TY - JOUR
AU - Möröy, Tarik
AB - Abstract The regulation of gene transcription is elementary for the function of hematopoietic stem cells (HSCs). The transcriptional repressor growth factor independence 1 (Gfi1) restricts HSC proliferation and is essential to maintain their self-renewal capacity and multipotency after transplantation. In addition, Gfi1−/− HSCs are severely compromised in their ability to compete with wild-type (wt) HSCs after transplantation. We now report that Gfi1 protects HSCs against stress-induced apoptosis, probably, by repressing the proapoptotic target gene Bax, since irradiated Gfi1−/− HSCs display higher expression of Bax and show a higher rate of apoptosis than wt HSCs. This protective function of Gfi1 appears to be functionally relevant since Gfi1−/− HSCs that express Bcl-2, which antagonizes the effects of Bax, regain their ability to self renew and to initiate multilineage differentiation after transplantation. Surprisingly, Gfi1−/−xBcl-2 transgenic mice also show a strong, systemic expansion of Mac-1+Gr-1− myeloid cells in bone marrow and peripheral lymphoid organs. These cells express high levels of the proleukemogenic transcription factor Hoxa9 and, in older mice, appear as atypical monocytoid-blastoid cells in the peripheral blood. As a result of this massive expansion of myeloid cells, all Gfi1−/−xBcl-2 mice eventually succumb to a myeloproliferative-like disease resembling a preleukemic state. In summary, our data demonstrate that Gfi1's ability to protect against apoptosis is essential for HSC function. In addition, our finding show that Gfi1 prevents the development of myeloproliferative diseases and provides evidence how Gfi1 deficiency could be linked to myeloid leukemia. Gfi1, Leukemia, Hematopoietic stem cells, Hoxa9, Apoptosis, Myeloproliferative disease Introduction Hematopoietic stem cells (HSCs) [1] are enriched in the Lin−, Sca1+, ckit+, Flt3−, (LSKFlt3−) bone marrow fraction [2–5]. The function of HSCs is coordinated by transcription factors [5, 6], of which the zinc finger protein growth factor independence 1 (Gfi1) is one example. We and others have described that Gfi1 deficiency leads to increased proliferation of LSK cells [7–10] and that no hematopoietic cells originating from Gfi1-deficient HSCs can be detected after a competitive transplantation of wild type (wt) and Gfi1−/− HSCs [7, 8, 10]. Hence, Gfi1−/− HSCs are deficient in self-renewal and in their ability to reconstitute hematopoietic lineages in a transplanted host [7, 8, 10]. Loss of Gfi1 also perturbs early B- and T-cell development, the function of mature B- and T-cells and affects myeloid differentiation [9–27]. A particular feature of Gfi1−/− mice is their increased number of myeloid precursors (CMPs and GMPs) and an increased expression of Hoxa9, a transcription factor involved in AML pathogenesis [1, 10, 16]. Moreover, Gfi1−/− mice show a prominent accumulation of atypical Mac-1+, Gr1lo monocytes, and a strongly reduced number of neutrophils, but never develop a myeloproliferative disease or a myeloid leukemia [16, 25]. Here, we show that Gfi1 protects HSCs against stress-induced apoptosis and that Gfi1 deficiency favors the emergence of a myeloproliferative-like disease. Materials and Methods Mice Gfi1−/− mice were generated in our laboratory as described in previous reports [23, 25]. Mice were housed at the animal facilities of the Institut für Zellbiologie, University of Essen Medical School or at the Institut de recherches cliniques de Montreal, under SPF conditions and according to local animal regulations. Two different strains of Bcl-2 transgenic mice (using the previously described vav-promoter and H2K-promoter [22, 28]) were used to ensure that the observed effects were solely due to Bcl-2 over-expression. No difference in phenotype or number of cells was observed between vav-Bcl-2 tg and H2K-Bcl-2 tg or vav- Gfi1−/−xBcl-2 tg and Gfi1−/−xH2K-Bcl-2 tg mice. Therefore, the mouse names used in this communication are: Bcl-2 tg Gfi1+/+ (wt), Bcl-2 tg, and the two Gfi1-deficient mouse strains Gfi1−/− and Gfi1−/−xBcl-2 tg. Flow Cytometry Analysis and Sorting of HSC-Enriched and Progenitor Populations Hematopoietic cells were analyzed on the LSR (Becton Dickinson) and were sorted with a MoFlo (Beckman Coulter) from adult mice as described [2, 29, 30]. The following antibodies were used: Sca1-PECy7 (BD Biosciences [Mississauga, ON, Canada, http://www.bd.com/ca/] 558162), c-kit APC (BD 553356), Gr-1 FITC (553127), Mac-1- PerCP-Cy5.5 (BD 550993), Lin (Lineage Cocktail kit BD 559971) and in addition, CD4 (BD 553045), CD8 (BD 553029), NK1.1 (E-biosciences [San Diego, CA, http://www.ebioscience.com/] 13-5941-85), IL-7R (e-bioscience 13-1271-85), CD5 (E-biosciences 13-0051-85), Strepavidin PerCP-Cy5.5 (BD 551419). Lasers with, emitting, the following wavelengths were used: 488 nm (used with FITC 530/30, PE 585/25, PerCP-Cy 5.5, PE-Cy7 [780/60] filters), 633 nm (used with APC 660/20), and UV-laser (Hoechst 450). Competitive Bone Marrow Transplantation Tester bone marrow cells (CD45.2+) were mixed with competitor (CD45.1+) bone marrow cells at a ratio of 2:1 or 10:1, and injected into CD45.1 recipient mice lethally irradiated with 9.6 Gy (n = 4 per group). Reconstitution of donor (CD45.2+) myeloid and lymphoid cells was monitored by staining peripheral blood cells with antibodies against CD45.2 and Mac-1 (CD11b), Gr-1, CD3, and CD19. The same experiments were performed using 250 FACS-sorted Lin−c-Kit+Sca-1+Flt3− cells mixed with 2 × 105 competitor CD45.1+ bone marrow cells. Real-Time Quantitative PCR Approximately, 5–10 × 103 LSKFlt3− cells wt or Gfi1−/− mice were collected and pooled. All real-time probes were purchased from Applied Biosystems. RT–PCR was performed according to the manufacturer's instructions on an ABI PRISM 7,900 (Applied Biosystems) or Mx-3005 (Stratagene) system. The expression of the gene of interest was calculated relative to the levels of GAPDH mRNA(Δct). The expression levels are presented as “fold induction” relative to values obtained with the respective wt control (set as “onefold”) and were confirmed with three independent preparations of cells. Determination of Cell Cycling The frequency of defined cell subsets in different cell cycle phases was determined by flow cytometric analysis of bone marrow cells staining with Hoechst (Hoechst 33342, Sigma-Aldrich [Canada Ltd. Oakville, Ontario, Canada, http://www.sigmaaldrich.com/canada-english.html]). To inhibit MDR activity, HSCs were treated with Verapamil (50 μM). Staining was done according to the manufacturer's instruction. Mitochondrial Potential Loss of mitochondrial potential (JC-1 mitochondrial potential kit, M34152, Invitrogen [Burlington, ON, Canada, http://www.invitrogen.com/site/us/en/home.html]) was measured 3 hours after irradiation according to the manufacturer's protocol. Statistical Analysis The Log-rank test was used for comparing survival rates of mice. The unpaired Student t test was chosen for analyzing the differences in the number of CLPs, LSKFlt3−s, number of blast cells, and cytological classification. All p values were calculated two-sided, and values of p < .05 were considered statistically significant. Statistical analysis was done with Graph-Pad Prism software (GraphPad software, Version 4 [La Jolla, CA, USA, http://www.graphpad.com/prism/]). Spectral Karyotyping of Mouse Bone Marrow Cells Metaphase preparations of mouse bone marrow specimens (wt, Bcl-2 tg, Gfi1−/−, and Gfi1−/−xBcl-2 tg) were performed according to standard cytogenetic procedures. Slide pretreatment, hybridization with the SkyPaint™ mouse probe mixture, and detection were performed with the protocol provided by Applied Spectral Imaging, Inc. (Vista, CA, USA, http://www.spectral-imaging.com/) with minor modifications. Spectral images were acquired with a SpectraCube® system (ASI) mounted on a Zeiss Axioplan II microscope (Carl-Zeiss [Toronto, ON, Canada, http://www.zeiss.ca/]) and analyzed using the SkyView version 1.6.1 software (ASI). Results and Discussion Gfi1 Protects HSCs Against Apoptosis Loss of Gfi1 increases apoptosis in thymocytes and peripheral T-cells [20, 23, 24, 31] probably due to a de-repression of the Bax promoter [31, 32]. To test whether Gfi1 restricts apoptosis in HSCs, we irradiated freshly isolated bone marrow cells and measured the rate of apoptosis by either measuring mitochondrial potential or staining with Annexin-V (Fig. 1A, 1B). We observed an increased rate of apoptosis in Gfi1−/− LSKs compared to wt LSKs in response to irradiation and an increased expression of the proapoptotic gene Bax in Gfi1−/− LSKFlt3− cells (Fig. 1C, 1D). As Bcl-2 interacts with Bax and impedes the Bax mediated apoptosis [33–36], a constitutive over-expression of Bcl-2 should prevent the increased cell death of Gfi1-deficient cells. Indeed, Gfi1−/− LSKFlt3− cells expressing a Bcl-2 transgene no longer showed elevated levels of Bax or increased apoptosis rates after irradiation compared to wt LSKFlt3− cells (Fig. 1A–1D). 1 Open in new tabDownload slide Gfi1 protects stem cells against apoptosis. (A): Bone marrow cells were explanted and irradiated. Loss of Gfi1 correlated with enhanced apoptosis in the LSKFlt3− measured by staining for Annexin V binding (n = 3–5 for each genotype; p ≤ .01). Shown is the percentage of Annexin V negative (i.e., live) LSKFlt3− cells normalized against untreated samples to take only the effects induced by irradiation into account (**, p ≤ .01) 6 hours after irradiation. (B): Bone marrow cells were explanted and irradiated. Loss of Gfi1 correlated with enhanced apoptosis in the LSKFlt3− measured by mitochondrial potential (n = 3–5 for each genotype; p ≤ .01). Shown are live LSKFlt3− cells normalized against untreated samples to take only the effects induced by irradiation into account (**, p ≤ .01) 3 hours after irradiation. (C): Bone marrow cells were explanted and irradiated. Expression level (mRNA) of the proapoptotic factor Bax in the different treated and untreated LSKFlt3− cells was measured 1 hour after irradiation (*, p ≤ .05). (D): Bone marrow cells were explanted and irradiated. Expression level (mRNA) of the antiapoptotic factor Bax in the different treated and untreated LSKFlt3− cells was measured 1 hour after irradiation (*, p ≤ .05). (E): Survival of mice after a sub lethal irradiation dose of 6 Gy. About 90% of all wt (Gfi1+/+) mice survived this dose, whereas only 40% of Gfi1−/− survived this dose (n = 13 for wt and n = 17 for Gfi1−/−p ≤ .01). A bone marrow failure was defined as Hb ≤8 g/l, platelets ≤200/fl, and leukocytes ≤1.5/fl. (F): Frequency of LSKFlt3− cells before and after γ-irradiation. Gfi1−/− mice show a more pronounced loss of LSKFlt3− cells than wt mice (**, p ≤ .01). (G, F): Number of platelets (E) and leukocytes (F) before and after γ-irradiation in wt and Gfi1−/− mice. Surviving Gfi1−/− mice (i.e., Gfi1−/− mice surviving more than 30 days after irradiation and thus showing initial recovery) display lower number of platelets (E) and leukocytes (F) after γ-irradiation, whereas irradiated wt mice recovered with normal number of platelets and leukocytes (n = 10 for irradiated wt) mice, n = 5 for irradiated Gfi1−/− mice, n = 11 for nonirradiated age matched wt (Gfi1+/+) mice, n = 14 for nonirradiated age matched Gfi1−/− mice; (*, p ≤ .01). Abbreviations: LSKFlt3−, Lin−, Sca1+, c-kit+ cells Flt3−. 1 Open in new tabDownload slide Gfi1 protects stem cells against apoptosis. (A): Bone marrow cells were explanted and irradiated. Loss of Gfi1 correlated with enhanced apoptosis in the LSKFlt3− measured by staining for Annexin V binding (n = 3–5 for each genotype; p ≤ .01). Shown is the percentage of Annexin V negative (i.e., live) LSKFlt3− cells normalized against untreated samples to take only the effects induced by irradiation into account (**, p ≤ .01) 6 hours after irradiation. (B): Bone marrow cells were explanted and irradiated. Loss of Gfi1 correlated with enhanced apoptosis in the LSKFlt3− measured by mitochondrial potential (n = 3–5 for each genotype; p ≤ .01). Shown are live LSKFlt3− cells normalized against untreated samples to take only the effects induced by irradiation into account (**, p ≤ .01) 3 hours after irradiation. (C): Bone marrow cells were explanted and irradiated. Expression level (mRNA) of the proapoptotic factor Bax in the different treated and untreated LSKFlt3− cells was measured 1 hour after irradiation (*, p ≤ .05). (D): Bone marrow cells were explanted and irradiated. Expression level (mRNA) of the antiapoptotic factor Bax in the different treated and untreated LSKFlt3− cells was measured 1 hour after irradiation (*, p ≤ .05). (E): Survival of mice after a sub lethal irradiation dose of 6 Gy. About 90% of all wt (Gfi1+/+) mice survived this dose, whereas only 40% of Gfi1−/− survived this dose (n = 13 for wt and n = 17 for Gfi1−/−p ≤ .01). A bone marrow failure was defined as Hb ≤8 g/l, platelets ≤200/fl, and leukocytes ≤1.5/fl. (F): Frequency of LSKFlt3− cells before and after γ-irradiation. Gfi1−/− mice show a more pronounced loss of LSKFlt3− cells than wt mice (**, p ≤ .01). (G, F): Number of platelets (E) and leukocytes (F) before and after γ-irradiation in wt and Gfi1−/− mice. Surviving Gfi1−/− mice (i.e., Gfi1−/− mice surviving more than 30 days after irradiation and thus showing initial recovery) display lower number of platelets (E) and leukocytes (F) after γ-irradiation, whereas irradiated wt mice recovered with normal number of platelets and leukocytes (n = 10 for irradiated wt) mice, n = 5 for irradiated Gfi1−/− mice, n = 11 for nonirradiated age matched wt (Gfi1+/+) mice, n = 14 for nonirradiated age matched Gfi1−/− mice; (*, p ≤ .01). Abbreviations: LSKFlt3−, Lin−, Sca1+, c-kit+ cells Flt3−. We next tested whether Gfi1 restricts apoptosis also in vivo, and irradiated Gfi1-deficient mice and controls with a sublethal dose of 6 Gy. Gfi1−/− mice succumbed with a high rate (60%) to bone marrow failure and the surviving animals exhibited significantly reduced numbers of LSKFlt3− cells, platelets, and leukocytes, whereas wt mice remained largely unaffected (Fig. 1E–1H). This suggested that Gfi1 protects HSCs or precursors against stress-induced apoptosis by controlling the expression of Bax. Thus, it is conceivable that Gfi1−/− HSCs loose their function upon transplantation, which represents a cellular stress because Gfi1 deficiency sensitizes them to stress-induced apoptosis. Bcl-2 Restores the Function of Gfi1−/− HSCs If this hypothesis was true, constitutive expression of a Bcl-2 transgene should restore the impaired function of Gfi1−/− HSCs. As the presence of a Bcl-2 transgene did not change the frequency of LSKFlt3− cells in Gfi1−/− mice, nor affected their cell cycle progression (Fig. 2A–2D; Supporting Information Fig. 1), the effect of Bcl-2 on HSC function could be studied separately from Gfi1's role in regulating cell cycle progression. Hence, we sorted LSKFlt3− cells from wt, Gfi1−/−, Bcl-2 tg, and Gfi1−/−xBcl-2 tg mice (CD45.2+) and transplanted them with wt CD45.1+ cells into irradiated syngenic hosts. Gfi1−/− HSCs were not detectable after transplantation, whereas Gfi1−/−xBcl-2 tg HSCs were present indicating a rescue of self-renewal of Gfi1−/− HSCs after transplantation by Bcl-2 (Fig. 3A) [7, 11]. This was confirmed by transplanting bone marrow cells from wt, Bcl-2 tg, Gfi1−/−, and Gfi1−/−xBcl-2 tg animals (CD45.2+) with CD45.1+ bone marrow cells at different ratios. Furthermore, whereas Gfi1−/− bone marrow cells could not contribute to different lineages after transplantation (Fig. 3B) [7, 10], Gfi1−/−xBcl-2 tg cells were able to do so (Fig. 3B), indicating that over-expression of Bcl-2 can at least partially restore the function of Gfi1−/− HSCs after transplantation very likely by protecting against apoptosis. In addition to the previously proposed model that enhanced cell cycling might exhaust Gfi1−/− HSCs [7, 10], our data suggest that Gfi1 protects HSCs against stress-induced apoptosis and that this function is critical to maintain their function after transplantation. 2 Open in new tabDownload slide Effect of Bcl-2 over-expression on percentage of HSCs and their proliferation. (A): Gating strategy for determining the frequencies of LSKFlt3− cells. (B): After gating on LSKFlt3− cells as demonstrated in (A), the cell cycle progression of the different LSKFlt3− was determined with the ModFit software (See also Supporting Information Fig. 1). (C): Frequency of LSKFlt3− cells in the bone marrow of mice with the indicated genotypes that are in S/G2/M phase determined by Hoechst staining. Forced expression of Bcl-2 does not alter significantly cell cycle progression in the LSKFLT3− subset. (p ≤ .4 between Gfi1−/− and **, p ≤ .01); wt (n = 4), Bcl-2 tg (n = 4), Gfi1−/− (n = 4), and Gfi1−/−xBcl-2 tg (n = 4). (D): Frequency of LSKFlt3− cells in the bone marrow of mice with the indicated genotypes. Forced expression of Bcl-2 does not alter the elevated proportion of LSKFlt3− cells observed in Gfi1−/− mice compared to wt mice (p ≤ .4 between Gfi1−/− and Gfi1−/−xBcl-2 tg and p ≤ .01 between wt and Gfi1−/−). wt (n = 7), Bcl-2 tg (n = 4), Gfi1−/− (n = 6), and Gfi1−/−xBcl-2 tg (n = 4) (**, p ≤ .01). Abbreviations: LSKFlt3−, Lin−, Sca1+, c-kit+ cells Flt3−. 2 Open in new tabDownload slide Effect of Bcl-2 over-expression on percentage of HSCs and their proliferation. (A): Gating strategy for determining the frequencies of LSKFlt3− cells. (B): After gating on LSKFlt3− cells as demonstrated in (A), the cell cycle progression of the different LSKFlt3− was determined with the ModFit software (See also Supporting Information Fig. 1). (C): Frequency of LSKFlt3− cells in the bone marrow of mice with the indicated genotypes that are in S/G2/M phase determined by Hoechst staining. Forced expression of Bcl-2 does not alter significantly cell cycle progression in the LSKFLT3− subset. (p ≤ .4 between Gfi1−/− and **, p ≤ .01); wt (n = 4), Bcl-2 tg (n = 4), Gfi1−/− (n = 4), and Gfi1−/−xBcl-2 tg (n = 4). (D): Frequency of LSKFlt3− cells in the bone marrow of mice with the indicated genotypes. Forced expression of Bcl-2 does not alter the elevated proportion of LSKFlt3− cells observed in Gfi1−/− mice compared to wt mice (p ≤ .4 between Gfi1−/− and Gfi1−/−xBcl-2 tg and p ≤ .01 between wt and Gfi1−/−). wt (n = 7), Bcl-2 tg (n = 4), Gfi1−/− (n = 6), and Gfi1−/−xBcl-2 tg (n = 4) (**, p ≤ .01). Abbreviations: LSKFlt3−, Lin−, Sca1+, c-kit+ cells Flt3−. 3 Open in new tabDownload slide Bcl-2 over-expression restores transplantation capacity of Gfi1-deficient hematopoietic stem cells (HSCs). (A): 250 cells, defined as CD45.2+Lin−Sca1+kit+Flt3−, were transplanted along with 1 × 105 CD45.1+ competitor bone marrow cells into CD45.1+ recipients. Gfi1−/−HSCs were unable to self renew in competition with wt HSCs and no Gfi1−/− HSCs were detected after 6 months in the recipient mice. In contrast, HSCs from Gfi1−/−xBcl-2 tg, could be detected after 6 months in the recipient mice. (B): Competitive repopulation assay with wt, Bcl-2 tg, Gfi1−/−, and Gfi1−/−xBcl-2 tg bone marrow cells. In all, 2 × 105wt or Gfi1−/− bone marrow cells (CD45.2+) were transplanted at a ratio of 2:1 (upper panels) or 10:1 (lower panels marked) with competitor CD45.1+ bone marrow cells into lethally irradiated CD45.1+ mice (n = 4). Peripheral blood was analyzed at various times after reconstitution for the presence of wt or Gfi1−/− bone marrow (CD45.2+)-derived myeloid (Mac-1+), B-lymphoid (CD19+), and T-lymphoid (CD3+) cells in transplanted recipients. Abbreviations: LSKFlt3−, Lin−, Sca1+, c-kit+ cells Flt3−. 3 Open in new tabDownload slide Bcl-2 over-expression restores transplantation capacity of Gfi1-deficient hematopoietic stem cells (HSCs). (A): 250 cells, defined as CD45.2+Lin−Sca1+kit+Flt3−, were transplanted along with 1 × 105 CD45.1+ competitor bone marrow cells into CD45.1+ recipients. Gfi1−/−HSCs were unable to self renew in competition with wt HSCs and no Gfi1−/− HSCs were detected after 6 months in the recipient mice. In contrast, HSCs from Gfi1−/−xBcl-2 tg, could be detected after 6 months in the recipient mice. (B): Competitive repopulation assay with wt, Bcl-2 tg, Gfi1−/−, and Gfi1−/−xBcl-2 tg bone marrow cells. In all, 2 × 105wt or Gfi1−/− bone marrow cells (CD45.2+) were transplanted at a ratio of 2:1 (upper panels) or 10:1 (lower panels marked) with competitor CD45.1+ bone marrow cells into lethally irradiated CD45.1+ mice (n = 4). Peripheral blood was analyzed at various times after reconstitution for the presence of wt or Gfi1−/− bone marrow (CD45.2+)-derived myeloid (Mac-1+), B-lymphoid (CD19+), and T-lymphoid (CD3+) cells in transplanted recipients. Abbreviations: LSKFlt3−, Lin−, Sca1+, c-kit+ cells Flt3−. Bcl-2 Overexpression Causes a Myeloproliferative-Like Disease in Gfi1−/− Mice Surprisingly, Gfi1−/−xBcl-2 tg mice featured an additional Mac-1+Gr-1− monocytic population that was not present in wt or Gfi1−/− mice (Table 1, Fig. 4A). This population expanded over time in the bone marrow, appeared in the bloodstream, and was also present at large numbers in the spleen of older Gfi1−/−xBcl-2 tg mice (Table 2, Fig. 4B–4E). This correlated with decreased numbers of platelets and increased numbers of white blood cells (Fig. 4F, 4G). Ultimately, all Gfi1−/−xBcl-2 tg mice succumbed to a myeloproliferative-like disorder, which had never been observed in wt, Gfi1−/−, or Bcl-2 tg mice (Fig. 4H). Cytogenetic examination did not provide evidence of a clonal expansion in the bone marrow of Gfi1−/−xBcl-2 mice, but a clonal disease cannot be completely ruled out at this time. Table 1 Myeloid maturation in bone marrow Open in new tab Table 1 Myeloid maturation in bone marrow Open in new tab Table 2 Blood composition of different mouse strains Open in new tab Table 2 Blood composition of different mouse strains Open in new tab 4 Open in new tabDownload slide Forced expression of Bcl-2 does not rescue loss of granulocytes in Gfi1−/− mice but induces a myeloproliferative-like disease. (A): Flow cytometric analysis of Mac-1 and Gr-1 surface expression on bone marrow cells from mice with the indicated genotypes. A new Mac1+Gr1− population (arrowhead) is detected in Bcl-2 tg mice that expands noticeably in Gfi1−/−xBcl-2 tg mice over time and eventually leads to a moribund state requiring euthanasia of the animals. (B): Quantification of cell numbers of the new Mac1+Gr1− population detected in A. Gfi1−/−xBcl-2 tg mice feature an over 10-fold increase of this population compared to wt or Gfi1−/− mice and an over fourfold increase compared to Bcl-2 tg mice (**, p ≤ .01 and *, p ≤ .05). This population further expands over time in Gfi1−/−xBcl-2 tg mice and is over 20-fold higher in moribund Gfi1−/−xBcl-2 tg mice compared to wt mice. Wt (n = 7), Bcl-2 tg (n = 4), Gfi1−/− (n = 9), healthy Gfi1−/−xBcl-2 tg (n = 4), and moribund Gfi1−/−xBcl-2 tg (n = 7) mice. (C): Quantification of leukocytes in the peripheral blood of animals with the indicated genotype. Bcl-2 tg and Gfi1−/−xBcl-2 tg presented with monocytoid-blastoid cells in peripheral blood (defined in an automated setting as large, peroxidase negative cells) and the number of these blast cells expands in moribund Gfi1−/−xBcl-2 tg mice (**, p ≤ .01 and *, p ≤ .05). Wt (n = 22), Bcl-2 tg (n = 4), Gfi1−/− (n = 18), Gfi1−/−xBcl-2 tg (healthy, n = 4), and Gfi1−/−xBcl-2 tg mice (moribund, n = 7). (D): Quantification of monocytoid-blastoid cells appearing in the peripheral blood of animals with the indicated genotype. Bcl-2 tg and Gfi1−/−xBcl-2 tg presented with monocytoid-blastoid cells in peripheral blood (defined in an automated setting as large, peroxidase negative cells) and the number of these blast cells expands dramatically in moribund Gfi1−/−xBcl-2 tg mice (**, p ≤ .01 and *, p ≤ .05). Wt (n = 22), Bcl-2 tg (n = 4), Gfi1−/− (n = 18), Gfi1−/−xBcl-2 tg (healthy, n = 4), and Gfi1−/−xBcl-2 tg mice (moribund, n = 7). (E): Quantification of monocytoid-blastoid cells appearing in the spleen of animals with the indicated genotype. Bcl-2 tg and Gfi1−/−xBcl-2 tg presented with monocytoid-blastoid cells in the spleen (**, p ≤ .01 and *, p ≤ .05). Wt (n = 4), Bcl-2 tg (n = 4), Gfi1−/− (n = 4), Gfi1−/−xBcl-2 tg (healthy and sick, n = 4), and Gfi1−/−xBcl-2 tg mice (moribund, n = 4). (F): Quantification of platelets in the peripheral blood of animals with the indicated genotype. Bcl-2 tg and Gfi1−/−xBcl-2 tg display lower number of platelets (**, p ≤ .01 and *, p ≤ .05). Wt (n = 22), Bcl-2 tg (n = 4), Gfi1−/− (n = 18), Gfi1−/−xBcl-2 tg (healthy and moribund, n = 11). (G): Quantification of red blood cells in the peripheral blood of animals with the indicated genotype. Bcl-2 tg and Gfi1−/−xBcl-2 tg display lower number of platelets (**, p ≤ .01 and *, p ≤ .05). Wt (n = 22), Bcl-2 tg (n = 4), Gfi1−/− (n = 18), Gfi1−/−xBcl-2 tg (healthy and moribund, n = 11). (H): Survival of animals (Kaplan Meier curve) with the indicated genotypes over time. All Gfi1−/−xBcl-2 tg mice become eventually moribund due to a myeloproliferative disease at a median age of 93 days. This disease phenotype was not observed in any of the other control groups (p ≤ .001) for survival between Gfi1−/−xBcl-2 tg (n = 7) mice and Gfi1−/− (n = 8), or Gfi1−/−xBcl-2 tg (n = 7) or Bcl-2 tg (n = 9) mice after 180 days (end of observation). 4 Open in new tabDownload slide Forced expression of Bcl-2 does not rescue loss of granulocytes in Gfi1−/− mice but induces a myeloproliferative-like disease. (A): Flow cytometric analysis of Mac-1 and Gr-1 surface expression on bone marrow cells from mice with the indicated genotypes. A new Mac1+Gr1− population (arrowhead) is detected in Bcl-2 tg mice that expands noticeably in Gfi1−/−xBcl-2 tg mice over time and eventually leads to a moribund state requiring euthanasia of the animals. (B): Quantification of cell numbers of the new Mac1+Gr1− population detected in A. Gfi1−/−xBcl-2 tg mice feature an over 10-fold increase of this population compared to wt or Gfi1−/− mice and an over fourfold increase compared to Bcl-2 tg mice (**, p ≤ .01 and *, p ≤ .05). This population further expands over time in Gfi1−/−xBcl-2 tg mice and is over 20-fold higher in moribund Gfi1−/−xBcl-2 tg mice compared to wt mice. Wt (n = 7), Bcl-2 tg (n = 4), Gfi1−/− (n = 9), healthy Gfi1−/−xBcl-2 tg (n = 4), and moribund Gfi1−/−xBcl-2 tg (n = 7) mice. (C): Quantification of leukocytes in the peripheral blood of animals with the indicated genotype. Bcl-2 tg and Gfi1−/−xBcl-2 tg presented with monocytoid-blastoid cells in peripheral blood (defined in an automated setting as large, peroxidase negative cells) and the number of these blast cells expands in moribund Gfi1−/−xBcl-2 tg mice (**, p ≤ .01 and *, p ≤ .05). Wt (n = 22), Bcl-2 tg (n = 4), Gfi1−/− (n = 18), Gfi1−/−xBcl-2 tg (healthy, n = 4), and Gfi1−/−xBcl-2 tg mice (moribund, n = 7). (D): Quantification of monocytoid-blastoid cells appearing in the peripheral blood of animals with the indicated genotype. Bcl-2 tg and Gfi1−/−xBcl-2 tg presented with monocytoid-blastoid cells in peripheral blood (defined in an automated setting as large, peroxidase negative cells) and the number of these blast cells expands dramatically in moribund Gfi1−/−xBcl-2 tg mice (**, p ≤ .01 and *, p ≤ .05). Wt (n = 22), Bcl-2 tg (n = 4), Gfi1−/− (n = 18), Gfi1−/−xBcl-2 tg (healthy, n = 4), and Gfi1−/−xBcl-2 tg mice (moribund, n = 7). (E): Quantification of monocytoid-blastoid cells appearing in the spleen of animals with the indicated genotype. Bcl-2 tg and Gfi1−/−xBcl-2 tg presented with monocytoid-blastoid cells in the spleen (**, p ≤ .01 and *, p ≤ .05). Wt (n = 4), Bcl-2 tg (n = 4), Gfi1−/− (n = 4), Gfi1−/−xBcl-2 tg (healthy and sick, n = 4), and Gfi1−/−xBcl-2 tg mice (moribund, n = 4). (F): Quantification of platelets in the peripheral blood of animals with the indicated genotype. Bcl-2 tg and Gfi1−/−xBcl-2 tg display lower number of platelets (**, p ≤ .01 and *, p ≤ .05). Wt (n = 22), Bcl-2 tg (n = 4), Gfi1−/− (n = 18), Gfi1−/−xBcl-2 tg (healthy and moribund, n = 11). (G): Quantification of red blood cells in the peripheral blood of animals with the indicated genotype. Bcl-2 tg and Gfi1−/−xBcl-2 tg display lower number of platelets (**, p ≤ .01 and *, p ≤ .05). Wt (n = 22), Bcl-2 tg (n = 4), Gfi1−/− (n = 18), Gfi1−/−xBcl-2 tg (healthy and moribund, n = 11). (H): Survival of animals (Kaplan Meier curve) with the indicated genotypes over time. All Gfi1−/−xBcl-2 tg mice become eventually moribund due to a myeloproliferative disease at a median age of 93 days. This disease phenotype was not observed in any of the other control groups (p ≤ .001) for survival between Gfi1−/−xBcl-2 tg (n = 7) mice and Gfi1−/− (n = 8), or Gfi1−/−xBcl-2 tg (n = 7) or Bcl-2 tg (n = 9) mice after 180 days (end of observation). Although Mac-1+Gr-1lo monocytic cells are present in the bone marrow, peripheral blood, and spleen of Gfi1−/− mice (Fig. 5A, 5B), the expansion of the new Mac-1+Gr-1− monocytic population is at least 10-fold to 100-fold higher in Bcl-2 tg Gfi1−/− mice and these cells show a peripheral infiltration into other organs (Fig. 5C). This indicates the emergence of a bona-fide myeloproliferative-like disease and argues against a simple circulation and redistribution of an aberrant cell population. Also, consistent with a myeloproliferative disease is the high expression of Hoxa9 in the Mac-1+Gr-1− population of Gfi1−/−xBcl-2 tg mice (Fig. 5D), since Hoxa9 has been described as causative for a Kirsten rat sarcoma viral oncogene homolog (KRAS)–induced myeloproliferative disorder in Gfi1-deficient mice [16]. Transplantation of bone marrow cells from moribund Gfi1−/−xBcl-2 tg affected by this disease led to re-appearance of Mac-1+Gr-1− cells in recipients indicating that the observed process is cell-autonomous (Fig. 5E). However, the disease course was much slower after transplantation (latency of 5–8 months) and only 20% of transplanted mice died as a result of the disease, suggesting that Gfi1−/−xBcl-2 tg mice succumb to a myeloproliferative-like disease but not a full blown leukemia. 5 Open in new tabDownload slide Combination of protection against apoptosis and higher expression of Hoxa9 induces a myeloproliferative-like disease in Gfi1−/− mice. (A): Cytospins of bone marrow cells from wt, Gfi1−/−, Bcl-2 tg, and Gfi1−/−xBcl-2 tg featuring the accumulation of aberrant monocytic cells in Gfi1−/− and Gfi1−/−xBcl-2 tg mice (see arrowheads, ×40 magnification, ×10 ocular, Northern Eclipse image software). (B): Histological spleen sections (H and E staining) from wt, Gfi1−/−, Bcl-2 tg, and Gfi1−/−xBcl-2 tg featuring the accumulation of aberrant monocytic cells in Gfi1−/− and Gfi1−/−xBcl-2 tg mice (see arrowheads, ×40 magnification, ×10 ocular, Northern Eclipse image software). (C): Histological liver sections (H and E staining) from wt, Gfi1−/−, Bcl-2 tg, and Gfi1−/−xBcl-2 tg featuring the accumulation of aberrant monocytic cells in Gfi1−/− and Gfi1−/−xBcl-2 tg mice (see arrowheads, ×20 magnification, ×10 ocular, Northern Eclipse image software). (D): Expression levels of Hoxa9 (mRNA) determined by q-PCR in sorted Mac-1+Gr1− cells from bone marrow of Gfi1−/− and Gfi1−/−xBcl-2 tg mice; n = 3 for all genotypes (**, p ≤ .01). (E): CD45.1+ recipient mice were transplanted with a mix of bone marrow cells consisting of CD45.1+ cells and either bone marrow from wt (CD45.2+) or moribund Gfi1−/−xBcl2-tg mice. Thirty weeks after transplantation, the transplanted mice were sacrificed and examined for the presence of Mac-1+Gr1− cells by flow cytometry. The previously observed aberrant Mac1+Gr1− population (arrowhead) can also be observed in the transplanted host, showing that the emergence of these cells in Gfi1−/−xBcl-2 tg mice is a cell autonomous feature. 5 Open in new tabDownload slide Combination of protection against apoptosis and higher expression of Hoxa9 induces a myeloproliferative-like disease in Gfi1−/− mice. (A): Cytospins of bone marrow cells from wt, Gfi1−/−, Bcl-2 tg, and Gfi1−/−xBcl-2 tg featuring the accumulation of aberrant monocytic cells in Gfi1−/− and Gfi1−/−xBcl-2 tg mice (see arrowheads, ×40 magnification, ×10 ocular, Northern Eclipse image software). (B): Histological spleen sections (H and E staining) from wt, Gfi1−/−, Bcl-2 tg, and Gfi1−/−xBcl-2 tg featuring the accumulation of aberrant monocytic cells in Gfi1−/− and Gfi1−/−xBcl-2 tg mice (see arrowheads, ×40 magnification, ×10 ocular, Northern Eclipse image software). (C): Histological liver sections (H and E staining) from wt, Gfi1−/−, Bcl-2 tg, and Gfi1−/−xBcl-2 tg featuring the accumulation of aberrant monocytic cells in Gfi1−/− and Gfi1−/−xBcl-2 tg mice (see arrowheads, ×20 magnification, ×10 ocular, Northern Eclipse image software). (D): Expression levels of Hoxa9 (mRNA) determined by q-PCR in sorted Mac-1+Gr1− cells from bone marrow of Gfi1−/− and Gfi1−/−xBcl-2 tg mice; n = 3 for all genotypes (**, p ≤ .01). (E): CD45.1+ recipient mice were transplanted with a mix of bone marrow cells consisting of CD45.1+ cells and either bone marrow from wt (CD45.2+) or moribund Gfi1−/−xBcl2-tg mice. Thirty weeks after transplantation, the transplanted mice were sacrificed and examined for the presence of Mac-1+Gr1− cells by flow cytometry. The previously observed aberrant Mac1+Gr1− population (arrowhead) can also be observed in the transplanted host, showing that the emergence of these cells in Gfi1−/−xBcl-2 tg mice is a cell autonomous feature. In myelodysplastic patients, low levels of Gfi1 are associated with a higher risk to develop AML. In addition, a Gfi1 variant is associated with AML and, neutropenia causing mutations of Gfi1 are associated with an elevated AML risk [37–41]. Moreover, Gfi1-deficient mice show expanded myeloid cell populations and up-regulated levels of Hoxa9. Despite all these elements, Gfi1−/− mice neither develop a myeloproliferative disease nor a myeloid leukemia. Our data could explain this conundrum since we show here that Gfi1 has several functions: it controls Hoxa9 expression and myeloid differentiation, but also protects against apoptosis. Hence, only in situations where protection against apoptosis coincides with Gfi1 deficiency and thus with elevated Hoxa9 levels and myeloid expansion, a myeloproliferative-like state can emerge and possibly also further develop into a myeloid leukemia. This may also explain clinical findings that myelodysplastic patients expressing low levels of Gfi1 have an increased risk to develop leukemia [38]. Here, the balance of the different functions of Gfi1 may have been tipped to favor of the expansion of monocytic cells and high Hoxa9 levels, while still maintaining an intact protection against apoptosis. Disclosure of Potential Conflicts of Interest The authors indicate no potential conflicts of interest. Acknowledgements We are indebted to Angelika Warda, Wojciech Wegrzyn, Inge Spratte, Mathieu Lapointe, and Rachel Bastien for technical assistance, Petra Plessow, Tomas Civela, Nancy Laverriere, Marlène Bernier, Marie-Claude Lavallée for excellent animal care, Eric Massicotte and Julie Lord for FACS and cell sorting. We thank Claude Rondeau (Leukemia Cell Bank of Quebec) for expert technical assistance in spectral karyotyping. M.C. is supported by a fellowship from the CIHR. J.H. was supported by a grant from the Cancer Research Network of Fonds de la Recherche en Santé du Québec. This work was supported by the Deutsche Forschungsgemeinschaft, DFG, and the Cancer Research Society, Canada. C.K. is supported by a fellowship from the Cole Foundation. J.H. was supported by a grant from the Cancer Research Network of Fonds de la Recherche en Santé du Québec. H.Z. is supported by an “overseas investigator program” grant from the Ministry of Human Resources and Social Security, China. 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Kosan: design of experiments, collection, interpretation and analysis of data, manuscript writing; M.-C.G. and U.D.: collection, interpretation, and analysis of data; H.Z.: design of experiments, interpretation, and analysis of data; J.H.: karyotype analysis; T.M.: conception of work, design of experiments, interpretation and analysis of data, writing of manuscript, financial support, and funding. Disclosure of potential conflicts of interest is found at the end of this article. First published online in STEM CELLSEXPRESS December 9, 2010. C. Khandanpour and C. Kosan contributed equally to this work. Telephone: +86-10-84322621. Fax: +86-10-84322606 Telephone: 514-987-5501; Fax: 514-987-5679 Copyright © 2010 AlphaMed Press This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Growth Factor Independence 1 Protects Hematopoietic Stem Cells Against Apoptosis but Also Prevents the Development of a Myeloproliferative-Like Disease
JF - Stem Cells
DO - 10.1002/stem.575
DA - 2011-02-01
UR - https://www.deepdyve.com/lp/oxford-university-press/growth-factor-independence-1-protects-hematopoietic-stem-cells-against-RyZSbAV9Sg
SP - 376
EP - 385
VL - 29
IS - 2
DP - DeepDyve
ER -