Glucocorticoid-regulated microRNAs and mirtrons in acute lymphoblastic leukemia.

Glucocorticoids (GCs) induce apoptosis in lymphoid lineage cells and are therefore used in the therapy of acute lymphoblastic leukemia (ALL) and related malignancies. MicroRNAs (miRNAs) and the related mirtrons are ~22 nucleotide RNAs derived from polymerase-II transcripts and implicated in the control of essential biological functions, including apoptosis. Whether GCs regulate miRNA-encoding transcription units is unknown. We investigated miRNA/mirtron expression and GC regulation in 8 leukemia/lymphoma in vitro models and 13 ALL children undergoing systemic GC monotherapy using a combination of expression profiling techniques, real time reverse transcription (RT)-PCR and northern blotting to detect mature miRNAs and/or their precursors. We found that mature miRNA regulations can be inferred from expression data of their host genes. Although a simple miRNA-initiated canonical pathway to GC-induced apoptosis or cell cycle arrest did not emerge, we identified several miRNAs/mirtrons that were regulated by GC in patients and cell lines, including the myeloid-specific miR-223 and the apoptosis and cell cycle arrest-inducing miR15 ~ 16 clusters. In an in vitro model, overexpression of miR15b ~ 16 mimics increased and silencing by miR15b ~ 16 inhibitors decreased GC sensitivity. Thus, the observed complex changes in miRNA/mirtron expression during GC treatment might contribute to the anti-leukemic GC effects in a cell context-dependent manner.

The BCL2 rheostat in glucocorticoid-induced apoptosis of acute lymphoblastic leukemia.

Glucocorticoid (GC)-induced apoptosis is essential in the treatment of acute lymphoblastic leukemia (ALL) and related malignancies. Pro- and anti-apoptotic members of the BCL2 family control many forms of apoptotic cell death, but the extent to which this survival 'rheostat' is involved in the beneficial effects of GC therapy is not understood. We performed systematic analyses of expression, GC regulation and function of BCL2 molecules in primary ALL lymphoblasts and a corresponding in vitro model. Affymetrix-based expression profiling revealed that the response included regulations of pro-apoptotic and, surprisingly, anti-apoptotic BCL2 family members, and varied among patients, but was dominated by induction of the BH3-only molecules BMF and BCL2L11/Bim and repression of PMAIP1/Noxa. Conditional lentiviral gene overexpression and knock-down by RNA interference in the CCRF-CEM model revealed that induction of Bim, and to a lesser extent that of BMF, was required and sufficient for apoptosis. Although anti-apoptotic BCL2 members were not regulated consistently by GC in the various systems, their overexpression delayed, whereas their knock-down accelerated, GC-induced cell death. Thus, the combined clinical and experimental data suggest that GCs induce both pro- and anti-apoptotic BCL2 family member-dependent pathways, with the outcome depending on cellular context and additional signals feeding into the BCL2 rheostat.

CD4(+)CD8(+)TCR(low) thymocytes express low levels of glucocorticoid receptors while being sensitive to glucocorticoid-induced apoptosis.

While signaling by either the TCR or glucocorticoid receptor (GR) can induce apoptosis in thymocytes, recent studies have shown that combining these signals results in survival of CD4(+)CD8(+) thymocytes. Although glucocorticoids (GC) in this way may directly affect T cell selection, no data are available addressing GR expression in thymocyte subsets and in individual cells within subsets. We studied GR expression by combining immunofluorescence cell surface staining for CD4, CD8 and TCR with intracellular staining of GR in four-color cytometry. Significant differences of GR expression were observed in various thymocyte subsets, although a homogeneous distribution of GR expression in individual thymocyte subsets emerged. The highest GR expression was found in CD4(-)CD8(-)TCR(-) thymocytes, and decreased during development via the CD4(-)CD8(+)TCR(-) subpopulation into the CD4(+)CD8(+)TCR(low) subset. Interestingly, the latter population, although expressing less than half the GR density of CD4(-)CD8(-)TCR(-) cells, is the most sensitive subset to GC-induced apoptosis. Up-regulation of TCR expression by the CD4(+)CD8(+)TCR(low) subset to CD4(+)CD8(+)TCR(high) cells was accompanied by a parallel increase in GR expression. The latter finding and the presence of a homogeneous distribution of GR in each thymocyte subset provides an experimental basis for the concept that GR can antagonize TCR-mediated signals at a constant rate relative to TCR expression.

Network of vascular-associated dendritic cells in intima of healthy young individuals.

In earlier studies, our group has established a new "immunological" hypothesis for atherogenesis supported by experimental and clinical studies showing that inflammatory immunological reactions against heat shock protein 60 initiate the development of atherosclerosis. In the present study, we describe the discovery of a so-far-unknown network of dendritic cells in the innermost layer of arteries, the intima, but not veins of healthy humans and rabbits. The number of these dendritic cells is comparable to that of Langerhans cells in the skin, and dendritic cells show a similar phenotype (CD1a(+) S-100(+) lag(+) CD31(-) CD83(-) CD86(-) and no staining for von Willebrand factor or smooth muscle cell myosin). These vascular-associated dendritic cells accumulate most densely in those arterial regions that are subjected to major hemodynamic stress by turbulent flow conditions and are known to be predisposed for the later development of atherosclerosis. These results open new perspectives for the activation of the immune system within the arterial wall.