Biomedical subjects

K T Wheeler

Publications and source records attributed to K T Wheeler.

At least 37 records · Page 2Linked to original sources

PubMed1992

Effect of administration schedules on the potentiation of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) by misonidazole in subcutaneous 9L tumors.

Our previous studies demonstrated that metabolism of misonidazole (MISO) by hypoxic cells is required to potentiate the cytotoxicity of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in sc 9L tumors. To determine the influence of administration schedules on this chemosensitization, tumors were either clamped to produce a reversible hypoxia or left unclamped. MISO (2.5 mmoles kg-1) was administered to rats with unclamped tumors simultaneously with BCNU (9 or 12 mg kg-1), 20 min before BCNU, or 2.5 hr before BCNU, and the drug pharmacokinetics and BCNU cytotoxicity were measured. MISO administered 20 min or 2.5 hr before BCNU increased the plasma elimination half-time (t1/2) of BCNU, but MISO administered simultaneously with BCNU did not change the plasma elimination t1/2 of BCNU. In unclamped sc 9L tumors, all administration schedules decreased the peak BCNU concentration and increased the initial BCNU elimination t1/2; however, the BCNU exposure dose (AUC0-infinity) calculated from these data did not change significantly. In agreement with the AUC calculations, none of the administration schedules altered the BCNU cytotoxicity in unclamped tumors. If the tumors were clamped for 5-120 min after the peak MISO concentration was reached, BCNU-induced cell kill was increased by a constant factor of 3 over the first hour of the clamping period and by an additional factor of 7 over the second hour of the clamping period. If the tumors were clamped for 2 hr after the peak MISO concentration was reached and then BCNU administered 0-60 min after the clamp was released, this chemosensitization remained at a constant factor of approximately 20 for the first 10 min, and then decreased rapidly to a factor of approximately 3 by 20 min after the clamp was released. These data indicate that in sc 9L tumors, (1) at least two biochemical mechanisms are involved in this MISO-BCNU interaction, one of which depends on the duration and extent of the metabolism of MISO by hypoxic cells, and (2) reoxygenation does not immediately eliminate the potentiation of BCNU by MISO. These data also suggest that MISO should be given 2-4 hr before BCNU to achieve the maximum chemosensitization in clinical trials.

Animals↗

PubMed1992

Radiation-induced DNA damage as a function of hydration. I. Release of unaltered bases.

The release of unaltered bases from irradiated DNA, hydrated between 2.5 and 32.7 mol of water per mole of nucleotide (gamma), was investigated using HPLC. The objective of this study was to elucidate the yield of the four DNA bases as a function of dose, extent of hydration, and the presence or absence of oxygen. The increase in the yield of radiation-induced free bases was linear with dose up to 90 kGy, except for the DNA with gamma = 2.5, for which the increase was linear only to 10 kGy. The yield of free bases as a function of gamma was not constant in either the absence or the presence of oxygen over the range of hydration examined. For DNA with gamma between 2.5 and 15, the yield of free bases was nearly constant under nitrogen, but decreased under oxygen. However, for DNA with gamma greater than 15, the yield increased rapidly under both nitrogen and oxygen. The yield of free bases was described by a model that depended on two factors: 1) a change in the DNA conformation from a mixture of the A and C conformers in vacuum-dried DNA to predominantly the B conformer in the fully hydrated DNA, and 2) the proximity of the water molecules to the DNA. Irradiation of the inner water molecules (gamma less than 15) was less efficient than irradiation of the outer water molecules (gamma greater than 15), by a factor of approximately 3.3, in forming DNA lesions that resulted in the release of an unaltered base. This factor is similar to the previously published relative efficiency of 2.8 with which hydroxyl radicals and base cations induce DNA strand breaks. Our irradiation results are consistent with the hypothesis that the G value for the first 12-15 water molecules of the DNA hydration layer is the same as the G value for the form of DNA to which it is bound (i.e., the pseudo-C or the B form). Thus we suggest that the release of bases originating from irradiation of the hydration water is obtained predominantly: (1) by charge transfer from the direct ionization of the first 12-15 water molecules of the primary hydration layer and (2) by the attack of hydroxyl radicals generated in the outer, more loosely bound water molecules.

Animals↗

PubMed1991

2-Nitroimidazole potentiation of nitrosourea induced cytotoxicity in subcutaneous implants of rat 9L brain tumor cells.

To determine if the 2-nitroimidazole (2-NI) and the nitrosourea (NU) in a brain tumor chemopotentiation trial should be selected on the basis of known structure-activity relationships (electron affinity, lipophilicity, alkylating activity, carbamoylating activity), s.c. implants of rat 9L brain tumor cells were treated with combinations of misonidazole (MISO) or etanidazole (SR-2508) administered under oxic and hypoxic conditions, and BCNU, CCNU or chlorozotocin (CLZ) administered under oxic conditions. Cell kill was assessed by an in vivo to in vitro colony formation assay. To mimic the 'preincubation effect', the 2-NI was injected i.p., and 30 min later the tumor was clamped. After 2 hr, the clamp was released, and the NU administered immediately. MISO (2.5 mmole/kg) and SR-2508 (3.75 mmole/kg) reached the same peak tumor concentration in 30 min. Both 2-NIs were metabolized at the same rate in the clamped tumors; however, metabolism of the 2-NIs by hypoxic cells over the 2 hr clamping period did not produce any measurable s.c. 9L cell kill. The relative effectiveness of the NUs for killing oxic s.c. 9L tumor cells was: BCNU greater than CCNU greater than CLZ. Clamping the tumor prior to NU administration did not change the NU cytotoxicity. No potentiation of the NU cytotoxicity by the 2-NIs was observed in oxic tumors. Although metabolism of MISO by hypoxic cells did not result in potentiation of CLZ cytotoxicity at any dose, it resulted in potentiation of BCNU cytotoxicity at all doses and CCNU cytotoxicity at high doses.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Characterization of nuclear matrices prepared without salt extraction.

The structure and composition of the nuclear matrices prepared from a mouse mammary adenocarcinoma cell (line 66) by digestion with DNase I and several proteases (PRT-matrices) were characterized by protein and DNA gel electrophoresis, flow cytometry, and scanning electron microscopy. The characteristics of these PRT-matrices were compared with the characteristics of conventionally prepared nuclear matrices that employ a high salt extraction step (HS-matrices) in order to select a preparation that can be used in biochemical and/or biophysical studies where salt extraction compromises either the analysis or the interpretation of the data. Of the characterized PRT-matrices, only those prepared with Type XIV protease (pronase) had most of the characteristics of HS-matrices. They, (i) maintained their structural integrity, (ii) had less than or equal to 5% of their nuclear DNA associated with the matrix, (iii) had no evidence of higher-order chromatin structure, and (iv) had a DNA size distribution in the range of 400-1100 bp. The major difference between the PRT-matrices and the HS-matrices was a decrease in the protein content of the PRT-matrices. Although the PRT-matrices may not be appropriate for studying the unique nuclear matrix associated proteins that are involved in functions such as replication, transcription, and differentiation, they are clearly suitable for studying the properties of the nuclear matrix associated DNA.

Adenocarcinoma↗

PubMed1991

Influence of proliferation on DNA repair rates in liver.

To test the hypothesis that the proliferative status of a mammalian cell determines the rate of removal of oxidative DNA damage, pre- and posthepatectomized livers in adult male Fisher 344 rats were irradiated in situ with 15.5 Gy of 137Cs-gamma-rays. At 10 and 45 min after irradiation, the livers were removed and dissociated into single cell suspensions, and the DNA damage in the isolated quiescent or proliferative liver cells was assayed by alkaline elution. Proliferative liver cells irradiated 20-24 h or 29-31 h after hepatectomy repaired their DNA damage faster than quiescent liver cells. A corresponding increase in the accessibility of the DNA to digestion by m. nuclease was observed for the post-hepatectomized liver cells. These data suggest that proliferative status is a major determinant of the rate of DNA repair in rat liver.

Animals↗

PubMed1991

Pharmacokinetics and cytotoxicity of RSU-1069 in subcutaneous 9L tumours under oxic and hypoxic conditions.

The acute toxicity, pharmacokinetics and hypoxic cytotoxicity of RSU-1069 were investigated using the subcutaneous (sc) rat 9L tumour model. The pharmacokinetics were studied after i.p. injection of RSU-1069 (20 mg kg-1 or 100 mg kg-1). For both doses, the elimination of RSU-1069 followed first-order kinetics in both plasma and unclamped tumours. After 100 mg kg-1, the peak plasma concentration of RSU-1069 was 40 micrograms ml-1; the elimination t1/2 was 39.3 +/- 11.1 min. After 20 mg kg-1, the peak plasma concentration was 3 micrograms ml-1; the elimination t1/2 was 47.8 +/- 6.3 min. In unclamped tumours, the peak concentration was 50 micrograms g-1 with an elimination t1/2 of 36.1 +/- 9.6 min for the 100 mg kg-1 dose, and 4 micrograms g-1 with an elimination t1/2 of 41.9 +/- 6.1 min for the 20 mg kg-1 dose. The tumour and plasma elimination half-times were not significantly different (P greater than 0.2) for the two doses. Clamping the tumour 30 min after administration of 100 mg kg-1 of RSU-1069 decreased the tumour elimination t1/2 to 10.9 +/- 1.4 min. After releasing the clamp, RSU-1069 returned rapidly to the unclamped tumour concentration. The unclamped tumour/plasma ratio reached a maximum of 4-6, then decreased to a constant value of about 2 for both doses, indicating that RSU-1069 accumulates in these 9L tumours. RSU-1069 kills hypoxic sc 9L cells more efficiently than oxic sc 9L cells; at a surviving fraction of 0.5, the SER was 4.8. For in vitro 9L cells, the SER was approximately 50 when the comparison was between those treated in 2.1% 0(2) and those treated in less than 7.5 x 10(-3)% 0(2); it was approximately 100 when the comparison was between those treated in 21% 0(2) and those treated in less than 7.5 x 10(-3)% 0(3). Tumours treated with RSU-1069 and clamped for various times exhibited biphasic cell-kill kinetics; at 50 mg kg-1, little additional cell kill was achieved after 40 min of clamping. Our data also indicate that RSU-1069 is 300-1000 fold more efficient than misonidazole or SR2508 for killing hypoxic sc 9L tumour cells in situ.

Animals↗

PubMed1991

Saturation of DNA repair measured by alkaline elution.

To determine whether the half-times (T1/2) of the DNA repair processes measured by alkaline elution increased in a dose-dependent manner, exponentially growing 9L/Ro rat brain tumor cells were irradiated with doses of 15-50 Gy, and their DNA repair kinetics was measured by alkaline elution. At 15 Gy, the DNA repair kinetics was biphasic with the fast phase having a T1/2 approximately 6 min and the slow phase having a T1/2 approximately 42 min. As the dose was increased to 50 Gy, the fast-phase T1/2 remained at approximately 6 min, but the slow-phase T1/2 increased to approximately 87 min. Although a dose-dependent increase in the T1/2 of the slow phase of DNA repair (saturation) was measured by alkaline elution, both the absolute value of the slow-phase T1/2 and the dependency of the slow-phase T1/2 on dose were less than those measured by alkaline sucrose gradient sedimentation in zonal rotors with slow reorienting gradient capability. Thus these two techniques appear either to depend on different hydrodynamic properties of the DNA or to have different coefficients of dependency for the same hydrodynamic properties of the DNA. The lower sensitivity for detection of the dose dependency of DNA repair makes it unlikely that the alkaline elution technique will be useful for quantitatively relating the shape of mammalian cell survival curves to the doses at which saturation of a DNA repair process occurs.

Animals↗

PubMed1990

Radiation-induced cytotoxicity, DNA damage and DNA repair: implications for cell survival theory.

The radiosensitivities and the kinetics for removal of radiation-induced DNA damage were compared for proliferative (P) and quiescent (Q) cells of the lines 66 and 67 derived from a mouse mammary adenocarcinoma. As determined from cell survival assays, the 66 and 67 Q cells were more radiosensitive than their 66 and 67 P counterparts. The rank order of their radiosensitivity was: 67 Q greater than 66 Q greater than or equal to 67 P greater than 66 P. Induction of radiation damage in the DNA of these cells, as measured by the alkaline elution technique, was identical for 66 and 67 P and Q cells. The repair of this DNA damage was biphasic for 66 and 67 P and Q cells. The half-times for the fast and slow repair phases in 66 Q cells were identical to those previously measured in 67 Q cells. The half-times of the fast and slow repair phases in 66 P cells were also identical to those previously measured in 67 P cells. However, the half-times for the fast and slow repair phases in 66 and 67 Q cells were longer than those measured in their 66 and 67 P counterparts. The 66 cell data are consistent with our previously published hypothesis that Q cells are more radiosensitive than their corresponding P cells because they repair their radiation-induced DNA damage slower. However, our results are not consistent with hypotheses that attempt to explain the radiosensitivity differences between lines 66 and 67 solely on the basis of measurable induction and repair of DNA damage.

Animals↗

PubMed1990

Chemosensitization of the nitrosoureas by 2-nitroimidazoles in the subcutaneous 9L tumor model: pharmacokinetic and structure-activity considerations.

Alterations of the pharmacokinetics and cytotoxic effects of the nitrosoureas, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and 1-(2-chloroethyl)-3-(cyclohexyl)-1-nitrosourea (CCNU) by the 2-nitroimidazoles, misonidazole (MISO) and SR-2508 were investigated using the subcutaneous (sc) 9L tumor model in male Fisher 344 rats. When 50 mg/kg of CCNU was given i.p., the peak plasma concentration of CCNU was about 3 micrograms/ml. CCNU was eliminated with biphasic kinetics that had a terminal half-time (T1/2) of approximately 47 min. When 2.5 mmole/kg of MISO was given i.p. 150 min before CCNU, the peak plasma concentration of CCNU was increased by approximately 63% with no change in the elimination kinetics. Clamping did not change the pharmacokinetics of CCNU in either plasma or tumors. MISO pretreatment increased the peak CCNU concentration in unclamped tumors by 3-fold, but had no effect on the CCNU pharmacokinetics in clamped tumors. With the exception of a decrease in the peak BCNU concentration in tumors similar to that observed with MISO, SR-2508 (3.75 mmole/kg, i.p.) did not change the pharmacokinetics of BCNU or CCNU in plasma and tumors. CCNU had no effect on the MISO concentration in plasma and unclamped tumors. However, in the clamped tumors, CCNU delayed the return of the MISO concentration to the unclamped tumor level by about an additional 60 min after the clamp was released. SR-2508 was eliminated from the plasma with biphasic kinetics having an initial and terminal T1/2 of approximately 11 and approximately 76 min, respectively. SR-2508 reached a peak tumor concentration of about 500 micrograms/ml in 30 min. The elimination T1/2 for SR-2508 in unclamped and clamped tumors was approximately 81 and approximately 42 min, respectively. When the clamp was released, the SR-2508 concentration returned to the level found in the unclamped tumors approximately 90 min after it reached its nadir; BCNU and CCNU had no effect on the kinetics of this process. MISO significantly potentiated the cytotoxicity of BCNU in clamped tumors at surviving fractions less than or equal to 0.5. MISO did not potentiate the cytotoxicity of CCNU until the surviving fraction reached 0.05. SR-2508 did not potentiate the cytotoxicity of either BCNU or CCNU.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1990

Formation of alpha-deoxyadenosine in polydeoxynucleotides exposed to ionizing radiation under anoxic conditions.

When poly(dA), poly(dA-dT), and salmon testis DNA were gamma-irradiated under nitrogen, the major deoxyadenosine damage product (excluding liberated adenine) was identified as the alpha-anomer of deoxyadenosine. The yields of alpha-deoxyadenosine from poly(dA), poly(dA-dT), and salmon testis DNA irradiated with a dose of 500 Gy under anoxic conditions were 1.5, 1.3, and 1.3%, respectively. No alpha-deoxyadenosine was detected after irradiation under oxic conditions. The presence of nucleotides with the alpha-configuration at the anomeric carbon atom in the DNA chain may have a significant effect on its tertiary structure and possibly modify its biological activity.

Chromatography, High Pressure Liquid↗

PubMed1989

Ability of the alpha and beta anomers of chlorozotocin to kill rat 9L tumor cells in vitro.

Chlorozotocin (CLZ), a nitrosourea synthesized in the hope that it would have little bone marrow toxicity, has been shown to be effective against animal tumors and tumor cells in culture. However, the clinical results with CLZ have been disappointing. The original report on the synthesis of CLZ indicated that alpha and beta anomers at the D-glucose moiety should be expected, particularly when CLZ is placed in aqueous solution. In this study, the alpha and beta anomers have been separated by high-performance liquid chromatography and characterized by UV spectroscopy, mass spectroscopy, and nuclear magnetic resonance. The equilibration and decomposition of the anomers in various physiological solutions were determined as a function of temperature, pH, and serum concentration. In Eagle's basal medium (pH 7.2) held at 25 degrees C, CLZ decomposed with a t1/2 of approximately 82 min; at 37 degrees C with serum, CLZ decomposed with a t1/2 of less than 10 min. In these two cases, the beta:alpha ratio reached 1 in approximately 48 min and less than 5 min, respectively. The maximum beta:alpha ratio obtained in all cases ranged from 1.25 to 1.5. After holding CLZ in tissue culture medium and compensating for its decomposition, 9L rat brain tumor cells were treated in vitro with CLZ having different ratios of the alpha and beta anomers. These experiments demonstrated that the beta anomer has little, if any, ability to kill 9L cells. Thus, this anomerization phenomenon may have been responsible for the disappointing clinical results with CLZ. Our data suggest that appropriate preparation, handling, and drug delivery procedures might be devised to minimize this problem in both experimental and clinical situations.

Animals↗

PubMed1989

Organization of DNA in cerebellar neurons of ageing unirradiated and irradiated rats.

Male Fischer 344 rats were either unirradiated or whole-brain irradiated with single doses of 10.83 or 17.16 Gy of X-rays at 4 months of age, and the organization of the DNA in permanently non-dividing cerebellar neurons examined as a function of age, dose and time after irradiation. In unirradiated rats and rats receiving a whole-brain dose of 10.83 Gy, there were no statistically significant changes in the organization of the bulk DNA and its association with the nuclear matrix as determined by: (a) the sensitivity of the DNA to digestion by micrococcal nuclease, (b) the sensitivity of the nuclear matrix-associated DNA to digestion by DNase I, (c) the relative DNA and protein content of undigested neuronal nuclei, and (d) the relative amount of DNA and protein that is tightly associated with the nuclear matrix after digestion with DNase I. In rats that were irradiated with 17.16 Gy at 4 months of age, there was a gradual decrease in the amount of nuclear proteins as a function of age (P less than 0.003). The amount of protein associated with the nuclear matrix in these irradiated aging rats was also consistently lower than that of their unirradiated counterparts (P less than 0.03). This decrease in the nuclear protein content of the cerebellar neurons in aging rats irradiated with 17.16 Gy may have caused a change in the overall organization of their neuronal DNA. Such a change in the organization of their neuronal DNA was indicated by a higher stainability of their bulk DNA by propidium iodide (P less than 0.03) and a higher sensitivity of the bulk DNA to digestion by m. nuclease (P = 0.087). Although these organizational changes in the neuronal DNA of aging rats irradiated with 17.16 Gy at 4 months of age are subtle, they might alter DNA repair processes or other neuronal functions that may be associated with the "natural" process of aging.

Animals↗

PubMed1989

Biodistribution of misonidazole and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in rats bearing unclamped and clamped 9L subcutaneous tumors.

The biodistribution of misonidazole (MISO) and 1,3bis(2-chloroethyl)-1-nitrosourea (BCNU) was studied using the subcutaneous (s.c.) 9L tumor model in male Fisher 344 rats. A transient hypoxia in these tumors was created by clamping the blood supply to the tumor. Reoxygenation occurred upon release of the clamp. The plasma and tumor concentrations of MISO and BCNU were quantitated by high pressure liquid chromatography. When 12 mg/kg of BCNU was given i.p. without MISO, the peak plasma concentration was about 6 micrograms/ml, and the elimination half-time was about 16 min. When 2.5 mmole/kg of MISO was given i.p. 150 min before the BCNU, the peak plasma concentration of BCNU increased by approximately 33%, and the plasma elimination half-time increased by approximately 57%. Clamping the tumor for 120 min did not significantly change the BCNU concentration in plasma, but in tumors the time to reach the peak level was delayed slightly, and the peak concentration was reduced when compared to that in the unclamped tumors. MISO pretreatment decreased the BCNU peak concentration in both unclamped and clamped tumors, but the decrease was more pronounced in the unclamped tumors. In both unclamped and clamped tumors, the BCNU concentration and its rate of disappearance were identical about 30 min after BCNU administration, with or without MISO pretreatment. The elimination half-time of MISO from the plasma (approximately 142 min) was identical for rats with unclamped or clamped tumors. The half-time for the disappearance of MISO from unclamped tumors was about 98 min. BCNU had no effect on the MISO concentration in plasma and unclamped tumors. MISO disappeared in the clamped tumors with a half-time of about 40 min. When the clamp was released, the MISO concentration returned to the level in the unclamped tumors after about 45 min. BCNU delayed the return of the MISO concentration to the unclamped tumor level by about 60 min. Two conclusions can be drawn from this study. First, the pharmacokinetics of each drug changed when the two drugs were combined. Second, the data indicate that alterations in the tumor BCNU pharmacokinetics are not the major mechanism responsible for the chemopotentiation previously measured in s.c. 9L tumors.

Animals↗

PubMed1989

Alterations of neuronal nuclear matrix and chromatin structure after irradiation under aerobic and anoxic conditions.

This study was undertaken to determine if structural alterations of the bulk chromatin and the amount of protein associated with the nuclear matrix in cerebellar neurons depend on radiation dose and a cell's state of oxygenation. After irradiation with 2.5 to 25.0 Gy under both aerobic and anoxic conditions, the sensitivity of the neuronal chromatin to m. nuclease digestion increase linearly with dose up to about 5 Gy, beyond which there was no further increase. The same increase in accessibility of chromatin to micrococcal nuclease digestion was observed when neuronal nuclei were irradiated at 4 degrees C. Neuronal nuclei were stained with propidium iodide (PI) for DNA and with fluorescein isothiocyanate (FITC) for protein, both before and after complete digestion with DNase I, and analyzed by flow cytometry. There was no change in either the PI (P greater than 0.4) or the FITC (P greater than 0.9) fluorescence of undigested nuclei after irradiation. For the DNase I digested nuclei, the PI fluorescence was unchanged after irradiation (P greater than 0.4), but the FITC fluorescence increased significantly (P less than 0.02). This increase in the FITC fluorescence was linear with dose up to about 5 Gy, beyond which there was no further increase. The flow cytometry results from DNase I digested nuclei were identical for neurons irradiated under aerobic or anoxic conditions, indicating that this phenomenon is oxygen independent. This increase in FITC fluorescence after irradiation was inhibited at ice-cold temperatures and probably reflects an increase in protein content at the nuclear matrix that requires metabolism. This may explain our previously observed resistance of nuclear matrix-associated DNA to digestion by DNase I. This protein increase at the nuclear matrix appears to follow "saturation" kinetics identical to that previously reported for repair of DNA strand breaks in cerebellar neurons. However, the exact molecular nature of this process and its role in DNA repair or cell survival remains to be determined.

Aerobiosis↗

PubMed1988

Regulation of DNA repair kinetics in proliferative and quiescent tumor cells.

The radiosensitivity and kinetics of repair of radiation-induced DNA damage were determined for proliferative (P) and quiescent (Q) cells of the mouse mammary adenocarcinoma line 67. 67 Q cells are more radiosensitive than 67 P cells. Radiation induced the same amount of DNA damage in both 67 P and 67 Q cells. Both 67 P and 67 Q cells repaired their DNA damage with biphasic kinetics, but the half-times for the fast and slow phase were longer in 67 Q cells. Q cell DNA appeared to be in a more compact or condensed chromatin structure and was less accessible to enzymatic digestion than P cell DNA. These data suggest that 67 Q cells are more sensitive to ionizing radiation than 67 P cells because they repair their radiation-induced DNA damage more slowly, perhaps as a result of their more condensed chromatin structure.

Adenocarcinoma↗

PubMed1988

Postirradiation alterations of neuronal chromatin structure.

Previous work from our laboratory suggested that neuronal chromatin structure may be altered immediately after exposure to ionizing radiation. In the present study, whole brains of 4-month-old male Fisher 344 rats were irradiated with a dose of 25 Gy. The kinetics of restoring the chromatin structure to its unirradiated state was investigated in rat cerebellar neurons using three different approaches: (1) measurement of changes in the DNA superhelical structure by the fluorescent halo assay, (2) measurement of changes in chromatin accessibility to digestion by micrococcal nuclease, and (3) measurement of changes in the accessibility of the nuclear-matrix-associated DNA to digestion by DNase I. Immediately after irradiation, the topological constraints on the DNA loops were altered, the chromatin was more accessible to m. nuclease digestion, and the DNA associated with the nuclear matrix was more resistant to digestion by DNase I. Return of the chromatin structure to its unirradiated state as measured by each of the three methods followed biphasic kinetics with the fast phase having a half-time of several minutes and the slow phase having a half-time of several hours. The kinetics are similar to that previously reported for repair of radiation-induced DNA damage in mammalian cells. Although the independent assays used in this study seemed to follow the same kinetics, their relationship at the molecular level remains to be determined.

Animals↗

PubMed1987

Saturation of a DNA repair process in dividing and nondividing mammalian cells.

We have reported that the half-time (T1/2) for the slow phase of repair of radiation-induced DNA lesions increased with dose from 12.5 to 50.0 Gy in both 9L rat brain tumor cells and cerebellar neurons. In this manuscript these studies have been extended to lower doses to determine at what dose this DNA repair process becomes unsaturated. Our alkaline sucrose sedimentation technique in zonal rotors limits determination of the slow phase T1/2 to doses of greater than or equal to 6 Gy for 9L tumor cells and greater than or equal to 4 Gy for cerebellar neurons. The slow phase T1/2 in cerebellar neurons appears constant at doses less than or equal to 6 Gy and then increases exponentially at higher doses; survival of whole brain irradiated rats does not begin to decrease until doses greater than 6 Gy. The slow phase T1/2 in 9L tumor cells is either saturated or just becoming saturated at 6 Gy; a dose just before the final slope of the 9L survival curve is reached. These data support the hypothesis that the shape of mammalian cell survival curves is related to the saturation of a DNA repair process.

Animals↗

PubMed1985

Relationship between DNA repair and cell recovery: importance of competing biochemical and metabolic processes.

The relationship between the inhibition of repair of radiation-induced DNA damage and the inhibition of recovery from radiation-induced potentially lethal damage (PLD) by hypertonic treatment was compared in 9L/Ro rat brain tumor cells. Fed plateau phase cultures were gamma-irradiated with 1500 rad and then immediately treated for 20 min with a 37 degree C isotonic (0.15 M) or hypertonic (0.50 M) salt solution. The kinetics of repair of radiation-induced DNA damage as assayed using alkaline filter elution were compared to those of recovery from radiation-induced PLD as assayed by colony formation. Hypertonic treatment of unirradiated cells produced neither DNA damage nor cell kill. Post-irradiation hypertonic treatment inhibited both DNA repair and PLD recovery, while post-irradiation isotonic treatment inhibited neither phenomenon. However, by 2 h after irradiation, the amount of DNA damage remaining after a 20 min hypertonic treatment was equivalent to that remaining after a 20 min isotonic treatment. In contrast, cell survival after hypertonic treatment remained 2 logs lower than after isotonic treatment even at times up to 24 h. These results suggest that the repair of radiation-induced DNA damage per se is not causally related to recovery from radiation-induced PLD. However, the data are consistent with the time of DNA repair as an important parameter in determining cell survival and, therefore, tend to support the hypothesis that imbalances in sets of competing biochemical or metabolic processes determine survival rather than the presence of a single class of unrepaired DNA lesions.

Animals↗