Rosemary S. L. Wong

Molecular Studies on the Hyperthermic Inhibition of DNA Synthesis in Chinese Hamster Ovary Cells

The hyperthermic inhibition of cellular DNA synthesis was studied to elucidate the mechanism involved in killing S-phase cells. A time-temperature dependence for inhibition of DNA synthesis similar to that seen for cell survival was found in CHO cells. This inhibition was primarily an effect on the replication process although some inhibition of both [3 H] TdR transport and endogenous synthesis of TTP was observed. Details of the inhibitory effect at the replicon level were studied for a 15-min-45.5°C treatment, which reduced both DNA synthesis and survival of S-phase cells by 70-90%. Alkaline sucrose gradient analysis of the heat effect on cellular chain elongation of

Analysis by Pulsed-field Gel Electrophoresis of DNA Double-strand Breaks Induced by Heat and/or X-irradiation in Bulk and Replicating DNA of CHO Cells

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Critical Steps for Induction of Chromosomal Aberrations in CHO Cells Heated in S Phase

DNA made immediately before treatment revealed a transient delay during heating and for about 5 min thereafter in the elongation and ligation of this nascent DNA into molecules of all sizes ranging from replicons to clusters. As incubation continued for up to 150 min after heating, a two- to threefold reductio...

Mechanism of killing Chinese hamster ovary cells heated in G1: effects on DNA synthesis and blocking in G2.

For a given amount of cell killing, heat alone (10-80 min, 45.5 degrees C) induced very few double-strand breaks (dsbs) compared with X-rays. Furthermore, 10 min at 45.5 degrees C immediately prior to X-rays caused only a 1.3-fold increase in the slope of the X-ray-induced dsb dose-response curve, i.e. 0.67 +/- 0.006 (95% confidence) dsbs/100Mbp/Gy for heated cells compared with 0.53 +/- 0.005 for unheated control cells. However, this same heat treatment caused > 5-fold inhibition in the rate of repair of dsbs induced by 60-Gy X-rays, with the degree of inhibition being much less in thermotolerant (TT) cells than in non-tolerant (NT) cells. This reduced inhibition of repair in TT cells correlated with the more rapid removal of excess nuclear protein from nuclei isolated from TT cells than from NT cells. These results plus a TT ratio of 2-3 for both heat-induced radiosensitization and heat-inhibition of repairing dsbs are consistent with the hypothesis that heat radiosensitization results primarily from heat aggregation of nuclear protein interfering with access of repair enzymes to DNA dsbs. The selective heat-radiosensitization of S-phase cells, however, may result from an increase in radiation-induced dsbs in or near replicating regions. For example, a preferential increase in dsbs in replicating DNA compared with bulk DNA was found following either hyperthermia alone (10-30 min, 45.5 degrees C) or a combined treatment (10 min, 45.5 degrees C before 60 Gy). A 30-min treatment at 45.5 degrees C induced dsbs equivalent to approximately 10 Gy in replicating DNA compared with 3-5 Gy in bulk DNA. When cells were heated immediately before irradiation, the increase in dsbs induced in the replicating DNA by 60 Gy was equivalent to 200 Gy. We hypothesize that the observed 2-fold increase in single-stranded regions in replicating DNA after heat resulted in radiation selectively inducing dsbs at or near the replication fork where the heat-induced increase in single-stranded DNA should occur. Thus, this preferential increase in dsbs in the replicating DNA by heat alone and especially when heat was combined with radiation may explain at least in part, the high sensitivity of S-phase cells to heat killing and heat radiosensitization.

Hyperthermic Radiosensitization of Thermotolerant Chinese Hamster Ovary Cells

The following four effects on DNA replication are observed in cells heated in S phase of the cell cycle: (1) inhibition of replicon initiation, (2) delay in DNA chain elongation into multicluster-sized molecules > 160S, (3) reduction in fork displacement rate, and (4) increase in single-stranded regions in replicating DNA. Since cells heated in S phase manifest chromosomal aberrations when they enter metaphase, whereas cells heated in G1 do not, we attempted to determine if the effects on DNA replication are critical for the induction of chromosomal aberrations by studying these same effects during DNA replication when synchronous CHO cells had been heated (10 min at 45.5 degrees C) in G1 phase. Following a heat-induced G1 block (12 h), we found previously that when the cells entered S phase, replicon initiation was functional and chain elongation into multicluster-sized molecules > 160S was delayed but completed during S phase. In the present study, we find that the fork displacement rate was near normal and that there was no increase in single-stranded DNA. Additionally, an increase in excess nuclear protein induced in the heated G1-phase cells returns to a normal level by about 12 h, just prior to when the cells enter S phase. Since the excess nuclear protein remains for many hours in heated S-phase cells, we hypothesize that the excess nuclear protein is responsible for the drastic reduction in the fork displacement rate and the associated increase in single-stranded DNA. Furthermore, we hypothesize that this persistent increase in single-stranded DNA during replication is a critical step for the induction of chromosomal aberrations in heated S-phase cells. Consistent with this hypothesis, we observed that aphidicolin (1-2 micrograms/ml) treatment of S-phase cells for 13-16 h, which results in a twofold increase in single-stranded DNA during the inhibition of DNA synthesis, also induces chromosomal aberrations. Possibly, endogenous endonucleolytic attack occurs opposite these sites of single-stranded DNA, thus creating double-strand breaks which either can remain unrepaired or are misrepaired to account for the chromatid breaks and exchanges, respectively, observed as cells complete their cell cycle and enter metaphase.

Recovery from Effects of Heat on DNA Synthesis in Chinese Hamster Ovary Cells

To determine where in the cell cycle Chinese hamster ovary cells die following heating in G1, a mild hyperthermia treatment, i.e., 10 or 11.5 min at 45.5 degrees C, resulting in 40-50% cell kill was used. After a 7-14-h delay in G1, the cells heated in G1 eventually entered S phase and replicated all their DNA. Both an autoradiographic analysis with tritiated thymidine and a bromodeoxyuridine-propidium iodide bivariate analysis by flow cytometry revealed that both clonogenic and nonclonogenic cells were delayed in progression through S phase for at least 4 h. Then they completed replication of all their DNA and entered G2. Alkaline sucrose gradient sedimentation analysis revealed that these heated cells could complete replicon elongation into cluster-sized molecules of 120-160 S which persisted for 2-12 h after heating. However, further replicon elongation into multicluster-sized molecules greater than 160 S required an additional 12 h in heated cells compared to the 4 h needed in unheated control cells. Our results when compared with the literature suggest that when G1 cells are heated to a survival level of about 50%, the nonclonogenic cells recover from a long delay in G1, traverse S at a reduced rate, and then die either in G2 or as multinucleated cells after an aberrant division.

DNA fork displacement rate measurements in heated Chinese hamster ovary cells

Synchronous G1 cells were given a priming dose of heat (45.5 degrees C for 15 min) and then heated and irradiated 6-120 h later. Compared to heat radiosensitization for cells irradiated 10 min after the priming heat dose (thermal enhancement ratio, TER of 2.6 for a 10-fold reduction in survival), heat radiosensitization 18-24 h after the priming heat dose was less (i.e., TER of 1.6 for radiation at 24 h compared with heat-radiation at 24 h). A thermotolerance ratio (TTR) at 24 h was calculated to be 2.6/1.6 = 1.6. TERs at 100-fold or 1000-fold reduction in survival and ratios of slopes of radiation survival curves also showed that the cells developed a similar amount of thermotolerance for heat radiosensitization at 18-24 h. Furthermore, since the TER for heat radiosensitization increased with heat killing either from the priming heat dose or the second heat dose in a similar manner for single or fractionated doses, the TER for nonthermotolerant and thermotolerant cells was the same when related to the heat damage (i.e., amount of killing from heat alone). When the radiation response of cells heated and irradiated 6-120 h after the priming heat dose was compared with the response of cells receiving radiation only, changes in TER as a function of time after the initial priming heat dose were shown to involve: recovery of heat damage interacting with the subsequent radiation dose, thermotolerance for heat radiosensitization, and redistribution of cells surviving the first heat dose into radioresistant phases of the cell cycle. In fact, redistribution resulted in a minimal TER at 72 h for heat-radiation compared with radiation alone, instead of at 24 h where maximal thermotolerance for heat killing was observed [P. K. Holahan and W. C. Dewey, Radiat. Res. 106, 111 (1986)]. These observations are discussed relative to clinical considerations and similar results reported from in vivo experiments.

Pulsed-field gel electrophoretic migration of DNA broken by X irradiation during DNA synthesis : Experimental results compared with Monte Carlo calculations

The hyperthermic inhibition of cellular DNA synthesis, i.e., reduction in replicon initiation and delay in DNA chain elongation, was previously postulated to be involved in the induction of chromosomal aberrations believed to be largely responsible for killing S-phase cells. Utilizing asynchronous Chinese hamster ovary cells heated for 15 min at 45.5 degrees C, an increase in single-stranded regions in replicating DNA (as measured by BND-cellulose chromatography) persisted in heated cells for as long as replicon initiation was affected. Alkaline sucrose gradient analyses of cells pulse-labeled immediately after heating with [3H]thymidine and subsequently chased at 37 degrees C revealed that these S-phase cells can eventually complete elongation of the replicons in operation at the time of heating, but required about six times as long relative to control cells which completed replicon elongation within 4 h. DNA chain elongation into multicluster-sized molecules was prevented for up to 18 h in these heated cells, resulting in a buildup of cluster-sized molecules (approximately 120-160 S) mainly because of the long-term heat damage to the replicon initiation process. Utilizing bromodeoxyuridine (BrdU)-propidium iodide bivariate analysis on a flow cytometer to measure cell progression, control cells pulsed with BrdU and chased in unlabeled medium progressed through S and G2M with cell division starting after 2 h of chase time. In contrast, the majority of the heated S-phase cells progressed slowly and remained blocked in S phase for about 18 h before cell division was observed after 24 h postheat. Our findings suggest that possible sites for where the chromosomal aberrations may be occurring in heated S-phase cells are either (1) at the persistent single-stranded DNA regions or (2) at the regions between clusters of replicons, because this long-term heat damage to the DNA replication process might lead to many opportunities for abnormal DNA and/or protein exchanges to occur at these two sites.

Glutathione depletion and cytotoxicity of buthionine sulphoximine and SR2508 in rodent and human cells.

DNA fork displacement rates (FDR) were measured in Chinese hamster ovary (CHO) cells heated at either 43.5 degrees C or 45.5 degrees C for various times. The inhibition of fork movement rate by heat was both time and temperature dependent, i.e., 10-20 min at 43.5 degrees C or 5 min at 45.5 degrees C was required to decrease the FDR to 20-30% of the control rate of 1 micron/min. Following heating, the reduced FDR was found to be constant for at least 75 min. The observed effects of heat on reduced rates of DNA replicon initiation and chain elongation and the increase in DNA with single-stranded regions could be explained by the heat sensitivity of the FDR. Any of these alterations in the DNA replication process may lead to many opportunities for abnormal DNA and/or protein interactions to occur which ultimately may lead to the observed formation of chromosomal aberrations.

Persistence of aneuploid immature/primitive hemopoietic sub-populations in mice 8 months after benzene exposure in vivo

Synchronous CHO cells were X-irradiated in G1 or mid-S phase with 30-750 Gy, and then the size distribution of DNA molecules resulting from DNA double-strand breaks (DSBs) was studied by pulsed-field gel electrophoresis (PFGE). Cells irradiated in S phase also were pulse-labeled with [3H]dThd for 15 min to compare the migration patterns of replicating DNA with those of DNA mass, measured by imaging with a CCD camera. When cells were irradiated immediately after pulse labeling, a large amount of the 3H-labeled replicating DNA was trapped in the plug, i.e. > 90% for doses < 100 Gy. As the dose increased, the percentage trapped decreased, i.e. to approximately 50% for 750 Gy. The same results were observed for DNA mass when cells were irradiated in S phase, except that much less of the DNA was trapped, i.e. approximately 60% for 70-100 Gy, which produced approximately 2-Mbp molecules, compared to approximately 10% for 750 Gy, which produced approximately 0.3-Mbp molecules. These results and the migration patterns of DNA released into the lane indicated that large molecules are trapped more readily than small molecules because they contain more replicating regions (bands with bubbles) of DNA than small molecules. Our interpretation is that as the dose increases, a greater fraction of the breaks occur between the replicating bands, thus releasing linear molecules that are not replicating. The relatively small amount of 3H-labeled replicating DNA that is released from the PFGE plug migrates aberrantly, with a small amount migrating like linear G1-phase molecules and a large amount, depending on dose, migrating much more slowly than the DNA mass from cells irradiated in G1 or S phase. To explain these results, a Monte Carlo computer program was written to introduce DSBs randomly into DNA that is configured according to a model of DNA replication that is developed in a related study (Dewey and Albright, Radiat. Res. 148, 421-434, 1997). In relating the experimental observations to the results of the Monte Carlo calculations, we assumed that (a) molecules containing replication bubbles with and without forks are trapped in the PFGE plug, (b) linear molecules and molecules with replication forks only that are < or = 8 Mbp are released into the lane, and (c) molecules having replication forks migrate more slowly than linear molecules.