The role of glutathione-dependent enzymes in drug resistance

Pharmac. Ther. Vol. 51, pp. 139-154, 1991 Printed in Great Britain.All fights reserved

0163-7258/91 $0.00+ 0.50 © 1991 PergamonPress pie

Specialist Subject Editor: K. D. TEw

THE ROLE OF GLUTATHIONE-DEPENDENT IN DRUG RESISTANCE

ENZYMES

STEPHENM. BLACKand C. ROLANDWOLF Imperial Cancer Research Fund, Molecular Pharmacology Group, Hugh Robson Building, George Square, Edinburgh EH8 9XD, U.K. Abstract--Glutathione and glutathione-dependent enzymes are ubiquitously distributed through nature. These enzyme systems appear to have evolved to protect cells from toxic and mutagenic environmental chemicals. There is now unequivocal evidence demonstrating that these enzymes play a role in chemical resistance in a variety of phylogeny including, bacteria, plants and insects. There is also increasing circumstantial, as well as genetic evidence which indicates that these enzymes are also a determinant in the sensitivity of tumor cells to anticancer drugs, particularly alkylating agents and those drugs whose toxic effects are mediated by free radicals. In this review some of the experimental data which leads to these conclusions is discussed.

CONTENTS i. Introduction 2. Glutathione-Dependent Enzymes 3. Glutathione S-transferases (GST) 3.1. GST and intrinsic drug resistance 3.2. GST and acquired drug resistance 3.3. Mechanisms of GST overexpression 4. Glutathione Peroxidase (GPX) 5. Glutathione Reductase (GRD) 6. Gamma Glutamyl Transpeptidase (GGT) 7. Concluding Remarks Acknowledgements References

1. I N T R O D U C T I O N In the following review we shall try to make an objective appraisal of the evidence implicating glutathione and glutathione-dependent enzymes in drug and carcinogen resistance. It is important to point out first, howeverl that drug resistance is a subjective term which refers to the loss of the therapeutic effect of a specific dose of chemotherapeutic agent. In cancer chemotherapy because the drugs are used at the limit of the tolerated dose only small changes in drug sensitivity in the target cell will generate what is termed the 'drug resistant' phenotype. In other uses of chemotherapy, for example in the treatment of bacterial infections, in spite of the ability to administer much higher antibiotic doses, drug resistance can still be observed indicating that a profound change in the sensitivity of the target cell has occurred. The genetic changes which therefore induce the drug resistance phenotype in cancer patients may be quite small. On this basis changes observed in human tumors before and after the onset of drug resistance may well both be difficult to measure and complex.

139 140 140 140 143 147 148 148 149 149 149 149

As part of their normal diet or living environment all organisms are constantly exposed to harmful chemicals (xenobiotics) which are of no functional value. Consequently, the development of protection mechanisms against the stress imposed by exposure to xenobiotics has been an essential part of the evolutionary process (see Hayes and Wolf, 1990). Glutathione-dependent enzymes are known to be involved in these protective reactions (Larsson et al., 1983; Hayes and Wolf, 1988). Such reactions however, are only part of many complex pathways of cellular protection which include for example, D N A repair (reviewed in Sancar and Sancar, 1988) altered drug transport (reviewed in Endicott and Ling, 1989), D N A replication (Hickson and Harris, 1988) and stress response proteins (reviewed by Lindquist, 1986). In the context of cancer chemotherapy fundamental processes which normally protect us from environmental chemicals are the ones which are responsible for intrinsic or acquired drug resistance. These appear to be of particular importance in the latter case where tumors responsive to a chemotherapeutic regimen become refractory to further treatment. This invariably involves resistance to a wide range of structurally diverse compounds and as a consequence 139

140

S.M. BLACKand C. R. WOLF

is referred to as multidrug resistance. Over the last few years the role of glutathione-dependent enzymes in this phenomenon has been extensively studied.

2. G L U T A T H I O N E - D E P E N D E N T ENZYMES There is now unequivocal evidence that cellular glutathione levels are a determinant in sensitivity to cytotoxic drugs as well as oxidative and radicalinduced damage. In relation to cytotoxic compounds the protection against the alkylating agents and drugs which act through the generation of free radical intermediates is particularly important. Glutathione in its oxidized or reduced form has been implicated in a host of important metabolic functions including cell division, D N A repair, regulation of enzyme activity, activation of transcription factors, modulation of calcium homeostasis and protection against oxidative damage (Dolphin et al., 1989). Many of these functions are probably directly related to the central role of this peptide in maintaining a reducing cellular environment. As a consequence of these multiple functions it is not easy to delineate which is the most important for a particular compound. There are now several reviews on the role of GSH in resistance to cytotoxic drugs (Arrick and Nathans, 1984; Wolf et al., 1987b). The evidence that this peptide is an important part of the drug resistance phenomenon is based on a variety of observations: (1) the levels of many enzymes involved in maintaining glutathione levels are elevated in bacteria following oxidative stress (Christman et al., 1985); (2) the levels of many glutathione-dependent enzymes, as well as enzymes involved in the maintenance of cellular GSH, are increased in cell lines resistant to alkylating agents or drugs which generate free radicals; (3) drug resistant bone marrow or lung cells as well as drug resistant preneoplastic foci in rat liver exhibit elevated GSH levels; (4) depletion of cellular GSH levels sensitizes cells to the toxic effects of a wide range of cytotoxic drugs. Such drugs include alkylating agents, cis-platinum and adriamycin; (5) augmentation of cellular GSH levels, both in vivo and in vitro, protects against the toxic effects of these compounds. As a direct consequence of the role of glutathione in cytoprotection it is expected that the enzymes which interact with this peptide, or are involved in its homeostasis, will play a role in drug resistance. Some of these enzymes are discussed below.

their initial indentification it was suggested that the role of GST and GSH was in the protection of cells from cytotoxic and mutagenic electrophilic chemicals (Boyland and Chasseaud, 1969). In GST catalyzed reactions the sulfur atom of GSH acts as nucleophilic substrate. The resulting GSHconjugate is then either excreted directly or further metabolized to yield a mercapturic acid (Jakoby, 1982), the normal excretion products of GSHconjugates in the urine (Mannervik, 1985). In addition to this function the GST can also act as peroxidases and drug or hormone binding proteins. Such properties may also be important in conferring the drug resistance phenotype. GST are found in multiple forms and in all organisms so far examined from bacteria to man. The presence of a large number of isoenzymes within a species together with the conservation of these enzymes through evolution suggests that GST carry out critical biological function(s). The apparently central role of these enzymes in catalyzing the detoxification of toxic electrophiles will explain why these proteins have diverged into multigene families such that each new enzyme exhibits a novel range of substrate specificities. This may also explain the presence of GST forms in the cytosolic, mitochondrial, nuclear and endoplasmic reticulum compartments of the cell. There have been many reviews published on the substrate specificity and nomenclature of the GST multigene family (Chasseaud, 1979; Morgenstern and De Pierre, 1984; Mannervik, 1985; Mannervik and Danielson, 1988; Sies and Ketterer, 1988; Coles and Ketterer, 1990; Hayes et al., 1990a) and in the context of this review it is sufficient to say that on the basis of sequence homology the GSTs have been divided in mammals into five gene families termed alpha, mu, pi and theta (cytosolic forms) or microsomal (Table 1). The number of genes within each family varies considerably and ranges between 5-10 for the alpha and mu class GST and one (or two) genes for the microsomal and pi class GST. The level of expression of specific GST subunits is cell and tissue specific and is dependent on both hormonal, environmental and in some cases genetic factors (see Sies and Ketterer, 1988). Some evidence indicating that the GST are involved in cellular drug resistance is shown in Fig. 1. Drug resistance can be either intrinsic or acquired (see Hayes and Wolf, 1990). The essential difference between these two phenomenon is that in the former case the majority of the target cells are already drug resistant at the onset of treatment. In the latter case the majority of cells are drug sensitive, but there is a small proportion of cells within the population with the drug resistant phenotype which are selected for, or are induced by mutation. 3.1. GST AND INTRINSICDRUG RESISTANCE

3. G L U T A T H I O N E S-TRANSFERASES (GST) The focus of the majority of work on glutathionedependent enzymes in relation to drug resistance has been on the GST. These are a multigene family of structurally diverse proteins which catalyze the formation of drug or foreign compound glutathione (GSH) conjugates (Booth et al., 1961). Subsequent to

Intrinsic resistance to cytotoxic agents is widely observed in drug therapy and is not limited to refractory human tumors commonly being observed in parasitic organisms. As mentioned above, significant GST activity has been found in almost all organisms including bacteria, nematodes (Kawalek et al., 1984), cestodes (Douch and Buchanan, 1978) and

Glutathione-dependent enzymes and drug resistance

141

°~

AA~

"1

¢xl o~

g~

~C

zz~ o

¢:

142

S.M. BLACKand C. R. WOLF

helminths (Smith et al., 1986) and may therefore play a role in the resistance of parasites to chemotherapy. Many intrinsically resistant human tumors have elevated levels of GST relative to the surrounding normal tissue (Table 2). For example GST pi has been shown to be expressed at high levels in tumors of the colon, stomach, pancreas, uterine cervix and lung (Kodate et al., 1986; Sato et al., 1987; Peters et al., 1989; Carmichael et al., 1988a; Moorghen et al., 1990). Immunoblotting and m R N A determinations have shown GST pi is also increased in adenocarcinoma of the breast and lung as well as nodular small cell lymphoma, mesothelioma, chronic lymphocytic leukemia, acute myelobastic leukemia and myelodysplastic syndrome (Shea and Henner, 1987; Moscow et aL, 1989a; Forrester et al., 1990; Shea et al., 1990; Holmes et al., 1990; Schisselbauer et al., 1990; Howie et al., 1990). For an excellent overview see Castro (1991). It has been suggested that the elevated GST pi levels are a marker for drug resistance (reviewed by Sato, 1989) however direct evidence that GST pi overexpression is a contributing factor in the failure of chemotherapy in cancer patients remains elusive. It should be noted that in

MAMMALIAN

1)

2)

3)

addition to GST pi many human tumors express significant levels of other GST subunits (see Tew et al., 1987; Carmichael et al., 1988a; Harrison et al., 1989; Howie et al., 1990; Forrester et al., 1990; Peters et al., 1990; Table 2). Although many groups, including ours, have reported the relative difference in GST content between tumor and surrounding normal tissue from the same patient the significance of any differences observed are unclear. This is because: (a) both the tumor and the normal tissue are heterogeneous cell populations and changes in GST expression may simply reflect this heterogeneity. The consistently observed elevation in GST expression in most tumors (excluding, liver, stomach and kidney, see Howie et al., 1990) however does indicate that elevated GST is an important part of the tumor phenotype; (b) for essentially all solid tumors, toxicity to the normal cells in the target tissue is not the limiting factor in chemotherapy. Changes in the normal to tumor ratio as it relates to drug resistance is of no meaning. However, comparison of the tumor GST level relative to that found in the bone marrow or another susceptible non-target tissue may be of importance. In Table 2

CELLS

Cell lines selected for resistance to

OTHER

1)

ORGANISMS

Plasmid encoded GST are

agents often show elevated

involved in antibiotic resistance

GST isoenzyme content.

to formycin.

GST and GSH are increased when

2)

Genetic evidence and gene transfer

normal cell populations (e.g. bone

experiments demonstrating the role of

marrow) are "primed" with a cytotoxic agent.

plant GST in herbicide resistance.

In rat hepatocarcinogenesls increases

3)

in GST levels are coincidental with the

Genetic evidence demonstrating a role for GST in insecticide resistance.

appearance of drug resistant hyperplastic

nodules. 4)

5)

Modulation of GST in vivo. with for example

4)

Circumstantial evidence

antioxidants, can cause a profound

demonstrating elevated GST in

change In susceptibility to toxins and

numerous examples of drug

carcinogens.

resistance in different phylogeny.

Inhibition of GST sensitizes cells to cytotoxins.

6)

Elevated GST levels have been observed in human tumour cell lines after the onset of clinical drug resistance.

7)

Transfection or transler of GST Into cells can result in increased resistance to cytotoxic agents.

FIG. I. Evidence for a role of GST in drug resistance. References for much of the work referred to here is reviewed in Hayes et aL, 1990b; Wolf et al., 1990

Glutathione-dependent enzymes and drug resistance

+

+

~++++ ++~+++ ++ ++ +

-4-

+

a summary of some of our studies on normal and tumor GST levels and the expression of specific subunits is presented. Certain tumor types which do respond to chemotherapy, such as chronic lymphocytic leukemia and breast tumors, do have lower GST content than tumors refractory to chemotherapy, such as liver and colon. There is, however, considerable individuality in tumor GST expression and studies correlating individual GST levels with patient response are still needed.

++++ +++ +

~+

+ + ~ +~++++

I ++++ + +

~+ +++++~+~ ..~ ....+

+ + ++ + ++1

I

+~7-+ + ++

+

3.2. GST AND ACQUIREDDRUG RESISTANCE

O'X~ o-~ o t~

+1

+

+ + +

++++~7

7+

+ + +

o2 X

+ ++ +++ ~.~+ + + + +++ ++ +

~~

=

~ ~.~ Z

oN=

O

.~ ~.- ~

z

JPT 51/l--J

143

There are now several models which have been used to study the role of GST in acquired resistance. These include insects, plants (reviewed in Hayes and Wolf, 1988, 1990) and bacteria (Arca et al., 1988). Such studies provide compelling evidence for the importance of the GST in cytoprotection and drug resistance• In addition to these examples changes in GST expression have been extensively studied during neoplastic development in the rat liver (Farber, 1976). Farber (1984) has proposed that in this case the development of resistance is a critical step in the events leading from a normal hepatocyte to primary hepatoma. It has been shown that initially after feeding with a hepatocarcinogen there is an overall increase in alpha and mu class enzymes within the liver hepatocytes, specifically subunits 1 and 3 (Kitahara et al., 1984; Buchmann et al., 1985). As the preneoplastic loci develop subunit 7 belonging to the pi class of GST is also found to be expressed at high levels (Sato et al., 1984; Neal et al., 1988). Although many of these loci redifferer~tiate into apparently normal hepatocytes, those that develop into nodules and hence into neoplastic tumors, are rich in GST isoenzymes. Initially these hepatomas have the same overall expression of subunits 1,3 and 7 as the nodules. However, as the tumor progresses all but subunit 7 are repressed (Ketterer, 1988). It is this phenomenon which has led to the extensive studies into the expression of GST pi in human tumors and its role in drug resistance. The role of GST in the progression of the hepatocyte to fully developed hepatoma has not as yet been elucidated• However, as drug resistance has been proposed as an important link in these events (Farber, 1984) the observed overexpression of these GST isoenzymes may give the preneoplastic foci a growth, and hence, selective advantage over their normal neighbors. It has been shown that rat liver epithelial cells (RLE) transfected with the viral oncogens V-H-ras or V - r a f d e v e l o p a multidrug resistant phenotype (Butt et al., 1988). These transformed rat liver epithelial cells were shown to be more resistant to the chemotherapeutic agents adriamycin and vinblastine as well as the procarcinogen 2-acetylaminofluorene. The ability to activate transcription of the rat GST pi gene by phorbol esters has led to the proposal that the overexpression of GST pi on transfection with V-H-ras is related to the presence of a phorbol ester responsive element (TRE) present in the 5' upstream region of the rat GST pi promoter (Okuda et al., 1987; Cowell et al., 1988). Transfection of RLE cells with V-H-ras leads to the transcriptional activation of the endogenous GST

144

S.M. BLACKand C. R. WOLF

pi gene and can also lead to the activation of another major protein implicated in drug resistance, P-glycoprotein. It is interesting to note that although the rat GST pi gene is activated by ras and by phorbol esters, this is not the case for the human GST pi gene (Dixon et al., 1989; Morrow et al., 1990). It therefore appears that either complex reactions are involved in its regulation in man or that other transcription factors are involved in its activation in drug resistance. In addition to changes in GST expression in tumor cells during the development of drug resistance, similar changes have also been observed in normal cell populations. The mouse bone marrow has been examined in this respect (Adams et al., 1985; Carmichael et al., 1986; Wolf et al., 1987c). The bone marrow is of special interest due to its susceptibility to the toxic effects of the drugs used in modern chemotherapy. Indeed the bone marrow is often the origin of drug-induced secondary malignancies. It is known that a low dose of a cytotoxic drug has the potential to protect the bone marrow against a normally lethal dose of the same compound (Millar et al., 1975). Following administration of such a 'priming dose' of either cyclophosphamide, 1-fl-Darabinofuranosylcytosine (ara C) or X-irradiation, DBA mice were challenged with a normally lethal dose of cyclophosphamide. It was found that 5-7 days post-treatment (when levels of resistance were maximal) a 2-3-fold increase in both GSH and GST could be measured. The increase in GST activity appeared to be due to elevated expression of the mu class GST in the granulocyte population (Carmichael et al., 1986; Wolf et al., 1987c). It was proposed that the increases in GST caused by these diverse treatments may be explained if these proteins are regulated as part of a general 'stress' response induced when cells are exposed to cytotoxic insult (Adams et al., 1985). Such cellular responses have been studied in some detail and it has been clearly demonstrated that the level of GSH and the expression of certain GSH-dependent enzymes are elevated in both mammals (Kimball et al., 1976) and other organisms (Christman et al., 1985; Morgan et al., 1986) following oxidative stress. Hyperoxic conditions (90% 02) have been shown to induce glutathione (2-fold), glutathione peroxidase (3-fold), glutathione reductase (2-fold) and superoxide dismutase in the rat lungs 7 days after treatment (Kimball et al., 1976). A hydrogen peroxide resistant mutant of S. typhimurium (Oxy RI) has also been shown to overproduce glutathione and glutathione reductase (4-fold). This protein was found to be among the 12 proteins initially activated as a protection against oxidative stress (Christman et al., 1985). The finding that glutathione-dependent enzymes change under oxidative stress in such diverse organisms strongly supports the important role of these enzymes in protecting cells from the stresses imposed by the environment. Changes in GSH, GST and other GSH-dependent enzymes have also been measured following the onset of acquired drug resistance in vivo. In these studies two cell lines were derived from the ascites of a patient with ovarian cancer before (PEO1) and after (PEO4) the development of clinical resistance to a combination therapy involving cis-platinum, chlorambucil and 5-fluorouracil (Wolf et al., 1987a). The

PE04 cell line was shown to be 3-fold more resistant to c i s - p l a t i n u m and chlorambucil but equally as sensitive to 5-fluorouracil as PE01. An overall 2.9fold increase in GST levels were measured in the PE04 cell line (Lewis et al., 1988a). However, the lack of cross-resistance to 5-fluorouracil is interesting since it demonstrates that although GST levels may be important in determining tumor resistance they do not confer resistance to all anticancer drugs and clearly other mechanisms of resistance are also important. Indeed, it would be naive to expect a single enzyme system or cofactor to be involved in the resistance mechanism of all cytotoxic agents (see Hosking et al., 1990). Some of the most compelling evidence indicating that GST overexpression may be part of a stress response, and as a consequence, be involved in drug resistance has come from the generation of drug resistant cell lines in vitro. The changes in GST expression have been most marked in cell lines made resistant to alkylating agents such as chlorambucil, melphalan, nitrosoureas and 1-chloro 2,4-dinitrobenzene or to redox cycling drugs such as adriamycin. However, such changes are also observed in cell lines made resistant to many other agents including vincristine and VPI6 (Whelan et al., 1989). In the above studies all three classes of cytosolic GST have been shown to be overexpressed in cell lines resistant to cytotoxic agents (reviewed in Wolf et al., 1990; Wang and Tew, 1985; Robson e? al., 1986, 1987; Batist et al., 1986; Cowan et al., 1986; Evans et al., 1987; Smith et al., 1989; and Table 3). Clapper and Tew (1989) have also reported the overexpression of a 29kDa microsomal GST in a Walker 256 rat mammary carcinoma cell line resistant to nitrogen mustards. However, since most work has involved the characterization of cytosolic GST these will be dealt with in detail. The initial studies demonstrating the elevation in GST expression in drug resistant cell lines came from the work of Tew and coworkers. The study of a previously established rat mammary carcinoma cell line selected for resistance to bifunctional nitrogen mustards (Tew and Wang, 1982) showed that the alpha class GST Yc (subunit 2) was overexpressed (Buller et al., 1987). Since this initial study the number of cell lines studied, and the selection agents used, has multiplied considerably (reviewed by Wolf et al., 1990; Table 3). The complexity of such studies is exemplified by the important papers of Batist et al. (1986) and Cowan et al. (1986) where a human breast carcinoma cell line, MCF7, was selected for resistance to adriamycin. This resistant line exhibited a 45-fold elevation in GST activity which was associated with an increase in the expression of the pi class GST. Further examination of this cell line showed that a large number of other biochemical changes had occurred in the drug resistant cells. An overall decrease in intracellular levels of adriamycin was measured associated with an increase in P-glycoprotein. The activity of phase 1 enzymes such as cytochrome P-450 was decreased as estimated by arylhydrocarbon hydroxylase activity, while other phase II enzymes, such as the UDP-glucuronyltransferases were increased 2-3-fold. Also glutathione peroxidase activity was elevated 12-fold and protein kinase C activity

Glutathione-dependent enzymes and drug resistance

145

TABLE3. S u m m a r y o f Changes in G S T Expression in Drug Resistant Mammalian Cells GST Class Resistance to:-

Fold change" in GST activity

~t

#

x

Cyclophosphamide

3-8

NC

T

NC

Carcinogens, adriamycin

--

TT

TT

TT

Cell lines MCF7 Hu breast

Adriamycin (100)

45

NC

NC

MCF7 Hu MCF7 Hu MCF7 Hu Walker rat

Ethacrynic Acid Vincristine (14) VP16(3) Chlorambucil (15)

2-3 7 6 1.5

NC NC NC TT

TT NC NC NC

Rat glioma CHO Chinese Hamster Ovary NCH H322 Hu lung

Nitrosoureas (3-4) Chlorambucil (24)

0.4 3

NC •TI"

I'T NC

CDNB (3)

2-3

i"TT

NC

Myeloma Yoshida rat sarcoma FLC mouse leukemia

Melphalan Cyclophosphamide Adriamycin

1.5 6 1.3

NC ND T

NC ND NC

Drug priming (mouse) Preneoplasia

breast breast breast mammary

References Adams et al. (1985) Wolf et al. (1987c) Farber (1984) Sato et al. (1987) Buchmann et al. (1985)

I"TT Batistet al. (1986) Cowan et al. (1986) NC Wolf et al. (1990) I"TT Whelan et al. (1989) 1"i"T Whelan et al. (1989) NC Bulleret al. (1987) Tew and Wang (1982) ~,~ Smithet al. (1989) NC Robson et al. (1987) Lewis et al. (1988) T Wolf et al. (1990) Wareing et al. (in preparation) (T) Gupta et al. (1989) ND McGowan and Fox (1986) NC Schisselbauer et al. (1989)

*Measured using CDNB, 2-chloro-2,4-dinitrobenzene as substrate. Values in parenthesis are fold resistance (ICs0)to the selectiveagent relative to wild type cells. C = No change, ND = Not determined. See also Wolf et al., 1990a. increased 7-fold (Fine et al., 1988). These studies exemplify the difficulty in associating drug resistance with any specific phenotypic change and implies that many factors are involved in conferring the overall level of resistance. It can be seen from the data presented in Table 3 that all the known classes of GST have the potential to become overexpressed in drug resistant cell lines. The finding that different selective agents can result in the overexpression of different GST subunits in the same cell line (e.g. in the case of the MCF-7 cell lines where both pi and mu class GST can become overexpressed and the CDNB resistant lung carcinoma cell line NCI H322 where both alpha and pi class GST are found in higher concentration; Table 3) indicates that the GST subunit which is actually changed may depend on a range of factors such as the type of selective agent, how the selection is carried out, and the nature of the target cell. The capacity to induce different GST, dependent on the selective agent, indicates that the GST are part of the protection mechanism and not simply a marker for a stress response effect. There is currently significant debate as to whether the GST are indeed part of the mechanism of resistance towards anticancer drugs or whether their overexpression is a consequence of a drug-induced stress. The fact that the GST appear to have evolved as part of an adaptive response to environmental stress provides strong evidence that they confer protection against some toxic environmental stimulus. The question is whether this includes the anticancer drugs. Support for such a role stems from the observation that GST genes are amplified in a cell line resistant to the anticancer drug chlorambucil (Lewis et al., 1988). The detection of amplified GST genes provides very strong evidence for a role in the resistance mechanism, as such amplification events only arise

and become fixed in the population as a consequence of selective pressure. The other direct evidence indicating the capacity of the GST to protect against the toxic effects of anticancer drugs comes from studies involving the transfection of GST cDNAs into a variety of different cell lines. Table 4 summarizes the data which has been published in the literature so far where GST have been expressed in either S - c e r e v i s i a e (Black et al., 1990) or transiently (Manoharan et al., 1987; Puchalski and Fahl, 1990) or stably (Moscow et al., 1989b; Leyland-Jones et al., 1991; Nakagawa et al., 1990) in mammalian cells. It should be borne in mind however that the data by necessity is simplified and values are expressed as fold changes in the IC50, IC37 or IC90 value i.e. the dose of drug required to kill a certain proportion of the cells. This manner of expressing the data is arbitrary and differences in drug sensitivity have been observed from essentially none to very marked. The steepness of the drug dose-response curve for any particular cell line may well also be a factor in the fold change which can be observed. cDNAs encoding for the human GST BIB l and GST pi (Kano et al., 1987) have been expressed in the yeast, S - c e r e v i s i a e . This is a convenient and versatile organism for such expression studies since the endogenous GST activity is extremely low, while the available vector systems allow a high level of protein to be produced within the host cell. The GST activities ranged between 100-1000 units (nmol CDNB conjugated per min per mg of cytosol), compared to approximately 1500 units in rat liver cytosol), an activity which results from the combined expression of a range of GST subunits. The use of yeast has the additional advantage in that genetic manipulation of the strains is easy and more than one GST subunit or indeed any combination of proteins can be expressed at one time.

146

S.M. BLACKand C. R. WOLF TABLE4. Effect o f Expression o f Recombinant G S T Subunits on Drug Sensitivity Yeast

Chemical Adriamycin Benzo[a]pyrene BCNU Benzo[a]pyrene7,8-diol-9,10-epoxide 1,Chloro-2,4dinitrobenzene Chlorambucil Cumene hydroperoxide Cis-platinum Dibromoethane Ethacrynic acid Melphalan

Mammalian cell lines

BIBI 1

Pi I

pi2(S)

pi3*(S)

8.0 1.0

2.1 -1.0

1.3 0.9 .

3.0 . .

--

--

1.4

--

1.3 4.5 5.0 -0.6 ---

0.5 2.2 . -1.0 ---

---

-1.1 . 0.8 . 7.9 1.1

.

. 1.1 . 4.0 0.9

B1BI4(S) 1.04 (1.33) . . .

B1BtS(S) pi6(T)f 1.0 . .

--

--

1.5

1.24 1.04 (1.19)

2.0 3.0-5.0 2.5 . . . --

. ---

1.0 .

(3.04) 0.92 (0.88)

Yb(rat)(T)1"

1.3

1.0

1.2

1.3

.

.

.

Ya(rat)(T)f

1.3

.

.

. -.

. .

.

---

. .

1.3

.

. 1.4

1.5

The GST subunits used were for the human enzymes unless otherwise stated. The values quoted are fold changes in LDso over control except for t which are LDg0 and * LD37. This table was produced from the work cited in (1) Black et al. (1990); (2) Moscow et al. (1989b); (3) Nakagawa et al., 1990 (Cell line RGN2); (4) Leyland-Jones et al. (1991); (5) Black et al. (in preparation); Manoharan et al. (1987); (7) Puchalski and Fahl (1990). S = Stable expression of reconbinant GST, T = Transient expression of the recombinant GST. In these experiments expression of the alpha or pi class G S T gave significant levels of resistance towards the anticancer drugs chlorambucil and adriamycin. At the highest doses of drug taken, the resistance observed was very marked i.e. 16.0- and 10.0-fold resistance to adriamycin and 9.0 and 5.0 resistance to chlorambucil in G S T alpha (B~B1) and pi expressing strains respectively. In these studies resistance to chlorambucil was reversed by treatment of the cells with the glutathione depleting agent buthionine sulfoximine indicating that detoxification by conjugation to glutathione may well be the mechanism of protection in this case. Similar studies with adriamycin did not give a clear result and the mechanism of GST-mediated protection against the toxic effects of this chemical are unclear. In addition to these experiments, it was interesting that the resistance of yeast strains expressing both the alpha and pi G S T subunits simultaneously, to adriamycin was very marked. Indeed, in these experiments doses of adriamycin which killed 78% of the control cells only killed 2 6 0 of the cells expressing the G S T subunits. The combined effect of the two subunits together appeared to be additive. It is important to note that the yeast strains expressing the G S T were sensitized to compounds such as halogenated alkanes and alkenes. These compounds, such as hexachlorobutadiene and dibromoethane are known to be activated by GST-mediated G S H conjugation (van Bladderen, 1988). These findings demonstrate that the resistance observed with adriamycin or chlorambucil are not due to a stress response induced in the cells as a consequence of expressing a foreign protein. In addition to the yeast system the effect of GST transfection on drug sensitivity has been evaluated in a variety of mammalian cells using a range of anticancer drugs (including adriamycin, chlorambucil, melphalan, BCNU), 1,chloro-2,4-dinitrobenzene (CDNB), cumene hydroperoxide (CHP), ethacrynic acid (EA) (GST substrates) as well as certain carcinogens and their metabotites such as benzo[a]pyrene7,8-diol-9,10-epoxide (BPDE). In an excellent study by Puchalski and Fahl (1990) Cos cells transfected

with rat alpha or mu class GST, selected for GST expression by F A C S sorting, were shown to have a low but significant resistance to melphalan and adriamycin. Resistance to adriamycin was also demonstrated in N I H 3T3 cells transfected with G S T pi (Nakagawa et al., 1990). In almost all the studies to date using mammalian cells the level of drug resistance observed has been low. There are a variety of explanations for this which include the heterogeneity of G S T expression in transfected cell lines, and the level of expression of the GST, which to date has been relatively low. Another explanation could be that the G S T do not play a major role in conferring resistance to anticancer drugs in mammalian cells or that their ability to do so depends on the concomitant expression of other factors; factors which may be constitutively expressed in yeast cells. The fold resistance obtained in transfected cell lines generally does not approach the resistance level in drug selected cell lines. The transfection data however, should be viewed in the context that in most studies where putative drug resistance genes have been transfected into mammalian cells the level of drug resistance obtained has been relatively low. Table 5 tabulates the available data on the level of resistance obtained following the expression of the multidrug resistance protein, P-glycoprotein, in mammalian cell lines. The levels of resistance achieved range between 2 to 20-fold, which is significantly less than the > 100-fold levels obtained in cell lines selected for resistance to cytotoxic agents. The C H R C 5 cell line for example is 300-fold resistant to colchicine (Ling and Thompson, 1978). Thus the overexpression of m d r 1 c D N A s (Gros et al., 1986; Croop et al., 1987; Ueda et al., 1987; Guild et al., 1988; Pastan et al., 1988; Choi et al., 1988) confers a level of resistance which is higher than that seen with G S T but in many cases the difference is not very marked. Taken in the context that a plethora of changes have occurred in drug selected cell lines (e.g. M C F - 7 adr R) it is inconceivable that a single protein will confer the same level of resistance. The exceptions to this premise is where the toxic selective agent

Glutathione-dependent enzymes and drug resistance has a highly specific target e.g. dihydroflotate reductase in the case of methotrexate resistance. Thus the expression of recombinant proteins such as GST and m d r 1 demonstrate the potential role a single protein will play in the resistance mechanism, if it has the capacity to function without interacting with other cell components. In the cancer patient it is likely that multiple genetic and nongenetic changes such as tissue vascularization, play a role in drug resistance and many important proteins undoubtedly remain to be identified. In view of the complexity of this phenomenon and the probability that small genetic changes can lead to drug resistance in cancer patients in vivo it will undoubtedly prove very difficult to find correlations between the level of expression of a single protein and patient response to therapy. This is exemplified by a recent report where the level of m d r 1 was studied in human lung cancer and no correlation with the level of drug resistance was observed (Lai et al., 1989). Evidence which indicates that the overexpression of GST may be causally linked to the drug resistance phenotype has come from the study of Yusa et al. (1988), who studied GST levels in a number of resistant and sensitive cell lines and found that neither the adriamycin resistant cell lines K562 (myelogenous leukemia), A2780 (ovarian tumor line) nor the CCRF-CEM colchicine resistant line overexpressed GST isoenzymes. More importantly an MCF-7 line selected for colchicine resistance exhibited a 70-fold increase in GST activity. However, a revertant of this line, which had parental susceptibility to colchicine, still overexpressed the GST. Bellamy et al. (1989) have shown that a myeloma cell line made resistant to adriamycin, a compound shown to cause GST overexpression in certain cell lines (Cowan et al., 1986; Whelan et al., 1989), exhibited no overall change in GSH, GST or peroxidase levels. These data indicate that the spectrum of biochemical changes seen using a specific selective agent will depend on the nature of the target cell and do not provide evidence against any particular resistance mechanism. Lutzky et al. (1989) have also shown that GSH and GST levels remain unchanged in an anthracycline resistant line

147

although the intracellular distribution of the GST was altered compared to the parental line. This suggests that resistance may also be determined by the precise localization of GSH/GST to specific intracellular compartments and not a priori by protein overexpression 3.3. MECHANISMSOF G S T OVEREXPRESSION

Relatively few studies to date have addressed the central issue of the mechanism of GST overexpression both in preneoplasia and cell lines resistant to cancer chemotherapeutic agents. In one case the high level of resistance to bifunctional alkylating agents was shown to be due to amplification of alpha class GST genes (Lewis et al., 1988b). There are now several examples where the elevation in GST expression is related to increased mRNA levels. Whether this is due to message stabilization or an increased rate of transcription has not been determined. In the case of a cell line resistant to CDNB it appears that neither transcriptional activation nor mRNA stabilization will explain the increased expression of the alpha class GST indicating that protein stabilization may be involved (Wareing et al., in preparation). One intriguing aspect of the studies in drug resistance both in cell lines, preneoplasia and in drug priming is the concomitant overexpression of a cluster of other proteins and cofactors. This indicates that a single transcription factor may be involved in the regulation of a number of genes, as in the bacterial stress response systems. The identification of such a transcription factor or regulating protein and whether it acts directly on the genes or whether it acts indirectly on other transcription factors remains intriguing and a critical area for further study. Several promoter elements have been identified in the 5' noncoding region of GST and m d r genes including phorbol ester response elements (Sakai et al., 1988), antioxidant responsive elements (ARE) and heat shock response elements. The recent identification of the ARE is of particular interest as it appears that this element is activated under conditions of oxidative stress (Rushmore et al., 1990).

TABLE5. The Effect o f P-Glycoprotein Expression on Drug Sensitivity Cell Line Resistant Actinomycin Adriamycin cis-platinum

Colchicine Daunomycin Puromycin Vinblastine

NIH3T3 (H) -2.5 . 5.0 -. 3.0

NIH3T3(A7)* (H)

NIH3T3(13)~" (M)

10.0 3.0 .

10.0 3.0 .

5.0 4.0 .

. 4.0 3.0

. 4.5

. 3.0

NIH3T3(USI7)* (M)

NIH3T3(CSIOTa)~" (M)

LR73~ (CH)

-1.7 . 2.1 -. 2.4

-5.8

->20.0

4.2 --

> 25.0 --

> 7.0

> 20.0

. .

*Transfected cell line not selected for colchicine resistance prior to cytotoxicity assay. ?Transfected cell line selected for colchicine resistance prior to cytotoxicity assay. ~:Values expressed as LDi0 of control. Values are expressed as fold resistance relative to controls The PGP cDNAs used in these studies were H = Human; M = Mouse and CH = Chinese hamster. The data presented is taken from Gros et al. (1986); Pastan et al. (1988); Ueda et al. (1987); Croop et al. (1987); Guild et aL (1988).

148

S.M. BLACKand C. R. WOLF 4. G L U T A T H I O N E PEROXIDASE (GPX)

Glutathione peroxidases protect cells from hydrogen peroxide and are thought to be involved in the protection of lipid membranes and the detoxification of their breakdown products. These include lipid hydroperoxides formed by the degradation of unsaturated fatty acids. Peroxidase reactions are catalyzed predominantly by the selenium dependent glutathione peroxidase and by the alpha class glutathione S-transferases (Landenstein, 1984). The activity of the selenium-dependent enzyme is completely dependent on the presence of selenocysteine which interestingly is encoded by a U G A codon in the m R N A which would normally encode a stop codon. This is effected by the use of a selenocysteinyl t-RNA (Reddy et al., 1988). The GPX protein has a peroxidase activity distinct from that encoded by the alpha class GSTs (Lawrence et al., 1978; Lawrence and Burk, 1978; Burk et al., 1978). The ability of GPX to detoxify hydroperoxides makes it a potentially important enzyme in drug resistance. This would be especially true for chemotherapeutic agents where their mechanism of action involves the production of free radicals. These compounds, such as adriamycin, can be bioactivated through a one electron reduction pathway by P-450 reductase or N A D P H dehydrogenase with the production of oxygen radicals (Powis and Prough, 1987). The subsequent generation of highly reactive hydroxyl radicals react with unsaturated membrane lipids to give reactive lipid hydroperoxides (Bachur et al., 1978). In spite of the potential importance of this enzyme and the observation that it is elevated in bacterial oxidative stress (Christman et al., 1985), there have been relatively few studies into its involvement in relation to drug resistance. MCF-7 adr R cells have a significantly elevated (12-14-fold) increase in GPX activity (Sinha et al., 1987, 1989). However, similar selection procedures in different cell lines do not result in increased GPX levels (Bellamy et al., 1989). This again stresses that drug resistance can be acquired by multiple mechanisns and the enzyme, or enzymes, involved are likely to be dependent on the cell line used. Thus the breast tumor cell lines used by Sinha et al. (1989) may contain the required elements for GPX expression, while those used by Bellamy et al. (1989) may not. The level of GPX has been examined in human tumors of the breast (Di Illio et al., 1985), lung (Di Illio et al., 1987; Carmichael et al., 1988a) and most recently the colon, stomach, kidney and liver (Howie et al., 1990). GPX levels have been measured in the tumor and compared to those in the normal surrounding tissue. In all cases, although there was found to be a wide interindividual and intertumor variaton, the level of GPX was found to be elevated within the tumor tissue. In most cases an elevation of around 2-5-fold was measured. However, in the case of two adenocarcinomas of the lung a 20-30-fold increase in GPX activity was measured (Carmichael et al., 1988a). It has been concluded that in most human tumors GPX is the predominant GSH-dependent peroxidase (Howie et al., 1990). However, it is not clear whether the observed increase in GPX

activity associated with these human tumors relates to drug resistance. More work is needed on this important enzyme.

5. G L U T A T H I O N E REDUCTASE (GRD) This flavoenzyme exists as a homodimer (MW = 100kDa) whose cellular function is in the maintenance of the G S H : G S S G ratio. G R D catalyzes the NADPH-dependent reduction of GSSG to GSH so that the overall ratio is kept at a value of 20:1 (GSH : GSSG). N A D P H + H + + GSSG --, N A D P + + 2GSH Although an important cellular enzyme it is not crucial for survival since E. coli mutants deficient in G R D have been isolated (Fuchs et al., 1983). G R D levels can be affected by a variety of treatments. They can be induced by TPA (Kumar et al., 1984), diethylnitrosamine (Pinto and Bartley, 1973) and selenium (Chung and Maines, 1981). While the anticancer agent, BCNU has been shown to inhibit the function of G R D (Frischer and Ahmad, 1977; Babson and Reed, 1978; Bilzer et al., 1984), G R D can also be suppressed in the intestine by starvation and iron depletion (Ogasawara et al., 1989). This has been shown to be associated with a depression of the intestinal GSH and has been proposed to result in a decrease in the resistance of the small intenstine towards exogenous dietary toxins (Ogasawara et al., 1989). The levels of G R D have been studied in the rat hepatocarcinogenesis model (Lewis, 1988) and shown to be elevated around 2-fold in the neoplastic loci as compared to control hepatocytes. This may be important because of the relationship between elevation of G R D and subsequent GSH increases and since acquired resistance has been proposed as an important link in tumor progression (Farber, 1984). GSH is known to be involved in the resistance to alkylating agents (Calcutt and Connors, 1963) and compounds which produce reactive oxygen intermediates (Wolf et al., 1987b). The level of G R D has also been measured in small cell lung (SCL) and nonsmall cell lung (NSCL) cancer cell lines derived from human lung tumors (Carmichael et al., 1988b). SCL is initially found to be responsive to chemotherapy with a later relapse while NSCL is intrinsically resistant. It was found that the levels of G R D measured were 1.5-fold higher in the NSCL lines, again suggestive of a role for G R D in the resistance mechanism. However, when the cell lines were subdivided into those derived from patients who had previously received chemotherapy and those which had received no treatment there was no correlation between increased G R D levels and previous therapy. It could be argued that G R D levels were not increased since no resistance had resulted. Similarly, resistant cell line models have been derived where GSH levels are increased, while G R D is unchanged (Mansouri et aL, 1989). Thus GSH can be increased in the absence of a similar elevation of G R D The results of Carmichael et al. (1988b) raise the question of the relevance of measuring

Glutathione-dependent enzymes and drug resistance GST-dependent enzymes (or P-glycoprotein etc.) in cell lines in relation to the situation in a human tumor. Carmichael et al. (1988b) suggest that the GSH levels measured in the cell lines may not be an accurate representation of those found in the tumor as the GSH concentration in some cultured ascites decrease to less than 30% of those found in the tumor within as little as 5 passages in tissue culture. Also, Chen et al. (1989) have shown that the levels of GSH and G R D in ovarian carcinoma tumors resistant to cis-platinum were significantly higher than in a sensitive counterpart. However, for the first 4 hr in culture GSH levels decreased with a subsequent increase. After 24 hr the levels in the sensitive line exceeded those found in the resistant cells. Over the same time period the difference in G R D levels were also lost; and after 24 hr were indistinguishable from the control. It is therefore important to establish clearly the relationship between cell line models and the in vivo tumors. The use of xenografts may provide a more relevant model to study changes in tumor enzyme levels which occur during chemotherapy. 6. G A M M A - G L U T A M Y L TRANSPEPTIDASE (GGT) This enzyme is unique in its ability to cleave intact GSH (Curthoys and Hughey, 1979) and catalyzes the transfer of the gamma-glutamyl moiety to a wide range of peptide and amino acid acceptors. G G T is found throughout the plant (Kasai et al., 1982) and animal (Tate, 1980) kingdoms. It is a membrane bound protein with the active site oriented on the outer surface of the cell membrane (Horiuchi et al., 1978). In mammals it is found in all tissues, particularly the kidney, biliary epithelium, lining of the small intestine, pancreas and seminal vesicle (Hanigan and Pitot, 1985). G G T has been shown to be elevated in a variety of tumors including induced hepatocarcinomas in the rat (Fiala et al., 1976) and mouse (Williams et al., 1980), squamous carcinomas of the skin (De Young et al., 1978), carcinomas of the hamster buccal pouch epithelium (Solt, 1981), adenocarcinomas of human lung (Dempo et al., 1981) and in rat mammary tumors (Jaken and Manson, 1978). Similarly in cell lines selected for resistance to cytotoxic agents elevations in G G T have been observed. For example a Chinese hamster ovary (CHO) cell line selected for resistance to chlorambucil (Robson et al., 1986) has a 3.5-fold increase in G G T (Lewis et al., 1988b). Also the PE01 and PE04 cell lines derived from a patient with adenocarcinoma before (PE01) and after (PE04) the onset of clinical resistance (Wolf et al., 1987a) the level of G G T is seen to be elevated 6-fold in the PE04 line (Lewis et al., 1988a). It is interesting that in all cases, elevated G G T activity is accompanied by an increase in intracellular GSH and it has been proposed (Hanigan and Pitot, 1985) that the increased level of G G T observed in preneoplastic and neoplastic lesions leads to a local increase in the hydrolysis of GSH from the serum, with a corresponding increase in the intracellular levels of the amino acids required to synthesize GSH. This may be especially important during the promotion step of hepatocarcinogenesis since these promoting agents are usually

149

electrophiles or they generate electrophiles, which will react with GSH and so reduce the level of damage caused to cellular macromolecules especially D N A and proteins. Thus an increase in G G T should decrease the stress imposed on a transformed hepatocyte, by increasing the intracellular levels of GSH. It will be intriguing to determine whether G G T is regulated by the same transcription factors as the GST and P-glycoprotein. The increase in intracellular GSH may also be important in cell division as Szent Gyorgyi (1987) has suggested that sulfydryl groups, such as those of GSH, play an important role in this process. Again this would result in a selective advantage for a G G T overexpressing cell. 7. C O N C L U D I N G R E M A R K S There is strong evidence that glutathione and its associated enzymes play a role in cellular resistance to anticancer drugs and environmental carcinogens. However, it is clear that these enzymes do not completely account for the resistance patterns observed in human tumors and cell lines selected for resistance to cytotoxic agents. Other enzymes, or proteins, involved include: P-glycoprotein, D N A repair systems, D N A topoisomerase II and probably other as yet, unidentified proteins. A current challenge in cancer chemotherapy is to devise therapies which minimize the risk of the development of resistance. This certainly requires additional model systems. Particularly as in most models the drug resistant cell lines exhibit resistance levels vastly in excess of those measured in human tumors and many of the early changes relevant to patient treatment are masked. Coregulation of mdr 1 and GST pi has already been shown (Burt et aL, 1988) with Ha-ras responsive sequences detected in the human GST pi promoter (Cowell et al., 1988) and possible heat shock responsive elements within the rndr 1 promoter (Chin et al., 1990). The study of the regulatory elements, such as the TRE and ARE, in these genes and the presence of these elements in other stress or chemical induced genes remains an essential way forward in this research area. It has recently been proposed (West, 1990) that the glutathione conjugates formed by the action of GST and GPX may be substrates for the P-glycoprotein drug efflux pump. Although this may not finally prove to be the case it exemplifies the fact that in many ways we still have a very limited understanding of the function of many GSH-dependent enzymes. Certainly an interesting time lies ahead to clearly delineate the role(s) of GSH-dependent enzymes in drug resistance and their interactions with other drug resistance proteins. thanks to L. McLelland and M. Hussey for help with Table 1 and Anne Ward for excellent secretarial assistance Acknowledgements--Many

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