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We present rank-based algorithms for making detection and comparison calls on expression microarrays. The detection call algorithm utilizes the discrimination scores. The comparison call algorithm utilizes intensity differences. Both algorithms are based on Wilcoxon's signed-rank test. Several parameters in the algorithms can be adjusted by the user to alter levels of specificity and sensitivity. The algorithms were developed and analyzed using spiked-in genes arrayed in a Latin square format. In the call process, p-values are calculated to give a confidence level for the pertinent hypotheses. For comparison calls made between two arrays, two primary normalization factors are defined. To overcome the difficulty that constant normalization factors do not fit all probe sets, we perturb these primary normalization factors and make increasing or decreasing calls only if all resulting p-values fall within a defined critical region. Our algorithms also automatically handle scanner saturation.

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Glucocorticoid and cyclic AMP increase tyrosine hydroxylase (TH) activity and mRNA levels in pheochromocytoma cultures. The transcriptional activity of the TH gene, as measured by nuclear run-on assay, is also increased when cultures are treated with the synthetic glucocorticoid dexamethasone or agents that increase intracellular cyclic AMP, such as forskolin and 8-BrcAMP. Both inducers effect transcriptional changes within 10 min after treatment and are maximal after 30 min for forskolin and after 60 min for dexamethasone. The 5' flanking sequences of the TH gene were fused to the bacterial gene chloramphenicol acetyltransferase (CAT), and the hybrid gene was transfected into pheochromocytoma cultures and GH4 pituitary cells. In both cell lines, a region of the TH gene containing bases -272 to +27 conferred induction of CAT by cyclic AMP, but not by glucocorticoid. The same results were found when a region of the TH gene containing -773 to +27 was used. Thus, the sequences required for induction of TH by cyclic AMP are contained within 272 bases of 5' flanking sequence, but sequences sufficient for glucocorticoid regulation are not contained within 773 bases.The rate of biosynthesis of specific neurotransmitters can be modulated by a variety of environmental, neuronal, and hormonal stimuli. In catecholamine biosynthesis, the rate of flow through the pathway is largely dependent on the activity of the initial enzyme, tyrosine hydroxylase (TH), which is expressed in the adrenal medulla, sympathetic ganglia, and certain defined nuclei of the brain. Activity of tyrosine hydroxylase can be modulated both by changes in the synthetic rate of new tyrosine hydroxylase polypeptide and by posttranslational modification of preexisting enzyme molecules.The synthesis of TH is influenced by a variety of factors; in vivo, TH is induced in response to environmental stimuli, such as stress (1), and this induction can be mimicked pharmacologically by treatment of animals with agents that deplete cellular catecholamine stores, such as reserpine (2). In cultures of adrenal chromaffin or pheochromocytoma cells, tyrosine hydroxylase activity is induced by a number of effectors, including glucocorticoid (3, 4), cyclic AMP (5, 6), epidermal growth factor (7), and nerve growth factor (6,8,9).Whether these effectors act through similar mechanisms and whether changes are modulated transcriptionally or posttranscriptionally is not known. Using a cloned cDNA probe for TH, we have previously shown increases in the mRNA for tyrosine hydroxylase following treatment of clonal rat pheochromocytoma cells from an adrenal medullary tumor with analogs of cyclic AMP and glucocorticoid (10). The TH RNA is also increased in adrenal glands and superior cervical ganglia in vivo when animals are subjected to cold stress or reserpine treatment (11-14). In this communication, we have extended those studies by demonstrating that both glucocorticoid and cyclic AMP stimulate the transcriptional activity of the TH gene. In addition, we report that the 5...

Exosomes are paracrine regulators of the tumor microenvironment and contain complex cargo. We previously reported that exosomes released from acute myeloid leukemia (AML) cells can suppress residual hematopoietic stem and progenitor cell (HSPC) function indirectly through stromal reprogramming of niche retention factors. We found that the systemic loss of hematopoietic function is also in part a consequence of AML exosome-directed microRNA (miRNA) trafficking to HSPCs. Exosomes isolated from cultured AML or the plasma from mice bearing AML xenografts exhibited enrichment of miR-150 and miR-155. HSPCs cocultured with either of these exosomes exhibited impaired clonogenicity, through the miR-150- and miR-155-mediated suppression of the translation of transcripts encoding c-MYB, a transcription factor involved in HSPC differentiation and proliferation. To discover additional miRNA targets, we captured miR-155 and its target transcripts by coimmunoprecipitation with an attenuated RNA-induced silencing complex (RISC)-trap, followed by high-throughput sequencing. This approach identified known and previously unknown miR-155 target transcripts. Integration of the miR-155 targets with information from the protein interaction database STRING revealed proteins indirectly affected by AML exosome-derived miRNA. Our findings indicate a direct effect of AML exosomes on HSPCs that, through a stroma-independent mechanism, compromises hematopoiesis. Furthermore, combining miRNA target data with protein-protein interaction data may be a broadly applicable strategy to define the effects of exosome-mediated trafficking of regulatory molecules within the tumor microenvironment.