Constitutional pericentric inversion of chromosome 9 and hematopoietic recovery after allogeneic stem cell transplantation.

Recent reports suggest that hemopoietic stem cells with constitutional pericentric inversion of chromosome 9 [inv(9)] may be related to delayed engraftment or hemopoietic defect after stem cell transplantation (SCT). We conducted a retrospective study on five allogeneic SCT in which constitutional inv(9) was detected either in the donor or the recipient. The results showed that hematologic recovery was within the expected time range for all our patients. However, one patient exhibited decreasing blood counts between day +45 and +272 after transplantation, possibly due to protracted cytomegalovirus (CMV) infection and gansiclovir and imatinib treatment. Our findings suggest that constitutional inv(9) may not be associated with delayed hemopoietic recovery after SCT.

Expression of different NF-kappaB pathway genes in dendritic cells (DCs) or macrophages assessed by gene expression profiling.

NF-kappaB/Rel transcription factors have been implicated in the differentiation of monocytes to either dendritic cells (DCs) or macrophages, as well as in the maturation of DCs from antigen-processing to antigen-presenting cells. Recent studies of the expression pattern of Rel proteins and their inhibitors (IkappaBs) suggest that their regulation during this differentiation process is transcriptional. To investigate differential gene expression between macrophages and DCs, we used commercially available gene microarrays (GEArray KIT), which included four of the NF-kappaB/Rel family genes (p50/p105, p52/p100, RelB, and c-rel) and 32 additional genes either in the NF-kappaB signal transduction pathway or under transcriptional control of NF-kappaB/Rel factors. To generate macrophages and DCs, human adherent peripheral blood monocytes were cultured with M-CSF or GM-CSF + IL-4 respectively for up to 8 days. DCs (and in some experiments, macrophages) were treated with lipopolysaccharide (LPS) for the last 48 h of culture to induce maturation. Cells were harvested after 7 days, cDNA was prepared and radiolabeled with alpha-(32)P-dCTP, then hybridized to gene arrays containing specific gene probes. beta-actin and GAPDH or PUC18 oligonucleotides served as positive or negative controls, respectively. The expression of all four NF-kappaB/Rel family genes examined was significantly upregulated in maturing DCs compared to macrophages. The strongest difference was observed for c-rel. RT-PCR determinations of c-rel, RelB, and p105 mRNAs confirmed these observations. Among the 32 NF-kappaB/Rel pathway genes, 14 were upregulated in mature DCs compared to macrophages. These genes were IkappaBalpha, IKK-beta, NIK, ICAM-1, P-selectin, E-selectin, TNF-alpha, TNFR2, TNFAIP3, IL-1alpha, IL-1R1, IL-1R2, IRAK, and TANK. By contrast, only mcp-1 (monocyte chemotactic protein 1) was upregulated in macrophages compared to DCs. NF-kappaB pathway genes upregulated in DCs compared to macrophages were constitutively expressed in monocytes then selectively downregulated during macrophage but not DC differentiation. LPS did not induce expression of most of these genes in macrophages but LPS did induce upregulation of IL-8 in mature macrophages. We conclude that NF-kappaB/Rel family genes, especially c-rel, are selectively expressed during differentiation of monocytes towards DCs. Moreover, this differential expression is associated both with activation of different NF-kappaB signal transduction pathways in DCs and macrophages and with expression of a unique subset of genes in DCs that are transcriptionally targeted by NF-kappaB/Rel factors. The results illustrate the ability of the NF-kappaB pathway to respond to differentiation stimuli by activating in a cell-specific manner unique signalling pathways and subsets of NF-kappaB target genes.

Expression of multiple genes regulating cell cycle and apoptosis in differentiating hematopoietic cells is dependent on iron.

OBJECTIVE: Iron plays critical roles in many biological processes including hematopoietic cell growth and differentiation. Iron is essential for the differentiation of HL-60 promonocytes. HL-60 cells stimulated with phorbol myristate acetate (PMA) undergo G1/S phase cell-cycle arrest and differentiate to monocyte/macrophages. With iron deprivation, PMA-induced HL-60 cells bypass differentiation and undergo apoptosis. To investigate the molecular basis underlying this observation, we used commercially available gene microarrays to evaluate expression of multiple genes involved in the regulation of cell cycling and apoptosis. METHODS: We treated HL-60 cells with PMA +/- desferrioxamine (DF), a potent iron chelator, to produce iron deprivation. Cells were cultured for 48 hours, and cDNA was prepared and radiolabeled with alpha-(32)P dCTP, then hybridized to gene arrays containing specific cDNA fragments. RESULTS: Expression of 11 of 43 genes was inhibited greater than 50% by iron deprivation. These genes were Rb; p21 (WAF1/CIP1); bad; cdk2; cyclins A, D3, E1; c-myc; egr-1; iNOS; and FasL. For each gene the microarray results were confirmed by RT-PCR and/or Northern or Western blotting. Nuclear transcription assays indicated that the role of iron in Rb expression was to support gene transcription. Addition of ferrioxamine (iron saturated DF) instead of DF to PMA-induced cells did not affect gene expression, indicating that diminished expression was due to iron deprivation, not nonspecific toxicity. CONCLUSION: Iron supports expression of multiple cell cycle-regulatory and apoptosis-related genes during HL-60 cell differentiation, and, in this way, is involved in regulation of a critical cell decision point-the decision to pursue a differentiation-related or apoptotic pathway.