International Journal of Radiation Biology
ISSN: 0955-3002 (Print) 1362-3095 (Online) Journal homepage: http://www.tandfonline.com/loi/irab20
2-Deoxy-d-glucose Inhibits Rejoining of Radiationinduced DNA Double-strand Breaks in Yeast M. Frankenberg-Schwager, R. Harbich, D. Frankenberg & V. Jain To cite this article: M. Frankenberg-Schwager, R. Harbich, D. Frankenberg & V. Jain (1992) 2Deoxy-d-glucose Inhibits Rejoining of Radiation-induced DNA Double-strand Breaks in Yeast, International Journal of Radiation Biology, 61:2, 185-190, DOI: 10.1080/09553009214550801 To link to this article: http://dx.doi.org/10.1080/09553009214550801
Published online: 03 Jul 2009.
Submit your article to this journal
Article views: 8
View related articles
Citing articles: 1 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=irab20 Download by: [UQ Library]
Date: 26 April 2016, At: 04:02
INT . J . RADIAT . BIOL .,
1992,
VOL .
61,
NO .
2, 1 8 5-190
2-Deoxy-D-glucose inhibits rejoining of radiation-induced DNA double-strand breaks in yeast M. FRANKENBERG-SCHWAGER j- , R. HARBICHt, D . FRANKENBERGt and V. JAIN*$
Downloaded by [UQ Library] at 04:02 26 April 2016
(Received 22 February 1991; revision received 24 May 1991; accepted 24 June 1991)
Abstract. Effects of 2-deoxy-D-glucose (2-DG) on radiationinduced DNA double-strand breaks (dsb) have been studied under non-growth conditions in a respiratory-deficient strain of the yeast Saccharomyces cerevisiae. Velocity sedimentation in neutral sucrose gradients was used to measure DNA dsb . Addition of 2-DG to the liquid-holding medium (67 mm phosphate buffer, pH 5, 30° C) at an equimolar concentration with glucose (50 mm) reduced the rate and extent of dsb rejoining . The inhibition of rejoining mediated by 2-DG is reversible for the majority-but not all-of the . radiation-induced dsb .
1. Introduction Cellular repair processes are important determinants of radiation sensitivity . 2-deoxy-D-glucose (2DG), has been shown in yeast to inhibit repair of potentially lethal damage (PLD) induced by ionizing (X-rays, y-rays, fast electrons) and non-ionizing (UVC, psoralen + UVA) radiation (Jain et al. 1973, 1976, 1977a, 1982, Holahan et al. 1988) . Inhibition of PLD repair in the presence of 2-DG has also been observed in X-irradiated Ehrlich ascites tumour cells of mouse (Jain et al. 1977b) . The mechanisms underlying 2-DG-induced inhibition of PLD repair are complex and not yet completely understood . 2-DG is known to strongly inhibit the glycolytic pathway at various stages, such as glucose transport, phosphorylation and isomerization (Woodward 1952, Wick et al. 1957, Heredia et al. 1964, Gupta et al. 1981), whereas the respiratory pathway is affected only to a small extent (Jain et al . 1982) . Thus, under respiratory-deficient conditions, the presence of 2DG leads to a considerable reduction in the rate of ATP production via the glycolytic pathway and thereby to a decrease of the cellular contents of high*Author for correspondence . t Institut fur Biophysikalische Strahlenforschung, Gesellschaft fur Strahlen-und Umweltforschung mbH, Paul-EhrlichStr. 20 D-6000 Frankfurt 70, Germany. $ Institute of Nuclear Medicine & Allied Sciences, Lucknow Marg, Delhi 110054, India .
energy phosphates such as ATP, GTP and UTP (Jain et al . 1973, 1982, Dwarakanath and Jain 1991) . Degradation of adenine nucleotides via deamination of AMP observed in the presence of 2-DG (Holmsen et al. 1974, Matsumoto et al. 1979) could lead to further perturbations in the cellular nucleotide pools. Under respiratory-proficient conditions the energy deficiency produced by the presence of 2DG is small since sufficient ATP can be generated by oxidative phosphorylation (Jain et al. 1985) . Thus, ten times higher concentrations of 2-DG were required to inhibit repair of PLD in wild-type yeast as compared to respiratory-deficient mutants (Jain et al. 1973) . An adequate supply of metabolic energy derived from the glycolytic and/or the respiratory pathway is necessary for the cellular repair of PLD (Jain and Pohlit 1972) . Since DNA is an important molecular target for radiation-induced PLD, it was suggested, as a working hypothesis, that reduction of cellular energy supply below a certain critical threshold induced by the presence of 2-DG could possibly inhibit repair of radiation-induced DNA lesions (Jain et al. 1973, 1982) . Postirradiation repair of DNA lesions assayed by the unscheduled DNA synthesis technique has indeed been observed to be inhibited by the presence of 2-DG in yeast (Jain et al. 1977a) and human cancer cells (Verma et al. 1982, Dwarakanath and Jain 1989) under conditions of respiratory deficiency . Lowering of cellular ATP levels, however, is not correlated in a simple manner with the inhibition of 2-DG-induced cellular and DNA repair (Jain et al. 1982, Verma et al. 1982) . Unscheduled DNA synthesis is supposed to monitor mainly the repair of DNA base damage and singlestrand breaks (ssb) via the excision repair pathway . In cases of ionizing radiations DNA double-strand breaks (dsb) are considered to be the most important DNA lesions responsible for chromosomal aberrations (Bender et al. 1974, Natarajan et al. 1980, Bryant 1984), mutations and cell death (Dewey et al. 1970, Dugle et al . 1976, Thacker 1986, Ho 1975, Resnick and Martin 1976, Frankenberg et al. 1981, Blucher and Pohlit 1982) . Furthermore, in yeast,
0020-7616/92 $3 .00 © 1992 Taylor & Francis Ltd
186
M . Frankenberg-Schwager et al .
repair of PLD induced by ionizing radiation has been demonstrated to be due to the rejoining of dsb (Frankenberg-Schwager et al . 1980, 1985, 1987) . Therefore, in the present work we investigated, in a respiratory-deficient yeast strain, the effects of 2-DG on the rejoining of radiation-induced dsb during incubation of cells in non-growth medium .
2. Materials and methods
Downloaded by [UQ Library] at 04:02 26 April 2016
2 .1 . Yeast cells A respiratory-deficient mutant strain 211 *B (ATCC 42607) of Saccharomyces cerevisiae auxotrophic for 2'-deoxy-thymidine 5'-monophosphate (5'dTMP) was used . This strain is capable of repairing dsb (Frankenberg-Schwager et al . 1980), it lacks mitochondrial DNA and its nuclear DNA can be specifically labelled with (3H)dTMP (Brendel and Fath 1974) .
2 .2. Growth of cells and labelling of DNA Cells were grown overnight at 30°C in a nutrient medium containing 4% glucose, 1 .3% yeast nitrogen base without amino acids, 0 .4% casamino acids supplemented with 7µg/ml 5'-dTMP (Boehringer, Mannheim) . After appropriate dilution into fresh medium, (methyl-3-H)dTMP (Amersham) was added to give 148 kBq/ml . The culture was rotated at 30°C until cells had reached the stationary phase .
2 .3 . Irradiation conditions Stationary cells were washed twice in phosphate buffer (pH 7 .0) and irradiated with 30 MeV electrons (Betatron) under continuous stirring at 4°C (dose-rate : 120 Gy/min) . The dosimetry was performed with an energy-independent ferrous sulphate dosimeter (Frankenberg, 1969) .
al . 1980) . In parallel experiments cells were resus-
pended immediately after irradiation in non-growth medium containing a mixture of glucose and 2deoxy-D-glucose (2-DG) at equimolar concentrations of 50 mm. Samples were held in this medium for 24 h (most experiments) or 48 h (one experiment, see Figure 1) to assess the effect of 2-DG on the rejoining of dsb. In another set of experiments 2-DG was washed out after a 24 h treatment, cells were resuspended in 2-DG-free non-growth medium and allowance for dsb rejoining was made by incubating cells for another 24 or 48 h at 30°C . After these intervals of time samples were taken for the measurement of dsb . These latter experiments were performed to test whether the effect of 2-DG on dsb rejoining is reversible .
2 .5. Measurement of DNA double-strand breaks Sedimentation of DNA in neutral sucrose gradients was used to measure dsb . Methods have been described in detail earlier (Frankenberg-Schwager et al . 1979) . Briefly, cell protoplasts were prepared and lysed on top of neutral sucrose gradients (5-20%) containing 0 . 1 M NaCl and 0 .02 M EDTA (pH 7 .0) in 13 ml cellulose nitrate tubes . The released DNA was sedimented at about 9000 rpm in an ultracentrifuge (Beckmann) at 20°C using the rotor SW 40 Ti . After centrifugation for about 21 h, gradients were fractionated onto glass-fibre filters . The filters were dried and the high molecular weight DNA precipitated with ice-cold 6% TCA, washed with ethanol and dried again . Radioactivity was counted in a liquid scintillation counter using a toluene-based cocktail . The number of radiation-induced dsb per average molar mass of DNA was calculated from the DNA profiles, i .e . from the distribution curves of the radioactivity as a function of the sedimented volume as described in detail earlier (FrankenbergSchwager et al. 1979, Frankenberg et al . 1981) . Briefly, the average number of dsb per average molar mass of DNA was calculated by computer simulation assuming random breakage of the DNA and by fitting these curves of the DNA profiles obtained from irradiated cells .
2 .4 . Postirradiation treatment of cells
After irradiation, cells were resuspended in nongrowth medium, consisting of phosphate buffer (67 mm, pH 5 .0) supplemented with 5'-dTMP (7µg/ml) and containing 100 mm glucose and incubated at 30°C. Under these conditions the respiratory-deficient strain 211 *B exhibits both PLD repair and repair of dsb (Frankenberg-Schwager et
3. Results 3 .1 . DNA sedimentation profiles DNA profiles from irradiated yeast cells (absorbed dose D = 1500 Gy) were obtained after liquid hold-
Inhibition of rejoining of DNA double-strand breaks
Downloaded by [UQ Library] at 04:02 26 April 2016
ing of cells in non-growth medium at 30 °C in the absence or presence of 2-DG (representative profiles are shown in Figure 1) . It was observed that after 48 h of incubation in the absence of 2-DG, the DNA sedimentation profile of irradiated samples shifts towards higher molecular DNA and does not differ very much from the unirradiated controls . This suggests that most of the dsb have been rejoined during this time . In contrast, the sample incubated in non-growth medium containing 2-DG retained a DNA sedimentation profile with a smaller shift towards the control, indicating inhibition of repair processes . It should be noted that even after a 48 h incubation period of irradiated cells in the presence of 2-DG no loss of cells was observed as judged by the same total radioactivity recovered from each sucrose gradient .
3.2 . The effect of 2-deoxy-D-glucose on the rejoining of double-strand breaks
The presence of 2-DG during a 24 h incubation results in the reduction of the rate of rejoining of
187
radiation-induced dsb in yeast, and rejoining seems to level off after this time (Figure 2) . After longer treatments with 2-DG the determination of dsb was difficult because most DNA profiles could not be fitted well after a 48 h treatment with 2-DG, and were therefore excluded from the quantitative evaluation. This effect was even more severe after a 72 h treatment with 2-DG, and therefore no determination of double-strand breakage was attempted . In order to test whether the 2-DG-mediated inhibition of dsb rejoining is reversible, 2-DG was washed out after 24 h of postirradiation treatment and cells were reincubated in 2-DG-free non-growth medium for another 48 h . During this treatment, samples were taken for the determination of dsb . The number of dsb remaining unrejoined after such a 2-DG treatment was, however, somewhat higher (significant for p=0 .1) than in cells which had not been treated with 2-DG (Figure 2) . This seems to indicate that the rejoining of some dsb may be irreversibly inhibited (i .e. `fixed') by 2-DG . To investigate this further we tried to enhance the possible yield of `fixed' dsb . For this purpose irradiated cells were incubated for 24 h solely in the presence of 2-DG (50 mm), i .e . without glucose . This
Figure 1 . DNA profiles obtained from cells immediately after irradiation with an absorbed dose of 1500 Gy (A) and after 48 h of liquid holding in nongrowth medium in the absence (0) and presence ( •) of 2-deoxy-D-glucose . The DNA profiles are representatives of a single experiment . The percentage of total radioactivity of DNA per fraction (A/A,) is plotted as a function of the fraction number. The direction of sedimentation is from left to right .
1 88
M. Frankenberg-Schwager et al .
N mil
4. Discussion
Downloaded by [UQ Library] at 04:02 26 April 2016
109Rg~mole
T
r
I 0
10
20
30
20
~0
60
70 hme/h
Figure 2 . Kinetics of rejoining of double-strand breaks observed in irradiated yeast (D=1500 Gy) during postirradiation incubation in normal non-growth medium (containing 100 mm glucose) (0) for 0 h (five experiments), 1 h (four experiments), 3 h (two experiments), 16h (two experiments), 24h (seven experiments), 48h (five experiments) and 72h (four experiments) . Rejoining was also measured after postirradiation incubation of cells in non-growth medium containing 2-deoxy-Dglucose (2-DG) and glucose at equimolar concentrations (50 mM) for 24h (14 experiments) and for 48h (one experiment, the profile of which is shown in Figure 1) (0) . Cells treated with 2-DG for 24h were washed and incubated in normal 2-DG-free nongrowth medium for another 24h (13 experiments) or 48h (8 experiments) (0) . Average values and their standard deviations are shown . N • m~ 1 : Average number of dsb (N) per relative molar mass of DNA (Mr)-
treatment should result in a greater number of `fixed' dsb which would not be rejoinable during subsequent incubation of cells in 2-DG-free medium . However, treatment of unirradiated cells with 2-DG alone or at higher than equimolar concentrations with glucose resulted in severe distortions of the DNA profiles (not shown) . In contrast, normal DNA profiles are observed for cells incubated for 24 h in non-growth medium containing only glucose or 2-DG and glucose at equimolar concentrations (50 mm) .
The results of the present studies show that rejoining of DNA dsb is inhibited in respiratorydeficient yeast cells incubated in non-growth medium in the presence of 2-DG (Figures 1 and 2) . Recombination processes between homologous chromosomes or sister chromatids, which have been implicated in the repair of DNA dsb in yeast (Resnick 1976), involve the coordinated functioning of many enzymes such as endo- and exonucleases, DNA polymerases and polynucleotide ligase . Activities of these enzymes, being energy-dependent, may be inhibited by a reduction in ATP supply and subsequent decrease in ATP concentration induced by the presence of 2-DG (Jain et al . 1973, 1982) . The excision repair pathway involved in the repair of DNA base damage and ssb also depends upon the proper functioning of similar types of enzymes, and has been observed to be inhibited by 2-DG in our earlier studies using the same strain of yeast (Jain et al. 1977a) . Similar observations have been made in cancer cells derived from human patients (Verma et al. 1982, Jain et al . 1985, Dwarakanath and Jain 1989) . Thus, it appears that both the excision and recombination repair pathways can be inhibited by 2-DG in cell systems that are highly dependent upon glycolysis for their energy supply . The kinetics of rejoining of DNA dsb induced by 30 MeV electrons has been shown to be biphasic in yeast held in nongrowth medium . A fast (t 112 =3 .8h) and a slow (4/2=10-11h) component could be distinguished (Frankenberg-Schwager et al. 1990) . In the present study a 2-DG-induced inhibition of dsb rejoining of about 45% was observed after the first 24h in cells irradiated with a dose of 1500 Gy . This inhibition could be due to a reduction of the rate of the fast and/or slow component . Further studies are required to clarify which of the repair components is more susceptible to inhibition by 2-DG . Figure 2 also indicates that the 2-DG-mediated inhibition of rejoining of the majority-but not allof the radiation-induced dsb is reversible under the present experimental conditions . Our previous experiments with a human cerebral glioma cell line have demonstrated that 2-DG-mediated inhibition of unscheduled DNA synthesis after irradiation could be reversed after removal of 2-DG under conditions of respiratory proficiency . In the absence of respiration, however, irreversible inhibition of DNA repair was observed (Dwarakanath and Jain 1989) . Experiments to demonstrate a possible irreversible 2-DG-mediated inhibition of rejoining of at least some of the dsb in yeast failed because a severe
Inhibition of rejoining of DNA double-strand breaks
Downloaded by [UQ Library] at 04:02 26 April 2016
distortion of the DNA profile was observed even in unirradiated cells incubated in non-growth medium in the presence of 2-DG (50 mm) alone or in combination at higher than equimolar concentrations with glucose (2-DG/G=2 .5 or 5 .0) . The distorted DNA profiles are flat and demonstrate an increase in the fraction of small as well as large DNA molecules . The reasons for this 2-DG-mediated fragmentation and enlargement of DNA molecules are not clear. It appears unlikely that depletion of the energy source may lead to this effect, since postirradiation incubation of these cells for 24 h in non-growth medium lacking glucose yielded undistorted DNA profiles (Frankenberg-Schwager et al . 1980) . Further studies to understand these phenomena are necessary .
Acknowledgement The support of the medical-biological collaboration with India by the BMFT is acknowledged .
References BENDER, M . A ., GRIGGS, H. G . and BEDFORD, J. S ., 1974, Mechanisms of chromosomal aberration production . III . Chemicals and ionizing radiation . Mutation Research, 23, 197-212 . BLOCHER, D . and POHLIT, W., 1982, DNA double strand breaks in Ehrlich ascites tumor cells at low doses of X-rays, II . Can all death be attributed to double strand breaks? International Journal of Radiation Biology, 42, 329-338. BRENDEL, M . and FATH, F . W., 1974, Isolation and characterization of mutants of Saccharomyces cerevisiae auxotrophic for 5'-dTMP. Zeitschrift fur Naturforschung, 29c, 733-738 . BRYANT, P . E ., 1984, Enzymatic restriction of mammalian cell DNA using Pvu II and Bam HI : evidence for the double-strand break origin of chromosomal aberrations . International Journal of Radiation Biology, 46, 57-65 . DEWEY, W . C ., FURMAN, S . C . and MILLER, H . H ., 1970, Comparison of lethality and chromosomal damages induced by X-rays in synchronized Chinese hamster cells in vitro . Radiation Research, 43, 561-581 . DUGLE, D. L ., GILLESPIE, C . J. and CHAPMAN, J. D., 1976, DNA strand breaks, repair and survival in x-irradiated mammalian cells. Proceedings of the National Academy of Sciences, USA, 73, 809-812 . DWARAKANATH, B . S . and JAIN, V. K ., 1989, Energy linked modifications of the radiation reponse in a human cerebral glioma cell line . International Journal of Radiation Oncology, Biology and Physics, 17, 1033-1040 . DWARAKANATH, B . S . and JAIN, V . K ., 1991, Effects of gammarays and glucose analogs on the energy metabolism of a cell line derived from human cerebral glioma . Indian Journal of Biochemistry and Biophysics, 28, 203-209 . FRANKENBERG, D ., 1969, A ferrous sulphate dosemeter indepen-
189
dent of photon energy in the range from 25 MeV up to 50 MeV . Physics in Medicine and Biology, 14, 597-605. FRANKENBERG, D., FRANKENBERG-SCHWAGER, M ., BLOCHER, D. and HARBICH, R ., 1981, Evidence for DNA doublestrand breaks as the critical lesions in yeast cells irradiated with sparsely or densely ionizing radiation under oxic or anoxic conditions . Radiation Research, 88, 524-532 . FRANKENBERG-SCHWAGER, M ., FRANKENBERG, D ., BLOCHER, D. and ADAMCZYK, C ., 1979, The influence of oxygen on the survival and yield of DNA double-strand breaks in irradiated yeast cells. International Journal of Radiation Biology, 36, 261-270 . FRANKENBERG-SCHWAGER, M ., FRANKENBERG, D ., BLOCHER, D . and ADAMCZYK, C ., 1980, Repair of DNA double-strand breaks in irradiated yeast cells under nongrowth conditions . Radiation Research, 82, 498-510 . FRANKENBERG-SCHWAGER, M ., FRANKENBERG, D . and HARBICH, R ., 1985, Potentially lethal damage, sublethal damage and DNA double-strand breaks . Radiation Protection Dosimetry, 13, 171-174 . FRANKENBERG-SCHWAGER, M ., FRANKENBERG, D . and HARBICH, R ., 1987, Potentially lethal damage repair is due to the difference of DNA double-strand break repair under immediate and delayed plating conditions . Radia-
tion Research, 111, 192-200. FRANKENBERG-SCHWAGER, M ., FRANKENBERG, D ., HARBICH, R . and ADAMCZYK, C ., 1990, A comparative study of rejoining of DNA double-strand breaks in yeast irradiated with 3 . 5 MeV a-particles or with 30 MeV electrons . International Journal of Radiation Biology, 57, 1151-1168 . GUPTA, I ., JAIN, V. K . and MISHRA, R . K ., 1981, Studies on the effects of 2-deoxy-D-glucose on glucose uptake and glycolysis in respiratory-deficient yeast cells . Indian Journal of Experimental Biology, 19, 231-237 . HEREDIA, C . F., DE LA FUENTE, G . and SoLS, A ., 1964, Metabolic studies with 2-deoxyhexoses . I . Mechanisms of inhibition of growth and fermentation in baker's yeast . Biochimica et Biophysica Acta, 86, 216-223 . Ho, K. S . Y ., 1975, Indication of DNA double-strand breaks by X-rays in a radiosensitive strain of the yeast Saccharomyces cerevisiae . Mutation Research, 30, 327-334. HOLAHAN, P. K ., KNIZNER, S . A ., GABRIEL, C . M . and SWENBERG, C . E ., 1988, Alterations in phosphate metabolism during cellular recovery of radiation damage in yeast . International Journal of Radiation Biology, 54, 545-562 . HOLMSEN, H ., SETKOWSKI, C. A . and JAMES, D. H ., 1974, Effects of antimycin and 2-deoxy-D-glucose on adenine nucleotides in human platelets . Biochemical Journal, 144, 385-396. JAIN, V. K . and POHLIT, W., 1972, Influence of energy metabolism on the repair of X-ray damage in living cells . I . Effect of respiratory inhibitors and glucose on the liquid holding reactivation in yeast . Biophysik, 8, 254-263 . JAIN, V . K., POHLIT, W. and PUROHIT, S . C ., 1973, Influence of energy metabolism on the repair of X-ray damage in living cells. II . Effect of 2-deoxy-D-glucose on the liquidholding reactivation in yeast . Biophysik, 10, 137-142 . JAIN, V. K ., BIswAS, R . K . and CHANDRA, P., 1976, Effect of 2deoxy-D-glucose on the liquid holding reactivation of yeast cells photodamaged by psoralen . Radiation and Environmental Biophysics, 13, 75-77 . JAIN, V. K., HOLTZ, G . W., POHLIT, W. and PUROHIT, S . C ., 1977a, Inhibition of unscheduled DNA synthesis and
190
Inhibition of rejoining of DNA double-strand breaks
repair of potentially lethal X-ray damage by 2-deoxy-Dglucose in yeast . International Journal of Radiation Biology, 32, 175-180 . JAIN, V. K ., PUROHIT, S. C . and POHLIT, W., 1977b, Optimization of cancer therapy : I . Inhibition of repair of X-rayinduced potentially lethal damage by 2-deoxy-D-glucose in Ehrlich ascites tumour cells . Indian Journal of Experimental Biology, 15, 711-713 . JAIN, V . K., GUPTA, I . and LATA, K ., 1982, Energetics of cellular repair processes in a respiratory-deficient mutant in yeast . Radiation Research, 92, 463-473 . JAIN, V . K ., KALIA, V . K ., SHARMA, R ., MAHARAJAN, V. and MENON, M ., 1985, Effects of 2-deoxy-D-glucose on glycolysis, proliferation kinetics and radiation response of human cancer cells. International Journal of Radiation Onco-
Downloaded by [UQ Library] at 04:02 26 April 2016
logy, Biology and Physics, 11, 943-950 . MATSUMOTO, S . S ., RAIvIo, K . O. and SEEGMILLER, J . E ., 1979, Adenine nucleotide degradation during energy depletion in human lymphoblasts . Journal of Biological Chemistry, 254, 8956-8962 . NATARAJAN, A . T., OBE, G ., VON ZEELAND, A . A ., PALITTi, F., MEIJERS, M . and VERDEGAAL-IMMERZELL, E . A . M ., 1980, Molecular mechanisms involved in the production of chromosomal aberrations . II . Utilization of Neurospora endonuclease for the study of aberration production by
X-rays in G I and GZ stages of the cell cycle . Mutation
Research, 69, 293-305 . RESNICK, M . A ., 1976, The repair of double-strand breaks in DNA : a model involving recombination . Journal of Theoretical Biology, 59, 97-106. RESNICK, M . A . and MARTIN, P ., 1976, The repair of doublestrand breaks in the nuclear DNA of Saccharomyces cereoisiae and its genetic control . Molecular and General Genetics, 143, 119-129 . THACKER, J ., 1986, The nature of mutants induced by ionizing radiation in cultured hamster cells . III . Molecular characterization of HPRT deficient mutants induced by grays or a-particles showing that the majority have deletion of all or part of hprt gene . Mutation Research, 160, 267-275 . VERMA, A., SHARMA, R. and JAIN, V. K., 1982, Energetics of DNA repair in UV irradiated peripheral blood leukocytes from chronic myeloid leukemia patients . Photochemistry and Photobiology, 36, 627-632 . WICK, A. N ., DRURY, D . R ., NAKADA, H . I . and WOLFE, J . B ., 1957, Localization of the primary metabolic block produced by 2-deoxy-D-glucose . Journal of Biological Chemistry, 224, 963-969. WOODWARD, G . E ., 1952, 2-deoxy-D-glucose an inhibitor in the aerobic glucose metabolism of yeast . Journal of the Franklin Institute, 254, 553-555 .
ISSN: 0955-3002 (Print) 1362-3095 (Online) Journal homepage: http://www.tandfonline.com/loi/irab20
2-Deoxy-d-glucose Inhibits Rejoining of Radiationinduced DNA Double-strand Breaks in Yeast M. Frankenberg-Schwager, R. Harbich, D. Frankenberg & V. Jain To cite this article: M. Frankenberg-Schwager, R. Harbich, D. Frankenberg & V. Jain (1992) 2Deoxy-d-glucose Inhibits Rejoining of Radiation-induced DNA Double-strand Breaks in Yeast, International Journal of Radiation Biology, 61:2, 185-190, DOI: 10.1080/09553009214550801 To link to this article: http://dx.doi.org/10.1080/09553009214550801
Published online: 03 Jul 2009.
Submit your article to this journal
Article views: 8
View related articles
Citing articles: 1 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=irab20 Download by: [UQ Library]
Date: 26 April 2016, At: 04:02
INT . J . RADIAT . BIOL .,
1992,
VOL .
61,
NO .
2, 1 8 5-190
2-Deoxy-D-glucose inhibits rejoining of radiation-induced DNA double-strand breaks in yeast M. FRANKENBERG-SCHWAGER j- , R. HARBICHt, D . FRANKENBERGt and V. JAIN*$
Downloaded by [UQ Library] at 04:02 26 April 2016
(Received 22 February 1991; revision received 24 May 1991; accepted 24 June 1991)
Abstract. Effects of 2-deoxy-D-glucose (2-DG) on radiationinduced DNA double-strand breaks (dsb) have been studied under non-growth conditions in a respiratory-deficient strain of the yeast Saccharomyces cerevisiae. Velocity sedimentation in neutral sucrose gradients was used to measure DNA dsb . Addition of 2-DG to the liquid-holding medium (67 mm phosphate buffer, pH 5, 30° C) at an equimolar concentration with glucose (50 mm) reduced the rate and extent of dsb rejoining . The inhibition of rejoining mediated by 2-DG is reversible for the majority-but not all-of the . radiation-induced dsb .
1. Introduction Cellular repair processes are important determinants of radiation sensitivity . 2-deoxy-D-glucose (2DG), has been shown in yeast to inhibit repair of potentially lethal damage (PLD) induced by ionizing (X-rays, y-rays, fast electrons) and non-ionizing (UVC, psoralen + UVA) radiation (Jain et al. 1973, 1976, 1977a, 1982, Holahan et al. 1988) . Inhibition of PLD repair in the presence of 2-DG has also been observed in X-irradiated Ehrlich ascites tumour cells of mouse (Jain et al. 1977b) . The mechanisms underlying 2-DG-induced inhibition of PLD repair are complex and not yet completely understood . 2-DG is known to strongly inhibit the glycolytic pathway at various stages, such as glucose transport, phosphorylation and isomerization (Woodward 1952, Wick et al. 1957, Heredia et al. 1964, Gupta et al. 1981), whereas the respiratory pathway is affected only to a small extent (Jain et al . 1982) . Thus, under respiratory-deficient conditions, the presence of 2DG leads to a considerable reduction in the rate of ATP production via the glycolytic pathway and thereby to a decrease of the cellular contents of high*Author for correspondence . t Institut fur Biophysikalische Strahlenforschung, Gesellschaft fur Strahlen-und Umweltforschung mbH, Paul-EhrlichStr. 20 D-6000 Frankfurt 70, Germany. $ Institute of Nuclear Medicine & Allied Sciences, Lucknow Marg, Delhi 110054, India .
energy phosphates such as ATP, GTP and UTP (Jain et al . 1973, 1982, Dwarakanath and Jain 1991) . Degradation of adenine nucleotides via deamination of AMP observed in the presence of 2-DG (Holmsen et al. 1974, Matsumoto et al. 1979) could lead to further perturbations in the cellular nucleotide pools. Under respiratory-proficient conditions the energy deficiency produced by the presence of 2DG is small since sufficient ATP can be generated by oxidative phosphorylation (Jain et al. 1985) . Thus, ten times higher concentrations of 2-DG were required to inhibit repair of PLD in wild-type yeast as compared to respiratory-deficient mutants (Jain et al. 1973) . An adequate supply of metabolic energy derived from the glycolytic and/or the respiratory pathway is necessary for the cellular repair of PLD (Jain and Pohlit 1972) . Since DNA is an important molecular target for radiation-induced PLD, it was suggested, as a working hypothesis, that reduction of cellular energy supply below a certain critical threshold induced by the presence of 2-DG could possibly inhibit repair of radiation-induced DNA lesions (Jain et al. 1973, 1982) . Postirradiation repair of DNA lesions assayed by the unscheduled DNA synthesis technique has indeed been observed to be inhibited by the presence of 2-DG in yeast (Jain et al. 1977a) and human cancer cells (Verma et al. 1982, Dwarakanath and Jain 1989) under conditions of respiratory deficiency . Lowering of cellular ATP levels, however, is not correlated in a simple manner with the inhibition of 2-DG-induced cellular and DNA repair (Jain et al. 1982, Verma et al. 1982) . Unscheduled DNA synthesis is supposed to monitor mainly the repair of DNA base damage and singlestrand breaks (ssb) via the excision repair pathway . In cases of ionizing radiations DNA double-strand breaks (dsb) are considered to be the most important DNA lesions responsible for chromosomal aberrations (Bender et al. 1974, Natarajan et al. 1980, Bryant 1984), mutations and cell death (Dewey et al. 1970, Dugle et al . 1976, Thacker 1986, Ho 1975, Resnick and Martin 1976, Frankenberg et al. 1981, Blucher and Pohlit 1982) . Furthermore, in yeast,
0020-7616/92 $3 .00 © 1992 Taylor & Francis Ltd
186
M . Frankenberg-Schwager et al .
repair of PLD induced by ionizing radiation has been demonstrated to be due to the rejoining of dsb (Frankenberg-Schwager et al . 1980, 1985, 1987) . Therefore, in the present work we investigated, in a respiratory-deficient yeast strain, the effects of 2-DG on the rejoining of radiation-induced dsb during incubation of cells in non-growth medium .
2. Materials and methods
Downloaded by [UQ Library] at 04:02 26 April 2016
2 .1 . Yeast cells A respiratory-deficient mutant strain 211 *B (ATCC 42607) of Saccharomyces cerevisiae auxotrophic for 2'-deoxy-thymidine 5'-monophosphate (5'dTMP) was used . This strain is capable of repairing dsb (Frankenberg-Schwager et al . 1980), it lacks mitochondrial DNA and its nuclear DNA can be specifically labelled with (3H)dTMP (Brendel and Fath 1974) .
2 .2. Growth of cells and labelling of DNA Cells were grown overnight at 30°C in a nutrient medium containing 4% glucose, 1 .3% yeast nitrogen base without amino acids, 0 .4% casamino acids supplemented with 7µg/ml 5'-dTMP (Boehringer, Mannheim) . After appropriate dilution into fresh medium, (methyl-3-H)dTMP (Amersham) was added to give 148 kBq/ml . The culture was rotated at 30°C until cells had reached the stationary phase .
2 .3 . Irradiation conditions Stationary cells were washed twice in phosphate buffer (pH 7 .0) and irradiated with 30 MeV electrons (Betatron) under continuous stirring at 4°C (dose-rate : 120 Gy/min) . The dosimetry was performed with an energy-independent ferrous sulphate dosimeter (Frankenberg, 1969) .
al . 1980) . In parallel experiments cells were resus-
pended immediately after irradiation in non-growth medium containing a mixture of glucose and 2deoxy-D-glucose (2-DG) at equimolar concentrations of 50 mm. Samples were held in this medium for 24 h (most experiments) or 48 h (one experiment, see Figure 1) to assess the effect of 2-DG on the rejoining of dsb. In another set of experiments 2-DG was washed out after a 24 h treatment, cells were resuspended in 2-DG-free non-growth medium and allowance for dsb rejoining was made by incubating cells for another 24 or 48 h at 30°C . After these intervals of time samples were taken for the measurement of dsb . These latter experiments were performed to test whether the effect of 2-DG on dsb rejoining is reversible .
2 .5. Measurement of DNA double-strand breaks Sedimentation of DNA in neutral sucrose gradients was used to measure dsb . Methods have been described in detail earlier (Frankenberg-Schwager et al . 1979) . Briefly, cell protoplasts were prepared and lysed on top of neutral sucrose gradients (5-20%) containing 0 . 1 M NaCl and 0 .02 M EDTA (pH 7 .0) in 13 ml cellulose nitrate tubes . The released DNA was sedimented at about 9000 rpm in an ultracentrifuge (Beckmann) at 20°C using the rotor SW 40 Ti . After centrifugation for about 21 h, gradients were fractionated onto glass-fibre filters . The filters were dried and the high molecular weight DNA precipitated with ice-cold 6% TCA, washed with ethanol and dried again . Radioactivity was counted in a liquid scintillation counter using a toluene-based cocktail . The number of radiation-induced dsb per average molar mass of DNA was calculated from the DNA profiles, i .e . from the distribution curves of the radioactivity as a function of the sedimented volume as described in detail earlier (FrankenbergSchwager et al. 1979, Frankenberg et al . 1981) . Briefly, the average number of dsb per average molar mass of DNA was calculated by computer simulation assuming random breakage of the DNA and by fitting these curves of the DNA profiles obtained from irradiated cells .
2 .4 . Postirradiation treatment of cells
After irradiation, cells were resuspended in nongrowth medium, consisting of phosphate buffer (67 mm, pH 5 .0) supplemented with 5'-dTMP (7µg/ml) and containing 100 mm glucose and incubated at 30°C. Under these conditions the respiratory-deficient strain 211 *B exhibits both PLD repair and repair of dsb (Frankenberg-Schwager et
3. Results 3 .1 . DNA sedimentation profiles DNA profiles from irradiated yeast cells (absorbed dose D = 1500 Gy) were obtained after liquid hold-
Inhibition of rejoining of DNA double-strand breaks
Downloaded by [UQ Library] at 04:02 26 April 2016
ing of cells in non-growth medium at 30 °C in the absence or presence of 2-DG (representative profiles are shown in Figure 1) . It was observed that after 48 h of incubation in the absence of 2-DG, the DNA sedimentation profile of irradiated samples shifts towards higher molecular DNA and does not differ very much from the unirradiated controls . This suggests that most of the dsb have been rejoined during this time . In contrast, the sample incubated in non-growth medium containing 2-DG retained a DNA sedimentation profile with a smaller shift towards the control, indicating inhibition of repair processes . It should be noted that even after a 48 h incubation period of irradiated cells in the presence of 2-DG no loss of cells was observed as judged by the same total radioactivity recovered from each sucrose gradient .
3.2 . The effect of 2-deoxy-D-glucose on the rejoining of double-strand breaks
The presence of 2-DG during a 24 h incubation results in the reduction of the rate of rejoining of
187
radiation-induced dsb in yeast, and rejoining seems to level off after this time (Figure 2) . After longer treatments with 2-DG the determination of dsb was difficult because most DNA profiles could not be fitted well after a 48 h treatment with 2-DG, and were therefore excluded from the quantitative evaluation. This effect was even more severe after a 72 h treatment with 2-DG, and therefore no determination of double-strand breakage was attempted . In order to test whether the 2-DG-mediated inhibition of dsb rejoining is reversible, 2-DG was washed out after 24 h of postirradiation treatment and cells were reincubated in 2-DG-free non-growth medium for another 48 h . During this treatment, samples were taken for the determination of dsb . The number of dsb remaining unrejoined after such a 2-DG treatment was, however, somewhat higher (significant for p=0 .1) than in cells which had not been treated with 2-DG (Figure 2) . This seems to indicate that the rejoining of some dsb may be irreversibly inhibited (i .e. `fixed') by 2-DG . To investigate this further we tried to enhance the possible yield of `fixed' dsb . For this purpose irradiated cells were incubated for 24 h solely in the presence of 2-DG (50 mm), i .e . without glucose . This
Figure 1 . DNA profiles obtained from cells immediately after irradiation with an absorbed dose of 1500 Gy (A) and after 48 h of liquid holding in nongrowth medium in the absence (0) and presence ( •) of 2-deoxy-D-glucose . The DNA profiles are representatives of a single experiment . The percentage of total radioactivity of DNA per fraction (A/A,) is plotted as a function of the fraction number. The direction of sedimentation is from left to right .
1 88
M. Frankenberg-Schwager et al .
N mil
4. Discussion
Downloaded by [UQ Library] at 04:02 26 April 2016
109Rg~mole
T
r
I 0
10
20
30
20
~0
60
70 hme/h
Figure 2 . Kinetics of rejoining of double-strand breaks observed in irradiated yeast (D=1500 Gy) during postirradiation incubation in normal non-growth medium (containing 100 mm glucose) (0) for 0 h (five experiments), 1 h (four experiments), 3 h (two experiments), 16h (two experiments), 24h (seven experiments), 48h (five experiments) and 72h (four experiments) . Rejoining was also measured after postirradiation incubation of cells in non-growth medium containing 2-deoxy-Dglucose (2-DG) and glucose at equimolar concentrations (50 mM) for 24h (14 experiments) and for 48h (one experiment, the profile of which is shown in Figure 1) (0) . Cells treated with 2-DG for 24h were washed and incubated in normal 2-DG-free nongrowth medium for another 24h (13 experiments) or 48h (8 experiments) (0) . Average values and their standard deviations are shown . N • m~ 1 : Average number of dsb (N) per relative molar mass of DNA (Mr)-
treatment should result in a greater number of `fixed' dsb which would not be rejoinable during subsequent incubation of cells in 2-DG-free medium . However, treatment of unirradiated cells with 2-DG alone or at higher than equimolar concentrations with glucose resulted in severe distortions of the DNA profiles (not shown) . In contrast, normal DNA profiles are observed for cells incubated for 24 h in non-growth medium containing only glucose or 2-DG and glucose at equimolar concentrations (50 mm) .
The results of the present studies show that rejoining of DNA dsb is inhibited in respiratorydeficient yeast cells incubated in non-growth medium in the presence of 2-DG (Figures 1 and 2) . Recombination processes between homologous chromosomes or sister chromatids, which have been implicated in the repair of DNA dsb in yeast (Resnick 1976), involve the coordinated functioning of many enzymes such as endo- and exonucleases, DNA polymerases and polynucleotide ligase . Activities of these enzymes, being energy-dependent, may be inhibited by a reduction in ATP supply and subsequent decrease in ATP concentration induced by the presence of 2-DG (Jain et al . 1973, 1982) . The excision repair pathway involved in the repair of DNA base damage and ssb also depends upon the proper functioning of similar types of enzymes, and has been observed to be inhibited by 2-DG in our earlier studies using the same strain of yeast (Jain et al. 1977a) . Similar observations have been made in cancer cells derived from human patients (Verma et al. 1982, Jain et al . 1985, Dwarakanath and Jain 1989) . Thus, it appears that both the excision and recombination repair pathways can be inhibited by 2-DG in cell systems that are highly dependent upon glycolysis for their energy supply . The kinetics of rejoining of DNA dsb induced by 30 MeV electrons has been shown to be biphasic in yeast held in nongrowth medium . A fast (t 112 =3 .8h) and a slow (4/2=10-11h) component could be distinguished (Frankenberg-Schwager et al. 1990) . In the present study a 2-DG-induced inhibition of dsb rejoining of about 45% was observed after the first 24h in cells irradiated with a dose of 1500 Gy . This inhibition could be due to a reduction of the rate of the fast and/or slow component . Further studies are required to clarify which of the repair components is more susceptible to inhibition by 2-DG . Figure 2 also indicates that the 2-DG-mediated inhibition of rejoining of the majority-but not allof the radiation-induced dsb is reversible under the present experimental conditions . Our previous experiments with a human cerebral glioma cell line have demonstrated that 2-DG-mediated inhibition of unscheduled DNA synthesis after irradiation could be reversed after removal of 2-DG under conditions of respiratory proficiency . In the absence of respiration, however, irreversible inhibition of DNA repair was observed (Dwarakanath and Jain 1989) . Experiments to demonstrate a possible irreversible 2-DG-mediated inhibition of rejoining of at least some of the dsb in yeast failed because a severe
Inhibition of rejoining of DNA double-strand breaks
Downloaded by [UQ Library] at 04:02 26 April 2016
distortion of the DNA profile was observed even in unirradiated cells incubated in non-growth medium in the presence of 2-DG (50 mm) alone or in combination at higher than equimolar concentrations with glucose (2-DG/G=2 .5 or 5 .0) . The distorted DNA profiles are flat and demonstrate an increase in the fraction of small as well as large DNA molecules . The reasons for this 2-DG-mediated fragmentation and enlargement of DNA molecules are not clear. It appears unlikely that depletion of the energy source may lead to this effect, since postirradiation incubation of these cells for 24 h in non-growth medium lacking glucose yielded undistorted DNA profiles (Frankenberg-Schwager et al . 1980) . Further studies to understand these phenomena are necessary .
Acknowledgement The support of the medical-biological collaboration with India by the BMFT is acknowledged .
References BENDER, M . A ., GRIGGS, H. G . and BEDFORD, J. S ., 1974, Mechanisms of chromosomal aberration production . III . Chemicals and ionizing radiation . Mutation Research, 23, 197-212 . BLOCHER, D . and POHLIT, W., 1982, DNA double strand breaks in Ehrlich ascites tumor cells at low doses of X-rays, II . Can all death be attributed to double strand breaks? International Journal of Radiation Biology, 42, 329-338. BRENDEL, M . and FATH, F . W., 1974, Isolation and characterization of mutants of Saccharomyces cerevisiae auxotrophic for 5'-dTMP. Zeitschrift fur Naturforschung, 29c, 733-738 . BRYANT, P . E ., 1984, Enzymatic restriction of mammalian cell DNA using Pvu II and Bam HI : evidence for the double-strand break origin of chromosomal aberrations . International Journal of Radiation Biology, 46, 57-65 . DEWEY, W . C ., FURMAN, S . C . and MILLER, H . H ., 1970, Comparison of lethality and chromosomal damages induced by X-rays in synchronized Chinese hamster cells in vitro . Radiation Research, 43, 561-581 . DUGLE, D. L ., GILLESPIE, C . J. and CHAPMAN, J. D., 1976, DNA strand breaks, repair and survival in x-irradiated mammalian cells. Proceedings of the National Academy of Sciences, USA, 73, 809-812 . DWARAKANATH, B . S . and JAIN, V. K ., 1989, Energy linked modifications of the radiation reponse in a human cerebral glioma cell line . International Journal of Radiation Oncology, Biology and Physics, 17, 1033-1040 . DWARAKANATH, B . S . and JAIN, V . K ., 1991, Effects of gammarays and glucose analogs on the energy metabolism of a cell line derived from human cerebral glioma . Indian Journal of Biochemistry and Biophysics, 28, 203-209 . FRANKENBERG, D ., 1969, A ferrous sulphate dosemeter indepen-
189
dent of photon energy in the range from 25 MeV up to 50 MeV . Physics in Medicine and Biology, 14, 597-605. FRANKENBERG, D., FRANKENBERG-SCHWAGER, M ., BLOCHER, D. and HARBICH, R ., 1981, Evidence for DNA doublestrand breaks as the critical lesions in yeast cells irradiated with sparsely or densely ionizing radiation under oxic or anoxic conditions . Radiation Research, 88, 524-532 . FRANKENBERG-SCHWAGER, M ., FRANKENBERG, D ., BLOCHER, D. and ADAMCZYK, C ., 1979, The influence of oxygen on the survival and yield of DNA double-strand breaks in irradiated yeast cells. International Journal of Radiation Biology, 36, 261-270 . FRANKENBERG-SCHWAGER, M ., FRANKENBERG, D ., BLOCHER, D . and ADAMCZYK, C ., 1980, Repair of DNA double-strand breaks in irradiated yeast cells under nongrowth conditions . Radiation Research, 82, 498-510 . FRANKENBERG-SCHWAGER, M ., FRANKENBERG, D . and HARBICH, R ., 1985, Potentially lethal damage, sublethal damage and DNA double-strand breaks . Radiation Protection Dosimetry, 13, 171-174 . FRANKENBERG-SCHWAGER, M ., FRANKENBERG, D . and HARBICH, R ., 1987, Potentially lethal damage repair is due to the difference of DNA double-strand break repair under immediate and delayed plating conditions . Radia-
tion Research, 111, 192-200. FRANKENBERG-SCHWAGER, M ., FRANKENBERG, D ., HARBICH, R . and ADAMCZYK, C ., 1990, A comparative study of rejoining of DNA double-strand breaks in yeast irradiated with 3 . 5 MeV a-particles or with 30 MeV electrons . International Journal of Radiation Biology, 57, 1151-1168 . GUPTA, I ., JAIN, V. K . and MISHRA, R . K ., 1981, Studies on the effects of 2-deoxy-D-glucose on glucose uptake and glycolysis in respiratory-deficient yeast cells . Indian Journal of Experimental Biology, 19, 231-237 . HEREDIA, C . F., DE LA FUENTE, G . and SoLS, A ., 1964, Metabolic studies with 2-deoxyhexoses . I . Mechanisms of inhibition of growth and fermentation in baker's yeast . Biochimica et Biophysica Acta, 86, 216-223 . Ho, K. S . Y ., 1975, Indication of DNA double-strand breaks by X-rays in a radiosensitive strain of the yeast Saccharomyces cerevisiae . Mutation Research, 30, 327-334. HOLAHAN, P. K ., KNIZNER, S . A ., GABRIEL, C . M . and SWENBERG, C . E ., 1988, Alterations in phosphate metabolism during cellular recovery of radiation damage in yeast . International Journal of Radiation Biology, 54, 545-562 . HOLMSEN, H ., SETKOWSKI, C. A . and JAMES, D. H ., 1974, Effects of antimycin and 2-deoxy-D-glucose on adenine nucleotides in human platelets . Biochemical Journal, 144, 385-396. JAIN, V. K . and POHLIT, W., 1972, Influence of energy metabolism on the repair of X-ray damage in living cells . I . Effect of respiratory inhibitors and glucose on the liquid holding reactivation in yeast . Biophysik, 8, 254-263 . JAIN, V . K., POHLIT, W. and PUROHIT, S . C ., 1973, Influence of energy metabolism on the repair of X-ray damage in living cells. II . Effect of 2-deoxy-D-glucose on the liquidholding reactivation in yeast . Biophysik, 10, 137-142 . JAIN, V. K ., BIswAS, R . K . and CHANDRA, P., 1976, Effect of 2deoxy-D-glucose on the liquid holding reactivation of yeast cells photodamaged by psoralen . Radiation and Environmental Biophysics, 13, 75-77 . JAIN, V. K., HOLTZ, G . W., POHLIT, W. and PUROHIT, S . C ., 1977a, Inhibition of unscheduled DNA synthesis and
190
Inhibition of rejoining of DNA double-strand breaks
repair of potentially lethal X-ray damage by 2-deoxy-Dglucose in yeast . International Journal of Radiation Biology, 32, 175-180 . JAIN, V. K ., PUROHIT, S. C . and POHLIT, W., 1977b, Optimization of cancer therapy : I . Inhibition of repair of X-rayinduced potentially lethal damage by 2-deoxy-D-glucose in Ehrlich ascites tumour cells . Indian Journal of Experimental Biology, 15, 711-713 . JAIN, V . K., GUPTA, I . and LATA, K ., 1982, Energetics of cellular repair processes in a respiratory-deficient mutant in yeast . Radiation Research, 92, 463-473 . JAIN, V . K ., KALIA, V . K ., SHARMA, R ., MAHARAJAN, V. and MENON, M ., 1985, Effects of 2-deoxy-D-glucose on glycolysis, proliferation kinetics and radiation response of human cancer cells. International Journal of Radiation Onco-
Downloaded by [UQ Library] at 04:02 26 April 2016
logy, Biology and Physics, 11, 943-950 . MATSUMOTO, S . S ., RAIvIo, K . O. and SEEGMILLER, J . E ., 1979, Adenine nucleotide degradation during energy depletion in human lymphoblasts . Journal of Biological Chemistry, 254, 8956-8962 . NATARAJAN, A . T., OBE, G ., VON ZEELAND, A . A ., PALITTi, F., MEIJERS, M . and VERDEGAAL-IMMERZELL, E . A . M ., 1980, Molecular mechanisms involved in the production of chromosomal aberrations . II . Utilization of Neurospora endonuclease for the study of aberration production by
X-rays in G I and GZ stages of the cell cycle . Mutation
Research, 69, 293-305 . RESNICK, M . A ., 1976, The repair of double-strand breaks in DNA : a model involving recombination . Journal of Theoretical Biology, 59, 97-106. RESNICK, M . A . and MARTIN, P ., 1976, The repair of doublestrand breaks in the nuclear DNA of Saccharomyces cereoisiae and its genetic control . Molecular and General Genetics, 143, 119-129 . THACKER, J ., 1986, The nature of mutants induced by ionizing radiation in cultured hamster cells . III . Molecular characterization of HPRT deficient mutants induced by grays or a-particles showing that the majority have deletion of all or part of hprt gene . Mutation Research, 160, 267-275 . VERMA, A., SHARMA, R. and JAIN, V. K., 1982, Energetics of DNA repair in UV irradiated peripheral blood leukocytes from chronic myeloid leukemia patients . Photochemistry and Photobiology, 36, 627-632 . WICK, A. N ., DRURY, D . R ., NAKADA, H . I . and WOLFE, J . B ., 1957, Localization of the primary metabolic block produced by 2-deoxy-D-glucose . Journal of Biological Chemistry, 224, 963-969. WOODWARD, G . E ., 1952, 2-deoxy-D-glucose an inhibitor in the aerobic glucose metabolism of yeast . Journal of the Franklin Institute, 254, 553-555 .
