Alterations in repair of alkylating agent-induced DNA damage in polyamine-depleted human cells

Cancer Lefters, 72 (1993) 83-90 Elsevier Scientific Publishers lreland

83 Ltd.

Alterations in repair of alkylating agent-induced DNA damage in polyamine-deipleted human cells Ronald D. Snyder and Sangita Bhatt Marion MerreN Dow Research Institute. Department of Cancer Biology, 2115 E. Galbraifh Road. Cincinnari. OH 452/S (USA) (Received 12 March 1993 I (Revision received 13 May 1993) (Accepted 14 May 1993)

Summary

Treatment of HeLa cells with the polyamine biosynthesis inhibitors difluoromethylornithine (DFMO) and/or methylglyoxal bis(guanylhydrazone) (MGBG) results in marked depression in levels of the cellular polyamines putrescine, spermidine and spermine. Cells in this polyaminedepleted state exhibited increased sensitivity to monofunctional alkylating agents, manifested as decreased cloning ability and retardation of the DNA excision repair process. DFMO treatment did not alter the initial level of interaction of radiolabeled alkylating agent with cellular DNA, but combined treatment with DFMO and MGBG reduced covalent binding, probably through effects on cell cycling. Polyamine supplementation had no effects on initial yield of DNA singlestrand breaks in d,rug-treated cells. The repair defect appeared similar to that observed previously in polyamine-depleted cells following Xirradiation and UV irradiation, namely retarded sealing of DNA strand breaks. It was not possible to reverse the effects of these inhibitors by short periods of polyamine loading, despite the fact that Correspondence to: Ronald D. Snyder, Marion Merrell Dow Research Institute, Department of Cancer Biology, 2110 E. Galbraith Road, Cincinnati, OH 45215, USA. Abbreviations: DFMO, u-difluoromethylornithine; DMS, dimethylsulfate; EMS, ethyl methanesulfonate; MGBG, methylglyoxal bis(guanylhydrazone); MMS, methyl methanesulfonate.

03043835/93/%06.00 0 1993 Elsevier Scientific Printed and Published in Ireland

Publishers

Ireland

all three polyamines could be restored to nearnormal levels. These findings provide the first demonstration of altered response of polyaminedepleted cells to monofunctional alkylating agents and contribute to our understanding of altered responses of polyamine-depleted cancer cells to a variety of DNA-reactive chemotherapeutic drugs.

Keywords: alkylating

agents; DNA; polyamine;

human cells Introduction

Examination of metabolic processes in cells depleted of polyamines by treatment with specific enzyme inhibitors has suggested a role for polyamines in nucleic acid structure, macromolecular synthesis and membrane structure and function [ 13 for review]. The loss of proliferative potential in cells depleted of polyamines by difluoromethylornithine DFMO [ 141 suggested that agents of this type might be useful as cancer chemotherapeutic agents, and, as reviewed by Sunkara et al. [29], some intriguing anti-tumor effects have been demonstrated, although the general applicability of this approach has been disappointing. However, DFMO has also been shown to alter the sensitivity of cultured cells to DNA-reactive chemotherapeutic drugs such as cisplatin [15,20], nitrosoureas [5,6,10,16-l&22-23] and adriamycin f 1,2 11, suggesting that polyamine Ltd.

84

depletion may be efficacious as an adjunct to chemotherapy. The mechanism by which polyamine-depleted cells acquire altered sensitivity to DNA-reactive drugs is not clear. A large body of evidence from in vitro studies suggests a role for polyamines in stabilizing DNA structure [12 for review], and altered chromatin structure has recently been observed in polyamine-depleted mammalian cells [2,25]. It is possible that this altered chromatin structure could affect DNA-drug interaction. Another possibility is that damage induced in cellular DNA by these chemotherapeutic drugs might be less susceptible to epair either because of modifications in chromatin structure or because of altered activity of polyamine-requiring DNA repair enzymes or processes. We have previously demonstrated that DFMO treatment of HeLa cells results in retardation of sealing of X-ray-induced DNA single-strand breaks [26] and a similar slowing of repair-dependent breaks following ultraviolet irradiation [28]. DNA damage induced by chemical alkylating agents is different from that induced by radiation exposure, as is the cellular response to that damage. Therefore the present studies were conducted to determine if DNA damage and repair induced by monofunctional alkylating agents were also modified in cells depleted of polyamines. It is demonstrated that a repair deticiency similar to that previously found in polyamine-depleted cells subjected to radiation is also seen following treatment with these DNAreactive drugs. Materials and Methods Cell culture

HeLa cells were maintained in Modified Eagle’s medium containing loo/o fetal calf serum. Growth was at 37°C in a CO?-controlled incubator. Polyamine depletion was accomplished by incubation of cells for 48 h with 2.5 mM DFMO with or without 5 PM MGBG (Sigma Chemical Company) added for the last 24 h, or with MGBG alone for 24 h. Putrescine, spermidine or spermine (Sigma Chemical Company) were added to cultures at 1 mM final concentration for l-3 h.

Polyamine

determinations

HPLC determination of cellular polyamine levels was as described in detail elsewhere [3]. Triplicate samples were analyzed with an associated error of lo-30%. Proliferation

studies

The ability of cells to proliferate was measured by their ability to form colonies when seeded at low density. Cells were trypsinized, counted and seeded at appropriate density into 60 mm tissue culture dishes. After 10 d growth, colonies were stained with Dif-Kwik. Untreated control cultures exhibited between 30 and 50% cloning efficiency using this protocol. Nucleoid breaks

sedimentation

analysis

of DNA

strand

DNA strand break determination was made as described in detail previously [26], utilizing neutral sucrose gradient sedimentation. Briefly, appropriately treated cells were applied to a lysis solution containing 1.9 M NaCl, 100 mM EDTA, 32 mM Tris (8.0) and 0.5% Triton-X 100 on top of a 15-30% linear neutral sucrose gradient. During a 20-min lysis period most of the chromosomal protein and RNA is removed, and the DNA ‘nucleoid’ unwinds to a degree dependent on the amount of DNA single-strand breakage. Sedimentation for 45 min at 18 000 revimin was followed by visualization of the nucleoid band under UV illumination. The distance sedimented by the treated cell nucleoids was compared to that observed with untreated cell nucleoids. DNA adduct studies

Cells were treated with [l-3H]EMS (Amersham, 3 Ci/mmol) or [methyl-3H]DMS (NEN. 2.4 Ci/mmol). After extensive washing to remove excess label, DNA was isolated from cells by standard phenol chloroform techniques. Radioactivity associated with DNA after repeated reprecipitation steps was quantitated. Results

As previously reported [25-281, inhibition of polyamine biosynthesis in HeLa cells by DFMO

85

results in nearly complete reduction of putrescine and spermidine, with little effect on spermine levels (Table I). When DFMO Is added with the inhibitor of S-adenosyl methionine decarboxylase, MGBG, spermine pools are also lowered. Addition of polyamines to untreated control cultures results in slight elevation of spermidine and spermine and an approximately 3-fold elevation in putrescine. DFMO-treated cells, on the other hand, are dramatically more responsive to exogenous putrescine ;and also display increased responsiveness to spermidine and spermine addition. Thus, cellular pools of putrescine and spermidine may be readily manipulated, allowing for analysis of DNA damage and repair as a function of polyamine content. Cells treated with either DFMO or DFMOMGBG exhibit increased sensitivity to the alkylat-

ing agents EMS and MMS in colony-forming assays, as shown in Fig. 1. The effects are more marked in DFMO-MGBG-treated cells. This sensitivity was not modulated by the addition of all three polyamines to cultures for 1 h before chemical treatment (not shown). Table II demonstrates that immediately after a l-h treatment with MMS or EMS, DNA strand breaks are seen. These breaks increase with dose of alkylating agent and represent true DNA singlestrand breaks rather than alkali-labile sites (such as abasic sites), since they are detectable under

A

60 Table I. Effects of inhibitors cellular polyamine levels.

of polyamine

biosynthesis

on 40

Putrescine (‘l/o)

Spermidine

Spermine

WU)

W)

Untreated + putrescine” + spermidine” + spermine” + all three, 1 h + all three, 2 h

100 340 98 93 181 1760

100 98 154 101 108 260

100 98 103 133 126 350

DFMO 2.5 mM 48 h + putrescineb + spermidine b + spermineb + all threeC + all threed

0.3 2250 0.4 0.1 1550 732

0.8 39 216 43 195 151

84 96 129 392 296 237

0.8 227

3 82

41 65

x C = g ? .E b (J_

20 0 100

c z

8o

8

60

DFMO + MGBG + all threeb

Numbers are derived from one set of triplicate determinations. Polyamine levels in untre,ated control cells were approximately 600 (putrescine). 6000 (spermidine) and 5700 (spermine) pmoles/106 cells. “Polyamines added for I h at 1 mM final concentration. bPolyamines added for the last hour of inhibitor treatment. C.dPolyamines added for 2 or 4 h, respectively, following removal of inhibitor.

0 0

5

10

15

20

25

30

35

40

45

50

55

EMS Dose (mM) Fig. 1. Colony-forming ability following alkylation agent treatment, Medium was removed from culture dishes and replaced with Hanks balanced salt solution containing MMS (panel A) or EMS (panel B) for 1 h at 37°C. Dishes were washed free of alkylating agent with PBS and the cells were harvested and reseeded at clonal density. Error bars represent standard error in three to five trials. 0. untreated; 0. DFMOtreated; A. DFMO-MGBG-treated.

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Table II. Production equivalents) following agent. Treatment

None EMS

MMS

2 mM 7mM I1 mM 14pM 112 pM

of DNA single-strand breaks (rad a l-h treatment of cells with alkylating

DFMO

Control

DFMO + MGBG

0 50+ 8 60 + 11 62* 7

0 80 + 14 90* IO 123zt 5

20 116 f 29 140 l 33 142 + 12

72* 95*

66 f 15 195 f 21

75 f 180*

4 3

19 8

X-ray doses from 20 to 300 rads produce a linear decrease in nucleoid sedimentation. 300 rads of X-irradiation reduces sedimentation 40-60%. Numbers are derived from two to six determinations.

neutral conditions. These breaks presumably arise from normal cellular repair processes, as they can be shown to increase when cells are incubated in the presence of repair inhibitors such as ara-C and hydroxyurea [24]. Cells treated with DFMO or DFMO-MGBG exhibit more DNA strand breaks at all alkylation agent doses tested. Polyamine addition to inhibitor-treated cells 1 h before EMS or MMS treatment did not result in a lowering of this initial level of breakage (not shown). Increased levels of DNA strand breaks could arise either from increased interaction of alkylating agent with cellular DNA, elevated incision frequency or retarded rate of gap tilling or ligation. To assess the first possibility, cells were incubated with radiolabeled EMS or DMS, DNA was isolated by phenol/chloroform extraction, and acidinsoluble radioactivity quantitated. Table III demonstrates that the initial yield of either DMS or EMS adducts in DFMO-treated cells was not significantly different from controls. DFMOMGBG-treated cells, on the other hand, showed a considerable reduction in covalent adduct formation, which did not appear to be reversible by polyamine addition before treatment with radiolabeled alkylating agent. Polyamine supplementation by itself also led to no difference in reaction of alkylating agent with cellular DNA,

Table III. Binding of radiolabeled untreated and polyamine-depleted ogenous polyamines.

-

alkylating agent to DNA in cells and the effect of ex-

Percentage

Control + 1 mM polyamines (2 h) DFMO DFMO

+ MGBG

+ I mM polyamines (2 h) HU

control

DMS

EMS

100 102 f 6 (n = 3) 97 f 2 (n = 2) 35.4 f 4 (n = 3) 41.2 f I5 (n = 2) n.d.

100 98 f 16 (n = 3) 93 f 8 (n = 3) 44.2 f 19 (n = 4) n.d.

46.7 f

1

(n = 2)

Pmoles of DMS incorporated per pg DNA in untreated cells ranged from 0.05 to 0.54, depending on initial amount of radiolabel added. This is approximately 2.5 x IO5 to 2.5 x lo6 adducts per cell. Pmoles of EMS incorporated per pg DNA ranged from 0.002 to 0.04 or 9.6 x IO3 to 1.9 x lo5 adducts per cell. n.d.: not determined.

even though cellular polyamine levels were elevated substantially under these conditions (Table I). Hydroxyurea (10 mM, 3 h) also resulted in decreased adduct formation. Hydroxyurea reduces the number of S-phase cells in the culture by about 50% (not shown), and we have previously demonstrated that DFMO-MGBG treatment results in a similar reduction in S-phase cells [25]. DFMO treatment at the dose and time periods employed in the present studies did not alter cell cycle kinetics (not shown). An examination of DNA strand breaks following treatment of cells with EMS or MMS revealed an apparent deficiency in the ability of polyaminedepleted cells to seal DNA single-strand breaks (Fig. 2). This deficiency was of nearly equal magnitude in DFMO and DFMO-MGBG-treated cells. In both cases strand breaks did eventually seal by 16 to 24 h after removal of the alkylating agent.

87

r 1

5

10

15

Time (h)

1

5

10

15

Time (h)

Fig. 2. Repair of DNA single-strand breaks following treatment with alkylating agents. Cells were treated with 112 PM MMS (panel A) or II mM EMS (panel B) for 1 h, as in the caption to Fig. 1. The agent was thoroughly washed off and replaced with complete medium. At various time; cells were harvested and subjected to nucleoid sedimentation analysis. Data is plotted as rad equivalent breaks (see Table II). Error bars represent standard error in three to five trials. 0, control; 0, DFMO; A, DFMO-MGBG.

Discussion

The data presented here demonstrates that pretreatment of cells with DFMO or DFMO together with MGBG results in an altered response to treatment ,with monofunctional alkylating agents. This is manifested as decreased cloning ability and accumulation of DNA single-strand breaks associated with repair. DFMO treatment alone was nearly as effective as DFMO-MGBG treatment in prolonging the occurrence of DNA strand breaks. This was also observed following ionizing and non-ionizing radiation [26,28] and suggests that depletion of putrescine and/or spermidine is sufficient for induction of this slow repair phenotype. Consistent with this interpretation, little or no repair inhibition is observed following treatment with MGBG alone, which re-

sults in an approximate 50% reduction in cellular spermidine and spermine pools but also leads to a massive increase in putrescine pools in HeLa cells (data not shown) [26,28]. Experiments in which polyamines are added back for short periods before the measuring of repair have led to confusing results. We have previously demonstrated that retarded sealing of X-ray and UV repair-induced DNA strand breaks is readily reversed by short periods of polyamine supplementation [26,28]. These findings led to the speculation that either polyamines were themselves involved in the DNA repair process or that perhaps they could rapidly stimulate repair enzymes or stabilize DNA structures. However, we have also demonstrated that restoration of normal X-ray repair in hyperthermically treated HeLa cells is ablated in polyaminedepleted cells, and that this cannot be reversed by

88

the addition of polyamines [27], suggesting an additional role for polyamines in chromatin structure. In the present study we were able to gather no compelling evidence that altered sensitivity could be reversed by polyamines. Thus, even though the defect in repair appears similar following X-ray, UV and alkylating agent treatment, the responsiveness to polyamines is different. This differential responsivenesss could relate to the distinctly different type of lesions formed or the kinetics or mechanism of repair of those lesions. Breaks induced by X-rays, for example, seal rapidly, and repair most probably requires insertion of very few nucleotides. In this case, polyamines may stabilize DNA ends and facilitate ligation. In the case of UV, however, we have demonstrated that the aborted repair sites are not ligatable [28], suggesting the existence of gaps at those sites. These sites seem somewhat less responsive to polyamine addition and even in uncompromised cells repair more slowly than X-ray lesions. The molecular nature of the slowly repairing sites in the present study has not yet been determined, but the fact that these sites are visualized by neutral sucrose sedimentation indicates that they are true strand breaks (resulting from excision repair) rather than apurinic (AP) sites (alkali-labile) resulting from glycosylase activity at the alkylated base. We are at present examining the possibility that either the distribution or repair of specific lesions within various chromatin compartments is altered in polyamine-depleted cells, since the number of DNA strand breaks in DFMO and DFMOMGBG-treated cells immediately after removal of alkylating agent (Table II) does not apparently correlate with DNA adduct levels in bulk DNA (Table III). Ryan et al. [19] have demonstrated that whereas the rate of removal of 7-methylguanine and 3-methyladenine appeared similar in all chromatin fractions, removal of 06-methylguanine proceeded much more rapidly in transcriptionally active chromatin and much more slowly in nuclear matrix-associated chromatin. As we have previously reported an apparent increase in that fraction of chromatin resembling transcriptionally active species in polyamine-depleted cells [25], these studies might prove enlightening. The present studies also demonstrates that

DNA in cells depleted of polyamines is not more susceptible to alkylation and that exogenous polyamine supplementation provides no apparent protection from alkylation. A similar lack of enhancement of adduct formation was previously observed in polyamine-depleted cells treated with a radiolabeled nitrosourea compound [ 181. The observed decreased interaction of radiolabeled alkylating agents with DNA from cells treated with DFMO-MGBG or HU may be at least partially explained by the fact that S-phase cells show a greater reactivity with chemical agents [4,8,1 l] and that this fraction is reduced by 41-50% by these treatments [25; unpublished results for HU]. DFMO-treated cells showed no decreased adduct formation and exhibited no statistically significant reduction in S-phase cells (not shown). Previous studies have suggested that DFMOtreated mammalian cells exhibit increased sensitivity to bifunctional [5,6,9,10,16- 18,20,22,23] but not monofunctional (6,171 alkylating agents. It was at first believed that this was related to delayed repair of DNA interstrand cross-links in DFMO-treated cells [5,7]. Studies utilizing cell lines deficient in the enzyme 06-alkylguanine DNA transferase (OAT), (designated mer-) - in which interstrand DNA cross-links accumulate and lines that possess normal levels of this enzyme (mer+) - in which these cross-links do not form - however, suggest that a lesion other than the DNA cross-link is responsible for enhanced sensitivity in polyamine-depleted cells [5,6,10]. The question of what lesion or lesions, if any, contribute to the enhanced sensitivity of polyaminedepleted cells to these bifunctional agents remains to be investigated. The present findings suggest that retarded repair of an unidentified lesion may be the underlying factor in the hypersensitivity of polyamine-depleted cells to chemotherapeutic DNA alkylating agents. References Bartholeyns, J. and Koch-Weser, J. (1981) Effects of alpha difluoromethylornithine alone and combined with adriamycin or vindesine on L1210 leukemia in mice, EMT6 solid tumors in mice and solid tumors induced by injection of hepatoma tissue culture cells in rats. Cancer Res., 41. 5158-5161.

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