Pharmac. Ther. Vol. 52, pp. 385-406, 1991 Printed in Great Britain. All rights reserved

0163-7258/91 $0.00+ 0.50 © 1992PergamonPress Ltd

Associate Editor: J. G. CORY

CISPLATIN RESISTANCE IN HUMAN CANCERS K. J. SCANLON,* M . KASHANI-SABET, T. TONE a n d T. FUNATO Biochemical Pharmacology, Department of Medical Oncology, Montana Building, City of Hope Medical Center, 1500 E. Duarte Road, Duarte, CA 91010, U.S.A.

Abstract---Cancer chemotherapeutic agents primarily act by damaging cellular DNA directly or indirectly. Tumor cells, in contrast to normal cells, respond to cisplatin with transient gene expression to protect and/or repair their chromosomes. Repeated cisplatin treatments results in a stable resistant cell line with enhanced gene expression but lacking gene amplification for the proteins that will limit cisplatin cytotoxicity. Recently, several new human cell lines have been characterized for cisplatin resistance. These cell lines have led to a better understanding of the molecular and biochemical basis of cisplatin resistance. The c-fos proto-oncogene, a master switch for turning on other genes in response to a wide range of stimuli, has been shown to play an important role in cisplatin resistance both in vitro and in patients. Based on these studies, new strategies have been developed to circumvent and/or exploit clinical cisplatin resistance.

CONTENTS I. Introduction 2. Mechanisms of Cisplatin Resistance 2.1. Cisplatin transport 2.2. Sulfhydryl peptides 2.3. Repair of cisplatin-DNA adducts 2.4. The signal transduction pathway 3. Clinical Resistance 3.1. Patient tumors 3.2. Early detection of drug resistance 4. Circumventing Cisplatin Resistance 4.1. Platin analogs 4.2. Modulators of cisplatin resistance 4.3. The fos oncogene in drug resistance 5. Exploiting the Mechanisms of Cisplatin Resistance 5.1. Thymidine salvage pathway 5.2. Nucleoside analogs 5.3. Pyrimidine pathway 6. Conclusions Acknowledgements References

1. I N T R O D U C T I O N There has been a rapid evolution in understanding the multistage process of cancer (Weinberg, 1989; Baker et al., 1990; Levine et al., 1991); unfortunately developing clinical strategies to exploit this finding have not followed as quickly (Antman, 1991; Perez et al., 1991a). In addition, both natural and acquired resistance to cancer chemotherapeutic agents have also limited the cure rates (Bertino, 1988). Patients treated with these agents have approximately a 50% response rate, but unfortunately, within 12 to 24 months the majority of these patients relapse

385 386 386 387 387 388 391 391 391 392 392 393 395 396 396 396 397 397 398 398

(DeVita, 1983; Frei, 1985; Louie et al., 1986). Cisdiamminedichloroplatin(II) (cisplatin) and its analogs are some of the most widely used cancer chemotherapeutic agents either alone or in combination with other agents in the treatment of cancer (Rosenberg, 1985). If one understands the molecular and biochemical basis for the development of cisplatin resistance then rational strategies to circumvent or exploit these properties could be developed for clinical protocols. During the past few years several reviews on cisplatin have been published (for an overview see Rosenberg, 1985), cisplatin structure (Sherman and 385

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Lippard, 1987); DNA repair in vitro (Fox and Roberts, 1987); DNA repair in patients (Scanlon et al., 1989a); clinical pharmacology (Loehrer and Einhorn, 1984; Ozois and Young, 1984; de Graeff et al., 1988; Muggia, 1991) cellular pharmacology (Reed and Kohn, 1990; Andrews and Howell, 1990); and for strategies to overcome cisplatin resistance (Scanlon et al., 1989b; Kelley and Rozencweig, 1989; Perez et al., 1990). The focus of this review will be to highlight new areas of cisplatin research that may provide novel treatment regimens to overcome cisplatin resistance in human tumors. This review will include only some references to cisplatin resistance in mouse model systems because of the poor correlation between cisplatin resistant properties in mouse cells (Sheibani and Eastman, 1990) when compared to human carcinomas, in vitro (Scanlon et al., 1989b) or patient tumors (KashaniSabet et al., 1990a). During the past few years a significant number of new human cell lines have been developed for studies on cisplatin resistance that reflect the type of patient tumors most widely treated with cisplatin. These new cisplatin-resistant human cell lines were made in vitro for the following types of tumors: ovarian carcinoma (Hamilton et al., 1985; Andrews et al., 1985; Kikuchi et al., 1986, 1990; Hill et al., 1987; Wolfet al., 1987; Lu et al., 1988; Kuppen et al., 1988; Hills et al., 1989; Terheggen et al., 1990; De Pooter et al., 1991; McLaughlin et al., 1991); head and neck carcinoma (Frei et al., 1985); prostate carcinoma (Metcalfe et al., 1986); lung carcinoma (Hospers et al., 1988, 1990b; Hong et al., 1988; Bungo et al., 1990; Kasahara et al., 1991); colon carcinoma (Scanlon et al., 1989c; Fram et al., 1990); breast carcinoma (Teicher et al., 1991b); bladder carcinoma (Bedford et al., 1987; Walker et al., 1990); fibrosarcoma (Chu and Chang, 1990); melanoma (Teicher et al., 1991b); testicular teratoma (Hill et al., 1990; Walker et al., 1990); and leukemia (Shionoya et al., 1986). These human cell lines resistant to cisplatin have been shown to have multifactorial mechanisms of resistance because these cells have had multiple types of cisplatin treatment. Administration of cisplatin has varied by either dose (high/low) and/or by exposure (continuous or pulse). Which mechanism is clinically relevant? Patients who are treated with cisplatin have been shown to attain serum levels of 10 to 50/~M with a clearance rate (T 1/2) of about 30 to 60 min (Rosenberg, 1985). Thus, human cell lines made resistant to cisplatin with one hour pulses of 50 # i cisplatin (Scanlon et aL, 1989b,c) may reflect the type of resistance found in patients treated with cisplatin (Kashani-Sabet et al., 1990a). Furthermore, unlike methotrexate resistance or the multidrug resistant phenotype where gene amplification and high levels of drug resistance (100-fold or greater) can be achieved in vitro, cisplatin resistance, in contrast, has relatively low levels of resistance (2- to 30-fold).

2. MECHANISMS OF CISPLATIN RESISTANCE 2.1. CISPLATINTRANSPORT Studies of cisplatin transport, efflux and accumulation in human cancer cells remain confusing because of terminology as well as the design of the transport experiments (Andrews et al., 1988; Andrews and Howell, 1990). Cisplatin, a highly reactive electrophile, interacts with the cellular molecules and this property of cisplatin will alter any interpretation of uptake and efflux measurements. Mammalian transport systems are usually characterized by non-metabolizable substrates, such as ct aminoisobutyric acid for the amino acid transport system (Shionoya et al., 1986). Unfortunately, an unreactive form of cisplatin is not available. Interpretation of cisplatin transport defects in resistant cells is further complicated by experiments using long incubation times (up to 24 hr) that do not discriminate between uptake from accumulation or the use of high concentrations of cisplatin (up to 1 mM) to measure uptake since patient tumors are rarely exposed to these concentrations of cisplatin. In spite of these limitations, several studies have suggested that a 25-75% decrease in cisplatin uptake contributes to cisplatin resistance in cell lines that have a 3- to 30-fold level of resistance. The clinical relevance of this small decrease in cisplatin accumulation as a mechanism of cisplatin resistance still remains unclear. Recently two laboratories have suggested that a membrane protein has been associated with cisplatin resistance. In one study with a specific type of mouse LI210 leukemia subline, a protein (200 kd) increased with a corresponding increase in cisplatin resistance (Kawai et al., 1990). The cell line from which this protein was isolated was 40-fold resistant to cisplatin but had only a 2-fold decrease in cisplatin accumulation. By contrast, in a second study, a protein was shown to decrease with increased cisplatin or methotrexate resistance (Bernal et al., 1990). The exact function of these proteins remains unknown. This is not the first time changes in membrane properties have been associated with cisplatin and methotrexate resistance (Scanlon et al., 1987; Kashani-Sabet and Scanlon, 1989). Several common pathways for methionine/folate metabolism and DNA synthesis/repair are activated with resistance to either cisplatin or methotrexate (see Sections 2.3 and 2.4). Thus, these proteins for cisplatin transport and/or efflux must be further characterized before any causal relationship can be established. Human ovarian carcinoma 2008 cells have also been made resistant to cisplatin (1.5- to 3.0-fold) in vivo by weekly courses of cisplatin (3 mg/kg) (Andrews et al., 1990). This resistant cell line was not stable and reverted to the sensitive phenotype within 20 to 40 days. A transport defect was suggested to explain that the resistance was associated with a 25% decrease in cisplatin accumulation. However, other

Cisplatin resistance in human cancers mechanisms of resistance were not investigated. Similar studies with the ovarian A2780 ceilswere also performed in athymic mice, but no mechanism of resistance was characterized (Rose and Bailer, 1990). In a third study, tumors were shown to develop mechanisms of cisplatin resistance which were found only in vivo (Teichcr et al., 1990), but not with in vitro mechanisms of cisplatin resistance (Teichcr et al., 1987). In contrast, tumors from ovary or colon patients who relapsed from cisplatinbased treatment have elevated levels of genes expressing enzymes associated with D N A repair (Kashani-Sabet et aI., 1990a) and these studies are consistent with data on ovarian and colon celllinesin culture (Scanlon et aI., 1989b). This topic will bc discussed in more detail in Section 3.1. 2.2. SULFHYDRYLPEPTIDES Glutathionine (GSH) is one of the most prevalent intracellular thiols and has been shown to be involved directly or indirectly with many cellular functions (Meister and Anderson, 1983). Human cells have been shown to have up to 3.3-fold increases in the level of GSH in cisplatin resistant cells; however these cells were also 30-fold resistant to cisplatin (Teicher, et al., 1987; Andrews and Howell, 1990; Perez et al., 1990). Only cisplatin resistant cells that have been exposed continuously to cisplatin (high dose or low dose) have shown these elevated levels of GSH. Correlating GSH levels with cisplatin resistance is problematic since there is such a relatively small concentration of cellular cisplatin in contrast to the relatively high levels of GSH in the cell. Clinically, the role of GSH in cisplatin resistance has also been inconclusive. The use of D,L-buthionine-(S,R)sulfoximine (BSO) has also shown to be effective in modulating melphalan-DNA damage but to a lesser extent cisplatin damage (Ozols et al., 1987). This will be discussed in more detail in Section 4.2. Metallothioneins are small proteins that are important in binding and detoxifying heavy metals such as zinc, cadmium and copper. Metallothionein gene expression can be modulated by the H-ras protooncogene (Hamer, 1986) and may be due to a responsive element in the promoter region of the metallothionein gene. Several reports (Andrews et al., 1987; Kelley et al., 1988; Schilder et al., 1990) have suggested that some but not all cell lines resistant to cisplatin may have increased expression for metallothioneins (for a review, Andrews and Howell, 1990). It has been difficult to correlate the level of metallothioneins and cisplatin resistance until recently when a human small cell lung carcinoma was shown to have elevated levels of metallothionein 1.6and 2.3-fold in 6.2- and 10.0-fold cisplatin resistant cells, respectively. However, no other mechanism of resistance was identified to account for the remaining resistance (Kasahara et ai., 1991). In contrast, 48 patients were recently analyzed for changes in metalJF'L" 52/3,--!

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lothionein levels in ovarian tumors before and after chemotherapy. The conclusion from these studies was that metallothionein content was not a major determinant of tumor sensitivity to chemotherapy (Murphy et al., 1991). There may be several reasons for this observation including the fact that the metallothionein gene expression is transient and under the control of several types of promoters, in particular its AP-I binding site (see Section 2.4). Cisplatin can elicit a cellular response along the signal transduction pathway to express genes necessary to protect itself. Metallothionein is one of these genes and depending on the cell line and the level of transient expression, this could explain the variable expression of this gene in response to cisplatin (Lazo and Basu, 1991). The section on signal transduction pathways (2.4) will discuss this concept in more detail. 2.3. REPAIROF CISPLATIN-DNA ADDUCTS

It has been well established that cisplatin binds to DNA and these adducts contribute to cellular toxicity. In most studies it is believed that the cell's ability to repair either Pt--GG or Pt-GA adducts contributes to cisplatin resistance (Ducore et al., 1982; Fichtinger-Schepman et al., 1985; Eastman, 1986; Sherman and Lippard, 1987; Bedford et al., 1988; Eastman and Schulte, 1988; Schwartz et al., 1989; Hospers et al., 1990a; Lemaire et al., 1991; Shellard et al., 1991; Eastman, 1991). There have been several indirect correlations suggesting DNA repair plays an important role in cisplatin cytotoxicity. Repair-deficient human xeroderma pigmentosum fibroblasts are more sensitive to cisplatin than normal fibroblasts, possibly due to their limited excision repair capacity (Dijt et al., 1988; Hansson et al., 1991). There also appears to be a correlation between repair of cisplatin-DNA damage in vitro and cytotoxicity (Roberts and Friedlas, 1987; Scanlon et al., 1990b) including data from clinical samples (Reed et al., 1987, 1990; Kashani-Sabet et al., 1990a). In a methotrexate-resistant Chinese hamster ovary cell line with elevated dihydrofolate reductase, cisplatin-DNA adducts were removed faster in the transcribed region of dihydrofolate reductase gene and c-myc gene than in the non-coding region of the f o s gene (Jones et al., 1991). These data suggest that selective removal of these adducts may contribute to the resistant phenotype. It will be interesting to learn the mechanism of this preferential repair. Recently, more indirect evidence for repair of cisplatin-DNA adducts has been demonstrated using cisplatindamaged plasmids in cell lines sensitive and resistant to cisplatin. In vitro replicating systems such as the pRSVcat plasmid (Sheibani et al., 1989; Chao et al., 1990), or pSVO plasmid (Heiger-Bernays et al., 1990) or the pBR322 plasmid (Hansson and Wood, 1989) have been used in cells or cell lysates to measure repair of specific cisplatin-DNA damage. The

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plasmid DNA is an artificial system for measuring repair and the question still remains, do these cells repair their own DNA in a similar manner? In a recent series of papers, it appears that cells possess a protein that can recognize only cisplatinGG or cisplatin-GA adducts (Chu and Chang, 1988). These proteins have been termed damage recognition proteins (Toney et al., 1989; Donahue et al., 1990) or xeroderma pigmentation binding factors (Chu and Chang, 1990). Identification of these proteins was based on demonstrating changes in gel mobility of labeled cisplatin-DNA adducts with nuclear extracts (Andrews and Jones, 1991). The specificity of these proteins appears to be unique for cisplatin-DNA adducts and not for other types of platin adducts or UV adducts (Fujiwara et al., 1990). Although these studies are interesting, several questions still need to be addressed: (i) Have cells recently evolved a binding protein that only recognizes cisplatin adducts? (ii) why is there a lack of correlation between the amount of these proteins and cisplatin cytotoxicity in different cell types? Obviously, these binding proteins must recognize a change in the conformational structure of DNA, not a cisplatin-DNA adduct and unfortunately the level of these proteins does not correlate with the level of cisplatin resistance. The ability to repair cisplatin-DNA adducts presupposes the availability of all four nucleotide triphosphates. The patch size for repair of cisplatin-DNA adducts appears to be about 140-bases from plasmid studies (Hansson and Woods, 1989). In addition, when cells are exposed to cisplatin, there is enhanced degradation of the DNA perhaps to protect the chromosomes from cisplatin (Scanlon et al., 1990a). This degraded (i.e. decoy) DNA could be used by the cell to detoxify cisplatin in a similar manner to that by which GSH or metallothionein protects the cell. Why does cisplatin selectively perturb the thymidine pools? Previous studies have shown that cisplatin alters methionine uptake into the cells which stimulates the dTMP synthase cycle and produces more methionine (Kashani-Sabet and Scanlon, 1989). The catabolism of homocysteine and serine results in the accumulation of both methionine as well as methylenetetrahydrofolate, a key co-factor for dTMP synthase and DNA synthesis (see Section 5.1 also). This was the biochemical basis for cisplatin/ 5-fluorouracil synergy in vitro and clinically (see Section 5.3). The enzymes necessary to repair cisplatin-DNA damage may involve the DNA polymerases a and fl (Lai, et al., 1988, 1989; Scanlon et al., 1989a,b,c). However, 2008 ovarian carcinomas did not have elevated mRNA levels for DNA polymerase ~, and fl 18 hr after cisplatin treatment (Katz et al., 1990). Other studies have shown enhanced levels of DNA polymerase fl between 3 and 9 hr after cisplatin treatment (Scanlon et al., 1990b) suggesting transient expression in response to cisplatin. The role for a second type of repair enzyme, ECRR-I/2 has been

implicated in cisplatin resistance but the data is also not conclusive due in part to its limited expression (Hoeijmakers and Bootsma, 1990). Dihydrofolate reductase, dTMP synthase and topoisomerases have also been implicated in cisplatin resistance (Scanlon and Kashani-Sabet, 1988; Scanlon et al., 1990b, 1991). What do all these enzymes have in common? There are several steps by which cells repair D N A damage: These enzymes mentioned above and others could exist as a multienzyme complex that respond to nuclear oncogene expression and repair the damaged chromosomes (Reddy and Pardee, 1980; Pardee, 1987). In addition, some of the genes have AP-I binding domains which can respond to c-fos oncogene expression (Scanlon et al., 1991). If the cells respond to cisplatin through a cascade involving nuclear oncogenes then genes having an AP-1 binding site would be targets for initiating repair. This will be discussed in more detail in the next section. Finally, if these repair processes are important in cisplatin resistance then by modulating expression of these enzymes one should be able to reverse drug resistance (see Sections 4 and 5). 2.4. THE SIGNALTRANSDUCTIONPATHWAY DNA-damaging agents induce the expression of specific genes and such transcriptional modifications can influence cell cytotoxicity (Scanlon et al., 1991). Cell growth and differentiation can be initiated by signal cascades (Edelman et al., 1987; Hunter, 1991). Cancer chemotherapeutic agents have been shown to disrupt this signal transduction pathway which may contribute to the evolution of drug resistant clones (Tritton and Hickman, 1990). In turn, the nuclear oncogene c-los shows increased expression in cisplatin resistant cells in culture (Scanlon et al., 1988, 1989c; Hollander and Fornace, 1989) and in patients (Scanlon et al., 1988, 1989b; Kashani-Sabet et al., 1990a). If this oncogene plays a role in cisplatin resistance then modulating c-los RNA expression should change cisplatin's cytotoxicity. The c-los oncogene has been shown to modulate gene expression by stimulating other genes that possess the AP-I binding domain (i.e. dTMP synthase, topoisomerase I and metallothionein). In addition, a l o s ribozyme (catalytic RNA) has also been shown to completely reverse cisplatin resistance by modulating these repair genes (Scanlon et al., 1991; see Sections 4.2 and 4.3). Thus, what is the relationship between cisplatin resistance, nuclear oncogene expression and the signal transduction pathway? Regulation o f l o s gene expression is mediated by combinations of short DNA sequences that provide binding sites for sequence-specific transcription factors. The interaction between individual trans- and cis-acting elements which determine the rate of gene transcription are thought to represent the ultimate steps in various signal transduction pathways (Fig. 1). Two of the major classes of regulatory elements that

389

Cisplatin resistance in human cancers Genes with A P responsive elements •

Extracellular stimuli

(I) Growth factors (EGF)-~ TPA

(1mE)

l

Ca=+ionophoros D metal ions 3

Ca=÷

PKC ~

I TGACTCAI c-fos/c-lun ~- leuane Zlpl~r ~

(II) uv

DNA-damaging agents

cA.p

PKA

I ar carCAC I

• •

c-los TS DNA pol o • Topoi • hMTIIA o • GST • MDR o

~TIF/CRE)

(III)

Genes with ras responsive elements o FIG. 1. Signal transduction pathways involved in cisplatin resistance. (See Section 2.4 for abbreviations and details.) Cisplatin

~" methionine ~"

Hras

contribute to transcriptional regulation by extracellular signals are the AP-I/TRE and ATF/CRE sequence motifs. The AP-1/TRE element was originally defined as the activator protein 1 (AP-I) binding site or the phorbol 12-O-tetradecanoate 13-acetate (TPA) responsive element (TRE) (Lee et al., 1987b); the ATF/CRE element was defined as the activating transcription factor (ATF) binding site or the cAMP responsive element (CRE). The AP-I/TRE site is recognized by a group of proteins including those encoded by the c-fos and c-jun gone families (Abate et al., 1991). The ATF/CRE site is recognized by a family of proteins referred to as ATF or CRE binding proteins (CREB) (Hai and Curran, 1991). This family of proteins has been implicated in cAMP-, calciumand virus-induced alterations in transcription. AP-1 is the collective name for a class of transcription factors (i.e. serum responsive elements) that has been characterized by the ability to bind to the promoter/enhancer elements containing the TGACTCA sequence. The members of this family include c-Fos, FosB, Fra-1, Fra-2, c-Jun, JunB and JunD. All of these proteins share a conserved region, consisting of a leucine repeat dimerization domain (ieucine zipper). The functional form of AP-I is a dimer composed of a permutation of one or more of these proteins. Several such elements have been previously described: e.g. The hormone responsive element (HRE), the metal responsive element of the human metallothionein IIA gone (MRE); the TPA responsive element (TRE), the cAMP responsive element (CRE) and the estrogen responsive element (ERE). However, individual genes often respond to more than one extracellular agent each of which may use a different signal transduction pathway. The human metallothionein IIA gone (see Section 2.2) is induced by heavy metals (cisplatin), glucocorticoid hormones, phorbol esters, interleukin 1, interferon, various serum factors and u.v. light (Lee et aL, 1987a;

Buscher et al., 1988; Sassone-Corsi et al., 1988). This, in principle, could be accounted for by two basic mechanisms. First, either the gone possesses distinct cis-acting genetic elements, each one responding to a different stimulus and recognized by a unique transacting factor; or several signal pathways converge and operate through a single cis element recognized by one or several trans-acting factors whose activity is subject to complex control. Recently, an AP-I site has also been shown to be located on the MDR gone (Teeter et al., 1991) and this AP-I site may explain the cyclosporin A (CSA) effect on a wide range of cancer chemotherapeutic agents (see Section 4.2). An example of the second possibility is the transcription factor AP-2 which mediates gone induction by agents which elevate intracellular cAMP and by agents which activate protein kinase A (Buseher et al., 1988). Both types of mechanisms are involved in the activation of the protooncogene c-fos, which responds to a particularly large number of extracellular agents. These agents include growth factors, phorbol esters, Ca2+-ionophores, thyrotropic hormones, metal ions (such as K +, Cd 2+, Zn 2+ and cisplatin), cAMP, heat shock proteins, DNA damaging agents and u.v. irradiation (Buscher et al., 1988). Cisplatin has also been shown to stimulate heat shock protein (HSP35) gone expression (Oesterreich et aL, 1991). Induction of the cAMP signal-transduction pathway significantly increases c-fos transcription. Several studies have demonstrated that the transcriptional activator cAMP-responsive element (CRE)-binding protein (CREB) is responsible for c-fos stimulation upon cAMP induction (Foulkes et aL, 1991; Hartig et al., 1991). CREB protein binds as a dimer to CREs and exerts its transcriptional regulatory function upon several genes when in a phosphorylated form. At least 10 different genes encode CRE-binding proteins which include the genes for CREB, ATF-I, CRE-BPI, ATF-~, ATF-3 and ATF-4. A link

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between the action of the EIA proteins and the cAMP-dependent consensus cAMP response element (CRE) 5'-TGACGTCA-3' is located upstream of the E1A-inducible adenovirus early genes E2A, E2, E3 and E4. Cellular factors that recognize these sequences have been identified, including the CREB/ATF and AP-I families. Moreover, the combination of E IA protein and cAMP activates the E1A and E4 genes synergistically, indicating that the cAMP-mediated and E1A-mediated responses are somehow linked or interactive. EIA also acts together with cAMP to regulate the level of transcription factor AP-1 (Engel et al., 1991). AP-I activity has been implicated in both positive and negative transcriptional regulation as well as in chromosomal DNA replication (Murakami et al., 1991; Ginsberg et al., 1991). The role of DNA polymerase fl in the cell has been linked to DNA repair gap-filling synthesis through inhibitor studies and the enzyme also could have a role in gap-filling DNA synthesis (Wang, 1991; see Section 2.3). The core promoter for human DNA polymerase fl contains one 10-residue DNA palindromic sequence G T G A C G T C A C at position -49 to -40 that is similar to the binding site for ATF/CREB proteins (Widen and Wilson, 1991; Kedar et al., 1991; Englander et al., 1991). In experiments with a fl-pol promoter, the integrity of this palindromic element was essential for full promoter activity, EIA/E1B transactivation, stimulation by activation of H-ras protein and in mediating response to the DNA damaging agents. This site is known to mediate E1A inducibility in several viral promoters and to confer response to elevated levels of cAMP in many cellular genes. In these promoters, the sequence TGAGTCA is referred to as the cAMP responsive element (CRE) or activating transcription factor (ATF) binding site. DNA polymerase fl and metallothionein also have an H-ras responsive element that responds to changes in H-ras gene expression (Schmidt and Hamer, 1986; Kedar et al., 1990). Sklar (1988) and others (Niimi et al., 1991) have shown a correlation with cisplatin resistance and H-ras gene expression. These studies have been confirmed in cisplatin resistant human cells in vitro and from patients (Scanlon and KashaniSabet, 1989; Scanlon et al., 1989b,c; Kashani-Sabet et al., 1990a). The H-ras oncogene has also been shown to influence the methionine requirement in H-ras transformed cells (Vanhamme and Szpirer, 1987). These studies further establish the link between methionine/folate metabolisms, DNA repair systems and proto-oncogenes (see Section 2.3). Furthermore, H-ras may also enhance transcriptional activity of c-jun via specific changes in the phosphorylation state of the Jun protein (Bin6truy et al., 1991). Protein kinase C (PKC) has a crucial role in signal transduction for a variety of substrate proteins putatively important for cell differentiation and proliferation (Adunyah et al., 1991). Cells treated by various growth factors or tumor promoters exhibited the

resultant altered PKC activity, leading to the phosphorylation of a number of proteins and a dramatic shift of gene expression (McDonnell et al., 1990). The exact pathways of signal transduction from activation of PKC to genetic regulation in the nucleus are not well understood. However, some portion of the PKC activity is present in the nucleus as well as in the plasma membrane; hence, nuclear proteins involved in transcription, replication and repair may be subjected to modification. Of particular interest was the finding that PKC activated DNA topoisomerase I (Pommier et al., 1990), as well as DNA polymerase /~. Thus, signal transduction from PKC may genetically regulate the expression of DNA polymerase (Tokui et al., 1991). The expression of this gene is also regulated by promoter sequences for a transcription factor that may be a Fos-related protein and that the product of the c-fos gene may repress expression of the DNA polymerase /~ gene (Tokui et al., 1991). The expression of c-myc gene is also regulated by several mechanisms, including transcriptional initiation, transcription elongation, mRNA stability and translation. Transcriptional regulation of c-myc is responsive to many physiologic signals, such as growth factors and to biological agents. Transcription of c-myc starts from at least three initiation sites, with distinct promoters. PI and P2 are the major promoters located upstream of exon I and within exon I, respectively. The transcription of c-myc from P1 and P2 is regulated by a composite of positive and negative elements located both upstream and downstream of these promoters. A negative transcriptional element, which acts on both PI and I)2, has been mapped between -350 and -294 bp relative to the transcriptional starting site, P1. There are two biochemically distinct factors that bind specifically to the 26 bp DNase I footprinted region of the c-myc negative element. One of the factors was identified as the fos/jun (AP-I) complex (Takimoto et al., 1989) and the other as a ubiquitous octamer-binding protein. Interestingly, the two complexes bind highly overlapping sequences. The presence of interdigitating binding sites for distinct factors may confer multiple functions on a single cis-element or allow cooperative interactions between trans-acting factors. Fos/jun (AP-I) and the ubiquitous octamer-binding factor are known to participate in transcriptional activation and/or DNA replication; these studies suggest that repression of c-myc expression may employ components common to transcriptional activation and DNA replication/repair. This has been also noted in cisplatin resistant A2780DDP cells with c-fos/c-myc modulation by cyclosporin A (KashaniSabet et al., 1990b) or by a f o s ribozyme (Scanlon et al., 1991). H-ras has also been shown to enhance the transcriptional activity of c-jun via changes in the phosphorylation state of the c-jun protein (Bin6truy et al., 1991). Platelet-derived growth factor (PDGF) and mitogens stimulate expression of the proto-oncogenes

Cisplatin resistance in human cancers c-myc and c.fos in their respective target cells (Hall and Stiles, 1987). Although much data have accumulated concerning the inducibility of c-myc and c-fos, the precise role of these proto-oncogenes in the proliferative response to growth factors and mitogens is unknown. Agents which activate protein kinase C stimulate c-myc expression, but are only weak mitogens. Down regulation of protein kinase C (by prolonged exposure to a phorbol-based tumor promoter) diminishes the ability of PDGF to induce c-myc, but has no effect on the ability of P D G F to induce growth. Other data tend to dissociate c-los from any direct role in cell growth or differentiation. This second body of data suggests that induction of the c-myc and c-los gene products is neither necessary nor sufficient for mitogen to stimulate cell proliferation. Phorbol esters induce the growth and differentiation of hematopoietic cells. The mechanism by which phorbol esters stimulate gene transcription has recently been shown to involve the activation of specific enhancer sequences including AP-1, AP-2, AP-3 and NF-KB. To activate the AP-1 enhancer sequence, phorbol esters induce an increase in c-jun and c-los transcription. The protein products of these genes, Jun and Fos, contain a leucine repeat region, (leucine zipper), which allows them to dimerize. Control of transcription by the c-jun mRNA also involves an AP-I binding site and autoregulation by a posttranslationally modified Jun protein. In contrast, c-los m R N A synthesis is regulated by multiple enhancer regions. Regulation of AP-1 enhancer activity is extremely complex involving multiple binding proteins and both posttranslational and transcriptional control mechanisms (Adunyah et al., 1991). Epidermal growth factors (EGF) have also been shown to stimulate the expression of the protooncogenes c-myc and c-los in epidermal cells (Bravo et al., 1985). The binding of EGF to the membrane initiates stepwise biochemical changes in calcium fluxes, amino acid uptake and activation of the protein kinases which can make the cells sensitive to cisplatin (Amagase et al., 1989; Christen et al., 1990). In turn, cisplatin-resistant cells have lost this responsiveness to EGF and have altered protein kinase responses. Thus, agents which interact with the signal transduction pathway may have an inhibitory or stimulatory effect depending on the close and exposure time. These agents have been used to modulate cisplatin cytotoxicity with varying degrees of success (see Section 4.2). 3. CLINICAL RESISTANCE 3.1. PATIENTTUMORS Due to a lack of tissue samples and several technical difficulties, only a small number of patient tissues have been analyzed directly for cisplatin resistance. Presently, tumors from fifteen ovarian and colon patients have been analyzed for changes in gene

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expression that are clinically resistant to cisplatin (Kashani-Sabet et al., 1988, 1990a; Scanlon et al., 1989a,b,c; Scanlon and Kashani-Sabet, 1989a; Kashani-Sabet et al., 1990a). These investigations have overcome the inherent difficulties in conducting in vivo drug resistance studies. These findings in patients corroborate the previous data on human ovarian and colon cell lines/n vitro (Scanlon et ai., 1989b). Human cells that become refractory to cisplatin based chemotherapy have enhanced expression of genes associated with DNA repair. In one study, multiple tumor samples from a patient treated with cisplatin were shown to have increased expression for oncogenes and enzymes associated with DNA synthesis and repair (Kashani-Sabet et al., 1990a). The polymerase chain reaction (PCR) assay has also overcome the problem of the limited number of patient cells that can be assayed to confirm these findings with conventional molecular biology assays. The PCR assay will be discussed in detail in the next section. Heterogeneity of the tumor sample still remains a problem especially if there are only quantitative changes rather than qualitative changes in gene expression. The use of normal tissue as a control is also problematic due to its non-proliferating properties. Finally, it is still difficult to uncouple the effects of tumor progression from drug resistance on gene expression in tumor cells in vivo. However, other mechanisms for cisplatin resistance have not yet been shown in fresh human tissue resistant to cisplatin. In a series of studies an ELISA assay was used to correlate increased cisplatin-DNA adducts in white blood cells of a patient treated with cisplatin or carboplatin with an increased patient response (Reed et al., 1986, 1987, 1988, 1990; Fichtinger-Sehepman et al., 1987; Parker et al., 1991). These studies had several limitations: (i) These were retrospective studies; (ii) the studies had a limited number of patient samples; (iii) adduct formation did not correlate with either the duration of the response or pharmacokinetits; and finally, (iv) specific adduct levels could not predict positive responses. In contrast, the PCR assay has become an indispensable tool in the laboratory, as well as for diagnostics. The use of the PCR assay for detection of drug resistance in patients will be discussed in the next section. 3.2. EARLYDETECTIONOF DRUG RESISTANCE Changes in mRNA for several genes are now known to predict drug resistance in patients. The appropriate screening for these tumor cell markers should be helpful clinically. However, multiple mechanisms of resistance can evolve in established malignancies; thus it would be important to test several genes for changes in expression in drug resistant cells. With the introduction of the Taq polymerase and an automated thermocycler, the PCR assay has become indispensable to most molecular biology laboratories (Guyer and Koshland, 1989; Erlich et al., 1991).

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Although a simple assay, PCR use as a diagnostic tool for human diseases prior to these advances was time consuming and tedious (Kashani-Sabet et al., 1988). For cancer diagnosis, the RNA PCR assay can identify which genes are expressed by the use of reverse transcriptase in a modified PCR assay. Studies in this laboratory with tumor samples from 18 patients (Kashani-Sabet et al., 1988; Scanlon and Kashani-Sabet, 1989; Scanlon et al., 1989a,b; Miyachi et al., 1991) have shown the PCR assay to be an effective device in detecting early failure of patients treated with cancer chemotherapeutic agents. This method has confirmed data obtained by conventional molecular biology techniques while circumventing the practical problems of time, sensitivity of detection and the small amount of patient tumor cells. In spite of these studies and others (Gilliland et al., 1990; Veelken et al., 1991), there has been scepticism about whether the PCR assay could quantitate mRNA analogous to Northern blots. A series of controls was introduced to quantitate RNA PCR: (i) Eliminating DNA contamination with DNase or primers that span an intron, (ii) use of an internal control gene that does not change its expression (i.e. phosphoglycerate kinase), (iii) standardizing the amount of RNA to demonstrate linearity for a specific number of PCR cycles, (iv) determining the authenticity of the DNA product by restriction enzyme digestion and (v) use of control cells whose gene expression does not change (Scanlon and KashaniSabet, 1989; Miyachi et al., 1991). Based on these criteria, we have quantitated gene expression in patient tumors for DNA synthesis genes (dihydrofolate reductase, dTMP synthase and DNA polymerase fl) and proto-oncogenes (c-fos, c-H-ras and c-myc). Changes in gene expression have also been shown by the PCR assay for the multidrug resistant phenotype. In a study with 202 patients, tumor samples from 26% of the patients were found to express this drug resistant gene (Noonan et al., 1990). In the future, more patient material will establish the PCR assay as a clinical diagnostic tool for patients with natural resistance or acquired resistance to these agents (Negrin and Blume, 1991). The advantage of measuring gene expression in contrast to gene amplification is that drug resistance in patients may not require gene amplification to achieve clinical resistance (Kashani-Sabet et al,, 1990a). 4. C I R C U M V E N T I N G CISPLATIN RESISTANCE 4.1. PLATINANALOGS Although cisplatin has proven effective against testicular, ovarian and bladder carcinoma, both host toxicity and natural/acquired resistance has limited its usefulness against some of the more common cancers, such as colon, lung and breast carcinomas. Thus, new cisplatin analogs have been actively syn-

thesized and tested alone or in combination with some success (Gill et ai., 1991; McKeage et al., 1991). Currently, the two most promising analogs from tissue culture studies are carboplatin and iproplatin but they may not be as effective as cisplatin against a wide spectrum of tumors (Harstrick et al., 1989). In the last few years, however, several new platin compounds have been synthesized and have shown promise in tissue culture against human carcinomas (Boven et al, 1985; de Graeff et al., 1988; Kuppen et al., 1988; Foster et al., 1990). Some of these agents may not trigger the cascade response induced by cisplatin (see Section 2.4). Tetraplatin (ormaplatin) has been shown to be more effective than cisplatin and less cross-resistant to cisplatin-resistant cells in several human cell lines, including ovarian carcinoma (Behrens et al., 1987; Scanlon et al., 1987, 1989b; Bhuyan et aL, 1991; Perez et al., 1991b); small cell lung carcinoma (Hospers et al., 1988); colon carcinoma and breast carcinoma (Scanlon et al., 1989b). This is due in part to a limited induction of the expression of genes involved in DNA synthesis and repair and a longer (2-6 hr) time required for this response (unpublished data). The d-transisomer of tetraplatin appears to be the more potent component from mouse studies (Bhuyan et al., 1991) and this agent is currently in phase I clinical trials. Oxalatoplatin, (1 R, 2R-1,2 cyclohexadiamine-N,N) oxalato(2-)-O,O')platinum, shows effectiveness that is similar to that of cisplatin against several transplanted murine tumors (Boughattas et al., 1989). Oxalatoplatin, at equally active doses of cisplatin, does not cause renal toxicity and has far less hematologic toxicity (Extra et al., 1990; Caussanei et al., 1990). Phase II clinical trials are underway using higher doses of oxalatoplatin resulting from optimization of circadian rhythm-based strategies and in combination with fluorouracil. Preliminary studies have shown good clinical responses to the 5-fluorouracil and oxalatoplatin combination (Levi et al., 1991). Bis(platinum) complexes [{cisPtCl2(NH3)}2 H2N(CH2)NNH 2 ] are a new series of anticancer agents in which two eis-diamine(platinum) groups are linked by an alkyidiammine of variable lengths. These complexes are potentially tetrafunctional and their interactions with DNA are significantly different from cisplatin (Roberts et al., 1989; Farrell et al., 1990). It appears that bis(platinum) adducts contain a high frequency of structurally novel interstrand cross links formed through binding of the two platinum centers to opposite DNA strands. In addition, bisplatinum complexes do not unwind supercoiled DNA, in a similar manner to cisplatin. Diastereoisomeric compounds [l,2-bis(2-hydroxyphenyl)ethylenediamine] dichloroplatinum(II) complexes, OL-3-PtC12 and meso-3-PtCl: compounds have been evaluated on the hormone-dependent, human MDA-MB231 breast cancer cell line, on the

Cisplatin resistance in human cancers cisplatin-sensitive and -resistant LI210 leukemia cell line, on the cisplatin-resistant human OVCAR3 ovarian cancer cell line and sensitive and resistant Ehrlich ascites tumors of the mouse. DL-3-PtCI2 produces a marked inhibitory effect on all tumor models. The diastereoisomer meso-3-PtC1 e is less active and more toxic than DL-3-PtClz. DL-3-PtC12led to a pronounced inhibition of all cisplatin-resistant tumors. At non-toxic concentrations DL-3-PtCI2 produces cytocidal effects on the NIH :OV-CAR 3 cell line. Due to the low toxicity and lack of cross resistance to cisplatin-resistant cells, DL-3-PtCi 2 is being further tested (Muller et al., 1990). A series of platinum(II) antitumor agents that are different from the classical structure-activity relationships established for platinum complexes have demonstrated activity against murine and human tumor systems cis[Pt(NH3)2(AM)CI] cations, in which AM is a derivative of pyridine, pyrimidine, purine, or aniline. They can inhibit simian virus 40 DNA replication in vitro and inhibit the action of DNA polymerases at individual guanine residues in replication mapping experiments. The platinumtriamine complexes do not produce the type of intrastrand cross-links on DNA that is characteristic of cisplatin and analogs with the general formula cis [Pt(amine)2X2]. These results indicate that cis[Pt(NH3)2(AMCi] + cations form monofunctional adducts on DNA. This is further supported by nuclear magnetic resonance spectroscopic and enzymatic digestion analyses of the products of the reactions of these triamine complexes with d(GpG) and dG, which also reveal monofunctional binding. The fact that trans[Pt(NH3)(4-Br-pyridine)Cl2] displays no anticancer activity, however, indicates that its formation from cis[Pt(NH3)2(4-Br-pyridine)Cl] + is not a significant component of the mechanisms of action of this platinum-triamine complex. Taken together, these findings again indicate that the cytotoxicity of cis[Pt(NH3)2(AM)C1] + complexes most likely arises from the formation of monofunctional adducts. The DNA binding properties associated with this new class of antitumor agents suggest that they may display an activity profile different from that of cisplatin and related analogs (Hollis et al., 1991). 4.2. MODULATORS OF CISPLATIN RESISTANCE As we elucidate the mechanisms of action for cisplatin, rational strategies will emerge to exploit or circumvent these phenotypic properties of cisplatinresistant cells. Several classes of biochemical modulators have been identified that modulate cisplatin acquired resistance by: (i) Interfering with the signal transduction pathway; (ii) inhibiting DNA repair; or (iii) acting through schedule-dependent cytotoxic combinations. Docosahexaenoic acid (DCHA) has been shown to change the lipid membrane composition of small cell lung carcinoma cells and enhance cisplatin activity in

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the cisplatin-resistant cells (Timmer-Bosscha et al., 1989). This 3-fold reduction in cisplatin cytotoxicity in the resistant cells was not associated with drug transport or changes in the GSH levels, but was associated with a decreased ability to remove cisplatin DNA adducts. These results suggest that DCHA may modify the signal transduction pathway and thus limit the cells' ability to repair their DNA. Both the calcium channel blocker, nifedipine (Onoda et aL, 1986) and the calmodulin antagonist, naphthalenesulfonamide (Hidaka and Tanaka, 1983; Kikuchi et al, 1987) have been shown to have synergistic toxicity when either are combined with cisplatin. Another inhibitor of calmodulin, trifluoperazine, has also been shown to modulate cisplatin cytotoxicity about 2-fold in human ovarian carcinoma cells (Charp and Regan, 1985; Perez et al., 1990). In addition, calmodulin antagonists have potentiated DNA damage from irradiation and u.v. light (Rosenthal and Hait, 1988). Conversely, calcium-free media or EGTA caused calcium depletion in the cells and protects against the cytotoxic action of cancer chemotherapeutic agents, hyperthermia and gamma radiation (Bertrand et al., 1991). Protein kinases have been shown to play an important role in the signal transduction pathway, which involves membrane ion fluxes, modulation of AP responsive elements and direct effects on DNA synthesis enzymes (see Section 2.4). Thus, it is not surprising that heavy metals (Zn 2+, Cd 2+ and Hg 2÷) have been shown to inhibit TPA binding and alter protein kinase activity (Speizer et al., 1989). The protein kinase A in cisplatin-resistant cells is not responsive to forskolin in contrast to sensitive cells in which forskolin enhances cisplatin cytotoxicity (Christen et al., 1990; Mann et al., 1991). TPA, however, is a dynamic modulator depending on the dose and length of exposure. TPA can act directly on responsive elements of the DNA synthesis genes such as topoisomerase I (Pommier et al., 1990); the repair gene, gadd45 (Papathanasiou et al., 1991); the metallothionein gene (Hamer, 1986); and the c-fos oncogene and c-jun/AP1 activation (Huang et al., 1991; see Section 2.4). If cells have a short term exposure to TPA a transient level of increased gene expression is noted. In contrast, long term exposure to TPA decreased PKC activity and gene expression in the signal transduction pathway. TPA has been shown to transiently modulate cisplatin toxicity with human ovarian carcinoma cells (2.5-fold) (Isonishi et al., 1990) or cause a 9-fold increase in cisplatin cytotoxicity in HeLa cells with 24 hr exposure of TPA (Basu et al., 1990). Cyclohexamide did not influence TPA activity; similarly cyclohexamide does not effect the dimer formation of Fos/Jun. Inhibitors of protein kinase C; lyngbyatoxin A, quercetin and staurosporine (Hofmann et aL, 1988; Basu et al., 1991) also effect cisplatin cytotoxicity. The protein kinase inhibitor, H-7, has been shown to inhibit c-fos mRNA accumulation and the transcriptional

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activation of c-fos (Shibanuma et al., 1987). This would suggest that H-7 could potentiate cisplatin cytotoxicity. Glucocorticosteroids may also have activity for modulating cisplatin toxicity. CSA, used primarily as an immunosuppressant in transplantation, has been shown to reverse resistance to a variety of chemotherapeutic agents, including etoposide, adriamycin, vincristine, daunorubicin and cisplatin (Osieka et al., 1986; Twentyrnan et al., 1987; Sweet et al., 1989; Kashani-Sabet et aL, 1990b). It is intriguing that CSA can restore sensitivity to cisplatin-resistant cells because of the potential clinical implications. It has been suggested that CSA interacts either with plasma membrane potentials (Damjanovich et al., 1986), calcium--calmodulin pathways (Colombani et al., 1985), or acts by suppressing nuclear oncogene expression (Kashani-Sabet et al., 1990b; Scanlon et al., 1990b). In lymphocytes, CSA inhibits PHA-stimulated gene expression of growth factors (IL-2) and may interfere with Ca2+-depen dent pathways that regulate several transcription factors (Baldari et al., 1991). CSA forms an inhibitory complex with peptidyl-prolyl cis-trans isomerase which may block transcription factors (Gunter et al., 1989; Mattila et al., 1990; Flanagan et al., 1991). How this works in non-T cells has yet to be determined. These studies further support the role of the signal transduction pathway in cisplatin resistance (Section

2.4). The role of GSH in resistance to cisplatin has been studied in several human cell lines (see Section 2.2) and data remains inconclusive. However, pretreatment of cisplatin resistant cells with BSO has been shown to reduce the repair capacity of the cells and enhance cisplatin cytotoxicity 2- to 4-fold (Hamilton et al., 1985; Fojo et al., 1987; Lai et aL, 1989; Meijer et al., 1990). BSO treatment may play a role in eliminating GSH from the cytosol and enhancing the formation of DNA-platin adducts. This, however, has not been seen with all cisplatin-resistant cell lines and high levels of BSO are sometimes required for modulation of GSH levels to effect cisplatin cytotoxicity. In contrast to cisplatin, it appears that tetraplatin cytotoxicity is modulated more by BSO in some human ovarian tumors (Mistry et al., 1991). These strategies have only partially reversed cisplatin resistance in contrast to CSA or ribozymes. Aphidicolin (APC), an inhibitor of DNA polymerase ~ and fl, can dramatically reduce the level of DNA synthesis (Sheaff et aL, 1991) if the cells are continuously exposed to APC (Sorscher and Cordeiro-Stone, 1991). However, APC is a reversible inhibitor and once removed from the media DNA strand growth rapidly resumes to near normal levels. In spite of these limitations several studies have suggested that APC increases cisplatin cytotoxicity (Masuda et aL, 1988; 1990) while other studies have shown no activity with APC (Fram et al., 1987). The combination of BSO and APC has been shown to be additive with a 4-fold increase in cisplatin cytotox-

icity in human ovarian cells (Lai et al., 1989). These results could be reversed with the addition of GSH and suggest that GSH depletion could potentiate cisplatin cytotoxicity. BSO has also been shown to restore radiation sensitivity to melphalan-resistant A2780 cells. However, BSO treatment of cisplatinresistant A2780 cells was less effective and the authors suggested that elevated levels of GSH may not be the only mechanism of cisplatin resistance (Louie et aL, 1985). A number of studies has suggested that single dose irradiation can produce a clone of tumor cells that survive the treatment and are transiently resistant to cancer chemotherapeutic agents (Schwartz et aL, 1988; Sharma and Schimke, 1989; Hopwood and Moulder, 1989; Hopwood et al., 1990). In contrast, multiple exposures of tumor cells to X-irradiation treatment produce clones collaterally sensitive to chemotherapeutic agents (Bedford et al., 1987; Hill et al., 1988; Mivechi et al., 1990; Dempke et al., 1991) or collaterally resistant to other agents (Hill et al., 1988, 1990; De Pooter et al., 1991). These apparently contradictory findings may be explained by understanding the signal transduction pathway (Fig. 1). Ionizing radiation has been shown to transiently activate the fos/jun complex in several human cell lines in a dose-dependent fashion (Stein et al., 1989; Sherman et al., 1990). This would stimulate APresponsive elements in genes associated with DNA synthesis and repair and cause transient collateral resistance to cancer chemotherapeutic agents (Scanlon et al., 1991). Conversely, pretreatment of cells with cisplatin can sensitize the cells to Xirradiation (Begg et al., 1986). In contrast to these findings, repeated treatment of human cells with X-irradiation suppresses gene expression for DNA repair and the cells become collaterally sensitive to anti-metabolites or hyperthermia (Hill and Bellamy, 1984; Mivechi et al., 1990; Dempke et al., 1991). There could be several explanations for this decrease in DNA repair activity (Dewit, 1987), however, it would appear that the final effect of this decreased enzyme activity may be due to repression of the promoter elements of the DNA polymerase fl gene. It would be interesting to have these observations confirmed clinically (Ensley et al., 1984; Ervin et al., 1987). In order to improve treatment of tumors, a trimodality therapy has been tested experimentally with radiation, hyperthermia and cisplatin in a fibrosarcoma (Herman and Teicher, 1988). It appears that the sequencing of cisplatin first, hyperthermia second and then X-irradiation results in the optimal tumor cytotoxicity. Similar results have also been shown with hyperthermia and X-irradiation for leukemia cells (Mivechi et al., 1990), mouse tumors (Wallner et al., 1986; Mansouri et al., 1989) and fibrosarcomas (Herman et al., 1990). A platin analog, platinum rhodamine 123, has been shown to have cytotoxic activity and is an effective radiosensitizing agent both

Cisplatin resistance in human cancers in vivo and in vitro (Herman et al., 1988). In addition, this compound has minimal whole animal toxicity and may have a different mechanism of action at DNA than cisplatin.

4.3. TI-mfos ONCOGENEIN DRUG RESISTANCE Previous studies in a variety of cisplatin-resistant cell lines have suggested the importance of the los oncogene in drug resistance (reviewed by Scanlon et al., 1989b). Cisplatin resistant cell lines both in culture (Scanlon et al., 1989c; Kashani-Sabet, et al., 1990b) and from patient samples (Kashani-Sabet et al., 1990a) have been shown to exhibit elevated c-los gene expression, concomitant with over expression of several enzymes involved in DNA synthesis and repair processes (e.g. dTMP synthase and DNA polymerase fl). The hypothesis thus has been developed that the Fos protein functions in drugresistance by activating transcription of genes with a direct role in repair synthesis of DNA. This was supported by experiments demonstrating that administration of cisplatin leads to a cascade of gene expression, beginning with that of c-los (an earlyresponse gene) (Greenberg and Ziff, 1984) within 1 hr of drug treatment followed by that of dTMP synthase (at 2 hr) and DNA polymerase fl (at 3 hr), presumably effecting the cellular response to cisplatin damage. Studies by other laboratories have defined the two important functions of the Fos protein: Transcriptional activation and DNA synthesis (Cohen and Curran, 1989). For instance, Fos combines with the c-jun oncogene product to facilitate transcription of several genes (Rauscher et al., 1988; Kouzarides and Ziff, 1989; Chiu et al., 1988; Lee et al., 1987b). Moreover, studies using fos antisense RNA have implicatedfos in DNA synthesis and cellular proliferation (Holt et al., 1986; Riabowol et al., 1988; Ledwith et al., 1990). However, it was not clear how the gene-regulating function of Fos contributed to DNA synthesis. Our studies suggested that this was achieved by activation of enzymes with a direct role in DNA synthesis. In order to strengthen this hypothesis, we designed a ribozyme to cleave c-fos mRNA and investigate its effects on signal transduction and cisplatin resistance. The discovery of catalytic RNAs (or ribozymes) has suggested these agents as a new modulator of gene expression. Ribozymes (reviewed by Cech and Bass, 1986) act as site-specific RNAses whose specificity is determined by sequences flanking the catalytic moiety which are complementary to the target RNA. A particular sub-group, known as hammerhead ribozymes, consists of three stems and a catalytic center (Forster and Symons, 1987) and cleaves 3' to any G U N site (N being A, C, or U) (Haseloff and Gerlach, 1988). DNA encoding a hammerhead anti-fos ribozyme was cloned into the dexamethasone-inducible

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pMAMneo plasmid and transfected into cisplatinresistant A2780DDP cells (Scanlon et al., 1991; Funato et al., 1992). The transformants demonstrated dramatic changes in morphology to resemble the parental drug-sensitive cell line. In addition, dexamethasone induction of the dbozyme completely reversed resistance to cisplatin in A2780DDP cells. Moreover, ribozyme induction and its effects on c-fos resulted in decreased expression of DNA synthesis enzymes, including dTMP synthase, DNA polymerase ~ and topoisomerase I, genes shown to be overexpressed in A2780DDP cells, hMTIIA, which has also been implicated in cisplatin resistance (Kelley et aL, 1988), is another known target of Fos/Jun action (Lee et aL, 1987a). Interestingly, hMTIIA expression is also lowered afterfos ribozyme activation in A2780DDP cells. These results, taken together, suggest that Fos is capable of controlling the cellular response to cisplatin-induced DNA damage by activation of several genes which have been separately implicated in cisplatin resistance. Importantly, dTMP synthase (Takeishi et aL, 1989) and topoisomerase I (Kunze et al., 1990) both contain AP-I binding sequences in their 5' regulatory sequences. DNA polymerase contains a ras-responsive element (Kedar et aL, 1990) andros has been shown to operate downstream of H-ras in signal transduction pathways (Ledwith et al., 1990). The fact that overexpression of several enzymes including dTMP synthase, DNA polymerase ~ and topoisomerase I is also implicated in resistance to 5-fluorouracil (Scanlon and Kasbani-Sabet, 1988), azidothymidine (Vazquez-Padua et aL, 1990) and camptothecin (Giovanella et al., 1989), respectively, broadens the spectrum of resistance of Fos action. The observation that both the GST gene (Friling et aL, 1992) and the MDR gene also contains an AP-I binding site (Teeter et aL, 1991) further reinforces the hypothesis that Fos may act as a master switch in the coordination of drug resistance (Scanlon and Kashani-Sabet, 1989), DNA repair and detoxification processes. It may require only a small modulation of Fos by the los ribozyme to have perterbations of the Fos/Jun complex which would either activate the wrong genes or inadequately turn on the correct genes. The aforementioned studies also introduce ribozymes as agents which can overcome drug resistance. That ribozymes can effectively curtail oncogene expression suggests that they may have roles either in reversing drug resistance or in acting as suppressors of tumor cell growth (Kasbani-Sabet et al., 1992). Ribozymes may have a useful advantage over antisense RNA for fos gene expression because of the short half-life of c-fos mRNA (Nishikura and Murray, 1987). These possibilities need to be further explored as a possibility for the development of tumor-selective chemotherapy. Recently a study (Isonishi et al., 1991) demonstrated that H-ras transfection into NIH3T3 cells resulted in an 8.1-fold resistance to cisplatin. The

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mechanisms of cisplatin resistance were only partially accounted for by a decrease in cisplatin transport (40%), a 17% decrease in cisplatin-DNA adduct formation, a 2-fold increase in metallothioneins and no change in glutathione content. There was no characterization of other H-ras responsive genes and/or c-fos oncogene expression. Surprisingly when the c-los oncogene was transfected into another mouse cell line, there was no change in cisplatin cytotoxicity. The 5-fold increase in the Fos protein had no effect on any f o s responsive genes. This was confusing since several genes with AP-I responsive elements that can modulate cisplatin cytotoxicity apparently did not respond to this increase in Fos (i.e. metallothionein, DNA polymerase fl, dTMP synthase), although they were not measured.

5. EXPLOITING THE MECHANISMS OF CISPLATIN RESISTANCE 5.1. THYMIDINESALVAGEPATHWAY Understanding the biochemical events that result in cisplatin resistance will likely provide targets for the development of schemes to circumvent resistance. Both the biochemical and tissue culture studies support the hypothesis that resistance to cisplatin and nucleotide analogs (fluorouracil, AZT) is a phenomenon related to the cells' capacity to repair DNA. Because DNA repair requires deoxyribonucleotides, the increased activity of deoxyribonucleotide metabolism was a likely component of enhanced DNA repair capacity (Scanlon et al., 1989b). In addition to expanded dNTP metabolism, it is likely that resistant cells have increased capacity for adduct excision, repair synthesis and ligation. In human colon carcinoma cells resistant to cisplatin (HCT8DDP), increased gene expression for DNA pol fl was observed (Scanlon et al., 1989c). These findings are similar to those obtained from several other cell lines resistant to cisplatin: mouse leukemia P388 cells, human leukemia K562 cells, human leukemia CEM cells and human ovarian carcinoma cells (Scanlon et al., 1989b). Based on these results, suicide substrates which are incorporated by DNA pol fl were incubated with cisplatin-resistant cells. Specifically, AZT, a derivative of thymidine metabolized via the thymidine metabolic pathway, as the triphosphate AZTTP acts as a suicide substrate causing chain termination (Scanlon et al., 1989c). DNA is degraded more rapidly in cisplatin-resistant cells than by the sensitive cells to remove the cisplatin-DNA adducts (Scanlon et al., 1990a). Repair gaps generated following cisplatin-DNA adduct removal would then provide sites for incorporation of suicide substrates, i.e. AZTTP. If cells were treated with cisplatin and then AZT, cisplatin-resistant cells are at a distinct disadvantage. HCT8DDP cells are prepared to repair cisplatin exposure effects due to the enhanced capacity to synthesize and repair cis-

platin-induced DNA damage, in contrast to the HCT8S cells. AZT may have additional advantages over other DNA synthesis inhibitors, such as aphidicolin. AZT is first phosphorylated by TK, which we have shown is overexpressed in cisplatin-resistant cells. Further, AZTTP can act as an inhibitor of DNA polymerase fl (similarly overexpressed in resistant cells), as well as DNA polymerase ct. AZT has advantages over dideoxythymidine (ddT), as ddT is poorly transported into human ovarian cells (Katz et al., 1990; Perez et al., 1990). Although A2780DDP cells have been shown to have enhanced machinery to repair cisplatin-induced DNA damage, it is still surprising that these cells can turn over 40% of the [t4C]-labeled DNA in the first 3 hr after cisplatin treatment (Scanlon et al., 1990a). The high turn over of labeled DNA in A2780DDP cells could have been due to this repair patch and/or the presence of thymidine rich patches. Consequently, the large quantity of degraded (i.e. decoy) DNA could have protected the A2780DDP cells from cisplatin. This concept has also been termed 'the cells tolerance to cisplatin'. Other studies suggest a similar role for metallothionein or glutathionine (Andrews and Howell, 1990; Scanlon et al., 1989b). An enhanced expression of genes necessary for DNA synthesis and repair has also been demonstrated in patients who failed cisplatin based chemotherapy. Thus, mechanisms for degrading DNA to protect drug resistant cells may have clinical relevance and could be easily exploited by this currently available nucleoside analog. 5.2. NUCLEOSIDEANALOGS Several DNA chain terminating nucleoside analogs have been developed in the past few years which have significant activity as antiviral agents. These analogs are activated by cellular enzymes to the triphosphate level. As the triphosphorylated derivatives, the modified nucleosides can serve as suicide substrates for DNA polymerases. Thus, increases in nucleoside metabolism in cisplatin resistant cells could potentially result in increased activation of these suicide substrates. Ganciclovir is an example used to inhibit DNA polymerase ct (Scanlon et al., 1989b). Increases in DNA polymerase levels in cisplatin-resistant cells could result in increased utilization of the drug and thus increased levels of truncated DNA molecules. Dideoxycytidine (ddC) is another nucleoside that has been studied in human ovarian carcinoma cells. A2780S cells have an ECs0 of 1.5 #M for continuous exposure to ddC. However, a suboptimal dose of cisplatin (1/~M) for l hr makes ddC eight times more potent in A2780S cells. Similar results have been obtained in human colon carcinoma HCT8 cells. In cisplatin resistant A2780DDP cells, cisplatin has only a small modulating effect on ddC, but in human colon carcinoma cisplatin-resistant cells, small doses of cisplatin are potent modulators of ddC (15-fold).

Cisplatin resistance in human cancers Additional studies to measure transport, metabolism and incorporation into DNA by sensitive and resistant cells are currently in progress. Studies with other nucleoside analogs which differ in their specificity as inhibitors of human polymerases are currently being investigated. These analogs offer another promising approach. By sequencing the order of administration of these analogs with cisplatin, synergistic combinations may lead to results similar to those described in the studies with fluoropyrimidines. The cytotoxic action of 1-fl-D-arabinofuranosylcytosine (araC, cytarabine) is complex and araCTP may exert its effect on multiple enzymes rather than just on DNA polymerase fl (Zittoun et al., 1989). Thus, demonstrating the interaction of araC with cisplatin has been confusing at times. Drug synergy has been reported for combinations of cytarabine and cisplatin in human carcinoma cells (Drewinko et al., 1976; Bergerat et al., 1981; Kingston et al., 1989) but this was not reproduced in human ovarian carcinoma 2008 cells (Howell and Gill, 1986) or in other laboratories (Fram et al., 1987). Kern and associates (1988) studied 55 human tumors for araC cytotoxicity and then optimized a 2 hr pretreatment of araC with cisplatin. Sequencing araC prior to cisplatin was most effective against melanoma and breast carcinomas. The reverse sequence decreased the synergism between araC and cisplatin. This observation was also confirmed by human colon cells in culture which showed a dependence on both the concentration and the exposure time for cisplatin to optimize the cytotoxicity of cisplatin with araC (Ozawa et al., 1989). Swinnen et al. (1989) expanded these studies of araC and cisplatin to include hydroxyurea (HU) in human colon carcinoma HT29 cells. This combination appeared to be effective due to the impaired repair capacity of HT29 cells. Carboplatin was substituted for cisplatin and these studies confirmed the clinical potential of this drug combination (Swinnen et al., 1991). This protocol was tested clinically and in the first of these studies 21 of 32 patients with previous treatment had a partial response. Nine of 32 patients and 3 of 8 patients with previous cisplatin treatment had significant responses (Albain et al., 1990). The combination of cisplatin and cyclophosphamide (CTX) has been well established in the treatment of ovarian carcinoma. In recent years, several clinical trials have been conducted comparing the efficacy of carboplatin with that of cisplatin given alone and in combination with CTX (Teeling and Carney, 1990) to patients with ovarian carcinoma. The 2-nitroimidazole radiosensitizer, etanidazole (ETA), is also a hypoxic-cell-selective cytotoxic agent (Teicher et al., 1991a) and a chemosensitizer or modulator of some antitumor drugs. Although the precise mechanism by which ETA acts as a chemosensitizer is not known, it appears to occur at the cellular level and may involve the interaction of a metabolite of the 2-nitroimidazole with the alkylating agent in the vicinity of DNA. Recent studies suggest

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that the addition of ETA to clinical protocols may be beneficial for platin/CTX based chemotherapy. In a clinical setting, cisplatin and etoposide have been shown to act synergestically, against lymphomas, small cell lung carcinoma, non-small cell lung carcinoma and testicular cancer (Hainsworth et al., 1985; Evans et al., 1985) and in animal models (Schabel et aL, 1979), while with cell lines in culture, it has not been possible to reproduce these findings consistently (Tsai et al., 1989). 5.3. PYRIMIDINEPATHWAY Cisplatin and 5-fluorouracil are widely used antineoplastic agents and each has a broad spectrum of antitumor activity. Studies both in vitro and in tumor bearing animals have shown marked therapeutic synergy for these drugs (Schabel et al., 1979; Scanlon et al., 1986). In addition, the combination of these drugs has been very effective for the treatment of head and neck cancer, cervix and upper digestive tract cancers (Etienne et al., 1991; Decker et al., 1983; Khansur and Kennedy, 1991). Because there have been conflicting studies, new types of protocols and schedule combinations with optimal dose sequences and exposure times for this drug regimen are necessary in order to accentuate the biochemical events that support this potentiation (Rowland et al., 1986; Galligioni et al., 1987; Paredes et al., 1988; Recondo et al., 1989; Schilsky et al., 1990; Kemeny et al., 1990). In vitro tissue culture studies (Rosowsky et al., 1987; Newman et al., 1988) have shown that carcinomas which are resistant to cisplatin are collaterally resistant to anti-metabolites because of enhanced dTMP synthase activity. The clinical protocols with cisplatin and 5-fluorouracil may have limited effectiveness in patients because of these changes in the dTMP synthase cycle.

6. CONCLUSIONS In the treatment of cancer there has been no clinical breakthrough to rival combination chemotherapy in the early 1960s. There have also been no major drug advances since the anthracyclines, platin analogs and the podophyllotoxins of the 1970s. The problem of drug resistant human tumors continues to be a challenge, but is slowly yielding to intense scrutiny as we gain a better understanding of the molecular basis of drug resistance in human tumors. Human cell lines have been shown to be good model systems to predict cisplatin resistance in patient carcinomas. Cisplatin initiates a cascade in the cell to protect chromosomes from the toxic effects of the drug (i.e. resistance). Cells respond to cisplatin with a transient response of gene expression to protect the cellular DNA. Repeated challenges of cisplatin established a cell line with stable resistance which has elevated levels of gene expression associated with

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