[CANCER RESEARCH 53, 5690-5696, December 1, 1993]

In Vivo Mutagenesis Induced by CC-1065 and Adozelesin DNAAlkylation in a

Transgenic Mouse Model

T h o m a s J. M o n r o e x a n d M a r k A . M i t c h e l l 2

Medicinal Chemistry Research, The Upjohn Company, Kalamazoo, Michigan 49001

ABSTRACT

Although considerable work has focused on characterizing the bonding chemistry and sequence selective alkylation of DNA by cyclopropylpyrro- ioindole compounds, little is known about the molecular consequence of their N-3-adenine addncts in whole animal systems. We have utilized a transgenic mouse system, harboring a ,~ phage shuttle vector, to assess the mutagenic potential of the antitumor compounds CC-1065 and adozelesin and, for the first time, to track the in vivo fate of their unique DNA modifications at the nucleotide level. Mice were inoculated with a single therapeutic dose of these agents and sacrificed at either 18 h, 3 days, or 15 days for extraction and analysis of liver DNA. Mutant frequencies ob- tained from drug treated and control animals were determined by in vitro packaging of the phage vector from genomic DNA followed by a colori- metric plaque assay to screen for phage in which the accompanying/acI repressor gene had mutated. Although undetectable at 18 h posttreatment, by 72 h a 3.fold increase in mutant frequency was observed in drug treated animals such that sequence analysis of drug induced mutations could be performed and a direct comparison made between in vitro and in vivo DNA alkylation. Base substitution involving guanine or cytosine accounted for 64% of the 41 mutations sequenced from drug treated animals. Only 7 of the mutations occurred at a cyclopropylpyrroloindole alkylation site while 23 occurred 1 to 4 nucleotides from a potentially alkylated adenine.

INTRODUCTION

CC-1065, an extremely potent cytotoxic antibiotic originally iso- lated from Streptomyces zelensis in 1978 by researchers at The Upjohn Company (1), has served as the prototype for a new and interesting class of DNA antitumor compounds. CC-1065 and its CPP containing analogues are therapeutically active against a variety of experimental tumors (2-6); however, CC-1065 itself was found to cause a lethal delayed toxicity in mice at therapeutic doses which precluded its clinical development (5). Over the past decade a number of highly efficacious analogues have been prepared which do not display the associated delayed death phenomenon (7). Clinical trials with adoze- lesin, the "first generation" CC-1065 analogue selected for human clinical evaluation, are currently under way.

In addition to the efforts undertaken to prepare analogues suitable for pharmaceutical development, considerable work has also focused on characterizing the sequence selective DNA alkylation (8, 9) and interesting bonding chemistry (8, 10-13) unique to this class of com- pounds. These CPI-containing agents alkylate adenine N-3 in the minor groove with high selectivity and considerable sequence speci- ficity. The biological consequences attributable to this unique DNA lesion are not yet fully understood.

CC-1065 and its analogues containing the alkylative cyclopropyl ring are mutagenic in both bacteria and mammalian cells (14). It has been postulated that the helix stabilizing effect and relatively nondis- tortive binding (8, 10) associated with CPI alkylation may pose an unusual problem for DNA repair pathways. Selby and Sancar (15)

Received 5/14/93; accepted 9/30/93. 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 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by The Upjohn Post-Doctoral Research Program. 2 To whom requests for reprints should be addressed. 3 The abbreviations used are: CPI, cyclopropylpyrroloindole; ENU, N-ethyl-N-nitro-

sourea.

have shown that the Escherichia coli ABC excinuclease system is capable of recognizing a CPI damaged template but it is uncleal: whether incisions can be efficiently induced to remove the adducted tract (16). Yeast rad52 mutants, which display extreme sensitivity to X-rays, are equally sensitive to CC-1065 when compared to the wild type strain (17), implying that recombination repair may also be ineffective in the recognition and/or resolution of the CPI lesion. The excision pathway used for efficient removal of UV photoproducts and a variety of other bulky adducts in human cells is also not effective when challenged with a CPI adduct (18). Thus, the CPI adduct may represent an intractable lesion to DNA repair processes resulting in primarily a genotoxic event (11). Lack of efficient repair would ex- plain the in vivo longevity of the CPI adduct (19), inhibition of DNA synthesis observed in cultured cells (3), and the acute cytotoxicity (3, 4) displayed by these compounds.

The lacl gene of E. coli has been used extensively as a target for analysis of both spontaneous and chemically induced mutation (20- 27). This gene codes for a transcriptional repressor of the Lac operon and thus enables one to rapidly screen for expression of/3-galactosi- dase in the absence of a functional repressor. Recently, a transgenic C57BL/6 mouse (Big Blue; Stratagene, La Jolla, CA), carrying the lacI gene as part of a multicopy A phage shuttle vector, has been developed as a short term mutagenesis assay which allows the recov- ery of the lacI target and characterization of mutations formed in vivo (28, 29). The Big Blue assay has been successfully applied to the characterization of induced mutations generated from a variety of well known mutagens (28, 29). In this study we have utilized the Big Blue mutagenesis system to address, for the first time, the in vivo fate of the CPI-N-3 adenine adduct at the DNA sequence level. Preliminary results from these studies have recently been presented (30).

MATERIALS AND METHODS

Assay Components. Big Blue (transgenic C57BL/6) mice, Transpack, E. coli strains and all other components of the Big Blue assay system were purchased under a licensing agreement from Stratagene.

Chemical Dosing. Male (6-8 weeks old) Big Blue mice were obtained from Stratagene and all protocols were approved by an independent animal use committee. CC-1065 was isolated and adozelesin was synthesized by scientists at The Upjohn Company (Kalamazoo, MI). CC-1065 and adozelesin were administered by tail vein injection at 50 and 36/xg/kg body weight, respec- tively. Both CPI compounds were dissolved and administered in a vehicle composed of 2% N,N-dimethylacetamide and 10% Emulphor EL620 (GAF Corp., Linden, NJ). This N,N-dimethylacetamide/Emulphor vehicle was also used in the mock dosing of control animals. ENU (Sigma Chemical Co., St. Louis, MO) was delivered by i.p. injection at 250 mg/kg body weight in a 1% ethanol solution.

Packaging and Plating. Liver DNA was isolated from drug treated and untreated mice using a protocol of Dounce homogenization, proteinase K digestion, phenol chloroform extraction, and ethanol precipitation (29). Pack- aging of the transgene vector, plating of rescued phage, identification of mutant plaques, and calculation of mutant frequency was performed according to the developer's instructions (Stratagene).

In lraro Phage Alkylation. Tritiated adozelesin was prepared by Chemsyn Science Laboratories (Lenexa, KS) by a method developed by R. C. Kelly, at The Upjohn Company. The cyclopropyl-CH2 group is specifically labeled with a single tritium atom. Tritiation in this position was found to be stable and was not labile after exposure to a variety of aqueous conditions. Phage DNA was

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CPI MUTAGENES1S

isolated from a batch culture of nonmutant phage recovered from control animals by standard protocols (31). Purified phage DNA (10 tzg) was treated with 10-fold dilutions of [3H]adozelesin covering a range from 1 drug/10 base pairs to 1 drug/105 base pairs. Noncovalently bound drug was removed from the reaction mixture by sequential phenol, phenol:chloroform (1:1), and chlo- roform extractions. Phage DNA was precipitated in ethanol, pelleted by cen- trifugation, rinsed in 70% ethanol, and resuspended in an appropriate volume of Tris-EDTA buffer (approximately 0.5/xg//xl). Each DNA sample was quan- titated by UV spectrophotometry and the levels of alkylation were calculated by liquid scintillation counting of a known amount of the treated DNA. 500 ng of each sample was incubated with a h phage packaging extract (Transpack; Stratagene), an aliquot corresponding to 25 ng was plated, and mutant fre- quency was determined as described above.

Sequence Analysis. Potymerase inhibition mapping of CC-1065 and adozelesin alkylation sites has been described (9). Dideoxynucleotide sequenc- ing of the lacI gene was performed using Sequenase version 2 (United States Biochemical, Cleveland, OH) on double stranded plasmid DNA obtained by excision from the phage genome (32). LacI sequencing primers were obtained from Stratagene (La Jolla, CA).

RESULTS

In Vivo Mutant Frequencies. Thirty animals were used in this study to assess the mutagenic potential of CC-1065 and the simplified analogue adozelesin. Incorporating several time points (18 h, 3 days, and 15 days) into the study we were able to determine the premuta- genic lag phase, time prior to mutation induction, and establish an appropriate interval at which to characterize the DNA damage induced by the CPI adduct. As stated previously, the CPI adduct is long lived in vivo (19) and therefore we anticipated an increase in the mutant frequency over time due to fixation of latent DNA damage. Two animals were inoculated i.p. with 250 mg/kg ENU and sacrificed after 3 days to serve as positive controls. This dose and time point were chosen because of previous work showing a significant mutation induction of approximately 6-fold over the spontaneous background (28). Four animals (twelve total), injected i.v. with vehicle only, were used as negative controls to monitor the spontaneous background mutant frequency at each of the three time points. Four animals were inoculated with a single i.v. dose of 50/xg/kg CC- 1065, a dose chosen to be slightly less than the maximum tolerated acute dose for mice, all of which were sacrificed on day 3. It should be noted that at this dose of CC-1065, a mean day of delayed death would have been expected approximately 37 days posttreatment (6). Because of our clinical interest in adozelesin, it was used to monitor the time course of mutation induction. Twelve animals were inoculated with a single therapeutic i.v. dose of 36/zg/kg (maximum tolerated dose, 45/zg/kg) and four animals were sacrificed at each of the three time points. The concentration of adozelesin was chosen to be equimolar with that of CC-1065. These two compounds display distinct physical and bio- logical properties which we thought might be reflected in either their induction frequencies or in their mutation spectra. Specifically, the properties we thought may play a role are the delayed death phenom- enon associated with CC-1065 and not adozelesin (5, 7), and the reversibility of the covalent adduct formed by adozelesin versus an essentially nonreversible CC-1065 adduct under physiological condi- tions (13).

All animals were sacrificed at the times indicated above and liver DNA was extracted for in vitro packaging of the h transgene and accompanying Iacl target. Liver was chosen as the target tissue for analysis because of the predominate hepatotoxicity effects observed with CC-1065 and thought to contribute to delayed lethality (5). Over 120,000 plaques were recovered from each animal and mutants, iden- tified as blue plaques when grown in the presence of 5-bromo-4- chloro-3-indolyl-/3-t>-galactopyranoside, were isolated and verified by

secondary plating. Mutant frequencies were calculated from the num- ber of blue plaques relative to the number of wild type clear plaques (Fig. 1).

The spontaneous mutant frequency determined from vehicle treated animals was very consistent at each of the three time points ranging from 2.58 _+ 0.47 (SEM) • 10 -s to 3.06 ___ 0.42 • 10 -s, which is in good agreement with frequencies reported in the literature (28, 29). Mutant frequencies obtained at 3 days from mice treated with 250 mg/kg ENU showed an induction of almost 4-fold (10.18 __ 1.27 • 10 -5) over background which also compares favorably to reported levels (28, 29, 33, 34). It is known that CPI alkylation reaches maxi- mal DNA adduct levels approximately 12 h after drug exposure before beginning a slow decline (19). Therefore, by inclusion of an early 18-h time point we would confirm that we were assaying damage resulting from CPI alkylation and not merely scoring the presence of premu- tagenic lesions on the DNA. At 18 h (0.75 days) posttreatment we did not observe a significant change in mutant frequency from adozelesin treated animals. This would indicate that although CPI alkylation reaches maximum levels within 12 h of exposure (19) the assay is insensitive to the presence of DNA adducts. After 3 days, CC-1065 and adozelesin displayed very similar levels of induction, 7.15 +__ 1.01 X 10 -s and 7.84 + 0.81 • 10 -5 respectively, which were nearly 3 times the spontaneous mutant frequency. Fifteen days following a single dose of adozelesin, we did not observe a significant increase in mutation frequency over that observed at three days. Thus, within 3 days of drug treatment a sufficient amount of erroneous replication/ repair has occurred such that representative mutant spectra could be obtained.

In trttro Phage DNA Alkylation. Knowing that CPI adducts are long lived in the mouse (19), we were concerned that adducts remain- ing on the DNA after extraction may be "fixed" as mutations during subsequent packaging and replication of the phage in the E. coli host. The E. coil strain (SCS8) used in the recovery of the lacI target from mouse DNA was recA- and therefore deficient in post replication repair and the induction of an SOS error prone response (35, 36). In the absence of SOS induction, single abasic sites and other nonin- structional lesions are extremely cytotoxic (37, 38). In addition, recA

deficiency has been shown to eliminate mutation induction by UV irradiation (23, 36, 39, 40) and alkylating agents (25, 41-44). Never- theless, to confirm that the frequencies we obtained were a true representation of the mouse mutation spectrum, aliquots of naked phage DNA were treated with various doses of tritium labeled adoze- lesin, subjected to in vitro packaging, and used to infect E. coti.

Mutant frequencies calculated from a minimum of 80,000 plaques did

12

11

10

9

8

7

6

5

4

3

2

1

o 0.75

l

3 15

Days Post Treatment

[ - - ] Control

Adozelesin

C C - 1065

Fig. 1. Mutant frequencies obtained from liver DNA isolated at either 18 h, 3 days, or 15 days posttreatment. Two mice were dosed with ENU at 250 mg/kg body weight. All other columns represent average mutant frequencies obtained from four animals. Control animals were inoculated with vehicle only to determine the spontaneous background frequency. CC-1065 and adozelesin were administered at 50 and 36/xg/kg body weight, respectively. Error bars represent standard error of the mean at 95% confidence.

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CPI MUTAGENESIS

not differ from the nontreated control DNA (data not shown). The spontaneous background observed in these experiments was similar to )t phage mutation rates in recA- host strains reported in the literature (39). These results suggest that the adozelesin-adenine adduct is not a mutable lesion in this E. colt strain. Importantly, we observed a dose dependent drop in the number of plaque forming units which corre- lated with the number of tritiated adozelesin adducts present on the DNA. By extrapolating from the number of expected phage progeny (approximately 200,000) obtained with nontreated control DNA, we found that the number of CPI adducts which reduced the number of viable progeny by 50% was 0.5 adduct/phage genome. The direct correlation between adduct levels and the reduction in subsequent phage production suggest that a single adduct is sufficient to block phage replication. This finding is significant in that the nonadducted strand is apparently also blocked in replication. A reasonable expla- nation is that a phage genome bearing a single adduct, after escaping the molecular filters of in vitro packaging and injection into the E. coil host, begins its replication cycle only to be blocked in an unresolved replication intermediate. This may reflect the inhibition of helicase progression at the replication fork as has been reported for CC-1065 and its CPI analogues (45, 46). Furthermore, in the event that a small fraction of the phage genomes bearing a single adduct are replication competent, there may be a replication bias in favor of the nonadducted strand resulting in only wild type phage (nonmutant) plaques being scored. Selective discrimination against the propagation of the strand on which a blocking lesion resides has been demonstrated (43). These studies suggest that, within the levels detectable by the assay, CPI adducted phage DNA would not significantly contribute to the muta- tion spectrum obtained from the mouse experiments, and more im- portantly, phage retaining CPI adducts would be effectively excluded from the plaque population being analyzed.

In Vitro CPI Alkylation Map. It is inherently difficult to discrimi- nate between induced and spontaneous mutations at only a 3-fold level of induction. In vitro polymerase inhibition mapping (9) of potential CPI alkylation sites did facilitate the alignment of the mutations with known sites of alkylation and suggested which mutations were prob- ably not associated with a CPI adduct. CPI binding maps were pro- duced for both CC-1065 and adozelesin which were indistinguishable at similar levels of alkylation. Although the IacI gene is not particu- larly AT rich (44%) approximately 170 sites were mapped along both strands of the gene. Sites of CPI alkylation that mapped in the vicinity of mutations isolated from drug treated animals are indicated in Fig. 2 and are used to categorize the mutations relative to the nearest CPI binding site.

Sequence Analysis of Spontaneous Mutations. Eleven mutants were sequenced from untreated control animals. These were com- posed exclusively of single base substitutions predominated by GC>AT transitions (90%) (Table 1). In contrast, single base substi- tutions account for only 11% (20 of 176) of lacI mutations in E. colt (24). Slipped mispairing in the lacI triple repeat sequence, 5'- TGGC-3' (21), accounts for 67% of spontaneous mutations of E. colt origin and deletions (13%) comprise the largest non-hot spot class (24). We did observe a single mutation at the TGGC hot spot, how- ever, it was isolated from a drug treated animal and the possible involvement of a CPI adduct could not be excluded (Fig. 2.,4, mutant C633).

CPI Induced Mutation. A total of 41 mutants (2 of which were double mutants) were sequenced from drug treated animais sacrificed 3 days posttreatment. It is interesting to note that while GC>AT transitions predominate in the control mutants (90%), mutations de- rived from drug treated animals display a much more diverse spectrum (Table 1). Many of the mutations from drug treated animals are suggestive of CPI induced damage. Several of these occur at or a few

bases removed from a potential site of alkylation (Fig. 2). These mutations consist of not only base substitutions but insertions and deletions as weI1 as a complex gene rearrangement (CC-1065 mutant CCC). Seventy three percent (30 of 41) of the mutations isolated :from drug treated animals appear to have arisen by either targeted (7 of 30) or locally targeted (within 4 bases) (23 of 30) events at the site of CPI alkylation (Fig. 2). This would be consistent with the 3-fold level of induction observed. Of the 30 potentially targeted and locally targeted mutations, l i can be attributed to the insertion of adenine into the nascent strand opposite a damaged template; 9 are transversions (pre-" dominantly GC>TA); 3 are transitions; and 7 are insertion/deletion events which could be the result of adduct enhanced misalignment of repetitive nucleotide tracts (47). Interestingly, two of these frameshift mutations involved the addition or the deletion of an A:T base pair at the same site by adozelesin and CC-1065 respectively (A1010 and C1010).

Targeted Events. Of the seven mutations that may have been targeted at the site of alkylation five involved base substitution, four of which (C78, C871a, C871b, A168) could be explained by the incorporation of adenine opposite the modified base, suggestive of the "A rule" for base incorporation (48, 49). In addition to these AT>TA transversions, an AT>GC transition (A324), a deletion of an alkylated AT base pair (CI010), and a potentially targeted gene rearrangement (CCC, described below), were observed as well.

Locally Targeted Events. The majority of the mutations isolated from drug treated animals (23 of 41) could be explained by polym- erase errors in the vicinity of the CPI adduct. Eighteen of these involved mutation on the 3 prime side of alkylation sites, one-half of which (C42, Cl16, C129, C847, A42a, A42b, A75, A281, A1010) appear to have arisen via events initiated at one nucleotide 3 prime to a predicted alkylated adenine. Mutations occurring one base 3 prime to the adduct would be analogous to the inhibition site of DNA polymerization observed in vitro with CPI adducts (9, 50). Two of these + 1 mutations (A281 and A1010) involved the insertion of an additional A:T base pair most likely due to misalignment of the damaged template. It is also interesting to note that with only one exception (A75) all the base substitutions at this + 1 position involved the sequence 5'-ACG-3' where cytosine was replaced on the 3 prime side of the modified adenine. In support of these observations, Sun and Hurley (50) studied the ability of Klenow fragment to elongate past the primary in vitro pausing site (+1 position) on a template 5'-ACG-3' and found that although CC-1065 and its analogues were strong blocks of replication, in the presence of excess dATP CC-1065 directed the misincorporation of adenine opposite the cytosine 3 prime to a site specific adduct. Sun and Hurley (50) also noted the influence of the 5 prime sequence on the position of polymerase termination. They showed that the presence of guanine in the third position 5 prime to the adduct would cause the polymerase to pause one base prior to the adduct with a minor termination site at the alkylated base. When guanine was located in the fourth position, polymerase progression was inhibited 2 bases before the adducted nucleotide and directed misincorporation at the + 1 site. Similarly, all of the + 1 base substi- tutions we observed from drug treated animals originated at CPI sites containing guanine either three or four bases 5 prime to the predicted site of alkylation. The predominance of mutations occurring one nucleotide 3 prime to a base adduct have also been observed for the N-(deoxyguanosin-8-yl)-2-aminofluorene adduct where 11 of 31 mu- tants were found adjacent to the modified base with the remaining distributed up to four nucleotides on either the 5 prime or 3 prime side of the adduct (47). Locally targeted mutations have also been reported for the 2-N-acetoxy-N-acetylaminofluorene adduct (51), pyrimidine dimers (52), cis-diamminedichloroplatinum(II) (53), and other bulky lesions (48) as well as at abasic sites (37).

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CPI MUTAGENESIS

Fig. 2. Sequence analysis of lacI mutations tabulated in table 1. Mutants are identified with a letter indicating from which treatment group they were isolated C (CC-I065), A (adozelesin), or N (non- treated control), and a number which designates the base position in the lacI sequence which was changed. The base pairs involved in the mutation are shown in bold capital letters with ten base pairs flanking the mutation in lower case. The sequence shown is the normal lacI sequence and the type of mutation is identified to the left. Drug binding sites identified by polymerase inhibition are shown above and below these sequences (-% ,-). The adduct formed by CC-1065 or adozelesin extends four bases 5 prime and overlaps one base pair on the 3 prime side of the covalently modified adenine. The alkylated adenine is shown 3' in the highlighted sequence covered by the drug. The mutations were categorized as targeted (at the adduct site), semi- targeted (within 4 bases), or other, based on CPI binding sites mapped in vitro.

A. CC-1065

Tarqeted

CCC

C78

C871a TA>AT C871b

C1010 -A:T

Semi-Tarqeted

C42 CG>TA

Cl16 GC>TA

C129 CG>AT

C131 CG>TA

C181 CG>AT

C221 GC>TA

C293 CG>TA

C369 TA>GC

C561 GC>TA

C633 +TGGC

C847 CG>AT

C882 CG>TA

C920 +G:C

Other

C56 GC>AT

C269 GC>AT

C383 -G:C

Deletion -126 .... +86 Inversion +87---+137

i AT>TA ggtgtctcttAtcagaccgtt

c c a c a g a g a a T a g t c t g g c a a

I acagctcatgTtatatcccgc t g t c g a g t a c A a t a t a g g g c g

] i gaaaagaaaaAccaccctggc cttttcttttTggtgggaccg

i aaaccagtaaCgttatacgat tttggtcattGeaatatgcta

ggccagccacGtttctgcgaa c c g g t c g g t g C a a a g a c g c t t

I I tctgcgaaaaCgcgggaaaaa agacgcttttGcgcccttttt

' l 1 tgcgaaaacgCgggaaaaagt acgcttttgcGccctttttca

1 ttcccaaccgCgtggcacaac aagggttggcGcaccgtgttg

gttgctgattGgcgttgccac caacg@ctaaCcgc@acggtg

i |

7 tcgcgccgatCaactgggtgc agcgcggctaGttgacccacg

i gtgcacaatcTtctcgcgcaa cacgtgttagAagagcgcgtt

gtcgcattggGtcaccagcaa c a g c g t a a c c C a g t g g t c g t t

i tggctggctggc,ataaatat a c c g a c c g a c c g ~ t a t t t a t a

i I

tagtgggataCgacgataccg atcaccctatGctgctatggc

1 tatatcccgcCgttaaccacc a t a t a g g g c g G c a a t t g g t g g

- - ! cctgctgggg-caaaecagcg ggacgacccc~gtttggtcgc

---7 atacgatgtcGcagagtatgc tatgctacagCgtctcatacg

| I gcaaattgtcGcggcgattaa cgttt@acagCgccgct@att

t , t_____

cgcgcaacgcGtcagtgggct gcgcgttgcgCagtcacccga

B,

Tarqeted

A168 AT>TA

A324 TA>CG

Semi-Tarqeted

A -15 GC>TA

A42a CG>TA A42b

A75 CG>AT

A84 CG>AT

AI80 GC>AT

A281 +A:T

A559 -G:C

A575 GC>CG

AI010 +A:T

Other

A56a GC>AT A56b A56C A56d

A93 GC>AT

A269 GC>AT

A381 GC>AT

A588 -G:C

Adozelesin

I gagctgaiai~Acattcccaac ctcgactt~T.~aagggttg

.......... gtgtcgatggTi~gaagc cacagctacc~i~i~igcttcg

ttcgeggtatGgcatgatagc aagcgcclai~iaC~gt~i~i~g

t ........... I

i aaacca~i~!i~Cgttatacgat t t t g g t c a t t G c ~ a ~ t l g c t a

. . . . f gccggtgtctC~tlatcagacc cggccacagaGaas

] tcttat!~i~igaCcgtttcr agaas

I , : ~ ............

. . . . ] attc~:~ia:~ccGcgtggcacaa taagggttggCgcaccgtgtt

........... i. cggcga~i~a~aytctcgcgcc gccget~!ai~!~!iE!Aagagcgcgg

tggtcgcattGggtcaccagc accagcgtaa~c~agtggtcg ] .........

ccagc~i~itcGcgctgttagc ggtcgtttagCgcg~i~icg

'I ] ga!a!aag~i~i~i~i, ccacectgg cttttctttttAggtgggacc

-,7 atacgatgtcGcagagtatgc tatgctacagCgtctcatacg

accgtttcccGcgtggtgacc tggcaaagggCgcaccactgg

'! ...... ] geaaattgtcGcggc~!s cgtttaa!cagcgccgctaa~

t. |

ctcgcgcaacGcgtcagtggg gagcgcgttgCgcagtcaccc

i ......... ! ctgt~!agcggGcc~!~agt gacaais

L

C,

N42a CG>TA N42b

N56 GC>AT

N83 AT>GC

N92 CG>TA

N94 GC>TA

N198 CG>TA

N270 CG>TA

N381 GC>AT

N530 CG>TA

N898 CG>TA

Control

] aaaccagtaaCgtta.tacgat tttggtcattGc~at~tgcta

atacgatgtcGcagagtatgc tatgctacagCgtctcatacg

i ctcttatcagAccgtttcceg gaga atlagt cTggc~ aia!g~gc

t ! ........

gaccgtttccCgcgtggtgaa ctggc~a:ggGcgcaccactt

accgtttcccGcgtggtgaac ...... tggcaaag~gCgcaceacttg

] r caacaactggCgggc:aiaacag gttgttgaccGcccgtttgtc

I l caaattgtcgCggcg~!~aa gtttaacagcGccgctaatts

ctcgcgcaacGcgtcagtggg gagcgcgttgCgcagtcaccc

agacggtacgCgactgggcgt tctgccatgcGctgacccgca

l cacca~aa~Caggattttcg gtggtagtttGtcct~aiaagc

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Table 1 Sumary of mutational spectra a

Mutation class CC-1065 Adozelesin Control

Transversion (10) 48% (5) 25% (1) 9% GC>TA (6) (3) (1) Gc>c~ (o) (t) (o) TA>AT (3) (1) (0) TA>GC (1) (0) (0)

Transition (6) 29% (11) 55% (10) 91% GC>AT (6) (10) (9) AT>GC (0) (1) (1)

Insertion (2) 9% (2) 10% (0) 0% Deletion (2) 9% (2) 10% (0) 0% Complex (1) 5% (0) 0% (0) 0%

a The number of mutants sequenced in each mutational class is shown in parentheses. Transversions and transitions are further subdivided showing the number of each possible base substitution.

Complex Mutation. Mutation CCC, derived from a CC-1065 treated animal, was unique in that it was the only complex mutation observed and was potentially initiated at a site of CPI alkylation (Fig. 3A). Close examination of the insert revealed that there was an in- version of a 51-base pair sequence immediately 5 prime to a strong polyadenylate alkylation site (Fig. 3B). This inversion was accompa- nied by a 212-base pair deletion in which a resulting set of direct repeats (TITCC-TrTCC) may have facilitated misalignment of the inverted sequence on the 5 prime side of the deletion. The junctions of this inversion/deletion both contain extensive secondary hairpin po- tential (Fig. 3C). It is conceivable that a CPI stalled replication com- plex or the initiation of adduct repair allowed formation of a poten- tially very stable hairpin (bases +87 to +137). Strand switching (54) at the base of the palindrome would then result in the synthesis of sequence templated from the complimentary strand (inversion). Reso- lution of this "inverted replication" after synthesis would then involve a second strand switching event accompanied by a misalignment with the direct repeat TITCC (pos. -126) resulting in the deletion of 212 base pairs. Formation of a second potential hairpin (-121 to -90) may have facilitated misalignment of the direct repeats by bringing them into close proximity such that one copy of the repeat could base pair on the complement of the other. Many spontaneous mutation hot spots involve DNA repeats (24) and hairpin structures such as these have

been shown to dramatically influence mutation spectra (40), however, rearrangements of the magnitude displayed here are extremely rare. Resolution of this type of mutation involving both a recombination and misalignment would be improbable in a recA- background (55) and is likely of mouse origin. The ability of DNA damage to potentiate mutagenic events occurring at palindromic structures is not without precedent (47, 54, 56). Todd and Glickman (57) suggested that UV induced mutation spectra may be related to potential palindromic structures in the DNA, where premutational lesions might be refrac- tory to error free repair and DNA replication in the vicinity of dimers becomes error prone (52). Such a mutation isolated from a small sampling and in immediate proximity to a strong alkylation site is very suggestive of a CPI induced event. It should also be noted that four other mutations (all base substitutions) from drug treated animals were mapped within this potential hairpin region. This sequence codes for a portion of the 59-amino acid DNA binding domain of the lacI repressor which has been shown to be the most mutationally sensitive region of the gene (58), Indeed, 50% (26 of 52) of all our mutations were found to be within this region, 7 of which involved bases +86 to + 137 encompassing the potential hairpin described.

DISCUSSION

Alkylation of DNA by CC-1065 or adozelesin produces an adduct in which the drug molecule lies within the minor groove covering four bases 5 prime and overlapping one base pair on the 3 prime side of the covalently modified adenine (59, 60). These compounds and related analogues are unique in that they are highly specific for the N-3 of adenine and display a considerable amount of sequence selectivity (8, 9). The distinctive features of the CPI adduct prompted us to address the in vivo consequences of these cytotoxic DNA lesions. The availability of the Big Blue assay system allowed us the opportunity to extend our knowledge of the in vitro manifestations of this unique adduct into a whole animal model. The system was attractive because it provided in vivo access to a target sequence (lacI) containing ap- proximately 1 CPI binding site for every 17 base pairs along the gene and thus lack of mutagenesis could not have been attributed to the lack of alkylation at mutationally sensitive sites. With a single therapeutic

Fig. 3. A, mutant CCC isolated from an animal treated with 50 ~g/kg CC-1065. This mutant in- volved the inversion of a 51-base pair sequence (+87 to +137) accompanied by a 212-base pair deletion (-126 to +86). In B, the polyadenylate tract on the 3' side of the inversion was identified by polymerase inhibition as being a strong site of CPI alkylation. In C, extensive hairpin potential may have facilitated this mutation event (see text).

A

C

- 1 2 6 +87 +1.37 I I I I

T C T T T C C . . . . . . . . G ' A C C a T T T C @ , > @@,~.A&AAG A G A A A G G . . . . . . . . C T G G C A A A G Q ~ C C T T T TTC

A G u 1 6 5 1 6 5 < 8 0 J k & & T T C : 5 '

GCCAGCCAGGT G T

ATT A T G C C C

T T C T C G A G

C c G T AGTGG TA AAAGC

CG GC TA CG AT GC TA CG TA CG C_.,,-C. CC-I

- 126 c G ' C - 8 4 + 8 3 TA

I GT I I TA _ 5' ~ T C T T T C C T A TTACCG . . . . . . . . . . . . . ACCGTA ~ A G - - 3'

r___J L-n +87 + 1 3 7

5694

3" �9 5 '

3'5'

5'3'

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CPI MUTAGENESIS

dose, both CC-1065 and adozelesin exhibited 3-fold increases in mutant frequency above spontaneous background levels.

The mutations we observed are very consistent with the known mechanism of action of these compounds. First, the ability to induce mutation at near maximally tolerated doses is only about 3-fold above the spontaneous background. Low induction frequencies might be expected from such highly cytotoxic agents (48, 61). If the primary consequence of the CPI adduct is its ability to cause a lethal block to DNA replication the majority of adducts would be expected to be genotoxic not premutagenic. Secondly, many of the mutations recov- ered could be associated directly with strong polymerase inhibition sites observed in vitro. Approximately two-thirds of the mutations characterized from drug treated animals could be associated with in vitro sites consistent with a 3-fold level of induction. Thirdly, targeted and locally targeted mutations, observed both 5 prime and 3 prime to the modified base, may reflect structural distortions of the DNA helix caused by covalent CPI modification (12, 62).

Mutant analysis would have been difficult without the aid of a CPI binding map produced by in vitro polymerase inhibition (9). The number of mutants analyzed is small and spectral differences between the two compounds could not be confidently assessed; however, sev- eral informative observations can be made. Many of the mutations can be explained by insertion of adenine, "A rule" (48, 49), opposite a noninformative nucleotide. Some of these can be classified as targeted at the site of alkylation (AT>TA transversions) while others could be explained by a semitargeted N > T substitution where the A rule may act opposite a locally distorted nucleotide. It is known that the CPI adduct can have profound effects on nucleotide recognition many bases removed from the site of covalent modification (12), thus, application of the A rule to base substitutions in the vicinity of the CPI lesion would be appropriate. AT>TA transversions have also been observed at the gpt locus in AS52 cells exposed to adozelesin at predicted CPI binding sites as well as deletions and insertions in the region surrounding these sites. 4 A similarly diverse mutation spectrum has also been observed in the aprt locus of Chinese hamster ovary cells treated with CC-1065. 5 where mutations were recovered at po- sitions slightly removed from the presumptive drug binding site.

In addition to the exceptional cytotoxicity displayed by cyclopro- pylpyrroloindole compounds, they have produced positive mutation induction in all mutagenicity assays in which they have been evalu- ated (14, 63-65). Interestingly, Harbach et al. (14) showed that the most mutagenic CPI analogue was one consisting solely of the reac- tive A subunit and in contrast to CC-1065 was mutagenic at nontoxic doses. This would indicate that covalent modification of N3-adenine is mutagenic yet the ultimate biological consequences are highly dependent on noncovalent associations of the compounds with DNA (66). Previous studies have also shown that these long lived adducts are very potent clastogens, producing chromatid and chromosome breaks and other complex rearrangements (63, 65). From these ob- servations one may have expected to see a rather exotic mutation spectrum; however, extreme chromosomal aberrations may be beyond the detection limits of the lacI target system (limited to deletions <5kb and insertions <8kb based on size constraints of packaging). The single mutant which did display this type of rearrangement (mu- tant CCC; Fig. 3) may well be representative of a class of mutations which occur on a larger scale.In conclusion, we propose that the molecular consequence of CPI induced DNA damage, although being primarily genotoxic, can direct both targeted and semi-targeted mu- tation during translesion bypass of a noninstructional N-3 modified

adenine. Furthermore, these targeted events are strongly influenced by the sequence context in which the premutagenic lesion resides.

4 K. R. Tindall, personal communication. 5 B. W. Glickman, personal communication.

ACKNOWLEDGMENTS

The authors thank Thomas Dekoning, Renae Ouding, Donna Waggoner, and Sally Couch for their technical assistance in the care, dosing, and necropsies of the animals.

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Mutagenesis Induced by CC-1065 and Adozelesin DNAIn Vivo

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