Ivan A. Bespalov

Characterization of a novel metabolic strategy used by drug-resistant tumor cells

Acquired or inherent drug resistance is the major problem in achieving successful cancer treatment. However, the mechanism(s) of pleiotropic drug resistance remains obscure. We have identified and characterized a cellular metabolic strategy that differentiates drug‐resistant cells from drug‐sensitive cells. This strategy may serve to protect drug‐resistant cells from damage caused by chemotherapeutic agents and radiation. We show that drug‐resistant cells have low mitochondrial membrane potential, use nonglucose carbon sources (fatty acids) for mitochondrial oxygen consumption when glucose becomes limited, and are protected from exogenous stress such as radiation. In addition, drug‐resistant cells express high levels of mitochondrial uncoupling protein 2 (UCP2). The discovery of this metabolic strategy potentially facilitates the design of novel therapeutic approaches to drug resistance.—Harper, M.‐E., Antoniou, A., Villalobos‐Menuey, E., Russo, A., Trauger, R., Vendemelio, George, A. M., Bartholomew, R., Carlo, D., Shaikh, A., Kupperman, J., Newell, E. W., Bespalov, I. A., Wallace, S. S., Liu, Y., Rogers, J. R., Gibbs, G. L., Leahy, J. L., Camley, R. E., Melamede, R., Newell, M. K. Characterization of a novel metabolic strategy used by drug‐resistant tumor cells. FASEB J. 16, 1550–1557 (2002)

Fluorescence detection of 8-oxoguanine in nuclear and mitochondrial DNA of cultured cells using a recombinant Fab and confocal scanning laser microscopy.

The presence of 8-oxoguanine (8-oxoG) in DNA is considered a marker of oxidative stress and DNA damage. We describe a multifluorescence technique to detect the localization of 8-oxoG in both nuclear and mitochondrial DNA using a mouse recombinant Fab 166. The Fab was generated by repertoire cloning and combinatorial phage display, and specifically recognized 8-oxoG in DNA, as determined by competitive enzyme-linked immunosorbent assays (ELISAs). In situ detection of 8-oxoG was accomplished using rat lung epithelial (RLE) cells and human B lymphoblastoid (TK6) cells treated with hydrogen peroxide (H(2)O(2)) or ionizing radiation, respectively. Using confocal scanning laser microscopy, we observed nuclear and perinuclear immunoreactivity of 8-oxoG in control cultures. The simultaneous use of a nuclear DNA stain, propidium iodide, or the mitochondrial dye, MitoTracker (Molecular Probes, Eugene, OR, USA), confirmed that 8-oxoG immunofluorescence occurred in nuclear and mitochondrial DNA. Marked increases in the presence of 8-oxoG in nuclear DNA were apparent after treatment with H(2)O(2) or ionizing radiation. In control experiments, Fab 166 was incubated with 200 microM purified 8-oxodG or with formamidopyrimidine DNA-glycosylase (Fpg) to remove 8-oxoG lesions in DNA. These protocols attenuated both nuclear and mitochondrial staining. We conclude that both nuclear and mitochondrial oxidative DNA damages can be simultaneously detected in situ using immunofluorescence labeling with Fab 166 and confocal microscopy.

Inhibition of oxidative DNA repair in cadmium-adapted alveolar epithelial cells and the potential involvement of metallothionein.

This study evaluated the effects of cadmium (Cd) adaptation in cultured alveolar epithelial cells on oxidant-induced DNA damage and its subsequent repair. Using the comet assay, we determined that lower levels of DNA damage occurred in Cd-adapted cells compared with non-adapted cells following treatment of cells with hydrogen peroxide (H(2)O(2)). This may be a consequence of increased thiol-containing antioxidants that were observed in adapted cells, including metallothionein and glutathione. Cd-adapted cells were, however, less efficient at repairing total oxidative DNA damage compared with non-adapted cells. Subsequently, we investigated the effect of Cd adaptation on the repair of particular oxidized DNA lesions by employing lesion-specific enzymes in the comet assay, namely formamidopyrimidine DNA glycosylase (Fpg), an enzyme that predominantly repairs 8-oxoguanine (8-oxoG), and endonuclease III, that is capable of repairing oxidized pyrimidines. The data demonstrated that adaptation to Cd results in significantly impaired repair of both Fpg- and endonuclease III-sensitive lesions. In addition, in situ detection of 8-oxoG using a recombinant monoclonal antibody showed that Cd-adaptation reduces the repair of this oxidative lesion after exposure of cells to H(2)O(2). Activities of 8-oxoG-DNA glycosylase and endonuclease III were determined in whole cell extracts using 32P-labeled synthetic oligonucleotides containing 8-oxoG and dihydrouracil sites, respectively. Cd adaptation was associated with an inhibition of 8-oxoG-DNA glycosylase and endonuclease III enzyme activity compared with non-adapted cells. In summary, this study has shown that Cd adaptation: (1) reduces oxidant-induced DNA damage; (2) increases the levels of key intracellular antioxidants; (3) inhibits the repair of oxidative DNA damage.

The Toll-Like Receptor 5 Agonist Entolimod Mitigates Lethal Acute Radiation Syndrome in Non-Human Primates

There are currently no approved medical radiation countermeasures (MRC) to reduce the lethality of high-dose total body ionizing irradiation expected in nuclear emergencies. An ideal MRC would be effective even when administered well after radiation exposure and would counteract the effects of irradiation on the hematopoietic system and gastrointestinal tract that contribute to its lethality. Entolimod is a Toll-like receptor 5 agonist with demonstrated radioprotective/mitigative activity in rodents and radioprotective activity in non-human primates. Here, we report data from several exploratory studies conducted in lethally irradiated non-human primates (rhesus macaques) treated with a single intramuscular injection of entolimod (in the absence of intensive individualized supportive care) administered in a mitigative regimen, 1–48 hours after irradiation. Following exposure to LD50-70/40 of radiation, injection of efficacious doses of entolimod administered as late as 25 hours thereafter reduced the risk of mortality 2-3-fold, providing a statistically significant (P<0.01) absolute survival advantage of 40–60% compared to vehicle treatment. Similar magnitude of survival improvement was also achieved with drug delivered 48 hours after irradiation. Improved survival was accompanied by predominantly significant (P<0.05) effects of entolimod administration on accelerated morphological recovery of hematopoietic and immune system organs, decreased severity and duration of thrombocytopenia, anemia and neutropenia, and increased clonogenic potential of the bone marrow compared to control irradiated animals. Entolimod treatment also led to reduced apoptosis and accelerated crypt regeneration in the gastrointestinal tract. Together, these data indicate that entolimod is a highly promising potential life-saving treatment for victims of radiation disasters.

Imaging techniques used for the detection of 8-oxoguanine adducts and DNA repair proteins in cells and tissues

The presence of 8-oxoguanine (8-oxoG) in DNA is considered a marker of oxidative stress and DNA damage. Numerous biochemical techniques have been described for its detection in cells or tissues. Although these approaches are quantitative, they do not provide insights into whether the lesion occurs in mitochrondrial versus genomic DNA. In addition, biochemical techniques are not amenable to the evaluation of individual cells or archival tissues. Antibodies have been raised against 8-oxoG, which may circumvent some of these issues. In this review, we described the use of in situ imaging techniques to detect oxidative DNA damage including the comet assay. We will review our previous work that describes the utility of an antibody fragment (Fab) engineered to recognize 8-oxoG in DNA. Furthermore, we will discuss the analysis of DNA repair enzymes in the assessment of oxidative DNA damage. Finally, advantages and potential concerns associated with immunodetection of 8-oxoG are discussed.

Detection of Oxidative DNA Base Damages

Antibodies to a variety of oxidized DNA bases have been generated in a number of laboratories including our own (see Table I). Most of these antibodies have been elicited using protein-conjugated haptens of interest. In general, the antibodies have reasonable affinity such that appropriate sensitivity in the various assays can be achieved. The difficulty with antibodies that recognize oxidized DNA bases is that the oxidized bases do not differ largely from their unoxidized derivatives. Thus, the specificity for detecting lesions in DNA must be high especially when one considers the low level of damaged compared to undamaged bases. Even if the sensitivity of the assay can be amplified, cross-reactivity of the antibody with the unoxidized base in DNA remains an obstacle to successful detection of low levels of the oxidized base. This particular problem is not seen as often when antibodies are elicited to the chemical adducts that have features that vary quite dramatically from the unadducted base. An additional consideration is that the lesion must be stable to the procedures used during preparation of the immunogen and during DNA denaturation. The latter is usually necessary since the antibodies often do not recognize the lesion as well in duplex DNA. Despite these shortcomings, antibodies to oxidized bases have been effectively utilized to detect these lesions in oxidized or ionizing radiation-treated DNA in vitro.