Increased adiposity is a feature of aging in both mice and humans, but the molecular mechanisms underlying age-related changes in adipose tissue stores remain unclear. In previous studies, we noted that 18-month-old normocalcemic vitamin D receptor (VDR) knockout (VDRKO) mice exhibited atrophy of the mammary adipose compartment relative to wild-type (WT) littermates, suggesting a role for VDR in adiposity. Here we monitored body fat depots, food intake, metabolic factors, and gene expression in WT and VDRKO mice on the C57BL6 and CD1 genetic backgrounds. Regardless of genetic background, both sc and visceral white adipose tissue depots were smaller in VDRKO mice than WT mice. The lean phenotype of VDRKO mice was associated with reduced serum leptin and compensatory increased food intake. Similar effects on adipose tissue, leptin and food intake were observed in mice lacking Cyp27b1, the 1alpha-hydroxylase enzyme that generates 1,25-dihydroxyvitamin D(3), the VDR ligand. Although VDR ablation did not reduce expression of peroxisome proliferator-activated receptor-gamma or fatty acid synthase, PCR array screening identified several differentially expressed genes in white adipose tissue from WT and VDRKO mice. Uncoupling protein-1, which mediates dissociation of cellular respiration from energy production, was greater than 25-fold elevated in VDRKO white adipose tissue. Consistent with elevation in uncoupling protein-1, VDRKO mice were resistant to high-fat diet-induced weight gain. Collectively, these studies identify a novel role for 1,25-dihydroxyvitamin D(3) and the VDR in the control of adipocyte metabolism and lipid storage in vivo.
Retroviral-mediated delivery of the P140K mutant O6-methylguanine-DNA methyltransferase (MGMTP140K) into hematopoietic stem cells (HSC) has been proposed as a means to protect against dose-limiting myelosuppressive toxicity ensuing from chemotherapy combining O6-alkylating agents (e.g., temozolomide) with pseudosubstrate inhibitors (such as O6-benzylguanine) of endogenous MGMT. Because detoxification of O6-alkylguanine adducts by MGMT is stoichiometric, it has been suggested that higher levels of MGMT will afford better protection to gene-modified HSC. However, accomplishing this goal would potentially be in conflict with current efforts in the gene therapy field, which aim to incorporate weaker enhancer elements to avoid insertional mutagenesis. Using a panel of self-inactivating gamma-retroviral vectors that express a range of MGMTP140K activity, we show that MGMTP140K expression by weaker cellular promoter/enhancers is sufficient for in vivo protection/selection following treatment with O6-benzylguanine/temozolomide. Conversely, the highest level of MGMTP140K activity did not promote efficient in vivo protection despite mediating detoxification of O6-alkylguanine adducts. Moreover, very high expression of MGMTP140K was associated with a competitive repopulation defect in HSC. Mechanistically, we show a defect in cellular proliferation associated with elevated expression of MGMTP140K, but not wild-type MGMT. This proliferation defect correlated with increased localization of MGMTP140K to the nucleus/chromatin. These data show that very high expression of MGMTP140K has a deleterious effect on cellular proliferation, engraftment, and chemoprotection. These studies have direct translational relevance to ongoing clinical gene therapy studies using MGMTP140K, whereas the novel mechanistic findings are relevant to the basic understanding of DNA repair by MGMT.
To examine the safety and efficacy of recombinant-methionyl human stem cell factor (r-metHuSCF), 38 patients with intermediate-grade or immunoblastic high-grade non-Hodgkin's lymphoma who were eligible for autologous transplantation were randomized to receive r-metHuSCF (5, 10, 15, or 20 μg/kg/d) plus Filgrastim (10 μg/kg/d) or Filgrastim (10 μg/kg/d) alone to mobilize peripheral blood progenitor cells. Subcutaneous administration of r-metHuSCF was well tolerated in conjunction with a multi-agent pre-medication regimen; local injection site reactions were the most commonly seen adverse event. The total mononuclear cell count, CD34+ cell content, granulocyte-macrophage colony-forming cells (GM-CFC), and burst-forming units-erythroid (BFU-E) per kilogram in the apheresis product was similar when all patients were analyzed by treatment cohort and mobilization regimen (Filgrastim or r-metHuSCF in combination with Filgrastim); however, when prior chemotherapy was taken into account in a supplementary analysis, clinically important differences were observed. Extensive prior therapy was defined as the amount of exposure to specific stem cell toxic chemotherapeutic agents that patients received. These agents include procarbazine, nitrogen mustard, melphalan, nitrosoureas (≥2 cycles of any of these drugs) or greater than 7.5 g of cytosine arabinoside. In these patients, there was an increased number of CD34+ cells (1.76 v 0.28 × 106/kg), GM-CFC (20.5 v 5.0 × 104/kg), and BFU-E (36.9 v 8.9 × 104/kg) in patients receiving r-metHuSCF and Filgrastim (N = 18) compared with Filgrastim alone (N = 5). These patients also had a decreased time to an untransfused platelet count of 20 × 109/L that was 10.5 days shorter in the patients who received r-metHuSCF and Filgrastim (12.5 v 23 days). These differences were not found to be statistically significant, possibly because of small size, but are clinically important.
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