Biomedical subjects

Erich Giedzinski

Publications and source records attributed to Erich Giedzinski.

7 recordsLinked to original sources

PubMed2006

Altered growth and radiosensitivity in neural precursor cells subjected to oxidative stress.

PURPOSE: To determine whether changes in oxidative stress could enhance the sensitivity of neural precursor cells to ionizing radiation. MATERIALS AND METHODS: Two strategies were used whereby oxidative stress was modulated endogenously, through manipulation cell culture density, or exogenously, through direct addition of hydrogen peroxide. RESULTS: Cells subjected to increased endogenous oxidative stress through low-density growth routinely exhibited an inhibition of growth following irradiation. However, cells subjected to chronic exogenous oxidative treatments showed increased sensitivity to proton and gamma-irradiation compared to untreated controls. Reduced survival of irradiated cultures subjected to oxidizing conditions was corroborated using enzymatic viability assays, and was observed over a range of doses (1 - 5 Gy) and post-irradiation re-seeding densities (20 - 200 K/plate). CONCLUSIONS: Collectively our results provide further support for the importance of redox state in the regulation of neural precursor cell function, and suggest that oxidative stress can inhibit the proliferative potential of cells through different mechanisms. This is likely to compromise survival and under conditions where excess exogenous oxidants might predominate, sensitivity to irradiation may be enhanced.

Adaptation, Physiological↗

PubMed2005

Efficient production of reactive oxygen species in neural precursor cells after exposure to 250 MeV protons.

The space radiation environment is composed of highly energetic ions, dominated by protons, that pose a range of potential health risks to astronauts. Traversals of these particles through certain tissues may compromise the viability and/or function of sensitive cells, including neural precursors found within the dentate subgranular zone of the hippocampus. Irradiation has been shown to deplete these cells in vivo, and reductions of these critical cells are believed to impair neurogenesis and cognition. To more fully understand the mechanisms underlying the behavior of these precursor cells after irradiation, we have developed an in vitro neural precursor cell system and used it to assess acute (0-48 h) changes in ROS and mitochondrial end points after exposure to Bragg-peak protons of 250 MeV. Relative ROS levels were increased at nearly all doses (1-10 Gy) and postirradiation times (6-24 h) compared to unirradiated controls. The increase in ROS after proton irradiation was more rapid than that observed with X rays and showed a well-defined dose response at 6 and 24 h, increasing approximately 10% and 3% per gray, respectively. However, by 48 h postirradiation, ROS levels fell below controls and coincided with minor reductions in mitochondrial content. Use of the antioxidant alpha-lipoic acid (before or after irradiation) was shown to eliminate the radiation-induced rise in ROS levels. Our results corroborate earlier studies using X rays and provide further evidence that elevated ROS are integral to the radioresponse of neural precursor cells.

Animals↗

PubMed2004

Cell-density-dependent regulation of neural precursor cell function.

Stress-induced reductions of neural precursor cells from the subgranular zone of the hippocampal dentate gyrus have been linked to impaired neurogenesis and cognitive dysfunction. Given the importance of redox state in regulating multiple damage-responsive pathways in the CNS, we hypothesize that oxidative stress plays a major role in affecting neurogenesis and subsequent cognitive function after cell injury/depletion. Using an in vitro system, we showed that the level of reactive oxygen species (ROS), which depend critically on changes in cell density, were significantly higher in neural precursor cells when compared with other primary and transformed cell lines. ROS were significantly elevated ( approximately 4-fold) under low- (<1 x 10(4) cells per cm(2)) versus high-density (>1 x 10(5) cells per cm(2)) conditions. Higher ROS levels found at lower cell densities were associated with elevated proliferation and increased metabolic activity. These ROS were likely a result of altered mitochondrial function that ultimately compromised the growth rate of cells. At high cell densities, intracellular ROS and oxidative damage were reduced in concert with an increased expression of mitochondrial superoxide dismutase 2. Our finding that DNA-damage-induced depletion of neural precursor cells in the subgranular zone of mice also led to increased ROS and altered proliferation validated our in vitro system. Increased ROS and proliferation associated with the reduction of precursor cell numbers both in vitro and in vivo could be reversed with the antioxidant alpha-lipoic acid. These data showed that neural precursor cells were predisposed to microenvironmental cues that regulate redox-sensitive pathways to control cellular proliferation after CNS damage.

Animals↗

PubMed2004

Radiation response of neural precursor cells: linking cellular sensitivity to cell cycle checkpoints, apoptosis and oxidative stress.

Therapeutic irradiation of the brain can cause a progressive cognitive dysfunction that may involve defects in neurogenesis. In an effort to understand the mechanisms underlying radiation-induced stem cell dysfunction, neural precursor cells isolated from the adult rat hippocampus were analyzed for acute (0-24 h) and chronic (3-33 days) changes in apoptosis and reactive oxygen species (ROS) after exposure to X rays. Irradiated neural precursor cells exhibited an acute dose-dependent apoptosis accompanied by an increase in ROS that persisted over a 3-4-week period. The radiation effects included the activation of cell cycle checkpoints that were associated with increased Trp53 phosphorylation and Trp53 and p21 (Cdkn1a) protein levels. In vivo, neural precursor cells within the hippocampal dentate subgranular zone exhibited significant sensitivity to radiation. Proliferating precursor cells and their progeny (i.e. immature neurons) exhibited dose-dependent reductions in cell number. These reductions were less severe in Trp53-null mice, possibly due to the disruption of apoptosis. These data suggest that the apoptotic and ROS responses may be tied to Trp53-dependent regulation of cell cycle control and stress-activated pathways. The temporal coincidence between in vitro and in vivo measurements of apoptosis suggests that oxidative stress may provide a mechanistic explanation for radiation-induced inhibition of neurogenesis in the development of cognitive impairment.

Adaptation, Physiological↗

PubMed2003

Persistent oxidative stress in chromosomally unstable cells.

Past work using the human-hamster hybrid line GM10115 has demonstrated that exposure to a variety of DNA damaging agents can lead to the persistent destabilization of chromosomes. To gain insight into the potential biochemical mechanisms involved in perpetuating the unstable phenotype, groups of clones characterized as stable or unstable were analyzed for indications of oxidative stress. All of the clones were derived from single progenitor cells surviving exposure to ionizing radiation or chemicals. Compared with their stable counterparts, unstable clones possessed elevated levels of reactive oxygen species (ROS) as measured by their enhanced ability to oxidize fluorogenic dyes. Fluorescence automated cell sorting analysis indicated that unstable clones had significantly higher mean fluorescence signals of approximately 2-fold and approximately 1.25-fold, respectively, as derived from the dyes 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate and dihydrorhodamine 123, respectively. To determine whether mitochondria might constitute a potential source of ROS, stable and unstable clones of cells were analyzed for mitochondrial content using nonyl acridine orange and function using rhodamine 123. Fluorescence automated cell sorting data indicated that compared with stable clones, unstable clones possessed an elevated number (15% increase in mean nonyl acridine orange fluorescence) of dysfunctional mitochondria (27% decrease in mean rhodamine 123 fluorescence). Interestingly, the consequences of elevated ROS did not translate to an increase in oxidative base damage in nuclear DNA. Analysis of nine different base damage adducts by gas chromatography/mass spectrometry did not reveal significant differences between stable and unstable clones. The data suggest that the perpetuation of many of the abnormal phenotypes associated with genomic instability may be linked to a state of chronic oxidative stress derived in part from dysfunctional mitochondria.

Aneuploidy↗

PubMed2001

UV-induced replication arrest in the xeroderma pigmentosum variant leads to DNA double-strand breaks, gamma -H2AX formation, and Mre11 relocalization.

UV-induced replication arrest in the xeroderma pigmentosum variant (XPV) but not in normal cells leads to an accumulation of the Mre11/Rad50/Nbs1 complex and phosphorylated histone H2AX (gamma-H2AX) in large nuclear foci at sites of stalled replication forks. These complexes have been shown to signal the presence of DNA damage, in particular, double-strand breaks (DSBs). This finding suggests that UV damage leads to the formation of DSBs during the course of replication arrest. After UV irradiation, XPV cells showed a fluence-dependent increase in the yield of gamma-H2AX foci that paralleled the production of Mre11 foci. The percentage of foci-positive cells increased rapidly (10-15%) up to fluences of 10 J.(-2) before saturating at higher fluences. Frequencies of gamma-H2AX and Mre11 foci both reached maxima at 4 h after UV irradiation. This pattern contrasts sharply to the situation observed after x-irradiation, where peak levels of gamma-H2AX foci were found to precede the formation of Mre11 foci by several hours. The nuclear distributions of gamma-H2AX and Mre11 were found to colocalize spatially after UV- but not x-irradiation. UV-irradiated XPV cells showed a one-to-one correspondence between Mre11 and gamma-H2AX foci-positive cells. These results show that XPV cells develop DNA DSBs during the course of UV-induced replication arrest. These UV-induced foci occur in cells that are unable to carry out efficient bypass replication of UV damage and may contribute to further genetic variation.

DNA Damage↗

PubMedDate not supplied

Induction of chromosomal instability by chronic oxidative stress.

Earlier studies using GM10115 cells analyzed the capability of different DNA-damaging agents to induce genomic instability and found that acute oxidative stress was relatively inefficient at eliciting a persistent destabilization of chromosomes. To determine whether this situation would change under chronic exposure conditions, the human-hamster hybrid line GM10115 was cultured under conditions of oxidative stress. Chronic treatments consisted of 1-hour incubations using a range of hydrogen peroxide (25-200 microM) or glucose oxidase (GO; 5-50 mU/ml) concentrations that were administered once daily over 10 to 30 consecutive days. The toxicity of chronic treatments was modest (- one log kill) and consistent with the low yield of first division aberrations (<5%). However, analysis of over 180 clones and 36,000 metaphases indicated that chronic oxidative stress led to a high incidence of chromosomal instability. Treatment of cells with 100 and 200 microM hydrogen peroxide or 50 mU/ml GO was found to elicit chromosomal instability in 11%, 22%, and 19% of the clones analyzed, respectively. In contrast, control clones isolated after mock treatment did not show signs of chromosomal destabilization. These data suggest that chronic oxidative stress constitutes a biochemical mechanism capable of disrupting the genomic integrity of cells.

Animals↗