Doreen Liebeskind

Immunoreactivity to antinucleoside antibodies in X-irradiated HeLa cells

Abstract Synchronized HeLa cells were stained with antibodies to purine and pyrimidine nucleosides by immunofluorescent and immunoperoxidase techniques. These antibodies react only with denatured or single-stranded regions of DNA. Nuclear attachment of antibody was seen only during the period of DNA synthesis as determined by 3 H-thymidine incorporation. Positive nuclear immunoreactivity was seen in approx. 15% of cells obtained by mitotic selection at a time corresponding to the G 1 phase. After exposure to ionizing radiation, 80% of the G 1 cells were reactive. Induction of immunoreactivity was dose dependent over the range of 100 to 1 000 rads. Treatment of irradiated G 1 cells with deoxyribonuclease completely eliminated the positive nuclear reaction. Exposure to ribonuclease had no effect. Incubation of the G 1 cells for 90 min at 37 or 0–4 °C after the administration of 1 000 rads resulted in a prompt decrease of immunoreactivity to control levels. However, in the presence of 0.04 μg/ml actinomycin D, positive nuclear staining remained at high levels. No such effect could be observed as a result of exposure to cytosine arabinoside or hydroxyurea. It is concluded that X-irradiation of G 1 HeLa cells produces single-stranded regions in nuclear DNA that can be detected by anti-pyrimidine and anti-purine antibodies.

DNA of HeLa Cells during Caffeine-promoted Recovery from X-ray Induced G2 Arrest

Progression of X-irradiated HeLa cells from G2 arrest through mitosis was promoted by 1mM caffeine. Caffeine promoted the return from abnormally high levels of radiation-induced immunoreactivity to antinucleoside antibodies, which indicates persistent DNA strand separation, to the low levels normally found in G2. With caffeine, the irradiated cells progressed through mitosis, producing daughter cells with the normal G1 content of DNA. Without caffeine, the DNA content of individual radiation-arrested cells retained G2 values and the abnormally high levels of immunoreactivity to antinucleoside antibodies.

Carcinogen-induced immunoreactivity to antinucleoside antibodies in HeLa cells

Abstract Immunoreactivity to fluorescein-labelled antinucleoside antibodies was induced in HeLa cell nuclei by the carcinogen N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). DNA strand breaks induced by MNNG were detected by measuring the influence of MNNG on the sedimentation in alkaline sucrose gradients of a fast sedimenting DNA-Containing complex. Dissociation of the complex and induction of immunoreactivity both proved to be sensitive, dose-dependent indicators of MNNG action in HeLa cells. Since antibodies directed against nucleosides react only with denatured or single-stranded DNA, it appears likely that MNNG-induced immunoreactivity results from exposure of single-stranded regions of DNA, providing a means for the rapid identification of carcinogen action by immunofluorescent techniques. N-Acetoxy-2-acetylaminofluorene also induced immunoreactivity and led to the dissociation of the DNA complex in HeLa cells. Seven other chemicals with known carcinogenic properties also induced immunoreactivity in this test. Immunoreactivity and dissociation of the complex were not induced by inhibitors of DNA synthesis such as hydroxyurea and cytosine arabinoside. Protein synthesis inhibitors such as cycloheximide induced immunoreactivity but did not dissociate the complex. Cycloheximide-induced immunoreactivity was rapidly and completely reversible following removal of the drug; the effects of MNNG were not reversible in our experiments. This novel application of immunofluorescent techniques provides a rapid quantitative means for detecting and studying the action of carcinogens in animal cells.

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

Abstract 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.

Abstract 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

Abstract 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.

Tightly bound chromatin proteins with rapid metabolic turnover suppress antinucleoside immunoreactivity in HeLa cell nuclei.

Abstract A class of non-histone chromatin proteins that were bound tightly to DNA and could not be dissociated from the chromatin by high salt and urea was isolated from HeLa cell nuclei and separated from DNA by DNase digestion. These ‘tight’ proteins retained their ability to bind to single- and double-stranded DNA as assayed by nitrocellulose filter binding. Polyacrylamide gel electrophoresis showed that the most prominent proteins possessed molecular weights of about 60 000 D. In asynchronously growing HeLa cell cultures about 1 3 of the cell nuclei were immunoreactive to fluorescein-labeled antinucleoside antibodies. The immunoreactive cells were the fraction in S phase. Cycloheximide treatment of the cultures raised the fraction of immunoreactive nuclei to over sol2 3 . Exposure of the fixed cycloheximide-treated cell to tight proteins prior to staining with the antibody reduced the fraction of immunoreactive cells to the normal S phase level. Immuno-reactivity induced by X-irradiation or by the intercalating mutagen hycanthone was also suppressed by tight proteins. Cycloheximide treatment preferentially reduced the cellular content of tight proteins, suggesting that these proteins undergo a metabolic turnover with a half-life of about 5 h.