The mutagenic properties of DNA minor-groove binding ligands

Fundamental and Molecular Mechanisms of Mutagenesis

ELSEVIER

Mutation Research 355 (1996) 141-169

The mutagenic properties of DNA minor-groove

binding ligands

Paul R. Turner, William A. Denny Cancer Research Luboruto~.

The Unicersie

Received 5 September

of Auckland, Prirate Bag 92OI9. Auckland 1000. New Zealand

1995; revised 1I December

1995; accepted

12 December

1995

Abstract This review summarises mutagenesis-related research on the major classes of DNA minor groove binding ligands. These compounds can bind to DNA covalently or non-covalently, and span a range of DNA sequence selectivities. Many of the non-covalent binders show effects on topoisomerase enzymes in mammalian cells, with the bisbenzimidazoles being the most active. Mutagenic effects consistent with topoisomerase inhibition are observed in vitro. Many of these compounds induce aneuploidy and polyploidy, properties which may also contribute to carcinogenic processes. Similarly, uvrA trapping by some minor groove binders may alter mutagenetic processes by inhibiting efficient repair. Distamycin has been shown to enhance the mutagenicity of ethidium bromide in bacteria by an undetermined mechanism. However, the inhibitory effects of minor groove binders on human DNA repair systems have not yet been reported. Hoechst 33258 and distamycin cause chromosome decondensation in both mouse and human cells particularly at heterochromatic regions which are rich in AT content. Various minor groove binders have been shown to induce fragile sites in cultured lymphocytes from susceptible individuals, which may have a propensity to develop particular cancers. Investigation of the relationship between fragile site inducing drugs and chromosomal rearrangements in fragile site carriers has not been investigated but may yield interesting results. Some DNA alkylating minor groove binders can generate lesions extremely toxic to mammalian cells (e.g.. CC-1065 and analogues), and induce a range of DNA sequence changes in vivo. both at the site of covalent bonding as well as at surrounding sequences. This may be typical of alkylating minor groove binders which have a binding site size of several base pairs, and which stabilise helical structure. Minor groove binders have effects on gene expression in vitro by inhibiting the sequence selective binding of various transcription factors to DNA. These effects may result in expression or repression of downstream genes also. This class of ligand thus offers the possibility of mutations targeted to specific genes or genomic regions. It will be interesting to determine whether such examples of targeted mutagenesis, as has already been observed with CC- 1065 and adozelesin, will result in an enhanced or in a lowered capacity to promote neoplastic disease. However it should be noted that pentamidine, a minor groove binder used in the treatment of AIDS-related PCP, has thus far shown no mutagenic effects in nuclear DNA and only a weak effect in mitochondrial DNA of yeast. These results suggest that minor groove binding does not necessarily lead to mutagenesis. Kewxwds:

Mutagenic property; Minor groove binding ligand

0027-5 107/96/S 15.00 0 1996 Elsevier Science BV PII SOO27-5107(96)00027-9

1. Introduction The notion that cancer is a multi-step process involving accrued genetic damage is now generally well accepted (Weinstein, 1988; Sugimura, 1992). Chemical carcinogens identified to date are many and varied but the majority have a common ability to react with cellular DNA. Chemicals which alkylate DNA have been used in the treatment of cancer since 1942 (Gilman and Phillips, 1946) and remain today as one of the most important classes of agent. The chemotherapy of cancer with such drugs is however a double edged sword. Ideally an anticancer drug should kill neoplastic tissue while leaving normal tissue unharmed, but the anticancer agents used clinically are not tumour specific and have a relatively narrow therapeutic window. Patients in effect undergo a controlled poisoning, the extent of which (in the case of alkylating agents) is often determined by myelosuppression. A balance must be carefully attained between antitumour effectiveness and both short-term and long-term toxicity. A major latter concern is genotoxicity, and a large number of papers have discussed the extent of chemotherapy-induced malignancies (reviewed in Boivin, 1990). For example. treatment with the MOPP protocol (mechlorethamine, procarbazine. vincristine and predinisone) can cure up to 70% of cases of Hodgkin’s disease, but at the same time increases the chance of secondary malignancies, including leukemia, by 2-107~. These secondary malignancies take 4-7 years to develop and are almost certainly the result of direct genetic damage by the chemotherapeutic agents used to treat the original disease. Although many cellular, bacterial and whole animal assays have been developed to investigate the mutagenic and carcinogenic potential of chemotherapeutic agents, particularly clinical drugs and those experimental ones showing clinical promise, such effects are inherently difficult to estimate. The time delay between mutagenic insult and its phenotypic expression complicates analysis, as does the common practice of using a combination of agents in therapy. Mutations induced by chemotherapeutic agents in tumour cells may also have deleterious effect on therapy by acceleration of the induction of drug resistance or enhancement of tumour progression. In

the treatment of terminal cancer the mutagenic potential of therapy is understandably of secondary concern, especially when the treatment is palliative. However in cases where treatment can be curative the potential mutagen effect of the drugs becomes more of a concern. Indeed it has been suggested treatment regimes in these cases should be reevaluated so that the risk of secondary neoplasms is kept to a minimum (Williams, 1990).

2. DNA minor groove binding ligands This review is concerned with the mutagenic potential of the general class of anticancer drugs which interact with DNA in the minor groove. Their mode of binding can be irreversible (by virtue of a covalent reaction with nucleophilic minor groove components such as the N3 of adenine or the ‘-amino group of guanine), or reversible via non-covalent interactions (electrostatic, hydrogen bonding and van der Waals contacts). The recent increase of interest in this group of compounds stems from their ability to interact in a sequence selective fashion at quite long DNA binding sites (up to 8 base pairs), suggesting the possibility of targeting specific DNA sequences within the genome. Structurally, minor groove binding ligands have a number of features that distinguish them from the more common DNA reversible intercalating agents and major groove alkylating agents. These features include an overall annular shape made up of aromatic rings which matches the curvature of the minor groove of DNA. Cationic charges provide affinity for the tunnel of negative potential in the groove and in addition many ligands possess H-bond donating or accepting atoms. These compounds tend to bind in the minor groove with relatively little distortion of the phosphate backbone, and in fact stabilise the regular B-DNA structure. Several classes of minor groove binders have received much investigation and development as antitumour agents and this review will focus primarily on these more well known classes. Mitomycin C is a clinically-used minor groove alkylating agent on which a large body of literature exists. and several analogues are now in clinical trial. CC- 1065 is another natural product alkylating agent,

P.R. Turner,

W.A. Denny/Mutation

for its extraordinary potency. Although CC1065 itself is not in clinical use, a number of synthetic analogues have reached clinical trial. The anthramycin family of pyrrolobenzodiazepines are a third family of natural product minor groove alkylators which have provided the basis for the design of DSB-120, a potent DNA interstrand crosslinker which has significant sequence selectivity. noted

OvNH2

H2N

H3C

0

Mitomycin C

H2N H3C

0

IO-Decarbamoyl Mitomycin C

“2N “3C

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In contrast to these compounds, which interact with DNA via covalent bonds, are a series of reversible minor groove binders. The most widely studied have been the polypyrroles, based originally on the natural products netropsin and distamycin, since extended to many synthetic analogues known collectively as the lexitropsins. These have been extensively analysed to obtain information on inherent sequence recognition motifs, and have been employed as carriers of alkylating moieties. Examples of such DNA-targeted alkylating agents are now undergoing clinical trial after showing promising results in vitro. Bisbenzimidazoles, alternatively known as Hoechst compounds, are used extensively as DNA specific dyes in the laboratory but in addition are now also being investigated for DNA sequence specific binding and antitumour activity. Additional interest has been fostered in these compounds following the recent discovery that they effectively inhibit mammalian topoisomerase I. A similar class of compounds, the polyamidines, were developed initially as anticancer drugs and several early examples underwent clinical trials (reviewed in Denny, 1988). These are also receiving renewed attention with the realisation that they are effective in the treatment of AIDS related pneumocystis carinii pneumonia. Finally, the class of bisquatemary ammonium salts were also first evaluated as anticancer drugs (Atwell and Cain, 1973) but are now being considered as antimalarial agents (Wilairat et al., manuscript in preparation). Various other minor groove binders have also been suggested as antimalarial compounds (Ginsburg et al., 1993; Carteau et al., 1994; Wang and Lown, 1992). Serious consideration should therefore be given to evaluating the mutagenic potential of these compounds as part of their development.

0

3. Mitomycin

EO-9 Fig. 1. Mitomycin

C and other pyrroloquinones.

C and other pyrroloquinones

Mitomycin C (MMC. Fig. 1) is a natural product first isolated from Streptomyces caespitosus (Hata et al., 1956), and is the best-known member of the pyrroloquinone family of compounds. It is used in combination therapies against various malignancies, but its effectiveness is limited by often unpredictable toxicities, including myelosuppression, renal and

144

P.R. Tunwr.

W.A. Drnn~/Muitrtion

pulmonary toxicity and a potentially fatal thrombus formation in small blood vessels of multiple organs (Black and Livingston, 1990; Erlichman. 1992). MMC requires activation before it can bind to DNA and exert its toxic effects (reviewed in Fisher and Aristoff, 1988). One electron reduction by cellular enzymes such as cytochrome P450 reductase generates the semiquinone radical anion, which may be further reduced to the hydroquinone. Oxygen can scavenge the semiquinone radical anion. reversing activation. Because of this MMC has been considered a hypoxia selective cytotoxin (Sartorelli et al.. 1994). Alternatively MMC can undergo direct reduction, primarily by the two-electron reductant DT diaphorase (EC 1.6.99.2) to yield the hydroquinone species (Siegel et al., 1990). This two electron reduction bypasses formation of the semiquinone radical anion and so enables activation of MMC in aerobic cells. A number of molecules based on MMC have been developed and have been tested clinically, both as hypoxia selective cytotoxins (e.g., porfiromycin (Sartorelli, 1988)) and as DT diaphorase-activated cytotoxins (e.g., E09 (Verweij et al., 1994)). Activated MMC alkylates B DNA at the 2-amino group of guanine position in the minor groove. Three adducts have been characterised and shown to form both in vitro and in vivo (Chawla et al., 1987). Two of these are monoadducts which differ only in the presence or absence of a carbamate group at the C-10 position. The third adduct consists of MMC linked to two guanine N2 atoms via the C 1 and C IO positions, indicative of the creation of either inter- or intrastrand crosslinks. The sequence specificity of the MMC/DNA adduct can influence the degree of DNA distortion. Using hydroxylamine as a probe for DNA distortion. MMC monoadducts at the sequence S-TG-3’ were shown to highly distort B DNA and labilise the complementary cytosine residue to hydroxylamine cleavage (Jolles and Laigle, 1995). All other 2-amino group guanine bound MMC adducts fit snugly in the minor groove and do not distort the B helix to any significant degree. Additionally the 2”-NHf groups of the molecule forms stabilising H bonds within the groove. These H bonds and van der Waals interactions with the walls of the DNA minor groove stabilise the B DNA structure and increase the melting temperature of the DNA (Chawla et al., 1987; Basu et al., 1993). Inter-

Rrsrurch 355 (1996) 14/L 169

strand crosslinks are formed at the sequence CpG while intrastrand crosslinks are formed at the sequence GpG. The sequence GpC is not crosslinked. The toxicity and antitumour activity of MMC has been ascribed to the interstrand crosslink. which is postulated to inhibit replication and DNA synthesis (Tomasz et al., 1987). Treatment of Ll210 murine leukemia cells with 10 pg/ml MMC completely inhibits DNA synthesis as assayed by [jH]thymidine uptake, but has no effect on RNA or protein synthesis (Yajima et al., 1990). It has also been shown, however, that the monoadducts of MMC cause extremely efficient blocks of DNA polymerase in vitro. The IO-decarbamoyl analog of MMC which cannot form crosslinks also efficiently blocks polymerase (Basu et al., 1993). IO-Decarbamoyl MMC is also toxic and possesses antitumor activity. suggesting that the monoadducts of MMC have relevant biological effects. MMC has been shown to bind B DNA sequences around Z DNA (a structural variation of B DNA is Z DNA which is favoured in certain sequences that have 5-methyl cytosines in CpG-rich regions), and inhibit the B + Z transition (Portugal and Sanchez-Baeza, 1995). The biological consequences of this action are unknown but the transcription of the c-myc proto-oncogene has been shown to be associated with Z DNA formation in certain regions of the gene. MMC is non-mutagenic in bacteria that cannot complete nucleotide excision repair (NER (Sancar and Tang, 1993)) by virtue of a mutation in the uvrB gene (uvrB_). However in bacteria that possess a functional NER system (uvrB+) and the resistance transfer factor plasmid pKM 10 1, MMC is mutagenic and its toxicity is reduced (Ferguson et al., 1988). Additionally, activation of MMC by rat liver S9 fraction lowers the mutagenicity of the compound (Seino et al.. 1978). MMC i, more toxic to Bacillus subtilis strains that lack the ret A protein which is integral to recombination mediated repair, than to strains that possess ret A (Mazza et al.. 1983). Interesting parallels can be drawn from these bacterial assays when compared with mammalian cells. Patients with Faconi’s anaemia have a predisposition to developing various neoplasms and are thought to have a defect in DNA repair of some form (Latt et al., 1975). Cultured lymphocytes of these patients also show an increased sensitivity to MMC as well

P.R. Turner, W.A. Denny/Mutation

as a decrease in the number of sister chromatid exchanges following MMC treatment, suggesting a lack of repair response. The repair response to MMC in Chinese hamster cells is complicated and involves numerous genes, not all of which have probably been identified. A recent report using complementation analysis of hyper-sensitive MMC mutants found eight distinct groups ranging from 4- to 30-fold in sensitivity to MMC (Jones, 1994). These results suggest that NER is only part of the cellular response to MMC damage and that other mechanisms are involved. The induction of gadd genes in mammals follows certain forms of DNA damage and the expressed proteins may be involved in inducing and/or maintaining growth arrest. This response has been shown to be independent of NER (Leuthy and Holbrook, 1992) and using a gadd promoter linked to the CAT gene in human HeLa cells expression of chloramphenicol acetyl transferase was shown to be induced by MMC (26 times control value expression with 5 kg/ml MMC). Similarly pretreatment of monkey cells with MMC followed by transfection with an UV irradiated SV40 based shuttle vector (pZ189) results in a 2-fold increase in UV mutations compared to control non-pretreated cells (Dixon et al., 1988). This suggests a change in the magnitude of response to DNA damage by MMC pretreatment resulting in enhanced mutagenesis. Cells lacking a NER system showed a similar response, again showing the involvement of alternative repair pathways. Sequence analysis of these mutants in the supF gene of the vector showed subtle differences. More misincorporation of bases at the site of damage and less in the vicinity of the damage were observed in pretreated cells and an increase in G . C + A . T transitions compared with G . C + T . A transversions were noted. These observations were attributed to a relaxation of the A rule (the preferential insertion of adenine opposite a damaged base by DNA polymerase) (Kunkel and Alexander, 1986). MMC has a number of effects on the chromosomes of mammalian cells and has been shown to be a carcinogen in rats (Preston et al., 1981). It also induces micronuclei in a variety of test systems. In Chinese hamster V79 cells at 1 pg/ml MMC induces a cell cycle delay 16 h after treatment and raises micronuclei levels 18 times that of controls (Krishna et al., 1989). Using measurements of the

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area of micronuclei induced in the bone marrow of mice after a 2 mg/kg dose Vanparys et al. (19901 suggested that MMC was a weak inducer of aneuploidy. Subsequently it was shown (Yajima et al., 1990) that 22% of micronuclei in V79 cells possessed a kinetochore, suggesting they encompassed whole chromosomes, while 79% had no kinetochore, suggesting they resulted from acentric chromatid fragments. In a clonogenic assay using L 12 10 murine leukemia cells, a dose of 1.35 p_g/ml for 1 h resulted in 37% survival (D3,) (Yajima et al., 1990). At 10 kg/ml DNA synthesis was completely inhibited whilst RNA and protein synthesis were unaffected. Crosslinked DNA was detected at 2.5 p_g/ml after 1 h by alkaline elution but was not detected 24 h later, suggesting efficient removal by repair systems. No single-stranded DNA breaks were detected over O-24 h. MMC is an efficient inducer of sister chromatid exchange in mammalian cells. Sister chromatid exchanges are a convenient way to monitor genetic damage and have been used extensively in this regard. In human cultured leukocytes at 1 pg/ml MMC causes aberrations in 62% of metaphases examined (Nowell, 1964). These aberrations include sister chromatid exchanges, chromosome and chromatid breaks and chromosome fragments. The persistence of MMC lesions causing SCEs has been investigated (Escalza et al., 1992) using a three way differential staining method. This technique allows metaphases to be identified as resulting from the lst, 2nd or 3rd division post treatment. At 0.3 p_g/ml there was a marked increase (28 times controls) in SCEs at the 1st division, at the 2nd division SCE levels were still elevated but only marginally (2 times controls). By the third post treatment division SCE values were the same as control values. These results suggest the majority of SCE inducing MMC lesions are repaired at the first cellular division. Another alkylating agent, ethyl methanesulphonate had persistent elevated levels of SCEs even after the 3rd post treatment division. MMC also induces 6thioguanine resistant mutations in Chinese hamster V79 cells at a rate of 280 per 10h viable cells/pg/ml (Nishi et al., 1984). SCEs are induced at a rate of 620 per cell/pg/ml thus giving a ratio of 6-TG mutants to SCEs of 0.45. A similar result is seen with decarbamoyl MMC which gives a ratio of

146

P.R. Turner. W.A. Denn~/Mutatiorz

0.52, suggesting monoadducts of MMC may be the main cause of SCEs. Studies of mutation of specific genes in mammalian cells has yielded much valuable information on the heritable changes induced by MMC. Using Chinese hamster ovary cells carrying the bacterial GPT gene (AS52 cells), MMC at 0.3 kg/ml was found to induce mostly putative point mutations by southern blotting (Tindall and Stankowski. 1987). Of these mutations, 25% were the result of a total deletion of the gene while 5% were the result of partial deletions. Importantly the AS52 cell line is hemizygous for the GPT gene and certain mutagenic mechanisms which require homologous sequences such as mitotic recombination or gene conversion cannot occur in this cell line. Human lymphoblastoid cell lines which are heterozygous at the thymidine kinase (tk) locus represent a useful tool for studying such events (Little et al.. 1987). The two alleles can be distinguished by restriction fragment length polymorphisms in combination with southern blotting. Similar results were found with MMC in this cell line also, about 67% of mutants resulted from putative point mutations, 27% from deletion of the gene and 6% from rearrangements. A range of yeast strains have been developed to examine the effects of various treatments on specific endpoints such as mitotic recombination, gene conversion and aneuploid induction. MMC has been shown to induce such events in virtually all strains examined (summarised in (Zimmerman et al., 1984)). Additionally the effects of drugs on the mitochondrial DNA of yeast can be determined using the petite mutagenesis assay (Ferguson and von Borstel, 1992). Yeast cells with damaged mitochondria form small colonies when grown on media containing a fermentable carbon source as opposed to normal cells. MMC does not induce these petite colonies in yeast and so is not considered to specifically damage mitochondrial DNA. Knowledge of the effects of MMC on the germ cells is of primary importance to patients undergoing chemotherapy, as induced genetic damage may lead to abnormal offspring. MMC can penetrate the blood-testis barrier (Ehling, 1977) and has various effects on developing sperm in mice. The LD,,, (dose resulting in 50% survival) of MMC in adult mice is 5 mg/kg while the LD,, for differentiated spermato-

Resrctrch 355 (1996) l-!-l69

gonia is 0.5 mg/kg. The LD,, for spermatogonial stem cells is 3 mg/kg (Meistrich et al., 1982). Thus differentiated sperrnatogonia are especially sensitive to MMC treatment while the spermatogonial stem cells which are more important with regard to continued sperm production are more resistant. There was also a potential increase in diploid spermatids. Similar results were obtained in another mouse study (Adler, 1974) designed to track the fate of MMC on sperm development. Spermatogonia were sampled

CC-1065

Adozelesin

Carzelesin

Bizelesin Fig. 2. CC- 1065 and analogues

for clinical trial

P.R. Turner, W.A. Denny/Mutation

for chromatid exchanges 24 h post treatment with 5 mg/kg MMC in mice and > 50% of these showed these aberrations. 90% of the aberrations were whole arm exchanges derived from breaks in the centromeric heterochromatin. Spermatocyte analysis revealed a drastic reduction in numbers which only returned to normal levels 50-60 days post treatment. Thus spermatocytes that were analysed 50-60 days post treatment were derived from spermatogonial stem cells and showed no chromosomal aberrations suggesting resistance of stem cells to damage. Another study, however, using rats and a different indicator of DNA damage (micronuclei) showed a significant increase in micronuclei 19-23 days post treatment with 0.8 mg/kg MMC (Channarayappa et al.. 1992) in spermatids. Overall these studies suggest the DNA damage induced by MMC in the male reproductive system may be transient due to the apparent resistance of the spermatogonial stem cells. Longer term effects of MMC on stem cells are necessary however before this can be taken for granted. There have been fewer studies on the effects of MMC on the female germ cells. In contrast to sperm production oocytes in most mammals do not replicate their DNA until they have been fertilised. MMC treatment of female mice has been shown to induce dominant lethality in the embryo at a dose of 5 mg/kg. Analysis of female pronuclei showed chromosome and chromatid aberrations notably premature chromosome condensation in nearly 90% of zygotes examined (Jacquet and Pire, 1984). MMC also showed a positive result in the mammalian cytogenetic oocyte and early embryo assay (Preston et al.. 1981). MMC thus causes a number of effects on cellular DNA, and would be predicted to be mutagenic in humans. However a serious study on second cancers following MMC treatment has not been undertaken.

4. CC-1065

and other

cyclopropylindoles

CC-1065 (Fig. 2) is a natural product isolated from Streptomyces zelensis which possesses extreme cytotoxicity (Hanka et al., 1978). CC-1065 never entered clinical trial, following the discovery that it caused delayed deaths in mice (McGovren et al.,

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1984). However, scientists at the Upjohn company subsequently developed analogues which lacked delayed toxicity but retained antitumor activity (Figure2). One of these, adozelesin, has undergone phase 1 clinical trial (Li et al., 1991; Shamdas et al., 1994) and while no responses were observed, adozelesin was generally well tolerated with the limiting dose dependent on myelosuppression. Two other analogues; carzelesin, a prodrug which is activated in serum (Li et al., 19921, and bizelesin (Walker et al.. 1994), a bifunctional agent. have showed good antitumor potential in human tumour xenografts and are expected to enter clinical trial soon (van-Tellingen et al., 1994; Walker et al., 1994). Much work has been done on the mechanism of action and sequence specific DNA binding of CC1065 and analogues, and it is necessary to examine this before considering their mutagenic effects. CC1065 alkylates DNA at the N3 of adenine residues via the cyclopropa[ c]pyrrolo[3,2-elindol-4-(5 H&one (CPI) subunit (Reynolds et al., 1985). Heat treatment of CC-1065-adducted DNA results in cleavage of the DNA backbone at the site of alkylation, enabling the sequence selectivity to be determined. Two sequences were shown to be preferentially alkylated; 5’-AAAA A-3’ and 5’-PuNTTA-3’ (Hurley and Needham-VanDevanter, 1986). Subsequently, 5’-A/T A/T A-3’ was determined to be the most important sequence for recognition. CC-1065 alkylates the 3’ adenine of these sequences and lies 5’ covering 5 base pairs. It should be noted that these studies were performed on naked DNA in vitro, which may give misleading results when compared to the in vivo situation. However studies using SV40 infected BSC-1 cells have shown viral DNA is alkylated at the same sites in whole cell systems as in vitro (McHugh et al., 1994). Additionally, similar adozelesin binding sites were detected in human repetitive alphoid DNA from drug treated prostatic carcinoma cell lines by a tuq polymerase stop assay (Bubley et al., 1994). In the same experiment, however, cisplatin and cyclophosphamide binding sites were mapped in cultured cells and compared to mononuclear cells from a patient undergoing chemotherapy with the same drugs, and found to differ in intensity at certain sites. Thus it cannot be taken for granted that drug binding sites even in cultured cells mirror the in vivo situation.

The planar shape of the CC- 106.5 molecule allows it to form close van der Waals contacts with the sides of the minor groove, which is narrower in AT-rich regions. On the basis of analogue studies it was postulated that the sequence selectivity of CC1065 was determined by the CPI subunit and modulated by the attached pyrroloindole subunits (Hurley et al., 1988). This view has recently been challenged (Boger and Johnson, 1995; Boger et al.. 19911 by studies of the sequence selectivity of alkylation of further analogues of CC-1065 and the related duocarmycins. These authors concluded that the sequence specificity of non-covalent interaction determined the specificity of alkylation. Irrespective of the precise mechanism involved in formation of the CC-1065 adduct, much is now known about the effects on DNA metabolism. CC- 1065 and analogues stabilise the structure of B-DNA as determined by an increase in melting temperature and the induced molar ellipticity of the adduct (Krueger and Prairie, 1992). Indeed the delayed death in mice has been attributed to the extreme long term stability of the CC-1065 adduct and its high bonding affinity. CC1065 also causes bending of the DNA helix 17-19” towards the minor groove (Lin et al., 19911 and a stiffening and winding of the helix 5’ to the adduct (Lee et al., 1991a; Sun and Hurley, 1992bl. Early studies on the mutagenesis of CC- 1065 and selected analogues (Harbach et al., 1988; Fig. 3) in V79 hamster cells revealed that at equitoxic doses the compounds fell into 3 classes. Compounds inducing 35-100 6-thioguanine resistant mutants/lO(’ survivors included CC-1065, U68415. U67786, U63360 and U66226. Compounds inducing 550 mutants/ IO’ survivors were U66694 and U66866. Finally the most mutagenic compound in the series was U62736, a CPI unit attached to a methyl sulphoxide (Fig. 3). In general, it seems that increasing the number of pyrroloindole subunits results in decreasing mutagenicity at equitoxic doses in V79 cells. Interestingly, the high rate of mutation with U62736 implicates the alkylation event as being mutagenic while the low rates of mutation with the non-alkylating U63360 and U66226 suggest that minor groove bind-

Fig. 3. CC-1065 analogues bath et al.. 1988.

used in the mutagenesis

i)H

U-63360 NHz

HO

U-66226

6

U-6841 5

U-67786, R=OCH3 ; U-66694, R=H

studies Har-

U-62736

P.R. Turner,

W.A. Denny/Muiatim

ing per se is not a particularly mutagenic event by this class of ligand. In the Ames test using strain TAI 00, CC- 1065, U68415, U66226 and U63360 were weakly mutagenic (1.6 times background) while the remaining compounds were significantly mutagenic (2.5-3 times background) (Harbach et al., 1988). Additionally, histidine revertants from TAlOO showed a strong dose-dependent response to CC- 1065, and induced mutants 19 times above background levels at 0.1 ng/ml (Harbach et al., 1986). Strain TA98 showed no mutagenic response to CC-1065. These early results suggested that the more DNA affinic compounds (with more subunits) had a lower mutagenicity and a higher toxicity. At 0.2 ng/ml (LD,,) CC- 1065 induces sister chromatid exchanges 4 times higher than background and chromatid and chromosome breaks. No single-strand DNA breaks or protein-DNA crosslinks were detected by alkali elution even at doses 50-500 times the LD,,. Micronuclei were induced in polychromatic erythrocytes from rat bone marrow at a frequency 20 times background with a CC-1065 dose of 400 kg/kg after 30 h (Harbach et al., 1986). Several lines of evidence now point to CC-1065like adducts being relatively persistent lesions which are very difficult for cells to deal with. The cell cycle effects of adozelesin in CHO, mouse and human ovarian carcinoma cell lines have been examined (Bhuyan et al., 1992). At similar toxicities (LD,,) the number of adozelesin molecules per pg of DNA varied, with ten times the number of adducts required to kill 90% of V79 cells compared to human cells. Mitotic cells had less [‘Hladozelesin uptake than G, or S phase cells and a transient slowing of progression through S phase was noted. All cells underwent a G? arrest but their response to this differed. Human and mouse cells remained arrested in G, and 48 h later a high background of fragmented DNA was observed by flow cytometry, suggesting perhaps that the cells had apoptosed. The CHO and V79 cells escaped G2 arrest either by dividing or becoming tetraploid. The difference in response to G1 arrest was suggested to depend on the degree of transformation of the cell line or on species related differences. It has been suggested (Tang et al., 1988) that the cytotoxicity of CC-1065 is related to a lack of

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efficient processing by the excision repair proteins. In bacteria NER is mediated by the excision repair proteins uvrA, B and C which make incisions 3’ and 5’ to DNA adducts. Specifically these researchers found uvr(A)BC mediated incision 5 to adducts with a corresponding lack of incision 3’ to the same adducts. Subsequent analysis however showed this not to be the case (Selby and Sancar, 1988) with (A)BC exinuclease incising the 8th phosphodiester bond 5’ and the 4th or 5th bond 3’ to the adduct except perhaps in one specific instance. In addition helicase II and DNA polymerase enhanced the incision activity. Taken together these results suggest that CC- 1065 induces sufficient structural distortion of the DNA to be recognised by the early components of the excision repair pathway (Sancar and Sancar, 1988). DNA repair is complicated by the fact that different regions of the genome are repaired with different efficiencies (Bohr et al., 1987) especially in the transcribed strand of active genes (Terleth et al., 1991; Selby and Sancar, 1993; Schaeffer et al., 1993; Sweder and Hanawalt, 1993). The persistence of CC-1065 adducts has been examined in CHO cells (Tang et al.. 1994) and were found to vary depending on genomic location. The amplified dihydrofolate reductase (DHFR) genes in these cells bind twice as much CC-1065 as the adenine phosphoribosyl transferase (APRT) gene contrasting to the in vitro situation where adduct concentration is virtually identical between these two regions. Aside from adduct concentrations, the repair of the APRT damage is 90% complete after 24 h. The damage to the DHFR coding and 3’ non-coding region is 65% and 40% repaired. respectively. after 24 h. In contrast, total genomic DNA has up to 85% bound CC- 1065 remaining. These results strongly suggest the involvement of transcription in the repair of CC-1065 adducts, a notion further supported by the observation that the transcribed strand of the APRT gene is repaired preferentially to the non-transcribed strand. The recent crystal structures (Chasman et al., 1993; Kim et al., 1993) of the TATA binding protein (TBP) with the TATA box have revealed intimate contacts of the protein with the minor groove of the DNA. CC-1065 has been shown (Chiang et al., 1994) to bind in this same region and can inhibit, or indeed displace, TBP. Adozelesin in contrast was

three orders of magnitude less effective. TBP (TFIID) is one of the first factors that binds to the promoter region of genes to activate transcription (Conaway and Conaway, 1993). In E. coli at least, an RNA polymerase stalled at a lesion recruits the uvr(A)BC exinuclease complex via a transcription repair coupling factor protein (Selby and Sancar. 1993). It is interesting in the context of these results that the transcription complex can form at all on the TATA box to permit transcription-coupled repair. It is likely that there is a second type of targeted repair to clear the promoter region of bound drug and permit complex formation. An example of this sort of phenomenon has been demonstrated previously in the 5’ region of the DHFR gene following UV induced damage (Bohr et al., 1986). Studies by Sun and Hurley have shown that CC1065 and adozelesin adducts inhibit the unwinding activity of helicase II (uvrD). Helicase II is thought to bind the incised strand complexed with uvrB and C and to displace the adducted strand along with uvrC. UvrB remains bound to the single tranded gap and is displaced by the action of DNA polymerase I (Sancar and Tang. 1993). As mentioned previously however, helicase II and DNA polymerase I were found to enhance the incision of CC-1065 adducts by uvr(AfBC (Selby and Sancar, 1988). Uvr D- mutants repair thymine dimers induced by UV with an efficiency of about 50% compared to wild-type cells. CC-1065 adducts in uvr D- cells are repaired normally (Tang et al.. 1988) suggesting that helicase II either is not essential for excision repair of CC-1065 adducts or is not inhibited in vivo. Quantitative measures of helicase II inhibition in vivo by CC- 1065 at concentrations that saturate uvriA)BC are necessary before definite conclusions can be made concerning its role in CC-1065 cytotoxicity. Nucleotide excision repair (NER) in humans and rodents is a more complex process than in prokaryotes and involves at least 8 and 11 (respectively) genes (Sancar and Tang, 1993). Poly(ADP-ribose) polymerase (Murcia and Murcia, 1994) binds to nicks in DNA and attaches poly(ADP-ribose) to selected nuclear proteins to modify their activity. Poly(ADP-ribose) is synthesised from nicotinamide adenine dinucleotide (NAD) pools within the cell. NAD levels have been shown to fall in cells treated with alkylating agents and poly(ADP-ribose) poly-

merase has been shown to bind to melphalan treated DNA (Bramson et al.. 1993). CC- 1065 also causes a depletion in NAD pools which is prolonged compared to other alkylating agents. The same response with CC-1065 is seen in repair defective xeroderma pigmentosum group C and D cells, suggesting perhaps another (defective) repair system is producing single-strand nicks in response to CC-1065 adducts (Jacobson and Twehous, 1986). Further investigation is needed with both rodent and human repair mutants to determine the role of NER in CC-1065 adduct repair. The mutational spectrum of a chemical agent is useful information to infer potential mechanisms of adduct processing. The ‘Big Blue’ mouse from Stratagene is a valuable tool in this respect. ‘Big Blue’ contains the E. coli. lac I gene which can be retrieved from the mouse following mutagen treatment and screened for mutations quickly and efficiently. Monroe and Mitchell (1993) used the ‘Big Blue’ system to investigate in vivo mutagenesis of CC- 1065 and adozelesin at doses of 50 and 36 kg/kg. respectively. Three days after treatment liver DNA was extracted and analysed for mutation. CC1065 and adozelesin had a mutant frequency three times that of the control while ethylnitrosourea had a mutant frequency four times the control value. Mutations attributable to CC- 1065 and adozelesin were classified as targeted (at covalently bound adenine) and locally targeted (within 4 base pairs of covalently bound adenine). Of the five targeted CC- 1065 mutations three were AT + TA transversions, one was an AT deletion and one was a complex rearrangement consisting of a 2 12 base pair deletion next to a 51 base pair inversion. Adozelesin targeted mutations consisted of one AT + TA transversion and one AT + GC transition. Locally targeted events were tentatively assigned to polymerase errors based on in vitro polymerase stop sites and misincorporations (Sun and Hurley. 1992a). Specifically, 18 mutations occurred one base 3’ to the alkylation site analogous to the polymerase termination site in vitro and 8 of these involved insertion of a base opposite cytosine in the sequence 5’-ACG-3’. Locally targeted mutations consisted of CG + TA transitions, TA + CG transitions. 1 base pair deletions and 1 and 4 base pair insertions (possibly due to polymerase slippage).

P.R. Turner. W.A. Dewy/Mutation

These results imply that unrepaired adducts cause polymerase errors 5’. 3’ and at the site of adenine alkylation. These results are interesting in that virtually all alkylating agents examined tend to induce mutations at the alkylated base rather than one or several nucleotides away. This seems to imply that mutagenesis can be the result not only of direct covalent bonding of a DNA base but also by non-covalent structural distortions resulting from minor groove occupancy. The implications of this are uncertain but suggest the mutagenesis of CC- 1065 and its analogues will be more complicated than simple alkylating agents.

Research 355 (1996) 141-169

151

NH2 Anthramycin

OH

5. Anthramycin and other pyrrolobenzodiazepines Sibiromycin The pyrrolo[ 1,4]benzodiazepines (Fig. 4) constitute a variety of structurally related compounds isolated from bacterial cultures. The most widely investigated compounds are anthramycin from Streptomyces refuineus var. thermotolerans (Tendler and Korman, 1963; Leimgruber et al., 1965) tomaymycin from Streptomyes tomaymyceticus (Arima et al., 1972) sibiromycin from Streptosporangium sibiricum (Gause et al., 1969) and neothramycin A and B from Streptomyes No. MC916C4 (Takeuchi et al., 1976). Neothramycin has seen limited use in the clinic notably in the treatment of bladder cancer (Tsugaya et al., 1986). Early studies (Hurley and Petrusek, 1979; Hurley et al.. 1977; Petrusek et al., 1981) suggested the 2-amino group of guanine was alkylated by anthramycin and this was later confirmed by NMR and X-ray crystallography experiments (Graves et al., 1985; Graves et al., 1984; Kopka et al.. 1994). The Cl 1 atom of anthramycin forms a covalent bond with the exocyclic ‘-amino group of guanine in double-stranded DNA. Anthramycin binds double-stranded DNA and increases the melting temperature in a relatively slow reaction taking about 1 h (Glaubiger et al., 1974). The sequence selectivity of anthramycin and tomaymycin has recently been investigated using uvriA)BC incision analysis combined with exonuclease III and X exonuclease digestion (Pierce et al., 1993). Anthramycin binds 5’-AGA-3’ and AGG pref-

Tomaymycin

HO CH30

Neothramycin A (R q=H, Rz=OH) Neothramycin B (R q=OH, R2=H) Fig. 4. Pymlo[

I .4]benzodiazepines

erentially to GGA and GGG while tomaymycin binds AGA and AGG preferentially to TGC and AGC. Thus there are subtle differences in sequence selectivity between the two structurally similar compounds. Similarly the degree of helical distortion is greater for anthramycin (bent 5.6-8.9”) than for

152

P.R. Turner, W.A. Derzn~/Mutation

tomaymycin (bent 8.2-14.5”) (Kizu et al.. 1993). Anthramycin also causes stiffening but not lengthening of double-stranded DNA (Glaubiger et al.. 1974). Clinical use of anthramycin and sibiromycin was prevented by cardiotoxicity and tissue necrosis at the site of injection (Cargill et al., 1974; Muraveiskaia. 1971). Cardiotoxicity has been related to the potential to undergo tautomerisation or oxidation to produce ortho-quinone imines (Hurley and Thurston. 1984). This process cannot occur with tomaymycin or the related neothramycins which lack the 7 or 9-phenolic hydroxyl groups and these compounds are not cardiotoxic. A DNA sequence specific interstrand crosslinker based on tomaymycin (DSB- 120) has been developed, and recognises the sequence 5’-PuGATCPy-3’ or S-PyGATCPu-3’ (Bose et al., 1992). DSB-I20 is .50-fold more effective than a major groove binder, at mechlorethamine, crosslinking DNA in vitro. An examination of the occurrence of the DSB-120 binding sites in 19 randomly selected oncogenes in the DNAstar database revealed some oncogenes possessed four times the expected (statistically random) number of occurrences while others possessed no binding sites (Neidle et al., 1994). The implications of these results are important in that minor groove binding drugs with significant sequence selectivity may be selective for neoplastic cells that are dependent on the expression of certain oncogenes while being non-selective for neoplastic cells expressing other oncogenes. The mechanism of antitumor activity of the pyrrolo[ 1.4lbenzodiazepines is considered to be the inhibition of DNA synthesis (Hurley et al.. 1977). The removal of [ 15-‘H] labelled anthramycin from human skin fibroblast cells is 86% complete after 72 hours while in excision deficient xeroderma pigmentosum (XP) cells over a similar time period only 49% of repair is completed (Hurley et al.. 1979). Anthramycin treatment in repair proficient cells is accompanied by persistent single-strand and doublestrand DNA breaks while repair deficient XP cells are more sensitive to anthramycin and don’t display single-strand and double-strand DNA breaks (Petrusek et al.. 1982). The bacterial nucleotide excision repair uvr(A)BC proteins recognise and excise anthramycin adducts on DNA apparently normally (Walter et al.. 1988) although certain sequences may prevent incision on

Research 355 (1996l 111-169

either side of the adduct similar to the situation with CC- 1065. Additionally it has been shown that uvrA in combination with uvrB but not uvrC can release intact anthramycin with no DNA strand scission in a process 6-7 times slower than excision with uvrC (Nazimiec et al.. 1992). The significance of this process to the overall repair of anthramycin adducts is unclear, however. Anthramycin is non-mutagenic in Salmonella Qpllimurium strains TA1535, TAIOO, TA1538 and TA98. S9 mix reduces the toxicity of anthramycin but does not alter the mutagenicity (Hannan et al.. 1978). In Sncdmromyes cerei,isine strain C 16- 1 1C anthramycin produced no forward mutations to adenine dependence even at concentrations resulting in 35% survival. Mutagenesis in another yeast strain XV 185- 13C. which carries three mutations (lys I - I. arg4- I7 and hisl-71, and thus requires lysine. arginine and histidine to grow normally has also been investigated. Mutations conferring lysine and arginine independence were shown to occur at a lower level than controls on anthramycin treatment by an unresolved mechanism. Mutations leading to histidine independence occurred at a frequency of 15.7 mutants/IO’ survivors above controls at a dose resulting in IO’% survival. The diploid yeast strain D7 showed an increase of mitotic crossing over of 1.92 crossovers/IO3 survivors at a dose resulting in 55% survival. Gene conversion in strain D7 similarly was increased to 543 convertants/ 10’ survivors at a dose resulting in 39% survival (Hannan et al.. 1978). An analysis of repair-deficient yeast strains showed hypersensitivity to anthramycin in radl, rad2. rad52. rad54. rad55, and to a lesser extent rad9 mutants (Hannan and Hurley, 1978). Rad53 and rad56 mutants did not display hypersensitivity to anthramycin. Tomaymycin had similar effects to anthramycin while sibiromycin had less of an effect in radl and rad2 mutants than anthramycin. Radl and 2 mutants are deficient in the removal of UV induced thymine dimers while rad52 and rad9 mutants are sensitive to ionising radiation and are deficient in recombination. Rad mutants 53-56 are ionising radiation sensitive mutants. Thus repair of anthramycin. tomaymycin and sibiromycin DNA adducts involves both excision repair and recombinational repair processes in yeast.

P.R. Turner. W.A. Denny/Mutation

Anthramycin induces sister chromatid exchanges in CHO cells 6 times above control values at 64 nM (Duncan and Evans, 1982). The major groove alkylatars methylmethanesulphonate, ethylmethanesulphonate and ethylnitrosourea were less effective than anthramycin on a molar basis at inducing SCEs. Similarly anthramycin induced SCEs about four times higher than control values in Indian Muntjac fibroblasts and the effect was potentiated by post-treatment with caffeine (Ved-Brat et al., 1979). Additionally 99% of the cells treated with anthramycin and caffeine were blocked at the first division. These synergistic effects of caffeine are thought to be due to the inhibition of post replication repair of DNA. Chromosomal abnormalities induced by anthramycin were also observed and included gaps, acentric fragments, dicentrics, triradials, quadriradials and multiradials. Sibiromycin has been shown to induce micronuclei in mouse polychromatic erythrocytes from bone marrow while anthramycin and tomaymycin were inactive (Gairola et al., 1983). Although petite mutagenesis data on pyrrolo[ 1,4]benzodiazepines is elusive, anthramycin and sibiromycin have been shown to inhibit H-strand synthesis in mitochondrial DNA (Gause and Dolgilevich. 1975). Taken together these results suggest anthramycin is recognised and repaired by excision repair as well as recombinogenic repair systems, and that both of these types of repair can lead to mutations. Gene conversion is a mechanism that may potentially lead to loss of heterozygosity in the progression of tumours (Fearon and Vogelstein. 1990). More mutagenesis data is needed before the extent and significance of these processes are known. especially with new agents like DSB-120.

6. Bisbenzimidazoles

and analogues

The bisbenzimidazoles Hoechst 33258 and Hoechst 33342 (Fig. 51, first synthesised in 1974 (Loewe and Urbanietz, 1974), have received much attention in the literature due to their use as DNA specific fluorochromes. Hoechst 33258 binds to AT rich DNA and covers four base pairs (Portugal and Waring, 1988). Binding of Hoechst 33258 to DNA elevates the melting temperature, especially with DNA of high AT content (Comings, 1975). The

Reseurch 355 (1996) 141-169

Hoechst 33258

Hoechst 33342 Fig. 5. Bisbenzimidazoles.

piperazine ring of Hoechst 33258 allows the acceptance of a GC base pair (Murray and Martin, 1994; Pjura et al., 1987; Spink et al., 1994). A recent study of bisbenzimidazoles differing in the number of cationic groups and benzimidazole subunits suggested that, although electrostatic interactions and hydrogen bonding provided some binding energy, the single most important factor for DNA binding was van der Waals interactions with the minor groove wall of B DNA (Czamy et al.. 1995). Hoechst 33258 (pibenzimol) has been tested in phase II trials against carcinoma of the exocrine pancreas but was found to be ineffective (Kraut et al., 199 1). It has selective toxicity against human melanoma MM9E cells in vitro. and also induces them to differentiate (Wong et al.. 19941. These effects have been attributed to selective inhibition of binding and also to displacement of previously bound transcription factors found in MM9E cells. Thus while Hoechst 33258 has a binding site only four base pairs long, its sequence selectivity has profound implications on the survival of certain cell types. It has been investigated as a dye for use in flow cytometric sperm sorting (Morrel and Dresser. 1989), but there was concern about its effect on sperm chromosomes and sorted sperm were used to inseminate various species to determine effects on offspring. However, no increase in any obvious congenital deformities were detected in pig, sheep, cattle or rabbits. Additionally several generations of interbred

rabbits from sorted sperm showed no adverse effects attributable to Hoechst 33342. Hoechst 33342 efficiently inhibits DNA synthesis in Chinese hamster V79 cells but DNA damage is only evident at supralethal concentrations as assayed by alkali elution (Durand and Olive. 1982). At a concentration of 50 p.M. it was minimally toxic but produced a G,/M phase arrest in the cell cycle. At SO FM it was also moderately mutagenic and induced about 30 6-thioguanine resistant mutants per 100000 cells. Studies with Hoechst 33258 in mouse LM cells and Chinese hamster ovary cells show elevated levels of endoreduplication and subsequent polyploidy (Kusyk and Hsu. 1979). Exposure to 20 Fg/ml for 26 h followed by incubation in drug free medium for 24 h resulted in 38% of metaphases displaying endoreduplication and 22% of metdphases displaying 4N-8N polyploidy in mouse cells. CHO cells showed similar effects albeit at shorter incubation times due to their faster cell cycle. Polyploidy is often observed in advanced tumours and is associated with a poor prognosis although measurements of true polyploidy are rarely undertaken (Mitchell et al.. 1995). Polyploidy as usually measured may in fact represent heteroploidy (not strict multiples of haploid number). Aneuploidy is generally considered to be more relevant to carcinogenesis and can lead to loss of heterozygosity resulting in altered gene expression (Mitchell et al.. 1995). Hoechst 33258 also induces aneuploidy in mouse L cells at 35 kg/ml with 24 h exposure and induces decondensation in the heterochromatic regions (Vig and Sweamgin. 1986). Micronuclei formed from this treatment occurred at a frequency of 7% and possessed chromosome fragments with or without kinetochores and also entire chromosomes. Chromosomes lying outside the mitotic spindle (laggards) were apparent in 72 out of 150 cells examined. The kinetochores appeared normal in these laggards but it was possible they lacked some essential component not identified by the antibody used. The repetitive DNA sequence (GGAATIn has been localised to human centromeric regions and also to the heterochromatic regions of chromosomes 1. 9. I6 and Y (Grady et al., 1992). Zoo-blot analysis has shown this sequence to be highly conserved and was found in all species examined including vertebrates. insects and plants and is estimated to be more than one

billion years old. Using a gel mobility shift assay GGAAT sequences were shown to bind with high specificity to a nuclear protein from HeLa cells (Grady et al., 1993). Subsequent studies using multidimensional NMR have shown GGAAT sequences to form stable stem-loop structures which utilise A G and G . G base pairing (Catasti et al.. 1994). It is possible that these stem-loop structures are formed by chromosome condensation and that hoechst 33258 may inhibit this process by stabilising the B-DNA structure. In this respect it is perhaps surprising that the laggards detected by Vig and Sweamgin showed no kinetochore abnormalites. Many of the observed effects on chromosomes may be related to anti topoisomerase activity. Hoechst 33258 and 33342 have recently been found to cause single-stranded calf thymus topoisomerase I mediated breaks in DNA (Chen et al.. 1993b3). Hoechst 33342 was found to be more effective than 33358 in vitro and is also more permeable to cell membranes. The two compounds were found to be at least as effective as camptothecin in inducing strand cleavage and were more sequence specific. Two sequences were identified as topoisomerase I/Hoechst cleavage sites. S-ATTTAAAACTT-CATTTTTAATTTAAAA-3’ and S-TTTTCAA-TA-TTTTTTTTATTC-3’. No effect with bacterial topoisomerase I or mammalian topoisomerase I1 was observed. Other researchers however have found Hoechst 33258. 33342 and analogues inhibit cleavable complex formation by known anti-topoisomerase drugs but do not promote cleavable complex formation in their own right. Beerman et al. ( 1992) examined a series of bisbenzimidazoles to determine structure-activity relationships for topoisomerase inhibition. Drug uptake was considered to play an important role as agents which inhibited topoisomerase in vitro were often inactive in whole cells. The seemingly contradictory results obtained by different researchers may retlect the concentrations of drugs used. For example. concentrations of Hoechst 33358 and 33342 exceeding 5 p.M tended to inhibit topoisomerase I mediated cleavage (Chen et al., 1993b), and much higher concentrations were used (Beerman et al.. 1992) to inhibit topoisomerase mediated cleavage of DNA by known anti-topoisomerase compounds. Hoechst 33342 has also been studied in human

P.R. Turner. W.A. Denny/Mut~~tion

cell lines (Chen et al., 1993a). Multi-drug resistant KB-VI cells were 222 times more resistant to Hoechst 33342 than their sensitive counterparts. However camptothecin-resistant CPT-KS cells were only 5 times more resistant to Hoechst 33342 compared with 400 times for camptothecin. CPT-KS cells have 3.8-fold less topoisomerase I enzyme as their sensitive counterparts, and also possess a mutation in the topoisomerase I gene. The authors proposed that the lack of significant resistance to Hoechst 33342 in these cells suggested the drug reacted equally well with mutant or normal enzyme and the lesser amount of topoisomerase I accounted for the 5fold difference in toxicity. Thus Hoechst 33342 and other bisbenzimidazole analogues may be useful in treating camptothecin-resistant tumours in the clinic. Various studies with bisbenzimidazole derivatives have shown that binding affinities to calf thymus DNA correlate positively with in vitro topoisomerase inhibitory potency or cytotoxicity (Beerman et al., 1992; Wang et al., 1994). Sequence selectivity was also considered to be relevant to inhibitory activity (Beerman et al., 1992). In addition to structure-activity studies altering the non-covalent DNA binding ability of bisbenzimidazoles (Bathini et al.. 1990; Bathini and Lown. 1990: Lee et al.. 199lb) these ligands have also been used as carriers to deliver alkylating moieties to DNA (Gravatt et al., 1994; Gupta et al., 1995). Analogues bearing either a monoor bifunctional nitrogen mustard linked to the phenol terminus by a short chain showed high in vitro cytotoxicity and good in vivo activity, while those linked via longer chains were inactive. Both the alkylating analogues and the non-alkylator Hoechst 33258 showed a weak mutagenic effect in Salmonella strain TA102, suggesting that minor groove binding of the chromophore was responsible rather than an alkylation event (Ferguson and Denny. 1995). Significantly none of the compounds showed any effect in strain TAl537 (unlike the parent mustard). suggesting that the pattern of DNA alkylation had been altered. The analogues which showed in vivo antitumour activity did not demonstrate mitotic recombination or mitochondrial mutagenicity effects in yeast, but the longer-chain compounds did show some promotion of mitotic crossing over and mitochondrial mutagenicity.

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155

A murine cell line (HoeR415) resistant to Hoechst 33258 and 33342, and cross-resistant to distamycin has been isolated (Debenham and Webb, 1986). In these cells the drugs are actively dissociated from the DNA, a process which is blocked by the topoisomerase II inhibitors novobiocin and etoposide (Smith et al., 1990). Preliminary results suggest no alteration in topoisomerase II expression or sensitivity to other anti-topoisomerase II drugs, and the mechanism of resistance remains unclear. Analysis of the topoisomerase I gene in HoeR415 cells would be interesting in this respect. Hoechst 33258 and other non-covalent binders such as the intercalators ethidium bromide and actinomycin D have been shown to inhibit the uvr(A)BC excinuclease by two different mechanisms. With an excess of undamaged DNA, Hoechst 33258 causes a non-specific uvrA binding (uvrA trapping). an effect that is reversed upon addition of more uvrA. It also inhibits the formation of pre-incision uvrAB complexes on UV damaged DNA, by forming non-specific uvrA complexes in close proximity to the damaged site (Selby and Sancar, 1991). These observations may be important, because any compound that inhibits cellular DNA repair systems may also alter mutagenesis. It is interesting to compare the biological effects of bisbenzimidazoles with known topoisomerase poisons. The topoisomerase I inhibitor camptothecin and various topoisomerase II inhibitors, including amsacrine and teniposide, caused Friend murine erythroleukemic cells to arrest in G, phase and inhibit chromosome condensation. There was also an induction of polyploid 4N-8N cells (Zucker et al., 199 I I. Topoisomerase II inhibitors also cause a slowing of progression through S phase and an increased frequency of sister chromatid exchanges. A topoisomerase II temperature-sensitive yeast strain fails to complete the first meiotic division (reviewed in Ferguson and Baguley, 1994). Additionally, the topoisomerase IT poisons teniposide and etoposide induce weak mutagenic responses in S. typhimurium strain TA102 at high drug concentrations as does Hoechst 33258. However, the mechanisms of revertant induction are unclear. These parallel observations with bisbenzimidazoles and known topoisomerase I and II inhibitors are interesting. but need to be interpreted cautiously until more direct evidence with cellular systems is available. The bisbenzimidazoles may

compare better with the topoisomerase I inhibitor camptothecin, as they have both been demonstrated to form cleavable complexes. No topoisomerase II minor groove binding ligand cleavable complexes have been identified to date, and these ligands would seem to act on topoisomerase II in a different manner to current anti topoisomerase drugs. It has been suggested that topoisomerase I ternary complexes with camptothecin and topoisomerase II ternary complexes are converted to the same type of lesion in vivo after collision with replication or transcription machinery (Anderson and Berger, 1994). The anti topoisomerase II epipodophyllotoxins have been linked to an increased risk of a distinct form of acute myelogenous leukemia in cancer patients. The latent period for this malignancy is 2-3 years as opposed to 4-5 years for alkylating agents. and frequently involves balanced translocations at 1 lq23 and 21q22 (Anderson and Berger. 1994). Bisbenzimidazoles and other topoisomerase active minor groove binders such as distamycin may also have the potential to induce serious long term consequences, and thus their development as antitumour agents should proceed with careful analysis of these considerations.

7. Polypyrrolecarboxamides (Iexitropsins)

and

analogues

Distamycin A and netropsin (Fig. 6) are natural polypyrrole antibiotics isolated from Streptomyces species (Arcamone et al., 1964; Finlay et al., 1951). Both compounds bind in the minor groove of B DNA, with binding sites of 4 (netropsin) and 5 (distamycin A) base pairs. Crystal structures of distamycin A (Co11 et al., 1987) and netropsin (Tabemero et al., 1993) show the compounds bound deep in the minor groove at AT-rich sequences. The specificity for such sequences depends on a variety of factors, notably the molecular electrostatic potential and steric and van der Waals interactions. Hydrogen bonding between the drug and DNA donor and acceptor atoms also provides stability of binding (Pullman. 1988). These observations of sequence selective binding spawned the idea of lexitropsins. molecules based on

Distamycin A Cl

Tallimustine

Netropsin Fig. 6. Polypyrrolecarhoxamides.

netropsin and distamycin A but designed to target defined DNA sequences. There is now a selection of lexitropsins available. differing in their groove binding backbone and their DNA sequence selectivities (Dervan. 1986; Krowicki et al., 1988b; Lown et al.. 1986; Rao et al.. 1990). The overall goal of this research (proposed by Dervan. 1986). the design of ligands capable of binding preferentially to any predetermined sequence on DNA. has not yet been realised. One of the most difficult problems has been to achieve selective binding to GC-rich regions of DNA, since such base pairs are usually tolerated in A-rich sequences rather than being selected for. However. recently ligands which bind relatively specifically to four consecutive G/C base pairs have been synthesised (Lee et al.. 1994). The general polypyrrolecarboxamide (lexitropsin) framework has been widely used as a carrier for alkylating agents. and a large number of such analogues have been synthesised (Gold et al., 199 I; Hartley et al.. 1994: Arcamone et al.. 1989; Krow-

P.R. Turner. W.A. Denny/Mutation Research 355 (1996) 141-169

icki et al., 1988a; Zhang et al., 19931. One of these (tallimustine. FCE 245 171, a bifunctional benzoyl mustard linked to a distamycin-derived carrier (Arcamone et al., 19891, showed good in vitro and in vivo antitumour activity in mice with human tumour xenografts, particularly against solid tumours (Pezzoni et al.. 1991). The sequence selectivity is very stringent, with the sequence 5’-TTTTGA-3’ being alkylated most strongly and S-TTTTAA-3’ alkylated less often (Broggini et al.. 1995). Tallimustine displays a similar range of transcription factor inhibition to distamycin. To explain the approximately 500-fold higher toxicity of tallimustine over distamycin, it has been postulated that the former may inhibit an unidentified transcription factor recognising the sequence TTTTGA (Broggini and D’Incalci, 1994). Tallimustine causes cells to arrest in Gz but its mechanism of action has yet to be determined. Tallimustine has undergone Phase 1 clinical trial (Sessa et al., 1994), and Phase 2 trials are in progress. Distamycin is not a particularly cytotoxic compound, with an IC,, of 488 p,M for murine L12 10 cells after a 72 h exposure (Pezzoni et al., 1991). Both distamycin and netropsin have been investigated as antiviral compounds (Cassaza et al., 1965; Chandra et al., 1976). Distamycin was subsequently found to inhibit viral DNA synthesis preferentially in H. simplex virus type-l infected KB cells, as measured by [3H]thymidine incorporation into DNA (Drach and Shipman Jr., 1977). Both netropsin and distamycin have been proposed to have a multitude of effects in the cell nucleus. Like CC-l 065. both inhibit the formation of the TBP/TATA box complex, albeit at a concentration about 200 times greater (50% inhibitory values of 160 and 240 nM for distamycin and netropsin, respectively. as assayed by mobility shift (Chiang et al., 1994)). In similar in vitro gel shift assays, distamycin inhibited binding of the transcription factor OTF-1 to its DNA recognition sequence on the y-globin gene, and the erythroid specific NFE-1 transcription factor on the human histone H2B promoter (Broggini and D’Incalci, 1994). Distamycin inhibits the binding of the anfennapedia homeodomain (Antp HD) to the sequence 5’ATTA-3’ and displaces preformed complexes of protein from DNA (Dom et al.. 1992). The Antp HD interacts mainly with the major groove but has the

157

N-terminal arm reaching into the minor groove. A similar homeodomain, the fushi tavu~u (ftz HD), has a mutant version with 6 amino acids missing from the N terminus (ftz(NTD)HD) (Percival-Smith et al., 1990). Ftz(NTD)HD is an order of magnitude less efficient in binding its cognate cis element but is inhibited and displaced by distamycin. This suggests that direct steric interference by distamycin in the minor groove with protein residues in the same groove is not obligatory for inhibition and displacement of binding. Distamycin also inhibits the cleavage of DNA by a variety of restriction endonucleases, with the effect being most notable with enzymes recognising distamycin binding sites. Flanking sequences also influence the ability of the enzyme to cleave DNA when inhibited by distamycin, suggesting a diffuse conformational change in DNA structure (Dom et al., 1992). These diffused conformational changes or allosteric effects on DNA structure were also thought to be involved in the specific activation of transcription (Bruzik et al., 1987). These authors used promoters based on P,, of phage A which possessed different DNA sequences in the spacer region between the -10 and -35 base pair sites. RNA polymerase contacts the -10 and -35 sites to form an open complex as a prelude to transcription. The 17 base pairs in between these sites is not contacted by RNA polymerase but variations in its sequence affect open complex formation. When a run of nine adenines were included in the 17 base pair spacer, distamycin and to a lesser extent netropsin enhanced formation of the open complex. Other spacer substitutions, including (AT),A, had little effect on complex formation. Distamycin also showed no inhibitory effect on transcript elongation by RNA polymerase in vivo on the lac L8-UV5 promoter in contrast to the intercalators actinomycin D and bis(daunomycin) (Phillips and Crothers, 1986). Distamycin is non-mutagenic in the Ames test with strains TA97, TA98, TAIOO, TA102, TA1535, TA 1537 and TA 1538. However distamycin increases the mutagenicity of ethidium bromide in strain TA98, an effect not seen with mitomycin C or adriamycin (Mazza et al., 1983). Investigation of the mechanism involved in the promotion of mutagenicity as well as the types of ligands involved would seem warranted in view of the common practice of using chemother-

apeutic agents in combination therapy. Distamycin does not induce point mutations, gene conversion or mitotic crossing over in various yeast strains and is not specifically toxic to ret A- strains of Bacillus mbtilis, suggesting recombinational repair mechanisms are not an important response to distamycin (Mazza et al., 1983). Nuclear effects of netropsin and distamycin are evident in cultured cells. Both netropsin and distamycin induce a G, arrest and polyploidisation in human diploid fibroblasts by an unknown mechanism possibly related to chromatin under-condensation (Poet et al., 1990). In V79-E cells, 10-j M netropsin does not produce a cell cycle delay but does induce chromosome elongation and enhances the frequency of sister chromatid exchanges from 0.352 to 0.459 per chromosome in control and drug treated cells respectively (Thust and Ronne, 1982). V79-E cells also display a pattern of light and dark bands on distamycin treatment, and human chromosomes from distamycin treated lymphoid cells show a general elongation but no distinctive banding patten (Ronne et al., 1982; Thust and Ronne, 1981). In a separate study with cultured human leukocytes, distamycin at 100 pg/ml caused despiralised areas in chromosomes I. 3. one of the C group chromosomes (probably 9) and the distal part of the long arm of the Y chromosome (Prantera et al., 1979). The affected areas of chromosomes I, 3 and 9 were concentrated at the centromeric areas and (as with the area affected on the Y chromosome) are heterochromatic sites. Other heterochromatic sites were unaffected however. It is interesting to note that chromosomes I, 9, 16 and Y (but not 3) possess the repetitive (GGAAT)n sequence in their heterochromatin but direct evidence of distamycin binding to this sequence in vivo is lacking. Parallel to these cellular studies, distamycin and netropsin binding to DNA in chromatin has been investigated (Melnikova et al., 1975). Chromatin from calf thymus was found to bind 897~ and 83% of distamycin and netropsin, respectively, compared to native DNA, as assayed by CD spectroscopy. A theoretical examination of DNA interaction with the nucleosomal core particle using a molecular modelling approach and analysis of molecular electrostatic potentials on the tyrT DNA fragment suggest netropsin binding can create a new energy minima if

the DNA rotates 180” (Perez and Portugal, 1990). This rotation should not disrupt the nucleosomal core particle as has been inferred from DNAase I footprinting studies (Low et al., 1986). Distamycin also shows more specific interactions with nuclear proteins such as histones. Scaffold attachment regions (SARS) on DNA have a high AT content and mediate the binding of DNA to the nuclear scaffold. SARs in vitro catalyse the preferential binding of histone HI in an all or nothing cooperative process (Kas et al., 1989). This process was found to be completely inhibited at a distamycin to DNA base pair ratio of 1 to 7.5. All SARs tested were equally affected, including the histone gene cluster, the heat shock protein 70 gene and several others from Drosophila. Distamycin similarly inhibits and displaces SAR containing DNA from purified nuclear scaffold. Structural effects on closed circular DNA have also been noted with distamycin. A 220 base pair DNA fragment from the kinetoplast DNA of the trypanosomatid Crithidia ,fikdatcr is bent almost 360” as visualised by electron microscopy. Distamycin at a drug to base pair ratio of 1 to 7 causes 82% of the molecules to straighten or to bend less than 90” (Griffith et al.. 1986). Similarly, netropsin is postulated to have a specific effect on the AT-rich circular mitochondrial DNA of yeast. S. cerecisiae strain D253-3C mitochondrial DNA synthesis is inhibited at 1 pg/ml netropsin, while there is no observable affect on nuclear DNA synthesis (Griddle and Short, 1976). Netropsin does not, however, induce petite colonies, although studies with petite inducers such as ethidium bromide in combination with netropsin demonstrate that netropsin is specifically lethal at some stage in petite formation. Distamycin and netropsin also inhibit topoisomerase I activity, but the effect is minor compared with Hoechst 33342 (Chen et al., 1993b). However: topoisomerase I inhibition cannot be excluded as a participant in mutagenesis by these compounds. In addition to these general chromosomal effects, some individuals possess specific distamycin inducible fragile sites on their chromosomes. To date five such rare sites have been identified in human lymphocytes fra(ll)(pl5.I), fra(16)(p12.1). fra(8)(q24. I). fra( 16)(q22) and fra( 17)(p 12) (Hori et al., 1988). All these sites are classified as rare, since they occur at a

frequency of less than 1% in the general population. All distamycin inducible sites are inherited in an autosomal co-dominant manner with complete penetrance (Hecht, 1988). Fragile sites are induced in susceptible lymphocytes to varying extents by different AT specific ligands in addition to distamycin, such as netropsin. Hoechst 33258 and berenil (Schmid et al.. 1987). Generally, lymphocytes from a donor are cultured in the presence of distamycin, blocked at metaphase using colcemid and then chromosome spreads prepared using Giemsa staining. Fragile sites are manifest as Giemsa-negative gaps and occasionally misalignment of the acentric fragment (Schmid et al., 1987). The precise molecular nature of these fragile sites is uncertain, but has been suggested to involve DNA amplification perhaps by unequal crossing over. Distamycin binding to these amplified sequences during replication may cause single or double-strand gaps in the DNA (Hori et al.. 1988). Consistent with this is a higher frequency of sister chromatid exchanges at fra( 16)(q22). which implies also that these sites may be recombination hot spots. It is interesting in this respect that CC- 1065 was found to alkylate cellular amplified DHFR DNA more efficiently than bulk genomic DNA or APRT DNA (Tang et al., 1994). Whatever the nature of these fragile sites, it seems they are involved in certain types of cancer. A recent Japanese study of distamycin inducible fragile sites in cancer patients found that there was no statistical increase in frequency of fragile sites among patients overall. However, when the frequency of occurrence of specific sites with specific cancers was examined a few striking examples emerged (Hari et al., 1988). Thus 16.7% of patients with polycythemia vera (PV) had fra( 17)(p 12) compared to 3.1% in controls, and a homozygous fra( 17)(p 12) PV patient also had a high rate of different cancers in her close relatives. Fra( 17)(p 12) was also found at a frequency of 9.1% in myeloproliferative disorders. Benign tumour patients also had a high frequency of distamycin inducible fragile sites. although not to a statistically significant degree. Cancer specific chromosomal rearrangements coincident with rare fragile sites (including folate and bromodeoxyuridine inducible sites) occurred at a frequency of 29.6%, and 6 of these were distamycin inducible. Two patients with acute

non-lymphocytic leukemia had the translocation t(7; 11)(p 13;~ 15) in their malignant cells and one also had the fra( 1 l)(p151 phenotype. Combined results from the Japanese study and others (Le Beau, 1986; Murata et al., 1987) showed that 18 cases of patients with acute myelomonocytic leukemia out of 27 carried the fra( 16)(q22) phenotype. Population incidences of this phenotype ranged from 1.4-5.1%, strongly suggesting a link between fra( 16Xq22) and leukemia. These observations may have implications in the treatment of cancer with AT sequence specific drugs, especially drugs like tallimustine which are based on distamycin. Fragile sites are sometimes observable without distamycin pretreatment, but distamycin will always induce these sites if they are present (Hungerford. 198 1). The mechanism of induction has not been definitively elucidated but these sites are implicated in cancer proneness. Two points should be noted however. Firstly distamycin is used to induce fragile sites so that they are observable in chromosome preparations. This involves 24 hours of exposure to the drug before chromosome preparation. This limited exposure time coupled with the immediate chromosome preparation may not allow the expression of complex chromosome alterations such as translocations. Mutation frequency with cell lines such as CHO AS52 require about a week post drug treatment before they reach their maximum level (Stankowski Jr. and Tindall, 1987). Secondly. studies on distamycin inducible fragile sites have focussed on the presence or absence of these sites in the general population and in cancer patients. The potential for distamycin to induce chromosomal aberrations in fragile site carriers’ lymphocytes has not been the purpose of these studies and even if such events were occurring they may have been overlooked. It would seem reasonable to investigate these considerations in vitro using a range of sequence specific drugs such as distamycin and Hoechst compounds. Additionally, lexitropsins with differing sequence selectivity may provide clues to the involvement of DNA sequence in fragile site induction. The technique of fluorescent in situ hybridisation (Pinkel et al., 1988) using probes specific for chromosomes of interest would seem an obvious mode of investigation into these phenomena.

8. Bisamidines The bisamidines (Fig. 7) are a large group of compounds, usually with terminal amidine groups, which bind reversibly to AT-rich DNA in the minor groove. Crystal structures have been reported for berenil, propamidine and pentamidine (Brown et al., 1990; Edwards et al., 1992; Nunn and Neidle. 1995). They were initially developed and used as antiprotozeal agents (Jensch, 1958) but interest has been revived recently with their use in the treatment of AIDS related Pneumocystis caritzii pneumonia (PCP). Tidwell et al. ( 1990) used a rat mode1 of PCP to test 31 new analogues of pentamidine. All of the analogues bound DNA to varying extents, and the most active compound had the strongest DNA binding ability. This compound was more effective against PCP than pentamidine in the rat mode1 and was also devoid of toxicity as measured by liver. spleen and kidney histology. Pentamidine is known to cause undesirable side effects such as hepatotoxicity. and nephrotoxicity and this was also reflected in the rat model. It has been shown that inhibition of transcription factor DNA binding by polyamidines results in modulation of gene expression (Gambari and Nastruzzi. 1994). Berenil has also been shown to inhibit the activity of topoisomerase II and to a small extent the decatenation of kinetoplast DNA (Portugal, 1994). Berenil. like distamycin and netropsin, causes a Gz arrest and polyploidisation in human diploid fibrob-

lasts and chromatin under-condensation (Poot et al., 1990). Additionally. polyamidines inhibit serine proteinases to different extents. although this ability is not correlated with antitumor activity. Mutagenicity data for these compounds is scarce but an early paper (Ferguson and Baguley, 1983) showed berenil to be an active mitochondrial mutagen in yeast. Pentamidine similarly has been shown to induce petite colony formation although only at growth inhibitory concentrations (Ludewig et al., 1994). The mutagenesis of pentamidine has been investigated in various strains of Salmotzella typhimurium and chromosomal effects studied in five human lymphoblastoid cell lines (Connor and Trizna. 1992). Pentamidine showed no significant mutagenicity in either of these assays. Additionally pentamidine did not induce micronuclei in polychromatic erythrocytes in mice (Phillips et al.. 1991). Despite pentamidine’s apparent lack of mutagenicity, it has been recommended (McDiarmid et al.. 1992) that a containment hood be used when administering the aerosolised drug to patients. Pentamidine’s effect on the uvr(A)BC excinuclease has not been investigated. but it is possible it will act in a similar fashion to the bisbenzimidazoles and other non-covalent minor groove binders. Its effects on human DNA repair systems also require investigation. However at the present time pentamidine would seem to be a relatively non-mutagenic DNA interactive compound, and as such provides impetus for the future design of other non-mutagenic therapeutic agents of this class.

9. Bisquaternary

Pentamidine

Berenil Fig. 7. Biaamidinea.

ammonium

salts

Bisquaternary ammonium salts (BQAH) are a class of synthetic DNA minor groove binding agents related to the polyamidines, but with quatemary ammonium heterocycles as the charged centres. Representative examples of this class are shown in Fig. 8. Early studies demonstrated the ability of BQAH to bind selectively to AT-rich regions of DNA. attributed to a combination of interactions. including ion-ion. DNA ion-drug dipole, induced dipole-dipole, van der Waal and hydrogen bonding (Baguley. 1982). Detailed NMR (Leupin et al.. 1986; Chen et al., 1992) and X-ray crystallographic studies (Gao et

P.R. Turner,

W.A. Denn~/Mutation

SN 18071

SN 4094

H3C’ SN 6999

NSC 60339

ri H3C

‘NT

(

>

C’-‘3

SN 24526 Fig. 8. Bisquaternary ammonium heterocycles.

al., 1993) on the representative quinolinium analogue SN 6999 (Fig. 8) showed the formation of specific stabilising hydrogen bonds from the drug amide protons. However the analogue SN 18071 (Fig. 8). which cannot form hydrogen bonds, shows that formation of these is not required for DNA binding, although SN 18071 does bind less strongly than SN 6999 (Zimmer et al., 1986). Although not strictly

Resecirch 355 (199cji 141-169

161

members of this class, phthalanilides are included here as they are structurally very similar and probably have a similar binding mode to the BQAH. Several phthalanilides have been investigated in man but were found to have severe toxic side effects although one (NSC 60339) showed a response against Burkitt’s lymphoma (Yesair and Kensler. 1975). The DNA binding and mutagenicity of a series of compounds related to the phthalanilides and bisquats, namely the polybenzamides, are under study in this laboratory. One example @N-24526, Fig. 8) is a sequence specific crosslinker, and has shown high potency and good antitumour properties in mice (Atwell et al., 1995). The BQAH were initially developed as anticancer drugs (Atwell and Cain, 1973), and structure-activity relationships showed an effect of lipophilicity and a strong positive correlation between anticancer potency and the degree of selective binding to AT regions in DNA (measured as the ratio of binding constants to poly . [dA-dT] and poly . [dG-dC] (Denny et al., 1979). While early examples (e.g., SN 4094) (Fig. 8) showed chronic toxicity in vivo (Atwell and Cain, 1973) this was overcome in later series, and a quinolinium analogue (NSC 1763 19) (Fig. 8) was considered for clinical trial but found too toxic (Plowman and Adamson, 1978). Recent studies have shown these compounds have high selectivity towards multi-drug resistant malaria parasites (Wilairat et al., manuscript in preparation). There are virtually no data on the mutagenic properties of the BQAH, with the only reported study being their ability to induce petite mutations in yeast (Ferguson and Baguley, 1983). All of the examples examined in this study, including the antitumour active agents SN 4094, SN 6999 and two phthalanilides, had strong petite-inducing ability at concentrations below the IC,, (the concentration resulting in 50% survival of L1210 cells). The petite inducing ability was not related to hpophilicity, but strongly correlated with the degree of selective binding to AT regions in DNA. If these compounds are to be further developed, further studies of their mutagenic potential in a range of different test systems are needed. The powerful mutagenic effects seen with related minor groove binders such as the Hoechst compounds suggest that the BQAH will not be benign as genotoxins.

10. Conclusions

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Minor groove binding ligands when considered as a whole induce a huge range of mutations from simple base sequence changes to deletions and ploidy changes. This is perhaps hardly surprising as minor groove binders are structurally diverse and encompass non-alkylating. mono-alkylating and bis-alkylating compounds. Additionally, a most important difference between these ligands is their DNA sequence selectivity. Clearly compounds must be considered on an individual basis when investigating mutagenic and biological effects. Most research in the development of minor groove binders is directed at sequence selectivity in an effort to target longer sequences more specifically. As specificity increases the role of mutagenesis may change. Currently, mutagenicity is considered a deleterious property in cancer treatment. However. if a drug possesses enough selectivity to bind sequences relevant to neoplasia such as over-expressed oncogenes, it is conceivable that inactivating mutations at these sequences may have therapeutic advantage. Sequence selectivity may also have implications for mutagenesis in different individuals. Inter-individual genetic variation may be sufficient to predispose certain individuals to specific mutations. Distamycin inducible fragile sites may represent valuable research tools in this respect. Finally, another obvious aspect in sequence selective mutagenesis is interspecies genetic variation. At the present time much research in mutagenesis is performed on bacteria, yeast and rodent cells. The DNA sequence differences between these organisms and humans is considerable and as drugs become more sequence specific test systems for investigating mutagenesis may need re-evaluation in order to detect fewer but possibly more important mutagenic events.

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