INT. J. RADIAT. BIOL., 1985, VOL.. 47, NO. 1, 103-112

Enhanced cell killing, inhibition ofrecovery from potentially lethal damage andincreased mutation frequency by 3-aminobenzamidein Chinese hamster V79 cells exposed to X-rays

ASHOK KUMARt, J. KIEFER, E. SCHNEIDERand N.E.A. CROMPTON

Strahlenzentrum der Justus-Liebig-Universitat,D-6300 Giessen, F. R. Germany

(Received 10 April 1984; revision received 5 July 1984; accepted 23 July 1984)

X-ray induced potentially lethal damage and its inhibition by the aromaticamide 3-aminobenzamide have been investigated in Chinese hamster V79 cells.3-Aminobenzamide (3-AB) is a known inhibitor of polyadenosine diphos-phoribose synthetase. With increasing concentrations of 3-AB an increasinginhibition of PLD repair was observed. Little inhibition of PLD repair wasseen when 3-AB was added 3 h following irradiation. Utilizing the 6-thioguanine mutation assay, the effect of poly(ADP-R) synthetase inhibitionunder conditions of PLD repair upon mutation frequency were also studied. Alarge increase in mutation frequency following 24 h post-irradiation recovery inthe presence of 3-AB was seen. These results favour a possible role of 3-AB inpreventing repair by facilitating early damage fixation before repair can occur,simultaneously reducing G2-arrest.

Indexing terms: potentially lethal damage (PLD), poly(ADP-R) synthetase,mutation, repair.

1. IntroductionPolyadenosine diphosphoribose (poly(ADP-R)) has been implicated in the

repair of DNA damage. Its exact function, however, is not known. It has beenshown that the synthesis of poly(ADP-R) in mammalian cells is greatly enhancedfollowing the production of DNA-strand breaks by chemical and physical agents,including X-irradiation (Benjamin and Gill 1980, Berger et al. 1978, 1979, 1980,Davies et al. 1978, Durkacz et al. 1980, Durrant and Boyle 1982, Jacobson et al.1980, Jurez-Salinas et al. 1979, McCurry and Jacobson 1981, Miller 1975, Ndukaet al. 1980, Nolan and Kidwell 1982, Skidmore et al. 1979, Sudhakar et al. 1979).Recently poly(ADP-R) has been reported to activate DNA ligase, suggesting aninvolvement in the last step(s) of the repair process(es) (Cleaver et al. 1983,Creissen and Shall 1982, Ohashi et al. 1983). This view is strengthened by datashowing that specific inhibitors of poly(ADP-R) synthesis enhance cell killing andinhibit DNA strand break rejoining with alkylating agents and with ionizingradiation (Durkacz et al. 1980, Nduka et al. 1980, Ben Hur et al. 1984b) but notwith ultraviolet radiation (Cleaver et al. 1983).

In the present study the effect of 3-aminobenzamide on cell survival, recoveryfrom potentially lethal damage and on the induction of mutants in Chinesehamster V79 cells exposed to X-rays was investigated. 3-Aminobenzamide has

tAlexander von Humboldt Fellow, present address: Department of Bio-Sciences, H. P.University, Summer-Hill, Simla, India.

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been shown to be a putative specific inhibitor of poly(ADP-R) synthetase (Purnelland Wish 1980). It has also been shown to inhibit de novo synthesis of DNApurines and thymidine (Cleaver et al. 1983). 3-AB also affects glucose metabolism(Milam and Cleaver 1984).

2. Materials and methods2.1. Cell culture

Chinese hamster V79 A cells were used and were routinely checked to be free ofmycoplasma using Hoechst 33528 stain, and examined under the fluorescentmicroscope. They were grown as monolayers in plastic Petri dishes (10cm) at37°C in a humidified atmosphere at 5 per cent CO2 and 95 per cent air inDulbecco's modified minimum essential medium (MEM) (Gibco), supplementedwith 7-5 per cent foetal calf serum (FCS) (Gibco).

2.2. Drug toxicity3-Aminobenzamide (3-AB; Sigma Chemical Company Ltd, Muinchen) was

dissolved in DMSO. The solvent controls were included in treatment regimes. Atotal of 105 cells were seeded into each well of 12 well dishes (Linbro) with 1 ml ofcomplete medium, containing 0, 1, 5, 10, 20 or 40mM of AB, and grown for 24h.At the end of this period cell numbers were determined by haemocytometer countsfor the AB-treated and untreated cultures after trypsinization.

2.3. X-irradiation and drug exposureExponentially growing asynchronous, V79A cells were irradiated with X-rays

(300kV, 10mA, Be window equivalent to 5 mm Al) at room temperature in air.The dose rate of 1 25 Gy/min ( 5 per cent) was determined by ferrous sulphatedosimetry and was monitored with Duplex ionization chamber (PTW, Dr.Pychlau, Freiburg, F.R.G.). The cells were irradiated as monolayers in Hank'sbalanced salt solution (HBSS). 3-AB was added 1 h before irradiation, cells wereincubated with various concentrations of 3-AB for 3 h after irradiation or cells werepre-incubated for 1 h and kept for a further 3 h after exposure. After that time thecells were washed in MEM twice and then incubated in MEM plus 75 per centFCS for 8 days to assess colony formation.

2.4. Determination of sublethal damage recoverySublethal damage recovery was estimated by the split dose method (Elkind and

Sutton 1959). Exponentially growing V79A cells were incubated with 20mM 3-ABjust after the first radiation dose (5 Gy). Immediately after receiving the secondradiation dose (5 Gy) given at various times after the first exposure, the cells werewashed and incubated in MEM plus 75 per cent FCS for colony formation. Controlcells were subjected to the same protocol but without the drug.

2.5. Determination of recovery from potentially lethal damageRecovery from potentially lethal damage was measured in density-inhibited

stationary phase cells. Some 105 cells and 1 ml MEM plus 7-5 per cent FCS(Gibco) were seeded into each well of 12 well plates (Linbro). The medium waschanged daily from the third day after seeding. On the seventh and eighth days thecells reached the density-inhibited stationary phase. Cell growth was monitored by

A. Kumar et al.104

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Inhibition and mutation by 3-AB

measuring the cell number per well; on day 8 it was 35 x 106 /well.The distribution of DNA in cells was measured by flow cytofluorimetry after

ethidium bromide mitramycine staining, and showed approximately 80 per cent ofthe cells having a DNA content equal to that shown by cells in G1 (F. Otto, privatecommunication). Such cultures were used because of their ability to repair PLDand because of the low fraction of cells in mitosis.

To determine the time course of inhibition of recovery from potentially lethaldamage (PLD), just before irradiation the medium was removed and HBSS wasadded to the stationary phase cells. Immediately after X-irradiation (10 Gy) theywere incubated with 0, 10, 20 or 40 mM 3-AB for 6, 18 or 24 hours before theywere plated to determine colony formation. To test whether 3-AB inhibits therecovery from PLD if added some time after irradiation, cells were treated with20mM 3-AB (sufficient to inhibit recovery from PLD) 3 h after irradiation for 6, 18or 24 hours and then replated for colony formation without the chemical.

For complete survival curves, stationary phase cells were irradiated withvarious doses of X-rays and either plated immediately (IP) or after a 24 h holdingperiod (DP) with or without 3-AB.

2.6. Mutation inductionInduction of forward mutations leading to 6-thioguanine (6-TG) resistance

was studied in stationary phase cells. Survival and mutation frequency wereassessed immediately after irradiation or after 6 and 24h post-irradiationincubation with or without 40 mM 3-AB.

For the determination of mutation frequency, 2 x 107 cells were plated in two14-cm Petri dishes (107 cells each). They were subcultured in MEM containing 7-5per cent FCS at 48-h intervals regularly for 8 days to allow phenotypic expression(O'Neill and Hsie 1979). At the end of the expression period, the cells weretrypsinized, pooled and plated at a concentration of 106 in two 14cm Petri dishes,with selection medium containing 6-TG at a concentration of 10/ug/ml. Anappropriate dilution of the cell suspension was also plated into drug-free mediumto assess the plating efficiency at the end of the expression period. Mutationselection plates were fixed after 10-12 days of incubation, the others after 7 days ofgrowth. Colonies with more than about 50 cells were scored as survivors. Themutation frequency was calculated by dividing the number of mutant colonies bythe number of cells plated multiplied by the plating efficiency. The mutationexperiments were done three times. Values were averaged from three independentexperiments, which also gave error limits.

3. Results3.1. Effect of 3-AB on the growth of V79 A cells

The table shows the effect of 3-AB on the proliferation of exponentiallygrowing V79A cells. Cells were grown in the presence of different concentrationsof 3-AB for 24h. No significant effect on cell growth was observed up to 20mM3-AB and only a slight inhibition at 40mM. The plating efficiency was also onlyslightly decreased. These results suggest that poly(ADP-ribose) synthesis maynot be essential for the normal cell cycle progression.

3.2. Enhanced cell killingIn cells exposed to 3-AB after irradiation there is an enhanced cell killing,

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A. Kumar et al.

Effect of 3-AB on the growth of V79 A cells

3-AB (mM) NAB/No PE (% )

0'0 10 83-51.0 1.0 80.050 099 800

100 097 760200 091 780400 086 750

Initial cell concentration 1 x 105 cells/mi. NAB, number of cells in 3-AB treated cultureafter 24 h incubation; No, number of cells in untreated control after 24 h incubation.

compared to cells exposed to X-irradiation only, in a concentration dependentmanner (figure 1). Pre-irradiation incubation with 3-AB caused a smallersensitization which was, however, additive to that caused by post-irradiationincubation.

3.3. Sublethal damage recoverySublethal damage recovery was not significantly affected by incubation with 3-

AB during the intervals between the two irradiations. The cells incubated with 3-AB showed full recovery but lacked the drop in survival seen at the 4 h interval incontrol cells (figure 2). All experiments were repeated three times. Each data pointis based on the values averaged from the three independent experiments. Errorbars represent the s.d. of the mean values.

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0.01

00012 4 6 8 10

DOSE/GY12

Figure 1. Survival of X-irradiated Chinese hamster V79 cells. Cells were treated with 3-AB1 h before irradiation, 3 h post-irradiation or 1 h before and 3 h post-irradiation.Error bars determined from three separate experiments.

106

* . )

t

o ONTROL

: 20mM AB (lhr PRE )

: 20mM A(3hr POST)

20mM AB(lhr PRE. 3hr POST)

O 40mM AB(3hr POST)

* 40mM AB(lhr PRE. 3hr.POT)

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Inhibition and mutation by 3-AB

In

z0

0.5

0.01I 2 3 /4

TIME/ hours

5 6 7

107

Figure 2. Split dose recovery with and without 3-AB (20mM). Two doses of 5 Gy wereseparated by the time interval given on the abscissa. The cells were washed free ofthe drug after the second fraction. Error bars determined from three separateexperiments.

3.4. Recovery from potentially lethal damageTo obtain information on the time course of 3-AB recovery inhibition, 3-AB

was added at different concentrations immediately after irradiation of stationary

phase cultures which were then plated at different times after irradiation. The

survival increase due to recovery from potentially lethal damage observed in thecontrol cells was gradually reduced as the 3-AB concentration increased and was

almost totally inhibited with 20mM 3-AB (figure 3). A further increase in the drug

concentration produced a further decrease in survival. However, when stationaryphase cultures were incubated with 20 mM 3-AB (concentration sufficient to inhibit

1.0

z0I-0

UL

z° 0.1

In

0.010 10 20 30

TIME /hours

Figure 3. Kinetics of recovery from potentially lethal damage. Cells were treated for I hbefore and various times after irradiation, as given on the abscissa, with a range ofdrug concentrations. With one set, the drug treatment was started 3 h after exposure.Error bars determined from three separate experiments.

CONTROL

20mM AB

I I I I I I I

o CONTROL

* 20 mM AB (3hr AFTER IRRAD)

o 10 mM AB ( hr PRE -POST)

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A. Kumar et al.

recovery from PLD) 3 h after irradiation, and the cells were plated 6, 18 and 24hours post-irradiation, there was no significant inhibition of recovery from PLD.This shows that 3-AB interferes with an early step of the recovery process.

Full survival curves with 24 h recovery period and various concentrations areshown in figure 4. At 20mM, the survival curve was almost the same as thatobtained for immediately plated control cells. For higher concentrations the maineffect appeared to be a reduction in shoulder width with little change in the finalslope. Thus the application of 3-AB resulted in an inhibition of recovery fromPLD, which influences the survival curve at low concentrations in a dosemodifying manner and at higher concentrations affects only the shoulder.

3.5. Mutation inductionMutation induction is shown in figure 5. Background mutation frequency in

unirradiated control was generally 5-6 per 106 viable cells. Values plotted in thefigure are the net mutation frequencies obtained after subtracting the background.To study the kinetics of repair of premutational damage, stationary phase cellsexposed to 10 Gy of X-ray were trypsinized and plated immediately or after 6 or24h of incubation in HBSS with or without 3-AB (40mM). Untreated cellssubjected to post-irradiation recovery from PLD showed an appreciable decreasein the mutation frequency. Therefore, in cells irradiated and plated immediately, alarger fraction of cells which survive, repair the damage by error-prone pathwayswhile the cells which undergo post-irradiation recovery do so by an error-freepathway. However, cells incubated with 40mM 3-AB and subjected to post-

2C

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27-Ur,

RADIATION DOSE GY

Figure 4. Survival curves for stationary phase V79A cells measured after irradiation and24 h treatment with different 3-AB concentrations. Survival curves are also shownfor cells plated immediately after irradiation (IP) and 24 h after irradiation-delayedplating (DP)-without chemicals. Error bars determined from three separateexperiments.

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Inhibition and mutation by 3-AB

0 6 12 18 24. 30

POST-IRRADIATIONINCUBATION TIME /hrs

Figure 5. Kinetics of mutation frequency in stationary phase V79A cells exposed to 10 Gyof X-rays, incubated with 40mM 3-AB to assess PLDR and then trypsinized andplated after different durations of incubation (). Untreated cells were plated insimilar conditions without chemical (0). Error bars determined from threeseparate experiments.

irradiation recovery showed increased mutation frequencies. 3-AB alone did not

induce any mutations.

4. DiscussionWe have observed that 3-AB significantly enhances radiation-induced cell

killing in a dose-dependent manner. Pre-irradiation incubation with 3-AB causes amuch smaller sensitization. Similar observations have been reported by Ben Hur etal. (1983) in Chinese hamster V79 cells using nicotinamide as an inhibitor ofpoly(ADP-R) synthetase. Ben Hur et al. (1984b) have also shown that enhancedcell killing by 3-AB and similar chemicals depends on their potential as ADPRT

inhibitors, thus implicating poly(ADP-R) in repair. The exact mechanism bywhich inhibitors of poly(ADP-R) synthetase enhance radiation cell killing is not

known. At least three mechanisms might be considered to explain the effect of 3-

AB (or the role of poly(ADP-R) polymerase in repair): (1) prevention of rejoining

of DNA strand breaks; (2) inhibition of activity of ligase; (3) reduction in the

radiation-induced G2 delay.Hypothesis (1) has contradictory support. Durkacz et al. (1980), Gray et al.

(1981) and Vikhanskaya and Martynova (1983) have reported that 3-AB inhibits

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A. Kumar et al.

radiation-induced single strand break rejoining. Zwelling et al. (1982) reportedthat rejoining of single strand breaks is not inhibited but only slowed down. Incontrast to these results, Bohr and Klenow (1981) found stimulation of DNAstrand break repair in human lymphocytes in the presence of ADPRT inhibitorsand Althaus et al. (1982) report an acceleration of DNA strand break removal.Lunec et al. (1984) reported that radiation-induced DNA strand breaks are fullyrepaired, although slowly, even under conditions of 95 per cent ADPRT inhibitionby 3-AB. The apparently complete repair of strand breaks might suggest that theseare unrelated to cell killing. Although this may be true we do not believe that thekinds of techniques used for measurement have enough resolution to permit theconclusion that every break is repaired (for example, enough single strand breaksmay remain open to be consistent with a double break model for cell killing).Further, misrepair of single or double strand breaks could not be detected. It maytherefore be postulated that inhibition of poly(ADP-R) synthesis results in misrepair(error prone). In support of this hypothesis is our observation on mutation inductionin stationary phase cells, in which we found that without drug treatment there is adecrease in mutation frequency when they are allowed to recover for certain post-irradiation times, whereas cells treated with 3-AB showed significantly highermutation frequencies. Furthermore, it has been reported by Natarajan et al. (1982)that there is an increase in the frequency of chromosome aberrations in X-irradiatedChinese hamster V79 cells after 3-AB-which may be associated with non-repair ormisrepair of double strand breaks in DNA (Bender et al. 1974, Natarajan et al. 1980).

Support for hypothesis (2) comes from Ohashi et al. (1983), who have shownthat ligase activity is inhibited by histones, but in the presence of ADP-ribosesynthetase an almost complete reversal of this inhibition takes place. If we acceptthat 3-AB could affect the final step of repair, through action on ligase, then thereshould be inhibition of double strand break repair. However, it has been reportedthat there is no inhibition of DSB-rejoining by 3-AB (Vikhanskaya and Martynova1983). Further, in our experiment we have observed that there is no inhibition ofrecovery from potentially lethal damage if 3-AB is added 3 h after irradiation, ascompared to significant inhibition of recovery from PLD observed if cells areexposed to 3-AB immediately after irradiation (figures 4 and 5). It has also beenreported by Ben Hur et al. (1983) that nicotinamide is not effective in cell killing ifadded 1 h after irradiation. However, it may be possible that the repair is a veryfast process and is completed within a very short period.

Enhanced cell killing may be explained by hypothesis (3). The G2 delay seemsto be very important for the repair of high-LET radiation damage (Lcke-Huhle1982). Rowley (1983) showed that the duration of radiation induced G2 -arrest wasmarkedly decreased by the presence of 3-AB or nicotinamide in a similar mannerto caffeine. Caffeine is known to inhibit poly(ADP-R) synthetase (Purnell andWish 1980). Brown et al. (1984) reported the order of the effect of these threeADP-R synthetase inhibitors to be caffeine>3-AB> nicotinamide. These dataimply that adenosine-ribosylation may be necessary for the induction of G2 -arrestby radiation. The action of 3-AB could then be explained by assuming that itprevents repair by facilitating early damage fixation before repair can occur. Themechanism thus implied does not postulate a direct interference with the repairprocess by the 'gating' of lesions into an error-prone pathway. This hypothesis isin line with our mutation results and may also explain why 3-AB is only effective ifpresent early after irradiation. Poly(ADP-R) has been shown to bind to histone

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Inhibition and mutation by 3-AB 111

H1, significantly impairing chromatin condensation (Poirier et al. 1982). The

relaxation of chromatin may facilitate DNA repair and could also play a role in the

induction of G 2 -arrest. Zolan et al. (1982) have reported a general rearrangement

of mammalian chromatin structure, seen as an increase in nuclease sensitivity,

following excision repair.In the present experiment recovery from potentially lethal damage was

significantly inhibited but there was no inhibition of sublethal damage repair. It

seems, therefore, that sublethal damage is not repaired by a poly(ADP-R)

dependent pathway, an observation consistent with the results of Ben Hur et al.

(1984 a).

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