Low-dose studies of bystander cell killing with targeted soft X rays

The Gray Cancer Institute ultrasoft X-ray microprobe was used to quantify the bystander response of individual V79 cells exposed to a focused carbon K-shell (278 eV) X-ray beam. The ultrasoft X-ray microprobe is designed to precisely assess the biological response of individual cells irradiated in vitro with a very fine beam of low-energy photons. Characteristic CK X rays are generated by a focused beam of 10 keV electrons striking a graphite target. Circular diffraction gratings (i.e. zone plates) are then employed to focus the X-ray beam into a spot with a radius of 0.25 microm at the sample position. Using this microbeam technology, the correlation between the irradiated cells and their nonirradiated neighbors can be examined critically. The survival response of V79 cells irradiated with a CK X-ray beam was measured in the 0-2-Gy dose range. The response when all cells were irradiated was compared to that obtained when only a single cell was exposed. The cell survival data exhibit a linear-quadratic response when all cells were targeted (with evidence for hypersensitivity at low doses). When only a single cell was targeted within the population, 10% cell killing was measured. In contrast to the binary bystander behavior reported by many other investigations, the effect detected was initially dependent on dose (<200 mGy) and then reached a plateau (>200 mGy). In the low-dose region (<200 mGy), the response after irradiation of a single cell was not significantly different from that when all cells were exposed to radiation. Damaged cells were distributed uniformly over the area of the dish scanned (approximately 25 mm2). However, critical analysis of the distance of the damaged, unirradiated cells from other damaged cells revealed the presence of clusters of damaged cells produced under bystander conditions.     

Mortality among patients with ankylosing spondylitis after a single treatment course with x rays.

Mortality was studied in 14 111 patients with ankylosing spondylitis given a single course of x-ray treatment during 1935-54. Mortality from all causes combined was 66% greater than that of members of the general population of England and Wales. There were substantial excesses of deaths from non-neoplastic conditions, but these appeared to be associated with the disease itself rather than its treatment. A nearly fivefold excess of deaths from leukaemia and a 62% excess of deaths from cancers of sites that would have been in the radiation fields ("heavily irradiated sites") were likely to have been a direct consequence of the radiation treatment itself. The excess death rate from leukaemia was greatest three to five years after treatment and was close to zero after 18 years. In contrast, the excess of cancers of heavily irradiated sites did not become apparent until nine or more years after irradiation and continued for a further 11 years. More than 20 years after irradiation the excess risk declined, but the fall was not statistically significant. The number of cancers of sites not considered to be in the radiation beams was 20% greater than expected. This excess, although not statistically significant, may also have been due to radiation scattered from beams directed at other parts of the body. The risk of a radiation-induced leukaemia or other cancer was related to the age of the patient at the time of treatment. Those irradiated when aged 55 years or more had an excess death rate from leukaemia more than 15 times that of those treated under 25 years of age, and a similar difference was apparent for cancers of heavily irradiated sites. The radiation dose to the bone marrow was estimated for the patients who died with leukaemia and for a 1 in 15 sample of the total study population. The excess risk of leukaemia varied erratically with radiation dose owing, perhaps, in part to the increase in the proportion of the cells in the bone marrow that are sterilised with increasing doses. A mathematical model using a linear leukaemia induction rate and exponential cell sterilisation fitted the data reasonably well, and the results suggested that for low radiation doses about two deaths from leukaemia would be induced per million people per rad of x rays per year for up to 20 years after exposure. Because of the failure to find a clear dose-response relationship this estimate must be regarded with caution, but it is in reasonable agreement with that derived from studies of the atomic bomb survivors.     

Cell death (apoptosis) in mouse intestine after continuous irradiation with gamma rays and with beta rays from tritiated water

Apoptosis is a pattern of cell death involving nuclear pycnosis, cytoplasmic condensation, and karyorrhexis. Apoptosis induced by continuous irradiation with gamma rays (externally given by a 137Cs source) or with beta rays (from tritiated water injected ip) was quantified in the crypts of two portions of mouse bowel, the small intestine and descending colon. The time-course change in the incidence of apoptosis after each type of radiation could be explained on the basis of the innate circadian rhythm of the cells susceptible to apoptotic death and of the excretion of tritiated water (HTO) from the body. For 6-h continuous gamma irradiation at various dose rates (0.6-480 mGy/h) and for 6 h after injection of HTO of various radioactivities (0.15-150 GBq per kg body wt), the relationships between dose and incidence of apoptosis were obtained. Survival curves were then constructed from the curves for dose vs incidence of apoptosis. For the calculation of the absorbed dose from HTO, the water content both of the mouse body and of the cells was assumed to be 70%. One megabecquerel of HTO per mouse (i.e., 40 MBq/kg body wt) gave a dose rate of 0.131 mGy/h. The mean lethal doses (D0) were calculated for gamma rays and HTO, and relative biological effectiveness values of HTO relative to gamma rays were obtained. The D0 values for continuous irradiation with gamma rays were 210 mGy for small intestine and 380 mGy for descending colon, and the respective values for HTO were 130 and 280 mGy, indicating the high radiosensitivity of target cells for apoptotic death. The relative biological effectiveness of HTO relative to 137Cs gamma rays for cell killing in both the small intestine and the descending colon in the mouse was 1.4-2.1.     

Growth delay in 9L rat brain tumor spheroids after irradiation with single and split doses of X rays

The response of 9L spheroids to irradiation with single and split doses of X rays has been investigated. Irradiation with single doses caused a dose-dependent decrease in spheroid growth rate, which eventually returned to the growth rate for unirradiated spheroids. This delay appeared to be related to cell survival. When spheroids were irradiated with two 4-Gy doses of X rays separated by various times the amount of growth delay was intermediate between that observed with single doses of 4 and 8 Gy. For relatively short times (15-90 min), recovery probably resulted from repair processes, but for longer times (up to 24 hr), recovery also appeared to depend on cellular redistribution and repopulation effects.     

Interaction function gamma(x) for Chinese hamster cells treated with hypertonic phosphate-buffered saline after irradiation

The repair of potentially lethal damage (PLD) in stationary-phase V79 Chinese hamster cells, which was expressible by a postirradiation treatment with hypertonic (0.5 M NaCl) phosphate-buffered saline (PBS), was analyzed within the framework of the theory of dual radiation action. The interaction function gamma(x) was estimated for cells permitted to repair PLD for various intervals of time. The experimental data indicated that 50-60% of the lethal lesions produced at the time of irradiation were repaired in 120 min. The repair of PLD was implicitly involved in the probability of the interaction of sublesions. That is, g(x,trep) was defined as the probability that two sublesions separated by distance x interact to produce a lethal lesion which will not be repaired until the fixation by treatment with hypertonic PBS at time trep after irradiation. It is concluded that the time dependence of the repair of PLD is not independent of the interaction distance x. Three conclusions are drawn: (1) The repair of a lesion produced by a long distance interaction is not detectable by postirradiation treatment with hypertonic PBS. (2) A lesion produced by a short distance interaction is rapidly repaired in about 20 min. (3) A lesion produced by the interaction of sublesions separated by a distance of about 100 nm is repaired slowly.     

Efficient carcinoma cell killing by activated polymorphonuclear neutrophils targeted with an Ep-CAMxCD64 (HEA125x197) bispecific antibody

Bispecific antibodies (bsAb) have attracted much attention over the past several years as a mean to improve immunotherapy of cancer. Due to their dual specificity, bsAb are able to redirect effector cells against tumor targets. In this study, the development and preclinical testing of a new quadroma-derived bsAb, HEA125x197, recognizing the tumor-associated Ep-CAM antigen and the high affinity Fc receptor for IgG, CD64, is reported. Using granulocyte-colony stimulating factor (G-CSF) and interferon-gamma (IFN-gamma)-stimulated polymorphonuclear neutrophils to induce CD64 expression, bsAb HEA125 x 197 elicited strong cytotoxic activity towards allogeneic and autologous ovarian carcinoma cells. The cytolytic efficiency of this antibody was comparable to that of a previously described bsAb, HEA125 x OKT3, targeting preactivated T lymphocytes against Ep-CAM-carrying tumor cells. Based on the pan-carcinoma specificity and the stable expression of Ep-CAM, bsAb HEA125x197 may broaden the spectrum of bispecific reagents for the treatment of epithelial malignancies.     

Laser microbeam irradiation of rat kangaroo cells (PTK2) following selective sensitization with bromodeoxyuridine and ethidium bromide

Ethidium bromide (l0 μg/ml) and bromodeoxyuridine (25 μg/ml) were used to sensitize selective cell organelles to visible wavelengths of an argon ion Her (488 and 514 nanometers). Ethidium bromide was shown to be salabtlve In sensitizing nucleoli, chromosomes, and the centriolar region of PTK₂ cells to the laser microbeam. Similarly, BrDU sensitized chromosomes to the microbeam irradiation. The lesions produced on the chromosomes when either agent was used appeared as a phase paling of the irradiated segment. Nucleolar lesions also appeared as a phase paling, and the centriolar region alteration appeared either as a phase paling or a phase darkening.     

Lack of cell death enhancement after irradiation with monochromatic synchrotron X rays at the K-shell edge of platinum incorporated in living SQ20B human cells as cis-diamminedichloroplatinum (II)

In this paper we describe the results of experiments using synchrotron radiation to trigger the Auger effect in living human cancer cells treated with a widely used chemotherapy drug: cis-diamminedichloroplatinum (II) (cisplatin). The experiments were carried out at the ID17 beamline of the European Synchrotron Radiation Facility, which produces a high-fluence monochromatic beam that is adjustable from 20 to 80 keV. Cisplatin was chosen as the carrier of platinum atoms in the cells because of its alkylating-like activity and the irradiation was done with monochromatic beams above and below the platinum K-shell edge (78.39 keV). Cell survival curves were comparable with those obtained for the same cells under conventional irradiation conditions. At a low dose of cisplatin (0.1 microM, 48 h), no difference was seen in survival when the cells were irradiated above and below the K-shell edge of platinum. Higher cisplatin concentrations were investigated to enhance the cellular platinum content. The results with 1 microM cisplatin for 12 h showed no difference when the cells were irradiated with beams above or below the platinum K-shell edge with the exception of the higher cell death resulting from drug toxicity. The intracellular content of platinum was significant, as measured macroscopically by inductively coupled plasma mass spectrometry. Its subcellular localization and particularly its presence in the cell nucleus were verified by microscopic synchrotron X-ray fluorescence. This was the first known attempt at K-shell edge photon activation of stable platinum in living cells with a platinum complex used for chemotherapy. Its evident toxicity in these cells leads us to put forth the hypothesis that cisplatin toxicity can mask the enhancement of cell death induced by the irradiation above the K-shell edge. However, K-shell edge photon activation of stable elements provides a powerful technique for the understanding of the biological effects of Auger processes. Further avenues of development are discussed.     

Cell killing, nuclear damage and apoptosis in Chinese hamster V79 cells after irradiation with heavy-ion beams of (16)O, (12)C and (7)Li

Chinese hamster V79 cells were exposed to high LET (linear energy transfer) (16)O-beam (625keV/mum) radiation in the dose range of 0-9.83Gy. Cell survival, micronuclei (MN), chromosomal aberrations (CA) and induction of apoptosis were studied as a follow up of our earlier study on high LET radiations ((7)Li-beam of 60keV/mum and (12)C-beam of 295keV/mum) as well as (60)Co gamma-rays. Dose dependent decline in surviving fraction was noticed along with the increase of MN frequency, CA frequency as well as percentage of apoptosis as detected by nuclear fragmentation assay. The relative intensity of DNA ladder, which is a useful marker for the determination of the extent of apoptosis induction, was also increased in a dose dependent manner. Additionally, expression of tyrosine kinase lck-1 gene, which plays an important role in response to ionizing radiation induced apoptosis, was increased with the increase of radiation doses and also with incubation time. The present study showed that all the high LET radiations were generally more effective in cell killing and inflicting other cytogenetic damages than that of low LET gamma-rays. The dose response curves revealed that (7)Li-beam was most effective in cell killing as well as inducing other nuclear damages followed by (12)C, (16)O and (60)Co gamma-rays, in that order. The result of this study may have some application in biological dosimetry for assessment of genotoxicity in heavy ion exposed subjects and in determining suitable doses for radiotherapy in cancer patients where various species of heavy ions are now being generally used.     

Chemical synthesis of a P(k)-antigenic globotriose analogue with a functionalized aglycon

6-Aminohexyl alpha-D-galactopyranosyl-(1-->4)-beta-D-galactopyranosyl-(1-->4)-beta-D-glucopyranoside (2), a globotriose analogue with a functionalized aglycon, was synthesized, using alpha-D-galactopyranosyluronic acid-(1-->4)-D-galactopyranosyluronic acid [di-GalA (3)] as the starting material, which is commercially available or can be readily prepared from pectin.     

Development of a Target cell-Biologics-Effector cell (TBE) complex-based cell killing model to characterize target cell depletion by T cell redirecting bispecific agents

T-cell redirecting bispecific antibodies (bsAbs) or antibody-derived agents that combine tumor antigen recognition with CD3-mediated T cell recruitment are highly potent tumor-killing molecules. Despite the tremendous progress achieved in the last decade, development of such bsAbs still faces many challenges. This work aimed to develop a mechanism-based pharmacokinetic/pharmacodynamic (PK/PD) modeling framework that can be used to assist the development of T-cell redirecting bsAbs. A Target cell-Biologics-Effector cell (TBE) complex-based cell killing model was developed using in vitro and in vivo data, which incorporates information on binding affinities of bsAbs to CD3 and target receptors, expression levels of CD3 and target receptors, concentrations of effector and target cells, as well as respective physiological parameters. This TBE model can simultaneously evaluate the effect of multiple system-specific and drug-specific factors on the T-cell redirecting bsAb exposure–response relationship on a physiological basis; it reasonably captured multiple reported in vitro cytotoxicity data, and successfully predicted the effect of some key factors on in vitro cytotoxicity assays and the efficacious dose of blinatumomab in humans. The mechanistic nature of this model uniquely positions it as a knowledge-based platform that can be readily expanded to guide target selection, drug design, candidate selection and clinical dosing regimen projection, and thus support the overall discovery and development of T-cell redirecting bsAbs.     

System of cell irradiation with a defined number of heavy ions (III).

A single cell irradiation system has been developed at JAERI-Takasaki to study radiobiological processes in single-ion-hit mammalian cells and bystander cells, in ways that cannot be achieved using conventional broad field exposures. Individual mammalian cultured cells are irradiated in the atmosphere on the cell dish, the bottom of which is made of ion-track-detector CR-39, with a single or defined numbers of 13.0 MeV/amu 20Ne and 11.5 MeV/amu 40Ar ions. Targeting and irradiation of the cells are performed automatically at the on-line microscope of the microbeam apparatus according to the positional data of the target cells obtained at the off-line microscope before irradiation. Using this system, Chinese hamster ovary (CHO-K1) cells were irradiated with counted number of 20Ne and 40Ar ions. Thereafter, the growth of the cells was observed individually and repeatedly during post-irradiation incubation. The cells hit by a single 40Ar ion on their nucleus showed strong growth inhibition. Meanwhile, the cells in the irradiated dish but not hit by the ion (bystander cells) showed limited cell growth. This might be a bystander effect caused by heavy ion hit cell co-existing in the same dish.     

Interaction of the alternating double stranded copolymer poly(dA-dT) x poly(dA-dT) with NiCl(2) and CdCl(2): solution behavior

The thermal denaturation of the synthetic high molecular weight double stranded polynucleotide poly(dA-dT) x poly(dA-dT) has been studied in aqueous buffered solution (Tris 1.0 mM; pH 7.8+/-0.2) in the presence of increasing concentrations of either Ni(2+) (borderline cation) or Cd(2+) (soft cation) at four different constant ionic strength values (NaCl), making use of UV and circular dichroism (CD) spectroscopies. The experimental results show that the B-type double helix of the polymer is stabilized against thermal denaturation in the presence of both cations at low concentrations, relative to the systems where only NaCl is present, in the same conditions of ionic strength and pH. The effect is more pronounced for Ni(2+) than for Cd(2+). At higher concentrations, both cations start to destabilize the double helix, with Cd cations inducing larger variations of T(m). In many cases, when denaturation starts, interstrand cross-linking occurs with formation of aggregates that precipitate.     

Postreplication repair of DNA in human fibroblasts after UV irradiation or treatment with metabolites of benzo(a)pyrene

We have examined the ability of normal fibroblasts and of excision-deficient xeroderma pigmentosum (XP) and XP variant fibroblasts to perform postreplication DNA repair after increasing doses of either ultraviolet (UV) irradiation or mutagenic benzo(a)pyrene derivatives. XP cells defective in the excision of both UV-induced pyrimidine dimers and guanine adducts induced by treatment with the 7,8-diol-9,10-epoxides of benzo(a)pyrene were partially defective in their ability to synthesize high molecular weight DNA after the induction of both classes of DNA lesions. This defect was more marked in XP variant cells, despite their ability to remove by excision repair both pyrimidine dimers and the diol epoxide-induced lesions to the same degree as observed in normal cells. The benzo(a)pyrene 9,10-oxide had no effect in any of the 3 cell lines. The response of the excision and postreplication DNA repair mechanisms operating in human fibroblasts treated with benzo(a)pyrene 7,8-diol-9,10-epoxides, therefore, appears to resemble closely that seen after the induction of pyrimidine dimers by UV irradiation.     

Acquisition of P-selectin binding activity by en bloc transfer of sulfo Le(x) trisaccharide to the cell surface: comparison to a sialyl Le(x) tetrasaccharide transferred on the cell surface

Sialyl Le(x), NeuNAcalpha2 --> 3Galbeta1 --> 4(Fucalpha1 --> 3)GlcNAcbeta --> R, is known to be a ligand for E-selectin in various assays. The sulfated counterpart of sialyl Le(x), sulfo Le(x), (Sulfo --> 3) Galbeta1 --> 4 (Fucalpha1 --> 3) GlcNAcbeta --> R, was also shown to be a ligand for E-selectin in solid-phase assays employing immobilized oligosaccharides. In order to determine whether sulfo Le(x) structure on the cell surface also works as E-selectin or P-selectin ligand, a novel approach for in vitro transfer of oligosaccharides (S. Tsuboi, Y. Isogai, N. Hada, J. K. King, O. Hindsgaul, and M. Fukuda (1996) J. Biol. Chem. 271, 27213-27216) was utilized. A synthetic GDP-fucose harboring sialyl Le(x) or sulfo Le(x) oligosaccharide was enzymatically transferred to Chinese hamster ovary (CHO) cells with a milk fucosyltransferase. The resultant cells, CHO-sialyl Le(x) and CHO-sulfo Le(x) were tested for adhesion to E-selectin. IgG or P-selectin. IgG chimeric protein coated on plates. The results indicate that CHO-sialyl Le(x) adhered efficiently to E-selectin, while adhesion of CHO-sulfo Le(x) was very poor despite the fact that near equal number of the ligands had been attached to the cell surface. In contrast, CHO-sulfo Le(x) adhered efficiently to P-selectin, while CHO-sialyl Le(x) adhered modestly to P-selectin. These results demonstrate that sialyl Le(x) and sulfo Le(x) structures on the cell surface differ substantially in their ability to adhere to E- and P-selectin.     

H+-ATPase activity of Escherichia coli F1F0 is blocked after reaction of dicyclohexylcarbodiimide with a single proteolipid (subunit c) of the F0 complex

Dicyclohexylcarbodiimide (DCCD) specifically inhibits the F1F0-H+-ATP synthase complex of Escherichia coli by covalently modifying a proteolipid subunit that is embedded in the membrane. Multiple copies of the DCCD-reactive protein, also known as subunit c, are found in the F1F0 complex. In order to determine the minimum stoichiometry of reaction, we have treated E. coli membranes with DCCD, at varying concentrations and for varying times, and correlated inhibition of ATPase activity with the degree of modification of subunit c. Subunit c was purified from the membrane, and the degree of modification was determined by two methods. In the "specific radioactivity" method, the moles of [14C]DCCD per total mole of subunit c was calculated from the radioactivity incorporated per mg of protein, and conversion of mg of protein to mol of protein based upon amino acid analysis. In the "high performance liquid chromatography (HPLC) peak area" method, the DCCD-modified subunit c was separated from unmodified subunit c on an anion exchange AX300 HPLC column, and the areas of the peaks from the chromatogram quantitated. The shape of the modification versus inhibition curve indicated that modification of a single subunit c per F0 was sufficient to abolish ATPase activity. The titration data were fit by nonlinear regression analysis to a single hit mathematical model, A = Un(1 - r) + r, where A is the relative activity, U is the ratio of unmodified/total subunit c, n is the number of subunit c per F0, and r is a residual fraction of ATPase activity that was resistant to inhibition by DCCD. The two methods gave values for n equal to 10 by the specific radioactivity method and 14 by the HPLC peak area method, and values for r of 0.28 and 0.30, respectively. Most of the r value was accounted for by the observed dissociation of 15-20% of the F1-ATPase from the membrane under ATPase assay conditions. When the minimal, experimentally justified value of r = 0.15 was used in the equation above, the calculated values of n were reduced to 8 and 11, respectively. The value of n determined here, with a probable range of uncertainty of 8-14, is consistent with, and provides an independent type of experimental support for, the suggested stoichiometry of 10 +/- 1 subunit c per F1F0, which was determined by a more precise radiolabeling method (Foster, D. L., and Fillingame, R. H. (1982) J. Biol. Chem. 257, 2009-2015).