SUMMARY Regulation of stem and progenitor cell populations is critical in the development, maintenance and regeneration of tissues. Here, we define a novel mechanism by which a niche-secreted ribonuclease, angiogenin (ANG), distinctively alters the functional characteristics of primitive hematopoietic stem/progenitor cells (HSPC) compared with lineage-committed myeloid-restricted progenitors (MyePro). Specifically, ANG reduces the proliferative capacity of HSPC while simultaneously increasing proliferation of MyePro. Mechanistically, ANG induces cell type-specific RNA processing events: tRNA-derived stress-induced small RNA (tiRNA) generation in HSPC and ribosomal RNA (rRNA) induction in MyePro, leading to respective reduction and increase in protein synthesis. Recombinant ANG protein improves survival of irradiated animals and enhances hematopoietic regeneration of mouse and human HSPC in transplantation. Thus, ANG plays a non-cell autonomous role in regulation of hematopoiesis by simultaneously preserving HSPC stemness and promoting MyePro proliferation. These cell type-specific functions of ANG suggest considerable therapeutic potential.

Drugs that target microtubules are thought to inhibit cell division and cell migration by suppressing dynamic instability, a "search and capture" behavior that allows microtubules to probe their environment. Here, we report that subtoxic drug concentrations are sufficient to inhibit plus-end microtubule dynamic instability and cell migration without affecting cell division or microtubule assembly. The higher drug concentrations needed to inhibit cell division act through a novel mechanism that generates microtubule fragments by stimulating microtubule minus-end detachment from their organizing centers. The frequency of microtubule detachment in untreated cells increases at prophase suggesting that it is a regulated cellular process important for spindle assembly and function. We conclude that drugs produce differential dose-dependent effects at microtubule plus and minus-ends to inhibit different microtubule-mediated functions.

Human brain and testis specific betaIII-tubulin was amplified from a cDNA library, modified to encode a C-terminal hemagglutinin antigen epitope tag, and cloned into a vector that allows tetracycline regulated expression in mammalian cells. Immunofluorescence analysis of transfected Chinese hamster ovary cells demonstrated that expressed HA-tagged betaIII-tubulin is able to assemble with endogenous tubulin into microtubules even though betaIII-tubulin is not a normal constituent of these cells. A stable G418-resistant clone with moderate HAbetaIII-tubulin expression displayed weak (1.5-2-fold) resistance to paclitaxel. A second clone with higher HAbetaIII-tubulin expression could not grow unless tetracycline was present to repress transcription of the transfected cDNA. Analysis of cellular microtubules in each of these clones indicated that incorporation of HAbetaIII-tubulin led to a significant expression-dependent decrease in assembled tubulin. Paclitaxel resistant cells were also directly selected from the transfected cell population using a paclitaxel concentration 4 times higher than the minimum toxic dose. Few cells were able to survive the selection and they grew very slowly. Western blot analysis of these resistant cells revealed very high HAbetaIII-tubulin expression that led to almost complete replacement of endogenous beta-tubulin at steady state. Transfected betaIII-tubulin with no epitope tag behaved in a very similar fashion indicating that presence of the HA tag had no discernible functional effect. The results demonstrate that betaIII-tubulin diminishes microtubule assembly, is toxic when present at high levels, but is able to confer weak resistance to paclitaxel when expressed at moderate levels in mammalian cells.