Immunoreactivity to antinucleoside antibodies persists during G2 arrest in X-irradiated HeLa cells

Arrest of HeLa cells in G2 after ionizing radiation is accompanied by persistent nuclear immunoreactivity to antinucleoside antibodies. The reactivity declined to the normal G2 level during escape from arrest and subsequent cell division.     

Immunoreactivity to antinucleoside antibodies in camptothecin treated HeLa cells

HeLa cells, incubated with camptothecin during the G 1 phase of the cell cycle, show nuclear fluorescence with fluorescein-labeled antinucleoside antibodies. If the G 1 cells are washed free of the drug, the cells no longer demonstrate nuclear fluorescence. Since these antibodies react only with single-stranded DNA, the positive staining in camptothecin-treated G 1 cells suggests that the drug induces denatured regions in DNA. Fluorescent antinucleoside antibodies may be a useful technique for the observation of drug-induced changes in DNA during the G1 phase of the cell cycle.     

Fast repair of anoxic radiation damage in HeLa cells detected by antinucleoside antibodies

The influence of anoxia on X-ray damage in HeLa cells was studied by observing effects on nuclear immunoreactivity to antinucleoside antibodies and on the sedimentation in alkaline sucrose gradients of their DNA “complexes”. The fraction of G1 HeLa cells which was immunoreactive to fluorescein labeled antinucleoside antibodies increased from control levels of 11% ± 3.5 S.E. to 71% ± 5.7 S.E. after 1 000 rads in air. In anoxia 1 000 rads increased this fraction to only 42% ± 3.1 S.E. After 1 000 rads in air the return to normal G1 levels of immunoreactivity required 90 min, but it required only 30 min after radiation in anoxia. If cells were held at 0 °C for 35 min before anoxic irradiation the rapid return to control levels of immunoreactivity during postradiation incubation at 37 °C was not observed. Cold shock did not increase the proportion of cells initially made immunoreactive by 1 000 rads in anoxia. Anoxia reduced the effect of 1 000 rads on the sedimentation properties of the DNA complex. Cold shock prior to anoxic radiation retarded the faster reconstitution of the DNA complex otherwise observed after anoxic radiation.     

Deficit in DNA content relative to histones in X-irradiated HeLa cells.

The DNA and histone content of HeLa S-3 cell cultures was measured by direct mass assays 21 hours after 1000 rad of X-irradiation, when the cells were arrested in G2 phase. The nuclear DNA content of such cultures was found to be deficient (73% of control values). In contrast, the synthesis of nuclear histones persisted, and the total histone content was close to 100% of control values. When synchronously-growing cultures were irradiated in mid-S phase and examined 3-5 hours later in G2 phase, both DNA and histone content were equal to control values.     

Monoclonal antibodies to the antigens of human HeLa tumor cells

A panel of 6 hybridomas XEJIMA producing monoclonal antibodies specific to HeLa cells is prepared. Monoclonal antibodies do not bind to antigens of human diploid fibroblasts, human continuous B- and T-lymphocytes and animal cell lines. The specificity of monoclonal antibodies to cellular antigens of 5 HeLa-like cell lines and 6 human tumour cells lines, not contaminated with HeLa cells, is determined. Antibody containing ascitic fluid and culture media of hybridomas XEJIMA-3, -12, -13, and -22 significantly decrease the attachment of HeLa cells to the surface of culture flasks. Monoclonal antibodies XEJIMA-11, -12 and -13 block the multiplication of HeLa cells. The effect depends on serum concentration in the nutrient medium.     

The action of caffeine on X-irradiated HeLa cells. IX. Hypothermic effects

Hypothermic enhancement of the lethal effect of 3.5 Gy of 220-kV X rays in the absence of caffeine as well as in its presence (4 mM) was examined at temperatures between 10 and 34 degrees C in monolayer cultures in the G1 phase of the cell cycle. Correction has been made for the toxicity of low temperatures, and of caffeine at low temperatures, by concomitantly measuring cell killing in unirradiated cells. In the absence of caffeine, incubation of irradiated cells for up to 34 h at temperatures in the range 15 to 30 degrees C (or possibly 34 degrees C) enhances killing compared to that observed at 38 degrees C; the amount of enhancement is about the same throughout this range, but is nil at 10 degrees C. The enhanced killing induced by caffeine at 38 degrees C decreases as the temperature is lowered to 15 degrees C; there is no enhancement at 10 degrees C. Less killing is manifested in the range 15 to 25 degrees C in the presence of caffeine than in its absence. Recovery (loss of sensitivity to caffeine) and fixation of potentially lethal damage were studied in late-S/G2-phase cells at reduced temperatures by delaying treatment with caffeine for increasing times after irradiation. As the temperature is progressively lowered to 20 degrees C, less recovery is manifested after 5 h of incubation; no recovery is detected in the range 10 to 20 degrees C. Despite extensive recovery at 34 degrees C, no fixation is observed at that (or any lower) temperature in G2-phase cells: the cells are able to recover essentially fully when returned to 38 degrees C. In addition, responses of unirradiated control series to incubation at low temperatures appear to differ from those reported by others for longer treatment times of different cell systems.     

On the identity of the damage expressed by treatment of X-irradiated HeLa cells with different agents

To determine whether different agents that enhance the expression of potentially lethal X-ray damage (PLD) interact with the same or different lesions (or spectrum of lesions), cell killing was measured in three kinds of experiments: (1) When cells were irradiated in G1 phase and treated with caffeine or hydroxyurea at concentrations that yield maximal response, the same survival plateaus were reached. (2) Treatment of cells irradiated in G1 phase either with caffeine or with hydroxyurea so as to yield survival levels that differed twofold after 4 h incubation, followed by treatment with caffeine to allow expression of PLD in G2 phase, resulted eventually in the same level of survival. (3) When cells were irradiated and treated with caffeine, hydroxyurea, or 9-beta-D-arabinofuranosyladenine (araA) after progressively longer delays, to trace the time course of recovery from the PLD, the responses obtained with caffeine and araA were similar; sensitivity to hydroxyurea was lost more rapidly. The results are consistent with the possibility that these three agents interact with the same lesions, but that different steps in the repair process are inhibited by caffeine or araA than by hydroxyurea.     

The action of caffeine on X-irradiated HeLa cells. VIII. Recovery from potentially lethal damage

Recovery from potentially lethal radiation damage in HeLa S3 cells has been studied by irradiating synchronous cultures with 4 Gy at selected ages in the cell cycle, initiating treatment with 4 mM caffeine, which prevents recovery, at progressively later times up to 24-30 h after irradiation, and determining the plateau level of survival after incubation with the caffeine until 36-40 h after mitotic collection. Cell recovery appears to begin immediately after irradiation at any time during interphase: an accelerating increase in survival gives way after several hours to a linear increase which lasts for an additional several hours. The median recovery time is approximately 13 h after irradiation at any time during G1, but is markedly shorter (5-7 h) after irradiation in S or G2. The rate of recovery is slightly depressed if DNA replication is inhibited with aphidicolin after irradiation and slightly enhanced if protein synthesis is inhibited with cycloheximide. Both the rate and the extent of recovery are dependent on the location of the cells in the cycle at the time of irradiation--both functions increasing with cell age from the beginning of S, but having different age dependencies in G1. Blocking cell progression with a DNA-synthesis inhibitor before irradiation halts the age-dependent changes.     

The action of caffeine on X-irradiated HeLa cells. VI. Damping of the structured age-survival function

Postirradiation treatment of synchronous HeLa S3 cultures with 4 mM caffeine until greater than or equal to 32 hr after mitotic collection, following exposure to 220-kV X rays at various times during interphase, severely damps the fluctuations in the age-survival curve. Not only does the dose-survival curve essentially lose its shoulder, as reported previously, but it becomes steeper and displays a virtually age-independent terminal slope (D0 congruent to 0.5 Gy). It becomes multicomponent, at least early in the cycle. The residual structure in the interphase age-survival curve, if any, appears to reflect mainly an age-dependent fluctuation in the size of a subpopulation of cells having marked sensitivity to X rays (D0 congruent to 0.25 Gy), though there might be small residual fluctuations in the size of the shoulder and the slope. Mitotic cells also respond to postirradiation treatment with caffeine; they yield a dose-survival curve whose slope is similar to that of the sensitive subpopulation seen in interphase. These findings indicate that most of the structure in the unperturbed age-survival function derives from repair of potentially lethal radiation damage.     

Immunofluorescent localisation of cytokeratin antigens in mitotic HeLa cells using monoclonal antibodies

The organisation of cytokeratin filaments in mitotic HeLa cells has been analysed by immunofluorescence microscopy using a monoclonal antibody which recognises proteins with apparent subunit molecular weights of 52 kDa and 57 kDa and which binds exclusively to cytokeratin-type filaments. Mitotic cells were prepared for microscopic analysis by hypotonic swelling, centrifugation onto glass slides, brief pre-extraction with 0.1% Triton X-100 and fixation in 80% ethanol. This procedure gave particularly good resolution of intermediate filaments and preservation of chromosome morphology. In prometaphase-metaphase cells the antigen was present in an anastomosing filament network which completely or partially enclosed the chromosomes, in filament fragments and in cytoplasmic aggregates. The epichromosomal filament network was absent from cells in anaphase or later stages of mitosis. In these cells non-filamentous antigen was often located in a narrow band defining the periphery of individual chromosomes and in variable numbers of cytoplasmic filaments or fragments. The results suggest that extensive disaggregation and reformation of cytokeratin filaments occurs during mitosis and that disaggregated cytokeratin proteins are frequently located adjacent to mitotic chromosomes.     

The action of caffeine on X-irradiated HeLa cells. X. Depressed recovery from potentially lethal damage in cells containing 5-bromodeoxyuridine

HeLa S3 cells were sensitized to the lethal action of 220-kV X rays by partially replacing the thymidine in their DNA with 5-bromodeoxyuridine (BrdU). To examine the expression of and recovery from potentially lethal radiation damage (PLD), both BrdU-grown and control cells were treated with 4 mM caffeine for increasing times up to 2 days, either immediately after irradiation or after increasing delays up to 28 h. When the same dose of X rays (3 Gy) was applied to BrdU-grown and control cells, the difference in survival that is found in the absence of caffeine disappeared after about 30 h of incubation in its presence; when isosurvival doses were applied (BrdU-grown cells, 2.5 Gy; control cells, 4 Gy), the control cells suffered more killing. When treatment with caffeine was delayed for progressively longer times after both groups of cells received 3 Gy, the control cells achieved a higher level of survival. These results indicate that the increased radiation sensitivity of cells containing BrdU derives from a decreased ability to repair PLD.     

Immunoreactivity of phage library-derived human single-chain antibodies to amyloid beta conformers in vitro

The pathogenesis of Alzheimer's disease involves conformational changes of A beta. A series of antibodies recognizing a distinct conformation of A beta (snapshot antibody) is useful for both understanding the mechanism of molecular conversion and identifying diagnostic and therapeutic reagents. As A beta with various conformations can be prepared in vitro under varying physicochemical conditions, snapshot antibodies can be isolated by directly binding to target molecules with antibody-displaying phages. We tested the feasibility of this idea. We show a feature of several A beta-reactive antibodies isolated from our human single-chain Fv antibody-phage library and particularly report the characteristics of an scFv clone, B6, selected from the fibrillar A beta 1-42-coated biopanning. B6 bound to fibrillar A beta 1-42 as well as globulomer A beta 1-42 but not to soluble A beta 1-42 or A beta 1-40. B6 inhibited A beta 1-42 fibril formation with 600 nM IC50 in spite of being the monovalent scFv form. Epitope analysis suggested that the binding site might be located at the beta2 sheet of the C-terminus of A beta 1-42. Although it is believed that N-terminus-recognizing antibodies tend to show the capability to inhibit A beta 1-42 fibrillation, B6 is the first human inhibitory antibody recognizing the C-terminus of A beta 1-42.     

The adhesion of protein A-bearing Staphylococcus aureus organisms to soluble-staphylococcal-antigens-coated HeLa cells mediated by specific antibodies

The adhesion of staphylococcal protein A (SpA)-bearing Staphylococcus aureus Cowan I organisms to HeLa cells was enhanced by pretreatment of HeLa cells with staphylococcal extracellular antigens and antibodies to them. The adhesion of HLj, an SpA-poor mutant derived from Cowan I, to HeLa cells was not enhanced by the same pretreatment of HeLa cells. Furthermore, the enhanced staphylococcal adhesion was inhibited by soluble SpA. The antigen(s) responsible for the enhanced staphylococcal adhesion was(were) heat stable. Pretreatment of HeLa cells with the mixture of staphylococcal extracellular antigens and antibodies to them also enhanced the adhesion of Cowan I. Similarly the adhesion of Cowan I was enhanced by pretreatment of HeLa cells with extracellular antigens of Pseudomonas aeruginosa and antibodies to them. These results indicated that cell-bound SpA mediated the binding of S. aureus to immune complexes composed of extracellular bacterial products and antibodies to them bound to the surface of HeLa cells, and suggested another role of cell-bound SpA as a co-adhesin with other factors in infections due to S. aureus.     

Split-dose recovery and protein synthesis in X-irradiated CHO cells

A temperature-sensitive mutant for protein synthesis, CHO-tsH1, has been compared to the wild-type (WT) cell, CHO-SC1, in single- and split-radiation-dose schemes. When the exponentially growing mutant and the wild-type cells were treated at 40 degrees C for up to 3 h prior to split doses of X rays, survival was progressively reduced in the mutant compared to the wild type. In addition, if a 2-h split-dose scheme was used with a treatment of 40 degrees C given before the first dose, between the dose fractions and after the second dose, the recovery from sublethal damage (SLD) was almost completely inhibited in the mutant cells. These observations implied that a pool of proteins was involved in the recovery from sublethal X-ray damage. However, if molecular repair was measured in the mutant cell by the alkaline-unwinding technique under the same time and temperature schemes as those demonstrating a reduction in the recovery from SLD, no difference in the kinetics of DNA strand rejoining was observed compared to similar measurements made under conditions permitting SLD recovery. Misrepair processes may permit restoration of DNA strand integrity but not allow functional repair. Split-dose experiments were also done using cycloheximide to chemically inhibit protein synthesis. Under conditions which mimicked those used in the temperature-shift experiments both cell lines showed a reduction in the recovery from sublethal damage comparable in magnitude to that observed in the mutant cells when they were treated with 40 degrees C. Both the chemical and thermal inhibition of protein synthesis substantiate its necessity for the recovery from sublethal damage.