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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. · 3.21 Impact Factor
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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.
Stem Cells 10/2008; 26(12):3218-27. · 7.78 Impact Factor
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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.Disclosure of potential conflicts of interest is found at the end of this article.
Stem Cells 09/2008; 26(12):3218 - 3227. · 7.78 Impact Factor
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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. · 5.48 Impact Factor
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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.
Methods in molecular biology (Clifton, N.J.) 02/2008; 438:135-50.
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ABSTRACT: 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 162bp deletion. Both transcripts were identified in NT2, hNT and adult human hippocampus. In vitro, MT5-MMPvar translated into a approximately 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.
FEBS Letters 01/2008; 581(30):5923-8. · 3.54 Impact Factor
