Heather H. Ross

Bromodeoxyuridine Induces Senescence in Neural Stem and Progenitor Cells

Bromodeoxyuridine (BrdU) is a halogenated pyrimidine that incorporates into newly synthesized DNA during the S phase. BrdU is used ubiquitously in cell birthdating studies and as a means of measuring the proliferative index of various cell populations. In the absence of secondary stressors, BrdU is thought to incorporate relatively benignly into replicating DNA chains. However, we report here that a single, low‐dose pulse of BrdU exerts a profound and sustained antiproliferative effect in cultured murine stem and progenitor cells. This is accompanied by altered terminal differentiation, cell morphology, and protein expression consistent with the induction of senescence. There is no evidence of a significant increase in spontaneous cell death; however, cells are rendered resistant to chemically induced apoptosis. Finally, we show that a brief in vivo BrdU regimen reduces the proliferative potential of subsequently isolated subependymal zone neurosphere‐forming cells. We conclude, therefore, that BrdU treatment induces a senescence pathway that causes a progressive decline in the replication of rapidly dividing stem/progenitor cells, suggesting a novel and uncharacterized effect of BrdU. This finding is significant in that BrdU‐incorporating neural stem/progenitor cells and their progeny should not be expected to behave normally with respect to proliferative potential and downstream functional parameters. This effect highlights the need for caution when results based on long‐term BrdU tracking over multiple rounds of replication are interpreted. Conversely, the reliable induction of senescence in stem/progenitor cells in vitro and in vivo may yield a novel platform for molecular studies designed to address multiple aspects of aging and neurogenesis.

Ethynyldeoxyuridine (EdU) suppresses in vitro population expansion and in vivo tumor progression of human glioblastoma cells

Thymidine analogs (TAs) are synthetic nucleosides that incorporate into newly synthesized DNA. Halogenated pyrimidines (HPs), such as bromodeoxyuridine (BrdU), are a class of TAs that can be detected with antibodies and are commonly used for birthdating individual cells and for assessing the proliferative index of cell populations. It is well established that HPs can act as radiosensitizers when incorporated into DNA chains, but they are generally believed not to impair normal cell function in the absence of secondary stressors. However, we and others have shown that HP incorporation leads to a sustained suppression of cell cycle progression in mammalian cells, resulting in cellular senescence in somatic cells. In addition, we have shown that HP incorporation results in delayed tumor progression in a syngeneic rat model of glioma. Here we examine ethynyldeoxyuridine (EdU), a newly developed and alkylated TA, for its anti-cancer activity, both in vitro and in vivo. We show that EdU, like HPs, leads to a severe reduction in the proliferation rate of normal and transformed cells in vitro. Unlike HPs, however, EdU incorporation also causes DNA damage resulting in the death of a substantial subset of treated cells. When administered over an extended time as a monotherapy to mice bearing subcutaneous xenografts of human glioblastoma multiforme tumors, EdU significantly reduces tumor volume and increases survival without apparent significant toxicity. These results, combined with the fact that EdU readily crosses the blood–brain barrier, support the continued investigation of EdU as a potential therapy for malignant brain tumors.

Production of Neurospheres from CNS Tissue

The relatively recent discovery of persistent adult neurogenesis has led to the experimental isolation and characterization of central nervous system neural stem cell populations. Protocols for in vitro analysis and expansion of neural stem cells are crucial for understanding their properties and defining characteristics. The methods described here allow for cell and molecular analysis of individual clones of cells--neurospheres--derived from neural stem/progenitor cells. Neurospheres can be cultivated from a variety of normal, genetically altered, or pathological tissue specimens, even with protracted postmortem intervals, for studies of mechanisms underlying neurogenesis, cell fate decisions, and cell differentiation. Neurosphere-forming cells hold great promise for the development of cell and molecular therapeutics for a variety of neurological diseases.

Transcriptional regulation of myotrophic actions by testosterone and trenbolone on androgen-responsive muscle

Androgens regulate body composition and skeletal muscle mass in males, but the molecular mechanisms are not fully understood. Recently, we demonstrated that trenbolone (a potent synthetic testosterone analogue that is not a substrate for 5-alpha reductase or for aromatase) induces myotrophic effects in skeletal muscle without causing prostate enlargement, which is in contrast to the known prostate enlarging effects of testosterone. These previous results suggest that the 5α-reduction of testosterone is not required for myotrophic action. We now report differential gene expression in response to testosterone versus trenbolone in the highly androgen-sensitive levator ani/bulbocavernosus (LABC) muscle complex of the adult rat after 6weeks of orchiectomy (ORX), using real time PCR. The ORX-induced expression of atrogenes (Muscle RING-finger protein-1 [MuRF1] and atrogin-1) was suppressed by both androgens, with trenbolone producing a greater suppression of atrogin-1 mRNA compared to testosterone. Both androgens elevated expression of anabolic genes (insulin-like growth factor-1 and mechano-growth factor) after ORX. ORX-induced increases in expression of glucocorticoid receptor (GR) mRNA were suppressed by trenbolone treatment, but not testosterone. In ORX animals, testosterone promoted WNT1-inducible-signaling pathway protein 2 (WISP-2) gene expression while trenbolone did not. Testosterone and trenbolone equally enhanced muscle regeneration as shown by increases in LABC mass and in protein expression of embryonic myosin by western blotting. In addition, testosterone increased WISP-2 protein levels. Together, these findings identify specific mechanisms by which testosterone and trenbolone may regulate skeletal muscle maintenance and growth.

Identification of a novel human MT5-MMP transcript variant in multipotent NT2 cells

Membrane‐type 5 matrix metalloproteinase (MT5‐MMP) expression is ubiquitous in brain development while restricted to regions of neuroplasticity in adult. In the multipotent NT2 model of neurogenesis and differentiation, MT5‐MMP is differentially expressed with significantly higher mRNA levels in the differentiated neuronal hNT cells. MT5‐MMP cDNA cloned from NT2 cells unexpectedly revealed a novel sequence (MT5‐MMPvar) characterized by a 162 bp deletion. Both transcripts were identified in NT2, hNT and adult human hippocampus. In vitro, MT5‐MMPvar translated into a ∼55 kDa protein. It was also detected in NT2, hNT and adult human hippocampus. These results suggest more than one human MT5‐MMP transcript may exist in the central nervous system.

Intraspinal transplantation of subventricular zone-derived neural progenitor cells improves phrenic motor output after high cervical spinal cord injury

ABSTRACT Following spinal cord injury (SCI), intraspinal transplantation of neural progenitor cells (NPCs) harvested from the forebrain sub‐ventricular zone (SVZ) can improve locomotor outcomes. Cervical SCI often results in respiratory‐related impairments, and here we used an established model cervical SCI (C2 hemisection, C2Hx) to confirm the feasibility of mid‐cervical transplantation of SVZ‐derived NPCs and the hypothesis that that this procedure would improve spontaneous respiratory motor recovery. NPCs were isolated from the SVZ of enhanced green fluorescent protein (GFP) expressing neonatal rats, and then intraspinally delivered immediately caudal to an acute C2Hx lesion in adult non‐GFP rats. Whole body plethysmography conducted at 4 and 8 wks post‐transplant demonstrated increased inspiratory tidal volume in SVZ vs. sham transplants during hypoxic (P = 0.003) or hypercapnic respiratory challenge (P = 0.019). Phrenic nerve output was assessed at 8 wks post‐transplant; burst amplitude recorded ipsilateral to C2Hx was greater in SVZ vs. sham rats across a wide range of conditions (e.g., quiet breathing through maximal chemoreceptor stimulation; P < 0.001). Stereological analyses at 8 wks post–injury indicated survival of ˜50% of transplanted NPCs with ˜90% of cells distributed in ipsilateral white matter at or near the injection site. Peak inspiratory phrenic bursting after NPC transplant was positively correlated with the total number of surviving cells (P < 0.001). Immunohistochemistry confirmed an astrocytic phenotype in a subset of the transplanted cells with no evidence for neuronal differentiation. We conclude that intraspinal transplantation of SVZ‐derived NPCs can improve respiratory recovery following high cervical SCI. HighlightsNeural progenitor cells (NPCs) were harvested from the forebrain sub‐ventricular zone (SVZ) in neonatal ratsNPCs were intraspinally delivered immediately caudal to acute C2 hemilesion in adult ratsNPC transplant rats had greater inspiratory tidal volume and phrenic nerve output after 8 wks vs. sham transplantsHistology indicated survival of ˜50% of transplanted NPCs with ˜90% of cells distributed in ipsilateral white matterIntraspinal transplantation of SVZ‐derived NPCs can improve respiratory recovery following high cervical spinal cord injury.