In this analysis, we conducted a cohort study of more than 500 female mice with a heterozygous GATA-1 gene knockdown allele (GATA-1.05/X). We found that these GATA-1.05/X mice suffer from leukemia with a very high incidence, but such a high incidence of leukemia was not observed in GATA-1-null/X and wild-type mice. The genetic enfeeblement of GATA-1 function was found to elicit two distinct types of leukemia. One is c-Kit+ nonlymphoid leukemia, and the other is CD19+ B-cell leukemia. Closer examination of the GATA-1.05/X leukemic mice revealed that almost all of the mutant mice first developed thrombocytopenia and dyserythropoiesis in their hematopoietic tissues. Some of the mutant mice then proceeded to the accelerating stage with a grossly enlarged livers and spleens, unregulated proliferation of monoclonal leukemic cells, and suppression of normal hematopoietic cells. This course of phenotypic changes shares high similarity to the transformation process of preleukemic myelodysplastic cells to overt leukemia in humans.
The cohort analysis further demonstrates unequivocally that GATA-1 expression at 5% of the normal level is insufficient to sustain normal erythroid differentiation, as GATA-1.05
homozygous and GATA-1.05
/Y pups have never been born (references 32
, and 37
and this study). Another interesting feature of GATA-1.05
/X mice is that they contain two types of hematopoietic cells, owing to the process of X inactivation (15
). In one cell type, the X chromosome bearing the GATA-1.05
allele is inactivated, but the one bearing the wild-type GATA-1
allele is active. These hematopoietic progenitors express normal amounts of GATA-1 and are able to differentiate into their appropriate cell types, including enucleated erythrocytes and platelets. In the other cell type, the X chromosome bearing the GATA-1.05
allele is active, but the one bearing the wild-type GATA-1
allele is inactive. In the latter case, the expression level of GATA-1 in erythroid and megakaryocytic progenitors is very low. This reduction appears to cause both arrest of differentiation and stimulation of proliferation (31
). Therefore, this type of progenitor remains in an immature stage.
Whereas the GATA-1.05/X embryos showed various degrees of anemia depending on the extent of inactivation of the X chromosome with the wild-type GATA-1 allele, most of the GATA-1.05/X mice acquired close to normal erythroid indices after birth. The expression of GATA-1 mRNA was severely repressed in the c-Kit+ leukemic cells. In contrast, GATA-1 mRNA was expressed at higher levels in hematopoietic tissues at the preleukemic stage of GATA-1.05/X mice than in those of wild-type mice. Thus, a compensatory expansion of hematopoietic progenitors with an active wild-type GATA-1 allele appears to take place in response to the anemia. These results further support the notion that the mouse hematopoietic system has the capacity to compensate for the substantial lack of erythroid and megakaryocytic progenitors caused by the heterozygous GATA-1.05 knockdown mutation.
In this regard, it is noteworthy that GFP+
cells, corresponding to late-stage erythroid progenitors (34
), have already accumulated in the livers of GATA-1.05
/X embryos. Histological analyses of the GATA-1.05
/X fetal livers suggest that these cells arise from the immature progenitors with the inactivated wild-type GATA-1
allele. In addition, the neo
gene is actively expressed in the GFP+
cells in type 1 leukemia mice. These results indicate that GFP+
cells are differentiation-arrested erythroid progenitors with an inactivated wild-type GATA-1
-null proerythroblasts are known to undergo apoptosis (42
). Upon examination of apoptosis, we found that there was a significant increase of TUNEL+
cells in the GATA-1
-null/X embryos. Consistent with the previous observations on the apoptosis of GATA-1
-null proerythroblasts in culture (42
), this result indicates that maturation-defective erythroid progenitors succumb to apoptosis in GATA-1
-null/X embryos. Importantly, this process is prevented effectively in the GATA-1.05
/X fetal livers, which display numbers of apoptotic cells similar to those observed in wild-type fetal livers.
We carried out in vitro ES cell differentiation analyses and found that the differentiation of hematopoietic cells derived from GATA-1.05/Y and GATA-1-null/Y mutant ES cells was severely arrested at the LEP stage. The analyses also delineated that erythroid progenitors derived from GATA-1.05/Y and GATA-1-null/Y mutant ES cells proliferate much more vigorously than those derived from the wild-type ES cells (data not shown). In contrast, these two mutant ES cell lines differ sharply in terms of their sensitivities to apoptosis. While hematopoietic cells derived from GATA-1-null/Y ES cells suffer extensively from apoptosis, GATA-1.05/Y ES cells do not suffer from apoptosis, suggesting that the low level of GATA-1 expression is sufficient to prevent hematopoietic cells undergoing apoptosis. These mechanisms are summarized in Fig. .
FIG. 8. Model for the development of leukemia in GATA-1.05/X mice. (A) GATA-1 function during erythroid-cell differentiation of ES cells in vitro. Wild-type ES cells develop into mature erythroid cells depending on the three distinct but cooperative functions (more ...)
Importantly, this conclusion shows excellent agreement with the in vivo analyses that we carried out in this study and strongly argues that the differentiation-arrested LEP erythroblasts are eliminated through active pressure toward apoptosis in the hematopoietic tissues of GATA-1
-null/X embryos. This determination is also in very good agreement with the previous observation that caspase-mediated down-regulation of GATA-1 prevents differentiation of normal proerythroblasts without inducing apoptosis (4
). Thus, the prevention of apoptosis, in conjunction with the differentiation arrest and stimulation of cell proliferation at the LEP stage, provokes the accumulation of erythroid progenitors in the livers of GATA-1.05
/X embryos. These accumulating progenitors provide a pool of cells that may progress into overt leukemia. Based on these observations, we conclude that GATA-1.05
/X mice are in a preleukemic condition from the embryonic stage of development (Fig. ).
Despite the limited number of mice in the GATA-1-null/X cohort, our findings provide important insight into the contribution of the residual amount of GATA-1 in GATA-1.05 cells to the transformation of LEP cells into overt leukemic cells. In GATA-1-null/X mice, this population of cells is rapidly eliminated through apoptosis, resulting in efficient prevention of leukemogenesis. Indeed, a large number of GATA-1-null/X mice have been maintained in one of the authors' institutes on average for 25 weeks; neither leukemia cases nor enlarged spleen cases have ever been noticed (S. Philipsen, unpublished observation). Taken together, these results suggest a scenario for the leukemogenesis in GATA-1 gene knockdown mice in which immature erythroid progenitors at the late stage accumulate because they are arrested in differentiation but still capable of proliferation. The accumulation of progenitors, which are resistant to apoptosis by virtue of low-level GATA-1 expression, is critical for the predisposition to leukemogenesis of GATA-1.05/X mice.
Whereas both GATA-1 and GATA-2 are known to play important roles in hematopoiesis, the GATA-1
genes show distinct expression profiles that are strictly regulated. The expression of GATA-1 increases as the differentiation of erythroid and megakaryocytic lineages continues (19
). In contrast, GATA-2 is expressed in hematopoietic stem and progenitor cells, and GATA-2 is essential for cell proliferation (33
). It has been reported that persistent expression of GATA-2 stimulates the production of immature progenitor cells that have the potential to differentiate into multiple lineages (2
). The expression of GATA-2 is elevated in the bone marrow of MDS patients, and the increase in the GATA-2/GATA-1 ratio correlates with the severity of the disease (6
). These data are consistent with our hypothesis that the expanded population of LEP cells in GATA-1.05
/X mice, in which GATA-2 expression is elevated, may be easily transformed through a “multistep hit” mechanism (39
). In fact, we observed in the course of this study a type 1 case in which p53 mRNA expression was dramatically decreased in leukemia cells compared to that in c-Kit+
cells in control adult spleens (data not shown). Interestingly, it has been reported that mutation of the p53 gene is required for the acute crisis in essential thrombocytemia (21
). Thus, these results suggest that the accumulation of GATA-1-knockdown progenitor cells, in combination with secondary genetic events, forms the molecular basis for the leukemic transformation in GATA-1.05
Finally, there remains the intriguing question of how defective GATA-1 function provokes leukemia in the B-cell lineage. We do not have any definitive answers to this question yet, but we propose three hypotheses that are not mutually exclusive. The first proposal is based on the observation that B-lineage cells substantially lack the expression of hematopoietic GATA factors (17
). The expression of GATA-2 in hematopoietic progenitors is downregulated after the commitment of cells to the lymphoid lineage, and the expression of GATA-3 becomes specific to T-lineage cells. Therefore, it is plausible that the targeted knockdown of GATA-1 creates a situation where the differentiation potential of the precursor cells is shifted from the erythroid to the B-lymphoid lineage. The second proposal is based on reports that GATA-1 represses PU.1 activity. Since PU.1 activity is important for B-cell development (22
), an increase of PU.1 activity in progenitor cells with aberrantly low GATA-1 activity may lead the cells to adopt a B-cell fate. Although the expression of PU.1 mRNA in type 2 leukemia cells is comparable with that of normal spleen cells (data not shown), we think that this possibility still exists since the GATA-1/PU.1 interaction is a posttranscriptional event. The third possibility is that the accumulation of erythroid precursor cells may create an environment that is permissive for the oncogenic transformation of normal B cells.
Many chromosomal mutations are implicated in human MDS and various leukemias (30
). The present study demonstrates that a simple genetic modification of a lineage-specific transcription factor can confer an unstable condition on progenitor cells, from which leukemias of two distinct lineages are provoked. In GATA-1.05
/X mice, the overall activity of GATA-1 is significantly decreased, but there is no mutation in the GATA-1 protein, unlike the situation with DS-AMKL and TMD. Thus, the GATA-1.05
knockdown mouse provides a prime example of a model system for the systematic analysis of the ontogeny of MDS and the transformation from MDS into overt leukemia.