drug resistance and dna repair

Cancer and Metastasis Reviews 6:261-281 (1987) © Martinus Nijhoff Publishers. Boston - Printed in the Nctherlands.

Drug resistance and DNA repair

Margaret Fox ~ and John J. Roberts ~- Paterson Institute for Cancer Research, Christie Hospital and Holt Radium Institute, Manchester :~,I20 9BX,

UK; 2 Institute of Cancer Research, Royal Cancer ttospital, Clifton A venue. Sutton, Surrey SM2 5PX, UK

Key words: DNA repair, drug resistance, molecular cloning

Summary

DNA repair confers resistance to anticancer drugs which kill cells by reacting with DNA. A review of our current information on the topic will be presented here. Our understanding of the molecular biology of repair of 06-alkylguanine adducts in DNA has advanced as a result of the molecular cloning of the E. coli ada gene but the precise role of this lesion in the cytotoxic effects of alkylating agents in mammalian cells is not completely understood. Less progress has been made in understanding the enzymology and molecular biology of DNA cross-link repair even though such lesions are important for the cytotoxic effects of the widely used bifunctional alkylating agents and platinum compounds. It is evident that drug sensitive or resistant phenotypes are as highly complex as are the effects of DNA damage on cell metabolism and various aspects of lhese effects are discussed. Few clear correlations have been made between quantitative differen-

ces in DNA repair capacity and cellular sensitivity but assays which were developed to measure fidelity and intragenomic heterogeneity in DNA repair are beginning to be applied. Such studies may reveal subtle differences between sensitive and resistant cell lines. The molecular cloning of human DNA repair genes by transfection into drug sensitive rodent cells has been attempted. Some success has been achieved in this area but the functions of the cloned genes have yet to be identified.

Introduction

In spite of recent advances in treatment of cancer as a result of the introduction of multidrug chemotherapy, the development of drug-resistant tumour cell populations is still a major factor in treatment failure. The basic premise on which the development of multidrug chemotherapy is based is that the acquisition of resistance (by somatic mutation) to a combination of many different drugs with different modes of action occurs at an extremely low frequency. Non-genetic factors make a considerable contribution to overall non-responsiveness of tumours [1]. Many features of the mode of action of the various drugs and drug combinations must be taken into consideration in order to understand the reasons for development of drug resistance at the cellular level.

Evidence now indicates that tumour cells arc inherently unstable genetically [2, 3] and it is important to remember that ionising radiation and alkylating agents commonly used in chemotherapy are in themselves mutagenic and clastogcnic [4]. Exposure of tumour cells to chemotherapy or radi- otherapy, in addition to selecting for intrinsically resistant cells, generates increased genetic diversity which may express drug resistance and increased metastatic potential. Changes in DNA repair capacity are only one of the many metabolic changes which may make a contribution to the drug resistant phenotype. The mechanisms of acquired and intrinsic resistance to all major classes of anticancer drugs have been recently reviewed [5].

Many antimetabolitc drugs and other inhibitors of DNA synthesis including alkylating agents cause

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significant alterations in gene expression 16-8] which may be related to the known capacity of the former class of drugs to induce differentiation [9]. In some cases altered genc expression has been correlated with alteration in methylation patterns [6]. In others it occurs by as yet undefined mechanisms. Such events may be causally related to the development of drug resistance or they may be cpiphenomena.

In the experimental setting, where single agents can be used and the development of non-responsive tumours monitored accurately by a variety of experimental techniques [10], resistancc develops significantly more rapidly than can bc accounted for by modcls which propose spontaneous mutation to drug resistance and consequent selection of drug resistant cells [11]. Although changes in DNA repair capacity may be demonstrated, they arc often accompanied by other changes which could contribute significantly to the resistant phenotypc.

Interactions between drugs can also contribute to drug resistance. For example, CHO cells exposed in vitro to ultraviolet irradiation or drug treatment (EMS or MNNG) prior to selection in methotrexate (MTX) show a transient increase in MTX resistant colonies of which a similar propor- tion are amplified at the dhfr locus irrespective of drug exposure [12].

The mechanism for induction of gene amplifica- tion is still not completely understood but one mechanism is thought to involve inhibition of fork progression and reinitiation of replication at a previously unused origin behind the blocked replication fork [12]. Inhibition of replication could be important in the evolution of drug resistant populations during combination chemotherapy. The efficiency of DNA repair may be crucial in this interaction as removal of the lesion blocking DNA synthesis prior to exposure to the second drug presumably prevents the increase in frequency of MTX ® colonies. The order of administration of drugs may also have important consequences. A variety of anticancer drugs increase the frequency of mutants at a number of genetic loci, hypoxanthine phosphoribosyl transferase (HPRT) [13- 15[, by induction of point mutations or major genetic re-arrangements [15] indicating that the use of

an antimetabolite prior to an alkylating agent is preferable to the other way around.

In an extensive review [16] compiled in 1982 it was concluded that in spite of the vast amount of published data there is little evidence that loss or acquisition of a specific DNA repair pathway is directly related to altered drug sensitivity in tumour cells. This conclusion was reached after per- forming experiments based on the biochemical techniques in use for the analysis of DNA repair capacity, which were reviewed in detail in 1981 [17]. The majority of these techniques measure quantitative differences in rates of DNA strand break rejoining, rates of repair of specific lesions or rate and total amount of unscheduled DNA synthesis or repair replication. However, few quantitative differences in repair have been reported which correlate with a drug sensitive or rcsistant phenotype. Assays have only recently been developed to measure the more qualitative aspects of DNA repair such as fidelity to the original or repair in transcriptionally active regions of DNA. An attempt will be made in this review to analyse the contribution of these newer techniques and other recent advances in molecular biology to our current understanding of the complexities of DNA repair and its role in intrinsic and acquired drug resistance in mammalian cells.

Use of transfected genes to assess repair fidelity

Agents which damage DNA produce a variety of lesions and it is thcrefore difficult to identify the lesion responsible for a particular cytotoxic effect. One approach to this problem is to analyse the functional inactivation of a dominant selectable marker after its transfcction into mammalian cells using appropriate vectors. An important feature of such vectors is that they do not code for their own repair enzymes, so any observed repair reflects that of the host cell. One transfection system uses the plasmid pSV2-gpt in which the bacterial gpt gcne has been engineered for expression in mammalian cells by splicing it between appropriate SV40 sequences [18]. Mammalian cells cannot normally utilise xanthine as a source of GMP, therefore gpt ÷

transfectants can bc identificd by their growth in media containing mycophenolic acid, aminopterin, thymidine, hypoxanthine and xanthine. Thus if a 7-irradiatcd or drug-treated plasmid is transferred into mammalian cells the ability of the gpt gene to function can be monitored after a suitable cxprcssion period by measuring colony formation in ap- propriatc sclective media. The effects of y- and UV-irradiation on pSV2-gpt and pSV2 neo, a plasmid containing the neo gene which confers resistance to the antibiotic G418. have recently been reviewed [191 .

The use of restriction endonucleases to generate DNA damagc in dcfincd sequences has also provided a new analytical method for assessing repair processes in normal or radiation-sensitivc cells. Radiosensitivc human ataxia tclangieclasia (AT) cells [19, 20] can rejoin endonuclease-generated breaks in the gpt gene in immortalised normal and AT-radiosensitive cells. The gene is inactivated by cuts at either the junction of the gene and its controlling sequences or within the gene itself. If the cut plasmid is rejoined with fidelity the genc will be expressed and confer resistance to selective drugs (G418 [neo] or mycophenolic acid [gpt]). Mis-repair involving some loss of DNA sequences commonly destroys gpt function. Cuts at sites further away from the coding frame produce less drastic effects. After cuts with one of several restriction enzymes normal cells have relatively high levels of gpt expression. In contrast, after cutting with the same enzymes AT cell expression is much rcduced. Cells which do not express the pgt gene are lost in this system and hence reasons for lack of function cannot be analysed.

In an extension of these studies a plasmid containing both neo and pgt and with only one Kpn 1 cut-site in the pgt coding frame is first selected for neo function and subsequently assessed for gpt function [20]. In experimcnts of this type 88% of normal cells express neo and gpt but only 12% of AT cells express both functions. Of 14 normal cell clones whose DNA was analysed after Southern transfer, 10 showed a DNA fragment of the correct size for the intact gpt gcne. One of 11 AT cells analysed had the appropriate fragment. These ana- lyses also revealed that manv cells had integrated

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multiple copies of the plasmid on which presumably there may be only one functional gp~. and several non-functional ones.

The radiosensitivity of AT cells is possibly explained by a disequilibrium between rejoining and exonuclease digestion at the sites of double strand scissions. The disequilibrium is not detectable by conventional analysis of rates and extents of repair of single and double strand breaks. It should be noted however that there are now 5 known complemcntation groups in AT cells [21] and only one cell linc has so far been examined in this way. How- ever, few defects in AT cells of other complementation groups have been detected by conventional biochemical assays, so most differences may be quite subtle.

Discquilibrum studies have been done on Walker 256 carcinoma cclls (WS) which are inherently sensitive to bifunctional alkylating agents. Resistant Walker cells (WR). on thc other hand, show sensitivity similar to that of conventional cell lines [22]. The comparative study is discussed in detail later.

Using a replica plating technique 4 X-ray sensitive mutants of V79 Chinese hamster cells have been isolated after screening 5000 ENU treated clones. Complementation by cell fusion has shown that the three most sensitive mutants are from different complemcntation groups and 2 of these are diffcrent from the X-ray sensitive Chinese hamster mutants xrs-1 and EM7-2 isolatcd by Jcggo and Kemp [23] and Thompson and co-workers [24] re- spcctively. The transfected gpt-neo plasmid tcch- nique revealed that the ability to correctly rejoin double strand DNA breaks is significantly reduced in one of the sensitive mutants [25]. No data arc vet available for other mutants, but subtle changes in ability to rejoin strand breaks are correlated with a radiation sensitive phcnotype. Another series of X-ray scnsitive mutants has been isolated after EMS mutagcnesis of CHO cells. All arc defective in double strand breaks rcjoining and are phe- notypically stable but revert with high frequency on treatment with 5-azactydine, suggesting that the xrs genc is under mcthylation control 126].

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Repair of monofunctional alkylation damage

O~-atkylguanine alkyl transferase

One of the best characterised repair enzymes, particularly in bacterial cells, is 0" alkylguanine alkyl transferase ((~-GAT), which removes methyl, ethyl and possibly higher alkyl groups from guanine by alkyl transfer to a cysteine residue in the protein [27]. The repair process appears essentially similar in both bacterial and mammalian cells. E. coli 06-alkylguanine alkyl transferase preferentially removes methyl groups but the enzyme is known to remove other alkyl groups in the order Me > Et > nPr > nBu > iPr > iBu. The enzyme is also known to dealkylate lYMeT but at a slower rate than 0~-MeG [28]. The gene (ada) coding for the E. coli enzyme has now been cloned by several groups of workers [2%31]. The ada protein has also been purified to homogeneity from E. coli [31], and the complete DNA sequence is known [32]. The gene ada has a molecular weight of 37 Kd with 12 cysteine residues, one of which (amino acid 321) ac- cepts the methyl group from 06-methyl guanine while another (cysteine residue between amino acids 6%72) acts as an acceptor for the S ster- eoisomer of methyl phosphotriesters. The latter specific methylated protein then acts as trigger for the adaptive response by binding to DNA 35 bases upstream from the transcriptional start of the gene and acting as a positive signal for further transcrip- tion [331.

Much less is known about the molecular biology of the mammalian enzyme. Repeated attempts to purify the protein to homogeneity from rat liver have so far been unsuccessful largely due to in- stability of the enzyme. Partial purification of the enzyme from a variety of sources has been reported [34]. Among the attempts at cloning of the mammalian genc is one report which suggests the suc- cessful transfection of human DNA from a mer ÷ cell line into a mer- human astrocytoma strain [35]. Protection against cytotoxicity induced by chloroethyl nitrosourea (CNU) shows that the human DNA complements the mer- phenotype. Meth- yltransferase levels, determined in transfectcd cells, arc increased relative to those in the mer-

strain. However it is possible that the endogenous human enzyme is re-expressed when cells are sub- jected to sevcral doses of MNNG which itself is a mutagen.

Saffhill, Margison and O'Connor [36] have reviewed thc varying levels enzyme activity of in different mammalian species and tissues and in normal and tumour derived cell lines in vitro. Be- cause they covered other aspects of repair of alkylation damage, particularly in vivo [36], we will concentrate on current status in cultured cells.

Some cstablished cell lines, c.g., V79 and CHO (both hamster derived) are completely deficient in 0~-GAT [37, 38] and are dcsignated mer-. The mer- phenotype was originally defincd as lack of capacity to reactivate adenovirus 5 when alkylated virus was grown in the host cell [38] but is now commonly used to describe any cell which lacks enzyme activity. However, in some cases the ability to reactivate viruscs can be demonstrated in the absence of detectable methyl transferase activity indicating that the two phenotypes are not neces- sarily identical. A number of reports indicate that some human tumour-dcrived cell lines have a mer- phenotype, but a number of primary tumours do not exhibit this phenotype [35, 36, 40]. Levels of enzyme activity are either normal or above normal in the majority of primary tumour samples. Such observations suggest that 06-GAT can be lost during establishment of cell lines in vitro. Transforma- tion of cells by SV 40 virus can lead to loss of enzyme activity and a single sample of lymphoblastoid cells from the same patient can show either mer ÷ or met- phenotype, suggesting that in vitro culture conditions may influence expression. The reasons for these differences in 06-GAT expression are not understood at present but indicate that great caution should be exercised in interpreting data on drug sensitivity of cultured human tumour cells as being indicative of responses of tumours in vivo. The current status of our knowledge of (~'-GAT expression has been rcviewed recently by D.B. Yarosh [40].

A novel approach has been made to understanding the roles of 06-methyl guanine methyl transferase activity in protecting mammalian cells from the cytotoxic and mutagenic effects of both mono-

and bifunctional alkylating agents which react at 0h-MeG. The E. coli gene coding for the 37Kd ada gene product and possessing both 06-GAT and methylphosphotriester methyl transferase ac- tivities has been engineered to contain only the protein coding sequences and inserted into a retro- virus-based electable expression vector [41, 42]. The vector was transfected into Chinese hamster V79 cells which lack detectable endogenous 06-GAT activity and clones expressing high levels of the bacterial enzyme selected. Rapid removal of 0~-MeG from alkylated DNA demonstrates the functional activity of the protein in transfected cells.

The survival rate of transfected cells is determined after exposure to increasing doses of N-methyl-N-nitrosourea (MNU), methyl methane sulphonate (MMS), chlorozotocin (CLZ), and nitrogen mustard (HN2). Those expressing the ada gene are clearly more resistant to MNU and CLZ but not to MMS or HN~. Both MNU and CLZ produce high levels of reaction at the 0G-position of guanine. CLZ gives 06-chloroethylguanine, which has been shown to be a substrate for 06-GAT in vitro. In contrast MMS produces very little 06 methylguanine (06meG/7McG ratio in DNA for MMS is 0.004) and no 06 substituted products have been detected in DNA after HN, exposure. These results in two cell lines which differ, so far as can be ascertained, only in ability to remove alkylation products from 06G and from phosphotriesters, indicate an important role for such lesions in the cytotoxic effects of certain drugs. It is not yet clear from these results whether 0C'-MeG or the methyl phosphotriesters are the major cytotoxic lesions or whether both contribute equally. If 0"MEG is a cytotoxic lesion then the question arises as to how it excrts its cytotoxic effects.

MNU and MNNG are considerably more toxic towards a line of HeLa cells than is MMS but no comparable differences are observed between the cffects of these three agents in hamster cells [43]. As neither HeLa nor V79 ceils are able to excise 0~'-MeG from DNA the vast difference in their sensitivity to the cytotoxic effects and MNU suggests that V79 cells possess some mechanism, which HcLa cells lack, whereby they are able to

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tolerate or bypass unexcised lesions in their DNA. The existence of such tolerance or bypass mechanisms also indicates that excision repair may be of secondary importance in some cell lines.

When the effects of MNU on DNA synthesis were compared some remarkable differences were observed between the two cell lines [43]. Exposure of HeLa cells to MNU in early S phase induces minimal initial depression of DNA synthesis indicating that the initial alkylation of DNA does not produce lesions which block DNA polymerasc. In the second post-treatment S phase, DNA synthesis is markedly depressed in a dose-dependent manner, suggesting the introduction of new damage to DNA. Exposure of synchronised Chinese hamster V79 cells to similarly toxic levels of MNU reveals a marked difference in response. An initial depres-

sion followed by an increase in the rate of DNA synthesis resulted in delay of the peak rate of DNA synthesis but no overall decrease in the amount of DNA synthesised during the first S phase after treatment.

Consideration of the nature of the secondary damage inducd in HeLa cells may give clues as to how 06MEG exerts its cytotoxic effects. The HcLa cell strain used appears to have a mcr- phenotype from 06/N ~ ratios (although mer- strains have been described) [44]. Therefore ceils entering the second S phase would still carry 06MEG lesions. If such lesions, although not initially constituting a block to the polymerase, arc rccognised by an cndo- nucleasc which incises close to the site of the lesion, then in a subsequent round of DNA synthesis double strand breaks could be produced.

Pertinent to the above discussion is the observation that in clone 8 cells (transfected with the ada gene and able to remove 06-MEG) MNU fails to induce sister chromatid exchanges at measurable frequencies above background [45]. In contrast, SCE frequencies increase approximately linearly with dose in clone 2 (transfected with the neo gene only). The generation of SCE is known to require some kind of recombination event [46] which also requires nicking at or near the sites of lesions. In the absence of lesions, (i.e.. in clone 8 where the majority are repaired) no nicking would occur and no SCE is generated.

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From levels of 0"-alkylguanine in V79 cells exposed to higher doses of ~4C MNU it can be esti- mated that with the doses used in this study there would be 104 (~'-MeG/cell [47]. Only 40-50 SCE/cell were observed, suggesting that the majority of lesions are not recognised and nicked to allow recombination to occur.

To pursue this line of reasoning, we suggest that V79 Chinese hamster cells which lack the ability to remove 06G have also lost the ability to incise the DNA at sites close to 0 ~ lesions. This hypothesis explains why no secondary damage is observed, and why V79 cells are considerably more resistant to MNU and MNNG. Reduced numbers of double strand breaks form, few chromosome exchanges or aberrations are produced and the chances of cell survival are enhanced. The production of aberrant chromosomes can lead to cell death.

Evidence in favour of an important role of alkylation at oxygen atom sites in cytotoxicity comes from the observation that the cytotoxicity of alkylating agents can be increased when endogenous levels of 06-GAT are depleted by pre-treatment of the cells with low (non-toxic) levels of alkylating agents or with the free base @ methylguanine [36, 40, 48]. However , this simple interpretation does not hold in all cases. Pre-treatment with equitoxic doses of MMS, MNU, MNNG, EMS, ENU and ENNG reduced survival in V79 Chinese hamster cells (which lack endogenous 06-GAT activity) to 75%. The V79 cells were then re-exposed to a range of doses of the same agent and their sub-

Table I. Survival ratios for pretreated and non-pretreated V79 cell population.

Drug Pre- Challcngc Recovery treatment dose mM ratio V79 dose mM

MMS 0.36 0.36 1.9 MNU 0.40 0.40 0.3 MNNG 0.001 0.001 0.12 EMS 8.0 8.0 1.41 ENU 1..0 1.0 1.35 ENNG 0.012 0.012 1.0

Recovery ratios are the ratio of survival at 96 hrs of pretreated and challenged population divided by survival of population given a challenge dose equal to the sum of the pre-treatment and challenge dose.

sequent survival determined. The ratios of survival of the pretreated compared with the non pretreated population were measured 24-96 hrs after pretreatment (data at 96 hrs arc shown in Table 1). Cells were not significantly sensitised by MMS pre- treatment or pre-treatment with cthylating agents even at early times, but MNU and MNNG pre- treatment did significantly sensitize. These results indicate that sensitization is not due to the depic- tion of 06-GAT and suggest that other persistent DNA lesions or alkylation of repair proteins by MNU, MNNG may play a role.

In another series of experiments V79 cclls on a different pre-treatment schedule with MNU show a transient rise in survival. This does not result from any alteration in the capacity of the cells to remove 0~-MeG as measured by loss of the radi- olabelled product from DNA [49]. Consistent with the suggestion that responses to pretreatment are very cell line dependant are the observations of Karran and Williams [50]. They were unable to sensitise mer + Raji cells or a mer- CNU sensitive derivative to the cytotoxic effects of MNNG or CNU by pre-treatment with {~-MeG at doses which significantly reduced 06-GAT levels. These results clearly imply that in this cell line adducts at the lY' position of G are not lethal lesions and that a defect other than lack of the enzyme is responsible for alkylation sensitivity in this met- strain as in the case of the mer- HeLa cell strain referred to earlier. These observations bear on other experiments designed to demonstrate an adaptive response on mammalian cells.

Adaptation

Several groups of workers have attempted to demonstrate an adaptive response in mammalian cells [40, 48] and the results of such experiments are conflicting even when nominally the same cell line is used. Data from one laboratory indicates increased survival and reduced SCE "after MNNG pre-treatment but there are several other reports of negative results. The mechanisms underlying the increased survival and reduced SCE frequency reported in V79 and CHO are unknown, but they seem unlikely to be related to changes in 0~-GAT

activity, as neither cell line expresses thc enzyme constitutively. It is conceivable that MNNG pre- treatment in the particular regime used induces expression of 0"-GAT by inducing hypomethylation [51], however, extensive treatment of V79 cells with azacytidine does not result in expression of 06GAT (Fox and IVlargison, unpublished data). In most other cell lines (with the exception of rat hepatoma cells [52, 53]) attempts to demonstrate adaptation by increase in survival or reduction in mutation have proved negative.

The demonstration of an adaptive response in cultured mammalian cells in vitro seems highly dependent on the cell line used, the precise prc-trcat- ment schedule, and the cultural conditions. The factors controlling these variables are far from being clearly understood, and the effects observed are obviously a balance between depletion of 06-GAT activity (resulting in sensitization) and its apparent induction by mechanisms yet to be elucidated. Several important factors may be determi- nants.

These are as follows: 1) The constitutive level of expression of ()6-1vleG

transferase 2) The rate of depletion of the cnzvme on exposure

to alkylating agents 3) The rate of resynthesis after treatment 4) Inducibility or otherwise of the enzyme

In human cells the differences in the rate at which various met- human cell lines regenerate ()~GAT activity have been correlated with different sensitivities to MNNG. Those showing a rapid regeneration capacity are classified rein + and those which regenerate levels more slowly rem- [40]. The apparent importance of regeneration rates implies that 0~-alkylguanine lesions may, not be toxic at the first round of DNA rcplication but can produce secondary damage during subsequent replication. a conclusion consistent with data discussed earlier.

Overall results available to date raise several important questions: 1) Is 0<methylguanine a major cytotoxic lesion in

some cell lines but not in others'? 2) In cells with constitutive enzyme activity what

are the precise levels of MNU or MNNG to completely saturate (l~'l~teG transferase activity?

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Other path ways of repair

The possible involvement of excision repair pathways in removal of 06-AG in cell lines which lack endogenous ff'-GAT activity is only just beginning to be appreciatcd. Human mer- cells and Chinese hamster cell lines V79 and V79/79 which lack 0~-GAT activity are all able to removc 0~'BudG from their DNA as shown by RIA of enzyme di- gests of DNA from cells exposed to BNU [54, 55]. In contrast, fibroblasts from xeroderma pigmentosum patients (complementation groups A and G) which show <5% unscheduled DNA synthesis after UV irradiation but are mer' fail to remove 0~'BudG, suggesting that 0~BudG is repaired in cells which lack the alkyl transferase by a process defective in XP cells (presumably nucleotide excision repair). Both Chinese hamster cell lines lost 0~-BudG at approximately equal rates but only V79/79 lost 06-MEG too, suggesting that the 06-BudG is recognised by excision repair endonucleases in both Chinese hamster cell lines and that 0<IVleG in some way distorts chromatin structure sufficiently in V79/79 cells to be recognised. Thus it is formally possible that increased loss of 0~-MeG observed in pre-treated cells (see earlier) is due not to enhanced 0O-GAT activity but is in some way due to up-regulation of excision repair bv mechanisms at present unknown.

A knowledge of the complexities of cellular responses to damage induced by monofunctional agents is important for understanding the mechanisms of acquired resistance to the bifunctional agents. Bifunctional agents which react at the 0~-position of guanine are generally more cytotoxic and are more commonly used clinically. CLZ BCNU, CCNU mitozolamide and many others are thought to act by a two-stage mechanism. The initial reaction places a chloroethvl group in the 0 ~ position of guanine, which then reacts further to form a DNA interstrand cross link [56]. Resistance to chloroethyl nitrosoureas appears to be consis- tently associated with the mer- phenotype, suggesting that removal of the monoadduct before cross link formation (which takes place only slowly) is an important protective mechanism for this class of drugs [57-59]. Evidence in support of

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this conclusion is provided by the observation that pre-treatment with 0~-McG sensitised the met ÷ cells to the cytotoxic effects of CNU (160] and Day, unpublished). This conclusion is further supported by recent rcsults (referred to earlier) from V79 cells transfected with and expressing bacterial 0"GAT in which the differential survival after exposure to chlorozotocin, mitozolamidc and methazolastone was far greater than aftcr exposure to MNU [42]. Similar conclusions have been reached from cx- periments with BCNU and other chloroethylating agents in mer + and mer- human fibroblasts and human lymphoblastoid ccll lines [48, 57, 58].

Bifunctional alkylating agents

The cytotoxicity of other classcs of bifunctional alkylating agents is generally thought to bc due to their ability to form interstrand and intrastrand cross-links in DNA. DNA-protein cross links are also tormed but these are not lethal events. The many aspects of the cytotoxic, clastogenic and mutagenic effects of this class of drugs have been revicwed extensively [5, 43, 61, 62].

Tumour cells can acquire resistancc (or losc sensitivity) to this class of alkylating agents by a number of diffcrent mechanisms and in many cases morc than one mechanism may be operating [62]. DNA cxcision repair has bccn studied extensivcly in several pairs of cell lines which exhibit large differences in sensitivity to bifunctional alkylating agents, but no clear-cut correlations bctwcen capacity to repair DNA : I)NA cross links and cellular sensitivity [16] have emerged. It is important to remember that when wild type tumor cells (c.g., Yoshida and Walker rat sarcomas), which show exquisite sensitivity to bifunctional alkylating agents, develop 'resistance' equivalent to that of Chinese hamster V79 cells, it is really a loss of sensitivity. Much evidence indicates that the majority of inter- or intra-strand DNA cross-links are repaired, but the detailed mechanisms have not yet been elucidated.

In a attempt to correlate othcr cellular rcsponses with sensitivity or resistance to nitrogen mustard induced cytotoxicity, the effects of the drug on cell

cycle progression in Raji and TK6 human lymphoblastoid cells are examined by flow cytomctry 163]. TK6 cells arc considerably more sensitive to HN~ than Raji. Delay in S phasc travcrsc is conccntra- tion-dependent in both cell lines and at a given concentration is two fold greater in TK6 than Raji. The number of cross-links increases linearly with dose and is approximatcly two fold higher in TK6 than in Raji. The difference in sensitivity between the two cell lines cannot be adcquatcly cxplained by differences in initial amounts of DNA damage, ratcs of rcpair of cross-links or differential S phase delay, but we are dealing with a complcx phenotype.

In the same study inhibition and recovery of DNA synthesis is measured by [3H]TdR incorporation and cvidence indicates that intraccllular dNTP pools wcrc pcrturbed over considerable periods after treatment, suggesting that ability to restore normal DNA metabolism is important in cellular recovery. However, no clcar differences were evident in the responses of the two cell lines. TK6 cells lack 06-GAT activity and are considerably more scnsitive to BCNU and MNU (but not to MMS) than arc Raji cells (Dean, Margison and Fox, unpublished data). This effect is unlikely to be related to their HN 2 sensitivity, as this drug as mcntiond earlier is not known to react with 06guaninc.

A similar study compares the responses to HN 2 of Fanconi anaemia cells and normal human fibroblasts. Two Fanconi anaemia cell lines exhibit a 6-10 fold greatcr sensitivity to HN2 than normal fibroblast stains but show no cell cycle delay even when cxposcd to high doses of HN 2 [64]. At equitoxic doses in normal human fibroblasts a marked S phase delay suggests that delay may bc protectivc in allowing timc for repair. No consistent association has been reported between cellular sensitivity and the ability of a cell to delay progress through S phase or any other phase of the cell cycle. This supports the suggcstion that thc delay response is a complex function resulting not only from DNA lesions which inhibit fork progression but also from complex change s in cellular dNTP pools and probably many other as-yet-unidentified changes which occur in response to DNA damage.

Other recent studies in HN~-sensitive and HN~-

resistant Walker rat tumour cells [65] attempt to correlate sensitivity with rates of rcpair of total and intcrstrand DNA cross-links and DNA protein cross links. No differences are dctccted in initial levels or in the ability of the cells to remove HN, or CLZ-induced [651 cross-links. The HN, resistant sub-line is collatcrally sensitive to CNU and shows reduced activity of 06-GA'I -, indicating the complexity of drug sensitive and resistant phenotypes in non-isogenic tumour cell lines [65].

Platinum compounds

Cis-diamminedichloroplatinum(lI) (Cisplatin, cis- DDP) is an antitumour drug particularly effective against testicular cancer, both alone and in combination with other drugs such as a vinblastine and bleomycin, and active against teratoma, ovarian carcinoma and squamous cell carcinoma of the head and neck [66]. Platinum drugs sccm to exert their cytotoxic and anti tumour effects through an interaction with DNA, although which of the many reactions with DNA is the critical lesion is still a matter for debate. Earlier comparative studies of cis-DDP and its trans isomer (trans-DDP). which is not an anti tumour agcnt and which is far less toxic to mammalian cells in culture, pointed to the likely importance of interstrand crosslinks in inducing cytotoxieity. The higher doses of trans-DDP required to produce toxic effects equal to those of cis- DDP produced higher levels of reaction with DNA but cqual extents of DNA interstrand crosslinking [13, 67, 68]. In addition, studies of both mouse leukemia cells and normal and transformed human cells of differing sensitivity to cis-DDP show that sensitivity correlates wcll with interstrand crosslink formation. It has also been shown that cells can be protcctcd from the toxic effects of cis-DDP (the formation of DNA crosslinks) by incubating cclls in the presence of thiourea immediately after treatment [69]. Potentiation of cell killing (accompanied by incrcascd crosslinking) can be achieved by post treatment with 1-[-3-D-arabinofuranosyl- cytosine (ara C) [701 and hyperthermia [71]. On the other hand, DNA-protein crosslinks do not similarly correlate with eytotoxicity and are thought to

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reflect the overall level of active drug metabolites in the cell [67, 68].

However, reactions resulting in DNA interstrand crosslinks account for only about 1%-2% of the total platinum adducts on cellular DNA and studies during the past five years have established that an intrastrand crosslink between the N-7 atoms of adjacent guanines is the principal adduct formed in DNA by cis-DDP. Sincc trans-DDP is stereoehemically unable to form such an adduct it has been proposed that the ability or inability to form such 1, 2- d(GpG) crosslinks adequately ac- counts for the difference in the antitumour activity of the cis and trans platinum compounds [72]. A difference in the reactions of the other two isomers is that end-labeled fragments of known base sequences are digested differently at the position of adjacent guanines with cxonuclease Ill whcn DNA is treated with cis-DDP as compared to trans-DDP [72, 73]. Somc additional insight into the possible importance of these various DNA adducts would be gained if it could be shown that cells which were either sensitive or resistant to cisplatin, and which did not differ with respect to the initial damage to D N A , differed in their capacity to repair either total or specific damage to DNA. Earlier studies established that bacterial strains deficient in DNA repair are lcss able to survivc cxposure to cis-DDP than wild type cells [74, 75]. More recently [76] it has been confirmed that proficiency in excision repair is required for survival of cis-DDP- but not apparently for trans-DDP- treated bacterial cells. Purified proteins coded by the uvr genes have been uscd to reconstitute the UVRABC nuclease and to study the incision step of the excision repair of DNA containing various lesions. The UVRABC nuclease cut plasmid DNA containing damage caused by either cis-DDP or trans-DDP. However, adducts caused by the two compounds are recognised differently by the nuclease. A specific cutting pattern involving incisions at the 8th phosphodiester bond to the 5' and at the 4th phosphodiester bond to the 3' of the adjacent GGs is observed for DNA damage by cis-DDP but not for damage by trans-DDP [77].

In mammalian cells the techniques of alkaline sucrose gradient sedimentation, alkaline elution

270

and DNA renaturation [68, 78, 79] have clearly demonstrated repair of DNA-protein and DNA- DNA crosslinks. Interstrand crosslinking is indi- cated by a shift in alkaline sucrose gradients of DNA molecules towards the high molecular weight end of the gradient, by a decrease in the rate of filter elution of DNA from X-irradiated cells or by an increase in the rapidly renaturing fraction of DNA in cell lysates. All of these drug induced phenomena reach a maximum 6-12 hours after drug treatment and then decline. The half-life of DNA interstrand crosslinks is usually between 12 and 24 hours. Since only minimal degradation of DNA occurs during this time and because crosslinks are stable in isolated DNA under physiological conditions, the reversal of drug-induced phenomena may be attributed to DNA repair of unknown mechanism.

The half-life of platinum bound to the DNA of cis-DDP-treated exponentially-growing Chinese hamster cells is approximately 28 hours [79, 80]. Again, since such DNA-bound platinum is stable chemically under physiological conditions its loss is presumed to represent excision repair of DNA adducts. A recent report claims that the different kinetics of accumulation and loss of platinum on the DNA of African green monkey CV-1 cells treated continuously with cis-DDP or trans-DDP is due to a difference in the repair of the respective DNA-bound adducts [81] and this difference pro- vides a rationale for the enhanced biological activity of cis-DDP over trans-DDP. However, trans- DDP-induced DNA bound platinum is not lost more rapidly than that induced by cis-DDP in either Chinese hamster or CV-1 cells. In Chinese hamster cells, the different kinetics of accumulation and loss of platinum on DNA could be adequately explained by the different effects of the two isomers on DNA replication and cell cycle progression [82].

Relationship between DNA excision repair and cytotoxicity

Despite correlations between the extent of drug- DNA interactions and cytotoxicity and the indica-

tions that cis-DDP-induced damage can be repaired, the relationship between excision repair of DNA damage and toxicity is not always clear. Cells taken from patients suffering from the rare skin condition Xeroderma pigmentosum (XP) are morc sensitive to UV-irradiation than normal cells and are deficient in excision repair of UV-induced damage. These repair deficient XP cells are also more sensitive than normal foetal lung cells to cis- DDP when the lethal effects of thc drug are expressed as a function of reaction with DNA rather than as a function of the dose of the reagent 1831. It has yet to be shown, however, that these cells are in fact defective in their ability to remove DNA- bound platinum. Cclls from paticnts with the genetic disease Fanconi's anaemia show unusual sensitivity to the cytotoxic and clastogenic effects of difunctional alkylating agcnts and arc also un- usually sensitive to cis-DDP. Such sensitivity is not the result of increased binding of platinum to DNA or, apparently, to a decreased abilitv to remove cis- DDP-induced DNA interstrand crosslinks (Pera and Roberts, unpublished results).

The role of DNA excision repair in protecting cells from the toxic effects of cis-DDP has emerged from studies on stationary phase cell cultures. It has been proposed that DNA synthesis on a damaged template is responsible for the toxic effects of cis-DDP [8(I] and that cells allowed to repair DNA prior to S-phase and cell division should show less toxicity than cells entering the proliferative cycle immediately after treatment. Stationary Chinese hamster cells are trcatcd with cis-DDP and their toxicity and platinum-DNA interaction are measured in the non-dividing state after various hold- ing periods. The cells slowly excise platinum and the survival of these cells (when subsequently plated) is increased. The relationship betwcen ccll survival and the amount of platinum bound to DNA with several doses of cis-DDP (determined immediately aftcr treatment) is similar to that determined after varying periods of recovery following treatment with one dose [79]. Human fibroblasts similarly recover from toxicity when held in a non-dividing state and excised platinum lesions from DNA with a half-life of 2.5 days and DNA- DNA and DNA-protein crosslinks with a half-life

of about 36 hours, and there was no evidence for any degradation of DNA. These results strongly support the hypothesis that damage present on the DNA template at the time of entry into the proliferative cycle is responsible for cellular toxicity. Further, the results show that the repair process is effective in achieving biological recovery: they do not however indicate that a particular lesion in DNA was specifically responsible for toxicity.

When, however, attempts are made to relate the inherent drug sensitivity of a given cell line to its repair capacity clear-cut results do not generally emerge. Walker 256 carcinoma cells display an tmusual sensitivity to a variety of bifunctional agents, including cis-DDP, but not UV- or X-irradiation or to monofunctional agents. A subline of this tumour shows a 30-fold relative resistance to cis-DDP. Nevertheless, following treatment with a given dose the two sublines bind the same amount of platinum to their DNA, they excise platinum lesions from their DNA at a similar rate, and they remove DNA-protein and DNA interstrand crosslinks from DNA at a similar rate [84]. A similar lack of correlation between crosslinking of DNA and cellular sensitivity has been found for lines of murine leukemia cells [85]. Another study of crosslink formation and removal indicates that while there is no significant difference between the crosslink removal rates in the parent and resistant lines, the kinetics are consistent with enhanced mechanism(s) for quenching the cis-DDP-DNA monoadduct in the resistant line [86]. Micetich, Zwelling and Kohn note that strand breaks do not accumulate in DNA during loss of Cisplatin-induced DNA adducts or DNA intcrstrand crosslinks. It can therefore be presumed that if, as seems likely, the loss of platinum or crosslinks is indicative of the operation of a cellular excision repair process, then subsequent steps in this repair process are equally efficient in both sensitive and resistant cells and restore the integrity of cellular DNA.

It is conceivable that the fidelity of DNA repair is defective in sensitive but not in resistant cells. This possibility can be studied by assaying for expression of bacterial genes that have been damaged in vitro prior to transfection into recipient cells. Knox et al. describe the mixed success of a study of DNA

271

repair in sensitive and resistant Walker tumour cells and reveal some possible difficulties with the system [22]. Expression of XGPRT is inhibited in a dose-dependent manner by cis-DDP treatment of pSV2gpt in both sensitive and resistant cell lines. Similar inhibition implies similar levels of plasmid repair in both cell lines, a result consistent with the findings in whole cells. More importantly, similar inhibition could imply comparable fidelity of repair in both cell lines. However. at a level of reaction with plasmid DNA that reduces the expression of XGPRT to less than 10% of the control level, fewer than 100 platinations were seen on the gpt region of the plasmid. Since DNA interstrand crosslinks occur with a frequency of only approximately 1% it follows that they cannot be the lesion in the plasmid DNA responsible for the inactivation of this genc: rather, the inactivating lesion is likely to bc the major product of reaction with the plasmid, namely an intrastrand crosslink. Therefore this transfection system is unable to compare the re- pairability of DNA interstrand crosslinks present in plasmid DNA by sensitive or resistant Walker cells. On the other hand it seems likely that it is the decreased ability of Walker sensitive cells to tolerate DNA interstrand crosslinks that is the basis for their unique sensitivity to difunctional agents [87].

The above considerations recall earlier observations in bacteria which showed that bacteriophage T7 and phage can be inactivated bv levels of plati- nation that do not produce, on average, one crosslinking event in their DNAs, and which therefore indicate that intrastrand erosslinks are likely inactivating lesions for these exogenous DNA molecules. Clearly the relevance of these in vitro sys- tems to the mechanism of the cytotoxic action of platinum compounds in mammalian cells must be questionable, but the transfection system still indicates the relative effcctivene~,s (in inactivating mammalian cells) of other (non-intcrstrand crosslinking) reactions with plasmid DNA. The level of reaction between the monofunctional platinum compound Pt(dien) and the above plasmid which was required to produce the same level of inactivation was approximately ten times that required with cis-DDP, paralleling the relative amounts of platinum bound to cellular DNA by

272

these compounds at equitoxic doses. These different levels of reaction are a reflection of a cell's ability to repair or tolerate certain types of damage to its DNA. It would therefore appcar that damage to exogenous DNA, introduced by some but not all agents, is rcpaired or toleratcd in a similar manner to that in genomic DNA. For such damaging agents it would seem possible to use the cxprcssion of transfected genes as a measure of the repair capacity of the recipient cell, as discussed carlier. How- ever further studies are required to validate this approach.

The relative sensitivities of Walker tumour cells and a derived resistant subline to cis-DDP and to a numbcr of other bifunctional agents is of thc order of twenty to thirty. Moreover it is apparent from the response of the resistant Walker cells that they have a sensitivity to cis-DDP and to various other bifunctional agents, such as sulphur mustard, that is essentially the same as that of a number of other normal or tumour cell lines. The responses of both cell lines to a number of monofunctional agents or to uv- or X-irradiation are about the same and similar to those of normal cells. Therefore the so-called resistant cells represent the prevalent phenotype, while the 'sensitive' cells are a mutant variant uniquely sensitive only to bifunctionai agents. Similarly, other experimental animal tumours such as the Yoshida sarcoma have the phenotype of mutant variants sensitive to specific types of DNA damagc [16]. In addition, a number of current studies [88, 89] compare the DNA repair characteristics of selected sensitive rodent cell variants (isolated by mutagcn treatment of normal cells followed by replica plating) with those of the parent cells. The sensitivities of these selected cells towards certain agents can even exceed that of the sensitive Walker cell to bifunctional agents. Such variants are potentially vital tools for the elucida- tion of DNA repair pathways.

Levels of complexity in mutagen sensitivity

The complexity of thc phenotype of some mutagen sensitive cell lines is illustrated by comparative studies on Chinese hamster V79 and its mutagen

sensitive derivative V79/79. This latter cell line is sensitive to X-rays, ultraviolet irradiation (UV) and bi and monofunctional alkylating agents. In contrast to the parental V79 line, 0~MeG was removed from DNA with a tL/2 of 23.2hrs but no 06-GAT activity was detectable. In the sensitive cell line (V79/79) various defects in DNA synthesis in both control and treated cells were detected using alkaline sucrose gradient centrifugation which overall suggested a defect in ligation of low to high molecular weight DNA [90]. In addition, after MMS and HN 2 exposure thc sensitive cells failed to dclay progress through the cell cycle [91]; delay in DNA synthesis appears to be associated with a tolerance mechanism to bypass DNA lesions prior to replication (see prcvious section).

Several differences exist between the sensitive V79/79 cell line and its parent, all of which presumably make varying contributions to its drug sensitive phenotype. In this rcspect it is important to remember that the V79/79 cell line was isolated as a slow growing survivor after X-irradiation and may thus carry multiple mutations and/or chromosome rearrangements.

A similar complex phenotypc has been described for UV-I isolatcd on the basis of hypersensitiviy to UV-radiation [88]. UV-1 is hyperscnsitive to some DNA methylating and cross-linking agcnts and moderately hypersensitive to other classes of mutagens. No differences in uptake and binding of la- bclled L~C MNU have been dctccted. Revertants of UV-1 are resistant to MNNG but still retain sensitivity to cross-linking agcnts. Fusion of UV-1 to 2 different UV sensitive CHO mutants results in hybrids resistant to mitomycin C but not to MMS. Methylation and cross-link sensitivity appear to be due to more than one genetic alteration. If this is so then it is difficult to understand how reversion occurs at measurable frequency unless the original variant is sensitive due to altered methylation patterns. In this context it is interesting to note that Xrs 'mutants' have been shown to revert to wild- type phenotype on exposure to 5-azacytidine, indicating that at least some repair genes are under methylation control [23].

Complex phenotypes are also evident in a series of 10 clones isolated as having greater than 5-fold

sensitivity to mitomycin C than wild type cells [89]. The mutants differ in their cross sensitivity to other DNA damaging agents (e.g., UV, cis-DDP, chlo- rambucil and melphalan). Only one mutant is UV sensitive and is also sensitive to the monofunctional decarbomyl mitomycin C. All clones MMC 1-5 have a recessive phenotypc and preliminary analysis indicates hat they fall into at least four complementation groups.

At least 130 UV and EMS sensitive mutants have bcen isolated [24, 92, 93] after mutagencsis of CHO cells with EMS and ICR 17(I. The UV sensitive mutants are assigned to 5 genetic complcmen- tation groups on the basis of their response to UV light, The majority (123"130) of mutants are assigned to groups I and 2 and the remaining 7 clones to another 3 complemcntation groups. The majority of the mutants show increased sensitivity to a varicty of othcr drugs, c.g., mitomycin C. An EMS sensitive mutant also shows a 10-fold increase in sensitivity to MMS. A 2-fold increasc in sensitivity was seen for ENU, MNNG and X-rays. When mutants from complementation groups 1-5 (UV sensitive) are fused with human lymphocytes the defects arc complemented by human chromosomes 19, 19, 2, 16 and 13 respectively. The complcmenta- tion of defects in groups 1 and 2 bv chromosomc 19 appears to be due to differcnt genes, as the phe- notypcs of these two groups differed markedly. Group I shows little or no hypcrsensitivity to DNA cross-linking agents, whcreas group 2 mutants are approximately 10-fold more sensitivc.

These studics in Chincse hamstcr cells parallel those in XP cells in which at lcast 8 complementation groups have now been identified indicating a level of complexity approaching that sccn in Sac- charomyces cerevisiae [94]. Indeed an even grcater level of complexity might be expected in mammalian cells than in lower eukaryotes when the higher order chromatin structure and thc possible preferential repair in some genes are taken into account. Some of the Chinese hamster UV-sensitive mutants have been used as recipients for transfection of human DNA in attempts to clone human DNA sequences which complcment the repair deficient phenotype. A cloned human repair gene ERCC-I has been located on chromosome 19 using

273

a panel of hamster x human hybrids and has been characteried at the molecular levcl [95-97]. It is 15 Kb long with 9 exons encoding two largely identical mRNAs of 1.0 and 1.1 Kb. These have been shown by sequencing the corresponding cDNAs to be largely generated by differential splicing. The function of the 297 amino acid protein encoded by the eDNA is still unknown. Although transfection of the eDNA in an expressing vector conferred UV and mitomycin C resistance to Chinese hamster cells it did not correct the repair defect in SV40 transformed XP cells (complementation groups A and F). The Chinese hamster mutants used are known to be excision defective. Thus although it seems to be possible to clone human DNA which complements repair defective hamster cell lines, the identification of the function of the gene is made more complex by the possibility that the recipient cell line carries multiple repair defects. We are obviously some wav from understanding the molecular biology of DNA repair.

Influence of chromatin structure on repair

Several lines of evidence now indicate that euk- aryotic DNA is arranged in higher order chromatin loops by attachment to the nuclear matrix [98]. Transcribing gene sequences are generally thought to be in close proximity to the nuclear matrix and biochemical and autoradiographic studies on single cells indicate that excision repair does not require the attachment of the DNA to the matrix. However in xeroderma pigmentosum cells belonging to group C(XPC) DNA near to nuclear matrix was prcferentially repaircd 199 i. This non-random distribution was not found in a number of other cell lines possessing low excision repair capacity (e.g.. XP group D. Syrian hamster embryo cells. HeLa cells) [98].

Other studies have demonstrated preferential repair of pyrimidine dimers induced by UV-irradiation [100, 101]. Both CHO and mouse cells remove few dimers from their genome overall. How- ever. dimers are removed efficiently from the dihydrofolic acid reductase gene (dhfr), the HMGCoA reductase gene in CHO cells and from

274

the transcribed c-abl, but not from thc silent c-mos genes in mouse cells [102]. In rcpair-proficient human cells thc removal of dimers appears much more rapid in the dhfr gcne than in the genomc as a whole. But in XPC cells repair is as deficicnt in the dhfr gene as in the genome as a whole.

The enhanced repair of essential active genes relative to the genome as a whole may play a significant role in overall survival of some cell lincs particularly in vitro where probably many genes are not actively transcribed. What happes in tumour cells in vivo is still unknown, as techniques for examining this problem are only just being developed.

q'hc subtleties of control of this apparently selective repair arc far from being understood, as are the possible roles such factors may have in the development of drug resistant cell lines. In several pairs of drug sensitive and resistant lines, in which no differences in the total amounts of DNA excision repair have bccn detected, it is possible that there have been redistributions of thc normal pattern of repair prefcrences or changes in chromatin structure which affcct the efficiency of repair. Now that probcs for a number of specific gcnes are available, it is technically feasible to look at pattern changes and repair preferences. The intragenomic heterogeneity in processing DNA damage is thought to be one of the factors that explain why Chinese hamstcr cells, which remove only 10% of UV-induccd pyrimidinc dimers, arc as resistant to UV induced cytotoxicity as the significantly more repair-proficient human cells [100]. The sequence specificity of DNA damage by alkylating agents is only just beginning to be explored. The relative extent of alkylation at any given guanine residue depends on the sequcnce flanking that site [103-105]. In the case of melphalan (phenyl alanine mustard) the alkylation of guanine is depressed by a 3' flanking cytosine. In a GpG sequence the 3' guanine is alkylated 2-3 times more than the 5'G except when in the sequence GpCpC when thc 5' guanine is preferentially alkylatcd. The GpG, Gp(3) and Gp(4) sequences are alkylated to a greater extent than isolated guanines by all the nitrogen mustards tested. Many of the 'housekeeping" genes (HPRT, dhfr, Tk) and a number of viral transforming genes

(c-Ha-ras 1 proto-oncogene) havc large G-C rich regions in their 5' flanking regions which could act as 'hot spots' for mutation in non-essential genes (HPRT) or cytotoxicity in essential genes (dhfr). Preferential alkylation of 5' flanking sequences in proto-oncogenes could result in mutation or al- tercd expression of such genes, which may be the molccular mechanism underlying increases in ma- lignancy observed in some cases when tumours become refractory to chemotherapy.

Superimposed on this specificity at the DNA level are factors operating at higher levels of DNA organisation which may also be important. Prefer- ential binding of a number of carcinogens to B DNA (aflatoxin BI) or Z DNA (N-hydroxy AAF) has been demonstrated, as has preferential binding to linker DNA as opposed to nucleosome DNA. The functional significance of such specific effects remains to be elucidated. DNA conformation can also modulate the extent of DNA repair as has been shown to be the casc for 0~-GAT. An alternating polynucleotide can undergo conformational transition from thc B to the Z form. The E. coli 06-GAT efficiently removed 0O-guanine residues when the polynucleotide was in the B form but repair activity was totally inhibited on transition to the Z form [106].

Cell metabolism and other factors which may influence sensitivity

The metabolic balance within the cell will also be of prime importance in its overall survival, and the precise mcchanisms involved in the coupling of DNA repair processes to DNA replication are still far from understood.

Intracellular pool sizes

Exposure of CHO-KI cells and other cell lines to chemical mutagens results in a rapid imbalance of cellular dCTP and dqq'P concentrations and recov- cry to normal Icvels takes several hours [107]. Dur- ing the recovery period dNTP pool imbalances prc- sumably make a contribution to the reduction of

semi-conservative replication. Addition of CdR (2 mM) results in a 2-3 fold greater depression of DNA synthesis and the time to rccoverv is corre- spondingly extended. The body of data revicwed in [108-110] indicates that post-treatment of cells with non-toxic concentrations of TdR (also known to produce imbalance of dNTP pools) can potentiate the lethal effects of monofunctional alkylating agents but not of UV or nitrogen mustard, suggesting that such effects could interfere with DNA repair.

Drugs other than thymidine, such as hydroxyurea, which inhibits ribonucleotide reductase, or 5-fluorouracil, which inhibits thymidylate synthetase, can also have profound effects on intracellular pool sizes and may potentiate or protect from the lethal effects of subsequent exposure to alkylating agents. Such interactions may be important in the development of non-responsive tumours. The importance of differences in the control of dNTP pools in drug resistant and sensitive cells is an area which is at present relatively unex- plored but is of obvious importance in some pairs of cell lines in which different rates of recovery from post-treatment DNA synthesis inhibition may reflect differences in pool sizes.

Activation of poly (ADP ribose) polymerase

Other metabolic perturbations which occur as a result of DNA damage (eithcr direct strand breaks or brcaks generatcd during excision repair [111, 112]) includc the activation of the chromatin bound enzyme (poly ADP ribose) polymerase. The cn- zyme uses NAD as substrate and its continued activation can lead to drastic depletion of NAI) and ATP pools. The activation of the polymerasc and utilisation of NAD arc dependent on the amount of DNA damage induced. The cffccts are reversible, however, if nucleotide pools arc sufficient to per- mit regencration of ATP. Cells of different lineage (normal and tumour cells) may show large diffcrcn- ces in their intrinsic ability to survive such metabolic alterations. Tumour cells with their greater genetic diversity and often polyploid karyotype may be better fitted to survive such metabolic dis-

275

turbances and this factor may contibute significantly to the rapid development of drug resistance in tumours.

Hypomethylation

The two carcinogens MNU and MNNG which methylate DNA at the (16 position of guanine have been shown to causc extensive hypomethylation of DNA of Raji human lymphoblastoid cells two days after addition of the drug. The lower level of methylation pcrsists for several cycles after treatment [113]. Treatment of K562 human erythroleukemia cells with CCRG 81045 an antitumour imidazo- tetrazine causes an increase in number of cells pro- ducing hemoglobin after 3 days and this incrcase is directly proportional to the decrease in 5Me cytosine content in [14] DNA. All ethyl analogue (CCRG 82019) failcd to cause any change in 5MedC content. These observations suggest a direct correlation between hypomethylation and the induction of a differentiated phenotype in K562 cells. One possible mechanism for hypomethylation of DNA is the insertion of thymidine in place of cytosine opposite I)6 alkylated guanines. In support of this hypothesis, evidence has been presented indicating that the ratio of incorporation of 5-(methyl) thymidine to cytidine rises significantly in CCRG 8103,5 treated cultures after 3 days. The critical result of such a transition is loss of potential sites of enzymatic methylation and hence disor- dered gene expression [112].

Alkylation of DNA may also impair the action of proteins which recognisc specific sequences in DNA. Reaction of DNA with MNU has been shown to impair the ability of restriction enzymes to cleave this substrate. Since enzymatic methylation is catalysed by sequence specific enzymes, alkylation in the parental strand may result in an aberrant methylation pattern in the progeny strand. It has been suggested that N-7 guanine alkylation, a far more frequent event than 0<al - kylation, may be an important lesion in this respect. This area has been reviewed recently [7.8]. Changes in DNA methvlation patterns have also been reported after exposure of cells to cytosine

276

arabinoside, hydroxyurea (hypermethylation) and UV irradiation (hypomethylation). UV irradiation has also been shown directly to activate the tran- scription of thc metaliothionine gene which, on the basis of experiments with azacytidine, is thought to be under methylation control [114]. Hypermcthyla- tion after cytosine arabinoside and hydroxyurea exposure appears to require direct inhibition of DNA synthesis. Indirect inhibition via cyclohcx- amide-mediated inhibition of protein synthesis or by depletion of growth factors by serum depriva- tion did not result in hypermethylation.

In cells exposed to alkylating agents and other drugs used in chemotherapy extensive changes in methylation patterns may lead to changes in gene expression, either silencing of transcriptionally active genes or activation of those previously re- pressed. Whethcr any of these changes contribute to drug induced cytotoxicity or to the development of drug resistant phenotypes and increased malig- nancy remains to be determined.

Conclusions

Drug resistance (intrinsic or acquired) is commonly considered to be of genetic origin. Available evidence indicates that defined identifiable genetic changes are involved in many types of drug resistance, particularly in the case of resistance to anti- metabolitcs where it can be due either to deletion or mutation of an enzyme necessary for incorporation of the drug into DNA (HPRT for resistance to 6-thioguaninc or 6-mercaptopurine) or amplifica- tion of a target enzyme (dhfr in the case of meth- otrexatc). In cell lines sensitive or resistant to alkylating agents or other drugs which covalently interact with DNA, available evidencc also suggests their mutational origin, that is, they show long term genetic stability in the absence of selective pressure. However, the exact mutations involved have yet to bc idcntified.

Exposure to DNA-damaging agents also triggers a highly complex chain of epigenetic events involving multiple changes in cell metabolism, including inhibition of DNA synthesis and DNA mcthylation with resultant changes in gene expression. Which

of these changes contribute to cell dcath is as yet unclcar; indeed it is probably the synergistic effect of many events which is ultimately lethal. This level of complexity has so far precluded any precise identification of a causal relationship between altered DNA rcpair capacity and altered cellular sensitivity. The correlations exist but most drug resistant (or sensitive) ccll lines exhibit other changes intrinsic or induced, transient or permanent which may contribute equally to their altered phenotypc.

It is important to know whether cells of a human tumour can possess a sensitivity to drugs which is comparable to that of the highly sensitivcr odent cell variants and whether tumours exhibit a comparable decrease in sensitivity following chemotherapy, due either to selection of pre-existing resistant cells or to mutational change. To our knowledge it has yet to be demonstrated that cclls of a primary human tumour can have a sensitivity to cytotoxic agents that cquates with that of the sensitive rodent variants, other, that is, than those occurring in individuals suffering from a genetically determined repair defcct. Likewise it has also yet to be demonstrated whether a so-called resistant human cell derived from an originally sensitive tumour has a sensitivity which equates with 'nor- real cells' or whether it is more resistant than normal cells due to acquisition of repair processes.

One of the main difficulties in assigning a defini- tive role to specific DNA lesions in the cytotoxic and mutagenic effects of DNA damaging agents is that comparisons of differentially sensitive cells are not comparisons of otherwise isogcnic cell lines. Similarly many of the UV and drug sensitive variants that are available have been isolated after mutagencsis and could carry many mutations other than those conferring the DNA repair defect selected for.

Thc response to and recovery from DNA damage is governed by a large number of genes involved not only in excision repair, but in DNA replication, control of poly ADP ribose activity and lcvcls of intraccllular DNA pools. Small differences in expression of one or more of these genes may make a profound difference to cellular sensitivity. The transfection of cloned DNA repair genes into freshly cloned cell lines of rodent or human origin

which lack specific repair funct ions may allow

more detai led analysis of such complex interac-

t ions.

It is to be hoped that the appl icat ion of the

powerful tools of r e combinan t D N A technology

will in the nea r future help to elucidate some of the

complexit ies of the drug resistant phcnotypes dis-

cussed, and that such knowledge when available

can be applied in the clinic.

Key unans~vered questions

I. How closely are excision repair and replicat ion

coupled and what is the mechanism of this coupling

at the molecular level?

2. What are the precise funct ions of the proteins

encoded by the m a m m a l i a n repair genes c loned to

date?

3. How many genes are involved in D N A repair in

m a m m a l i a n cells?

4. Is the cytotoxicity of D N A damaging agents dttc

directly to lesions in D N A and as a corollary, is

resistance duc to more efficient repair or do other

events , e .g. , dcmethv la t ion of D N A , and per turba-

t ions of cellular pools play an impor tan t role?

5. Are D N A repair genes, in part icular that for

06-(}A'I" inducible in m a m m a l i a n cells?

6. Do sensit ive and resistant cells differ in their

abil i ty to tolerate damage to D N A and what is the

precise role of D N A repair?

Acknowledgements

The authors are suppor ted by grants from the Med-

ical Research Counci l and the Canccr Rcscarch

Campaign .

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Addre.~,~ .for oJfprinLs: M. Fox. Paterson lnstilule for Cancer Research, Christie Hospital amt Holt Radium Institute. Man- chester M20 9BX. United Kingdom

drug resistance and dna repair

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