Neutral filter elution detects differences in chromatin organization which can influence cellular radiosensitivity.

We have shown previously that the neutral filter elution assay is dependent not only on the number of DNA double-strand breaks present in a mammalian cell but also on the way in which DNA expands on the filter following lysis. Results in this study indicate that the rate of DNA elution appears to be dependent upon the proximity of the DNA in relation to the replication complex. The rate of elution for DNA analyzed immediately after a 30-min labeling period with [14C]thymidine was about five times slower than the rate of elution for bulk-labeled DNA. However, the rate was increased a few hours later when the recently replicated DNA had matured and was likely to be farther from replication-associated attachment sites on the nuclear protein matrix. About one cell cycle after pulse labeling, when the labeled DNA was replicated again, DNA underwent similar changes in elution rate. For the four cell lines examined here, the elution rate 3-4 h after pulse labeling correlated with cellular radiosensitivity. Changes in rate of elution caused by altering EDTA concentration or pH may also be explained by DNA structural changes which occur during lysis. We conclude that the neutral filter elution method is sensitive to differences in chromatin organization which may also play a role in cell sensitivity to ionizing radiation.

Factors influencing DNA migration from individual cells subjected to gel electrophoresis.

Alkaline and neutral gel electrophoresis of individual mammalian cells allows detection of DNA single- and double-strand breaks, respectively. For both the alkaline and the neutral assays, lysis conditions influence how much DNA migrates, and factors in addition to DNA size play a role in migration. In particular, the tight packing of DNA in individual nuclei appears to reduce the ability to detect double-strand breaks in all of the genome. Tangling of DNA molecules is probably also responsible for the presence of "wings" associated with each nucleus after application of pulsed-field gel electrophoresis; these wings were aligned in the directions of the pulsed field, not along the resultant vector of the fields as was expected. The choice of fluorescent staining methods (propidium iodide, Hoechst 33342, or antibodies against bromodeoxyuridine) did not influence sensitivity for detecting DNA damage.

Comparison between pulsed-field and constant-field gel electrophoresis for measurement of DNA double-strand breaks in irradiated Chinese hamster ovary cells.

Pulsed-field gel electrophoresis (PFGE) is one of the most sensitive methods for detecting DNA double-strand breaks in mammalian cells. However, it has been observed that constant-field gel electrophoresis (CFGE), when optimized, can detect breaks with equal efficiency. The migration of DNA from the well and the separation of DNA molecules according to size appear to be different processes; only the latter requires the application of PFGE. CFGE is very sensitive and can detect DNA damage produced by less than 5 Gy of radiation. Low voltage (ca. 0.6 V/cm) during electrophoresis appears to be essential for the migration of the largest fraction of DNA from the agarose plug containing the cells; the electrophoresis run time, cell density in the plug, agarose concentration, nature of detergent and extent of radiolabelling are less important. It is concluded that CFGE is equally sensitive but more rapid and economical than PFGE for the measurement of DNA damage.

DNA double-strand breaks measured in individual cells subjected to gel electrophoresis.

Microscopic examination of individual mammalian cells embedded in agarose, subjected to electrophoresis, and stained with a fluorescent DNA-binding dye provides a novel way of measuring DNA damage and more importantly, of assessing heterogeneity in DNA damage within a mixed population of cells. With this method, DNA double-strand breaks can be detected in populations of cells exposed to X-ray doses as low as 5 Gy. The radiation dose-response relationship for initial formation of double-strand breaks was identical for cell lines irradiated in G1, regardless of their sensitivity to killing by ionizing radiation. However, for cells irradiated in S phase, DNA migration was significantly reduced. For Chinese hamster V79 cells, Chinese hamster ovary cells, WiDr human colon carcinoma cells, and L5178Y-R mouse lymphoblastoid cells, S-phase DNA appeared to be about 3 times less sensitive to X-ray damage than DNA from other phases of the cell cycle. However, for the very radiosensitive L5178Y-S cells, the migration of replicating DNA was reduced only slightly. For Chinese hamster V79 and Chinese hamster ovary cells, damage was repaired at a similar rate in all cells of the population, and 85% of the breaks were rejoined within 2 h after irradiation. The radiosensitive L5178Y-S cells repaired damage more slowly than V79 or Chinese hamster ovary cells; 2 h after exposure to 50 Gy, approximately 50% of the damage was still present.

Physical basis for detection of DNA double-strand breaks using neutral filter elution.

Results using neutral filter elution are difficult to explain if this method detects only DNA double-strand breaks (DSBs). In an attempt to understand neutral filter elution, the size of DNA pieces eluted from filters was measured using pulsed-field gel electrophoresis. Contrary to expectation, the size of the pieces was independent of radiation dose and time of elution, and much smaller (approximately 460 kb) than anticipated based on the expected number of DSBs induced. Shearing of the DNA molecule, the presence of nonspecific nucleases, and the influence of DNA-associated proteins were examined but could not explain our results. Consequently, we propose that cell lysis causes swelling of the DNA gel, and the exposed fraction of DNA on the surface of the gel is then sheared as the elution solution flows through the filter. We suggest that the rate of DNA elution measured using neutral filter elution is dependent upon the number of DSBs present, the composition of the eluting solution, especially with regard to the presence of molecules which can influence chromatin swelling on the filter, and the conformation or "packaging" of DNA before lysis.

Cell cycle-mediated structural and functional alteration of P85gag-mos protein kinase activity.

We investigated cell cycle-dependent regulation of protein kinase activity encoded by the viral mos gene by using a normal rat kidney cell line (NRK-6m2) chronically infected with a temperature-sensitive mutant (ts110) of Moloney murine sarcoma virus, which produces the P85gag-mos transforming protein. In elutriation experiments, in which cells in various phases of the cell cycle are separated based upon size, a twofold increase in the specific activity of the P85gag-mos protein kinase was observed as cells progressed from G0/G1 through S and G2/M. A three- to fourfold increase in gas-mos protein kinase specific activity relative to unsynchronized cells was observed in mitotic NRK-6m2 cells synchronized by treatment with thymidine followed by colcemid or with nocodazole alone. Interestingly, the gag-mos protein was structurally altered in mitotic cells generating a protein species moving slower than P85gag-mos in SDS-polyacrylamide gels. Our findings indicate that viral mos protein kinase activity is regulated during the cell cycle via phosphorylation. We propose that the mos transforming protein functions in a pleiotropic manner, affecting both cytoplasmic and nuclear targets.

Differential response of excision proficient and deficient L5178Y cells to UVC irradiation and benzamide.

L5178Y-R and L5178Y-S cells differ in sensitivity to UVC radiation (D0 values: 2.8 and 9.0 J m-2 respectively, exposure in Fischer's medium). The UVC sensitivity is related to the excision repair ability. Benzamide (Bz), an inhibitor of adenosine diphosphoribosyl transferase (ADPRT), does not modify the lethal effect of UVC radiation in L5178Y-R cells, whereas it sensitizes L5178Y-S cells. The content of NAD+ after irradiation decreases only in the latter cells and this decrease can be prevented by 2 mM Bz treatment. In agreement with the survival data, in L5178Y-R cells neither the proportion of abnormal cells nor the frequency of chromatid aberration are affected by 2 mM Bz treatment, in contrast with L5178Y-S cells. Bz slightly reverses inhibition of 3H-thymidine incorporation only in L5178Y-S cells, but it does not affect the proportions of cells in the different phases of the cell cycle in either cell strain after UVC exposure. These data could be taken as an indirect indication of the involvement of ADPRT in DNA repair in UVC-irradiated L5178Y-S cells. However, the increase in the number of DNA strand breaks in UVC-exposed, Bz-treated cells compared with UVC-exposed untreated cells is the same in both L5178Y strains.

The relationship of DNA and chromosome damage to survival of synchronized X-irradiated L5178Y cells. I. Initial damage.

We investigated the role of initial DNA and chromosome damage in determining the radiosensitivity difference between the variant murine leukemic lymphoblast cell lines L5178Y-S (sensitive) and L5178Y-R (resistant) and the difference in cell cycle-dependent variations in radiosensitivity of L5178Y-S cells. We measured initial DNA damage (by the neutral filter elution method) and chromosome damage (by the premature chromosome condensation method) and compared them with survival (measured by cloning) for both cell lines synchronized in G1 or G2 phase of the cell cycle (by centrifugal elutriation) and irradiated with low doses of X rays (up to 10 Gy). The initial yield of DNA and chromosome damage in G2 L5178Y-S cells was almost twice that in G1 L5178Y-S cells and G1 or G2 L5178Y-R cells. In all cases DNA damage expressed as relative elution corresponded with chromosome damage (breaks in G1 chromosomes, breaks and gaps in G2 chromosomes). Also we found that the initial DNA and chromosome damage did not determine cell age-dependent radiosensitivity variations in L5178Y-S cells, as there was less initial damage in the more sensitive G1 phase than in the G2 phase. L5178Y-R cells showed only small changes in survival or initial yield of DNA and chromosome damage throughout the cell cycle. Because survival and initial damage in sensitive and resistant cells irradiated in G2 phase correlated, the difference in radiosensitivity between L5178Y-S and L5178Y-R cells might be determined by initial damage in G2 phase only.

The relationship of DNA and chromosome damage to survival of synchronized X-irradiated L5178Y cells. II. Repair.

The purpose of this study was to investigate the role of DNA and chromosome repair in determining the difference in radiosensitivity between a radiosensitive murine leukemic lymphoblastoid cell line, L5178Y-S, and its radioresistant counterpart, L5178Y-R. Populations of cells in the G1 or G2 phase of the cell cycle were obtained by centrifugal elutriation and irradiated with X-ray doses up to 10 Gy and allowed to repair at 37 degrees C for various periods. The kinetics of DNA double-strand break repair was estimated using the DNA neutral filter elution method, and the kinetics of chromosome repair was measured by premature chromosome condensation. L5178Y-S cells exhibited decreased repair rates and limited repair capacity at both the DNA and chromosome level in both G1 and G2 phases when compared to L5178Y-R cells. For the repair-competent L5178Y-R cells, the rate of DNA repair was similar in G1 and G2 cells and exhibited both fast and slow components. While the kinetics of chromosome break repair in G1 cells was similar to that of DNA repair, chromosome repair in G2 cells had a diminished fast component and lagged behind DNA repair in terms of fraction of damage repaired. Interestingly, concomitant with a diminished repair capacity in L5178Y-S cells, the number of chromatid exchanges in G2 cells increased with time, whereas it remained constant with repair time in L5178Y-R cells. These results suggest that the basis for the exceptional radiosensitivity of L5178Y-S cells is a defect in the repair of both DNA double-strand breaks and chromosome damage.

Differential effect of benzamide on NAD+ content and the frequency of chromatid aberrations in X-irradiated L5178Y-R and L5178Y-S cells.

The effect of treatment with benzamide, an inhibitor of adenosine diphosphate (ADP) ribosyl polymerase (ADPRP) was studied in cells of two strains of L5178Y (LY) murine lymphoma exposed to ionizing radiation. Continuous 2 mmol/l benzamide (Bz) treatment increased the frequency of chromatid aberrations in the radiation sensitive LY-S strain, but not in the resistant LY-R strain. This result is in agreement with the previously found, enhanced by Bz, killing effect of roentgen irradiation in LY-S cells. Also, the decrease in the cellular NAD+ content in these cells after irradiation was more pronounced than in LY-R cells; this may indicate an increased ADPRP activity upon infliction of DNA damage, or a difference in poly(ADP-ribose) turnover.

The repair of double-strand DNA breaks correlates with radiosensitivity of L5178Y-S and L5178Y-R cells.

To better understand the basis for the difference in radiosensitivity between the variant murine leukemic lymphoblast cell lines L5178Y-R (resistant) and L5178Y-S (sensitive), the production and repair of DNA damage after X irradiation were measured by the DNA alkaline and neutral elution techniques. The initial yield of single-strand DNA breaks and the rates of their repair were found to be the same in both cell lines by the DNA alkaline elution technique. Using the technique of neutral DNA elution, L5178Y-S cells exhibited slightly increased double-strand breakage immediately after irradiation, most significantly at lower doses (i.e., less than 10 Gy). Nevertheless, even at doses that yielded equal initial double-strand breakage of both cell lines, the survival of L5178Y-S cells was significantly less than that of L5178Y-R cells. When the technique of neutral DNA elution was employed to measure the kinetics of DNA double-strand break repair, both cell lines exhibited biphasic fast and slow components of repair. However, the double-strand repair rate was much lower in the radiosensitive L5178Y-S cells than in the L5178Y-R cells (T1/2 of 60 vs 16 min). This difference was more pronounced in the fast-repair component. These results suggest that the repair of double-strand DNA breaks is an important factor determining the radiosensitivity of L5178Y cells.

Effects of irradiation and inhibition of adenosine diphosphate ribosyl transferase in radiation sensitive and resistant mouse lymphoma (L5178Y) cells.

The effects of treatment with benzamide, an inhibitor of adenosine diphosphate (ADP) ribosyl transferase, were studied in two strains of L5178Y lymphoma cells of differential sensitivity to ionizing radiation. Continuous 2 mmol/l benzamide treatment enhanced the killing effect of roentgen irradiation in the sensitive cells, but not in the resistent cells. Three hours' treatment with 2 to 15 mmol/l benzamide sensitized irradiated cells of both strains to a similar extent. Rejoining of DNA breaks was delayed by 2 mmol/l benzamide in sensitive cells more than in resistant cells.

Heat-induced inhibition of DNA synthesis in L5178Y-S cells is reversed by caffeine.

Heating L5178Y cells for 15 min at 43 degrees C caused a decrease in [3H]thymidine incorporation, which could be reversed by post-treatment with 0.75 mM caffeine in an L5178Y-S (radiation-sensitive, heat-resistant) but not in an L5178Y-R (radiation-resistant, heat-sensitive) strain. The reversal was accompanied by a sparing effect of the treatment: survival of L5178Y-S cells increased by a factor of 1.5. The effect of combined (heat + caffeine) treatment of L5178Y-R cells was cumulative.

No radiosensitivity-related change in plasma membrane properties in X- or gamma-irradiated L5178Y-R and L5178Y-S cells.

Two strains of murine lymphoma cells, L5178Y-R (LY-R) and L5178Y-S (LY-S) differ in radiosensitivity (D0 ca 1 and 0.5, respectively), in Na+/K+ and Mg2+-ATPase activities and susceptibility to heat; their fatty acid composition is also slightly different. Nevertheless, neither Na+-dependent amino acid uptake, nor membrane fluidity change after X- or gamma-irradiation (10 Gy under aerobic conditions, 27 Gy under extreme hypoxia). Although in LY-S cells there is a decrease in partition coefficient in a two-phase system, which indicates a late (24 h after irradiation) change in surface charge, this change is not related to viability, membrane fluidity, amino acid transport and survival.

ADP-ribosylation and post-irradiation cellular recovery in two strains of L5178Y cells.

Two strains of murine lymphoma cells, L5178Y-R (LY-R) and L5178Y-S (LY-S) differ in radiosensitivity (D0 ca 1.0 and 0.5 Gy) In this cellular system we tested the influence of poly (adenosine diphosphoribose) transferase (ADPRT) inhibitors, benzamide and caffeine, in combination with X or gamma irradiation under aerobic conditions, on the cellular response. We found a small difference between LY-R and LY-S cell strains in incorporation of 3H from [3H]-NAD (nicotinamide dinucleotide) into permeabilized cells. The incorporation was related to DNA degradation initiated by the permeabilization procedure. The post-irradiation inhibition of DNA synthesis was reversed only in LY-S cells by 2mM benzamide or 2mM caffeine treatment. Changes in the cell cycle, as determined by flow-cytometry, were of similar character in both LY-R and LY-S cells, within 12 h after irradiation, whereas the effect of treatment on survival differed: only LY-S cells were radiosensitized by benzamide; both LY strains were sensitized by caffeine, but to different extents. From these observations it followed that there was a pattern of relations between the extent of postirradiation DNA synthesis, duration of mitotic delay and recovery.

Ion content and the response of L5178Y-R and L5178Y-S cells to X rays and ionophore A23187.

We examined the response of L5178Y-S (radiosensitive, LY-S) and L5178Y-R (radioresistant, LY-R) lymphoblasts to X-irradiation with concomittant treatment with divalent cation ionophore, A23187 (3 h or 5 h, 5 micrograms/ml). Cells treated with A23187 alone progressed through the cell cycle more slowly than the untreated cells and their cloning efficiency was reduced. In both cell strains the ionophore prolonged duration of the postirradiation mitotic delay. Radiation-induced inhibition of DNA synthesis was reversed by A23187 in LY-S but not in LY-R cells. Cells subjected to the ionophore treatment survived X-irradiation in almost the same way as untreated cells, as if the effect of A23187 treatment were reversed by irradiation. There was also a reversion in the ion content: A23187 caused a marked increase in Na+ content and a decrease in K+ content, irradiation itself did not change the ion content, whereas in the A23187-treated cells it restored almost the same pattern as that found in the control cells. We found less Mg2+ ions in LY-S cells after treatment with A23187 and A23187 + X than in LY-R cells, in relation to untreated (control) cells. These observations point to the possible importance of ion transport for recovery from radiation damage.

Effect of ionizing radiation and low temperature on L5178Y-R and L5178Y-S cells. II. Cell survival and DNA strand breaks after roentgen or gamma irradiation.

The repair of DNA strand breaks and the effect on the cell survival after cold treatment or combined treatment with cold+irradiation was investigated in two strains of murine L5178Y lymphoma cells. Cold treatment alone had a slight and similar effect on the survival of L5178Y-R and L5178Y-S cells. No effect of cooling was found on the repair of DNA strand breaks. When L5178Y-R and L5178Y-S cells were cold-treated for various time intervals before irradiation with 2 Gy roentgen rays a slight gradual decrease of survival was found, a little more marked in L5178Y-S cells. No effects on the rejoining of DNA strand breaks could be seen. When the cells were first irradiated and then cold-treated the survival data showed that combined treatment gives slightly less than additive results. Five hours' cold treatment after gamma irradiation did not change the kinetics of DNA strand breaks rejoining.

DNA repair and replication in radiation-sensitive and -resistant mouse lymphoma cells gamma-irradiated under aerobic and hypoxic conditions.

Repair of DNA damage induced by gamma-rays was studied by the DNA unwinding technique in two strains of murine L5178Y lymphoma cells. These strains differ in sensitivity to low LET radiation by a factor of 1.4. No difference was found in the rate of DNA strand break rejoining during the first 3 hours after irradiation with 2 or 10 Gy of gamma-rays under aerobic conditions either at 0 degrees C or at room temperature. Also, the lesions inflicted by irradiation with 5 Gy of gamma-rays under hypoxia at room temperature were repaired by both strains at the same rate. The final level of unrejoined DNA strand break was the same in both strains, under all conditions examined, within the limits of resolution of the DNA unwinding technique. The rates of DNA chain elongation were also similar in both strains after irradiation under hypoxic or aerobic conditions.

Effect of ionizing radiation and low temperature on L5178Y-R and L5178Y-S cells. I. DNA strand breaks induced by decays of incorporated 3H and 125I.

The induction and repair of DNA strand breaks induced by disintegration of 3H and 125I incorporated into DNA was examined by the DNA unwinding technique in two strains of murine L5178Y lymphoma cells. The L5178Y-S cells are extremely sensitive to decay of 3H incorporated into DNA, L5178Y-R cells have rather 'normal' sensitivity. The number of strand breaks induced during a cold treatment was about 2.2 per 3H decay and 4 per 125I decay. No differences were found between the two strains. During the labelling period accumulation of unrepaired DNA strands occurred. About 80 per cent of all 125I induced DNA strand breaks was left unrepaired after a 21 h labelling period. No differences occurred between the two cell strains. It thus seems that the marked difference between the two cell strains in sensitivity to decay of the nuclides incorporated into DNA is not due to differences in the rejoining capacity of DNA strand breaks.