Purpose: Primary medulloblastoma and glioblastoma multiforme tumor cells that express the surface marker CD133 are believed to be enriched for brain tumor stem cells because of their unique ability to initiate or reconstitute tumors in immunodeficient mice. This study sought to characterize the radiobiological properties and marker expression changes of CD133+ vs. CD133- cells of an established medulloblastoma cell line.Methods and Materials: Daoy and D283 Med cell lines were stained with fluorescently labeled anti-CD133 antibody and sortedinto CD133+ and CD133- populations. The effect of oxygen (2% vs. 20%) on CD133 expression was measured. Both populations were analyzed for marker stability, cell cycle distribution, and radiosensitivity.Results: CD133+ Daoy cells restored nearly native CD133+ and CD133- populations within 18 days, whereas CD133cells remained overwhelmingly CD133-. Culturing Daoy cells in 2% oxygen rather than the standard 20% oxygen increased their CD133 expression 1.6-fold. CD133+ Daoy cells were radioresistant via the beta-parameter of the linear-quadratic model relative to CD133- Daoy cells, although their a-parameters and cell cycle distributions were identical.Conclusions: Restoration of the original CD133+ and CD133- populations from CD133+ Daoy cells in serum is further evidence that CD133+ cells are functionally distinct from CD133- cells. The radioresistance of CD133+ compared with CD133- Daoy cells is consistent with better repair of sublethal damage. Enlargement of the CD133+ sector is a new feature of the hypoxic response. (c) 2007 Elsevier Inc.

Measurement of infrequent DNA double-strand breaks (DSB) in mammalian cells is essential for the understanding of cell damage by ionizing radiation and many DNA-reactive drugs. One of the most important assays for measuring DSB in cellular DNA is filter elution. This study is an attempt to determine whether standard concepts of fluid mechanics can yield a self-consistent model of this process. Major assumptions of the analysis are reptation through a channel formed by surrounding strands, with only strand ends captured by filter pores. Both viscosity and entanglement with surrounding strands are considered to determine the resistance to this motion. One important result is that the average elution time of a strand depends not only on its length, but also on the size distribution of the surrounding strands. This model is consistent with experimental observations, such as the dependence of elution kinetics upon radiation dose, but independence from the size of the DNA sample up to a critical filter loading, and possible overlap of elution times for strands of different length. It indicates how the dependence of elution time on the flow rate could reveal the relative importance of viscous and entanglement resistance, and also predicts the consequences of using different filters.

Lipopolysaccharide (LPS) is a primary agent of sepsis that damages the vascular endothelium. Endothelial cell proliferation is key to the repair of damaged endothelium, and drugs that counteract the antiproliferative impact of LPS on endothelial cells should be beneficial. Because LPS exerts much of its cytotoxicity by generating reactive oxygen and nitrogen intermediates, it would be helpful to know whether therapeutic antioxidant thiols maintain cell proliferation in injured endothelium. In this study, it was found that LPS inhibited bovine aortic endothelial cell proliferation by inducing apoptosis and by decreasing DNA synthesis. Because of its benefit to irradiated endothelial cells, we then treated the cells with a radio- and chemoprotective aminothiol, WR-1065 ([N-2-mecaptoethyl]-1-3-diaminopropane, the active form of Amifostine(R)/Ethyol(R)). WR-1065 attenuated the inhibition of DNA synthesis caused by LPS exposure. The disulfide of WR-1065, WR-33278, was tested and shown to both promote DNA synthesis and inhibit apoptosis. The effectiveness of the disulfide suggests that the reduction of cytotoxicity does not necessarily result from the scavenging of free radicals. These findings demonstrate a novel role for aminothiols in promoting DNA synthesis and lowering apoptosis in endothelium injured with LPS.

During the past 30 years, several authors have formulated models of tumor control probability (TCP) by radiotherapy based on the principle that every tumor clonogen must be killed to achieve tumor control. Attempts to fit these models to clinical data have so far implied tumor clonogen radiosensitivities much smaller than those measured in vitro, and tumor clonogen numbers orders of magnitude smaller than the total number of metabolically active cells in the tumor. Here we incorporate a term for repopulation into the TCP model, and show that it correctly predicts the dependence of TCP on pretreatment volume for one series of head and neck cancers, assuming mean values of radiosensitivity measured in vitro. This model also implies that the tumor clonogen density is a large fraction of the total tumor cell density.

The apparent biological significance of DNA double-strand breaks (DSBs) has stimulated considerable effort toward quantification of this lesion. The neutral (or nondenaturing) filter elution assay at pH 7.2 or 9.6 has long been a standard method for the measurement of double-strand breakage and rejoining in eukaryotic cells, with a threshold dose for detection of DSBs of 5-10 Gy. Agarose gel electrophoresis, either pulsed- or constant-field, can detect DSBs induced by as little as 1 Gy of ionizing radiation, but electrophoresis assays may have inherent problems in measurement of break rejoining, and may be more susceptible than elution to factors other than break frequency, such as cell cycle stage or bromodeoxyuridine substitution. We report here that filter elution performed at pH 11.1 can detect DSBs produced by only 1 Gy of ionizing radiation, but is insensitive to the single-strand breaks that are formed when cells are exposed to hydrogen peroxide. Double-strand breaks produced in permeabilized cells by the restriction endonuclease HaeIII were used to demonstrate that the increase in the pH of the eluting solution from 9.6 to 11.1, although increasing assay sensitivity by a factor of five, converts few additional alkali-labile sites to DSBs. Thus validated, the pH 11.1 filter elution assay was applied to a low-dose measurement of induction and rejoining of DSBs in 9L cells.

Normal tissue toxicity limits radiation therapy and could depend on the extent of damage to the vascular endothelium Aminothiols such as WR-1065 [N-(2-mercaptoethyl)-1,3-diaminopropane] provide radioprotection for normal tissues, but little is known about how the aminothiols specifically affect the endothelium. Bovine aortic endothelial cells in culture were exposed to WR-1065 for 2 h before irradiation (137Cs gamma rays, 1 Gy/min). Alone, WR-1065 demonstrated an antiproliferative effect that was related to dose (0.5-4 mM) and was evident by lowered counts of adherent cells 48 h after exposure. WR-1065 was clearly radioprotective when assessed by colony formation and incorporation of [3H]thymidine. However, when the number of adherent cells was evaluated, radioprotection appeared to be slight and evident only in logarithmically growing cells. WR-1065 at 2 mM suppressed single-strand DNA breaks after 3 Gy by 22% and double-strand breaks after 9 Gy by 47%. Also in the irradiated cells, WR-1065 more than doubled the rate of progression of cells from G1 to S phase. WR-1065 pretreatment elevated cellular glutathione (GSH) content more than twofold. Although pretreatment with buthionine sulfoximine inhibited the elevation of GSH, the radioprotective impact of WR-1065 on total DNA strand breaks and colony formation was unaffected. These results suggest that WR-1065 may enable tissue recovery from irradiation by promoting the replication of endothelial cells, possibly by mechanisms independent of GSH.

In this paper we explore the feasibility of using DNA molecules as a biophysical radiation dosimeter. Supercoiled phi X174 bacteriophage DNA molecules were irradiated with different gamma radiation doses. The strand breakage produced by ionizing radiation within supercoiled double-stranded DNA molecules (RFI) yields relaxed circular DNA molecules (RFII) and linear DNA molecules (RFIII) as a result of single-strand breaks and double-strand breaks, respectively. The irradiated samples were subjected to electrophoresis on agarose gels to separate the three forms. A proprietary fluorescent dye was used to detect DNA bands within the gel, which was photographed under UV transillumination. The negative was scanned with a computerized imaging densitometric system for DNA band quantitation. The relative fractions of the three molecular forms are dose dependent, and can be modeled mathematically with five parameters. The values of the parameters were determined by optimizing the fit of the model to the data, using a nonlinear regression procedure of a commercial statistical analysis package. Once the parameters of DNA breakage have been determined, absorbed dose can be measured by this technique, which we have termed supercoil relaxation dosimetry. The average accuracy of dose determination for our system over the range of 1-40 Gy was about 5%. Supercoil relaxation dosimetry may be well suited to certain difficult dosimetric problems.

The nucleoside 4‐thiouridine, present in some bacterial tRNA species, is known to be a chromophore and a target for near‐UV light‐induced growth delay and also mediates both photoprotection and near‐UV cell killing in various bacterial strains. To investigate the photoreaction of 4‐thiouridine with DNA or its precursors, we irradiated aqueous mixtures of thymine and 4‐thiouridine with 334 nm light and then separated photoproducts using two or more stages of reversed‐phase high performance liquid chromatography. The two equally abundant major photoproducts were analyzed by UV absorbance spectrophotometry, fast‐atom bombardment and electron‐impact mass spectrometry, and 1H‐ and 13C‐NMR spectroscopy, and have been identified as two diastereomers of 6‐hydroxy‐5‐[1‐(β‐D‐erythro‐pentofuranosyl)‐4′‐pyrimidin‐2′‐one]dihydrothymine (o6hThy[5‐4]Pdo), of molecular weight = 370.32. These two diastereomers, although stable at room temperature or below, are interconvertible by heating (90d̀C for 5 min) in aqueous solution. The possible biological significance of this photoproduct is discussed, and an application as a crosslinker for oligonucleotides to selectively block replication is suggested.

The alkaline and neutral (or nondenaturing) filter elution assays are popular methods for the measurement of DNA strand breakage and its repair in eukaryotic cells. In both alkaline and neutral elution, it is recommended practice to wash the filter support after removal of the filter and to analyze the DNA recovered by this procedure together with that remaining on the filter as uneluted DNA, although it is not obvious why the DNA in the filter support wash should be so interpreted. We have observed that the sum of the DNA on the filter and that recovered in the filter support wash is approximately constant when the pH of the alkaline filter elution assay for total strand breaks is increased from 12.1 to 12.6, whereas the fraction on the filter itself is markedly smaller at the higher pH. This behavior characterized DNA elution from undamaged cells, as well as from cells treated with various DNA-damaging agents. These findings are consistent with the "tug-of-war" mechanism that has been proposed for alkaline elution, but are inconsistent with the simplest mechanism of the "sieve" class. In the neutral filter elution assay for double-strand breaks, by contrast, the distribution of DNA between the filter and the filter support wash is pH-independent. This suggests that single- and double-stranded DNA segments traverse a filter by different physical mechanisms. Our observations underscore the importance of carrying out the filter support wash and the analysis of the DNA it contains as uneluted DNA in alkaline elution, while indicating that a different analysis of this DNA might be appropriate for neutral elution.

Neutral elution is a sensitive and convenient method for measuring double-strand breaks in cellular DNA, but results obtained with this method are controversial, particularly as regards form of dose-response relationship. We pointed out in a recent publication (1) that explanation for different results obtained by neutral elution compared with neutral sedimentation remains obscure. The possibility exists that types of radiation damage other than double-strand breaks might affect neutral elution behavior. In a recent Letter to Editor, Hutchinson (2) stated that single-strand breaks might alter neutral elution because of configurational changes in DNA and/or increased susceptibility to shear but the necessary experiments to determine effect of single-strand breaks on neutral elution have not been performed. These necessary experiments must test whether single-strand breaks can modulate elution behavior of DNA containing a significant number of double-strand breaks, and we have performed such experiments. We induced double-strand breaks in DNA of human P3 epithelioid cells using 125I decay exactly as described previously (3), then, after allowing for accumulations of specific numbers of decays, we induced various numbers of single-strand breaks in cells by using treatments known to produce many single-strand breaks and few or no double-strand breaks: H202 (4) and bromodeoxyuridine photolysis (5), as well as y radiation. In no case (three measurements for each single-strand breakinducing treatment at different levels of induced singleand double-strand breaks) did extra single-strand breaks have any measurable effect on elution profiles (manuscript in preparation). Thus we have experimental evidence that neutral elution is not affected by concomitant singlestrand breaks, and this explanation for discrepancy between neutral sedimentation and neutral elution appears to be invalid. With respect to effect of protein crosslinked to DNA, more rigorous DNA lysis and cleaning procedures were described by Okayasu and Iliakis (6), who obtained double-strand break dose responses without a shoulder and, in some cases, straight elution profiles following these treatments. We have repeated those experiments as closely as possible using P3 cells, but saw neither exponential profiles nor dose responses without shoulders that Okasayu and Iliakis obtained. Therefore, DNA-to-protein crosslinking does not appear to be a determinant of neutral elution behavior in all types of cells. Thus question of different form of dose response measured by elution and sedimentation is still not resolved. Hutchinson's critique of neutral elution did not mention several positive aspects of assay or generally negative aspects of neutral sedimentation covered very thoroughly by van der Schans (7). The validity of elution assay is supported by fact that singleto doublestrand break ratios caused by different agents (which may differ by more than two orders of magnitude) detected by neutral elution correspond well with results obtained by other methods. Neutral sedimentation is beset with problems of irreproducibility caused in part by susceptibility of DNA to shear (7), as well as insensitivity. (Equivalent sensitivity to that of neutral elution has been achieved (8) but only by using extraordinarily laborious sample-handling procedures and 4-day sedimentation times.) Although neutral sedimentation, unlike neutral elution, is based upon mathematical theory, extent to which sedimentation theory is both untested and problematic in range of molecular weights relevant to low-dose studies in mammalian cells (9) should not be ignored. Despite unresolved probms, neutral elution remains only practical means of determining double-strand breaks at relatively low radiation doses.

We have used nondenaturing filter elution performed at both pH 7.2 and pH 9.6 to measure the induction of double-strand breaks (DSBs) in the DNA of Chinese hamster V79 cells by 60Co gamma-radiation doses between 10 and 120 Gy. The absolute DSB yields as measured by this assay were determined by using our recent calibration of the assay based upon disintegrations of 125I incorporated into the DNA. An analysis of the dose-response relationship for the induction of DSBs by 60Co gamma rays showed that the number of DSBs induced per dalton of DNA was proportional to the square of the applied dose throughout the dose range used. The contribution made by the dose to the first power was small at pH 9.6 and negligible at pH 7.2. These results suggest that DSB induction in cells by gamma rays may be entirely a two-hit event.

A covalently closed, circular, supercoiled plasmid was exposed to singlet oxygen by a separated-surface sensitizer. For each exposure, the quantity of single oxygen entering the DNA target solution was estimated by its oxidation of histidine. After singlet oxygen exposure, some DNA samples were treated to disclose occult lesions. Agarose gel electrophoresis was then used to resolve the unrelaxed supercoils from the relaxed circular and linear species, and all bands were quantitated fluorometrically. Exposure of supercoiled plasmid DNA to singlet oxygen induced frank DNA strand breaks, alkali-labile sites (pH 12.5, 90 degrees C, 30 min), and piperidine-labile sites (0.4 M, 60 degrees C, 30 min), all in a dose-dependent manner. Yields of alkali-labile and piperidine-labile sites ranged from one to four times the frank strand break yield. Replacement of buffered H2O by buffered D2O as the DNA solvent for singlet oxygen exposures increased DNA lesion yields by a factor of 2.6 (averaged over lesion classes). Our data for the detection of frank strand breaks is at variance with published results from studies in which singlet oxygen was derived from a thermolabile endoperoxide dissolved in the DNA solution.

We labeled the DNA of Chinese hamster lung V79 cells with 125I in the form of iododeoxyuridine and subsequently measured the elution of the DNA through polycarbonate filters at pH 9.6 and pH 7.2. Since decay of incorporated 125I produces predominantly double-strand breaks (DSB) in DNA at a rate close to one DSB per 125I decay, this measurement provides an absolute calibration for the assay of DSBs by neutral filter elution. Neutral elution profiles are not first order with respect to elution time; thus we have examined the relationships between accumulated 125I decays and several functions of retention of DNA on the filter at various times during the elution process. At both pH 9.6 and pH 7.2 there were linear relationships between accumulated decays and certain retention functions. The retention function most closely correlated to 125I decays for both pH values was the logarithm of the ratio of the retention of control DNA to that of 125I-labeled DNA, both evaluated at the 9th fraction (13.5 h of elution). The linear relationship between this ratio and 125I decays allows DSB induction to be determined directly from retention values. The calibration was used to measure DSBs induced by X rays.

A supercoiled plasmid of 7300 base pairs was isolated and exposed in various aqueous environments to 60Co gamma-radiation. Conversion of the supercoiled form to the relaxed circular and linear forms was monitored by agarose gel electrophoresis and quantified by fluorescence scanning of the gel. Acetate, which has been reported to affect the conformation of DNA in solution, decreased the radiosensitivity of the supercoil in a concentration-dependent manner. Acetate, formate, and azide anions, as well as mannitol, all protected the supercoil from relaxation in approximate proportion to the rate at which their solutions quench the hydroxyl radical. At concentrations greater than 300 mmol dm-3, however, the efficiency of acetate radioprotection is reduced. Disodium ethylenediaminetetraacetate protected the supercoil more efficiently than would be expected from the published value of its rate constant for quenching the hydroxyl radical.