radiation-induced apoptosis in a murine t-cell hybridoma...in 5-gy-exposed cells, a...

(CANCER RESEARCH 52. 883-890. February 15, 1992]

Radiation-induced Apoptosis in a Murine T-Cell Hybridoma

Raymond L. WartersDepartment of Radiology, University of Utah Health Sciences Center, Salt Lake City, Utah 84132

ABSTRACT

Induction of an apoptotic cell death was studied in a mouse T-cellhybridoma. Apoptosis was induced in these cells following exposure todexamethasone, X-radiation, 43Â°Cheat shock, .â€¢(.,,light, and hydrogenperoxide. In 5-Gy-exposed cells, a radiation-induced G2 phase cell cycleprogression block was maximum by 8 h. The cells began to escape thisprogression block by 10 h. Nuclear DNA fragmentation and uptake ofthe vital dye trypan blue began at 12 and 14 h, respectively, and werecomplete by 28 h. X-radiation-induced cell death was diminished whencells were irradiated in the presence of dimethyl sulfoxide, indicating thatcell death was induced by oxidative cell damage. Substitution of nuclearDNA with bromodeoxyuridine enhanced death in cells exposed to eitherX-radiation or ,-(,,(>light, indicating that apoptosis could be induced byDNA damage. The results are consistent with radiation-induced apoptosisbeing stimulated by oxidative DNA damage. DNA damage stimulates along-lived signal which controls the expression of apoptosis. Apoptosisis expressed in the (., phase of the cell cycle subsequent to the cellirradiation.

INTRODUCTION

Ionizing radiation induces at least two characteristically different modes of cell death currently termed necrosis and apoptosis (1, 2). Necrosis, or reproductive cell death, is observed inproliferating cell populations. At lower radiation doses at whichsurvival is detectable, the onset of cell death or lysis occursfollowing multiple cell divisions (3,4). At the cellular level, thismode of death is characterized by increased plasma membranepermeability, a decline in protein synthesis, swelling of thematrix of the mitochondria, dissolution of ribosomes and ly-sosomes, and irreversible cell swelling and autolysis (2, 5). Thisis the predominant mode of radiation-induced cell death inmitotic fibroblast cultures in vitro in which the single cellsurvival response is characterized by a low-dose quasithresholdconsistent with a potentially toxic cell lesion which is repairable(6). The correlation between the extent of reproductive celldeath and both DNA lesion induction (7, 8) and chromosomeaberration induction (9, 10) indicates that this mode of radiation-induced cell death results from the failure of irradiatedcells to faithfully, or fully, repair some DNA lesions, ultimatelyresulting in imbalances in biochemical or metabolic reactionsin nonsurvivors.

In contrast, a second mode of radiation-induced cell death,originally described in lymphocytes, has been termed interphasedeath, programmed cell death, or apoptosis (1, 2, 5). It ischaracterized by a reduction in cell volume, condensation ofthe cytoplasm, alterations in membrane permeability, and anonrandom degradation of nuclear DNA into oligonucleosome-sized fragments (2, 5). In irradiated thymocytes, the onset ofapoptosis is rapid with DNA degradation and loss of membranepermeability accompanied by uptake of vital dyes completewithin 6-7 h (11-13). Inhibition of both DNA degradation andvital dye uptake by protein synthesis inhibitors and chelatingagents has been taken to indicate that the process may involve

Received 7/8/91; accepted 12/3/91.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

the de novo synthesis of a divalent cation-dependent endonucle-

ase(13, 14).While radiation-induced apoptosis has been considered to be

an interphase death in nonmitotic cells recovered from animalssuch as lymphocytes, a characteristically similar cell death hasbeen reported in a number of proliferating cell lines and tumorcells (15-18). Use of a cell population proliferating in tissueculture would broaden the range of experimental approachespossible in any study of the mechanism(s) involved in radiation-induced apoptosis. In this study, the response of a mousethymoma-activated T-lymphocyte hybridoma to ionizing radiation was studied. As has been shown for thymocytes (15, 19),this cell line apoptoses in response to exposure to the glucocor-ticoid dexamethasone.1

MATERIALS AND METHODS

Cell Culture and Labeling. Mouse Hb 8.3 cells (provided by Dr. B. A.Araneo, Department of Pathology, University of Utah) were maintainedin suspension culture in exponential growth between 1 and 5 x 10*

cells/ml in RMPI medium 1040 (GIBCO) supplemented with 10%fetal bovine serum (GIBCO). V79 Chinese hamster lung fibroblasts(provided by Dr. L. A. Dethlefsen, Department of Radiology, University of Utah) were maintained in monolayer culture in the same mediumwith a doubling time of 12 h. To label nuclear DNA, cells were exposedto medium containing [methyl-'^C] thymidine (0.15 Â¿iCi/ml)(New

England Nuclear) for 20 h. In some experiments, nuclear DNA wassubstituted with BrdUrd2 (Sigma) by exposure to 0.1-6.0 x IO"5 MBrdUrd for 20 h at 37Â°C.BrdUrd was dissolved to IO'3 M in PBS andstored at -20Â°C until used. For flow cytometry studies, cells wereresuspended to IO6 cells/ml in 70% ethanol and allowed to fix inethanol at 4Â°Cfor at least 24 h before staining. Fixed cells were stained

with propidium iodide (25 Mg/ml) for at least 30 min prior to analysis.Fixed and stained cells were analyzed with an Ortho CytofluorographII flow cytometer at the Utah Regional Cancer Center. A computercurve-fitting program (MCYCLE) was used to estimate the fraction ofcells in the various cell cycle phases using the method of Dean and Jett(20). Exponentially growing cells had a doubling time of 10 h with 35Â±2, 53 Â±2, and 12 Â±1% (average Â±1 SE; n = 6), respectively, of cellsin the GÃ¬,S, and G2 + M phases of the cell cycle.

Vital Dye Exclusion Estimates. Trypan blue (Sigma) was broughtinto solution to 0.2% by boiling in PBS. The dye solution was filteredthrough a 0.2-^m pore-sized filter and stored at ambient temperature.Cells to be assayed were either pelleted and resuspended to IO6cells/

ml in PBS or taken directly from tissue culture flasks. Dye exclusionassay results were the same regardless of whether cells were in wholemedium or PBS. An equal volume of cell suspension and 0.2% trypanblue solution were mixed, placed under a coverslip, and viewed with anOlympus inverted microscope (x200 total magnification). At least 400total cells were counted for each data point, and the fraction of totalcells counted which stained with trypan blue was calculated.

Cell Irradiation. Cells suspended in whole medium (1-2 x 10 /ml)were irradiated at 25Â°Cwith a Philips T-250 instrument operating at

250 kV, 15 mA, with 3 mm Al filtration, HVL 0.5 mm Cu, at a doserate of 5.0 Gy/min. In some experiments cells were exposed to increasing concentrations of DMSO (Fisher Scientific Co.) in whole mediumat 25Â°Cfor 30 min prior to and during irradiation. After irradiation,

1B. A. Araneo, unpublished results.2The abbreviations used are: BrdUrd, bromodeoxyuridine; PBS, phosphate-

buffered saline; DMSO, dimethyl sulfoxide; DEX, dexamethasone; SSF, strandscission factor; II)..,,. lethal dose for 50% of cells examined.

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RADIATION-INDUCED APOPTOSIS

the cells were centrifugea out of DMSO-containing medium, resus-pended in whole medium, and replaced at 37Â°C.Cells lightly attached

to T flasks were placed directly above an Ultraviolet Products modelC-63 transilluminator or a Cole-Parmer 15-W A}12light source. Theintensity at the surface of the T flask was 0.25 and 2.0 J/nr/s at A^oand AMO,respectively, above the transilluminator and 0.04 and 0.5 J/nr/s at A2io and A,,0, respectively, above the A,,2 light source. Alternately, cells suspended in 1 ml of ice-cold PBS in an open Petri platewere placed below a Cole-Parmer 15-W /4254light source. The intensityat AIM and /4310at the surface of the Petri plate was 0.5 and 0.025 J/m2/s, respectively. UV light intensity was measured with an Ultra-Violet Products model UVX digital radiometer using a UVX-25 (/


radiated cells (Fig. \A, â€¢¿�).Exposure to 5 Gy of X-radiationslowed cell growth, and cell number barely doubled within 28h at 37Â°C(Fig. IA, D). When analyzed by flow cytometry (Fig.

IB), the fraction of 5-Gy-exposed cells in G, or S phase of thecell cycle decreased between 1 and 8 h, while the fraction of G2cells increased to a maximum. Between 8 and 12 h, the fractionof cells in G2 phase began to decline, and the fraction of cellsin GÃ¬phase began to increase. By 12 h, the DNA content in asignificant fraction of cells had declined to less than observedin G i cells, and by 13-14 h this decline in cell DNA contentwas so pronounced that the cell cycle distribution of the population could not be modeled.

Between 5 and 10% of an exponentially growing, unirradiatedpopulation of Hb 8.3 cells stained with the vital dye trypan blue(Fig. 2, O). The fraction of cells which stained with the dyeincreased to 15% as control cells moved into a nongrowingculture. Dye uptake in cells exposed to 5 Gy of X-radiation(Fig. 2, D) remained at control levels for up to 12 h postirradia-tion. At 14 h, the fraction of stained cells began to increase andreached a maximum by 28 h. By 14 h, an increasing fraction ofthe irradiated cells exhibited membrane blebbing. The majorityof trypan blue-stained cells also exhibited a decreased cellvolume, detectable both by microscopic examination of the cellsand during cell counting with a Coulter model Z counter. Nosignificant increase in dye uptake occurred within this time inV79 Chinese hamster lung fibroblasts exposed to 5 Gy of X-radiation (Fig. 2, A).

Since the time (12-14 h) at which the cells began to apoptosecorresponded to the time at which irradiated cells began toescape from the radiation-induced G2 progression block (Fig.IB), we determined the timing of apoptosis in the presence ofcaffeine, a drug which reduces radiation-induced ( Â¡progressiondelay (24). When Hb 8.3 cells were exposed to 5 HIMcaffeinefor 15 min prior to, and continuously after, exposure to 5 Gyof X-radiation, apoptosis began, and was completed, soonerthan in the absence of caffeine (Fig. 2, â€¢¿�).In the irradiated andcaffeine-treated population, 50% of the cells became stainedwith trypan blue by 10 h, 10 h earlier than in cultures notexposed to caffeine. Exposure to caffeine itself induced a significant amount of cell death, with 20 and 70% of 5 mMcaffeine-exposed, unirradiated cells stained with trypan blue by12 and 25 h, respectively. Addition of 25 Mg/ml cycloheximide9 or 14 h after exposure to 5 Gy of X-radiation did not inhibitthe subsequent increase in trypan blue uptake in irradiatedcultures. Exposure to both 5 mM caffeine and 25 tig/ml cycloheximide 15 min prior to irradiation (Fig. 2, 0) inhibited thesubsequent uptake of trypan blue relative to cultures exposedto 5 mM caffeine only prior to irradiation (Fig. 2, â€¢¿�).In culturesexposed only to cycloheximide 15 min prior to and after irradiation, trypan blue uptake remained at control cell levels forup to 8 h after irradiation (results not shown). Incorporation of'H-amino acids into acid-insoluble cellular material in expo

nentially growing cultures exposed to 25 Mg/ml cycloheximidewas reduced to 0.03 Â±0.01 (n = 3) of incorporation observedin control cells.

When Hb 8.3 cell DNA was analyzed by electrophoresisthrough an agarose gel, which resolved 1-7 x IO6base pairs of

DNA, only a small fraction (approximately 5%) of DNA wasfound to exit the well (Fig. 3A, lane 2). In cells exposed to 5Gy of X-radiation, this value increased to 15% (lane 3). Theincreased fraction of irradiated cell DNA which entered the gel,presumably due to the induction of DNA strand breaks, decreased by 1-2 h of 37Â°Cincubation, presumably due to DNA

i.o-

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Fig. 2. Vital dye uptake by irradiated cells. Unirradiated (O) or S-Gy-irradiatedcells (LI,â€¢¿�,'.inwere placed at 37Â°Cin whole medium (I 1)or in medium containing

either 5 mM caffeine (â€¢)or S IHMcaffeine and 25 Â«


A.

Fig. 3. DNA degradation in irradiated Hb8.3 cells. In A, the DNA of unirradiated cells(lane 2), cells exposed to 5 Gy only (lane 3),or cells exposed to 5 Gy and incubated at 37'Cfor 1 (lane 4) or 2 h (lane 5) was electropho-resed through 0.8% Megarose gel as describedin "Materials and Methods." Lane 1 includes

chromosomal DNA of S. pombe. In B, theDNA from unirradiated cells (lane 2) or 5-Gy-irradiated cells incubated at 37'C for 12, 14,16, 20, 24, or 28 h (lanes 3-8) was electropho-resed through a 1.5% LE agarose gel in 0.5xTBE buffer at 200 V for 5 h with the electricalfield alternating with a ramp of 1-4 s. Lane Iincludes a 1-kilobase pair ladder.

. 0.6-,q0

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Fig. 4. The size distribution of fragmentedDNA. DNA from cells exposed to 5 Gy andincubated at 37'C for 20 h (lane 4) or 24 h

(lane 3) was electrophoresed through a 1% LEagarose gel at 150 V for 7 h with the electricalfield alternating every 0.8 s. Lanes I and 2include, respectively, high molecular weightDNA size markers and a 1-kilobase pair ladder(both from Bethesda Research Laboratories).The gel was stained with ethidium bromideand photographed by IV transillumination.The photographic negative was scanned witha Bio-Rad model 620 video densitometer. Therelative i (ordinate) across lane 4 is plottedversus DNA length (10'base pairs; abscissa)

taken from lanes 1 and 2.

and 10% of irradiated cells produced colonies containing >50cells after exposure to 3, 6, and 9 Gy, respectively.

Incubation of Hb 8.3 cells with increasing concentrations ofDMSO for 30 min prior to irradiation resulted in a DMSOconcentration-dependent decrease in radiation-induced vitaldye stainability (Fig. SB, O), growth delay (D)>and DNA single-strand break induction (â€¢).Exposure to up to 3 M DMSO for60 min induced no detectable apoptosis within 30 h at 37Â°C.

When the DNA of Hb 8.3 was substituted with BrdUrd byexposure to 4 x IO"5 M BrdUrd for 24 h prior to irradiation,

vital dye uptake 24 h after X-irradiation was enhanced (Fig.5A, â€¢¿�).A significant fraction (10-15%) of the Hb 8.3 cellsfailed to apoptose regardless of radiation dose up to 20 Gy,indicating the possible presence of a radiation-resistant subpop-ulation of these cells. The cells were exposed to 1-5 Gy of X-radiation and allowed to regrow. When the regrown, previouslyirradiated cells were again exposed to increasing radiation dosesand the extent of apoptosis was determined, the response curvesuperimposed the radiation-response curve (Fig. 5A) observedfor naive, unirradiated cells. Thus, no radiation-resistant sub-

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Fig. 5. Radiation dose-dependent vital dye uptake. In A, Hb 8.3 cells (O), \T)hamster fibroblasts (D), or Hb 8.3 cells substituted in their DNA with 4 x 10~!M BrdUrd (â€¢)were exposed to increasing radiation doses, placed at 37'C for 24h, and monitored for trypan blue stainability as described in "Materials andMethods." In B, Hb 8.3 cells were incubated in increasing concentrations ofDMSO for 30 min at 25'C and exposed to 5 Gy of X-radiation. The fractionaldecrease in DN A single-strand break induction as a function of increasing DMSOconcentration (â€¢)was calculated as the ratio of the SSF in cells incubated inDMSO prior to irradiation divided by the SSF in cells which were not incubatedin DMSO prior to irradiation. Alternately, DMSO was removed from the cellsand were incubated at 37'C for 24 h. The fractional decrease in trypan blue

stained cells (O) was calculated as the ratio of trypan blue stained cells in DMSOtreated cells divided by untreated cells. The fractional decrease in growth delay(D) was calculated as the number of cells/ml in DMSO-treated cells divided byuntreated cells.

CONCENTRATION Mio'10io"8io"61.0-0.8-2

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Fig. 6. Induction of apoptosis by various traumas. In. I, Hb 8.3 cells in T flaskswere exposed to A:.Â¡light from below from a transilluminator (â€¢)or cells in anopen Petri plate were exposed to A2,4 light from above from an /)JM light source(O). Alternately, cells substituted in their DNA by exposure to 4 x 10"' M BrdUrd

for 24 h (â€¢)or unsubstituted cells (O) were exposed t light in BrdUrd-substituted cells (lane 7) was

isolated from cells by proteinase K digestion, phenol extraction, and ethanolprecipitation as previously described (21). DNA was electrophoresed through a0.5% agarose-3.0% acrylamide composite gel, stained with ethidium bromide,and photographed by transillumination. Lanes I and 8 include, respectively, a 1-kilobase pair and a 123-base pair ladder.

887




2.0-,

1.5-

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0.5-

0.0

1.0

TIO 20 O 40 80

DOSE (Gy or J/m2)

\120 180

Fig. 8. Radiation-induced DNA strand breakage. Hb 8.3 cells were exposed to4 x 10"' M BrdUrd for 24 h at 37'C (O, â€¢¿�)or left unsubstituted (O). The cells

were exposed to increasing doses of X-radiation (circles) or . IM, light (squares)and washed into ice-cold PBS to 5 x 10' cells/ml. Cell DNA was analyzed byfilter elution at pH 12.2 for the presence of single-strand breaks (A) or at pH 7.2for the presence of double-strand breaks (B) as described in "Materials andMethods." In both cases, a SSF was calculated at 11 ml of total elution andplotted versus dose. Point, average; bar, Â±1SE; n = 3.

exposure to X-radiation in unsubstituted cells produced 1000single- and 40 double-strand breaks/Gy (8), we estimate that atLD5(j exposures of X-radiation in unsubstituted (1.75 Gy) orBrdUrd-substituted cells ( 1.0 Gy) or in BrdUrd-substituted cellsexposed to AÃ¬Ã¬alight (45 J/m2/s), approximately 1.75 x 10\1.74 x 10', and 5 x IO4single-strand breaks and 70, 73, and 0

double-strand breaks were induced. Thus, at an LD50 exposure,there were equivalent frequencies of single- and double-strandbreaks in X-irradiated cells regardless of whether their DNAwas substituted with BrdUrd. An LD50 exposure to A3to lightin cells substituted in their DNA with BrdUrd induced onlyDNA single-strand breaks.

To assess the level of nuclease activity in Hb 8.3 cells, nucleiwere isolated both from unirradiated cells and from cells 14 hafter exposure to 5 Gy of X-radiation. Nuclei were resuspendedin a buffer containing 25 mM NaCl-1 mM CaCl2-0.5 HIMATPand placed at 37Â°C.As expected, nuclear DNA from cells 14 h

after irradiation was partially fragmented (Fig. 9A, lane 6),while nuclear DNA from control cells remained unfragmented(Fig. 9A, lane 3). During 37Â°Cincubation, DNA in nuclei from

both control (lanes 4 and 5) and previously irradiated cells(lanes 7and 8) was progressively fragmented into a distributionof DNA lengths predominantly between 50 and 300 kilobasepairs but not to shorter DNA lengths. DNA in permeabilizedcontrol cells (Fig. 9Ã„,lanes 2-4) and previously irradiated cells(lanes 5-7) was rapidly degraded during 37Â°Cincubation into

a distribution of DNA lengths between 0.1 and 10 kilobasepairs. The results were consistent with control and previouslyirradiated cells containing the same amounts of two nucleaseactivities: an endonuclease which was tightly bound withinnuclei which degraded nuclear DNA into 50- to 300-kilobasepair fragments and a soluble nuclease which degraded nuclearDNA into lengths comparable to those (Fig. 35) observed inapoptosing cells. Since both control and previously irradiatedcells contained comparable nuclease activities, the results wereconsistent with the regulation, not the levels, of endogenousnuclease activities being different in apoptosing Hb 8.3 cells.

DISCUSSION

Radiation-induced apoptosis typically has been studied innondividing thymic lymphocytes recovered from rodents. Since

a wider range of experimental approaches could be taken in aproliferating cell population, we determined the effect of ionizing radiation on a proliferating T-cell hybridoma. Apoptosiscan be induced in T-cell hybridomas or thymocytes by activation(25-28) or by exposure of these cells to glucocorticoids (15,19). Similar treatments had been found to induce apoptosis inthe Hb 8.3 T-cell hybridoma used in this study.' If ionizing

radiation also induces apoptosis in these cells, it would beexpected to induce a number of responses characteristic ofapoptosing cells (5) including (a) a reduction in cell volumeaccompanied by an increase in cell permeability (vital dyeuptake) and density, convolution, and blebbing of the cellsurface, (b) chromatin condensation associated with the activation of an endogenous nuclease, and (c) dependence of theprocess on active protein synthesis. These elements were observed in irradiated Hb 8.3 cells. With respect to time afterirradiation, DNA degradation, observed both by gel electropho-resis of cell DNA and by flow cytometry, preceded cell surfaceblebbing, the uptake of a vital dye, and a decrease in cell volume,all three occurring in quick succession. At early times afterirradiation, the process was also inhibited by the addition ofcycloheximide, a protein synthesis inhibitor. By these criteria,increasing exposures of ionizing radiation did induce increasingfractions of Hb 8.3 cells to die by apoptosis. Thus, radiation-induced apoptosis can be induced in certain proliferating cellsand is not exclusively an interphase cell death limited to non-dividing lymphocytes. A similar response was not observed inirradiated Chinese hamster fibroblasts, indicating that radia-

Fig. 9. Endogenous nuclease activity in Hb 8.3 cells. In A, nuclei were isolatedfrom control cells (lanes 3-5) or from cells 14 h after exposure to 5 Gy of X-radialion (lanes 6-8), placed into a low-salt buffer (25 mM NaCI-1 mM Tris, pH7.4-1 mM CaClj-0.5 mM ATP) and incubated at 37'C for 0 (lanes 3 and 6), 30

(lanes 4 and 7), or 60 min (lanes 5 and 8). Nuclear DNA was electrophoresedthrough a I % agarose gel in O.Sx TBE buffer for 15 h at 200 V with the electricalfield alternating every 60 s. Lanes I and 2 contain, respectively, chromosomalDNA from S. cerevisiae and a high molecular weight DNA marker. In B, controlcells (lanes 2-4) or cells 14 h after irradiation (lanes 5-7) were permeabilized inlow-salt buffer containing 0.1% Triton \ 1(10. Permeabilized cell suspensionswere taken to 37'C for 0 (lanes 2 and J), 15 (lanes 3 and 6), or 30 min (lanes 4

and 7). DNA was electrophoresed through a 1% agarose gel in 1x TAE buffer at150 V for 7 h with the electrical field alternating every 0.8 s. Lane I contains a1-kilobase pair ladder.

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tion-induced apoptosis may be limited to cells previously "programmed" genetically to respond in this manner to certain

environmental stimuli. In contrast to the V79 fibroblast, theHb 8.3 hybridoma may have retained this capacity to apoptosefrom the T-cell lymphocyte from which it was derived.

It is not clear what subcellular change initiates apoptosis. Inaddition to activation of T-cell hybridomas or thymocytes andexposure of these cells to glucocorticoids, apoptosis can beinduced by withdrawal of growth factors from dependent celllines (29) and lysis of target cells by cytotoxic T-cells (30, 31).Apoptosis has also been found to be induced in thymocytes andsome tumor cell lines by exposure to heat shock, 5-fluorouracil,vincristine, l,3-bis(2-chloroethyl)-l-nitrosourea, and mel-phalan (32, 33). We found that apoptosis could be induced ina T-cell hybridoma exposed to X-radiation, /f254 light, 43Â°C

heat shock, H2O2, and DEX (Figs. 5 and 6). The diversity oftreatments which induce this cell death process does not readilysuggest a single subcellular change common to all stimuli.

The subcellular events which initiate radiation-induced apoptosis are somewhat more clear. Consistent with a previousreport using cysteamine as a radical scavenger (34), we foundthat the presence of the radical scavenger DMSO (35) duringcell irradiation could virtually eliminate radiation-inducedapoptosis, as well as other radiation-induced cell changes (Fig.5.6). Thus, the earliest subcellular change required in radiation-induced apoptosis is the formation of radicals resulting fromwater radiolysis. Presumably, some form of intracellular oxi-

dative damage produced by these radicals induces apoptosis.The presence of radical scavengers during cell irradiation potentially could protect all subcellular macromolecules to a similar extent. A recent study concluded that radical damage to theplasma membrane induced apoptosis in irradiated B-lympho-cytes (34). In contrast, our results indicate that the inductionof DNA damage may stimulate radiation-induced apoptosis ina T-cell hybridoma. Exposure to A^0 light alone induced no

apoptosis (Fig. 6A). In contrast, exposure of cells substitutedin their DNA with BrdUrd to A},0 light induced both DNAstrand breakage and apoptosis. Substitution of cell DNA withBrdUrd also enhanced both radiation-induced DNA strandbreakage (Fig. 8) and apoptosis (Fig. 5A) to a similar extent.While the results indicate a correlation between DNA damageinduction and radiation-induced apoptosis, there was no clearcorrelation between the frequencies of any general class of DNAlesion and the level of apoptosis. At a 50% cytotoxicity level,no DNA double-strand breaks were produced by photolysis ofBrdUrd containing DNA with Ain>light, indicating that DNAsingle strand breakage may be sufficient to induce apoptosis.However, at a 50% cytotoxicity level, photolysis of BrdUrd-containing DNA produced approximately 25-fold more DNAsingle-strand breaks than were observed in X-irradiated cells ata similar cytotoxicity level. Thus, either the strand breaksproduced by these two treatments differ in their capacity tostimulate apoptosis or some other DNA lesion produced atequivalent frequencies by both treatments at 50% cytotoxicitylevels is responsible. One such DNA lesion might be a DNAabasic site. In a previous study, we found that 43Â°Cheating

produced few frank DNA single- or double-strand breaks butdid produce approximately 15 abasic sites/min of 43Â°Cheating(23). Thus, at a 43Â°Ctreatment inducing 50% cytotoxicity (75-80 min, Fig. 65), we would expect between 1.1 and 1.2 x 10'

total abasic sites to have been produced. Since equivalent numbers of DNA single-strand breaks and abasic sites are producedin irradiated cells (23), we would expect similar frequencies of

abasic sites to have been produced in the DNA of Hb 8.3 cellsat LDcn exposures of X-radiation and 43Â°Cheating.

While oxidative DNA damage appears to be the initiatingstimulus for apoptosis in irradiated Hb 8.3 cells, the continuedpresence of neither radicals nor DNA damage appears to benecessary for the ultimate expression of apoptosis. The half-life of radicals produced in irradiated cells ranges from 10~9to10~6s (36). The half-lives of single-strand breaks (22), double-

strand breaks (22), and thymine base damage (37) in irradiatedmammalian cells are approximately 5, 30, and 5 min, respectively. In contrast, the time required for half-maximum apoptosis in irradiated Hb 8.3 cells is 20 h (Fig. 2). By 20 h of 37Â°C

incubation, we would expect few, if any, of the original, radiation-induced DNA lesions to persist. Thus, while oxidativeDNA damage may serve as an initiating stimulus, it must berecognized and induces a secondary signal. This secondarysignal, which may be the recognition factor itself, must be longlived since it remains active in irradiated Hb 8.3 cells for >12h at 37'C.

The expression of apoptosis in irradiated Hb 8.3 cells alsoappears to be cell cycle phase dependent since it does not occuruntil cells overcome the radiation-induced, G2 phase progression block. The association in time between the initiation ofapoptosis (Fig. 2) and recovery from the G2 phase progressionblock (Fig. \B) does not appear to be coincidental since continuous culture of irradiated Hb 8.3 cells in 5 mM caffeine, whichdepresses the G2 phase progression block (24), also causesapoptosis to be expressed 10 h early (Fig. 2). Apoptosis appearsto occur in the G, phase subsequent to irradiation, not duringmitosis, since the Hb 8.3 cell population doubles (Fig. \A) overthe period during which the cells apoptose (Fig. 2). Thus, thesecondary signal induced by oxidative DNA damage must persist in irradiated cells until they mitose and enter the subsequentGÃ¬phase, a period of up to 20-22 h in irradiated G, cells. Afterirradiated cells move into the subsequent d phase, the long-lived signal must activate the apoptosis process itself. Sincesimilar levels of comparable nuclease activities are detectedboth in control and apoptosing cells, nuclear DNA degradationby these enzymes must be inhibited until the irradiated cellsreach the subsequent G, phase of the cell cycle. Thus, radiation-induced apoptosis in a T-cell hybridoma appears to be regulatedat a number of levels. The process requires recognition ofoxidative DNA damage. Recognition of DNA damage activatesa long-lived secondary signal. Activation of the apoptotic proc

ess is cell cycle phase dependent and occurs only upon entryinto the (., phase subsequent to cell irradiation.

Radiation-induced apoptosis in the mitotic Hb 8.3 cell lineis distinctly different from both the interphase cell death whichoccurs in nonmitotic thymocytes and the mitotic-linked celldeath which occurs in fibroblasts such as the V79 cell line. Inthymocytes, an apoptotic type cell death occurs within 6-10 hin the G! phase of the cell cycle without transit through mitosis(11-14). Thus, noncycling thymocytes must retain the subcellular machinery and genetic programming to respond in thismanner. In contrast, transit through mitosis into the subsequentd phase is required for expression of apoptosis in the Hb 8.3T-cell hybridoma. While these cells retain the capacity to respond in a similar manner to appropriate environmental stimuli, the full expression of apoptosis must require genetic repro-gramming during mitosis. Cell death occurs in subsequent cellgenerations in mitotic fibroblasts such as the V79 cell line (4).This mode of cell death, however, is not accompanied bysubcellular events characteristic of apoptosis. Thus, mitotic

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fibroblasts must not contain the genetic programming necessaryto respond actively to irradiation and ultimately die due tobiochemical and metabolic imbalances.

REFERENCES

1. Okado, S. Radiation-induced death. ///.- K. I. AItnum. G. G. Gerber, and S.Okada (eds.), Radiation Biochemistry, Vol. 1, pp. 247-307. New York:Academic Press, Inc., 1970.

2. Duvall, E., and Wyllie, A. H. Death and the cell. Immunol. Today, 7: 115-119, 1986.

3. Hofer, K. G. Radiation effects on death and migration of tumor cells in mice.RadiÃ¢t.Res., 43:663-678, 1970.

4. Hopwood, L. Â£.,and Tolmach, L. J. Manifestations of damage from ionizingradiation in mammalian cells in the postirradiation generations. In: }. T.Lett and H. Adler (eds.), Advances in Radiation Biology, Vol. 8, pp. 318-362. New York: Academic Press, Inc., 1979.

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1992;52:883-890. Cancer Res Raymond L. Warters Radiation-induced Apoptosis in a Murine T-Cell Hybridoma

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