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Increasing the susceptibility of the rat 208F fibroblast cell line toradiation-induced apoptosis does not alter its clonogenicsurvival dose-response

Citation for published version:Aldridge, DR, Arends, MJ & Radford, IR 1995, 'Increasing the susceptibility of the rat 208F fibroblast cellline to radiation-induced apoptosis does not alter its clonogenic survival dose-response', British Journal ofCancer, vol. 71, no. 3, pp. 571-7.

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Increasing the susceptibility of the rat 208F fibroblast cell line toradiation-induced apoptosis does not alter its clonogenic survivaldose -response

DR Aldridgel2, MJ Arends3 and IR Radford'

'Research Division, Peter MacCallum Cancer Institute, Melbourne 3000, Australia; 2 Walter and Eliza Hall Institute of MedicalResearch, Parkville 3052, Australia; 3Cancer Research Campaign Laboratories, Department of Pathology, University MedicalSchool, Teviot Place, Edinburgh EH3 8AG, UK.

Sm.mary Recent studies have suggested a correlation between the rate and incidence of apoptosis and theradiation response of particular cell lines. However, we found that increasing the rate of induction ofapoptosis in the fibroblast line 208F, by transfecting it with human c-myc, did not lead to a change in itsclonogenic survival dose-response for either '-?irradiation or "5I-induced DNA damage. It was also found thatexpression of mutant (T24) Ha-ras in the 208F line appeared to decrease the level of apoptosis per mitosisafter irradiation and inhibited the formation of nucleosomal ladders, but did not affect either the onset of themorphological features of apoptosis or the clonogenic survival dose-response of the cells to either '--irradiation or "5I-induced DNA damage. Our findings suggest that it may be incorrect to make predictionsabout the radiosensitivity of cells based only on knowledge of their mode of death.

Keywords: apoptosis; fibroblasts; radiosensitivity; myc; ras; endonuclease

The sensitivity of both normal and transformed cell types tokilling by ionising radiation can differ markedly. For exam-ple, haemopoietic cell types are often radiation sensitive,while hepatocytes are relatively radiation resistant (Hendry,1985). Similarly, lymphomas are typically responsive toirradiation, while melanomas often respond poorly (Steel,1989). Although such generalisations about the dependenceof radiation response on cell lineage have been useful indevising tumour radiotherapy regimens, a significant con-founding factor is inter-individual differences in normal tissueand tumour response (Rofstad, 1986; Burnet et al., 1992).Accordingly, there is considerable interest in understandingthe mechanisms that produce such differences and thus devis-ing assays that will more accurately predict tumour radio-responsiveness.

Recent work has focused attention on the possibility thatsusceptibility of normal or transformed cells to radiation-induced apoptosis may be an important indicator of radio-sensitivity. Using a panel of mouse lymphoid or myeloid celllines, all of which underwent apoptosis after irradiation, acorrelation between the rapidity of induction of apoptosisand the clonogenic survival dose-response of a particularcell line was shown (Radford, 1994a). The greater sensitivityof these haemopoietic lines to radiation-induced DNAdouble-strand breaks as compared with fibroblast lines suchas V79, which die by necrosis, suggested that radiosensitivitymay be related to the mode of death (Radford, 1991, 1994a).Similarly, in vivo studies with transplantable murine tumoursshowed that elevation of both spontaneous and radiation-induced apoptosis correlated positively with growth delayand negatively with TCD50 (dose to cure 50% of animals)(Meyn et al., 1993). However, these studies also showed acorrelation between the incidence of apoptosis and tumourtype, with several adenocarcinomas found to display in-creased apoptosis and radiosensitivity (longer growth delayand lower TCD%) when compared with several sarcomas.Interestingly, one sarcoma was highly radiation sensitive inspite of showing no apoptosis following irradiation.

Because the cell lines used in the above studies havedifferent origins, we were interested in investigating the rela-tionship between mode and rapidity of cell death andradiosensitivity in cell lines with a common onrgin. Accord-ingly, we have examined the response to y-ray and DNA-associated "2I decay-induced damage of two transfectants ofthe rat lung fibroblast line 208F which express either humanc-myc (cell line RBM7) or activated Ha-ras (cell line TI).Expression of these oncogenes has been shown to differenti-ally alter susceptibility to both 'spontaneous' and serumwithdrawal-induced apoptosis, with activated Ha-ras reduc-ing the incidence of apoptosis over a 48 h period and c-mycincreasing the incidence of apoptosis over this time (Arendset al., 1993). A similar pattern of high and low levels ofapoptosis was seen in vivo using solid fibrosarcomas formedby the RBM7 and TI cell lines (Arends et al., 1994). In-creased levels of apoptosis following growth factor with-drawal have also been demonstrated in Rat-l fibroblastsexpressing c-myc (Evan et al., 1992) and in murine myeloidcells constitutively expressing c-myc (Askew et al., 1991). Itwas therefore of interest to determine how the expression ofthese genes affected the mode and rapidity of cell deathfollowing irradiation, and whether cellular sensitivity toDNA damage was altered.

Materials and methods

Cell lines: growth conditions andp53 status

The derivation of the 208F, TI and RBM7 cell lines used inthis study is described elsewhere (Spandidos and Wilkie,1984; Arends et al., 1993). Cells were grown in the alphamodification of Eagle's medium (ICN/Flow) supplementedwith 10% fetal calf serum (Commonwealth Serum Labora-tones, Melbourne, Australia). Cells were incubated at 37TC insealed flasks that had been flushed with 5% carbon dioxide,5% oxygen and 90% nitrogen. Cells were subcultured bytreatment with pronase (Calbiochem). All experiments wereperformed with asynchronous cell cultures in log-phasegrowth. Under these conditions, the cell lines had populationdoubling times of 16 (208F), 16 (T1) and 17 h (RBM7).The p53 status of each of the cell lines was examined by

immunoprecipitation as described in Radford (1994b). A

Correspondence: DR Aldridge, Peter MacCallum Cancer Institute,Melbourne 3000, AustraliaReceived 9 May 1994; revised 2 September 1994; accepted 18October 1994

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positive result was found for extracts of all three cell linesusing PAb 421 antibody, which binds to both wild-type andmutant p53 protein, whereas a negative result was found withPAb 240 antibody, which binds specifically to mutant p53(Gannon et al., 1990).

y-Irradiation, "I labelling and clonogenic assayCultures were irradiated at room temperature in a _3Cssource at a dose rate of approximately 0.9 Gy min-'. Prior toirradiation, cultures were rinsed to remove any dead cells andfresh growth medium was added. Cell monolayers for clono-genic assay were treated with 0.03% pronase in phosphate-buffered saline (PBS) plus 0.2 mM EDTA for 5 min at roomtemperature and then dispersed by pipetting. After washing,cell suspensons were counted using a Coulter counter andappropriate cell numbers were plated to give 50-100 coloniesin each of five replicate Petri dishes. Colonies of > 50 cellswere scored after 8 days' incubation at 37C. Mean (± s.e.)cloning efficiencies for 208F, TI and RBM7 were respectively0.33 ± 0.03, 0.42 ± 0.05 and 0.40 ± 0.04.

'25I labelling was performed by incubating cultures forapproximately 24 h in growth medium containing around2.5 kBq ml- ' ['OIIiododeoxyuridine (NEN/DuPont) and2.5 gLM thymidine. Cells were then washed and incubated ingrowth medium containing 20 pM thymidine and 20 gMdeoxycytidine for 3 h. After chasing, incorporated "2I wasmeasured by pelleting cells and counting radioactivity in aCompugamma CS (LKB) gamma counter. Pellets were thenresupended in a known volume and aliquots were taken forcounting to determine the number of "2I decays per cell perday. Cells were then aliquoted and frozen (at -1C per minin a controlled-rate freezer) in growth medium plus 10%dimethylsulphoxide (DMSO). Cell aliquots were removedfrom liquid nitrogen storage at various times, after knownnumbers of "2I decays per cell had occurred, and assayed forclonogenic survival. Further details are given in Radford(1991). Freezing and thawing did not significantly affect thecloning efficiency of these cell lines as evidenced by values of0.33 ± 0.02, 0.35 ± 0.01 and 0.40 ± 0.02 for 208F, TI andRBM7 respectively.A least-squares fit, using the criterion (SO- SE)2ISO, where

SE is the estimated survival and So is the observed survivalfor each experimental point, was obtained for cell survivaldata using the KaleidaGraph (Abelbeck Software) programon a Macintosh computer. The tray survival data were fittedto the equation S= exp l- (aD + AD2)] where S is survival,D is dose and a and P are constants. '"I-decay survival datawere fitted to the simple exponential S= AOexp(- DIDo),where S is survival, D is the number of 'lI decays and AOand Do are constants.

Electron microscopy and gel electrophoresis

At 24 h intervals after irradiation, non-adherent cells werecollected by giving flasks several sharp taps, removing thegrowth medium, and then rinsing the monolayer once withPBS-EDTA. The growth medium and PBS-EDTA washwere pooled and centrifuged and the cell pellets were thenfixed on ice for 30 min in growth medium (without serum)containing 0.25% glutaraldehyde and 45 min in 2.5% glutar-aldehyde before post fixation with osmium tetroxide. Cellswere then embedded in Spurr's resin and sectioned. Sectionsof control cells were obtained by fixing and embedding cellsin situ on glass coverslips.DNA degradation samples were processed as described

previously (Radford et al., 1994). Approximately 2 Lg ofDNA from each sample was electrophoresed on a 1.5%agarose gel using SPP-l/EcoRI DNA (Bresatec, Adelaide,Australia) as size markers. Pulsed-field gel electrophoresis(PFGE) was carried out on a CHEF apparatus with a hex-agonal array of electrodes (Chu et al., 1986) for 24 h at150V with a pulse time of 80s. Plugs were prepared bymixing 1 x 10' (unless otherwise stated) non-adherent oradherent cells (removed from flasks by pronase treatment as

descnbed above) in growth medium with an equal volume of1% low gelling temperature agarose (SeaPlaque, FMC) inbalanced salt solution (BSS). Plugs were then placed in NDS(10mm Tris, 0.5 EDTA, 1% lauroylsarcosine, pH 9.5) plus1 mg ml-' proteinase K for 60 min on ice followed by over-night incubation at 37C before eectrophoresis. Yeastchromosomes from Saccharomyces cerevisiae strain YP148(Pyle et al., 1988) and A DNA (BRL) were used as markers.Following electrophoresis, gels were stained with ethidiumbromide.

Meaurement of apoptosis, population expansion and mitoticfraction

Non-adherent cells were collected over 24 h intervals as de-scribed above. They were then pelleted and counted using ahaemocytometer. The level of apoptosis was determined bymixing equal volumes of non-adherent cell suspension andgrowth medium containing #g ml-1 ethidium bromide and3 ig ml-' acridine orange and then scoring approximately500 cells for apoptotic nuclear morphology by fluorescencemicroscopy. The number of adherent cells present was deter-mined by treating the monolayer with pronase and Coultercounting the suspension. The fraction of non-adherent cellswhich was apoptotic was multiplied by the total number ofnon-adherent cells, divided by the total number of cells inboth adherent and non-adherent fractions, and then multi-plied by 100 to give the percentage apoptosis.

After irdiation, all flasks were rinsed and had freshgrowth medium added at 24 h intervals. The non-adherentcells collected thus represented cells released from themonolayer over a 24 h period. A combination of 24 hmedium changes and seeding flasks at appropriate cellnumbers ensured that cell death was attributable to theeffects of irradiation and not to medium depletion. Popula-tion expansion was defined as the number of cells in themonolayer at a given time divided by the number at the timeof irradiation.The level of post-irradiation mitotic activity was deter-

mined by incubation of cultures with the mitotic spindlepoison nocodazole at 0.1 agml-' for 3h and then scoringthe fraction of metaphase-arrested cells. Further details aregiven in Radford and Murphy (1994).

Resus

Clonogenic suival dose-response is not markedly changed byover-expression ofc-myc or T24-ras

Clonogenic survival curves were obtained for the parent andtransfected cell lines exposed to either 7-irradiation or DNA-associated "2I decays. The -tray survival curves suggest thatthe overexpression of c-myc or an activating mutation ofHa-ras in these cells does not markedly affect their radiosen-sitivity (Figure 1). The similarity in the response of these celllines is particularly evident at high doses. At low doses, theHa-ras transfectant TI may show increased resistance leadingto a slightly larger shoulder region as compared with theparent line. An activated ras-induced change in the shoulderregion of the survival curve of a transfected line has beenreported by others (Hermens and Bentvelzen, 1992).

In order to quantify more readily the level of DNAdamage required for cell kIilling, the sensitivity of the threecell lines to DNA-associated "tI decays was measured.Radioactive decay of "zI atoms incorporated into cellularDNA produces high lnear energy transfer (LET)-type DNAdamage (Charlton, 1986) and results in approximately oneDNA double-strand break per decay event (Krisch andSauri, 1975). The "2I data shown in Figure 2 suggest thatthere is no significnt difference in the number of DNAdoubl-strand breaks required to produce a lethal event ineach of the cell lines. Do values of 50 ± 2.5, 53 + 1.4 and56 ± 2.5 "2I decays were obtained for 208F, TI and RBM7respectively. These Do values suggest that around 50-56 "2I

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Fugwe 1 Clonogenic survival of 208F (--), Ti (--O--)and RBM7 (--O--) fibroblasts as a function of t-radiation dose.Data were fitted to a quadratic function of dose as described inMaterials and methods. The alculated values of a(Gy-') andP(Gy-') were: 208F, 0.40 ± 0.05 and 0.012+ 0.004; TI, 0.27 ±0.03 and 0.019 ± 0.003; RBM7, 0.38 ± 0.003 and 0.009 ± 0.004.Each data set was obtained from at least four independentexperiments.

Decays per cell

Fum 2 Clonogenic survival curves of 208F (-- ), TI(--O--) and RBM7 (--O--) fibroblast cells that had accumu-lated DNA-associated "5I decays during liquid nitrogen storage.The data were fitted to a simple exponential function ofaccmulated decays and Do values of 50 ± 3, 53 ± 1 and 56 ± 3"2I decays were obtained for 208F, TI and RBM7 respectively.Each data set was obtained from at least three independentexperiments.

Fuge 3 Ekctron micrographs of control (unirradiated) and -tirradiated (12 Gy plus incubation at 3TC for 24 h) cells. (a) 208Fcontrol, (b) 208F irradiated, (c) TI controL (d) TI irradiated, (e) RBM7 control and (f) RBM7 irradiated. Scale bars represent5 pm.

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574decay-induced DNA double-strand breaks are required toproduce a lethal event in each of these cell lines.

All cell lines used show apoptotic death following irradiationThe morphology associated with cell death in each of thethree cell lines, following exposure to a -tray dose (12 Gy)that would reduce clonogenic survival to 0.5% of the controlvalue, was determined. Both light microscopy of stainedsections and fluorescence microscopy (after staining withethidium bromide and acridine orange) suggested that dyingcells showed cytoplasmic shrinkage and condensation andmargination of nuclear chromatin (data not shown). Thesefeatures are diagnostic for apoptosis (Arends and Wylie,1991). The concusion that radiation induces apoptosis ineach of the three cell lines was confirmed by electron micro-scopy (Figure 3). These eltron micrographs showed charac-teristic features of apoptosis such as chromatin condensationand margmation to the nuclear periphery (Figure 3f), con-volutions of the nuclar membrane (Figure 3b) and eventualcellular break-up into apoptotic bodies (Figure 3d). Cellscolcted over 0-24 or 48-72 h after irradiation were alsoexamined by electron microscopy for features characteristicof necrotic cell death, such as cellular or organelle swelling.Over these time intervals, no evidence indicative of necroticcell death was found in any of the three cell lines.The echanism of cell death after iradiation was further

examined by analysis of the pattern of DNA dgradationoccurring in dying cells. One of the features often associtedwith apoptosis is the digestion of nuclear DNA into frag-ments that are multiples of the 180 bp nucleosome unit (Wyl-lie, 1980; Arends et al., 1990). When eletrophoreticallyseparated on an agarose gel, these DNA fragments produce achaaeristic 'ladder' pattern. At various time points afterirradiation, DNA was extrcted from control (non-irradi-ated) monolayers and separately from the adherent and non-adherent cell fractions of iradiated culures. The parent ine208F and the c-myc-transfected ine RBM7 both showedclear DNA ladders charactersti of apoptosis in the non-adherent cell fraction 24 h after irradiation (Figure 4a). How-ever, the Ha-ras-transfected ie TI showed no ladder after24h (Figure 4a). DNA extracted and analysed from non-adherent Tl cells at 36 or 48 h after irradiation still did notshow laddering of the DNA (Figure 4b). DNA was alsoextracted from all three cell lines at time points prior to 24 hpost irradiation, but ladders were not d (data notshown). DNA degradation in T cells was then examinedfurther usng PFGE of cells incubated for 24 or 48 h at 3TCafter 12 Gy of t-irradiation. II each case, the DNA from1 x 106 cells was examined, except for the 24 h non-adherentfraction where only 0.5 x 10' ceils were available. The adhe-rent fraction of the irradiated cell cultures, and to a lesserextent the control sample, showed a broad range of DNAsizes, indicating random gradation. DNA from the non-adherent fraction of irradiated TI cultures incubated for48h, showed more marked degradation and the possibleappearance of a lOO1kbp intermediate that is subsequentlydegraded further (Figure 4c).

Elevated expresmion of c-myc increases the incidence ofapoptosisfollowing 7-trradiationPrevious work using these cell lines had shown a difference inthe incidence of apoptosis at 48 h after serum withdrawal(Arends et al., 1993). It was therefore of interest to examinethe relative time of onset of apoptosis for asynchronouscultures of each of the cell lines following a 0.5% clonogenicsurvival ?-ray dose. At 24 h periods following irradiation,non-adherent cells were colleted, counted, and the percent-age of cells undergoing apoptotic death was scored by fluor-escence microscopy. Apoptotic cells were not found in adhe-rent cell populations and non-adherent, non-apoptotic cellswere found to account for less than 3.5% of non-apoptoticcells. The results of this experiment showed that the c-myc-trnsfected cell line RBM7 had a markedly greater icidence

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of radiation-induced apoptosis, over the time intervalexamined, than the other lines (Figure 5a). However, theexpression of activated Ha-ras (TI) did not alter the fre-quency of apoptosis compared with the parent line (208F) upto 96 h post irradiation (Figure 5a). Adherent cells were alsocounted and RBM7 was found to maintain a similar numberof cells on the monolayer as the parent line in spite of itshigher rate of apoptosis. Cell numbers of TI, however, con-tinued to increase up to 72 h post irradiation (Figure 5b).

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In order to determine the level and time of resumption ofmitotic activity, replicate cultures of the three cell lines wereincubated with the mitotic spindle poison nocodazole forsuccessive 3 h intervals following 12 Gy of y-irradiation(Figure 5c). Following an initial marked depression ofmitotic activity, a wave of mitosis was noted, which sug-gested a partial cell cycle-synchronising effect of irradiationon each of the lines. Cultures of both the myc- and ras-transfected lines (RBM7 and TI) resumed cycling morerapidly and showed a higher fraction of cells entering mitosisthan the parental 208F line. In loto, the data in Figure 5suggest that, relative to the parental cell line, the level ofapoptosis per mitosis is similar in the myc-transfected linebut might be dereased in the ras-transfected line. Assumingthat radiation-induced apoptosis occurs after mitosis in thesecell lines, the increased incidence of apoptosis in the myc-transfected line would then be a consequence of its higher(relative to the parental line) level of post-irradiation mitoticactivity. Time-lapse cinemicroscopy studies will be requiredin order to confirm these conclusions.

575

0-2 hr 24-4enh 4-2nh 2-9 n The 208F rat fibroblast line and its m1'c- and ras-transfectedderivatives RBM7 and TI all undergo radiation-inducedapoptosis as evidenced by morphology. This response isbroadly in keeping with previous observations of 'spon-

b taneous' cell death of these cell lines in culture by apoptosis,5 (albeit at high and low levels respectively for RBM7 and TI)

(Arends et al., 1993) and also the pattern of death observed4- in solid tumours: RBM7 fibrosarcomas showed a high level

of apoptosis and very little necrosis, but TI demonstratedwidespread necrosis with low levels of apoptosis (Arends et

3- - - b--0-X~~ al., 1994). This occurrence of radiation-induced apoptosis in2- ~ ~~-- - - - - -all three fibroblast lines examined in this study differs from

2-- --0 L _ ° some reports in which normal and transformed fibroblasts-----* were found to undergo necrosis after irradiation (e.g.

..- . O Afanas'ev et al., 1986; Radford, 1991). However, Tomei et al.(1988) using C3H-IOTl/2 mouse fibroblasts and Lowe et al.

: (1988) using adenovirus EIA gene-expressing mouse embry-o l l onic fibroblasts both reported induction of apoptosis after0 24 48 72 96 irradiation.

Time (h) after irradiation In an effort to explain differences in response to irradiationbetween cell types, many groups have looked at the possiblelink between oncogene expression and radiosensitivity(reviewed in Kasid et al., 1993). The consequences of trans-

C fection of members of the ras family of oncogenes have been

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IMA1. IV3JWL. L^_FIV33V UMUaVS IH 41UaLrWC W VIoverexpression of normal ras have been reported to increasethe radioresistance of mouse 3T3 fibroblasts and of rat rhab-domyosarcoma cells (Sklar, 1988; Samid et al., 1991;Hermens and Bentvelzen, 1992), while studies using trans-fected normal or immortalised human cells found that ex-pression of activated ras does not, by itself, lead to anincrease in radioresistance (Mendonca et al., 1991; Su andLittle, 1992). We found that expression of activated Ha-ras inthe TI derivative of the 208F fibroblast line had no markedeffect on its clonogenic survival dose-responses for y-ray or

0 10 20 30 4() 1i decay-mduced uINA damage. Activated ras dd notappear to alter the fraction of the total cell population

Time (h) after irradiation undergoing apoptosis up to 96 h after irradiation as com-pared with the parent line. However, irradiated cultures of

5 (a) Fraction of cell population showing apoptotic mor- the Ti line showed greater residual proliferation, suggestingy (determined by fluorescence microscopy of ethidium that activated ras protein may decrease the level of apoptosise/,acridine orange-stained cells) after 7-irradiation (12 Gy) per mitosis. This effect may be related to the inhibition ofcubation at 37C for the indicated time periods. =, serum withdrawal-induced apoptosis and to the decrease in-, TI; B/. RBM7 (b). Increase in adherent cell susceptibility to apoptosis under both in vitro and in vivo

rs after 7-irradiation (12 Gy) relative to number present at growth conditions observed previously for this cell lineie of irradiation. (c) Entry of t-irradiated (12 Gy) cell grow t al., 1993 e1994c ltions into mitosis as determined by incubation with (Mends et at., 1993, 1994).zole for successive 3 h intervals. Data points represent the The expression of activated Ha-ras inhibited the endo-± s.d.) of two determinations. Error bars have been omit- nuclease activity responsible for the 180 bp nucleosomal lad-m c for the sake of clanrty. 0-, 208F; --U--, TI; der that is commonly associated with apoptosis (Arends etRBM7. al., 1990). This finding is consistent with that of Arends et al.

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(1993), who showed that this endonuclease(s) is down-regulated by the expression of actvted ras. Despite lackingdetectable nucleosomal ladder-producing endon seactivity, irradiated TI cells showed extesive DNA degrada-tion to relatively high molkular weight fragments and nor-mal apoptotic morphology. Apoptosis without the appear-ance of nucleosomal ladders has been reported previously(Cohen et al., 1992), and it has been shown that the charac-teristic apoptotic nuclear morphology is associated withinital cleavage of nuclear DNA into 300 kbp fragments andthen into 50 kbp fragmets and does not require the produc-tion of 180 bp nudeosomal ladders (Brown et al., 1993;Oberhammer et al., 1993).The RBM7 cell ine, which overexpresses c-myc, showed an

increase in the incidence of radiation-induced apoptosis, overthe time period examined, with respect to the parent line.This finding is consistent with the observations of Arends etal. (1993, 1994) that the RBM7 line shows increased suscep-tibility to serum withdrawal-induced apoptosis and increasedintrinsic apoptosis when growing as a solid fibrosarcoma orin culture, and with the hypothesis that c-myc expressioninduces a state in which cells are 'primed' for apoptosis(Arends and Wylie, 1991). The increase in apoptosis mayreflect a more rapid post-irradiation resumption of mitoticactivity in the RBM7 cell line.

However, despite their inceased rate of i-irradiation-induced apoptotic death, the sensitivity of RBM7 cells toeither ?-irradiation or DNA-associated '2I decay-induced cellkilling is not significantly different from that of the parentline. This result contrasts with previous data from mouselymphoid lines, which revealed a correlation between rate ofradiation-induced apoptotic death and radiosensitiity (Rad-ford, 1994a). However, it should be noted that, even in theRBM7 line, apoptosis is induced considerably more slowly inirradiated fibroblasts than in the more radiosensitive lym-phoid lines. For example, apoptotic cells were not det inRBM7 cultures until at least 8 h after irradiation (data notshown), as compared with 1-2 h in radiosensitive lymphoidines (Radford, 1994a). The comparatively lengthy timeperiod available to irradiated RBM7 cells, prior to possibleinduction of apoptosis, may be adequate for DNA repair.

The reason for the difference in the rate of induction ofapoptosis between irradiated fibroblasts and some lymphoidlines is currently unclear. Studies with mouse lymphoid ineshave s ed that rapid induction of apoptosis afteriradiation is dependent upon the presence of wild-type p53protein (Radford, 1994b). Although all three fibroblast linesused in this study appear to contain non-mutant p53 protein,a definitive conclusion awaits DNA sequencing (see Materialsand methods). Studies by other investigators, although notdefining the mode of cell death occurring, have also generallyconcluded that overexpression of c-myc does not alterradioresistance (reviwed in Kasid et al., 1993).Tese findings lead us to question the hypothesis that a

cell's radiosensitivity can be directly related to its mode ofdeath. Indeed, the number of "5I decay-induced DNAdouble-strand breaks required to produce a lethal event inthe 208F ine and its transfected derivatives was smilar tothe nunmber required to kill the V79 fibroblast line(Do = 61 ± 2), which undergoes necrotic cell death (Radford,1991). This sugests that differences in radiosensitivitybetween cell lines may be related more to the intrinsic charac-teristics of the cell type of origin than to mode of death andthat radiosensitivity is a phenotypic property distinct fromsusceptibility to apoptosis and that it may be independentlygenetically influenced. However, there is a need to study therelationship between radiosensitivity and mode of cell deathin a wider range of cell types before such conclusions can beconfirmed. It should also be noted that the significantdifference in residual post-irradiation proliferation betweenthe cell lines studied may complicate the extrapolation of ourresults to the in vivo situation.

DA was supported by a Peter MacCallum Cancer Institute PhDstudentship and the work was funded in part by a grant from theAnti-Cancer Council of Victora. The author are grateful to DrJohn Radley and Manuela Palatsides for the production of electronmicrographk and to Dr Alan Harrs (Walter and Eliza Hall Instituteof Medical Research) for reading the manuscnpt.

R Ier es

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ARENDS MJ AND WYLLIE AH. (1991). Apoptosis: nisms androes in pathology. It. Rev. Exp. Padl., 32, 223-251.

ARENDS M, MORRIS RG AND WYLLIE AHl (1990). Apoptosis: therole of the endonuelas. Am. J. Pathal., 136, 593-608.

ARENDS MJ, MCGREGOR AH, TOFT NJ, BROWN EJH AND WYLLIEAL (1993). Susceptibility to apoptosis is differentiajly regulatedby c-myc and mutated Ha-ras oncognes and is aiated withendonue availability. Br. J. Cancer, 60, 1127-1133.

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