6-Aminonicotinamide – 8 bromo camp

Radiosensitization by 6-aminonicotinamide and 2-deoxy-D-glucose in human cancer cells

R. VARSHNEY, B. S. DWARAKANATH & V. JAIN Institute of Nuclear Medicine & Allied Sciences, Delhi, India (Received 6 July 2004; accepted 13 April 2005)

Abstract

The aim was to exploit simultaneous inhibition of glycolytic and pentose phosphate pathways of energy production for radiosensitization using 2-deoXy-D-glucose (2-DG) and 6-aminonicotinamide (6-AN) in transformed mammalian cells. Two human tumour cell lines (cerebral glioma, BMG-1 and squamous carcinoma cells 4197) were investigated. 2-DG and/ or 6-AN added at the time of irradiation were present for 4 h after radiation. Radiation-induced cell death (macrocolony assay), cytogenetic damage (micronuclei formation), cell cycle delay (bromodeoXyuridne (BrdU) pulse chase), apoptosis (externalization of phosphotidylserine (PS) by annexin V), chromatin-bound proliferation cell nuclear antigen (PCNA) and cellular glutathione (GSH) levels were investigated as parameters of radiation response. The presence of 2-DG (5 mM) during and for 4 h after irradiation increased the radiation-induced micronuclei formation and cell death, and caused a time- dependent decrease in GSH levels in BMG-1 cells while no signiﬁcant effects could be observed in 4197 cells. 6-AN (5 mM) enhanced the radiosensitivity of both cell lines and reduced the GSH content by nearly 50% in gamma-irradiated 4197 cells. Combining 2-DG and 6-AN caused a profound decrease in the GSH content and enhanced the radiation damage in both the cell lines by increasing mitotic and apoptotic cell death. Further, the combination (2-DG + 6-AN) enhanced the radiation- induced G2 block, besides arresting cells in S phase and inhibited the recruitment of PCNA. The combination of 2-DG and 6-AN enhances radiation damage by modifying damage response pathways and has the potential for improving radiotherapy of cancer.

Introduction

Resistance of tumour cells to therapeutic doses of radiation and normal tissue tolerance are the two main factors that limit the success of radiotherapy (Weich- selbaum et al. 1988, Suit et al. 1989, Kasid et al. 1993).Previously, we developed an approach based on the energy-linked modiﬁcations of DNA and cellular repair processes exploiting differences in the pattern of glucose-dependent energy metabo- lism between tumour and normal tissues (Jain & Pohlit 1972, Jain et al. 1973). Using this approach,

This has prompted the development of approaches to selectively enhance radiation damage in tumours using radiosensitizers (Adams 1973, Jain et al. 1973) and/or improve the normal tissue tolerance using radioprotectors (Jain et al. 1979, Yuhas et al. 1980, Varshney & Kale 1990, 1996). However, on examina- tion in clinical studies, most radiomodiﬁers have proven to be less effective/toXic at therapeutic doses lacking the ability to act differentially on tumour versus normal tissues. Therefore, there is considerable interest in developing approaches using combinations inhibitor of glucose transport and glycolytic ATP production (Woodward & Cramer 1952, Wick et al. 1975), can inhibit repair of DNA damage in highly glycolytic cells and differentially enhance radiation- induced cell death in cancer cells in vitro (Jain et al. 1977a, 1985, Dwarakanath & Jain 1987, 1989, 1991, Jha & Pohlit 1992, Aft et al. 2002) and in vivo (Jain et al. 1977b, Purohit & Pohlit 1982) while reducing radiation damage in normal cells (Jain et al. 1979, 1985, Kalia et al. 1982, Singh et al. 1990).

Radiosensitization induced by 2-DG, however, obtain synergistic or additive effects. varies considerably among different human tumour
cell lines (Dwarakanath et al. 1999, 2001) and is partly reversible in the presence of respiratory metabolism (Dwarakanath & Jain 1989, Jha & Pohlit 1993), while irreversible in the absence of respiratory ATP production. It has been demon- strated that combinations of 2-DG with agents that inhibit respiratory metabolism reduce the energy supply further and enhance radiation damage in human tumour cell lines (Dwarakanath et al. 1999). In a solid tumour, the glucose and oXygen supply are heterogeneous and the effects of 2-DG on well- oXygenated cells are likely to be less and may not induce signiﬁcant levels of radiosensitization. It is suggested that further biochemical modulation to suppress energy levels in these cells could enhance 2-DG-induced radiosensitization, augmenting tu- mour regression resulting in a larger therapeutic gain. One approach currently being investigated is the combination of 2-DG with 6-aminonicotina- mide (6-AN) (Sharma et al. 2000, Varshney et al. 2003, 2004).

6-AN, an analogue of nicotinamide, inhibits the pentose phosphate pathway (PPP), an alternate metabolic pathway involved in energy production and biosynthesis of nucleotides (Ames et al. 1996). 2-DG interferes with cellular energetics by reducing glucose ﬂuX through competitive inhibition of glucose transport and subsequent phosphorylation by hexokinase. 2-deoXy-D-glucose 6-phosphate (2- DG-6-P) thus formed is not metabolized further. Accumulated 2-DG-6-P reduces glycolysis by in- hibiting phosphoglucose isomerase, thereby suppressing the conversion of glucose 6-phosphate to fructose 6-phosphate (Wick et al. 1975). On the other hand 6-AN reduces PPP by inhibiting 6- phosphogluconate dehyrogenase resulting in reduc- tion of NADPH and accumulation of 6- phosphogluconate (6-PG) (Keniry et al. 1989, Street et al. 1996, 1997). 6-PG additionally decreases glycolytic ﬂuX by inhibiting phophoglucose isomer- ase (Kahana et al. 1960). Both 6-AN and 2-DG thus lead to reductions in glycolytic energy production, but the primary sites of action are different. Our previous studies have shown that combining 6-AN with 2-DG drastically and irreversibly reduced ATP levels in oXygenated Ehrlich ascites cells (Sharma et al. 2000). Therefore, it was suggested that the combination of 2-DG with 6-AN may enhance the radiosensitivity of tumours by irreversibly inhibiting cellular repair and recovery processes (Sharma et al. 2000). Indeed, the combination has been found to enhance the local tumour control to a signiﬁcantly higher degree than either 2-DG or 6-AN (Varshney et al. 2004). The present studies were carried out to investigate in detail the mechanism underlying enhanced radiosensitization in human glioma (BMG-1) and squamous carcinoma (4197) cell lines. Cell survival (macrocolony assay), cell cycle progres- sion, cytogenetic damage (micronuclei formation), cellular glutathione as well as PCNA levels and apoptosis were investigated as treatment-induced cellular responses.

Materials and methods
Chemicals

Dulbecco’s modiﬁed Eagles medium (DMEM), N- 2-hydroXyethyl piperzine-N’-2-ethanesulfonic acid (HEPES), streptomycin, penicillin G, nystatin, O- phthalaldehyde (OPT), propidium iodide (PI), diamidino-2-phenyldihydrochloride (DAPI), RNase, and foetal calf serum (FCS) were from Sigma Chemical Co. (St Louis, MO, USA). Anti-BrdU (bromodeoXyuridine), anti-PCNA (proliferation cell nuclear antigen), FITC-conjugated antimouse IgG and annexin V FITC-labelled antibodies were obtained from Beckton Dickenson (San Jose, CA, USA). All other chemicals and reagents were of analytical grade and were from BDH and SRL (Mumbai, India).

Cells

BMG-1 cells (plating efﬁciency (PE) = 0.82) estab- lished by our group from a human cerebral glioma (Dwarakanath 1988) and 4197 cells (PE = 0.45) derived from a squamous cell carcinoma, and which was kindly provided by Dr Zolzer, University of Essen, Germany (Zolzer et al. 1995), were main- tained as monolayer cultures in DMEM, supplemented with 10 and 20% FCS respectively, 10 mM HEPES and antibiotics (30 mg ml 7 1 penicillin G, 50 mg ml 7 1 streptomycin and 2 mg ml 7 1 nystatin).

Experimental procedure and irradiation

Monolayer BMG-1 or 4197 cells were grown for 24 h (exponential growth phase) before treatment in DMEM containing 10 or 20% FCS, respectively. All treatments were carried out under liquid holding conditions to facilitate repair. Hanks’ balanced salts solution (HBSS) containing 5 mM glucose, which does not support growth, was used for this purpose. 6-AN and/or 2-DG were added to liquid-holding media (HBSS) just before irradiation and removed 4 h later by washing. A Co60 gamma-ray source (Gamma Cell, Atomic Energy of Canada Ltd (AECL) Chalk River, Ontario, Canada) was used for irradiation at a dose-rate of 2.8 Gy min 7 1. Cells were grown at 378C following treatment for various time intervals to study micronuclei, macrocolony formation and cell cycle perturbations.

Macrocolony assay

Cells (150 – 600, depending on the treatment) were plated in 60-mm Petri dishes and incubated at 378C in a 5% CO2 humidiﬁed atmosphere for 8 – 10 days to allow colony formation. Colonies were ﬁXed with methanol and stained with 1% crystal violet. Colonies with more than 50 cells were counted; plating efﬁciency (PE) and surviving fraction (SF) were calculated. Differences between the means of data from different groups were tested for statistical signiﬁcance using the student’s t-test.10 000 events using a ﬂow cytometer (FACS- Calibur, Becton Dickinson) and plotted as two parameter scatter grams. Non-speciﬁcally labelled cells were excluded by choosing appropriate win- dows.

Micronuclei analysis

Air-dried slides of acetic – methanol-ﬁXed cells were stained with a DNA-speciﬁc ﬂuorochrome DAPI (in disodium phosphate (0.045 M) buffer containing 10 mg ml 7 1 citric acid (0.01 M) and 0.05% Tween-20 detergent).

Cell cycle analysis

Following treatment, cells were incubated in growth medium for varying periods, harvested by trypsini- zation and counted using a haemocytometer. Flow cytometric measurements of cellular DNA content analysed from duplicate slides. Since radiation as well as modiﬁers are known to alter the rate of cell proliferation which inﬂuences the expression of micronuclei, cell numbers and the percentage of cells with micronuclei were determined as a function of time up to two to three population doublings after were performed with ethanol (70%)-ﬁXed cells irradiation. Data were analysed by obtaining inte-using the intercalating DNA ﬂuorochrome PI as described earlier (Dwarakanath et al. 1999). Brieﬂy, the cells (0.5 6 106) were washed in phosphate- buffered saline (PBS) after removing ethanol and treated with ribonuclease-A (200 mg ml 7 1) for 30 min at 378C. Subsequently, cells were stained with PI (50 mg ml 7 1) in PBS. Measurements were made with an argon laser-based ﬂow cytometer (FACS-Calibur, Becton Dickinson, San Jose, CA, USA) using the blue line (488 nm) for excitation. Distribution of cells in different phases of cell cycle was calculated from the frequency distribution of DNA content by using the Modﬁt program (Variety Software, CA, USA).

Cell proliferation kinetics

The BrdU-DNA double-labelling technique was used to monitor the progression of S phase cells as described previously (Schutte et al. 1987, Gilbertz et al. 1993). Brieﬂy, cells were pulse labelled with 10 mM BrdU for 15 min at 378C. Immediately after treatment, the BrdU was removed by two washes with complete medium. Cells resuspended in com- plete media were allowed to progress through the cell grated values with respect to cell numbers. The frequency of cells with micronuclei, called the M fraction (MF), was calculated as : MF(%) = Nm/Nt 6 100,where Nm is the number of cells with micronuclei and Nt is the total number of cells analysed. A Modifying factor rx was calculated to evaluate the radiomodifying effects of the radiomodiﬁer x, by taking into account the effects of various treatments on cell proliferation: rMF ¼ ½ðMFÞx þ g — ðMFÞc]=ðMFÞg — ðMFÞc]; where x is the modiﬁer 6-AN, 2-DG or the combination, (MF)c is the micronuclei frequency of the unirradiated control and (MF)g is the micronuclei frequency after irradiation.

Annexin V binding

Apoptotic cells were detected by the labelling of externalized phosphatidylserine using annexin-V- FITC in unﬁXed cells (Vermes et al. 1995). Follow-70% chilled ethanol and stored at 48C until preparation for analysis. For immunostaining, cells
were incubated with pepsin (0.5% in 0.05% N HCl) to isolate nuclei and treated with 2 N HCl partly to denature the DNA. Subsequently, cells were labelled with mouse anti-BrdU antibody at a dilution 1:20 followed by ﬂuorescein (FITC)-conjugated anti- mouse IgG antibody diluted in PBS with 1% bovine serum albumin (BSA). The DNA was counter-stained by PI (50 mg ml 7 1). Green (FITC) and red (PI) ﬂuorescence were recorded from at least and aliquots of 105 cells resuspended in 100 ml binding buffer (10 mM HEPES/NaOH, pH 7.4; 140 mM NaCl; 2.5 mM CaCl2) and 5 ml annexin- V-FITC and 10 ml PI (50 mg ml 7 1) were added. After 15 min at room temperature, 400 ml binding buffer were added to each sample and analysed by ﬂow cytometry. The percentages of annexin V- positive and -negative cells were estimated by
applying appropriate gates and using regional statis- tics analysis facility provided in the Cell Quest software (Becton Dickenson, San Jose, CA, USA).

Assay of GSH

GSH levels in cell lines were determined by the method of Hissin & Hilf (1976) using O-phthalalde- hyde (OPT) as the ﬂuorescent reagent. Brieﬂy, cells were washed once with PBS and resuspended at 2 6 106 ml 7 1 in cold lysis buffer consisting of 5%
trichloroacetic acid (TCA):1 mM EDTA:0.1 M HCl (1:1:1, v/v/v). Following lysis, non-soluble material was removed by centrifugation at 2500g for 25 s at 48C. For the measurement of GSH, 100 ml supernatant were then mixed with 1.8 ml 0.1 M phosphate/5 mM EDTA buffer (pH 8) and 0.1 ml OPT stock (1 mg ml 7 1 in methanol). Fluorescence was measured at 420 nm with excitation at 350 nm.

Measurement of chromatin-bound PCNA

Cells were washed twice with PBS after treatment and incubated in lysis buffer (10 mM Tris-HCl, 2.5 mM MgCl2, 1 mM phenyl methyl sulfonyl ﬂuoride (PMSF), 0.2% NP-40) for 10 min at 48C and washed with PBS before ﬁXing with chilled 99.5% methanol then kept at – 208C for 20 min. Methanol was removed by centrifugation at 800g and nuclei were washed twice with PBT (1% BSA and 0.2% Tween- 20 in PBS). The pellet was gently resuspended in 200 ml anti-proliferating cell nuclear antibody (PCNA) solution (diluted 1:50 in PBT) and incu- bated at room temperature for 3 h. Primary antibody containing solution was removed by centrifugation and the pellet resuspended in 200 ml FITC-conjugated anti-mouse IgG (diluted 1:100 in PBT). After incubation at room temperature for 30 min in the dark, cell were washed with PBS and resuspended in PBS containing PI (10 mg ml 7 1). After 30 min of incubation, samples were analysed on ﬂowcytometer (FACS Calibur, Becton Dickenson).

Results
Cell survival

Effects of 2-DG (5 mM) equimolar concentration with glucose and 6-AN (5 mM) present during the liquid holding (HBSS; 4 h) before and after irradia- tion on the dose – response of exponentially growing BMG-1 cells are shown in ﬁgure 1. The values of Dq (quasi threshold dose), which is a measure of shoulder region, was reduced from 0.90 to 0.50 Gy by both 2-DG or 6-AN, while the combination (2- DG + 6-AN) further reduced Dq to 0.20 Gy, with no signiﬁcant changes being observed in Do (slope of exponential region of survival curve reﬂecting the dose required to reduce survival to 37% also referred to as D37). Neither 2-DG nor 6-AN (alone) had any signiﬁcant effects on the clonogenicity of unirradiated cells (data not shown). Reduction of shoulder in dose – response curve suggests that a decrease in the protection or inhibition of repair processes could be responsible for enhanced cell kill by these agents. The concentration dependency of radiosensitiza- tion by 6-AN was studied in BMG-1 cells at a constant 2-DG concentration of 5 mM. 6-AN alone or in combination with 2-DG enhanced the radia- tion-induced cell death signiﬁcantly even at 2.5 mM. A further increase in the concentrations of 6-AN up to 10 mM did not show any additional cell death (ﬁgure 2).

Figure 1. Dose – response of gamma-ray-irradiated exponentially growing BMG-1 cells in the presence of equimolar concentrations of 2-DG (5 mM) with glucose or 6-AN (5 mM), or 2-DG plus 6- AN present during radiation and a 4-h post-radiation liquid holding. Values are the mean ( + SD) of ﬁve experiments. All treatments groups are signiﬁcantly different from 2.5 Gy (p 5 0.04).

To investigate the possible variability in the radio- modifying effects of the combination (6-AN and 2- DG), the responses of BMG-1 cells were compared with a human squamous carcinoma cell line (4197) at a therapeutically relevant dose of 2.5 Gy (table 1). While both 2-DG and 6-AN decreased the survival of gamma-ray-irradiated BMG-1 cells by the same extent (18%), a signiﬁcantly higher effect (45%) was observed with 6-AN alone in case of 4197 cells. Irradiation of 4197 cells in the presence of 2-DG (5 mM) had no effect on cell survival in agreement with our previous observations (Dwarakanath et al. 2001). However, the combination (2-DG + 6-AN) enhanced the cell death of both BMG-1 and 4197 cells by nearly 35 and 50% respectively (table 1).

Apoptosis

Since, radiosensitivity of cells could be determined by measuring the mitotic and interphase (apoptotic) death (Akagi et al. 1995), we investigated the effects of 2-DG and 6-AN on radiation-induced apoptosis in BMG-1 cells. EXternalization of phosphatidylser- ine on account of membrane asymmetry changes during apoptosis was probed using FITC-labelled annexin V (Vermes et al. 1995). Signiﬁcant changes in the annexin V-positive cells could not be observed at 24 h after irradiation as compared with the unirradiated cells. The presence of 6-AN or 2- DG also did not show any statistically signiﬁcant increase on the formation of annexin V-positive cells at this time interval. However, when cells were treated with the combination 6-AN + 2-DG, more than 50% of cells were observed to be annexin V positive at this time (ﬁgure 3a), indicating that combined treatment with 6-AN + 2-DG consider- ably enhances the apoptotic cell death in irradiated cells.

Figure 2. Effects of varying concentrations of 6-AN (0 – 10 mM) and a constant concentration of 2-DG (5 mM) on cell survival studied by macrocolony assay in exponentially growing BMG-1 cells following gamma-ray (D = 2.5 Gy) irradiation. Data have been normalized to the zero dose in 6-AN irradiated cells. Values are the mean ( + SD) of ﬁve experiments.

Figure 3. Effects of 2-DG (5 mM) and 6-AN (5 mM) on (a) the fraction (%) of micronuclei (M) formation and (b) the per cent of annexin V positive BMG-1 cells observed 24 or 48 h following irradiation. Acetic methanol ﬁXed cells were stained with DAPI for the detection of micronuclei and live cells were labelled with annexin V-FITC for the detection of externalization of phospho- tidylserine as a function of time up to two to three doublings after irradiation.

Micronuclei induction

The induction of mitotic death was studied by investigating the effects of the modiﬁers (2-DG and 6-AN) on radiation-induced micronuclei expression (MN) in BMG-1 cells. Both 2-DG (5 mM) and 6- AN (5mM) enhanced radiation-induced micronuclei formation by nearly 50%, while approXimately 90% increase could be obtained with the combination (2- DG + 6-AN) at 24 h (ﬁgure 3b). Neither 2-DG nor 6-AN alone or in combination induced any signiﬁ- cant change in micronuclei expression in the unirradiated cells (data not shown).

GSH levels

The effects of 2-DG and 6-AN on total GSH levels in exponentially growing BMG-1 and 4197 cells were studied as a function of time following irradiation. Signiﬁcant post-irradiation differences between the effects of 2-DG on the two cell lines BMG-1 and 4197 were observed (ﬁgure 4). The presence of 2-DG or 6-AN alone in unirradiated cells did not inﬂuence the GSH levels in BMG-1 cells. In BMG-1 cells, the levels of GSH decreased continuously with time up to 4 h following radiation (ﬁgure 4a). In contrast, in 4197 cells, which show higher basal GSH levels than BMG-1 cells, the initial decrease in GSH content after irradiation was followed by a recovery that was nearly complete by 4 h following irradiation (ﬁgure 4b). The presence of 2-DG slightly reduced the recovery of GSH levels; while the presence of 6-AN completely inhibited the recovery over 4 h in irradiated 4197 cells (ﬁgure 4b). It is noteworthy that the combination of 6-AN with 2-DG resulted in a profound decrease in the GSH levels of both the cell lines that did not recover till 4 h following irradiation.

Figure 4. Fluorometric measurements of GSH level in cell lines by using O-phthaldehyde as a ﬂuorescent reagent. Fluorescence was measured at 420 nm with excitation set at 350 nm. Modiﬁcations of cellular GSH levels were measured in gamma-ray-irradiated (a) BMG-1 and (b) 4197 cells by 2-DG (5 mM) and 6-AN (5 mM). The inhibitors were present during irradiation and for 4 h during liquid holding. Values are the mean ( + SD) of four experiments.

Growth delay and cell cycle perturbations

Treatment-induced division delay and cell cycle perturbation has been shown to inﬂuence the radiation response of mammalian cells. To investi- gate the role of altered cell cycle perturbations in the radiosensitization induced by these modiﬁers, the effects of 2-DG and 6-AN on radiation-induced growth inhibition and cell cycle perturbations were studied in BMG-1 cells. An absorbed dose of 2.5 Gy delayed the progression of cells slightly through the cell cycle and resulted in a marginal growth inhibi- tion (about 15%) at 24 h following irradiation (table 2). Proliferation was delayed further when cells were irradiated in the presence of 2-DG or 6-AN. The presence of 6-AN resulted in a 20% growth inhibi- tion, whereas 35% growth inhibition was observed in the presence of 2-DG. Interestingly, the combination (2-DG + 6-AN) induced signiﬁcantly greater (70%) inhibition in the growth (table 2) with considerable effects on the progression of cells through the cell cycle (table 2). Under the present experimental conditions, 2-DG (5 mM) and 6-AN (5 mM) en- hanced the radiation-induced G2 block besides a marginal accumulation of cells in S phase almost to the same extent. However, the combination signiﬁ- cantly enhanced the accumulation of cells in S phase and decreased the number in G2/M phase at 24 h, implying a considerable inhibition in the DNA synthesis under these conditions. To investigate these disturbances further, cells labelled with BrdU were chased immediately following treatment for the next 24 h at 4-h intervals. Pulse-chase measurements clearly showed that the combination of 6-AN and 2- DG induced a considerable amount of delay in the progression of cells from S to G2/M and G1 phase following irradiation (ﬁgure 5). For example, while more than 70% of the irradiated S-phase cells (BrdU-positive cells) had entered G1 phase by 12 h, nearly 50% cells treated with either 2-DG or 6-AN entered the G1 phase and less than 30% of the cells treated with the combination could enter the G1 phase at this time (table 3). Further, an excess of nearly 50% of the labelled cells was still in the early S phase following the combined treatment of 2-DG + 6-AN (22%) as compared with irradiated cells (14%) at 4 h.

Figure 5. Effects of 2-DG (5 mM) or 6-AN (5 mM) or a combination of 2-DG + 6-AN on the distribution of BrdU-positive cells in different phases of the cell cycle chased following gamma-ray irradiation (2.5 Gy) of exponentially growing BMG-1 cells. Cells were pulse labelled with 10 mM BrdU for 15 min at 378C immediately after treatments; after washing, cells were resuspended with complete media, allowed to progress through the cell cycle and collected at different time intervals for analysis by ﬂow cytometery.

PCNA and radiation response

PCNA is one of the key adapter proteins involved in the regulation of a number of nuclear transactions including the modulation of DNA repair and DNA synthesis as a part of cellular response to radiation damage. We therefore investigated the effects of these metabolic modiﬁers on the chromatin bound PCNA following irradiation of exponentially growing BMG- 1 cells. Our results show an elevation in the level of chromatin bound PCNA following irradiation imply- ing its recruitment in response to radiation damage, which remained high till 24 h post-irradiation (ﬁgure 6). However, the recruitment of PCNA was signiﬁcantly reduced in the presence of 2-DG or 6-AN, with the levels decreasing profoundly in cells treated with both the metabolic modiﬁers (6-AN + 2-DG).

Figure 6. Alterations in chromatin-bound PCNA induced by 2- DG (5 mM) and 6-AN (5 mM) in BMG-1 cells observed 24 h after irradiation. Nuclei were isolated and incubated with anti- PCNA antibody at room temperature for 3 h then analysed with ﬂow cytometery by using FITC-conjugated anti-mouse IgG as secondary antibody.

Discussion

Our studies have shown that while 2-DG, an inhibitor of glucose transport and glycolysis, enhanced the radiosensitivity of the glioma cell line maintain a proper redoX state. Note that a 50% BMG-1, it had no signiﬁcant effect in 4197 cells under similar conditions (table 1). On the other hand, 6-AN, which inhibits the pentose phosphate pathway (PPP), enhanced the radiosensitivity of both the cell lines, with the combination (2-DG + 6-AN) showing a moderately synergistic effect. Present data suggest that the combination of 2-DG with inhibitors of the alternate pathway (PPP) involved in the generation of different types of metabolic energy decrease in surviving fraction and a severe depletion of GSH content was observed in 4197 cells when irradiated in the presence of 6-AN, while 2-DG had no inﬂuence on either the survival or GSH content of irradiated 4197 cells (table 1 and figure 4). There- fore, it appears that due to the diverse mechanisms by which radiation affects cell survival and the multiple functions in which GSH is involved, its depletion below a critical value enhances the therapy. The differences in the efﬁcacy of this combination in BMG-1 and 4197 cells could be attributed at least partly to the variations in glucose metabolism of the two cell lines particularly, relative contributions of glycolytic and pentose phosphate pathways.

AN caused a remarkable decrease in GSH levels of irradiated cells (ﬁgure 4). Severe depletion of GSH might contribute to the increased radiosensitivity of cells due to the stimulation of the processes of apoptosis resulting in loss of clonogenicity. The mechanisms by which GSH depletion by these modiﬁers could enhance radiosensitivity include impairment in the regulation of speciﬁc gene products involved in DNA repair. Recent studies in murine and human tumour cell lines have shown a reduction of Ha-, Ki and N-ras expression after GSH depletion by post-transcriptional mechanism (Miller et al. 1993).

The present results suggest that the relative contribution of PPP with respect to glycolysis is expected to be higher in 4197 cells as compared with BMG-1. This is indicated from the observations in 4197 cells where the level of GSH decreased immediately after irradiation, followed by a rapid recovery resulting in restoration of GSH to a pre- irradiation level within 4 h. This suggests that in these cells (4197) the intracellular reducing environ- ment is maintained due to a higher contribution and activation of PPP. NADPH regenerated by PPP is used by glutathione reductase to convert GSSG to GSH. In contrast, due to the lower contribution and activation of PPP, BMG-1 cells may be unable to radiation in combination with metabolic modiﬁers.

Cells treated with 6-AN are unable to produce NADPH from PPP. NADPH depletion due to PPP inhibition may have a signiﬁcant role in the enhancement of radiation damage by 6-AN. The formation of NADPH by PPP is also required for the maintenance of reduced GSH levels. Glutathione functions as a reducing agent for the maintenance of Reduction in NAD+ levels due to competitive synthesis of 6-aminoNAD in the presence of 6-AN has been proposed as one of the mechanisms responsible for enhanced radiosensitivity. The me- chanism suggested for the enhancement of the radiation effect by 6-AN is inhibition of poly-ADP ribose polymerase (PARP) activity (Hunting et al. 1985). PARP is a chromosomal enzyme that catalyses the transfer of adenosine diphosphoribose moieties from NAD+ into poly-(ADP-ribose). Treatments that cause DNA damage and strand breaks stimulate the activity of PARP and the time repair of DNA damage (Mitchell & Russo 1987). Cellular redoX status regulates cell function, includ- ing activation, proliferation and differentiation, and possibly DNA repair (Morales et al. 1998). Our data clearly show that the combination of 2-DG and 6- with the unscheduled DNA repair of DNA breaks. Further, inhibitors of PARP impair the ability of cells to rejoin DNA strand brakes and/or repair alkaline- labile DNA damage (Jeggo 1998). 6-AN has been shown to inhibit PARP activity in permeabilized L1210 cells (Smulson et al. 1977) and therefore may impair PARP-dependent repair/recovery processes leading to enhanced cell death.

Radiation-induced DNA and non-DNA damage elicit cellular responses by way of DNA repair, cell cycle perturbations and cell death through multiple processes by inducing alterations in signal transduc- tion pathways involved in all three responses. These responses are mutually non-exclusive and have many common regulators. PCNA is an essential compo- nent of eukaryotic chromosomal DNA replisome and has the ability to interact with multiple partners, which are involved in several metabolic pathways such as DNA repair, translesion DNA synthesis, chromatin remodelling and cell cycle regulation (Maga & Hubscher 2003). Cell cycle check points (G1 and G2 blocks) purporting to facilitate cellular recovery lead to enhanced survival under favourable (such as lower levels of DNA damage, optimal cellular energy and thiol status) conditions. In the present studies it was observed that 2-DG or 6-AN and (2-DG + 6-AN) enhanced the radiation-induced G2 block, while the combination enhanced the accumulation of cells in S phase implying a delay in progression of DNA synthesis under severe energy-limiting conditions. PCNA is known to interact with proteins (cyclins and cyclin-dependent kinase (cdk) inhibitors) required for cell cycle control. It also plays an important role in DNA synthesis both during replication and the repair of DNA damage and it coordinates a number of cellular processes at the chromatin level in a spatiotemporal manner (Prosperi 1997, Paunesko et al. 2001). A nearly twofold increase in the fraction of chromatin- bound PCNA observed following irradiation (ﬁgure 6) suggests its involvement in the response of BMG- 1 cells to radiation damage. Since the recruitment of PCNA is known to be an energy-dependent process (Majka et al. 2004) incubation of cells with 2-DG or 6-AN is expected to inﬂuence its recruitment for repair, with the combination severely compromising this process. Therefore, the present data suggest that reduced energy-linked binding of PCNA to the template could be one of the contributing factors for the inhibition of DNA repair pathways and translesion DNA synthesis leading to enhanced ﬁXation of lesion and cell death induced by 6-AN + 2-DG besides the delay in S phase progression observed earlier under conditions of energy deﬁciency.

Necrosis and apoptosis are the two major death processes induced by a variety of cytotoXic agents that contribute to the loss of clonogenicity, while mitotic death is a major contributor following ionizing radiation (Hendry & West 1997). Indeed a lack of correlation between the induction of apopto- sis and loss of clonogenicity (cell survival) has been demonstrated in variety of tumour cell lines follow- ing irradiation, suggesting that other death processes (such as mitotic death) also contribute to the loss of survival (Steel 2001). The present results suggest that the combination 2-DG + 6-AN could facilitate radiation-induced cell death through multiple path- ways. Necrotic/mitotic cell death is linked to the induction of cytogenetic damage in the form of chromosome aberrations in the metaphase and micronuclei formation in the post-mitotic daughter cells (Krepinsky & Heddle 1983, Paglin et al. 1997). An increase in radiation-induced micronuclei for- mation observed here suggests that enhanced mitotic death is partly responsible for the sensitization observed in the presence of 6-AN and 2-DG (ﬁgure 5). Further, increases in the fraction of annexin V- positive cells following irradiation observed under energy-limiting conditions indicate a higher level of apoptotic cell death as well. Therefore, under these conditions, it appears that both mitotic and inter- phase (apoptosis) death are enhanced, thereby leading to a higher level of cell kill.

Implication for improving tumour radiotherapy

Earlier attempts to exploit the metabolic effects of 2- DG or 6-AN to enhance the efﬁcacy of ionizing radiation met with limited success because of the toXicity associated with continuous infusion required to maintain adequate drug levels for a sufﬁciently long time. The half-life of 2-DG in peripheral blood is approXimately 90 min (Mohanti et al. 1996), whereas 6-AN was found to clear rapidly, with two half-lives of 7.4 and 31.3 min in mice (Walker et al. 1999). However, the present approach of exploiting energy-linked modulation of repair and recovery processes requires the modiﬁers to be present only for a few minutes for inhibition of repair processes in tumours thus minimizing side-effects. Transient CNS disturbances mainly related to hyperglycaemic and cellular glucopenia have been observed following the administration of high doses of 2-DG (Landau et al. 1958, Meldrum & Horton 1973, Thompson et al. 1981), while with 6-AN vitamin B1 deﬁciency syndrome in addition to transient neurological disturbances have been reported (Herter et al. 1961). The data presented here indicate that exposure to low doses of 6-AN along with 2-DG before irradiation would be more effective to enhance the effect of radiation in vitro in cell systems showing a high glucose ﬂuX through glycolysis or the PPP. Oral administration of pharmacologically re-
levant doses of 2-DG in human cancer patients (200 – 300 mg kg 7 1 bw) in combination with radio- therapy indicate a slight beneﬁt in Phase I/II studies and dose escalation studies for the management of malignant glioma patients (Mohanti et al. 1996, Singh et al. 2004). The doses of 2-DG and 6-AN required for radiosensitization when used in combi- nation are expected to be lower than the doses required when either one of them is used individually and therefore could reduce the undesirable toXicity. Preliminary observations in Ehrlich ascites tumour- bearing animals have shown that combination of 6- AN (2 mg kg 7 1 bw) and 2-DG (2 mg kg 7 1 bw) results in a cure rate (tumour-free survival) of more than 80%. Indeed, a similar cure rate was obtained even when 6-AN dose was reduced to 1 mg kg 7 1 bw (Varshney et al. 2004). Further, the dose of 6-AN used in the present studies (2 mg kg 7 1 bw) is much lower than the LD50 dose (35 mg kg 7 1 bw) as well as the dose of (16 mg kg 7 1 bw) that was used earlier Dwarakanath BS, Zolzer F, Chandana S, Bauch T, Adhikari JS, Muller W, Streffer C, Jain V. 2001. Heterogeneity in 2-deoXy-tumours in nude mice (Life Sciences Update 2001). These observations have important therapeu- tic implications as the dose-related toXicity associated with 2-DG can be reduced signiﬁcantly when used in combination with 6-AN. Further studies to evaluate the radiosensitivity of various dose combinations in tumour models are warranted before contemplating clinical trials with the combi- nation of 2-DG + 6-AN. It is therefore expected that the combination of 2-DG with 6-AN (at a low dose) could further improve the therapeutic efﬁcacy of radiotherapy in the treatment of malignant tumours.

Acknowledgement

The authors are grateful to Lt Gen. T. Ravindranath, Director, INMAS, Delhi, for support and constant encouragement; and to Ms Divya Khaitan for technical support during experimentation. Work was carried out as part of a project supported by Grant INM-301 from the Government of India.