Side population (SP) cells in cancers, including multiple myeloma, exhibit tumor-initiating characteristics. In the present study, we isolated SP cells from human myeloma cell lines and primary tumors to detect potential therapeutic targets specifically expressed in SP cells. We found that SP cells from myeloma cell lines (RPMI 8226, AMO1, KMS-12-BM, KMS-11) express CD138 and that non-SP cells include a CD138-negative population. Serial transplantation of SP and non-SP cells into NOD/Shi-scid IL-2γnul mice revealed that clonogenic myeloma SP cells are highly tumorigenic and possess a capacity for self-renewal. Gene expression analysis showed that SP cells from five MM cell lines (RPMI 8226, AMO1, KMS-12-BM, KMS-11, JJN3) express genes involved in the cell cycle and mitosis (e.g., CCNB1, CDC25C, CDC2, BIRC5, CENPE, SKA1, AURKB, KIFs, TOP2A, ASPM), polycomb (e.g., EZH2, EPC1) and ubiquitin-proteasome (e.g., UBE2D3, UBE3C, PSMA5) more strongly than do non-SP cells. Moreover, CCNB1, AURKB, EZH2 and PSMA5 were also upregulated in the SPs from eight primary myeloma samples. On that basis, we used an aurora kinase inhibitor (VX-680) and a proteasome inhibitor (bortezomib) with RPMI 8226 and AMO1 cells to determine whether these agents could be used to selectively target the myeloma SP. We found that both these drugs reduced the SP fraction, though bortezomib did so more effectively than VX-680 due to its ability to reduce levels of both phospho-histone H3 (p-hist. H3) and EZH2; VX-680 reduced only p-hist. H3. This is the first report to show that certain oncogenes are specifically expressed in the myeloma SP, and that bortezomib effectively downregulates expression of their products. Our approach may be useful for screening new agents with which to target a cell population possessing strong tumor initiating potential in multiple myeloma.
Multiple myeloma (MM) is characterized by the accumulation of a population of malignant plasma cells within the bone marrow and its microenvironment. A hypoxic niche is located within the microenvironment, which causes myeloma cells to become quiescent, anti‐apoptotic, glycolytic, and immature. Cell heterogeneity may be related to distinct gene expression profiles under hypoxic and normoxic conditions. During hypoxia, myeloma cells acquire these phenotypes by downregulating interferon regulatory factor 4 (IRF4), an essential transcription factor in myeloma oncogenesis. To identify essential microRNAs and their targets regulated under hypoxic conditions, we undertook microRNA and cDNA microarray analyses using hypoxia‐exposed primary MM samples and myeloma cell lines. Under hypoxia, only miR‐210 was highly upregulated and was accompanied by direct downregulation of an 18S rRNA base methyltransferase, DIMT1. This inverse expression correlation was validated by quantitative RT‐PCR for primary MM samples. We further determined that DIMT1 has an oncogenic potential as its knockdown reduced tumorigenicity of myeloma cells through regulation of IRF4 expression. Notably, by analyzing gene expression omnibus datasets in the National Center for Biotechnology Information database, we found that DIMT1 expression increased gradually with MM progression. In summary, by screening for targets of hypoxia‐inducible microRNA‐210, we identified DIMT1 as a novel diagnostic marker and therapeutic target for all molecular subtypes of MM.
Summary:We have examined the reconstitution of ␥␦ T cell repertoire diversity after human allogeneic hematopoietic cell transplantation using a polymerase chain reaction (PCR)-based complementarity-determining region (CDR) 3 size spectratyping and DNA sequencing. The CDR3 complexity in the variable region of the T cell receptor (TCR)-␦ chain was different amongst the individuals studied. Furthermore, CDR3 size distribution patterns of allogeneic hematopoietic cell transplant recipients were almost completely recovered by a few months after transplantation. In some patients, clonal predominance of the TCRDV1+ T cells became evident during the period after transplantation. In one particular donor/recipient pair, clonal predominance of TCRDV1+ T cells was already present in blood lymphocytes of the donor, and was also observed in the recipient after transplantation. Using this donor/recipient pair, we have questioned whether ␥␦ T cell regeneration occurs via the peripheral expansion of mature T cells in the graft. In the donor lymphocytes, two expanding ␥␦ T cell clones, which were demonstrated by CDR3 sequences of the TCR-␦ chain, were recognized. These two clones were identified in the T cells from the recipient post transplant, but not before transplantation. One of the two clones was still detectable 1. years after the transplant procedure. These results strongly suggest that peripheral expansion of mature T cells in the graft is the principal pathway of ␥␦ T cell regeneration after allogeneic hematopoietic cell transplantation in adults. Bone Marrow Transplantation (2000) 26, 177-185. Keywords: T cell receptor; ␦-chain; diversity; transplantation High-dose chemoradiotherapy depletes T cells in blood and marrow transplant recipients and adequate T cell regeneration is essential for successful transplantation. The repertoire of T cells requires to be sufficiently diverse in order to recognize a wide range of microorganism-derived antigens. ␣ T cells are almost exclusively thymus-dependent
We report downregulatory effects of granulocyte colony-stimulating factor (G-CSF) on allogeneic immune responses in vitro. G-CSF did not affect the proliferative response of peripheral blood mononuclear cells (PBMC) against allogeneic Daudi cells but did inhibit tumor necrosis factor (TNF)-alpha secretion. In contrast with G-CSF, granulocyte- macrophage (GM)-CSF and interleukin (IL)-3 enhanced alloactivation- induced TNF-alpha production. G-CSF-mediated suppression of TNF-alpha production was not affected by fixation of stimulators. G-CSF did not inhibit TNF-alpha mRNA expression or accelerate mRNA degradation, whereas pentoxifylline inhibited the expression of TNF-alpha mRNA. These results indicate that G-CSF acts directly on responder cells and modulates TNF-alpha production at posttranscriptional levels. Suppression of TNF-alpha secretion was accompanied by an increase of intracellular cyclic adenosine monophosphate (cAMP) concentration in alloactivated PBMC. The cell-permeable cAMP analogue, dibutyryl cAMP, suppressed TNF-alpha secretion without affecting TNF-alpha mRNA expression. G-CSF showed an inhibitory effect on the development of cytotoxic effector cells against allogeneic Daudi cells. Anti-TNF-alpha monoclonal antibody (MoAb) also inhibited the induction of cytolytic activity, and the inhibitory effects of G-CSF and anti-TNF-alpha MoAb on killer activity generation were overcome by adding exogenous TNF- alpha. Hence, impaired generation of cytolytic effector cells by G-CSF is believed to be the result of reduced TNF-alpha production. Collectively, the results described above suggest that G-CSF downregulates allogeneic immune responses by posttranscriptionally inhibiting TNF-alpha production.
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