, WT1 may function downstream and be involved in mediating the response to the activation of the pathway. Genes such as p21 and Pbx1 may be direct WT1 targets or may participate in the same signaling pathways involved in hematopoiesis. The transcriptional role of WT1 in hematopoiesis is likely to be complex. In a study of ectopic expression of WT1 in human cord blood progenitors and leukemic cells, WT1 expression appears to impact primitive hematopoietic precursors and lineage-specific progenitors.20 Our observations on WT1-deficient mice are consistent with this view. The targets of WT1 in committed lineage progenitors may differ from the targets of WT1 in primitive hematopoietic stem cells, adding levels to the understanding of the impact of WT1 on hematopoiesis. These findings on WT1-deficient murine hematopoietic stem cells permit some suggestions to be made regarding the role of WT1 in leukemia. WT1 expression appears to activate genes that confer a competitive advantage to hematopoietic stem cells. WT1 expression in leukemic cells may play a significant role during the initial expansion of a leukemic clone or may be required for the effective preferential propagation of the leukemic clone. The occurrence of inactivating mutations to WT1 in some leukemias may reflect the impact of WT1 on the differentiation of later progenitors, permitting the leukemic clone to be maintained in a less differentiated state. Detailed understanding of the impact of WT1 expression in each of the contexts will be of great interest in establishing the usefulness of WT1 expression as an indicator of leukemic status and as a potential target for therapy in leukemia.
We thank Dr Rudolf Jaenisch for support during the early stages of this work. We also thank Glenn A. Paradis of the MIT Flow Cytometry Laboratory for assistance with flow cytometry.
Submitted June 5, 2002; accepted October 30, 2002.
Prepublished online as Blood First Edition Paper, December 5, 2002; DOI 10.1182/blood-2002-06-1656.
Supported by National Institutes of Health grant 2-PO1-CA42063-17, National Center for Human Genome Research (NCHGR) grant HG00061 (G.M.S.), National Cancer Institute Individual Research Service Award grant CA60464 (J.A.A.), and the Charles A. King Trust Medical Foundation.
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: David Housman, Center for Cancer Research, Massachusetts Institute of Technology, 77 Main St, Cambridge, MA 02139; e-mail: dhousman{at}mit.edu.
1. Scharnhorst V, van der Eb AJ, Jochemsen AG. WT1 proteins: functions in growth and differentiation. Gene. 2001;273:141-161[CrossRef][Medline] [Order article via Infotrieve]. 2. Baird PN, Simmons PJ. Expression of the Wilms' tumor gene (WT1) in normal hemopoiesis [see comments]. Exp Hematol. 1997;25:312-320[Medline] [Order article via Infotrieve].
3.
Bergmann L, Miething C, Maurer U, et al.
High levels of Wilms' tumor gene (wt1) mRNA in acute myeloid leukemias are associated with a worse long-term outcome.
Blood.
1997;90:1217-1225 4. Bergmann L, Maurer U, Weidmann E. Wilms tumor gene expression in acute myeloid leukemias. Leuk Lymphoma. 1997;25:435-443[Medline] [Order article via Infotrieve]. 5. Im HJ, Kong G, Lee H. Expression of Wilms tumor gene (WT1) in children with acute leukemia. Pediatr Hematol Oncol. 1999;16:109-118[CrossRef][Medline] [Order article via Infotrieve]. 6. Inoue K, Ogawa H, Sonoda Y, et al. Aberrant overexpression of the Wilms tumor gene (WT1) in human leukemia. Blood. 1997;89:14051412. 7. Menssen HD, Renkl HJ, Rodeck U, et al. Presence of Wilms' tumor gene (wt1) transcripts and the WT1 nuclear protein in the majority of human acute leukemias. Leukemia. 1995;9:1060-1067[Medline] [Order article via Infotrieve]. 8. Menssen HD, Renkl HJ, Rieder H, et al. Distinction of eosinophilic leukaemia from idiopathic hypereosinophilic syndrome by analysis of Wilms' tumour gene expression. Br J Haematol. 1998;101:325-334[CrossRef][Medline] [Order article via Infotrieve]. 9. Menssen HD, Schmidt A, Bartelt S, et al. Analysis of Wilms tumor gene (WT1) expression in acute leukemia patients with special reference to the differential diagnosis between eosinophilic leukemia and idiopathic hypereosinophilic syndromes. Leuk Lymphoma. 2000;36:285-294[Medline] [Order article via Infotrieve]. 10. Sako M, Ogawa H, Okamura J, et al. Abnormal expression of the Wilms' tumor gene WT1 in juvenile chronic myeloid leukemia and infantile monosomy 7 syndrome [letter]. Leuk Res. 1998;22:965-967[CrossRef][Medline] [Order article via Infotrieve]. 11. Algar E, Blackburn D, Kromykh T, Taylor G, Smith P. Mutation analysis of the WT1 gene in sporadic childhood leukemia. Leukemia. 1997;11:110-113[CrossRef][Medline] [Order article via Infotrieve]. 12. Carapeti M, Goldman JM, Cross NC. Dominant-negative mutations of the Wilms' tumour predisposing gene (WT1) are infrequent in CML blast crisis and de novo acute leukaemia. Eur J Haematol. 1997;58:346-349[Medline] [Order article via Infotrieve]. 13. Hosoya N, Miyagawa K, Mitani K, Yazaki Y, Hirai H. Mutation analysis of the WT1 gene in myelodysplastic syndromes. Jpn J Cancer Res. 1998;89:821-824[CrossRef][Medline] [Order article via Infotrieve].
14.
King-Underwood L, Renshaw J, Pritchard-Jones K.
Mutations in the Wilms' tumor gene WT1 in leukemias.
Blood.
1996;87:2171-2179
15.
King-Underwood L, Pritchard-Jones K.
Wilms' tumor (WT1) gene mutations occur mainly in acute myeloid leukemia and may confer drug resistance.
Blood.
1998;91:2961-2968 16. Miyagawa K, Hayashi Y, Fukuda T, Mitani K, Hirai H, Kamiya K. Mutations of the WT1 gene in childhood nonlymphoid hematological malignancies. Genes Chromosomes Cancer. 1999;25:176-183[CrossRef][Medline] [Order article via Infotrieve]. 17. Mori N, Okada M, Motoji T, Mizoguchi H. Mutation of the WT1 gene in myelodysplastic syndrome and acute myeloid leukaemia post myelodysplastic syndrome. Br J Haematol. 1999;105:844-845[CrossRef][Medline] [Order article via Infotrieve]. 18. Pritchard-Jones K, King-Underwood L. The Wilms tumour gene WT1 in leukaemia. Leuk Lymphoma. 1997;27:207-220[Medline] [Order article via Infotrieve]. 19. Algar EM, Khromykh T, Smith SI, Blackburn DM, Bryson GJ, Smith PJ. A WT1 antisense oligonucleotide inhibits proliferation and induces apoptosis in myeloid leukaemia cell lines. Oncogene. 1996;12:1005-1014[Medline] [Order article via Infotrieve]. 20. Ellisen LW, Carlesso N, Cheng T, Scadden DT, Haber DA. The Wilms tumor suppressor WT1 directs stage-specific quiescence and differentiation of human hematopoietic progenitor cells. EMBO J. 2001;20:1897-1909[CrossRef][Medline] [Order article via Infotrieve]. 21. Deuel TF, Guan LS, Wang ZY. Wilms' tumor gene product WT1 arrests macrophage differentiation of HL-60 cells through its zinc-finger domain. Biochem Biophys Res Commun. 1999;254:192-196[CrossRef][Medline] [Order article via Infotrieve].
22.
Inoue K, Tamaki H, Ogawa H, et al.
Wilms' tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells.
Blood.
1998;91:2969-2976
23.
Smith SI, Weil D, Johnson GR, Boyd AW, Li CL.
Expression of the Wilms' tumor suppressor gene, WT1, is upregulated by leukemia inhibitory factor and induces monocytic differentiation in M1 leukemic cells.
Blood.
1998;91:764-773
24.
Smith SI, Down M, Boyd AW, Li CL.
Expression of the Wilms' tumor suppressor gene, WT1, reduces the tumorigenicity of the leukemic cell line M1 in C.B-17 scid/scid mice.
Cancer Res.
2000;60:808-814 25. Svedberg H, Chylicki K, Baldetorp B, Rauscher FJ 3rd, Gullberg U. Constitutive expression of the Wilms' tumor gene (WT1) in the leukemic cell line U937 blocks parts of the differentiation program. Oncogene. 1998;16:925-932[CrossRef][Medline] [Order article via Infotrieve]. 26. Svedberg H, Chylicki K, Gullberg U. Downregulation of Wilms' tumor gene (WT1) is not a prerequisite for erythroid or megakaryocytic differentiation of the leukemic cell line K562. Exp Hematol. 1999;27:1057-1062[CrossRef][Medline] [Order article via Infotrieve].
27.
Yamagami T, Sugiyama H, Inoue K, et al.
Growth inhibition of human leukemic cells by WT1 (Wilms tumor gene) antisense oligodeoxynucleotides: implications for the involvement of WT1 in leukemogenesis.
Blood.
1996;87:2878-2884 28. Yamagami T, Ogawa H, Tamaki H, et al. Suppression of Wilms' tumor gene (WT1) expression induces G2/M arrest in leukemic cells [letter]. Leuk Res. 1998;22:383-384[CrossRef][Medline] [Order article via Infotrieve]. 29. Gaiger A, Schmid D, Heinze G, et al. Detection of the WT1 transcript by RT-PCR in complete remission has no prognostic relevance in de novo acute myeloid leukemia. Leukemia. 1998;12:1886-1894[CrossRef][Medline] [Order article via Infotrieve]. 30. Gaiger A, Linnerth B, Mann G, et al. Wilms' tumour gene (wt1) expression at diagnosis has no prognostic relevance in childhood acute lymphoblastic leukaemia treated by an intensive chemotherapy protocol. Eur J Haematol. 1999;63:86-93[Medline] [Order article via Infotrieve].
31.
Inoue K, Sugiyama H, Ogawa H, et al.
WT1 as a new prognostic factor and a new marker for the detection of minimal residual disease in acute leukemia.
Blood.
1994;84:3071-3079
32.
Inoue K, Ogawa H, Yamagami T, et al.
Long-term follow-up of minimal residual disease in leukemia patients by monitoring WT1 (Wilms tumor gene) expression levels.
Blood.
1996;88:2267-2278 33. Ogawa H, Tsuboi A, Oji Y, et al. Successful donor leukocyte transfusion at molecular relapse for a patient with acute myeloid leukemia who was treated with allogenic bone marrow transplantation: importance of the monitoring of minimal residual disease by WT1 assay. Bone Marrow Transplant. 1998;21:525-527[CrossRef][Medline] [Order article via Infotrieve]. 34. Schmid D, Heinze G, Linnerth B, et al. Prognostic significance of WT1 gene expression at diagnosis in adult de novo acute myeloid leukemia. Leukemia. 1997;11:639-643[CrossRef][Medline] [Order article via Infotrieve]. 35. Sugiyama H. Wilms tumor gene (WT1) as a new marker for the detection of minimal residual disease in leukemia. Leuk Lymphoma. 1998;30:55-61[Medline] [Order article via Infotrieve].
36.
Tamaki H, Ogawa H, Inoue K, et al.
Increased expression of the Wilms tumor gene (WT1) at relapse in acute leukemia [letter].
Blood.
1996;88:4396-4398 37. Tamaki H, Ogawa H, Ohyashiki K, et al. The Wilms' tumor gene WT1 is a good marker for diagnosis of disease progression of myelodysplastic syndromes. Leukemia. 1999;13:393-399[CrossRef][Medline] [Order article via Infotrieve].
38.
Gao L, Bellantuono I, Elsasser A, et al.
Selective elimination of leukemic CD34(+) progenitor cells by cytotoxic T lymphocytes specific for WT1.
Blood.
2000;95:2198-2203
39.
Ohminami H, Yasukawa M, Fujita S.
HLA class I-restricted lysis of leukemia cells by a CD8(+) cytotoxic T-lymphocyte clone specific for WT1 peptide.
Blood.
2000;95:286-293 40. Oka Y, Elisseeva OA, Tsuboi A, et al. Human cytotoxic T-lymphocyte responses specific for peptides of the wild-type Wilms' tumor gene (WT1) product. Immunogenetics. 2000;51:99-107[CrossRef][Medline] [Order article via Infotrieve].
41.
Oka Y, Udaka K, Tsuboi A, et al.
Cancer immunotherapy targeting Wilms' tumor gene WT1 product.
J Immunol.
2000;164:1873-1880
42.
Fraizer GC, Patmasiriwat P, Zhang X, Saunders GF.
Expression of the tumor suppressor gene WT1 in both human and mouse bone marrow [letter].
Blood.
1995;86:4704-4706
43.
Maurer U, Weidmann E, Karakas T, Hoelzer D, Bergmann L.
Wilms tumor gene (wt1) mRNA is equally expressed in blast cells from acute myeloid leukemia and normal CD34+ progenitors [letter].
Blood.
1997;90:4230-4232 44. Maurer U, Brieger J, Weidmann E, Mitrou PS, Hoelzer D, Bergmann L. The Wilms' tumor gene is expressed in a subset of CD34+ progenitors and downregulated early in the course of differentiation in vitro. Exp Hematol. 1997;25:945-950[Medline] [Order article via Infotrieve].
45.
Menssen HD, Renkl HJ, Entezami M, Thiel E.
Wilms' tumor gene expression in human CD34+ hematopoietic progenitors during fetal development and early clonogenic growth [letter].
Blood.
1997;89:3486-3487 46. Pritchard-Jones K, Renshaw J. Regarding "expression of the Wilms' tumor gene (WT1) in normal hemopoiesis" by P.N. Baird and P.J. Simmons Experimental Hematology 1997;25:312-320[Medline] [Order article via Infotrieve][letter; comment]. Exp Hematol. 1997;25:1311-1312. 47. Timens W, Kamps WA. Hemopoiesis in human fetal and embryonic liver. Microsc Res Tech. 1997;39:387-397[CrossRef][Medline] [Order article via Infotrieve]. 48. Phelan SA, Lindberg C, Call KM. Wilms' tumor gene, WT1, mRNA is down-regulated during induction of erythroid and megakaryocytic differentiation of K562 cells. Cell Growth Differ. 1994;5:677-686[Abstract].
49.
Sekiya M, Adachi M, Hinoda Y, Imai K, Yachi A.
Downregulation of Wilms' tumor gene (wt1) during myelomonocytic differentiation in HL60 cells.
Blood.
1994;83:1876-1882 50. Kreidberg JA, Sariola H, Loring JM, et al. WT-1 is required for early kidney development. Cell. 1993;74:679-691[CrossRef][Medline] [Order article via Infotrieve].
51.
Yang JT, Rando TA, Mohler WA, Rayburn H, Blau HM, Hynes RO.
Genetic analysis of alpha 4 integrin functions in the development of mouse skeletal muscle.
J Cell Biol.
1996;135:829-835 52. Moore AW, McInnes L, Kreidberg J, Hastie ND, Schedl A. YAC complementation shows a requirement for Wt1 in the development of epicardium, adrenal gland and throughout nephrogenesis. Development. 1999;126:1845-1857[Abstract]. 53. Shirane M, Sawa H, Kobayashi Y, et al. Deficiency of phospholipase C-gamma1 impairs renal development and hematopoiesis. Development. 2001;128:5173-5180[Medline] [Order article via Infotrieve]. 54. Scott EW, Fisher RC, Olson MC, Kehrli EW, Simon MC, Singh H. PU.1 functions in a cell-autonomous manner to control the differentiation of multipotential lymphoid-myeloid progenitors. Immunity. 1997;6:437-447[CrossRef][Medline] [Order article via Infotrieve].
55.
Cheng T, Rodrigues N, Shen H, et al.
Hematopoietic stem cell quiescence maintained by p21cip1/waf1.
Science.
2000;287:1804-1808
56.
Englert C, Maheswaran S, Garvin AJ, Kreidberg J, Haber DA.
Induction of p21 by the Wilms' tumor suppressor gene WT1.
Cancer Res.
1997;57:1429-1434 57. Mackarehtschian K, Hardin JD, Moore KA, Boast S, Goff SP, Lemischka IR. Targeted disruption of the flk2/flt3 gene leads to deficiencies in primitive hematopoietic progenitors. Immunity. 1995;3:147-161[CrossRef][Medline] [Order article via Infotrieve]. 58. Russell ES. Hereditary anemias of the mouse: a review for geneticists. Adv Genet. 1979;20:357-459[Medline] [Order article via Infotrieve].
59.
Alexander WS, Roberts AW, Nicola NA, Li R, Metcalf D.
Deficiencies in progenitor cells of multiple hematopoietic lineages and defective megakaryocytopoiesis in mice lacking the thrombopoietic receptor c-Mpl.
Blood.
1996;87:2162-2170 60. Bernad A, Kopf M, Kulbacki R, Weich N, Koehler G, Gutierrez-Ramos JC. Interleukin-6 is required in vivo for the regulation of stem cells and committed progenitors of the hematopoietic system. Immunity. 1994;1:725-731[CrossRef][Medline] [Order article via Infotrieve]. 61. Escary JL, Perreau J, Dumenil D, Ezine S, Brulet P. Leukaemia inhibitory factor is necessary for maintenance of haematopoietic stem cells and thymocyte stimulation. Nature. 1993;363:361-364[CrossRef][Medline] [Order article via Infotrieve].
62.
Kimura S, Roberts AW, Metcalf D, Alexander WS.
Hematopoietic stem cell deficiencies in mice lacking c-Mpl, the receptor for thrombopoietin.
Proc Natl Acad Sci U S A.
1998;95:1195-1200
© 2003 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  K. M. Kirschner, P. Hagen, C. S. Hussels, M. Ballmaier, H. Scholz, and C. Dame The Wilms' tumor suppressor Wt1 activates transcription of the erythropoietin receptor in hematopoietic progenitor cells FASEB J, August 1, 2008; 22(8): 2690 - 2701. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. M. Kirschner, N. Wagner, K.-D. Wagner, S. Wellmann, and H. Scholz The Wilms Tumor Suppressor Wt1 Promotes Cell Adhesion through Transcriptional Activation of the {alpha}4integrin Gene J. Biol. Chem., October 20, 2006; 281(42): 31930 - 31939. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Rong, L. Cheng, H. Ning, J. Zou, Y. Zhang, F. Xu, L. Liu, Z. Chang, and X.-Y. Fu Wilms' Tumor 1 and Signal Transducers and Activators of Transcription 3 Synergistically Promote Cell Proliferation: A Possible Mechanism in Sporadic Wilms' Tumor Cancer Res., August 15, 2006; 66(16): 8049 - 8057. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Dame, K. M. Kirschner, K. V. Bartz, T. Wallach, C. S. Hussels, and H. Scholz Wilms tumor suppressor, Wt1, is a transcriptional activator of the erythropoietin gene Blood, June 1, 2006; 107(11): 4282 - 4290. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. J. Walter, J. S. Park, R. E. Ries, S. K. M. Lau, M. McLellan, S. Jaeger, R. K. Wilson, E. R. Mardis, and T. J. Ley Reduced PU.1 expression causes myeloid progenitor expansion and increased leukemia penetrance in mice expressing PML-RAR{alpha} PNAS, August 30, 2005; 102(35): 12513 - 12518. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2003 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
/
4, CFU-GM × 10