Biomedical subjects

R L Warters

Publications and source records attributed to R L Warters.

At least 19 recordsLinked to original sources

PubMed1999

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 cell's DNA to the induction of DSBs.

Binding Sites↗

PubMed1997

Modulation of radiation-induced apoptosis by thiolamines.

Exposure to the thiolamine radioprotector N-(2-mercaptoethyl)-1,3-propanediamine (WR-1065) induced apoptosis in the mouse TB8.3 hybridoma after a 60-min (LD50 = 4.5 mM) or during a 20-h (LD50 = 0.15 mM) exposure. In contrast, a 20-h exposure to 17 mM L-cysteine or 10 mM cysteamine was required to induce 50% apoptosis within 20 h. Apoptosis was not induced by either a 60-min or 20-h exposure to 10 mM of the thiazolidine prodrugs ribose-cysteine (RibCys) or ribose-cysteamine (RibCyst). Thiolamine-induced apoptosis appeared to be a p53-independent process since it was induced by WR-1065 exposure in human HL60 cells. Exposure to WR-1065 (4 mM for 15 min) or cysteine (10 mM for 60 min) before and during irradiation protected cells against the induction of both DNA double-strand breaks and apoptosis, while exposure to RibCys (10 mM for 3 h) did not. Treatment with either WR-1065, cysteine, RibCys or RibCyst for 60 min beginning 60 min after irradiation did not affect the level of radiation-induced apoptosis. In contrast, treatment with either cysteine, cysteamine or RibCys for 20 h beginning 60 min after irradiation enhanced radiation-induced apoptosis. Similar experiments could not be conducted with WR-1065 because of its extreme toxicity. Our results indicate that thiolamine enhancement of radiation-induced apoptosis is not involved in their previously reported capacity to reduce radiation-induced mutations.

Animals↗

PubMed1997

Free radicals generated by ionizing radiation signal nuclear translocation of p53.

The p53 tumor suppressor is a transcription factor that regulates several pathways, which function collectively to maintain the integrity of the genome. Nuclear localization is critical for wild-type function. However, the signals that regulate subcellular localization of p53 have not been identified. Here, we examine the effect of ionizing radiation on the subcellular localization of p53 in two cell lines in which p53 is normally sequestered in the cytoplasm and found that ionizing radiation caused a biphasic translocation response. p53 entered the nucleus 1-2 h postirradiation (early response), subsequently emerged from the nucleus, and then again entered the nucleus 12-24 h after the cells had been irradiated (delayed response). These changes in subcellular localization could be completely blocked by the free radical scavenger, WR1065. By comparison, two DNA-damaging agents that do not generate free radicals, mitomycin C and doxorubicin, caused translocation only after 12-24 h of exposure to the drugs, and this effect could not be inhibited by WR1065. Hence, although all three DNA-damaging agents induced relocalization of p53 to the nucleus, only the translocation caused by radiation was sensitive to free radical scavenging. We suggest that the free radicals generated by ionizing radiation can signal p53 translocation to the nucleus.

Animals↗

PubMed1995

DNA double-strand break repair in two radiation-sensitive mouse mammary carcinoma cell lines.

The capacity of two radiation-sensitive clones (SX9 and SX10) of the mouse mammary carcinoma cell line SR1 to rejoin radiation-induced DNA double-strand breaks (DSBs) was determined by pulsed-field agarose gel electrophoresis. DSBs were produced with equivalent efficiency in all three cell lines. Both the SX9 and SX10 cell lines demonstrated a significantly diminished capacity to rejoin radiation-induced DSBs. The fraction of the original DNA DSB damage remaining in the DNA of 20 Gy-exposed SR1, SX9 and SX10 cells after 6 h of 37 degrees C incubation was estimated to be 14, 82 and 54%, respectively. In addition the SX10 cell line exhibited enhanced cytotoxicity when exposed to the DNA topoisomerase II poison mitoxantrone. The results indicate that both the SX9 and SX10 cell lines are DNA DSB repair mutants.

Animals↗

PubMed1995

Thiazolidine prodrugs of cysteamine and cysteine as radioprotective agents.

The need for protection against the toxic effects of ionizing radiation comes from many different directions: occupational exposure, nuclear accidents, environmental sources and protection of normal tissue during the therapeutic irradiation of cancer. Sulfhydryl-containing compounds, including cysteamine and L-cysteine, have long been known to possess radioprotective properties, but their therapeutic utility is limited by their side effects at radioprotective doses. To avoid this drawback, thiazolidine prodrugs of cysteamine and L-cysteine were prepared by the condensation of each thioalmine with the aldose monosaccharides, D-ribose and D-glucose, producing RibCyst, GlcCyst, RibCys and GlcCys. The prodrugs were designed to liberate the parent thiolamine nonenzymatically, after ring opening and hydrolysis, which is then available to function as a radioprotective agent. Cysteamine's inherent toxicity, measured using Chinese hamster V79 cells growing in culture, was completely eliminated, even at concentrations as high as 25 mM, by providing the thiolamine in the form of a prodrug. Good protection against radiation-induced lethality was demonstrated by the cysteamine prodrugs using a clonogenic assay. Protection against radiation-induced DNA single-strand breaks, as measured by alkaline elution, was also shown by both RibCyst and GlcCyst; this activity was higher than that exhibited by either cysteamine or WR-1065. The L-cysteine prodrugs, RibCys and GlcCys, also possessed radioprotective abilities under most of the conditions studied. Protection against DNA damage was comparable between L-cysteine, WR-1065 and RibCys.

Animals↗

PubMed1994

Heat sensitivity of HeLa S3 cell DNA topoisomerase II.

The sensitivity of HeLa DNA topoisomerase II to 45 degrees C heat shock was measured both in the intact cell and in vitro. In the intact cell, DNA topoisomerase II activity was estimated by measuring the formation and reversal of enzyme-DNA cleavable complexes by alkaline filter elution of cells exposed to the enzyme poison 4'-(9-acridinylamino)methanesulfon-m-anisidide). In vitro enzymatic activity was estimated by measuring changes in the topological state of plasmid and kinetoplast DNA produced by sonicates of nuclei from previously heated cells. The capacity of the enzyme to form, or reverse, enzyme-DNA cleavable complexes was inactivated during 45 degrees C heating with a reciprocal slope of 120 or 15 min, respectively. In vitro estimates of the activity of the enzyme from previously heated cells indicated that the enzyme was inactivated with a reciprocal slope of 99, 45, and 21 min after 45, 46 and 47 degrees C heating, respectively. DNA topoisomerase I activity was inactivated with a reciprocal slope of 130 min at 45 degrees C. The cumulative results indicate that during 45 degrees C heat shock, thermal inactivation of neither DNA topoisomerase I nor II is rate limiting for either cell survival or for DNA replication. While DNA topoisomerase II is resistant in situ to heat inactivation, in vivo assays indicate that the enzyme's capacity to function in the intact cell may be compromised by hyperthermic changes in the enzyme's environment.

Amsacrine↗

PubMed1993

Nuclear protein redistribution in heat-shocked cells.

An increase was observed in the total protein mass of nuclei isolated from Chinese hamster ovary cells heated at 45 degrees C or 45.5 degrees C. An increase in the fractional recovery of DNA polymerase alpha and beta, and of DNA topoisomerase activity coincided with this increase in the protein mass of nuclei from heated cells. Nuclear protein mass which was soluble in 2.0 M NaCl decreased 0.5 fold, while DNA-associated and nuclear matrix-associated protein mass increased 2.2 and 3.4 fold, respectively. The results indicate that the increase in nuclear protein mass observed in nuclei from heated cells is due in part to an increased binding, or precipitation, of nuclear proteins onto the cell's DNA and nuclear matrix.

Animals↗

PubMed1993

DNA-damage processing in a radiation-sensitive mouse cell line.

The induction and repair of radiation-induced DNA damage was assessed in 3 mouse cell lines, including the parental L cell line, a radiation-sensitive, SL3-147 mutant cell line and the H5 revertant to radiation resistance. The yield of neither radiation-induced DNA single- nor double-strand breaks could explain the variable sensitivity of the 3 cell lines. Closure of DNA single-strand breaks proceeded at a similar rate in both the L and SL3-147 cell lines. Closure of DNA double-strand breaks however was significantly slower and less complete in the SL3-147 cell line than in either of the radiation-resistant cell lines. The results are consistent with the increased radiation sensitivity of the SL3-147 cell line resulting from a defect in their ability to repair radiation-induced DNA double-strand breaks.

Animals↗

PubMed1993

Persistence of radiation-induced double-strand breaks in the DNA of heated CHO cells.

Chinese hamster ovary cells were either heated at 45 degrees C for 15 min or left unheated immediately prior to irradiation and incubation at 37 or 41 degrees C for 5 h. When cellular DNA was analysed by electrophoresis of double-stranded DNA through agarose gels 5 h after irradiation, DNA fragments presumably resulting from unrepaired DNA double-strand breaks (dsbs) were observed in the DNA of all cells. The frequency of the putative unrepaired dsbs was greater in cells heated at 45 degrees C for 15 min before, or incubated at 41 degrees C for 5 h after irradiation, than in unheated, control cells. Gel electrophoresis results were consistent with a failure of irradiated cells to rejoin dsb completely when heated at 45 degrees C before, or incubated at 41 degrees C for 5 h after irradiation. In contrast, nuclear DNA accessibility studies using either an exogenous or an endogenous endonuclease detected no change in the accessibility of DNA in nuclei from 41 degrees C-heated cells. These DNA accessibility studies indicate that the dsbs observed in the DNA of 41 degrees C-heated cells may not result from an actual failure of irradiated cells to repair radiation-induced dsbs during incubation at 41 degrees C.

Animals↗

PubMed1993

Makaluvamines, marine natural products, are active anti-cancer agents and DNA topo II inhibitors.

The makaluvamines were isolated from a sponge of the genus Zyzzya by following bioactivity against the human colon carcinoma cell line, HCT 116. These compounds have considerable cytotoxic activity. The makaluvamines appear to be acting through inhibition of DNA topoisomerase II. The compounds show enhanced toxicity toward a topoisomerase II-cleavable complex-sensitive cell line, they inhibit topoisomerase II decatenation of kinetoplast DNA in vitro. Makaluvamine C was shown to produce protein-linked DNA double-strand breaks, and makaluvamine A produced DNA double-strand breaks by neutral filter elution in a dose-dependent fashion similar to 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA). The makaluvamines also increased the life span of nude mice bearing solid tumors of human ovarian cancer cells.

Animals↗

PubMed1993

Induction and repair of double-strand breaks in the replicating DNA of HeLa cells.

The induction and closure of double-strand breaks produced by X rays were measured in the replicating DNA of HeLa S3 cells using the techniques of neutral (pH 7.2) filter elution and pulsed-field agarose gel electrophoresis. In whole cell DNA the apparent yield of double-strand breaks in pulse-labeled DNA was approximately half that observed in bulk DNA as estimated by both techniques. In contrast, when nuclear DNA was reduced to sub-replicon-cluster lengths prior to irradiation, the yield of radiation-induced double-strand breaks was the same in both replicating and bulk DNA. During incubation of pulse-labeled whole cells at 37 degrees C, the sensitivity of pulse-labeled DNA to strand break induction approached that observed in bulk DNA with a half-time of approximately 105 min. The results indicate that double-strand breaks are produced at a similar frequency per DNA mass in both replicating and bulk DNA. The structure of replicating DNA obscures length reduction in whole cell DNA when estimated by either filter elution or gel electrophoresis. Closure of double-strand breaks proceeded at a similar rate in both replicating and bulk DNA.

DNA↗

PubMed1992

Radiation-induced apoptosis in a murine T-cell hybridoma.

Induction of an apoptotic cell death was studied in a mouse T-cell hybridoma. Apoptosis was induced in these cells following exposure to dexamethasone, X-radiation, 43 degrees C heat shock, A254 light, and hydrogen peroxide. In 5-Gy-exposed cells, a radiation-induced G2 phase cell cycle progression block was maximum by 8 h. The cells began to escape this progression block by 10 h. Nuclear DNA fragmentation and uptake of the vital dye trypan blue began at 12 and 14 h, respectively, and were complete by 28 h. X-radiation-induced cell death was diminished when cells were irradiated in the presence of dimethyl sulfoxide, indicating that cell death was induced by oxidative cell damage. Substitution of nuclear DNA with bromodeoxyuridine enhanced death in cells exposed to either X-radiation or A310 light, indicating that apoptosis could be induced by DNA damage. The results are consistent with radiation-induced apoptosis being stimulated by oxidative DNA damage. DNA damage stimulates a long-lived signal which controls the expression of apoptosis. Apoptosis is expressed in the G1 phase of the cell cycle subsequent to the cell irradiation.

Animals↗

PubMed1992

Repair of DNA strand breaks at hyperthermic temperatures in Chinese hamster ovary cells.

The repair of DNA double-strand breaks was measured by pH 7.2 filter elution in cells incubated at 25-45 degrees C either before or after X-irradiation. Exposure to 45 degrees C for 15 minutes immediately prior to X-irradiation significantly increased both the half-time for DNA double-strand break closure and the number of DNA double-strand breaks remaining in nuclear DNA 180 minutes after irradiation. Exposure to temperatures between 41 and 45 degrees C immediately after X-irradiation accelerated DNA double-strand break closure and resulted in no increase in the number of DNA double-strand breaks remaining in the cell's genome 180 minutes after irradiation. The results indicate either that the radiosensitization produced by the administration of hyperthermic temperatures before and after irradiation result from two characteristically different molecular mechanisms, or that neither the rate of DNA strand break closure nor the number of DNA strand breaks remaining in nuclear DNA after irradiation accurately predict hyperthermic radiosensitization. These conclusions assume that no DNA strand breaks are below the resolution of this DNA damage assay and that a comparison between cytotoxicity and DNA repair after exposure to high radiation doses is valid.

Animals↗

PubMed1992

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.

Animals↗

PubMed1991

Topoisomerase II activity in a DNA double-strand break repair deficient Chinese hamster ovary cell line.

Topoisomerase II activity was measured in wild-type, Chinese hamster ovary K1 cells, and in the DNA double-strand break repair deficient xrs-6 cell line. Total topoisomerase II activity in a high salt, nuclear extract was found to be the same in both cell lines, as measured by decatenation of kinetoplast DNA networks and catenation of plasmid pBR322 DNA. While at low drug concentrations m-AMSA-induced enzyme cutting of nuclear DNA was 25% less in xrs-6 cells, the frequency of DNA breaks at high concentrations of the drug, and thus the frequency of the topoisomerase II enzyme, was the same in both cell lines. Despite the presence of equivalent enzyme levels in both cell lines, the xrs-6 cell line was 3 times more sensitive to drug-induced cytotoxicity. These results may be due to the fact that, as with X-radiation-induced DNA damage, xrs-6 cells are deficient in the capacity to rejoin topoisomerase II-induced DNA double-strand breaks.

Amsacrine↗

PubMed1991

Topoisomerase II activity in the replicating DNA of irradiated hamster cells.

The activity of DNA topoisomerase II in the replicating DNA of irradiated Chinese hamster ovary cells was estimated by determining protein-linked DNA double-strand breaks generated in the presence of the DNA intercalative drug 4'-(9-acridinylamino) methanesulfon-m-anisidide. In the presence of this drug, DNA double-strand breaks were produced at the same rate, and with the same overall frequency, in both the bulk and the newly synthesized DNA of control cells and cells irradiated with 10 Gy. The results indicate that DNA topoisomerase II is fully active in the replicating DNA of irradiated cells and is distributed at a frequency similar to that in parental DNA.

Animals↗

PubMed1990

Inhibition of replicon cluster ligation into chromosomal DNA at elevated temperatures.

The rate-limiting enzymatic step for DNA replication in HeLa cells incubated at 43.5 degrees C was the ligation of clusters of replicons into the cell's genome. At 43.5 degrees C the reciprocal slope for inhibition of DNA chain (replicon) initiation, or of the ligation of replicon clusters into the genome, was 18 or 7 min, respectively. The failure of replicon clusters to be ligated into chromosomal DNA was not a consequence of the failure of histone proteins to be deposited onto replicating DNA, or of chromatin replicated at 43.5 degrees C to be organized into fully condensed chromatin. In addition it was not due to the failure of fully active topoisomerase II to be deposited at a normal frequency along replicating chromatin DNA. The failure of replicon clusters to be ligated into the genome resulted in the persistence of single, but not double, DNA strand breaks in the cell's genome 24 hours after cell heating.

Amsacrine↗

PubMed1990

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.

Animals↗