SUMMARY The importance of the p53 protein in the cellular response to DNA damage is well known, but its function during steady-state hematopoiesis has not been established. We have defined a critical role of p53 in regulating hematopoietic stem cell quiescence, especially in promoting the enhanced quiescence seen in HSCs that lack the MEF/ELF4 transcription factor. Transcription profiling of HSCs isolated from wild type and p53 null mice identified Gfi-1 and Necdin as p53 target genes and using lentiviral vectors to upregulate or knockdown the expression of these genes, we show their importance in regulating HSC quiescence. Establishing the role of p53 (and its target genes) in controlling the cell cycle entry of HSCs may lead to therapeutic strategies capable of eliminating quiescent cancer (stem) cells.

Lethal 3 malignant brain tumor 1 (L3MBTL1), a homolog of the Drosophila polycomb tumor suppressor l(3)mbt, contains three tandem MBT repeats (3xMBT) that are critical for transcriptional repression. We recently reported that the 3xMBT repeats interact with mono-and dimethylated lysines in the amino termini of histones H4 and H1b to promote methylation-dependent chromatin compaction. Using a series of histone peptides, we now show that the recognition of mono-and dimethylated lysines in histones H3, H4 and H1.4 (but not their trimethylated or unmodified counterparts) by 3xMBT occurs in the context of a basic environment, requiring a conserved aspartic acid (D355) in the second MBT repeat. Despite the broad range of in vitro binding, the chromatin association of L3MBTL1 mirrors the progressive accumulation of H4K20 monomethylation during the cell cycle. Furthermore, transcriptional repression by L3MBTL1 is enhanced by the H4K20 monomethyltransferase PR-SET7 (to which it binds) but not SUV420H1 (an H4K20 trimethylase) or G9a (an H3K9 dimethylase) and knockdown of PR-SET7 decreases H4K20me1 levels and the chromatin association of L3MBTL1. Our studies identify the importance of H4K20 monomethylation and of PR-SET7 for L3MBTL1 function.

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The transcriptional circuitry that regulates the quiescence of hematopoietic stem cells is largely unknown. We report that the transcription factor known as MEF (or ELF4), which is targeted by the t(X;21)(q26;q22) in acute myelogenous leukemia, regulates the proliferation of primitive hematopoietic progenitor cells at steady state, controlling their quiescence. Mef null HSCs display increased residence in G0 with reduced 5-bromodeoxyuridine incorporation in vivo and impaired cytokine-driven proliferation in vitro. Due to their increased HSC quiescence, Mef null mice are relatively resistant to the myelosuppressive effects of chemotherapy and radiation. Thus, MEF plays an important role in the decision of stem/primitive progenitor cells to divide or remain quiescent by regulating their entry to the cell cycle.

Purpose: Lymph vessel density (LVD) and microvessel density (MVD) correlate with the malignant potential of tumors and patient survival. Vascular endothelial growth factors (VEGF)-A, VEGF-C, and VEGF-D could modulate LVD and MVD. We investigated the clinical and prognostic significance of LVD and MVD on lymphangiogenic and angiogenic function of VEGF-A,VEGF-C, and VEGF-D in human bladder cancer. Experimental Design: We reviewed tissue samples from patients with nonmetastatic bladder cancer who had undergone transurethral resections (n = 126). The densities of D2-40-positive vessels (LVD) and CD34-positive vessels (MVD) were measured by a computer-aided image analysis system. Expression of VEGF-A, VEGF-C, and VEGF-D was examined by immunohistochemistry; survival analyses and their independent roles were investigated using multivariate analysis models. Results: LVD was associated with tumor grade but not with pTstage. LVD was associated with metastasis-free survival (log rank P = 0.039), but was not an independent prognostic factor. Although MVD affected survival, the combination of high LVD and high MVD in tumors was an independent predictor of metastasis-free survival. Although VEGF-C expression was positively associated with both LVD and MVD,VEGF-D was associated only with LVD.VEGF-A expression was associated with MVD in univariate analysis, however, it was not an independent factor. Conclusions: Lymphangiogenesis and angiogenesis influence metastasis-free survival, and are regulated by VEGF-C and/or VEGF-D. Our results suggest that LVD and MVD are useful tools for the selection of postoperative management and treatment strategies in patients with bladder cancer.Metastatic dissemination of the primary tumor is an important factor that negatively affects the prognosis in most malignancies and neovascularization (angiogenesis) plays a critical role in tumor growth and systemic dissemination of cancer cells (1). As such, much attention has been focused on the pathologic significance and detailed mechanism of the vascular system and angiogenesis in cancers. In addition to dissemination of cancer cells via the bloodstream, the lymphatic system is also thought to play an important role in tumor cell dissemination.Indeed, metastatic spread to regional lymph nodes is an early step in the systemic dissemination of tumors, and lymph node metastasis is generally associated with poor survival (2, 3). However, the clinical significance of the de novo formation of lymphatic capillaries (lymphangiogenesis) and its regulation in cancer remains unclear, largely because specific endothelial markers for lymphatic vessels are unknown and lymphatic vessels cannot be detected in human cancer tissues (4). In recent years, several new specific antibodies for lymphatic endothelial cells have been developed and used to investigate the clinical and pathologic significance of lymphangiogenesis in various cancers (5).Bladder cancer is the second most common malignant tumor of the urogenital region. This tumor is associated with fr...

Mesenchymal stem cells (MSCs) have emerged as a new therapeutic modality for reconstituting the hematopoietic microenvironment by improving engraftment in stem cell transplantation. However, the availability of conventional bone marrow (BM)-derived MSCs (BMSCs) is limited. Recent studies showed that a large number of MSCs can be easily isolated from fat tissue (adipose tissue-derived MSCs [ADSCs]). In this study, we extensively evaluated the hematopoiesis-supporting properties of ADSCs, which are largely unknown. In vitro coculture and progenitor assays showed that ADSCs generated significantly more granulocytes and progenitor cells from human hematopoietic stem cells (HSCs) than BMSCs. We found that ADSCs express the chemokine CXCL12, a critical regulator of hematopoiesis, at levels that are three fold higher than those with BMSCs. The addition of a CXCL12 receptor antagonist resulted in a lower yield of granulocytes from ADSC layers, whereas the addition of recombinant CXCL12 to BMSC cocultures promoted the growth of granulocytes. In vivo cell homing assays showed that ADSCs facilitated the homing of mouse HSCs to the BM better than BMSCs. ADSCs injected into the BM cavity of fatally irradiated mice reconstituted hematopoiesis more promptly than BMSCs and subsequently rescued mice that had received a low number of HSCs. Secondary transplantation experiments showed that ADSCs exerted favorable effects on long-term HSCs. These results suggest that ADSCs can be a promising therapeutic alternative to BMSCs.

The zinc finger transcription factor GATA-2 plays a critical role in the survival and proliferation of hematopoietic stem cells. This study examined the interaction of GATA-2 with histone deacetylases (HDACs) to define the involvement of HDACs in the regulation of GATA-2 function. GATA-2 directly associates with HDAC3 but not with HDAC1. Consistent with this, HDAC3 suppressed the tran-scriptional potential of GATA-2, whereas HDAC1 did not affect GATA-2-dependent transcription. Results further demonstrated that GATA-2 and HDAC3 colocal-ized in the nucleus. These results identify GATA-2 as a nuclear target for HDAC3-mediated repression. Furthermore, GATA-2 also directly associated with HDAC5 but not with other class II HDACs examined, that is, HDAC4 and HDAC6. This is the first demonstration that a tissue-specific transcription factor directly and selectively interacts with HDAC3 and HDAC5 among HDAC family members. (Blood. 2001;98:2116-2123)

The objective of this prospective clinical trial (JALSG-STIM213, UMIN000011971) was to evaluate treatment-free remission (TFR) rates after discontinuation of imatinib in chronic myeloid leukemia (CML). CML patients who received imatinib treatment for at least 3 years and sustained deep molecular response for at least 2 years were eligible. Molecular recurrence was defined as loss of major molecular response (MMR). Of the 68 eligible patients, 38.2% were women, the median age was 55.0 years, and the median duration of imatinib treatment was 97.5 months. The 12-month TFR rate was 67.6%. Patients who lost MMR were immediately treated with imatinib again; all re-achieved MMR. Three-year treatment-free survival (TFS) was estimated as 64.6% using the Kaplan-Meier method. Undetectable molecular residual disease (UMRD) was defined as no BCR-ABL1 in > 100,000 ABL1 control genes using international scale polymerase chain reaction. UMRD at the study baseline was found to be predictive of continuation of TFR. Our findings suggest that CML patients who meet all the eligibility criteria that have commonly been used in the TFR trials are able to discontinue imatinib use safely. TFR may thus be valuable as a new goal for CML treatment in Japan.