[Show abstract][Hide abstract] ABSTRACT: 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 6 weeks 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.
[Show abstract][Hide abstract] ABSTRACT: A cervical (C2) hemilesion (C2Hx), which disrupts ipsilateral bulbospinal inputs to the phrenic nucleus, was used to study diaphragm plasticity after acute spinal cord injury. We hypothesized that C2Hx would result in rapid atrophy of the ipsilateral hemidiaphragm and increases in mRNA expression of proteolytic biomarkers. Diaphragm tissue was harvested from male Sprague-Dawley rats at 1 or 7 days following C2Hx. Histological analysis demonstrated a reduction in cross-sectional area (CSA) of type I and IIa fibers in the ipsilateral hemidiaphragm at 1 but not 7 days post-injury. Type IIb/x fibers, however, had reduced CSA at both 1 and 7 days. To examine the mechanisms of C2Hx-induced diaphragm atrophy, a targeted gene array was used to screen mRNA changes for genes associated with skeletal muscle myopathy and myogenesis; this was followed by qRT-PCR validation. Changes in diaphragm gene expression suggested that profound myoplasticity is initiated immediately following C2Hx including activation of both proteolytic and myogenic pathways. We conclude that an immediate myoplastic response occurs in the diaphragm after C2Hx with atrophy occurring in ipsilateral myofibers within 1 day.
[Show abstract][Hide abstract] ABSTRACT: In vitro exposure of neural progenitor cell (NPC) populations to reduced O(2) (e.g. 3% versus 20%) can increase their proliferation, survival and neuronal differentiation. Our objective was to determine if an acute (<1hr), in vivo exposure to intermittent hypoxia (AIH) alters expansion and/or differentiation of subsequent in vitro cultures of NPC from the subventricular zone (SVZ). Neonatal C57BL/6 mice (postnatal day 4) were exposed to an AIH paradigm (20×1 minute; alternating 21% and 10% O(2)). Immediately after AIH, SVZ tissue was isolated and NPC populations were cultured and assayed either as neurospheres (NS) or as adherent monolayer cells (MASC). AIH markedly increased the capacity for expansion of cultured NS and MASC, and this was accompanied by increases in a proliferation maker (Ki67), MTT activity and hypoxia-inducible factor-1α (HIF-1α) signaling in NS cultures. Peptide blockade experiments confirmed that proteins downstream of HIF-1α are important for both proliferation and morphological changes associated with terminal differentiation in NS cultures. Finally, immunocytochemistry and Western blotting experiments demonstrated that AIH increased expression of the neuronal fate determination transcription factor Pax6 in SVZ tissue, and this was associated with increased neuronal differentiation in cultured NS and MASC. We conclude that in vivo AIH exposure can enhance the viability of subsequent in vitro SVZ-derived NPC cultures. AIH protocols may therefore provide a means to "prime" NPC prior to transplantation into the injured central nervous system.
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Neuro-Oncology 06/2011; 105(3):485-98. · 3.12 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: The thymidine analog bromodeoxyuridine (BrdU) is incorporated into newly synthesized DNA and has been shown to increase the susceptibility of incorporating cells to ionizing radiation. However, in the absence of secondary stressors, BrdU is thought to substitute relatively benignly for thymidine and is commonly used to "birth-date" proliferative cells. We report a novel antiproliferative effect of BrdU on cancer cells, which is independent of its role in radiosensitization. A single, brief in vitro exposure to BrdU induces a profound and sustained reduction in the proliferation rate of all cancer cells examined. Cells do not die but variably up-regulate some senescence-associated proteins as they accumulate in the G1 phase of the cell cycle. Bromodeoxyuridine also impairs the proliferative capacity of primary tumor-initiating human glioma cells and may therefore represent a means of targeting cancer stem cells. Finally, conservative in vivo BrdU regimens--in the absence of any other treatment--significantly suppress the progression of gliomas in the highly aggressive, syngeneic RG2 model. These results suggest that BrdU may have an important role as an adjunctive therapeutic for a wide variety of cancers based on new insights into its effect as a negative regulator of cell cycle progression.
Neoplasia (New York, N.Y.) 09/2008; 10(8):804-16. · 5.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.