Raymond L. Warters

Excision of X-ray-induced thymine damage in chromatin from heated cells.

Experiments were performed to distinguish between two possible modes of hyperthermia-induced inhibition of thymine base damage excision from the DNA of CHO cells: (1) heat denaturation of excision enzyme(s) or (2) heat-induced alteration of the substrate for damage excision (chromatin). While hyperthermia (45/sup 0/C, 15 min) had no apparent effect on the capacity of the excision enzymes to excise damage from DNA it had a dramatic effect (ca. 80% inhibition) on the ability of chromatin to serve as a substrate for unheated enzymes. These results suggest that hyperthermia-induced radiosensitization of CHO cells may be due primarily to lesions in the cellular chromatin.

Heat Shock (45°C) Results in an Increase of Nuclear Matrix Protein Mass in HeLa Cells

SummaryThe nuclear matrix from HeLa cells heated at 45°C was isolated to determine the effect of thermal shock on its composition and structure. The matrix from unheated cells contained about 10 per cent of total cell protein and was observed to be a spherical particle with a diameter ranging from 3 to 5 μm with the major constituent polypeptides having molecular weights of 45, 47, 55, 57, 59 and 65 kilodaltons. The nuclear-matrix protein mass increased linearly with increasing exposure time at 45°C with no observable change in its size or shape. The additional proteins were observed in general to have molecular weights greater than 45 kilodaltons, with marked increases in polypeptides of 28·5, 38·5, 60, 66, 75, 81, 88, 100 and 115 kilodaltons. An exponential relationship was observed between heat-induced cytotoxicity and the nuclear matrix protein mass increase. A 15 per cent increase in matrix protein mass was sustained prior to the onset of cytotoxicity, while a 35 per cent increase in matrix protein c...

Variation in Radiation-Induced Formation of DNA Double-Strand Breaks as a Function of Chromatin Structure

The influence of chromatin structure on induction of DNA double-strand breaks (DSBs) by X radiation was studied in DNA from CHO cells. Whole cells, nuclei with condensed or relaxed chromatin, and deproteinized DNA in agarose plugs were irradiated and DSB formation was measured as a decrease in the length of DNA by nondenaturing, pulsed-field, agarose gel electrophoresis. The yield of DSBs in deproteinized DNA (2.3 x 10(-10) DSBs Da-1 Gy-1) was observed to be 70 times greater than the yield of DSBs (3.1 x 10(-12) DSBs Da-1 Gy-1) observed in DNA in the intact cell nucleus. Organization of DNA into the basic nucleosome repeat structure and condensation of the chromatin fiber into higher-order structure protected DNA from DSB induction by factors of 8.3 and 4.5, respectively. An additional twofold protection of DNA in fully condensed chromatin occurred in the intact cell nucleus. Since this protection did not appear to involve chromatin structure, we speculate that this additional protection may result from the association of soluble protein and nonprotein sulfhydryls with DNA in the intact cell nucleus. The results are consistent with the organization of nuclear DNA into both basic nucleosome repeat structure and higher-order chromatin structure providing significant protection against DSB induction.

The Effects of Hyperthermia on DNA Replication in HeLa Cells

The extent of heat-induced inhibition of DNA replication in HeLa cells was assayed at temperatures between 43 and 48 degrees C. During hyperthermic exposure replicon initiation, as well as elongation of replicons into larger replicative fragment sizes, was rapidly inhibited. Elongation of nascent DNA into replicons continued at a normal rate for up to 45 min at 45 degrees C. Heated cells, replaced at 37 degrees C, elongated nascent DNA at a reduced rate and elongation was incomplete for up to 36 hr. Nascent DNA, not fully elongated 24 hr after hyperthermic exposure, was observed in replicative fragment sizes as small as replicons. The extent of heat-induced inhibition of DNA elongation increased with increasing time-temperature exposure with an activation energy of 122 kcal/mole of DNA. When pulsed cells were incubated at 37 degrees C for various times prior to heating, the extent of heat-induced inhibition of DNA elongation decreased with a half-time of 20-25 min, suggesting that the heat-sensitive structure is associated with replicative fragments having sizes less than 140-150S.

Hyperthermia and the Cell Nucleus

Recent reviews document that hyperthermic shock inhibits the major nuclear activities, including semiconservative DNA replication (1, 2). These inhibitions may result either from a direct effect of heat on the cell nucleus or as a consequence of changes in other cell compartments [e.g., cell membrane damage (3)]. Here we review recent results concerning heat-induced changes in mammalian cell nuclear structure and function, principally DNA replication. These data are discussed in terms of a current model of nuclear organization in order to relate these heat-induced alterations in the nucleus to known cellular effects of heat, such as cytotoxicity and alterations in DNA synthesis.

Detection of Ionizing Radiation-Induced DNA Double-Strand Breaks by Filter Elution Is Affected by Nuclear Chromatin Structure

Chinese hamster ovary cells were irradiated with 250 kVp X rays and analyzed for the presence of DNA double-strand breaks using either polycarbonate filter elution or pulsed-field agarose gel electrophoresis at neutral pH. Reduction in DNA length detected by filter elution was produced as a nonlinear function of increasing radiation dose, with a quasi-threshold at low total dose, and as a first-order function of increasing radiation dose as detected by gel electrophoresis. The quasi-threshold observed with filter elution was eliminated when nuclei were isolated from irradiated cells and their chromatin relaxed in a buffer containing low-molarity monovalent cation prior to analysis by filter elution. The results suggest either that the chemical structure of the DNA double-strand breaks produced by low-LET radiation necessitates a DNA relaxation step before they can be detected accurately by filter elution, or that at low total radiation dose a DNA complex forms on the polycarbonate filter.

The nuclear matrix is a thermolabile cellular structure

Abstract Heat shock sensitizes cells to ionizing radiation, cells heated in S phase have increased chromosomal aberrations, and both Hsp27 and Hsp70 translocate to the nucleus following heat shock, suggesting that the nucleus is a site of thermal damage. We show that the nuclear matrix is the most thermolabile nuclear component. The thermal denaturation profile of the nuclear matrix of Chinese hamster lung V79 cells, determined by differential scanning calorimetry (DSC), has at least 2 transitions at Tm = 48°C and 55°C with an onset temperature of approximately 40°C. The heat absorbed during these transitions is 1.5 cal/g protein, which is in the range of enthalpies for protein denaturation. There is a sharp increase in 1-anilinonapthalene-8-sulfonic acid (ANS) fluorescence with Tm = 48°C, indicating increased exposure of hydrophobic residues at this transition. The Tm = 48°C transition has a similar Tm to those predicted for the critical targets for heat-induced clonogenic killing (Tm = 46°C) and thermal radiosensitization (Tm = 47°C), suggesting that denaturation of nuclear matrix proteins with Tm = 48°C contribute to these forms of nuclear damage. Following heating at 43°C for 2 hours, Hsc70 binds to isolated nuclear matrices and isolated nuclei, probably because of the increased exposure of hydrophobic domains. In addition, approximately 25% of exogenous citrate synthase also binds, indicating a general increase in aggregation of proteins onto the nuclear matrix. We propose that this is the mechanism for increased association of nuclear proteins with the nuclear matrix observed in nuclei isolated from heat-shocked cells and is a form of indirect thermal damage.

Radioprotection of Human Cell Nuclear DNA by Polyamines: Radiosensitivity of Chromatin is Influenced by Tightly Bound Spermine

The polyamines putrescine (PUT) and spermine (SPM) were examined for their ability to protect human cell DNA against the formation of radiation-induced double-strand breaks (DSBs). As observed previously, under conditions where polyamines were shown to be almost completely absent, association with nuclear matrix protein into a nucleoid, and organization into chromatin structure, protected DNA from induction of DSBs by factors of 4.5 and 95, respectively. At concentrations below 1 mM, PUT or SPM provided equivalent levels of protection to deproteinized nuclear DNA, consistent with their capacity to scavenge radiation-induced radicals. At constant ionic strength, 5 mM SPM protected deproteinized DNA and nucleoid DNA and DNA in nuclear chromatin by factors of 100 and 26, respectively. At 5 mM, SPM provided 15 times greater protection of deproteinized DNA than did PUT. Under physiologically relevant conditions, 5 mM SPM protected DNA in the intact nucleus from the induction of DSBs by a factor of 2 relative to DNA in the absence of SPM. Studies of SPM binding during cellular fractionation revealed that a significant fraction of the cellular SPM is tightly bound in the nucleus but can be removed by extended washing. Thus the association of SPM with nuclear chromatin appears to be a significant contributor to the resistance of the cells DNA to the induction of DSBs.

Nucleosome structure in chromatin from heated cells.

The effect of hyperthermia (40 to 80/sup 0/C) on the nucleosome structure of mammalian chromatin was determined using the enzyme micrococcal nuclease. At equivalent fractional DNA digestion it was found that neither the size of DNA nor the total fraction of cellular DNA associated with nucleosome structure is altered by heat exposure up to 48/sup 0/C for 30 min. It is proposed that this heat-induced reduction in the accessibility to nuclease attack of DNA in chromatin from heated cells is due to the increased protein mass associated with chromatin.

Measurement of Radiation-Induced DNA Damage Using Gel Electrophoresis or Neutral Filter Elution Shows an Increased Frequency of DNA Strand Breaks after Exposure to pH 9.6

The filter elution technique using nondenaturing conditions is widely used to assay DNA double-strand break (DSB) induction and repair. It has been reported that in the measurement of strand breaks higher rates of elution and of initial rejoining are obtained at pH 9.6 compared to pH 7.2. In the present experiments neutral elution at pH 7.2 and 9.6 were compared in the assay of damage to DNA induced by X rays, 125I decay, and restriction enzyme digestion, in an effort to explain this discrepancy and to determine whether the higher rate of elution observed at pH 9.6 corresponds to a greater number of DSBs. X-ray damage to cellular DNA resulted in significantly different elution profiles at the two pH values. In contrast the elution profiles of the DSB induced by intragenomic 125I decays or restriction endonuclease were independent of the pH of the elution buffer. When gamma-irradiated SV40 DNA was exposed to pH 7.2 or 9.6 elution buffer prior to analysis by gel electrophoresis, a significantly greater number of DNA DSBs were detected in the DNA exposed to pH 9.6. We conclude that X and gamma radiation produce lesions (pH 9.6-labile lesions), in proportion to dose, that have the potential of becoming measurable DSBs following incubation under the mildly alkaline condition of pH 9.6. The data suggest that these lesions may result from single-hit events.