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Biomedical subjects

R L Warters

Publications and source records attributed to R L Warters.

At least 55 records · Page 3Linked to original sources
PubMed1985

DNA damage repair in quiescent murine mammary carcinoma cells in culture.

Murine mammary carcinoma cells (line 67) were grown in unfed cultures for up to 9 days. In cultures (day 2-3) in which cells were proliferatively active and in day 3-5 (transition) cells, a large fraction of nuclear DNA was retained on polycarbonate filters when assayed by the alkaline filter elution technique. In contrast, the fraction of DNA retained on filters was significantly reduced for nonproliferating (Q, quiescent) cells from unfed 7-9 day cultures. The increase in endogenous DNA breaks followed both the decrease in proliferative state and clonogenicity in these cells. When day 7 Q cells were refed these endogenous DNA breaks were removed with a half-time of about 2.5 h. When the cells were exposed to X-irradiation and the integrity of their nuclear DNA measured by the alkaline filter elution assay, as much as a 2-fold greater frequency of radiation-induced DNA breaks was produced in Q versus P cells. DNA breaks were also removed from irradiated Q cells at a rate which was 0.23 that observed in P cells. We suggest that the depressed capacity for DNA damage removal in Q cells is responsible for their greater radiosensitivity, and the impaired DNA damage repair is probably due to a reduced level of energy sources in these unfed Q cell cultures.

Animals↗
PubMed1985

Fluorescence studies of Hoechst 33342 with supercoiled and relaxed plasmid pBR322 DNA.

The fluorescence properties of Hoechst 33342 (HO 33342) were examined with plasmid pBR322 in the supercoiled (Form I) or relaxed covalently closed circular (Form Io) conformation in order to determine whether qualitative or quantitative differences in fluorescence properties might provide an assay for topological states of DNA. It was found that HO 33342 exhibited a 30% greater fluorescence intensity with Form I pBR322, independent of the dye or DNA concentration. As the dye to DNA ratio was increased, a red shift of approximately 8 nm was observed for HO 33342 complexed with Form I or Form Io. The red shift in fluorescence emission occurred at higher HO 33342 concentrations with Form I vs. Form Io DNA; however, when Form I and Form Io were mixed in various proportions, neither the fluorescent intensity differences nor the HO 33342 concentration at which the wavelength shift occurred could be used to quantitate the relative proportions of topological states present. These results suggest that although the fluorescence properties of HO 33342 complexed with Form I DNA are different than those of HO 33342 complexed with Form Io DNA, the fluorescence assay is not sufficiently sensitive to quantitatively discriminate among a mixture of DNA in various topological states.

Benzimidazoles↗
PubMed1985

DNA damage production in CHO cells at elevated temperatures.

Induction of DNA lesions in the nucleus of Chinese hamster ovary (CHO) cells was observed at hyperthermic temperatures using the alkaline filter elution and the alkaline sucrose gradient sedimentation methods. These lesions were observed principally at temperatures greater than 45 degrees C with an activation energy of 140 kcal/mole. On alkaline sucrose gradients the cell genome was reduced to a 140 S or 2 X 10(8) dalton subunit of DNA independent of increasing exposure time at temperatures above 45 degrees C. The large thermal activation energy and the limited DNA size reduction suggest the possible involvement of thermal denaturation of a nuclear polypeptide in the production of these nuclear lesions.

Animals↗
PubMed1985

Cell lethality after selective irradiation of the DNA replication fork.

It has been suggested that nascent DNA located at the DNA replication fork may exhibit enhanced sensitivity to radiation damage. To evaluate this hypothesis, Chinese hamster ovary cells (CHO) were labeled with 125I-iododeoxyuridine (125IUdR) either in the presence or absence of aphidicolin. Aphidicolin (5 micrograms/ml) reduced cellular 125IUdR incorporation to 3-5% of the control value. The residual 125I incorporation appeared to be restricted to low molecular weight (sub-replicon sized) fragments of DNA which were more sensitive to micrococcal nuclease attack and less sensitive to high salt DNase I digestion than randomly labeled DNA. These findings suggest that DNA replicated in the presence of aphidicolin remains localized at the replication fork adjacent to the nuclear matrix. Based on these observations an attempt was made to compare the lethal consequences of 125I decays at the replication fork to that of 125I decays randomly distributed over the entire genome. Regardless of the distribution of decay events, all treatment groups exhibited identical dose-response curves (D0: 101 125I decays/cell). Since differential irradiation of the replication complex did not result in enhanced cell lethality, it can be concluded that neither the nascent DNA nor the protein components (replicative enzymes, nuclear protein matrix) associated with the DNA replication site constitute key radiosensitive targets within the cellular genome.

Animals↗
PubMed1985

Inhomogeneity of the nucleus to 125IUdR cytotoxicity.

Synchronized suspension cultures of Chinese hamster ovary (CHO) cells were used to determine the lethal effects produced by the decay of 125I incorporated into different subfractions of the nuclear genome. Such a shift in nuclear incorporation pattern was achieved by using the drug aphidicolin, which inhibits 95% of all nuclear DNA synthesis, is nontoxic to cells in a colony-forming assay, and does not modify the radiation response of CHO cells to X irradiation. In addition to shifting incorporation of 125I to only 5% of the nuclear genome, both nuclease digestions to characterize the molecular location of 125I and electron microscope autoradiography show an inhomogeneous distribution of sites of 125I incorporation in the presence of 5 micrograms/ml aphidicolin. These data in combination with survival curves of CHO cells labeled with 125I-iododeoxyuridine (125IUdR) either with or without aphidicolin showed a dramatic change in the survival response (DO: 30 decays/cell and 96 decays/cell, respectively). It is concluded, therefore, that the nucleus is not a homogeneous target for radiation-induced cell death because when subfractions of the nuclear genome are labeled, radically different levels in cell survival are obtained.

Animals↗
PubMed1984

Histone protein and DNA synthesis in HeLa cells after thermal shock.

Exposure of suspension-cultured HeLa cells to a 45 degrees thermal shock resulted in cell inactivation and inhibition of both protein and DNA synthesis. DNA synthesis was inhibited in a biphasic manner with a more sensitive (Do = 7 min) and a less sensitive (Do = 20 min) phase. The less sensitive process was demonstrated to be DNA chain elongation. Transport of thymidine into intracellular pools was significantly less sensitive to thermal shock (Do in excess of 200 min). When HeLa cells were heated at 45 degrees for 15 min there was an 80% inhibition of incorporation of precursors into both DNA and protein with little effect on precursor transport into cellular pools. While the rate of synthesis of whole cell and histone protein (H2a, H2b, H3, and H4) and DNA chain elongation recovered by 6 h after cell heating, total precursor incorporation into DNA was only 0.4 of control levels. The long-term depression of the DNA synthetic rate could not be explained by a cell cycle redistribution, a depression in the total fraction of S phase cells synthesizing DNA, or by a depression in the rate of DNA chain elongation. We conclude that thermal shock results in a long-term depression in the fraction of cell replicons involved in DNA replication.

DNA↗
PubMed1984

The sedimentation coefficient and buoyant density of nucleosomes from replicating chromatin in heated cells.

The sedimentation coefficient and buoyant density of mononucleosomes from the replicating chromatin of heated (45 degrees) and unheated HeLa cells were determined. It was observed that both physical parameters were the same (S = 10.8 and p = 1.165 g/cm3) for nucleosome particles from replicating and mature chromatin in heated and unbeated cells. The size of DNA in the nascent and mature nucleosome from heated or unheated cells was observed to be 145 base pairs of double-stranded DNA. A significant fraction of nucleosomal DNA from heated cells was observed at subnucleosomal sizes, principally at 125 base pairs of DNA. It is concluded that 45 degrees thermal shock does not alter appreciably the subunit structure of chromatin at the replication fork.

Centrifugation, Density Gradient↗
PubMed1983

Macromolecule synthesis in HeLa cells after thermal shock.

The incorporation of radioactivity into HeLa cell polypeptides and DNA after exposure to 45 degrees C heating for 15 min was measured by continuous exposure to radiolabeled precursors. Both polypeptide and DNA synthesis were inhibited by thermal shock. The rate of incorporation of radiolabeled amino acids into whole cell, nuclear, or histone protein recovered to control levels by 5 to 8 hr after thermal shock. The rate of incorporation of radiolabeled thymidine into DNA did not recover to a control level within the first 8 hr after thermal shock. Thermal effects on amino acid and nucleotide precursor pools could not explain the inhibition of either protein or DNA synthesis. Since histone protein synthesis recovers prior to DNA synthesis, we conclude that the inhibition of histone protein synthesis after thermal shock is not responsible for the depression in synthesis of cellular DNA.

DNA↗
PubMed1983

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.

Cell Division↗
PubMed1982

Heat protection by glycerol in vitro.

Heating of either Chinese hamster ovary or HeLa cells in medium containing glycerol protected against thermal killing. Above glycerol concentrations of 100 mM, protection of Chinese hamster ovary cells increased in a concentration-dependent manner. Exposure to glycerol only, before or after heating at 45 degrees, did not protect against cell killing. Glycerol protection against thermal damage was also expressed at the subcellular level. The fractional increase in the protein:DNA ratio for nuclei from heated HeLa cells (15 min, 48 degrees) was 1.6 with heating in 1 M glycerol, compared to 2.0 for medium controls. Both glycerol (1 M) and heat-induced thermotolerance (4 hr, 41.5 degrees) partially reversed the sensitizing effects of pH 6.4 and stepdown heating at 41.5 degrees. The partial deprivation of nutrients achieved by incubating cells in Hanks' balanced salt solution-sensitized Chinese hamster ovary cells against 45 degrees hyperthermia. Glycerol reversed this sensitization but only when nutrient deprivation was short term (75 min). With a long-term, 8.5-hr exposure to glycerol in Hanks' balanced salt solution, cells were significantly more sensitive to heat killing at 41.5 degrees than were cells heated for an equal period in Hanks' balanced salt solution alone. The similarities in the characteristics of glycerol protection and heat-induced thermotolerance suggest a common mechanistic basis for the two phenomena, although the ability of glycerol to act as a sensitizer in nutrient-deprived cells is not understood.

Animals↗
PubMed1982

DNA degradation in chinese hamster ovary cells after exposure to hyperthermia.

Chinese hamster ovary cells grown in suspension showed a progressive reduction in the size of their nuclear DNA to 50 to 60S fragments after hyperthermia (43-48 degrees). This DNA degradation was not a homogeneous response but was observed only in cells incapable of attaching to a substratum after acute heating. The DNA degradation was associated with the inability of cells to exclude the vital stain, trypan blue. The degradation process appeared to be a result of nucleolytic enzyme digestion which accompanies cell necrosis. A similar phenomenon was observed in heated monolayer cells but only after significantly greater time-temperature exposures. Our results show that cellular subpopulations can be separated after hyperthermia and that these subpopulations are biochemically distinct and characterized by different viability.

Animals↗