Polymorphic Radiation Sensitivity of Human Natural Killer Activity: Possible Role of DNA Strand Breakage*

Bernice Schacter, Susan Hansal, Janet Arno, and Mark J. LeVine

ABSTRACT: Natural killer (NK) activity of human mononuclear cells is sensitive to inhibition by radiation, under the control of polymorphic X linked genes. In order to define the mechanism of this inhibition, we have evaluated the ability of treatments known to damage D N A to inhibit NK activity. The alkylating agents streptozotocin (SZ) and N-methyl-N'-nitro-N-nitrosoguani- dine (MNNG) were potent inhibitors of N K activity. Further. a specific competitive inhibitor of adenosine diphosphoribosyl polymerase (ADPRP), 3-aminobenzamide, was able to prevent inhibition by 7-radiation, UV radiation, and the two alkylating drugs, SZ and M N N G , suggesting the ADPRP, known to be activated by D N A strand breakage, mediates the inhibition by these treatments. NK activity of radioresistant subjects was somewhat more resistant to in- hibition by SZ or UVR when compared to radiosensitive NK activity but neither of these treatments gave the clear phenotypic distinction of 7-radiation, suggesting that chemical strand breakage does not precisely model 7-radiation and also that the mechanism of UVR inhibition may differ from that ofT-radiation. These results indicate a role for activation of ADPRP in the inhibitory effect of UV and 7-radiation on human NK activity and suggest that the biochemical basis for polymorphism in the sensitivity of NK activity to 7-radiation will be found in the sensitiz ity to ADPRP activation or the level of activation of this enzyme, known to be the key to DNA repair.

A B B R E V I A T I O N S ADPRP adenosine diphosphori- mJ/cm 2 millijoules/square

bosylpolymerase centimeter 3AB 3 aminobenzamide krads killorads DMSO dimethylsulfoxide LGL large granular lymphocytes E/T effector/target M N N G N-methyl-N'-ni t ro-N- Fcs fetal calf serum nitrosoguanidine HRPMI hepes buffered RPM11640 NK natural killer IFN interferon SZ streptozotocin IL-2 interleukin-2 UVR ultraviolet radiation

I N T R O D U C T I O N

Natural ki l le r ( N K ) activity, a p r o p e r t y o f a small p r o p o r t i o n o f m o n o n u c l e a r cells charac te r i zed by m o r p h o l o g y , the absence o f T and B cell surface markers ,

*This work was supported in part by U.S. Public Health Service Grant CA36189. Received January 15, 1985; accepted April 9, 1985. From the Departments of Pathology and Medicine. Case Western Reserve University. Cleveland, Ohio

44106. Address reprint requests to Bernice Schacter, Bristol-Myers Company, P.O. Box 4755. Sy'racuse, NY

13221.

Human Immunology 14, 49-58 (1985) © Elsevier Science Publishing Co., Inc., 1985 52 Vanderbik Ave., New York, NY 10017

49 0198-8859/85/$3.30

50 B. Schacter et ai.

and a receptor for the Fc portion of lgG, is measured by the ability of such cells to lyse selected tumor targets in vitro [1-4] . The precise biological functions of this activity m vlvo are uncertain, but N K activity has been implicated in sur- veillance against tumors, the response to viral infections, suppression of primary T independent immune responses, and graft vs. host disease [5-13] . N K activity is augmented by at least two lymphokines, interleukin-2 (IL-2) and interferon (IFN), and is subject to the influence of several environmental and genetic factors [5 ,7 ,14-17] . We have previously reported that human NK activity is sensitive to inhibition by both gamma and ultra-violet radiation [18,19]. The sensitivity to 3000 rads of gamma radiation is a polymorphic trait in humans, under control of a diallelic gene on the X chromosome.

The dose and wavelength dependency of the sensitivity of N K activity to ultraviolet radiation (UVR) suggested that inhibition occurred at environmentall y achievable doses of UVR and that the chromophore mediating UVR inhibition is a nucleic acid [19]. The major cytotoxic effects of radiation are mediated by reactive oxygen intermediates generated by the radiolysis of water and by primary and secondary effects o f direct damage to nucleic acid by radiation [20,2 l]. The distinction between these mechanisms may be artificial in that these effects may act synergistically to cause cell damage. Utilizing alkylating drugs that are known to cause damage to DNA, we have tried to determine whether D N A damage is responsible for the radiation-induced inhibition of N K activity. D N A damage by alkylating agents or radiation leads to strand breakage which causes activation of the enzyme adenosine diphosphoribosyl polymerase (ADPRP). This enzyme uti- lizes nicotinamide dinucleotide as a substrate and forms a polymer o f adenosine diphosphoribosyl units covatently linked to nuclear proteins [22-25] . The con- sequences of activation o f ADPRP which occur rapidly after strand breakage, include a depletion of N A D and ATP as well as the modification of the nuclear proteins [26-28] . A highly specific inhibitor of ADPRP, 3-aminobenzamide (~- AB) can be used to test for a role for this enzyme [29-31] .

The polymorphism of the sensitivity of NK acuvity to radiation suggests that the underlying mechanism has some normal physiological counterpart with a role in the control of N K activity. Further, activation of N K activity by IL-2, but not by IFN, is associated with the acquisition o f the radioresistant phenotype by cells from subjects of all genotypes ([32] and unpublished observations). Definition of the biochemical basis for the polymorphic sensitivity of spontaneous NK actiwty to radiation may allow us to define the physiological conditions under which this polymorphism is of relevance and thus to aid in the identification of the function of N K activity.

MATERIALS A N D METHODS

Effector cells. Effector cells were prepared by Ficoll-Hypaque density centrifu- gation of blood drawn f rom healthy subjects and anticoagulated with :heparin. The per ipheralmononucIear cells were washed twice with Hepes buffered RPM! 1640 (M,A. Bioproducts, Watkersville, MD), [HRPMI] and resuspended at 1.5 million per ml in HRPMI with 10% fetal calf serum. In some experiments, adherent cells were depleted by passage of the cells over a nylon wool column, as previously described [ 19].

Radiation. Effector cells were treated to ~ radiation by exposure to. a 6°Cobalt source, emitting at a rate of 2500 rads/min. The cells, in H R P M I in 10% fetal calf serum in 12 × 75 mm polystyrene culture tubes (Fisher Scientific, Pittsburg, PA), were lowered into the radiation cylinder for measured periods of time to

DNA Damage and Inhibition of NK Activity 51

vary radiation dose. Unirradiated control cells were held at room temperature. The effector cells were plated without washing for N K assay. U V R was achieved with a high-intensity Xenon arc lamp (Schoeffel, 2.5 kW), as previously described [19]. Effector cells were suspended in Hanks Balanced Salt solution, without phenol red (Grand Island Biolocal Co., Grand Island, NY) at a concentration of 5 million/ml. Thirty tzl of the cell suspension was placed in replicate round bot tom microti ter wells of a 96-well tray (W. Sarstedt, Inc., Princeton, NJ), and the wells were exposed to the simulated sunlight, the dose of radiation varied by the length of exposure. Control cells were similarly plated and held in the dark during the radiation period. After radiation, 100 bd of H R P M I with 10% fetal calf serum was added to the wells and the N K assay per formed as usual.

N K assay. The target cell was the erythroleukemia line K562 maintained in the continuous suspension culture in H R P M I in 1640 with 15% fetal calf serum. Two million K562 were washed twice in H R P M I and resuspended in 0.1 ml saline containing 100/~Ci of NaS*CrO4, with a specific activity of 400 mCi/mg (New England Nuclear, Boston, MA). The cells were incubated for 1-1.5 hr at 37°C in 5% CO2 in air, washed three times with HRPMI , and resuspended in H R P M I with 10% fetal calf serum at a concentration of 100,000 per ml. One hundred microliters of effector cells, at concentrations of 1.5 to 0.375 million per ml, were placed in triplicate wells of round bot tomed 96-well microti ter trays. Fifty microliters of target cells were added to each well. Spontaneous release of 5~Cr was determined with wells in which H R P M I with 10% fetal calf serum substituted for the effector cells, and total releasable chromium was determined by substituting 1% sodium dodecyl sulfate for the effector cells. The tray was spun for 10 min at 50 ×g, incubated for 4 hr at 37°C in 5% CO, in air, spun for 10 min at 150 ×g, and 50 bd of supernatant harvested into glass tubes for counting in a well type gamma counter.

Cytotoxicity is expressed as:

cpm(experimental) - cpm(spontaneous)/cpm(total release).

Spontaneous release was always < 2 0 % of the total releasable cpm and effects of solvents or drugs on spontaneous release were measured and used in calcu- lation. Cytotoxicity was linear over the range effector-to-target ratios used and unless noted is given for an E/T ratio of 30/1.

Streptozotocin (SZ) was dissolved in RPMI 1640 with 10% FCS immediately prior to use and held on ice during addition to cells in the microtiter trays. The inhibitor 3-aminobenzamide (3AB) was prepared as a stock solution of 100 mM in RPMI 1640 with 10% FCS. N-methyl -N' -n i t ro-N-ni t rosoguanidine ( M N N G ) was dissolved in 100% D M S O at 480 tzg/ml just prior to use and diluted 1:16 in RPMI 1640. Serial dilutions were made of this 30/~g/ml stock and 100 ill of the stock and of the dilutions were added to 0.9 ml of effector cells to achieve the final concentrations of M N N G . This resulted in a concentration of D M S O of 0 .6% which was included in the suspensions of control cells for the M N N G experiments. In all cases, spontaneous release from K562 was measured in the presence of added solvents or drugs and cytotoxicity corrected for any alteration in spontaneous release caused by drug or solvent. In no case was spontaneous release altered more than 20% by drug or solvent.

R E S U L T S

We have previously repor ted that three drugs that interact with D N A and inhibit replication in response to mi togens - -mi tomycin C, acridine orange, and Adria-

52 B. Schacter et al.

mycin are poor inhibitors of N K activity [18]. Two other drugs were chosen. N-methyl -N ' -n i t ro-N-ni t rosoguanid ine ( M N N G ) and streptozotocin (SZ), both known to alkylate D N A and lead to D N A strand breakage [33-35] . Inhibition of N K activity to 50% of control levels was obtained with approximately 0.5 ~g/ml of M N N G (Figure 1A) and 3 -5 mg/ml of SZ (Figure 1BL Thus both alkylating drugs were effective inhibitors of N K activity at concentranons known to cause D N A strand breakage and A D P R P activation [28,34].

I f the inhibition of N K activity caused by the alkylating agents were mediated by D N A strand breakage leading to the activation of ADPRP, then an inhibitor of that enzyme would be expected to protect from such inhibition. A highly specific, competi t ive inhibitor o f ADPRP, 3-aminobenzamide (3AB), a structural analogue of N A D , was used to test this hypothesis [29-31] . Inhibition of N K activity by ~ radiation, UVR, SZ, or M N N G was significantly reduced by the inclusion of 1 mM 3AB in the cell suspension exposed to these treatments (Figure 2A,B,C, Table 1). A protect ive effect against radiation was obtained with as little as 10 ~zM 3AB. The protection was afforded only if the 3AB were present during radiation or added to the cells immediately after; addition 30 rain after radiation did not prevent or alter the effects o f radiation indicating that the radiation- induced damage occurred rapidly and that the effect of 3AB was on the effector cell and not on the target cell (Figure 3A). Incubation of the cells with 3AB and removal by centrifugation prior to radiation also did not provide protection of the N K activity from the gamma radiation (Figure 3B).

We evaluated next the potential contributions of D N A damage to the poly- morphic sensitwlty of N K activity to ~/radiation. Groups of unrelated subjects whose radiation sensitivity phenotype had been previously determined were tested for the sensitivity of their N K activity to graded doses of SZ (Table 2). The dose. required to cause a 50% inhibition was determined for each subject from the dose response curve. In all cases the 50% inhibition point was bracketed by experimental points, and the inhibition was linear over at least five concentrations tested. The 50% inhibitory dose was determined from a line plotted by the least- squares method. While heterogeneity was noted, the dose required to cause 50e/r

FIGURE 1 (A) Dose dependent inhibition of NK activity by MNNG. MNNG was added as indicated to suspensions of nonadherent lymphocytes and the cells were plated at 150,000 per well in three wells for each concentration. 5~Chromium labeled K562 were

-7o . added to achieve E/T of 30:1 and cytotoxicity measured after 4 hr incubation at 3, ( . The DMSO concentration in MNNG welts was 0.6% and control wells contained 0.6% DMSO. Shown are representative experiments with two subjects, both radiosensitive. (B) Dose-dependent inhibition of NK activity by Streptozotocin. Experiment performed as described for Figure 1. SZ was dissolved in HBSS just prior to use and kept on ice during the addition to cells. Shown are results with two Subjects, both radiosensitive.

ioo I - - c~ 80

z 6 0

~ 4 0

N 2O ILl n,"

I.o 2.0 310- 4.0

(A ) [MNNG] ug/ml

I..- 125

t 00~ <

~ 7 5 Z

_.1

'~ 5 0

,,, 2 5 n,-

(B)

f ~ . . . . 3

STREPTOZOTOClN mg/ml

D N A Damage and Inh ib i t ion of N K Activi ty 53

(A)

J o 3 krads

- - I I I 10-5 10 -4 10 -3

DAB3 Molar

40

35

3q ~_c5 > x 25

< ~0 20 >.

' 4 0 1 5

z o ~ 1 0

5,

4O >-

58 ~S 20 - ' b z I0

(B)

e 3AB (ImM) OL

I I ~ I i 5 I0 15 20 25 50

UV ( m J/crn z )

I00 ~ • 3AB ~ ~ T R O L

~§60 o ,, 40 ~-o >-

~ 20

I 2 5

STREPTOZOTOC IN (C) mg/ml

F I G U R E 2 Abili ty of 3 -aminobenzamide to p reven t or reduce inhibi t ion of N K activity by (A) gamma-radia t ion , (B) U V B radiat ion; and (C) s t reptozotocin . In each case nona- d h e r e n t m o n o n u c l e a r cells were incubated with 3AB for 30 min pr ior to exposure to radia t ion or SZ. In A, 3AB concen t r a t ion was varied, in B and C, 3AB concen t ra t ion was 1 mM. N K activity in all cases was assayed at E/T ratio of 30 : 1 in 4 hr ~ ~Chromium release assay with K562 as target.

i n h i b i t i o n in six r a d i o r e s i s t a n t s u b j e c t s was s ign i f i can t ly h i g h e r t h a n t h e d o s e r e q u i r e d to c a u s e 5 0 % i n h i b i t i o n o f cel ls f r o m six r a d i o s e n s i t i v e s u b j e c t s , u s i n g a p a i r e d t tes t . H o w e v e r , t h e c l ea r d i s t i n c t i o n s e e n w i t h r a d i a t i o n b e t w e e n all s e n s i t i v e a n d all r e s i s t a n t s u b j e c t s was n o t o b s e r v e d . W e f u r t h e r d e t e r m i n e d t h e r e l a t i v e s e n s i t i v i t y o f N K ac t iv i ty o f g a m m a r a d i o r e s i s t a n t a n d r a d i o s e n s i t i v e

T A B L E 1 3 A B r e d u c e s i n h i b i t i o n o f N K ac t iv i ty by M N N G

Addition

NK activity ~ NK activity" ('7/c cytotoxicity ((:; cytotoxicity

+ S.D.) +- S.D.) Exp. l Exp. 2

0 19.7 +- 1.8 32.6 ± 1.1 1 /~g/ml M N N G 7.0 +- 2.0 5.2 ± 2.0 5 p~g/ml M N N G nt 1.0 ± 0.7 1 ~g/ml M N N G + 1 mM 3AB 19.2 + 1.0 15.4 -+: 2.4 5 ~ g / m I M N N G + 1 mM 3AB nt 4. l -+: 0.7 1 mM 3AB 24.9 -+ 2.3 28.9 ~: 1.2

~Percent cytotoxicity at E/T of 30: l against K562. Effector cells were incubated with 3AB fi~r ~0 rain prior to addition of MNNG and ~ICR labeled target cells, nt = not tested.

54 B. Schacter et al.

[ ] 0 3AB 0.I mM

E~ 3AB 0.1 mM

~ ~ I001__ ( ~ - ~ 3AB 30' prior I washed out

,-P 4Oao 3AB 3o'°,,e, 40 o , , , ,o

O 0 3

(A) KRADS (B) o RADS 3 KRADS

FIGURE 3 3AB must be present at time of y radiation in order to provide protection. In Figure 3A the time of addition of 3AB to effector ceils was staggered in relation to radiation with 3 krad and NK activity was assayed as described in the legend for Figure 2. In Figure 3B, mononuclear cells at 1.5 × 10 c' in RPMI 1640 with 10% FCS were' incubated for 30 rain with 3AB at 0.1 mM. An aliquot of treated cells was washed !5 by centrifugation and resuspended at 1.5 × 10C'/ml. Treated, treated and washed, and control cells were divided in half and assayed as described in the legend fi)r Figure 2 for NK activity with and without exposure to 3 krads of y radiation.

subjects to 6 mJ/cm 2 of simulated sunlight. The percent N K activity remaining after treatment at this dose o f UVR, was compared to the residual activity after 3 krad gamma radiation (Table 3). While a correlation is noted between sensitivity to the two forms of radiation, there is more heterogeneity with the UVR than with 7 radiation, especially for the sensitive subjects.

D I S C U S S I O N

These results indicate that human N K activity, which is radiosensitive under the control of an X-linked gene, is also sensitive to two drugs known to cause D N A strand breakage, SZ and M N N G . We had previously reported that three other D N A interactive drugs--mitomycin C, Adriamycin, and acridine orange, are poor inhibitors o f N K activity [19]. The relatively greater inhibitory activity of SZ

T A B L E 2 Inhibition by SZ of N K activity of radioresistant and radiosensitive subjects ~

Experiment

Dosage required to give 50% inhibition b (mg/ml)

Radioresistant subject Radiosensitive subject

1. 0.92 t):'79

2. 1.29 0,95 3. 1.41 (}.98 4, 2.02 0.96

5. 1.91 t 3 5 0, 4.88 [ 7 8

Mean -+ S.D. 2.07 _+ 1.43 1.33 r_ 0.37

Up < 0.05, paired t test, 5 dr.

qs0 calculated by !east-squares method from cytoroxicity for K562 at E/T of 30:1 after exposure ot cells to SZ over concentration range as shown in Figure 2.

DNA Damage and Inhibition of NK Activity 55

T A B L E 3 Relation between UVR and 7-irradiation inhibition of N K activity

Res idua l N K activity (C/` )"

D o n o r Sex Af te r 6 mJlcm 2 3000 rads

1 F 19 11

2 F 39 15

3 M 28 10

4 M 55 14

5 M 87 12 6 M 69 82 v M 85 99

8 M 98 t26 9 M 83 117

10 M 129 110

r : 0 .76

~Residual NK activity is expressed as a r:[ of control unirradiated activity at an E/T of ~,0: I.

and M N N G may be related to relatively lower levels of actual (or apparent) D N A strand breakage caused by mitomycin C, Adriamycin, and acridine orange [36,37]. Inhibition by 5' radiation, UVR radiation, SZ and M N N G was significantly re- duced by 3AB, a competitive inhibitor of ADPRP, suggesting that ADPRP ac- tivity mediates the inhibition by these treatments. Milam and Cleaver have cau- tioned that nicotinamide analogues such as 3AB may have metabolic effects in addition to inhibition of ADPRP, but such effects were not observed by these authors at the low (1 mM) concentration of 3AB we utilized [38]. Further, the ability of 3AB at such low concentrations to prevent inhibition by such diverse D N A damaging agents as ionizing, and UV radiation as well as alkylation agents, is consistent with a role in the response to D N A damage and not the secondary effects seen at high 3AB concentrations. We have evaluated the relative sensi- tivities of N K activity from radioresistant and radiosensitive subjects to inhibition by SZ and by UVR. While SZ was slightly more effective against radiosensitive than radioresistant N K cells, the marked distinction between subjects of the two phenotypes seen with 7 radiation was not observed with this drug. This lack of clear distinction between resistant and sensitive subjects was also noted for the inhibition caused by UVR. These results suggest that the expression of the polymorphic sensitivity to gamma radiation involves mechanisms other than those modeled by either the drugs known to induce strand breaks or UVR individually. The ability of 3AB to completely prevent the inhibition of y radiation at 3 krads suggests that ADPRP activity is crucial to the inhibitory effect of radiation. Because 3AB has been shown to inhibit D N A repair and increase the level of strand breakage detectable with a given dose of alkylating agent or radiation, these data also suggest that D N A strand breakage per se is not detrimental to N K activity [39]. While the major metabolic effects of ADPRP activation include rapid and profound alterations in N A D and ATP concentrations, the role of ADPRP implied by the ability of 3AB to prevent or reduce inhibition of N K activity by radiation and alkylation drugs may be through effects other than alteration of ATP and/or N A D concentration. ADPRP has been implicated in D N A repair but the precise role of poly(ADP-ribose) synthesis in the D N A repair process is not entirely clear [30,39]. Cleaver has reported that inhibition of ADPRP by 3AB consistently causes an increase in detectable strand breaks after exposure to alkylating strand breakers such as methylmethane sulfonate

56 B. Schacter et al.

(MMS) and M N N G , in all cell types, and this is consistent with a role for poly(ADP- ribose) in the regulation of the D N A ligase, as reported by Creissen and Shall [39,40]. Such a consistent change was not noted for UVR and suggests that the role o f ADP-ribosylation in the control of D N A repair may vary with the nature of the damage done, particularly with the level of strand breaks [25,39]. Two other enzymes of relevance to D N A damage and repair may be considered: the Ca + +, Mg ++ dependent endonuclease and the topoisomerases. Activation of the Ca + +, Mg + + dependent endonuclease leading to extensive D N A degradation has been associated in murine cells with radiation treatment and cell death me. diated by cytotoxic T cells [ 4 i - 4 3 ] . The topoisomerases, which catalize the interconversion of topological isomers of D N A via the transient formation of strand breaks, have been implicated in the repair of D N A damage after UVR or treatment with a!kylating agents [44-46] . Both the endonuclease and type ! topoisomerase have been shown to function as acceptors of poly-ADP-ribose, leading to loss of enzymatic activity [47,48]. Thus these activities may interact in the repair of D N A damage. We are evaluating the activity of these three enzymes - -ADPRP, endonuclease, and topoisomerase~in N K cells following radiation.

ACKNOWLEDGMENTS We would like to thank Drs. Helen Evans and Nathan Berger for helpful discussions, Karen Larson for testing the effect of 3AB on UVB inhibition, and Sandy Wehmann and Wendy Morey for preparation of the manuscript.

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