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We examined expression profiles of hematopoietic tissue-specific microRNAs (miRNAs; miR-142, miR-155, miR-181 and miR-223) in 17 commercially available malignant hematopoietic cell lines and compared to those in highly purified normal human B, T, monocytic and granulocytic lineages. Although malignant cell lines examined showed miRNA expression patterns similar to normal human hematopoietic lineages, the levels of miRNA expression among cell lines and normal cell lineages were considerably different, indicating the significance of miRNAs in human hematopoietic diseases. Further our results showed differences in miRNA expression between mouse and human hematopoietic cells, suggesting important regulatory roles of miRNAs in human hematopoiesis and oncogenesis.

Haematopoiesis is the process by which blood and immune cells are replenished from a finite number of resident bone marrow (BM) haematopoietic stem cells (HSCs). Regulatory molecules within the BM microenvironment contribute developmental signals to an interactive network capable of ensuring ordered biological processes. Many bioactive molecules contribute to the network through G protein-coupled receptors (GPCRs). GPCRs are seven-transmembrane receptors that, following ligand binding, signal by activating coupled heterotrimeric G proteins. This review focuses on those bioactive molecules that regulate haematopoietic development through GPCRs. Chemokines (SDF-1alpha, MIP-1), opioids and tachykinins (SP, NK-A) are important G protein-coupled haematopoietic regulators. Their biology in normal and diseased haematopoiesis is discussed below, as well as their potential as therapeutic targets.

MicroRNAs (miRNAs) are an important class of endogenously derived, small approximately 22 nucleotide noncoding regulatory RNAs that have recently become implicated in development, cell regulation and cancers of various tissues. Here we report a nonisotopic Northern analysis method for miRNA detection using 3'-digoxigenin (DIG)-labeled RNA oligo probes. Northern blot analysis was performed using miRNA or total RNA fractions extracted from human leukemic cell lines, and blots were hybridized with either 32P- or DIG-labeled RNA probe for miR-181, miR-155 or miR-16. A labeled probe for U6 small nuclear RNA served as an internal control. The use of DIG-labeled RNA probes was equally sensitive compared to 32P-labeled probes in detecting miRNA quantities as low as 50 ng. The ability to use nonisotopic methods and yet obtain sensitive and reliable results offers an advantage to investigators who prefer to avoid isotopes.

Breast cancer remains the cancer with the highest mortality among women in the United States. Peptides derived from the oncogenic Tac1 gene (full transcript: betaPPT-A) stimulate the proliferation of breast cancer cells (BCCs) via seven-transmembrane G protein-coupled neurokinin 1 (NK1) and NK2 receptors. The NK1 gene could generate full-length (NK1-FL) and truncated (NK1-Tr) transcripts. NK1-Tr lacks 100 residues in their cytoplasmic end, could couple to G proteins, and shows reduced efficiency with respect to internalization and desensitization. This study reports on a role of NK1-Tr in the transformation of nontumorigenic breast cells, and investigates whether Tac1 expression is linked to the generation of NK1-Tr. Western blots and Northern analyses showed coexpressions of NK1-Tr and NK1-FL in BCCs (cell lines and primary cells from patients with different stages of breast cancer). Stable transfections of betaPPT-A or NK1-Tr expression vectors in nontumorigenic cells showed each induces the expression of the other, consequently resulting in a transformed phenotype. Analyses with microarrays indicate similar patterns of cytokine production by NK1-Tr transfectants and BCCs, but not NK1-FL transfectants. These observations indicate tumor-promoting properties by NK1-Tr, but not NK1-FL. Overall, the oncogenic property of Tac1 in breast cells involves concomitant expression of NK1-Tr and vice versa, consequently leading to the production of cytokines with growth promoting functions.

Despite intensive therapy, approximately 60-80% of children who are diagnosed with metastatic neuroblastoma (NB) succumb to the disease. NB preferentially metastasizes to the bone marrow (BM). In the present study we used SY5Y and CHP212 (NB cell lines) to study the roles of the preprotachykinin-I (PPT-I) gene and the natural receptors for PPT-I peptides, neurokinin-1 (NK-1) and NK-2, in the biology of NB. PPT-I, NK-1 and NK-2 were constitutively expressed in the NB cells. Functional studies, with specific NK receptor antagonists, showed that PPT-I peptides mediate autocrine proliferation of the NB cells through both NK-1 and NK-2 receptors. Full-length and truncated NK-1 receptors were detected in NB cells. Since there is one copy of the NK-1 gene, we used NK-1-specific siRNA to suppress the expression of NK-1. The NK-1-deficient NB cells showed phenotypes consistent with cell differentiation. Suppression of NK-1 did not appear to cause cell death, as demonstrated by trypan blue exclusion and by undetectable active caspase. NK-1 suppression reduced the proliferation of the NB cells beginning by 10-fold at day 1 and reached a 10(5)-fold reduction by day 10. The NK-1 deficient cells did not proliferate when they were placed as cocultures with BM stroma, which suggests that NK-1 signaling is important for the survival of NB cells in the BM. The results show potential roles for NK receptors in the proliferation of NB.

Pure red cell aplasia (PRCA) is a rare hematological syndrome characterized by the lack of red cell progenitors in an otherwise normocellular bone marrow. Many agents and mechanisms have been implicated in the pathophysiology of PRCA, including immune-mediated dysfunctions. This report describes three patients with PRCA with unknown underlying cause and showed that for each, increases in CD8+ cells blunted the maturation of early erythroid (BFU-E). Each patient subsequently responded to immunosuppressive therapy. Peripheral blood mononuclear cells from age- and sex-matched healthy controls showed comparable distribution of CD3, CD4 and CD16, but significant increase in CD8 and decreased CD19. The distribution of lymphocyte subsets correlated with mitogen responses, but showed no difference in allogeneic responses when compared to controls. The adherent population in PRCA is important for mediating the hyper-immune state of patients, when IL-2 levels were used as readout. There was a trend for decreased BFU-E in patients, but marked reduction for late erythroid progenitors (CFU-E). CD8+ cells from PRCA blunted the maturation of BFU-E, despite increasing erythropoietin concentrations. These results strongly suggest that there are defects in the lymphoid compartment that feedback on the erythroid lineage of PRCA.