Effects of radioprotectors on DNA damage and repair, proteins, and cell-cycle progression

Pharmac. Ther. Vol. 39, pp. 133 to 137, 1988 Printed in Great Britain.

0163-7258/88 $0.00+0.50 Pergamon Press plc

Symposium Editors: J. F. WEISS and M. G. SIMI¢

EFFECTS OF RADIOPROTECTORS ON DNA DAMAGE AND REPAIR, PROTEINS, AND CELL-CYCLE PROGRESSION DAVID J. GRDINA,* WILLIAM H . GUILFORD,* CURTIS P. SIGDESTAD*t and CAROL S. GIOMETTI*

*Argonne National Laboratory, Argonne, Illinois 60439-4833, U.S.A. tUniversity of Louisville, Louisville, Kentucky 40292, U.S.A.

1. INTRODUCTION The impetus for investigating the application of phosphorylthioate drugs for use in cancer therapy arose from the early observation by Yuhas and Storer (1969) that these drugs might differentially protect normal as compared to tumor tissue. Recent reports now suggest that these agents may have a more general role with respect to the cancer problem, especially in the area of cancer prevention. Following the initial observation by Milas et al. (1984) that S-2(3-aminopropylamino)ethylphosphorothioic acid (WR-2721, ethiofos) significantly protected rodents against the induction of tumors by irradiation, a series of reports have appeared in the literature that describe the antimutagenic (Grdina et al., 1985a; Nagy et al., 1986; Nagy and Grdina, 1986), antitransformation (Hill et al., 1986), and the anticarcinogenic properties (Grdina et al., 1985b) of WR-2721 and its corresponding free thiol 2-(3-aminopropylamino)ethanethiol (WR-1065). These studies have helped to stimulate a strong interest in characterizing selected molecular and cellular end points affected by radiation such as DNA damage and repair, effects on proteins, and cell-cycle progression.

2. THE EFFECT OF WR-1065 ON DNA DAMAGE AND REPAIR Since damage to DNA is clearly the most important end point implicated in the processes of cell death, mutagenesis, transformation, and carcinogenesis, a series of studies have been initiated to characterize the effect of WR-1065 on the induction and repair of single- (SSB) and double- (DSB) strand-break damage in DNA following exposure to ionizing radiation. Of the many techniques developed to assess DNA damage following relatively low doses of radiation (e.g. biologically relevant with respect to mutation and transformation induction), the procedure of alkaline elution, as described by Kohn (1979) and Meyn and Jenkins (1983), offers a relatively rapid and quantitative method with which to measure the formation and repair of DNA SSB damage. Using this procedure, Grdina and Nagy (1986) demonstrated that WR-1065 (at a concentration of 4 mM) was effective, if present during irradiation (i.e. doses ranging from 6 to 10 Gy), in protecting against the induction of SSB by radiation. Using a modified version of the neutral elution technique of Bradley and Kohn (1979), Sigdestad et al. (1987) reported that WR-1065 significantly protected against DNA damage, presumably DSB formation. Relative protection factors were determined for WR-1065 against both types of DNA damage and are presented in Table 1 for comparison. While protection by WR-1065 against the formation of these types of DNA damage was not surprising, its effect on DNA-strand rejoining (e.g. a measure of DNA repair) was unexpected. Grdina and Nagy (1986) observed that when WR-1065 was added immediately following irradiation (10 Gy), the magnitude and the rate of DNA-strand rejoining was reduced by about a factor of 3 (see Table 2). Twenty-four hours following exposure to radiation, DNA-strand rejoining was complete. Sigdestad et al. (1987), however, observed no inhibitory effect by 133

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TABLE 1. Protection Against the Formation of Radiation-induced DNA Strand Breaks DNA lesion

SSB DSB

Radiation protector WR-1065 WR-1065

Protection factor 1.5 1.8

Reference Grdina and Nagy (1986) Sigdestad et al. (1987)

TABLE 2. Effect of WR-1065 on DNA Strand-break Rejoining DNA lesion

SSB SSB DSB DSB

Presence of radiation protector

Half time of strand rejoining (min)

+ +

27 88 102 102

Reference Grdina and Nagy (1986) Grdina and Nagy (1986) Sigdestad et al. (1987) Sigdestad et al. (1987)

WR-1065 on the rejoining of DSB. This latter process reflects a more complex repair system since both strands of DNA must be rejoined. These data are summarized in Table 2.

3. THE EFFECT OF WR-1065 ON PROTEINS It has been proposed that an ionic association between WR-1065 and phosphate groups from DNA could account for its concentration in the nucleus (Smoluk et al., 1986). It has also been suggsted that WR-1065 might bind covalently to DNA at selected sites (Fahey et al., 1983). The binding of thiol compounds to histone proteins through disulphide interactions can also account for its concentration within the nucleus (Jellum, 1966). Using lac-labeled WR-1065 (2-[(3-aminopropyl)amino] [ 1,2-14C]-ethanethiol dihydrochloride) (16/zCi/mmol, 0.62 mCi) obtained from the Chemistry and Life Sciences Division, Research Triangle Institute, Research Triangle Park, North Carolina (Contract No. DAMD 17-84-C-4009), we investigated the ability of the radioprotector to bind to DNA and/or nucleoproteins in irradiated (10 Gy) and unirradiated cells. 14C-Labeled radioprotector was dissolved in 3 ml of 0.06 N HC1, lyophilized, and again dissolved in 0.06 N HCI to achieve a concentration of 50 #Ci/ml. The drug was divided and stored into aliquots of 25 #Ci, sealed in glass ampules and kept frozen at - 7 5 ° C until needed. Prior to use, the ampules were defrosted, and the protector was neutralized with 0.1 M NaOH. To assess the ability of the labeled protector to bind to DNA and nucleoproteins in irradiated (10 Gy) and unirradiated cells, concentrations of WR-1065 ranging from 0.10 to 0.35 #Ci/ml were added to respective cell suspensions for 10 min. In these studies, the protector was not present during irradiation. Nucleoproteins and DNA were extracted by standard protocols (Crampton et al., 1954; Johns, 1964), and the resulting data are presented in Fig. 1. While labeled WR-1065 had little affinity for binding to unirradiated DNA, a slight increase in lac activity was routinely observed associated with DNA extracted from irradiated cells. Compared with the level of 14Cbinding to DNA, there was a significant increase in 14C activity associated with nucleoproteins. The largest amount of activity was associated with irradiated cells, i.e. a factor of 4 increase over corresponding unirradiated control cells. The effect of time of incubation of cells with labeled WR-1065 on 14C-associated activity with nucleoproteins and DNA from cells exposed to 10 Gy irradiation was also determined, and these data are presented in Fig. 2. lac activity associated with DNA was relatively low and did not appear to increase as a function of time of exposure. In contrast, binding of the radioprotector to nucleoproteins appeared to increase in both a time- and concentration-dependent manner. These results suggest that concentration of the radioprotector near DNA in vivo may be a function of both association with DNA and sulfhydryl-containing nucleoproteins such as F3

DNA damage and repair, proteins, and cell-cycleprogression

135

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o NP + I000 RAD NP DNA + IOOO RAD • DNA •

o

G 0

OI

02

03

04

/.~Ci (14C)WR- 1065/mr FIG. 1. 14C-LabeledWR-1065 bindingto DNA and nucleoproteinsin irradiated and unirradiatedcells. Irradiated cells were exposed to 10 Gy of 6°Co irradiation. All cells were exposed at 37°C to labeled radioprotector for 10 rain. Immediatelythereafter, nucleoproteinsand DNA were extracted and the associated 14C activity was determinedusing standard liquid scintillationtechniques (Cramptonet al., 1954; Grdina and Nagy, 1986; Johns, 1964).

histone. The specificity of the observed interactions and their role in the radioprotective function of WR-1065 remain to be determined.

4. T H E E F F E C T OF WR-1065 ON CELL PROGRESSION It has been reported that cysteamine can inhibit DNA polymerase-directed repair synthesis (Billen, 1983). While the exact mechanism of this inhibition is at present unclear, thiols of low molecular weight have been reported to possess a high affinity for chelating selected metal ions

tO =L ,t i O

~

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DNA n5

Exposure

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45

to /.¢Ci/m[ W R - 1 0 6 5

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prior to irmd. FIG. 2. Effect of time of incubationof labeledWR-1065 on n4C-associatedactivity with nucleoproteins and DNA from cells exposed to 10 Gy of 6°Co irradiation. Cells were exposed at 37°C to 0.3 tLCi/ml of labeled WR-1065 for 15, 30 and 60 min prior to irradiation. Immediatelyfollowing irradiation, nucleoproteinsand DNA were extracted and the associated n4C activity was determined(Cramptonet al., 1954; Grdina and Nagy, 1986; Johns, 1964).

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TABLE 3. Effect of WR-1065 on V79 Cell-cycle Progression* Time of exposure

Percentage of cells in:

(min)

GI

S

G2 + M

0 30 60 9O 120 150 180

50 44 35 26 25 18 22

35 38 51 56 58 60 58

15 18 14 18 17 22 20

*The percentages of cells in GI, S, and G 2 + M were obtained from flow cytometric profiles obtained from DAPI-stained cells using a PARTEC PASII particle analyzing system (PARTEC AG, Basel, Switzerland) and a computer program obtained from TECHNO SYSTEM-GmbH (Darmstadt, West Germany). The coefficient of variation of the G l peak obtained using unperturbed cell samples routinely ranged from ! .5 to 2.0%.

(Jellum et al., 1973). Since many polymerases require metal cofactors for activity (Kornberg, 1980), it may be that this property of thiols can be a factor in affecting DNA polymerase activity in cells. An agent capable of affecting enzymes involved in DNA synthesis should also be effective in perturbing cell-cycle progression in exponentially growing cell populations. Using flow cytometry, this phenomenon was observed by Grdina and Nagy (1986). V79 cells were exposed to 4 mM WR-1065 for up to 3 hr. While the protector exhibited no cytotoxic effect under these conditions, it significantly altered cell-cycle progression. As indicated in Table 3, cells accumulated in S phase as a function of time of exposure to the radioprotector. This effect is, however, reversible. The doubling time of cells exposed in this fashion to WR-1065 increased from about 11 to 18 hr (Grdina and Nagy, 1986). Clearly, the perturbing effect of WR-1065 on cell-cycle progression and delay is significant. It has long been known that cell division is an important step in the fixation of mutational and/or transformational events (Chu and Mailing, 1968; Farber, 1984). Agents that can prolong the time required for cell division without the induction of a concomitant cytotoxic effect might be expected to be effective as antimutagens or anticarcinogens.

5. CONCLUSION Radioprotectors such as WR-2721 and its free thiol WR-1065 have been extensively investigated because of their potential for increasing the therapeutic gain of cancer therapy as a consequence of their ability to differentially protect normal as compared to neoplastic tissues. The potential mechanisms by which these agents express their protective effects are deafly numerous. However, emphasis for future studies should not be directed only toward investigating their ability to reduce initial radiation/chemical damage but, rather, consideration should also be given to better understanding their ability to affect postirradiation processes involved in mutagenesis, transformation, and carcinogenesis.

Acknowledgements--This work was supported in part by the U.S. Department of Energy, Office of Health and Environmental Research, under Contract No. W-31-ENG-38, and Public Health Service Grant No. R01 CA37435, awarded by the National Cancer Institute, Department of Health and Human Services.

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