Novel concepts in modification of radiation sensitivity

Int. J. Radiation

Oncology

Pergamon

Biol. Phys., Vol. 29. No. 2, pp. 249-253. 1994 Copyright 0 I994 Elsevier Science Ltd Printed in the USA. All rights reserved 0360-3016/94 $6.00 + .OO

0360-3016(93)EOOll-T

??Chemo- and Radiosensitivity

NOVEL

CONCEPTS

and Biochemical Modulation

IN MODIFICATION

OF RADIATION

SENSITIVITY

A.

BuMP,PH.D.,*SUSANJ.BRAUNHUT,PH.D.,+SANJEEWANIT.PALAYooR,PH.D.,* DIANE MEDEIROS,+LEON L. LAI,B.A.,* BETH A.CERCE,B.A.,*RUTH E. LANGLEY,M.B.,B.S.* ANDC.NORMANCOLEMAN,M.D.* EDWARD

Center for Radiation Therapy, Harvard +Departments of Surgery and Ophthalmology,

*Joint

Medical School and Dana-Farber Children’s Hospital and Harvard

Cancer Institute, Boston, MA 02 115: Medical School, Boston, MA 02 115

Purpose: To determine whether biological effects of radiation, such as apoptosis, that differ from classical clonogenic -killing, can be modified with agents that would not be expected to modify classical clonogenic cell killing. This would expand the range of potential modifiers of radiation therapy. Methods and Materials: EL4 murine lymphoma cell apoptosis was determined by electrophoretic analysis of deoxyribonucleic acid (DNA) fragmentation. DNA was extracted 24 h after irradiation or addition of inducing agents. Modifiers of radiation-induced apoptosis were added immediately after irradiation. The effects of radiation on wounded endothelial monolayers were studied by scraping a line across the monolayer 30 min after irradiation. Cell detachment was used as an endpoint to determine the protective effect of prolonged exposure to retinol prior to irradiation. Results: EL4 cell apoptosis can be induced by tert-butyl hydroperoxide or the glutathione oxidant SR-4077. Radiationinduced EL4 cell apoptosis can be inhibited with 3-aminobenzamide, an agent that sensitizes cells to classical clonogenic cell killing. Radiation-induced endothelial cell detachment from confluent monolayers can be modified by pretreatment with retinol. Conclusion: These results raise the possibility that radiation could induce apoptosis by an oxidative stress mechanism that is different from that involved in classical clonogenic cell killing. These and other recent findings encourage the notion that differential modification of classical clonogenic cell killing and other important endpoints of radiation action may he possible. Ionizing radiation, Tert-hutyl hydroperoxide, SR-4077, Endothelial cells, Apoptosis.

INTRODUCTION The principal mechanism of action of ionizing radiation is believed to be the production of double-strand DNA lesions by clusters of ionizations, leading to chromosome aberrations, rendering cells incapable of sustained cell division (4,29). For the purpose of this report, we will define this endpoint as “classical” clonogenic cell killing. In this narrow definition, the factors that contribute to cell death are restricted to the radiochemical events that occur within milliseconds of irradiation (26), and the enzymatic repair of the resulting DNA damage (1). Most modifiers of radiation effects are designed to interfere with one of these two mechanisms (5). Recent developments have indicated that some effects of radiation that are mechanistically distinct from classical clonogenic cell killing may also be biologically important.

For example, radiation-induced apoptosis is now recognized to be a significant factor in the radiation response of a number of cell types (34). This creates a new opportunity for the development of modifiers for radiation therapy since differential modification of various endpoints should be achievable by taking advantage of differences in the mechanisms of their induction. It has long been known that thymocytes undergo apoptosis following low dose irradiation (33,46). More recently this has been found to occur in serous acinar cells (35), intestinal crypt cells (16), C3H- 10T,,2 cells (37), murine ovarian carcinoma, and hepatocarcinoma cells (34), a murine T-cell hybridoma (43), EL4 murine lymphoma cells (27), F9 murine teratocarcinoma cells (2 l), and human melanoma xenografts (9). Apoptosis is a normal cellular response which is invoked during development and during organ involution (18). The mechanism by which

Reprint requests to: Edward A. Bump, Joint Center for Radiation Therapy, Harvard Medical School, 50 Binney St., Boston, MA02115. Acknowledgement-Supported by grants CA 4239 1, CA-46776

from the National Cancer Institute, Department of Health and Human Services, USPHS, and AICR-6 1 from the American Institute for Cancer Research. Accepted for publication 13 October 1993. 249

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radiation can trigger this response is not known, but appears to be different from the mechanism of classical clonogenic cell killing because the process can be interrupted with various agents (22) which would not be expected to modify classical clonogenic cell killing, and the endpoint is normally apparent within one cell cycle of irradiation. The vascular response to radiation is another example of a biological response that is mechanistically distinct from classical clonogenic cell killing. Changes in vascular permeability can be observed immediately after irradiation at doses as low as 2 Gy (19). Irradiated endothelial cells release increased amounts of mitogenic factors (45). Irradiated endothelial cells exhibit a wide range of metabolic and phenotypic alterations including cell detachment, hypertrophy and disorganization of the cytoskeleton (11, 23, 31, 32, 41). A recent report by Fuks (12, 15) indicates that radiation can induce apoptosis in endothelial cells and that bFGF, which is produced by irradiated endothelial cells, can prevent this effect. Retinoids are important modulators of endothelial cell shape and growth factor responsiveness (3). Retinol is present in serum at a concentration of about 1 PM (42). We have used retinol pretreated cells to model normal physiological conditions. METHODS

AND MATERIALS

EL4 cell apoptosis EL4 murine T-lymphoma cells were maintained in suspension (27) in RPM1 medium’ supplemented with 10% fetal bovine serum, 20 mM HEPES, 2 mM glutamate, 60 PM 2-mercaptoethanol and antibiotics. Cells (4 X 1OS/ ml, 10 ml/flask) were incubated with, t-butyl hydroperoxide (t-BOOH), diazene dicarboxylic acid bis (N,N’-piperidine) (SR-4077),* or etanidazole (SR-2508)3 for 24 h, or were harvested 24 h after irradiation; 3-aminobenzamide was added immediately after irradiation and was present during the entire 24 h postirradiation incubation. Cells were centrifuged at 800 g for 5 min at room temperature, resuspended in Dulbecco’s phosphate-buffered saline, centrifuged again, and lysed for 20 min at 0°C in 10 mM Tris containing 3 mM EDTA (TE) and 0.2% triton X- 100 (27). Samples were centrifuged at 12,000 g and low molecular weight DNA fragments were extracted from the supernatant in phenol:chloroform:isoamyl alcohol (25: 24: 1). DNA was precipitated with ethanol at -70°C overnight, recovered by centrifugation and pellets were washed with ethanol. After air drying for 10 min, the pellets were resuspended in TE containing ribonuclease A (200 pgl

’Unless otherwise specified, reagents were obtained

from

Sigma Chemical Co., St. Louis, MO. 2 SR-4077 was provided by SRI International, Menlo Park, CA. 3 Obtained from the Division of Cancer Treatment, National Cancer Institute, DHHS, Bethesda, MD.

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ml). Following the RNase treatment (37”C, 10 min, 65 “C, 5 min), 10 kg of DNA was loaded on a 1.8% agarose gel. DNA fragments were separated by electrophoresis (75 volts for 1 h) and visualized with ethidium bromide (0.5 pg/ml).

Endothelial cells Microvascular endothelial cells were derived from bovine adrenals4 (10, 13) and were used before passage 12. Cultures were examined for contaminating cell types by immunofluorescent determination of homogeneous expression of Factor VIII antigen (17). Cultures were grown to confluency on 35 mm tissue culture dishes5 in Dulbecco’s modified Eagle’s medium containing 5% calf serum, 2 mM glutamine and antibiotics.6 Confluent monolayers were incubated for 6 days with or without 1 PM all-trans retinol (solubilized in degassed ethanol) prior to irradiation. The medium was changed 3 days before irradiation (with or without retinol). This preincubation was designed to allow the formation of a stable extracellular matrix. The medium was changed immediately after irradiation. Monolayers were wounded 30 min after irradiation by scraping the dish with a plastic pipette tip (single line across the plate). Detached cells were counted at indicated times after irradiation and parallel plates were trypsinized and counted to determine total number of cells per plate. Controls were sham treated. RESULTS EL4 cell apoptosis Both t-butyl hydroperoxide and SR-4077 caused internucleosomal DNA fragmentation characteristic of apoptosis in EL4 cells (Fig. 1). t-butyl hydroperoxide is expected to act by initiating lipid peroxidation following metal ion catalyzed decomposition to alkoxyl and methyl free radicals (24). SR-4077 is a reagent for oxidation of cellular glutathione (6). The concentration dependence for induction of internucleosomal DNA fragmentation by SR4077 is similar to that seen for other biological effects of thiol oxidants: little or no effect at low concentration, as the cell effectively counters the oxidative perturbation, and an abrupt response over a narrow concentration range as the reductive capacity of the cell becomes limiting (30). The induction of apoptosis by ionizing radiation was inhibited by 3_aminobenzamide, with progressive inhibition up to 20 mM 3-aminobenzamide (Fig. 2). 3-aminobenzamide (20 mM) induced some DNA fragmentation by itself (Fig. 2). Etanidazole-induced DNA fragmentation was enhanced by 3-aminobenzamide (Fig. 2). We were

4 Microvascular endothelial cells were obtained from Dr. Judah Folkman and Ms. Catherine Butterfield, Children’s Hospital, Boston, MA. ’ Costar Corp., Cambridge, MA. 6 Tissue culture reagents for endothelial cell culture were obtained from Grand Island Biological Co.. Gaithersburg, MD.

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irradiation would be expected to produce 1 PM radicals per Gy and up to 32 PM H202 did not induce apoptosis in EL4 cells, and neither catalase nor superoxide dismutase inhibited radiation induced apoptosis (results not shown).

kb

Endothelial cell detachment Radiation ( 10 Gy) caused cell detachment from wounded endothelial cell monolayers (Fig. 3). There appeared to be a threshold for this effect, since 4 Gy did not cause cell detachment. Only l-2% of the cells in monolayers treated with 10 Gy detached and the monolayers remained relatively intact. About 5% of the cells exhibited altered morphology, remaining firmly attached (could not be dislodged by vigorous pipetting) but appearing more birefringent and occupying a focal plane that was above

- 1.0 - 0.5 - 0.3

Fig. 1. Oxidative stress-induced internucleosomal DNA fragmentation in EL4 cells. Fragmented DNA was extracted from EL4 cells and separated by electrophoresis on a 1.8% agarose gel ( 10 gg DNA/lane) 24 h after addition of reagents or irradiation with a cesium irradiator. DNA was visualized with 0.5 pg/ml

A Y

--I + RETINOL

12 -

8

ethidium bromide. The series of bands (ladders) on the gel represent series of DNA fragments of sizes that are approximately multiples of 185 bp. Lane I: untreated cells; Lane 2: 20 FM tBOOH; Lane 3: 50 FM t-BOOH; Lane 4: 80 PM t-BOOH; 5: 200 PM SR-4071; Lane 6: 300 FM SR-4077.

Lane

concerned that the radiolytic production of O? ’ and Hz02 in the medium could result in artifactual induction of apoptosis, since the volume of the medium is about 1000 times the volume of the cells-a situation that would not occur in vivo. This did not appear to be the case, since

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Fig. 2. Effect of 3-aminobenzamide on radiation-induced internucleosomal DNA fragmentation in EL4 cells. Fragmented DNA was extracted from EL4 cells and separated by electrophoresis on a 1.8% agarose gel (10 wg DNA/lane) 24 h after irradiation (8 Gy) with a cesium irradiator. 3-aminobenzamide was added after irradiation. DNA was visualized with 0.5 pg/ml ethidium bromide. The series of bands (ladders) on the gel represent series of DNA fragments of sizes that are approximately multiples of 185 bp. Lane 1: untreated cells; Lane 2: 20 mM 3-aminobenzamide; Lane 3: 8 Gy; Lane 4: 8 Gy + 5 mM 3-aminobenzamide; Lane 5: 8 Gy + 10 mM 3-aminobenzamide; Lane 6: 8 Gy + 20 mM 3-aminobenzamide; Lane 7: 5 mM etanidazole, 24 h; Lane 8: 5 mM etanidazole + 20 mM 3-aminobenzamide, 24 h.

2

3

4

POST-IRRADIATION

14 , RETINOL

4

12 -

?---------

10 -

,’ /’

0:

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1 DAYS

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4

POST-IRRADIATION

Fig. 3. Effect of radiation on detachment of endothelial cells from wounded monolayers. Bovine adrenal microvascular endothelial cells were maintained for 6 days as confluent monolayers in the presence (A) of absence (B) of 1 mM retinol prior to radiation. Monolayers were wounded 30 min after irradiation with a 250 kVp x-ray machine: no irradiation (0), 4 Gy (A), 10 Gy (O), and detached cells were counted at the indicated times (cumulative total f SE). The total number of cells per dish, regardless of treatment, was 6.75 X IO5 f 20%.

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the monolayer. These phenotypic changes were progressive over a period of 24-72 h. Radiation did not cause cell detachment when endothelial cells were grown for 6 days in the presence of alltrans retinol prior to radiation (Fig. 3). Retinol also prevented the morphological changes described above.

DISCUSSION The initiating event responsible for classical clonogenic cell killing by ionizing radiation is believed to be the production of double-strand DNA lesions by clusters of ionizations (4, 29). Other biological effects of radiation, such as increased vascular permeability ( 19. 23) clastogenic factor production (8) production of a diffusible agent that intensifies the oxidative burst in activated phagocytes (39), changes in membrane protein conformation, as determined by Raman spectroscopy (38). induction of early response genes (44). etc. could result from other initiating events. Since all molecules are at risk of free radical damage by ionizing radiation, it is possible that biologically active products could be formed that could initiate a cellular response at very low concentrations. For example, Lands and Pendleton (20) have reported that 20 nM organic peroxides can activate cyclooxygenase. Similarly. phospholipase A2 has been implicated in the mechanism of cell killing by H202 (14). It is particularly likely that some radiation effects on endothelial cells may be mediated by altered lipid products. There have been a number of reports of perturbation

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of arachidonate metabolism by radiation. Both increases and decreases in prostaglandin formation have been reported, in vifro and in viro (28). Irradiation of bovine aortic endothelial cells results in the appearance in the medium of a neutrophil chemoattractant of the lipoxygenase pathway (7). There is evidence for activation of phospholipase A2 and cyclooxygenase (25) and inhibition of PG 15OH dehydrogenase (40). The ability of t-BOOH and SR-4077 to induce apoptosis (Fig. 1) suggests that radiation could induce apoptosis by a mechanism that is different from the mechanism of classical clonogenic cell killing. Zhong et ul. (47) have reported that t-BOOH and menadione can induce apoptosis in conditionally immortalized nigral neural cells. We have previously reported that t-BOOH and SR-4077 can induce apoptosis in F9 murine teratocarcinoma cells (22). The inhibitor of poly (ADP-ribose) polymerase, 3-aminobenzamide, inhibited the induction of apoptosis by radiation in EL4 cells (Fig. 2). Inhibition of apoptosis by 3aminobenzamide has been reported in connection with poly (ADP-ribose) polymerase dependent depletion of NAD (2). An interesting alternative explanation is suggested by recent results that biological effects of cyclicADP-ribose, a Ca’ +-mobilizing second messenger, can be blocked with 3-aminobenzamide (36). The ability of retinol to protect against endothelial cell detachment from irradiated wounded monolayers suggests that effects other than classical clonogenic cell death in endothelial cells may be biologically modifiable, and that this could be a consideration in the design of radiation therapy protocols.

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