PU.1, a hematopoietic transcription factor, is absolutely required for development of myelo-lymphoid cells from hematopoietic stem cells (HSC). PU.1-deficient mice fail to develop common myeloid ...progenitors (CMPs) or common lymphoid progenitors (CLPs), resulting in complete loss of dendritic cells (DC) in addition to mature myeloid and lymphoid cells. In this study, by disrupting PU.1 specifically at the mature DC stage, we here show that PU.1 is necessary for maintenance of mature DC pool. By crossing PU.1 floxed/floxed mice with a mouse line harboring the Cre transgene driven by the CD11c-BAC, we disrupted PU.1 at the CD11c+ DC stage. In these mice, development of DC precursors such as Lin−c-KitloFLT3+MCSFR+ DC progenitors, FLT3+ CLP and FLT3+CMP were not affected. The number of CD11c+B220− DCs, however, significantly reduced in all lymphoid tissues including the thymus, the spleen, the lymph node and the skin, down to <30%, <10%, <10% and <5% of DC numbers in control mice, respectively. In contrast, mice possessed normal numbers of granulocytes/monocytes, B cells, and naïve, effector or regulatory T cells. These mice have not developed any significant hematological or immune disorders at least until 6 months after birth. These results clearly show that PU.1 is required not only for DC development but also for maintenance of the peripheral DC pool. We are currently trying to elucidate the underlying mechanism for PU.1 to maintain mature DC numbers in peripheral organs.
In hematopoietic stem cell development, the expression of critical genes is precisely regulated in a stage specific manner, which supports normal hematopoietic development through adequately ...regulating timing of cell division, self-renewal, and lineage commitment. Regulation of gene expression is known to take place at least at transcriptional level. In addition to the transcriptional regulation, there are growing evidences that post-transcriptional control of critical genes may play an important role, suggesting an interesting possibility that post-transcriptional control may also play a role in hematopoiesis. Here, we provide the evidence that the expression of Notch1, a key factor in lymphoid development, is controlled at post-transcriptional level in hematopoietic stem cell (HSC). By quantitative PCR, Notch1 mRNA is substantially expressed at HSCs as well as common lymphoid progenitors (CLPs) or double negative (DN) thymocytes. However, Notch1 protein is detected at very low level in HSCs compared to CLPs or DN thymocytes, suggesting that Notch1 expression is regulated at post-transcriptional level in HSC. To investigate the effect of 3′UTR (untranslated region) on post-transcriptional regulation, we prepared a retrovirus sensor vector, in which 3′UTR of target gene is placed between the GFP coding region and the retrovirus 3′LTR, and found that induction of the sensor vector with the 3′UTR sequence of Notch1 showed marked suppression of the GFP intensity at the HSC stage. This effect was not observed when we introduced the vector into DN thymocytes. Suppression of Notch1 by its 3′UTR was further confirmed by using a retrovirus vector which has two distinct markers of YFP and GFP-3′UTR fusion genes under bi-directional EF1 promoter. Deletion mutant analysis showed that the responsible region required for this post-transcriptional suppression is confined to 120-bp sequence within Notch1 3′UTR so far. These data suggest that the expression of Notch1 should be regulated at post-transcriptional level by its 3′UTR at the HSC stage and our data provide the first evidence that the stage-specific translational regulation can play an important role in organization of hematopoietic development.
PU.1 is an Ets family transcription factor, which is important for differentiation of both myeloid and lymphoid lineages. In the Friend leukemia model, the failure of PU.1 down-regulation in ...erythroblasts reportedly results in differentiation arrest, leading to erythroleukemia. In mice conditionally knocked-out of the 3.5 kb length of enhancer region located in14 kb 5′ upstream of the PU.1 gene, PU.1 is down-regulated in myeloid cells and B cells to 20% of that of wild type, and such mice develop acute myeloid leukemia and CLL-like diseases. Since the 3.5 kb enhancer region contains a suppressor region for PU.1 expression in T cells, such mice ectopically express PU.1 in T cells and develop T cell lymphoma. Thus, the failure of proper expression of PU.1 in certain differentiation stages for certain cell lineages appears to result in hematological malignancies.
We recently reported that human PU.1 is down-regulated in a majority of myeloma cell lines through the methylation of the promoter and enhancer region located in17 kb 5′ upstream of the PU.1 gene which is homologous to that in14 kb 5′ upstream of murine PU.1 gene. Conditionally expressed PU.1 induced cell growth arrest and apoptosis of two PU.1 low-negative myeloma cell lines, U266 and KMS12PE, suggesting that the down-regulation of PU.1 is essential for myeloma cell growth. We have also reported that PU.1 is expressed in normal plasma cells and PU.1 is down-regulated in myeloma cells of certain myeloma patients, who appear to have poor prognosis. In the present study, to elucidate the mechanisms of the cell growth arrest and apoptosis in PU.1-conditionally expressing myeloma cells, we performed DNA microarray analysis to compare gene expression levels before and after PU.1 expression, utilizing Illumina Sentrix® Human-6 Expression BeadChip. Of 47,296 genes, 479 genes were up-regulated (>2fold) and 1,697 genes down-regulated (<0.5 fold), either 1 or 3 days after PU.1 expression in U266 cells. Among cell-cycle related genes, p21WAF1/CIP1 was found up-regulated in U266 cells, which was confirmed at protein levels. Among apoptosis related genes, TRAIL was highly up-regulated in both U266 and KMS12PE cell lines. Stably expressed siRNA for TRAIL inhibited apoptosis of PU.1-expressing U266 cells, suggesting that TRAIL may have a crucial role in the PU.1- induced apoptosis. We subsequently examined how TRAIL was up-regulated in such PU.1-expressing myeloma cells. We performed chromatin immunoprecipitation assay and found that PU.1 directly binds to the promoter region of the TRAIL gene in U266 cells. We also determined the PU.1 binding site using electrophoretic mobility shift assays. An introduction of mutations in the PU.1 binding site abolished the binding. We also subcloned the TRAIL promoter and exon1 5′ non-coding sequence into pGL4.26 vector and performed reporter assays. Induction of PU.1 by removal of tetracycline in U266 cells induced 4-fold up-regulation of luciferase activity compared to that without PU.1 expression (28.7-fold compared to that with pGL4.26 vector alone) and mutations in the PU.1 binding site completely abolished the up-regulation. Taken together, we hereby conclude that PU.1 can directly transactivate the TRAIL gene in myeloma cells, leading to apoptosis.
Dendritic cells (DCs) have been shown to arise from both myeloid and lymphoid pathways, by evaluating the in vivo reconstituting potential of myeloid- and lymphoid-committed progenitor populations. ...However, evaluation of DC development after conventional adoptive transfer experiments may not correctly represent normal DC development including lineage contribution toward the formation of the DC pool. By crossing RAG1-Cre knockin with yellow fluorescence protein (YFP)-floxed reporter lines, we developed a mouse line in which cells with a history of RAG activation should be permanently marked with YFP as a result of lymphoid commitment. Lymphoid-derived DCs were successfully marked as YFP+ in vivo. We found that only ∼10% of conventional DCs (cDCs) and ∼20% of plasmacytoid DCs (pDCs) were YFP+, even in the thymus. This is a formal evidence that the majority of DCs in steady-state hematopoiesis originate from stages that have committed to the myeloid lineage. The lineage origin of DCs did not affect their functional abilities, including antigen-presentation and cytokine production. We then profiled the gene expression of the YFP+ and YFP- DCs at a genome wide level by DNA microarray analysis. Unexpectedly, the gene expression profiles of lymphoid and myeloid-derived DCs were virtually identical, irrespective of their organs. In contrast, the genetic programs were apparently different between splenic and thymic DCs, regardless of their lineage origin. Moreover, thymic DCs contained a number of more activated genes, such as MHC class II-related ones, as compared to splenic DCs. These results suggest that lymphoid and myeloid DCs might use a common developmental program that can be activated even after lymphoid or myeloid commitment. Thus, DCs are a unique cell population independent of other conventional lineage cells. Lineage-restricted DCs might be merged into an identical pool, distributed to various tissues, and finally fated by the microenvironment of their sites.
Cyclin A, the first cyclin ever cloned, is thought to be an essential component of the cell cycle engine. Mammalian cells encode two A-type cyclins, testis-specific cyclin A1 and ubiquitously ...expressed cyclin A2. Here we tested the requirement for cyclin A function using conditional knockout mice lacking both A-type cyclins. We found that acute ablation of cyclin A in fibroblasts did not affect cell proliferation, but led to prolonged expression of cyclin E across the cell cycle. However, combined ablation of all A- and E-type cyclins extinguished cell division. Hence, in fibroblasts cyclins A and E play redundant roles in cell proliferation. In contrast, ablation of cyclin A in bone marrow obliterated hematopoiesis. We found that cyclin A function was essential for proliferation of hematopoietic and embryonal stem cells. In these compartments cyclin A-Cdk complexes are expressed at particularly high levels, which may render stem cells dependent on cyclin A.
Objective: Patients with acute myelogenous leukaemia (AML) show co‐existing frequently internal tandem duplications of FLT3 (FLT3‐ITD) and mutations of nucleophosmin (NPM1‐Mt). We investigated the ...biological and clinical significance of FLT3‐ITD and/or NPM1‐Mt in this context.
Methods: We analysed 89 AML patients according to whether NPM1 and FLT3‐ITD were single mutants, double mutants, or wild type for both.
Results: FLT3‐ITD was detected in 19 of 89 patients (21.3%), while NPM1‐Mt was detected in 19 of 89 patients (21.3%); eight of 89 patients (9.0%) carried both FLT3‐ITD and NPM1‐Mt. By multivariate analysis, white blood cell count and peripheral blood blast cell count at diagnosis were significantly higher in patients with FLT3‐ITD but not in those with only NPM1‐Mt. NPM1‐Mt was significantly related to female gender, normal karyotype, and M4 or M5 disease according to French–American–British criteria. In addition, leukaemic blast cells with NPM1‐Mt, FLT3‐ITD, or both expressed CD34 less frequently than wild‐type blasts (P < 0.0001 and P = 0.005 respectively), while myelomonocytic markers such as CD11b and CD14 were expressed more frequently in patients with NPM1‐Mt.
Conclusion: FLT3‐ITD may increase potential for cell proliferation to produce a leukaemic population; NPM1‐Mt may cause cells to develop along the myelomonocytic lineage. Extensive analyses and detailed experiments will be required to clarify how NPM1 and FLT3 mutations interact in leukaemogenesis.
Mcl-1 (myeloid cell leukaemia-1) is an anti-apoptotic member of the Bcl-2 family protein, originally identified through the analysis of differentiating myeloid cells. We have recently reported that ...disruption of murine MCL-1 in adult murine hematopoiesis resulted in a complete deficiency of hematopoietic cells in the bone marrow, and that MCL-1 expression was activated in response to activation of c-Kit tyrosine kinase receptor signaling (Science 307;1101, 2005). Here we propose that human MCL-1 also plays a critical role in maintenance of normal and malignant human hematopoiesis. The analysis of FACS-sorted human HSCs and myeloid progenitors demonstrated that, as in case of murine hematopoiesis, HSCs expressed hMCL-1 at the highest level, and that its expression gradually downregulated in committed myeloid progenitors such as CMPs and GMPs. We also found that unlike murine long-term HSCs that express c-Kit but not FLT3 receptor tyrosine kinase, human long-term HSCs express both of these receptors. Ligation of FLT3 receptor in purified human HSCs immediately induced the expression of MCL-1, suggesting that FLT3 supports human HSC survival through stimulating MCL-1 expression. We then evaluated whether hMCL-1 is expressed in leukemic stem cells. Thirty patients with acute myeloid leukemia (AML) were enrolled in this study. Within the immature CD34+ blast population, the CD34+CD38− fraction expressed higher levels of MCL-1 than those in more differentiated CD34+CD38+ fraction. In most cases, CD34+CD38− AML cells expressed 2 to 10-fold higher levels of MCL-1 transcripts as compared to normal HSCs. Interestingly, the group of samples expressed highest levels of MCL-1 constituted mostly of FLT3/internal tandem duplications (ITDs) positive AML, suggesting that constitutive activation of FLT3 signaling by FLT3 mutations might induce overexpression of MCL-1. A high level expression of MCL-1 was also found in MOLM-13 and MV4-11, FLT3/ITD positive AML cell lines. Treatment of MOLM-13 and MV4-11 with the small molecule tyrosine kinase inhibitor PKC 412, resulted in induction of apoptosis, which was associated with decreased expression of MCL-1 transcripts and proteins. Based on these data, MCL-1 might play a critical role in maintenance of normal and malignant HSCs in human at least through FLT3 signaling. Our data also suggest that constitutive activation of FLT3 signaling by FLT3 mutation might contribute to leukemic transformation through enforcing survival of AML stem cells.
In hematopoietic stem cell development, the expression of critical genes is precisely regulated in a stage specific manner, which supports normal hematopoietic development through adequately ...regulating timing of cell division, self-renewal, and lineage commitment of hematopoietic stem cells. Regulation of gene expression is known to take place at transcriptional level. In addition to the transcriptional regulation, there are growing evidences that translational control of critical genes may play a role, especially in embryogenic development, suggesting an interesting possibility that translational control may also play a role in hematopoiesis. Here, we provide the evidence that the expression of a key transcription factor in lymphoid development, Notch1, is controlled at translational level in hematopoietic stem cell. To examine whether translation of mRNAs of hematopoietic major factors can be regulated at each stage of hematopoietic development, we established a retrovirus sensor vector, in which 3′UTR (untranslated region) of target genes is placed between the GFP coding region and the retrovirus 3′LTR. Since the transcribed mRNA from this vector is a fusion mRNA of GFP and 3′UTR from the gene of interest, this vector allows us to visualize the effect of 3′UTR on translational control of particular genes by GFP as a reporter. We cloned 3′UTR fragments from PU.1, GATA-1, GATA-2, C/EBPα and Notch1 genes into the sensor vector. Then, we introduced these retrovirus vectors into prospectively-purified hematopoietic stem and progenitor cells, including HSC, CMP and GMP, and their GFP intensities were monitored by a flowcytometer. We found that induction of the sensor vector with the 3′UTR sequence from Notch1 showed marked suppression of the GFP intensity at the HSC stage. The 3′UTR sequences from the other 4 transcription factors did not show such intensive inhibitory effect. Suppression of Notch1 transcription by its 3′UTR was further confirmed by using a retrovirus vector which has two distinct markers of YFP and GFP-3′UTR fusion genes under bi-directional EF1α promoter. These data suggest that the expression of Notch1 should be regulated at translational level by its 3′UTR at the HSC stage as well as at transcriptional level. Our data provide the first evidence that the stage-specific translational regulation can play an important role in organization of hematopoietic development.
Dendritic cells (DCs) have been shown to arise from both myeloid and lymphoid pathways, by evaluating the in vivo reconstituting potential of myeloid- and lymphoid-committed progenitor populations. ...It has been suggested that these “myeloid” or “lymphoid” DCs may constitute independent DC compartments with different functions. However, evaluation of DC development after conventional adoptive transfer experiments may not correctly represent normal DC development including lineage contribution toward the formation of the DC pool. In order to accurately evaluate the distribution and the function of DCs originated from each lineage, it is ideal to mark their origin in vivo in steady-state hematopoiesis. By crossing RAG1-Cre knockin with yellow fluorescence protein (YFP)-floxed reporter lines, we developed a mouse line in which cells with a history of RAG activation should be permanently marked with YFP as a result of lymphoid commitment. The YFP expression started at the common lymphoid progenitor (CLP) stage (~35%), and the percentages of YFP+ cells progressively increased as they differentiate into mature lymphocytes. More than 99.5% of mature T and B cells were marked by YFP, while <0.5% of granulocyte/monocyte progenitors (GMPs) or granulocytes were YFP+, indicating that this system efficiently and exclusively marks cells of lymphoid origin. We found that only 4.1, 4.6, and 9.3% of DCs were YFP+ in the spleen, the lymph nodes, and the thymus, respectively. This is the formal evidence that the vast majority of DCs in steady-state hematopoiesis originate from stages that have committed to the myeloid lineage, even in the thymus. We then profiled the gene expression of the YFP+ and YFP- DCs at a genome wide level by DNA microarray analysis. Unexpectedly, the gene expression profiles of myeloid and lymphoid-derived DCs are virtually identical. These results collectively suggest that myeloid and lymphoid DCs might use a common developmental program that can be activated even after myeloid or lymphoid commitment. Thus, the DC developmental program is unique, which should be independent of either myeloid or lymphoid commitment program.
Human herpesvirus‐8 (HHV‐8) encodes viral homologues of cellular genes, including viral interleukin 6 (vIL‐6), which induces endogenous human IL‐6 (hIL‐6) secretion. Unregulated overproduction of ...hIL‐6 in lymph nodes (LN) is thought to be responsible for the systemic manifestations of multicentric Castleman's disease (MCD). In the present study, we assessed the presence of HHV‐8 and HHV‐8‐encoded viral homologues in LN and peripheral blood mononuclear cells (PBMC) from adult Japanese patients with MCD. HHV‐8 DNA was amplified by nested polymerase chain reaction (PCR) and was detected in LN from 13 out of 16 MCD patients (81%). HHV‐8 DNA was also detected in PBMC from six out of seven patients (86%) whose LN were positive for HHV‐8 DNA. Because mRNA could not be successfully extracted from LN sections that were either formalin‐fixed or embedded in paraffin, we examined the expression of mRNA for HHV‐8‐encoded viral homologues, such as vIL‐6, vBCL‐2, vCyclin‐D and viral G‐protein‐coupled receptor (vGPCR) by nested reverse transcription (RT)‐PCR in PBMC from 10 MCD patients. However, mRNA of these HHV‐8‐encoded viral homologues was not detected in any patients tested. Although our results do not indicate a role for HHV‐8‐encoded viral homologues in the pathogenesis of MCD, they do suggest that HHV‐8 infection may be associated with MCD in adult Japanese patients.
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