Keynote address: Mechanisms of cellular resistance to cytotoxic drugs and X-irradiation

Inr. J Radiation Oncology Eiol Phys., Vol. 20, pp. 197-202 0360-3016/91 S3.00 + .OO Printed 8” the U.S.A. All rights reserved. Copyright 0 1991 Pergamon Press plc

l Session 1

KEYNOTE ADDRESS: MECHANISMS OF CELLULAR RESISTANCE TO CYTOTOXIC DRUGS AND X-IRRADIATION

JAMES CARMICHAEL, M.D.’ AND IAN D. HICKSON, PH.D.~

‘Imperial Cancer Research Fund, Department of Clinical Oncology, Churchill Hospital, Headington, Oxford OX3 7L.l; and ‘Imperial Cancer Research Fund, Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DU, U.K.

Lack of response to X-irradiation and cytotoxic chemotherapy is the major cause of treatment failure in patients with cancer. However, “resistance” to these modalities may be considered a normal cellular response. The ability to cure patients with particular tumor types may be related to hypersensitivity to these modalities caused by loss or abnormality of certain normal cellular constituents such as enzymes involved in DNA repair. It is likely that the initial chemo or radiosensitivity of a tumor will broadly reflect the intrinsic resistance of the tissue type from which the tumor arose. There are many cellular biochemical mechanisms responsible for this relative resistance to drugs and radiation. Many of these mechanisms, although present in normal tissues, may be inducible and can result in enhanced resistance to DNA damaging agents. Although certain resistance mechanisms would appear to be specific for drug resistance or for radiation resistance, there are other resistance mechanisms that potentially affect both modalities. In particular, the study of DNA repair genes in mammalian cells may give us greater insight into common mechanisms of resistance to these modalities.

Chemotherapy resistance, Radiation resistance, Detoxification, DNA repair, Topoisomerases.

INTRODUCTION

The outlook for the majority of patients who develop common solid tumors remains bleak despite the use of intensive combined modality approaches. Significant im- provements in cure rates have been seen in particular his- tological types such as germ cell neoplasms and Hodgkin’s disease, but the results of treatment of more common malignancies such as lung cancer or carcinoma of the gastrointestinal tract have been much less dramatic. Al- though in the majority of cases the main cause of death is the failure to control the growth of metastatic disease, in approximately 30% of cases failure to control the disease at the primary site is the major cause of death despite surgery and/or radiotherapy. Thus “resistance” to chemotherapy and/or radiation therapy is a major clinical problem in the control of tumor cell growth. However, it should be emphasised that most normal tissues are also resistant to these modalities, with specific organ failure a rare event. Many of the mechanisms described are present in normal tissues, offering a protective function. It is likely that these resistance mechanisms may be inducible both in normal and tumor cells following exposure to various stresses.

A number of mode1 systems have been established to investigate the basic cellular mechanisms involved in the development of resistance to these treatment modalities. The majority of studies addressing this issue have used in vitro mode1 systems, although a limited number of in vivo models have also been studied. Large numbers of cell lines are available which exhibit a wide range of sensitivity to cytotoxic drugs. They range from hypersensitive mutants to cell lines highly resistant to a wide range of cytotoxic drugs.

Studies of cellular responses to radiation are more complex and more difficult to define. In contrast to in vitro derived drug resistance, where high levels of drug resistance can be achieved, induction of radiation resistance has been almost uniformly unsuccessful. Courtenay described a resistance mode1 using continuous low dose irradiation in L5 178Y cells, where 5-fold resistance was observed, although it should be emphasised that the parental cell line was extremely sensitive to X ray therapy (8). Other groups have been less successful. There are many human tumor cell lines which exhibit some vari- ability in radiation sensitivity, although marked “resistance” has not been observed. However, with multiple fractionation schedules these differences could be clinically

Presented at the Third International Conference on the In- teraction of Radiation Therapy and Systemic Therapy, Mon- terey, California, March 1990.

Reprint requests to: Dr. J. Carmichael. Accepted for publication 24 August 1990.

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198 I. J. Radiation Oncology 0 Biology 0 Physics February 199 1, Volume 20, Number 2

significant. Hypersensitivity to X-irradiation is more clearly defined. Ataxia telangiectasia (AT) is the only human genetic disease consistently conferring cellular hypersensitivity to X rays. This cancer-prone disease is apparently caused by an abnormality in DNA repair, although the precise defect has not been identified. In addition to AT cell lines, there are laboratory-isolated rodent cell mutants with an AT-like phenotype, both in terms of X ray sensitivity and radio resistant DNA synthesis. Defects in the ‘AT’ genes lead to sensitivity to a number of free radical generating drugs, including the antitumor agent, bleomycin.

There are many factors that can influence the response of tumor cells to cytotoxic drugs and radiation, as listed in Table 1. This review focuses on the biochemical mechanisms involved in the resistance phenotype.

DISCUSSION

Cellular biochemical mechanisms Resistance to anti-metabolites. There are many poten-

tial mechanisms for the development of resistance to an- timetabolites, including altered intracellular transport, altered or amplified target enzymes, altered drug metabolism, or alteration of intracellular nucleotide pools. These mechanisms have been summarized recently (18).

Multidrug resistance. Multidrug resistance (MDR) is an important mechanism in the resistance of cells to a wide range of chemotherapeutic agents. This resistance is associated with the expression of a high molecular weight protein p-glycoprotein in the cell membrane. This protein acts as an energy dependent efflux pump for a range of cytotoxic drugs such as the anthracyclines and epipodo-

Table I. Factors involved in the response to svstemic treatment

1. Host factors (a) Patient performance status (b) Tolerance of normal tissues (c) Pharmacokinetics of drug administered (d) Dose intensity of drug administered (e) Chemotherapy sanctuary sites (f) Vascular supply-drug access

2. Tumor-related factors (a) Tumor size (b) Tumor cell growth fraction (c) Hypoxic fraction (d) Tumor pH

3. Biochemical mechanisms affecting response (a) Anti-metabolites, e.g.:-altered transport

-amplification or alteration of target enzymes

-salvage pathways (b) Multidrug resistance-p-glycoprotein expression (c) Detoxification mechanisms-GSH/GST;

Merallothioneins (d) Topoisomerases (e) DNA repair (f) Hypoxia (g) Inducible responses

phyllotoxins. High levels of expression have been observed in intrinsically resistant tissues, and the possibility that acquired resistance relates to induction of MDR has been suggested (19, 32). However, in isolation, MDR appears to have no specific role in resistance to many other anti- cancer drugs such as bleomycin, cis-Platinum or to radiation (27).

Detoxljication mechanisms- glutathione and glutathione transferases. Glutathione (GSH) has been implicated in resistance to a number of cytotoxic drugs, in particular alkylating agents and cisplatinum. There are a number of potential mechanisms by which GSH may affect cellular response to drugs and radiation. These include conjugation of electrophilic compounds, frequently cat- alyzed by the glutathione transferases (GST). In addition, GSH can detoxify oxygen-induced free radicals and or- ganoperoxides using GSH peroxidases (33). Modulation of GSH levels has been shown to affect cellular response to many cytotoxic drugs, with elevation of levels normally associated with increasing resistance and lowering of GSH levels by various techniques shown to increase sensitivity to many drugs (28). The involvement of GSH in the response to radiation is less clear cut. GSH depletion has been shown to radiosensitize hypoxic cells and can en- hance the hypoxic radiosensitization induced by nitroimidazoles. However, variable responses have been observed with aerobic cultures, as summarized by Mitchell et al. (29).

The GST’s are a multigene family of isoenzymes which have been implicated in resistance to a wide range of toxic compounds and carcinogens in many species (22). A number of GST genes have now been cloned and studies are at present underway to evaluate whether high expression of particular GST isoenzymes produces resistance to specific drugs in transfected cells. The role of GST’s in resistance to X-irradiation remains at best in doubt. It has been suggested that GST’s may be involved in the repair of thymine hydroperoxides and a rat nuclear GST with high activity against DNA hydroperoxides has been isolated (24).

DNA topoisomerases. DNA topoisomerases are nuclear enzymes that catalyse the inter-conversion of topological forms of single and double-stranded DNA. They are essential for several processes in DNA metabolism, including DNA replication, transcription, and recombination.

Topoisomerase II. The type II topoisomerases in bacteria and yeast (respectively termed DNA gyrase and DNA topoisomerase II) are ATP-dependent enzymes essential for cell growth and cell division. The mammalian cell counterpart, DNA topoisomerase II, is presumably also required for proliferation. This is because of its apparently essential function in the segregation of daughter chro- mosomes (41). The normal catalytic activity of topoisomerase II can be viewed as comprising several distinct steps (30).

There has been considerable interest in the role of topoisomerase II as a target for certain classes of antitumor

Mechanisms of cellular resistance 0 J. CARMICHAEL AND 1. D. HICKSON 199

agent. These agents include anthracyclines, amino acri- dines, anthracenediones, and the epipodophyllotoxins. These drugs act by freezing the normal catalytic activity of the enzyme, producing the so-called ‘cleavable complex’. This complex consists of topoisomerase II mono- mers bound covalently to the 5’ termini of nicked DNA. The drugs consequently prevent strand passage and reli- gation. These protein-associated DNA lesions may be directly toxic, but it is more likely that an interaction with DNA replication or some other process in DNA metabolism is required. This is suggested by the S-phase speci- ficity of killing by the topoisomerase II inhibitors (26).

The initial response of tumors to topoisomerase II inhibitors will depend upon a number of factors, including presumably topoisomerase II enzyme activity. Cell types expressing high levels of topoisomerase II are in general more sensitive than cell types with low levels, and it is suggested that estimation of levels in tumors may at least partially predict response to inhibitors.

The phenomenon of resistance to topoisomerase inhibitors in tumors has only recently been studied in any detail, although it is recognized that most of these drugs are transported by p-glycoprotein. Obviously, because topoisomerase II is directly involved in the formation of DNA strand breaks and is an essential component of the cell killing mechanism by these drugs, it presents a very attractive target for antitumor therapy. It follows that un- derstanding the relationship between activity and cell killing is important. Davies et al. (10) showed that the level of expression of topoisomerase II in Chinese hamster ovary cells directly correlates with their resistance to inhibitors. In mutant overexpressing topoisomerase II, it was found that higher levels of DNA damage were induced by topoisomerase II inhibitors leading to hypersensitivity compared to parental cells. A number of rodent and human tumor cell lines with resistance to topoisomerase II inhibitors have been described. The resistance mechanism, where it has been studied, appears to be the result of a decrease in the activity of topoisomerase II (9, 17, 43). Reduced levels of topoisomerase activity in cells can occur by a variety of different mechanisms, including abnormalities in gene expression due to methylation (37) mutations in the topoisomerase II gene which result in abnormal expression of the enzyme (1 l), or reduced enzy- matic activity of topoisomerase II (36). These mutants have been isolated relatively easily by a number of different laboratories; therefore, it is likely that mutations in the topoisomerase II gene may arise in tumors during clinical treatment. It may be possible to upregulate the expression of topoisomerase II, thereby potentially increasing the sensitivity of tumors. This may be achieved by several mechanisms including changing the degree of topoisomerase II gene methylation, or induction of topoisomerase II gene expression (e.g., by estrogen stimu- lation in breast cancer cell lines).

DNA topoisomeruse 1. DNA topoisomerase I is also a potential target for chemotherapy. The range of antitumor

agents that act via this enzyme is restricted, with camp tothecin the only agent characterized in any detail. Camptothecin is a plant alkaloid that inhibits topoisomerase I at the cleavable complex stage, although in this caSe the enzyme remains bound to the 3’ end of the DNA. Camptothecin was briefly tested in Phase I clinical trials some years ago, but dose limiting toxicity was observed. However, a recent paper by Giovanella et al. ( 16) indicated that DNA topoisomerase I may be an important target for chemotherapy. These authors found that an analog of camptothecin, 9-amino camptothecin, could induce re- missions in human colon cancer xenografts. This was suggested to be a consequence of increased topoisomerase I levels. A limited number of camptothecin resistant cell lines have been developed. Detailed studies have shown that these cells express an abnormal camptothecin resistant form of topoisomerase I (3, 20).

Treatment of cells with ionizing radiation induces a variety of lesions in DNA. Of particular interest are single and double-strand breaks, DNA protein cross-links, and DNA base damage. These lesions are regarded as being potentially cytotoxic because cells held under non-growth conditions recover viability, possibly as the result of in- ducing DNA repair functions. This so-called potentially lethal damage repair (PLDR) leads to a significant increase in survival of irradiated cells over several hours. A number of studies have indicated that the repair of double-strand breaks in DNA is an important component of PLDR. PLDR capacity is an important determinant of the radiation sensitivity of cells in culture, and presumably of malignant tumors. A number of agents have been reported to reduce cellular capacity to carry out PLDR and among these are two compounds that interact with topoisomerase I; camptothecin and @-lapachone (6). The fact that both drugs can inhibit DNA repair capacity in cells (and radiosensitize cells), is evidence that topoisomerase I is involved in the repair of DNA strand breaks induced by ionizing radiation. However, these compounds act by apparently different mechanisms (&lapachone activates the enzyme), and therefore it is not clear at this stage which function of topoisomerase I is crucial for DNA repair, or whether topoisomerase I acts directly or indirectly in DNA repair.

If it is confirmed that DNA topoisomerase I is both an important target for anti-tumor agents and intimately involved in cellular response to radiotherapy, it may be possible to use topoisomerase I inhibitors both as chemotherapeutic agents in their own right and as radiosensi- tizers.

DNA repair as a mechanism of drug and radiation resistance. A variety of studies have shown that DNA repair is a crucial factor in cellular survival following X-irradiation. Despite this, it has been shown that enhancing the DNA repair capacity of cells to produce X ray resistant variants occurs either at very low frequency or not at all. However, there is evidence that the DNA repair capacity of cells can be modulated and most of this work has involved the use of the anti-tumor agent cis-Platinum.

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Resistance to cis-Platinum has been shown to develop via a number of different mechanisms. Reduced accu- mulation and alteration in cellular thiol (GSH or metal- lothionein) levels have been implicated in several studies (5, 14, 25, 31, 39). However, certain platinum resistant cell lines show no apparent abnormality in either of these factors and accumulate identical levels of DNA platination to that seen in their sensitive counterparts (12, 34). This suggests that the resistant cells are either more efficient at DNA repair or able to tolerate platinum lesions in the DNA. Eastman and Shulte (12) described a murine leukemia L 12 10 cell line resistant to the toxic effects of cis- Platinum. The resistant L12 10 cells were able to tolerate 50-times more DNA platination and removed four times more platinum adducts during the rapid phase of DNA repair than did the parental sensitive cells. In these resistant cells, a clearly enhanced rate of repair of intra-strand cross links at GG pairs was demonstrated. Intra-strand cross links form the bulk of adducts induced by cis-Plat- inum and are implicated in the cytotoxic action of the drug. Enhanced repair of, presumably, intrastrand cross linked adducts was also reported by Behrens et al. (5) in a cis-Platinum resistant human ovarian cell line. This was shown indirectly by assessing unscheduled DNA synthesis which reflects repair patches generated after excision of bulky adducts.

If the DNA repair capacity of cells is a significant factor in the acquired resistance of tumor to cis-Platinum, it follows that inhibition of DNA repair may be a useful adjunct to platinum therapy. In support of this suggestion, Hamilton et al. (21) found that aphidicolin could overcome resistance in the human ovarian cell line described above. Aphidicolin is an inhibitor of DNA polymerases (Y and 6, with the latter probably being the important target for inhibition of repair synthesis following cis-Platinum adduct excision.

There are suggestions that the inherent DNA repair capacity of human cells in vivo is an important factor in their sensitivity to cisPlatinum. Bedford et al. (4) showed that highly cisPlatinum sensitive testicular tumor cells had a reduced capacity to repair platinum adducts than relatively cis-Platinum resistant bladder tumor cells. Pre- liminary data indicate almost complete deficiency in excision repair of platinum adducts in cell free extracts from at least one testicular tumor cell line (Harnett and Hick- son, Jan 1990, unpublished results). This would be in line with the known sensitivity of testicular cancer to cis-Plat- inum containing protocols.

Where it is being studied, it does not appear that the c&Platinum resistant cell lines exhibiting enhanced repair show cross resistance to other DNA damaging agents which produce bulky chemical adducts. This suggests that the observed enhanced repair is not the result of a general improvement in excision repair capacity but some far more specific repair event relevant to platinated DNA. Eastman and Shulte ( 12) suggested that a mutation in a specific repair enzyme, perhaps in an enzyme subunit

crucial for platinum adduct recognition, may be responsible; this remains to be confirmed.

Hypoxia. It is well recognized that hypoxia confers cellular resistance to X-irradiation. It was hoped that the nitroimidazoles would overcome hypoxic cell radiation resistance, but normal tissue toxicity has prevented their widespread use. There is also evidence that hypoxia can modulate response to cytotoxic drugs. Alternatively, certain bioreductive drugs can be activated in hypoxic conditions, such as the quinone mitomycin C, the nitroim- idazole RSU 1069, and the benztriazene-di-N-oxide SR4233. These represent prototype compounds in three distinctly different structural classes that may offer a degree of selectivity against tumor cells.

Inducible responses. In bacteria and lower eukaryotes, DNA repair is an inducible phenomenon, the capacity being enhanced markedly following cellular insult. The SOS response in E. cofi affects numerous genes and in- volves increased production of DNA repair enzymes and other proteins involved in the maintenance of cellular integrity. In yeast, UV irradiation results in increased transcription of several DNA repair genes, including RADZ, RAD52, RAD54, and CDC9, which encodes DNA ligase (15). The SOS response can be activated by a wide range of DNA damaging agents or drugs which inhibit DNA replication. In contrast, the so-called adap- tive response is elicited by a far narrower range of DNA damaging agents, being largely specific for alkylating agents. This response leads to the enhanced synthesis of enzymes involved in cellular tolerance to DNA alkylation, including 3-methyl-adenine DNA glycosylase II and O6 methylguanine DNA methyltransferase (40).

Whether similar inducible responses to DNA damaging agents occur in mammalian cells is controversial. It has been suggested that 06-methyl transferase is inducible, and the recent cloning of the human gene should allow this question to be answered (38). It has been demonstrated clearly that the expression of several genes is increased following exposure to DNA damaging agents. However, unlike the analogous responses in bacteria in yeast, the vast majority of these inducible genes do not appear to code for enzymes involved directly in DNA repair. In contrast, DNA polymerase /3 is inducible by a limited range of agents, particularly monofunctional alkylating agents and peroxides, which fits with its known role in the repair of base damage induced by alkylating agents and peroxides (13). As mammalian repair genes are isolated, it is likely that more will be found to be inducible.

Growth factors/oncogenes. The expressions of various growth factor receptors and oncogenes have been associated with a poor prognosis in a number of clinical studies. In particular, studies evaluating neu expression in breast cancer have revealed a subgroup of patients at high risk of early recurrence (42). Preliminary studies show short relapse-free survival suggesting a potential lack of response to chemotherapy in these patients. In addition,

the use of blocking monoclonal antibodies to a growth factor receptor, the EGF receptor, resulted in enhanced sensitivity to cis-Platinum (1). Recent experimental studies have shown that transfection of certain activated oncogenes, such as ras and raf, resulted in increased resistance to cis-Platinum and was also suggestive for X-irradiation (23, 35). The degree of resistance achieved in these studies has been relatively minor, but further evaluation of these interactions may give us greater insight into the mechanisms of resistance and potential targets for drug development.

Heat shock. It is well known that exposure to thermal injury results in the synthesis of a number of proteins collectively termed the heat shock proteins. This results in protection not only from heat but from a range of other stresses, with adriamycin resistance frequently associated. Heat shock stress has been associated with increased expression of mdr 1 (7). Similar protection mechanisms are evident in the protection from repeated exposures to toxins such as cadmium, H202 and ozone, and in the priming response using cyclophosphamide (2), although resistance to radiation is not seen following these stresses.

CONCLUSIONS

Many mechanisms are involved in cellular resistance to cytotoxic drugs and to X-irradiation. P-glycoprotein and topoisomerase II are important proteins in resistance to cytotoxic drugs, but have no apparent role in radiation resistance. Resistance, although observed in many solid tumors, is also seen in many normal tissues, associated with normal levels of DNA repair enzyme expression. Abnormalities of DNA repair are associated with cancer proneness and drug and radiation hypersensitivity. Whether drug resistance is associated with enhanced expression of DNA repair enzymes remains unanswered. The role of topoisomerase I in resistance is unclear, but preliminary reports suggest an involvement in radiation resistance, and this protein may prove to be an important new drug target. This list is by no means comprehensive, and is merely an indication of the multitude of factors involved in the development of resistance to DNA damaging agents. Further studies into resistance mechanisms are indicated to identify new targets and develop novel treatment strategies.

Mechanisms of cellular resistance 0 J. CARMICHAEL AND 1. D. HICKSON 201

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Keynote address: Mechanisms of cellular resistance to cytotoxic drugs and X-irradiation

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