Explaining Differences in Sensitivity to Killing by Ionizing Radiation Between Human Lymphoid Cell Lines David R

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(CANCER RESEARCH 58, 2817-2824. July I. 1998] Explaining Differences in Sensitivity to Killing by Ionizing Radiation between Human Lymphoid Cell Lines David R. Aldridge and Ian R. Radford1

Sir Donald and Lady Trescowihick Research Laboratories, Peter MacCallum Cancer Institute, St. Andrews Place, East Melbourne, Victoria 3002, Australia

ABSTRACT sensitivity. These studies suggested that sensitivity to clonogenic killing by y-rays or DNA-associated 12il decays correlated closely We surveyed five human hematopoietic cell lines (IISlt-2, MOI, I -4. with how rapidly apoptosis was induced after irradiation (2, 3). Such Reh, CEM, and III,-60) to determine whether any simple correlates with differences in timing reflected whether induction occurred soon after sensitivity to killing by y-irradiation might be revealed. The clonogenic irradiation ("rapid interphase" death), following arrest in the G2 phase survival y-ray dose-response curves for these cell lines cover a wide range ("delayed interphase" death), or after cell division ("mitotic" death). of sensitivities. Consistent with previous results for murine hematopoietic cell lines, there was a clear correlation between the rapidity with which Cell lines could show different proportions of death at these times irradiation induced apoptosis and clonogenic radiosensitivity of a cell line, dependent upon the radiation dose they had received. It was specu although the relationship between timing of apoptosis and radiosensitivity lated that DNA dsb2 directly initiates the destruction of cell lines differed between human and murine cell lines. Flow cytometric determi susceptible to rapid interphase death and that chromosomal aberra nation of cell cycle distribution after irradiation showed that differences tions can trigger delayed interphase or mitotic apoptosis (4). The between human hematopoietic cell lines, in the rate of induction of apop occurrence of rapid interphase apoptosis appeared to be dependent tosis, were generally related to the functioning of cell cycle checkpoints. Whereas the rapidly dying and radiosensitive 1-1SB-2cell line underwent upon the presence of wild-type p53 in these cells (5). apoptosis from different points in the cell cycle, the more slowly dying cell There have been relatively few comparative radiobiological studies lines showed a variety of cell cycle arrest profiles and initiated apoptosis of human hematopoietic cell lines. The studies performed have sought after accumulation of cells in the G2 phase. The lag-phase between arrest to correlate radiosensitivity with differences in capacity for sublethal in ( ;, and induction of apoptosis was comparable for MOI ,1-4, Reh, and damage repair (6, 7). differences in lymphoid lineage maturation or CEM; however. III,-6(1 cells showed a markedly longer G2 arrest that various biochemical parameters (8), or differences in the rate of DNA correlated with their greater radioresistance. The results suggest that the dsb repair (9). However, aside from noting that the most radiosensi total length of time available for DNA damage repair (irrespective of tive lines were derived from immature lymphoid cells [we came to a whether this time accrues as blockage in G,, S, or G2), prior to potential similar conclusion from studies with murine lymphoid lines (3)], these activation of apoptosis, is a critical determinant of radiosensitivity in studies have been unable to explain the differences in radiosensitivity human hematopoietic cell lines. Comparison of the p53 status of these cell lines suggested that mutations in the 77*53 gene are contributing to the among the cell lines examined. delay of induction of apoptosis seen in the more radioresistant cell lines. The relationship between p53 status, the kinetics of induction of The sensitivity of MOLT-4 and HL-60 cells to killing by DNA-associated apoptosis, and radiosensitivity has been studied in human lymphoid I25I decays was determined and was found to correlate with the relative lines, although to a more limited extent than for mouse cell lines. sensitivity of these lines to y-irradiation. The highly localized deposition of Human studies have compared the TK6 (wild-type p53) and WI- energy by I25I decays argues that DNA damage is a potent initiator of L2-NS (mutant p53) cell lines, which were both derived from the apoptosis in these cell lines. The results presented suggest that differences Burkitt's lymphoma WI-L2 cell line. WTK1 (a clonal derivative of in the radiosensitivity of the cell lines examined reflect differences in the WI-L2-NS, which is also a p53 mutant) showed delayed induction of rapidity of induction of apoptosis and that radiation-induced cell death in apoptosis and was correspondingly more resistant to killing by X- hematopoietic cells can be explained as a response to DNA damage. irradiation than was TK6 (10, 11). Olive et al. (12) also showed a correlation between slower induction of apoptosis and increased ra INTRODUCTION dioresistance in WI-L2-NS as compared with TK6. although, this correlation did not hold when TK6 cells in different phases of the cell Tumorigenesis frequently involves genetic changes that impair the cycle were compared. More recent work has cast doubt on whether the control of cell cycling and/or the regulation of apoptosis. Such differences seen in the WI-L2 system are due solely to the expression changes can also alter radioresponsiveness, and there is now extensive of mutant p53 protein. Yu et al. (13) disrupted p53 function in the literature on the involvement of genes such as p53, c-myc, and bcl-2 TK6 cell line by transfection with the human papillomavirus E6 gene in determining the radiosensitivity of tumor cells. The impact on and showed that this had a relatively small effect on the kinetics of radiosensitivity of mutation or change in the level of expression of induction of apoptosis and no effect on the clonogenic survival such genes can, however, be variable and cell type dependent (re dose-response for y-irradiation. The rate of induction of apoptosis in viewed in Ref. 1), presumably reflecting the complexity of the path irradiated WI-L2-derived cell lines is, however, significantly slower ways that lead to cell death and the multiplicity of the regulatory than is seen for many of the murine cell lines that we have examined. biochemical interactions that together determine the fate of an irradi O'Connor et al. (14) examined a panel of Burkitt's lymphoma lines ated cell. We have examined different aspects of the radiation re and found that cells containing wild-type p53 were generally more sponse of murine lymphoid and myeloid cell lines to determine sensitive to radiation-induced growth inhibition. whether there are any simple, general correlates with relative radio- There are various lines of evidence that support the assumption that differences in hematopoietic cell line radiosensitivity are related in Received 12/9/97; accepted 4/23/98. some way to the cell's response to DNA damage. For example. 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 in accordance with Warters (15) demonstrated that bromodeoxyuridine labeling enhanced 18 U.S.C. Section 1734 solely to indicate this fact. the sensitivity to X-ray-induced killing of a murine T-cell hybridoma. 1To whom requests for reprints should be addressed, at Research Division, Peter MacCallum Cancer Institute, Locked Bag No. 1, A'Beckett Street. Melbourne. Victoria 8006. Australia. Phone: 61-3-9656-1291; Fax: 61-3-9656-1411; E-mail: i.radford@ 2 The abbreviations used are: dsb. double strand break; IL, interleukin; BrdUrd, pmci.unimclb.cdu.au. bromodeoxyuridine; ECL. enhanced chemiluminescence. 2817

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1998 American Association for Cancer Research. RADIOSENSmVITY OF HUMAN LYMPHOID CELLS and we have shown that murine lymphoid cell lines can be exquisitely mulated decays at day "t" (At) were determined using the equation sensitive to killing by DNA-associated 125Idecays and that, in gen A, = 86.56/l0(l - i'-0-01155Â»),where Aa represents the number of decays per eral, their sensitivity to such radioactive decays correlates closely with cell per day at day 0. their sensitivity to external beam irradiation (2, 3). The DNA-centric Survival assays were performed using single-cell suspensions that were view has, however, been challenged by investigators who assert a obtained by gently pipetting the cultures approximately 10 times through a 5-ml glass pipette. Dilutions of this cell suspension were then counted using a crucial role for ceramide production, following activation of sphin- Coulter counter. For each of the cell lines except HSB-2, clonogenic survival gomyelinase at cell membranes, in the induction of apoptosis by was determined by plating cells in growth medium plus 0.3% Noble agar irradiation (16). Consistent with the latter hypothesis, it was shown (Difco) and 0.4% (packed cell volume) sheep RBCs (Amadeus International, that acid sphingomyelinase-deficient human or murine lymphoid cells Melbourne, Australia). After 14 days, plates were fixed and stained by the showed reduced levels of radiation-induced apoptosis (17). Recently, addition of 0.01% crystal violet in 1.5% acetic acid. Colonies containing >50 a correlation between the level of postirradiation ceramide production cells were then scored. The HSB-2 cell line could not be cloned in soft agar. and the relative radiosensitivity of a panel of human lymphoid cell Consequently, the survival of this cell line was assayed by plating cells in lines has been adduced as further support for the importance of growth medium in 96-well, U-bottomed microtiter plates (Greiner) and check membrane damage and for the inferred irrelevancy of DNA damage ing for growth after 14 days. Microtiter plate wells were scored as positive if they contained greater than 50 cells. Cell numbers added per well were (18). adjusted to give â€”¿50%positivewells. In control platings, this corresponded to Accordingly, we wished to determine whether the correlations, around three cells per well. The cloning efficiency of HSB-2 cells was found using murine cell lines, between external beam radiosensitivity significantly improved by the addition of 10% pooled human AB serum and and the rate of induction of apoptosis and between sensitivity to 5% conditioned medium from the IL-2-secreting gibbon leukaemic cell line killing by external beam radiation and DNA-associated 125I decays MLA 144 (data not shown). Although Guizani et al. (21) demonstrated that are also observed in human hematopoietic cell lines that show a broad HSB-2 does not constitutively express the IL-2 receptor, growth factors present range of radioresponsiveness. For these purposes, we have used a in either the human serum or the conditioned media may be inducing expres panel of four human lymphoid (CEM, HSB-2, MOLT-4, and Reh) and sion of IL-2 receptor. Alternatively, other factors secreted by MLA-144 cells one myeloid (HL-60) cell line. may be contributing to the improved growth. Results for HSB-2 cells were calculated using the equation Ce = [-ln(AO]/Cw, where cloning efficiency (Ce) is equal to the negative natural log of the fraction of wells without cell growth MATERIALS AND METHODS (N) divided by the number of cells plated per well (Cw). Clonogenic -y-ray dose-response survival curves for CEM cells plated in either soft agar or Cell Lines. All cell lines were obtained from the American Type Culture Collection. Cells were grown in the a modification of Eagle's medium (ICN) microtiter wells showed no significant difference (data not shown). plus 10% PCS (CSL, Melbourne, Australia) at 37Â°C,using sealed flasks that Survival data were fitted to equations using a computer-generated least- squares fit. The fit was minimized according to the criterion of Â£Â¡(0^â€”¿Â£Â¡)2/VÂ¡, had been flushed with 5% CO2, 5% O2, and balance N2. All studies were performed using asynchronous, log-phase cultures. The cell lines were rou where O is observed value, E is calculated value, and V is variance. The statistical errors associated with the survival data were calculated, after the tinely checked for Mycoplasma contamination using a PCR method (19). The method proposed by Boag (22), by assuming a simple Poisson variance on the origin and some of the characteristic features of each of the cell lines used are total number of control and test colonies counted. presented in Table 1. y-Irradiation, 125IIncorporation, Clonogenic Survival Assay, and Sta Determination of Apoptotic Morphology. Cell samples, taken at intervals after irradiation, were pelleted by centrifugation at 1000 rpm, gently resus tistical Treatment of Survival Data. Cell cultures were irradiated at room temperature in a '37Cs -y-ray source at a dose rate of approximately 0.9 Gy/min. pended in a small volume of residual growth medium, and then mixed with an I25I labeling was performed by incubating cells for greater than 1.5 popu equal volume of growth medium containing 10 fig/ml of ethidium bromide and lation doubling times in growth medium containing 50-100 Bq/ml of 3 p.g/ml of acridine orange. At least 500 cells were then scored for apoptotic [125I]iododeoxyuridine (NEN/DuPont) and 2.5 Â¿IMthymidine. Cells were then nuclear morphology under a fluorescence microscope. Cell Cycle Distribution. Cell cultures were labeled with 10 JXMBrdUrdfor washed, and unincorporated label was chased by incubation in growth medium 30 min and then analyzed using the anti-BrdUrd antibody technique (23). containing 20 /UMthymidine and 20 /J.Mdeoxycytidine for 3 h. After chasing, incorporated '~5I was measured by pelleting the cells and counting the level of Cellular DNA content was determined by staining samples with propidium radioactivity in a Compugamma CS (LKB) gamma counter. Counts were iodide. Samples were processed using a Becton Dickinson FACStar Plus flow corrected to decays per minute using the method of Eldridge (20). Cell pellets cytometer, and the data were analyzed using the WinMDI version 2.5 program. Cells were counted as apoptotic if they had a sub-G, or sub-G2 DNA content were then resuspended in a known volume of growth medium, aliquots were taken, and cell number was determined by Coulter counting. The mean number and lacked BrdUrd incorporation. This assignment was checked by cell sorting of I25I decays occurring per cell, per day was then calculated. After slow and microscopic examination. Levels of apoptosis determined by flow cytom- addition of an equal volume of growth medium plus 20% DMSO, the cell etry were generally lower than values obtained by scoring morphology (pre suspension was aliquoted, frozen at -PC/min in a controlled-rate freezer sumably due to overlap between the DNA content of apoptotic and G, or G2 [Cryologic (Melbourne) model CL3000 with Cryogenesis V3 software] to cells); however, the two methods gave qualitatively similar results (compare â€”¿60Â°Cand then transferred to liquid nitrogen for storage. Cell aliquots were Figs. 2 and 4). Western Blotting. Protein was extracted from each cell line by lysing IO7 removed from liquid nitrogen storage at various times, after known numbers of I25I decays per cell had occurred, and assayed for clonogenic survival. Accu- cells in 0.5 ml of 50 mm Tris (pH 7.5) plus 2% SDS. To reduce sample viscosity, DNA was sheared by 20 passages through an 18-gauge needle. Samples were then boiled for 3 min, cooled on ice, and spun for 5 min at 10,000 rpm in a microcentrifuge to remove insoluble material. Supematants Table 1 General properties of human hematopoielic cell lines were assayed for protein concentration using bicinchoninic acid and then stored at â€”¿70Â°C.Prior to electrophoresis, buffer containing bromphenol blue efficiency status"Wild was added to give a final concentration of 10% glycerol, 10 HIMDTT, and 50 CelllineHSB-2 time(h)29 (% Â±SE)15 mM Tris (pH 7.5). Samples were boiled for 3 min and allowed to cool Â±3 type immediately prior to loading. MOLT-4 Pre-T Pseudotetraploid 23 63 Â±7 Wild type/mutant Equal amounts of total protein (100 /j,g unless stated otherwise) were Early B Pseudodiploid 41 Â±3 Reh 21 ^Mutant separated on a 12.5% SDS-PAGE gel and then transferred to a polyvinylidene CEM T Pseudodiploid 2233Cloning48 Â±8 HL-60OriginPre-TPre-myelocyleKaryotype"PseudodiploidPseudodiploidDoubling 50 Â±5TP53Deleted difluoride membrane (Millipore) for 1 h at 50 V in transfer buffer containing " Data from American Type Culture Collection catalog. 25 mM Tris base, 192 mM glycine, and 20% methanol at pH 8.3 (24). The * Data from other studies (see "Results" for references). membrane was then allowed to dry overnight at room temperature, stained for 2818

tion are presented in Fig. 1. The a and ÃŸvalues derived from fitting the data to a linear-quadratic function of dose are given in the figure legend. The results show clearly that HSB-2 and HL-60 are. respec

(0 tively, the most radiosensitive and the most radioresistant of the cell > .1 lines surveyed, with approximately a 200-fold difference between the lines in the level of survival after a dose of 4 Gy. MOLT-4 cells show 3 CO a comparable, although somewhat more radioresistant, response to I .01 that of HSB-2. Reh and CEM are considerably more radioresistant than HSB-2 and MOLT-4. Both show similar clonogenic dose re 0) Â§> sponses, although CEM is significantly more radioresistant than Reh at lower radiation doses. Our dose responses for MOLT-4 and HL-60 o .001 are comparable with those of previous studies (7, 27). o Rate of Induction of Cell Death after y-Irradiation Correlates with Radiosensitivity. Based on the y-ray survival dose-responses .0001 presented in Fig. 1. cultures of the different cell lines were given equitoxic radiation doses sufficient to reduce clonogenic survival to 0.5% of the control value and were then incubated at 37Â°C.The timing of occurrence of cell death was then determined in culture samples Dose (Gy) taken at various times after irradiation. All five cell lines showed induction of apoptotic nuclear morphology after irradiation. HL-60 Fig. I. Clonogenic survival of different human hemalopoietic cell lines as a function of -y-ray dose. Data were fitted to the equation S = exp - [aD + ÃŸD2].where S is cultures showed some necrotic death, although apoptosis was the survival, D is dose, and a and ÃŸare constants. Calculated values of a and ÃŸ:HSB-2 predominant form of death over the time interval examined (data not (1.75 Â±0.06 and 0.087 Â±0.029); MOLT-4 (1.49 Â±0.05 and 0.064 Â±0.018); Reh (1.06 Â±0.01 and 0.005 Â±0.005); CEM (0.61 Â±0.03 and 0.081 Â±0.006); and HL-60 shown). As shown in Fig. 2, the rate of induction of apoptosis after (0.62 Â±0.02 and 0.029 Â±0.004). Each data set is from at least two separate experiments. equitoxic doses of y-irradiation varied greatly between the five cell lines examined, and the relative ordering of these responses reflected total protein with Ponceau-S (Sigma), and blocked with 3% skim milk powder, the clonogenic survival dose responses. For example, HSB-2 is the 2% gelatin, and 0.1% Tween 20 in PBS (pH 7.4). After incubation for 2 h at most radiosensitive of the cell lines examined and also shows the most room temperature, fresh blocking solution containing the primary antibody rapid induction of apoptosis. The more radioresistant MOLT-4 cell (AC 21 (Santa Cruz Biotechnology) for Bcl-2 or PAb 421 for p53) was added, line shows a pronounced delay of â€”¿4-6h before commencement of and the membrane was incubated overnight at 4Â°C.The membrane was then induction of apoptosis at a similar rate to that of HSB-2. Reh cultures washed three times in PBS plus 0.2% Tween 20, followed by incubation with are more radioresistant than MOLT-4 and show a somewhat longer peroxidase-labeled secondary antibody for l h at room temperature. After delay before initiating apoptosis. The subsequent entry of Reh cells four X 15-min washes in PBS plus 0.2% Tween 20 at room temperature, the into apoptosis occurs at a much slower rate than is seen for MOLT-4 membrane was incubated for 1 min in ECL buffer [0.4 mg/ml luminol, 0.1 or HSB-2. CEM cells are more resistant to y-irradiation than Reh and mg/ml 4-iodophenol, and 1 /Â¿I/mlH2O2 (36%) in 60 mM Tris buffer, pH 7.5], and proteins were then visualized on Fuji Rx medical X-ray film using show a significantly longer delay prior to the induction of apoptosis. exposure times varying from 30 s to 5 min. ECL provides qualitative, but not Once apoptosis has begun in irradiated CEM cultures, the rate of quantitative, information about protein levels. accumulation of apoptotic cells appears to be similar to that of Reh.

RESULTS

Optimizing Clonogenic Assays. Our initial studies suggested that, 1.0 when plated in growth medium plus semi-solid agar, the human cell lines chosen generally had low cloning efficiencies that showed a high o O 0.8 level of inter-experimental variability. This result was of concern u given claims that, at least for some cell lines, there is a correlation between cloning efficiency and radiosensitivity (25). The control 0.6 cloning efficiencies of CEM, HL-60, MOLT-4, and Reh were, how o o. ever, found to be greatly increased by the addition of sheep RBCs to the medium. For example, the cloning efficiency of HL-60 increased from 1% to ~50% in the presence of sheep RBCs. The ability of sheep RBCs to increase the cloning efficiency of hematopoietic cells "'So was first demonstrated by Bradley et al. (26). Even with the addition 0.2 of conditioned media, sheep RBCs, or lethally irradiated feeder cells, SS IL we were unable to clone HSB-2 cells in soft agar. This cell line was, however, found to be clonable in microtiter plates as described in "Materials and Methods." Our inability to clone HSB-2 cells in soft 10 20 30 40 agar may indicate a requirement for cell-to-cell contact or that some aspect of the cloning methodology is lethal to this cell line. Control Time (h) cloning efficiencies, obtained for each of the cell lines using the above Fig. 2. Rate of induction of apoptosis. as determined by nuclear morphology, in methods, are shown in the Table. The values obtained are, in general, â€¢¿y-irradiatedhumanlymphoid cell lines. Cell cultures were given equitoxic doses (HSB-2. 3 Gy; MOLT-4. 3.5 Gy; Reh, 5 Gy; CEM. 5 Gy; and HL-60. 6.5 Gy) and then incubated significantly greater than those reported in previous studies. at 37Â°C.The response of the murine ST4 lymphoid cell line after 3 Gy of -y-irradialion is Clonogenic Survival y-Irradiation Dose Responses. Dose-re included for comparison [data from Radford et al. (36)|. Each data set was pooled from sponses for the five human hematopoietic cell lines under investiga- at least two separate experiments. 2819

BrdUrd at intervals after exposure to equitoxic y-ray doses (sufficient to reduce clonogenic survival to 0.5% of the control value) and were then analyzed by flow cytometry. Cell cycle distributions, as a func tion of time after irradiation, for each of the five cell lines are shown in Fig. 4.

HL-60 Flow cytometric analysis of irradiated HSB-2 cultures showed no (31.211.9) marked accumulation of cells in any phase of the cell cycle prior to an MOLT-4 increase in the levels of apoptosis. Irradiation has rapid and profound (22.110.6) .1 effects on these cells as evidenced by an almost total suppression of mitotic activity after 3 Gy of y-irradiation and a marked decrease in BrdUrd incorporation into S-phase cells within 2 h after irradiation (data not shown). Flow cytometric profiles of cultures that had been incubated for 4 or more hours after irradiation, showed a significant increase in the level of cells with sub-G, or intermediate to G, and G2 (but lacking BrdUrd incorporation) DNA content (Fig. 5), suggesting .01 that the induction of apoptosis in HSB-2 cells can occur at different 20 40 60 80 100 points in the cell cycle and is not linked to one checkpoint. In contrast to the results obtained for HSB-2, the other more rapidly dying cell 125Idecays/cell line, MOLT-4, shows an accumulation of cells in G2 that parallels and precedes the induction of apoptosis. Consistent with MOLT-4 cells Fig. 3. Clonogenic survival responses of MOLT-4 and HL-60 cells exposed to DNA-associated 125Idecays. Data were fitted to the equation S = exp - [DIDâ€ž\,where undergoing apoptotic death after blockage in G2, flow cytometric S is survival, D is dose, and Ã¶â€žisa constant. Calculated values for D0 and its associated profiles showed a progressive increase, with time after irradiation, in error are shown on the figure. Each data set is from two separate experiments. the proportion of cells showing a DNA content intermediate to G, and G2 (but lacking BrdUrd incorporation; Fig. 5). Similar to MOLT-4, the increase in the fraction of apoptotic Reh cells follows and occurs The apparent decline in the numbers of apoptotic CEM cells at â€”¿20 in parallel with the accumulation of cells in the G2 phase. This h after irradiation is an artifact caused by the fragility of apoptotic accumulation occurs more slowly than is seen for MOLT-4, appar CEM cells (i.e., the true kinetics of appearance of apoptotic CEM cells ently due to a more prolonged blockage of Reh cells in Gl and/or S. are probably more comparable with those of Reh than to HL-60). Consistent with induction of apoptosis in G2, flow cytometric profiles HL-60 is clearly the most radioresistant cell line examined and also from irradiated Reh cultures undergoing apoptosis showed increased shows the most pronounced delay prior to the induction of apoptosis. levels of cells with a DNA content intermediate to G, and G2 (but Accumulation of apoptotic cells was not observed in HL-60 cultures lacking BrdUrd incorporation; see Fig. 5). Although Reh and CEM until at least 24 h after irradiation. It is of interest to note that even the have comparable sensitivities to killing by y-irradiation, their cell most rapidly dying of the human cell lines (HSB-2) shows markedly cycle responses are significantly different (Fig. 4). Unlike Reh, CEM slower kinetics of induction of apoptosis than does a murine lym- cells show no postirradiation blockage in the G, phase, but they do phoma such as ST4 (Fig. 2). show a pronounced blockage in mid- to late-S phase that is followed Sensitivity to Killing by DNA-associated 125I Decays. Because by a rapid exit from S and accumulation in the G2 phase. As with of the continuing debate over the importance of DNA damage in MOLT-4 and Reh, the latter event precedes the appearance of apop initiating radiation-induced apoptotic cell death and because the level totic cells in culture. Consistent with the death of CEM cells in G2 of DNA damage induced per unit dose of y-irradiation can vary phase, flow cytometric profiles of apoptosing cultures showed in between cell lines (28), we were interested in comparing the sensi creased levels of cells with a DNA content intermediate to G( and G2 tivity of our panel of cell lines to killing by DNA-associated I25I (but lacking BrdUrd incorporation; data not shown). HL-60, the most decays. radioresistant and the slowest dying of the cell lines in our panel, lacks The technique used for I25I decay experiments involves freezing a marked G, arrest but shows prolonged blockage of cells in G2 prior cells and then storing them over liquid nitrogen for periods up to 2-3 to the appearance of apoptosis in the culture (Fig. 4). The timing of months to allow the accumulation of DNA damage without interfer occurrence of apoptosis in irradiated HL-60 cells also coincides with ence from DNA repair activities. The cells must then show reasonable the resumption of mitotic activity in these cultures (data not shown). cloning efficiencies after thawing. Our attempts to determine the This result and the flow cytometric data suggest that death of these sensitivity of these cell lines to DNA-associated 125I decays were, cells occurs both in G2 and during or around the time of mitosis. however, generally unsuccessful due to low and variable cloning p53 and Bcl-2 Status. The p53 protein is an important modulator efficiencies after freezing and thawing. MOLT-4 and HL-60 were the of the cell cycle and a determinant of radiosensitivity in lymphoid only lines to show reasonable cloning efficiencies after freezing and cells. Accordingly, we sought to correlate the status and expression of thawing (30 Â±9% for MOLT-4 and 6.7 Â±0.4% for HL-60). The the TP53 gene with the radiosensitivity and cell cycle response of the clonogenic survival curves for these cell lines, after accumulation of cell lines examined. The TP53 gene of the HSB-2, MOLT-4, CEM, 125I-induced DNA damage, are shown in Fig. 3. As for the y-ray and HL-60 cell lines has been sequenced by other investigators, and survival results, HL-60 cells (D0 = 38.2 Â±1.9 decays) were found to the results are summarized in Table 1. Similar sequence data for Reh be far more resistant to 125I decay-induced DNA damage than are cells could not be found in the literature. We tested for the presence MOLT-4 cells (D0 = 22.1 Â±0.6 decays). of p53 protein in each of the cell lines by Western blotting. The blots Effect of y-Irradiation on Cell Cycling. Studying the effect of were probed with the PAb 421 monoclonal antibody, which is specific y-irradiation on subsequent cell cycling can indicate whether induc for an epitope in the COOH-terminal region of the protein (29), and tion of apoptosis is related to cell cycle events (e.g., cell cycle reactive proteins were detected using ECL. Expression of p53 protein blockage or mitosis) and how differences in radiosensitivity might be was found in all of the cell lines, except for HL-60 (Fig. 6). The latter related to these events (4). Cell cultures were pulse labeled with result is consistent with the finding that HL-60 has a large deletion 820

1.0

o -

0 2 4 602468 10 0 8 12 16 20 24 Fig. 4. Effect of -y-irradiation on cell cycle progression of human lymphoid lines. The distribution of cells be tween the different phases of the cycle (G,, S. and G2) Time (h) Time (h) Time (h) and the level of apoptosis (Api were determined by flow cytometry using cultures given equitoxic doses and then incubated at 37Â°C.Each data set was pooled from two 1.0 CEM (5 Gy) HL-60 (6.5 Gy) separate experiments.

.2 .8

8.2

4 8 12 16 20 240 10 15 20 25 30 35

Time (h) Time (h) within the TP53 gene that prevents protein expression (30). The two DISCUSSION most rapidly dying and radiosensitive cell lines, HSB-2 and MOLT-4, have both been shown to express wild-type p53 (31, 32). In addition We have examined whether the correlation between the rate of to a wild-type gene, the MOLT-4 cell line also contains a TP53 induction of apoptosis and the clonogenic survival y-ray dose- mutation that leads to aberrant splicing of the mRNA, causing a frame response, observed for murine hematopoietic cell lines, is also seen in shift and truncating the protein before translation of exons 10 and 11 human hematopoietic cell lines. The results presented in this report (32). Of the more slowly dying cell lines, none are known to express clearly demonstrate, for the cell lines examined, a correlation between wild-type p53. Both copies of the TP53 gene in CEM cells contain the rapidity with which apoptosis is induced after irradiation and the single base substitutions that produce missense mutations (31), and as clonogenic survival dose-response. For example, the HSB-2 cell line, mentioned above, the HL-60 cell line does not express p53 protein due which shows the most rapid induction of apoptosis after y-irradiation, to gene deletions. Reh does express p53 protein (Fig. 6): however, we was also found to be the cell line most sensitive to clonogenic killing, do not know whether this protein is mutant or wild type. whereas MOLT-4, Reh, CEM, and HL-60, which show progressively Members of the Bcl-2 family of proteins are important modulators longer delays prior to the induction of apoptosis after an equitoxic of apoptosis and have been shown to influence radiosensitivity (33). radiation dose, are correspondingly more resistant to killing by Western blotting, using a polyclonal antibody to Bcl-2 protein, y-irradiation. showed that all of the cell lines used in this study expressed a reactive Although a general correlation between the relative rate of induc protein of Mr -26,000 (Fig. 6). Reh and HL-60 differed from the tion of apoptosis and the clonogenic survival y-ray dose-response is other lines in showing an additional reactive band of slightly higher seen for both human and murine hematopoietic cell lines, one notable molecular weight. The additional band might represent expression of difference is that although the survival dose-response curves for the a Bcl-2-related protein, such as Bcl-x,. which has a molecular weight more radiosensitive human and murine cell lines are similar, equitoxic of 29.000 (34), or a phosphorylated form of the protein. A variant of radiation doses induce apoptosis far more rapidly in the murine lines. HL-60 has been shown to express Bcl-xL (35). For example, we found that at least 90% of ST4 cells (a murine pre-T 2821

relevant that Bcl-2 protein is readily detectable in the human cell lines HSB-2 (Control) HSB-2 (3 Gy + 5 hrs) that we have used. Irradiation of cells can induce blockage at checkpoints in different phases of the cell cycle, including p53-dependent arrest in G, (37) and p53-independent blocks in S (38) and G2 (39). These blocks are ultimately caused by inhibition of cyclin-dependent kinases that are necessary for cycle progression. In the cell lines that we have exam ined, the rate of induction of apoptosis, after y-irradiation, generally appears to be related to the functioning of these cell cycle checkpoints. HSB-2 may be an exception in this regard, because it shows no clear evidence for accumulation at checkpoints and appears to undergo MOLT-4 (Control) 2 MOLT-4 (3.5 Gy + 10 hrs) induction of apoptosis in different phases of the cell cycle. Consistent with previous observations that the more rapidly dying and radiosen u sitive murine lymphoid cell lines contain wild-type p53 (5), HSB-2 O encodes wild-type TP53 (31), and we have shown that it expresses O p53 protein. It may also be relevant to the radiosensitivity of HSB-2 3 that, due to homozygous deletion of the Rb gene (40), this cell line 'E does not express pRb. Expression of pRb was associated with p53- independent protection against radiation-induced apoptosis in both a human osteosarcoma and a lymphocytic cell line (41, 42). When O 2 MOLT-4 or Reh cells were exposed to a y-ray dose that reduces m m survival to 0.5% (unlike HSB-2), both showed induction of apoptosis O Reh (Control) O Reh (5 Gy + 24 hrs) after accumulation of cells in G-,. Due apparently to blockage earlier in the cycle, Reh cells accumulated in G2 much more slowly than did MOLT-4 cells and showed a corresponding increase in the time between irradiation and the appearance of apoptotic cells that corre lated with their greater radioresistance. The molecular basis for this O difference is unclear. Both MOLT-4 and Reh express pRb (40, 43) and, as shown in this study, p53. Although MOLT-4 cells express both wild-type and a truncated p53 (32), the status of the p53 expressed by Reh cells is not known to us. The relative slowness of the induction of apoptosis in Reh cells suggests that they contain mutant p53. Like MOLT-4 and Reh, irradiated CEM cells appeared to die after block DNA Content age in G2. CEM cells differed, however, in showing a prominent Fig. 5. Dot-pio! distributions for BrdUrd incorporation (ordinale) versus DNA content blockage in mid- to late-S phase that preceded accumulation in G2. (abscissa) of cells from control and irradiated MOLT-4 and Reh cultures. Regions Consistent with their slow kinetics of induction of apoptosis and their corresponding to cells in G, and G2 phases of the cell cycle are indicated. radioresistance, CEM cells express pRb (40) and mutant p53 (31). Finally, the response of HL-60 to a y-ray dose that reduces survival to cell line), exposed to a y-ray dose sufficient to reduce clonogenic 0.5% is characterized by transient arrest in S and a marked and survival to 0.5%, showed apoptotic morphology 3 h after irradiation prolonged blockage in G2. Other authors have also described radia (36). In contrast, the most rapidly dying human cell line used in this tion-induced G-, arrest and apoptosis of HL-60 cells (39, 44). Al study (HSB-2) does not reach this level of apoptosis until at least 24 h though MOLT-4, Reh, and CEM cultures showed an increase in the after receiving an equitoxic y-ray dose, despite having a similar level of apoptosis that lagged about 4 h behind the accumulation of clonogenic survival y-ray dose-response to ST4. Unfortunately, be cells in G2, our results suggest that HL-60 cells can remain arrested in cause we were unable to determine the sensitivity of HSB-2 cells to G2 for much longer times before undergoing apoptosis. The basis for killing by DNA-associated 125Idecays, we cannot rigorously compare this difference has not been examined, although in other cell types the response to DNA damage of ST4 and HSB-2. If we assume that expression of a ras oncogene was associated with a decrease in cyclin there are similar y-ray dose-responses for DNA lesion induction in the Bl mRNA levels and a lengthening of G2 arrest (45). two cell lines, then perhaps the simplest explanation for the difference in timing is that, after the detection of lethal cellular injury, the HSB-2 MOLT-4 Reh CEM HL-60 biochemical events that lead to the triggering of apoptosis occur much more slowly in HSB-2 than in a murine line such as ST4. Two factors that may be relevant to this human/murine difference are: (a) there is p53 a marked difference in the population doubling times of cell lines such as ST4 and HSB-2 (9 h versus 29 h). The data of Olive et al. (12), however, suggest that human TK6 cells have a relatively rapid dou bling time (around 12 h) and yet show induction of apoptosis after irradiation at a comparable rate to HSB-2 cells; and (b) the human cell lines that were used in this study all derived from tumors that relapsed bcl-2 after intensive radiotherapy and/or chemotherapy of the primary tu mor, whereas the derivation of murine lines like ST4 did not involve exposure to cytotoxic agents. Such treatment may well have selected Fig. 6. Western blotting analysis of p53 and Bcl-2 protein expression in human for cells with down-regulated apoptotic responses, and it is perhaps hematopoietic cell lines. 2822

The importance of the duration of postirradiation arrest at the G, Evans. H. H.. Ricanati. M.. Horng. M. F.. Jiang. Q.. Menci. J.. and Olive. P. DNA double-strand break rejoining deficiency in TK6 and other human B-lymphoblast cell and G2 checkpoints in determining radiosensitivity has been tested in lines. RadiÃ¢t.Res.. 134: 307-315. 1993. various nonlymphoid cell types that undergo apoptosis. There is 10.Amundson, S. A., Xia. F.. Wolfson. K.. and Liber. H. L. Different cytotoxic and evidence to suggest that although the p53-dependent G, checkpoint tnutagenic responses induced by X-rays in two human lymphoblastoid cell lines derived from a single donor. MutÃ¢t.Res.. 286: 233-241. 1993. may not be critical in this regard (46), the length of the G2 delay may Xia. F., Wang, X.. Wang. Y. H.. Tsang. N. M.. Yandell. D. W., Kelsey. K. T.. and be directly related to radiosensitivity (45). Consistent with our previ Liber, H. L. Altered p53 status correlates with differences in sensitivity to radiation- induced mutation and apoptosis in two closely related human lymphohlasl lines. ous results for murine hematopoietic cell lines (4), the data presented Cancer Res., 55: 12-15, 1995. in this paper suggest that the total length of time available for DNA Olive, P. L., Banath. J. P., and Durand. R. E. Development of apoptosis and damage repair (irrespective of whether this time accrues as blockage polyploidy in human lymphohlast cells as a function of position in the cell cycle at the time of irradiation. Radial. Res.. 146: 595-602. 1996. in G|, S, or G-,), prior to potential activation of apoptosis, is a critical Yu. Y.. Li. C. Y.. and Little. J. B. Abrogation of p53 function by HPV16 Â£6gene determinant of radiosensitivity in human hematopoietic cell lines. delays apoptosis and enhances mutagenesis hut does not alter radiosensitivity in TK6 human lymphoblast cells. Oncogene. 14: 1661-1667. 1997. We have assumed that the explanation for the differences in radio- O'Connor, P. M., Jackman. J.. Jondle. D.. Bhatia. K.. Magralh. !.. and Kohn. K. W. sensitivity between hematopoietic cell lines lies in understanding the Role of the p53 tumor suppressor gene in cell cycle arrest and radiosensitivity of Burkitt's lymphoma cell lines. Cancer Res.. 5.?: 4776-4780. 1993. response of the cell to DNA damage. Consistent with this view, we Warters. R. L. Radiation-induced apoptosis in a murine T-cell hybridoma. Cancer have shown in this report a correlation between the sensitivity of Res., 52: 883-890, 1992. MOLT-4 and HL-60 to y-irradiation and to DNA-associated 125I Haimovitz-Friedman. A.. Kan. C. C.. Ehleiter. D.. Persaud. R. S., McLoughlin, M . decays. It is interesting to note that the D0 values for I25I decay- Fuks. / and Kolesnick. R. N. loni/ing radiation acts on cellular membranes to generate ceramide and initiate apoptosis. J. Exp. 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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1998 American Association for Cancer Research. Explaining Differences in Sensitivity to Killing by Ionizing Radiation between Human Lymphoid Cell Lines

David R. Aldridge and Ian R. Radford

Cancer Res 1998;58:2817-2824.

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