Recent developments in drug resistance and apoptosis research

Critical Reviews in Oncology/Hematology 28 (1998) 181–205

Recent developments in drug resistance and apoptosis research

Martin Clynes a,*, Carmel Daly a, Roisın NicAmhlaoibh a, Deirdre Cronin a, Cathal Elliott a,Robert O’Connor a, Toni O’Doherty a, Lisa Connolly a, Anthony Howlett b, Kevin Scanlon b

a National Cell and Tissue Culture Centre, Dublin City Uni6ersity, Glasne6in, Dublin 9, Irelandb Berlex Biosciences, 15049 San Pablo A6e., P.O. Box 4099, Richmond, CA 94804-0099, USA

Accepted 20 June 1998

Contents1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182

2. Apoptosis: a final common pathway for cancer drug cytotoxicity?. . . . . . . . . . . . . . . . 1832.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1832.2. JNK Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1832.3. Growth factor receptor and oncogene signalling and resistance to apoptosis . . . . . . . 1842.4. Cell death signaling via Fas and TNF receptors . . . . . . . . . . . . . . . . . . . . . . . 185

3. Bcl proteins in the regulation of apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1853.1. Function of Bcl-2/Bcl-XL, Bax and related proteins and their mechanism of action in

antagonising apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1853.2. Western blot analysis of Bcl family members . . . . . . . . . . . . . . . . . . . . . . . . . 1863.3. Phosphorylation of Bcl-2/Bcl-XL and Bad . . . . . . . . . . . . . . . . . . . . . . . . . . . 1863.4. Bcl-X gene structure and alternative splicing . . . . . . . . . . . . . . . . . . . . . . . . . 187

4. Bcl proteins in the regulation of cancer chemotherapeutic drug-induced apoptosis . . . . . . 1874.1. Bcl-XL, like bcl-2, can protect cancer cells in vitro against chemotherapeutic drug-

induced apoptosis; the presence of antagonistic proteins (Bax) is a crucial factor . . . . 1874.2. Enhancement of chemosensitivity in vitro and of cancer cell apoptosis in vivo by over-

expression of bcl-XS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1884.3. Role of bcl-2 in modulation of telomerase activity . . . . . . . . . . . . . . . . . . . . . . 188

5. Other proteins which may modulate apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . 1885.1. Bag-1 co-operates with Bcl-2 in promoting cell survival and also activates Raf-1 . . . . 1885.2. Bad can heterodimerise with Bcl-XL displacing Bax and promoting cell death. Bad

antagonises Bcl-XL rather than Bcl-2 activity . . . . . . . . . . . . . . . . . . . . . . . . . 1895.3. Galectin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1895.4. MCL-1, BFL-1, survivin, 1AP-like protein. . . . . . . . . . . . . . . . . . . . . . . . . . . 1895.5. Bcl-XL/Bcl-2-interacting proapoptotic proteins . . . . . . . . . . . . . . . . . . . . . . . . 189

6. Bcl-X expression in vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1906.1. Expression of bcl-X in various tissues, and physiology of bcl-x, bcl-2- and bax-

deficient mice. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1906.2. Expression of bcl-2, bcl-X and bax in tumours . . . . . . . . . . . . . . . . . . . . . . . . 190

* Corresponding author. Tel.: +353 1 7045720; fax: +353 17045484; e-mail: [email protected]

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M. Clynes et al. / Critical Re6iews in Oncology/Hematology 28 (1998) 181–205182

7. Recent developments in understanding the role of MRP in drug-resistance . . . . . . . . . . . 1907.1. MRP expression in cell lines and in human tumours. . . . . . . . . . . . . . . . . . . . . 1907.2. MRP gene structure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1917.3. MRP protein structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1917.4. MRP transport activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1927.5. Modulation of MRP mediated drug resistance . . . . . . . . . . . . . . . . . . . . . . . . 1927.6. MRP as a member of the ATP-binding cassette superfamily . . . . . . . . . . . . . . . . 193

8. Clinical studies on reversal of multi-drug resistance in cancer . . . . . . . . . . . . . . . . . . 1948.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1948.2. Problems associated with chemosensitisation . . . . . . . . . . . . . . . . . . . . . . . . . 1948.3. Circumvention of P-170 overexpression . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1948.4. Resistance due to DNA repair mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . 1968.5. New strategies to overcome drug resistance . . . . . . . . . . . . . . . . . . . . . . . . . . 196

8.5.1. Ribozymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1968.5.2. Cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196

8.6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196

9. Conclusion/prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198

Reviewer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198

Biography. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205

1. Introduction

Chemotherapy can cure many human cancers, butresistance—intrinsic or acquired—is a problem formany patients, especially in solid tumours. Combina-tion chemotherapy was developed in order to circum-vent specific resistance mechanisms, (as well as toallow higher overall doses of anti-tumour chemicalsby combining agents with different side-effects/organtoxicity). Unfortunately, many tumours can developresistance to a wide range of drugs with differentcellular targets.

Many changes have been associated with multipledrug resistance in cell culture systems and in tumoursin vivo [1–3]. Alterations in drug accumulation at thecellular level, and in intracellular drug distribution,seem to play an important role [4,5] and P-glyco-protein/P-170, the product of the mdr-1 gene, hasbeen thoroughly studied at the molecular [6,7] and atthe clinical [8] levels. The more recently describedefflux pump MRP (discussed in detail in Section 7below) may also be important in drug transport andsubcellular distribution. LRP, the major protein of arecently described subcellular organelle, the vault [9]may be associated with multidrug resistance in celllines and in clinical samples [10]. Chauffert et al. [11]have drawn attention to the phenomenon of kineticresistance to chemotherapy, and the proportion ofcells at different stages of the cell cycle in a tumour

cell population may play a significant role in responseof that tumour to drug exposure; altered levels of thecyclin-dependent kinase inhibitor p27-Kip1 may be in-volved in kinetic drug resistance [12]. Altered levels/activity/isoenzyme profile of protein kinases may beimportant in drug resistance at least in part by modu-lating activity of proteins directly involved in resis-tance, such as the drug efflux pumps [13]. Changes inlevels or substrate affinity of Topoisomerases I and IIseem to be important also in MDR, although thedrug menu involved is often different from that ofclassical MDR [14–17]. Alterations in DNA repairand in glutathione-related pathways [18,19] may be ofparticular importance in cross-resistance betweenalkylating agents [20–22].

Multidrug resistance in vivo may not always be dueto alterations at the level of the tumour cell. Thevascularisation of a tumour may be critical, both insupply of drug to the tumour, and to deliver oxygen,since hypoxia can make tumours more resistant todrugs (see [23], for review). Expression of effluxpumps in blood vessels of brain tumours may form a‘blood-brain barrier’ conferring resistance on inher-ently sensitive tumours [24].

This review attempts to cover in some detail twoareas where there have been many recent develop-ments, and which may impact on improved under-standing and treatment of resistant cancers; theseareas are the relationships between drug resistance

M. Clynes et al. / Critical Re6iews in Oncology/Hematology 28 (1998) 181–205 183

and apoptosis, and the biology of MRP. There is alsoa brief discussion on clinical approaches to overcom-ing MDR, since improved treatment of cancer pa-tients is the goal of MDR research, and new scientificadvances should be seen as opportunities to developnew treatment strategies.

2. Apoptosis: a final common pathway for cancer drugcytotoxicity?

2.1. Introduction

Most interest in multidrug resistance (MDR) overthe past decade has centered on drug-efflux pumpssuch as P-170 and MRP, but so far results with P-170 modulators have been somewhat disappointing,and in any event the range of drugs pumped by P-170 and MRP is considerably narrower than themenu of drugs involved in MDR in vivo. Some invitro models for wider range MDR are now available[25]. With this historical background, the idea that afinal cell-killing pathway common to all anticancerdrugs may exist has caused considerable interest.

The discovery of the bcl family of proteins whichcan apparently alter the threshold of recognition ofcell damage as a cell death signal suggests novelmechanisms of MDR and new approaches to thera-peutic intervention.

The bcl-2 (B-cell leukaemia/lymphoma 2) gene wasdiscovered by Tsujimoto and Croce [26]; the bcl-2gene is translocated in many follicular B-celllymphomas from its normal 18q21 position to 14q32where its location adjacent to enhancers in the im-munoglobulin H gene [27] leads to high level expres-sion. Alternative splicing yields two proteins, bcl-2a

(239 amino acids) and bcl-2b (205 amino acids), dif-fering only at their C terminus. Most publicationsrefer to bcl-2 (without specifying a or b) and whereWestern blots are reported, the a form appears to bepredominant. Vaux et al. [28] found that transfectionof immature pre-B cells with bcl-2 expression vectorsprotected against cell death due to IL-3 deprivation,thus indicating a role in antagonizing apoptosis.Transfection [29,30] and antisense [31] experimentsconfirmed an important role for bcl-2 in resistance toapoptosis induced by chemotherapeutic drugs. Bcl-2 iswidely expressed in human tumours, and by inhibitingcell death and thus altering the normal cell deathversus cell division ratio it may allow tumour cells toaccumulate the mutations which will later cause thecells to become invasive and metastatic. More recentresearch indicates a key role for bcl-XL, as well asbcl-2, in apoptosis regulation in follicular lymphoma[32].

Investigation of genes involved in programmed cell

death in the nematode Caenorhabditis elegans hasprovided many important leads to an understandingof apoptosis in mammalian cells, since many of thesegenes have been conserved between these distantly-re-lated species [33]. In order of sequence of action inthe cell death pathway the most important CEDgenes include CED-9 (bcl-2 homologue), CED-4(mammalian bcl-XL and caspases can interact withCED-4 protein) and CED-3 (homologue of caspaseenzymes).

It is now becoming clear that bcl-2 is just one com-ponent of a large and complex family of proteinswhich determine how readily particular cells die inresponse to particular physiological or pharmacologi-cal environments (e.g. growth factor deprivation, drugexposure). Programmed cell death may be particularlyimportant in determining organ shape and size duringdevelopment [34]. Some of the current knowledge inthis area is reviewed here, with particular emphasison how cancer cells can develop resistance to apopto-sis induced by cancer chemotherapeutic drugs.

Programmed cell death or apoptosis is mediated bydegradation of DNA and a number of important cel-lular proteins. This in turn is dependent on activationof certain members of the caspase family of prote-olytic enzymes (e.g. conversion of 32 kDa procaspase-3 to active 17 kDa caspase 3 [35]). This activation ismediated by release of activating factors, includingcytochrome c, from mitochondria (Section 3.1). Thepathways linking cellular damage induced by toxicagents such as anti-cancer drugs to release of mito-chondrial procaspase activators are still poorly under-stood, but some information on them is nowemerging.

2.2. JNK Pathway

The JNK/SAPK (c-Jun N-terminal kinase/stress-ac-tivated protein kinase) pathway may be one impor-tant signalling pathway linking exposure to drugs andactivation of caspases (JNK is a member of the mito-gen-activated protein kinase (MAPK) family). Seimiyaet al. [36] showed that reduction of JNK1 proteinlevels in U937 cells (by using JNK1 antisense, but notsense, oligodeoxynucleotides) reduced the rate ofapoptosis induced by etoposide or camptothecin;there was a corresponding reduction in caspase-3 acti-vation. Caspase-3 does not have an appropriateMAPK family phosphorylation site [37] so that inter-mediaries (perhaps release of mitochondrial media-tors) must be involved. The upstream regulatorslinking DNA damage to JNK activation are not com-pletely defined, but the tyrosine protein kinase c-Ablis a JNK activator. Antisense experiments indicatethat Lyn Kinase, a src family tyrosine kinase may


also have this function [38]) and SH-P1 protein ty-rosine phosphatase may be a negative feedback regu-lator [39,40]. In CML cells, however, use of antisenseoligonucleotides directed against the bcr–abl tran-script enhanced cell killing, via apoptosis, by mafos-famide, the treatment being relatively non-toxic tonormal bone marrow cells [41]; further antisense ex-periments by [42] indicate a role for Grb2 and Crk1proteins in a bcr–abl-mediated signalling pathway.Activation of JNK is achieved by a protein kinasecascade, being activated by a MAPKK (MAP kinasekinase) which in turn is activated by a MAPKKK.Activated JNK phosphorylates the c-Jun transcriptionfactor (and other transcription factors including p53);the phosphorylated c-Jun mediates transcription of avariety of AP-1-dependent genes, including c-Jun it-self. Although the details are obscure, it appears thatthe JNK pathway plays an important role in trans-duction of apoptotic signals following exposure to awide range of chemotherapeutic drugs including vincaalkaloids, anthracyclines, mitomycin C, etoposide,camptothecin and cisplatin [39,43,44]. Fas ligand sig-nalling (Section 2.4) may also activate JNK, via alinker protein, Daxx, which mediates a FADD-inde-pendent Fas-associated (Section 2.4) apoptotic path-way [45]. Seimiya et al. [36] isolated a TPA-resistantvariant of U937 cells, which showed resistance toapoptosis induction by anticancer drugs (includingetoposide and camptothecin) and which showed afailure in drug-induced JNK1 activation, in spite ofDNA damage (measured by DNA-protein crosslink-ing) equivalent to that of parental cells. This observa-tion may point to a new class of drug-resistancemechanism, i.e. a failure of signalling pathways link-ing drug-induced cellular damage to apoptosis.

2.3. Growth factor receptor and oncogene signallingand resistance to apoptosis

The c-erbB2 gene encodes a 185 kDa transmem-brane glycoprotein which is the preferred het-erodimerisation partner for the other, homologous,members of the erb family (EGF Receptor, erbB3and erbB4) after they have bound their ligands [46].The actual ligand for c-erbB2 is still unknown. C-erbB2 gene amplification and overexpression is seenin about a quarter of breast carcinomas and expres-sion seemed to correlate with sensitivity to CMF (cy-clophosphamide, methotrexate and 5-fluorouracil)according to studies by Gusterson et al. [47], Klijn etal. [48] and Tetu and Brisson [49]. Muss et al. [50]found that higher doses of adriamycin were needed,but were effective, in patients expressing high levels ofc-erbB2. Bacus et al. [51] reported that tumours over-expressing c-erbB2 are less responsive to CMF, butdo respond to high doses of CAF (A=adriamycin).

Bottini et al. [52] found no correlation between c-erbB2 expression and CMF response in breast cancerpatients. A recent report on cross-reactivity (using theP-170 monoclonal antibody C219) between P-170 andc-erbB2 could be relevant in assessing studies involv-ing immunohistochemical measurements [53]. Tsai etal. [54] found a correlation between the level of c-erbB2 expression in cell lines and resistance to cellkilling by a range of chemotherapeutic drugs (e.g.adriamycin, melphalan, mitomycin C, VP-16 and cis-platin); they also found that c-erbB2 cDNA transfec-tion conferred resistance to these drugs. Yu et al. [55]found that c-erbB2 protein expression in a panel ofbreast cancer cell lines correlated with levels of resis-tance to taxol and taxotere, and that transfection ofc-erbB2 cDNA conferred resistance on a humanbreast carcinoma line (MDA-MB-435) with low-levelexpression of p185. While increased levels of EGFReceptor have been reported in some multidrug-resis-tant cells, and combination chemotherapy with cis-platin and an anti-EGFR-monoclonal antibodyshowed synergistic activity in a human tumour xeno-graft system [56], downregulation of EGFR with anti-sense oligonucleotides resulted in increased cisplatinresistance in the human breast carcinoma line MDA-MB-468 [57]. It remains unclear why the effect ofoverexpression of these receptors has variable effectson chemotherapy-induced apoptosis and preciselywhat the signalling pathways involved are.

Blocking of ‘survival signals’ (e.g. use of antibodieswhich block the interaction of b1 integrin with ECM)in normal but not in malignant breast cells [58] mayalter the balance of ‘death’ and ‘survival’ signals inthe intracellular signal transduction pathways, result-ing in cell death. It has recently been shown that thesurvival/anti-apoptotic signal delivered by IL-3 oper-ates via sequential activation of phosphoinositide 3-kinase and Akt (a serine-threonine protein kinase).Activated Akt can phosphorylate and thus inactivateBAD, a pro-apoptotic protein [59]. It remains to beestablished if this is a common pathway also relevantto other survival signals such as those mediated viaintegrins. Rodeck et al. [60] have shown that block-ade of the EGF receptor or forced suspension culture(preventing cell-substratum adhesion) downregulatesbcl-XL rendering the cells more susceptible to apopto-sis.

The STAT-1 (Signal transducer and activator oftranscription) deficient cells are resistant to TNF-ainduced apoptosis, and have low expression of cas-pases, both properties being restored by transfectionof STAT-1a cDNA [61]. STAT-1 may act as a tran-scriptional activator of caspases [62]. These discover-ies may point to a new potential mechanism by whichcells could become multidrug resistant.


Oncogenes such as c-fos, H-ras and K-ras are im-portant modulators of drug resistance, partly becauseof their role in controlling expression levels of mR-NAs for efflux pumps, repair enzymes and enzymesfor detoxification, as demonstrated by ribozyme ex-periments [63–66]; their role in regulation of apop-totic pathways deserves further study.

2.4. Cell death signaling 6ia Fas and TNF receptors

The Fas/Fas L signal transduction system forapoptosis induction has been reviewed by Nagata[67]. Briefly, Fas (APO-1/CD95) is a cell-surface re-ceptor which, following ligand (Fas L) binding andoligomerisation, induces a cell death pathway involv-ing caspase activation. Other receptor-ligand path-ways (TNF-1 receptor/TNF-a) with similar activitiesexist. Fas and similar receptors contain a homologous80 amino acid cytoplasmic region, called the ‘deathdomain (DD)’ which is essential for transduction ofcell killing signals. Yeast two-hybrid experiments ledto identification of FADD (Fas-associating proteinwith death domain), also called MORT1; and anotherprotein called TRADD (TNFR-1 associated deathdomain protein). FADD contains a C-terminal DDwhich binds to the Fas DD; it also contains an N-terminal ‘death effector domain (DED)’ (also calledthe MORT1 domain). DED, on activation, interactswith and activates Caspase 8 (also called FLICE(FADD-like ICE) or MACH (MORT1-associatedCED-3 homologue)), which contains two DED do-mains. Activated Caspase 8 appears to have substratespecificity similar to that of Caspase 3. (TRADD mayact by direct interaction with FADD, since it has noDeath Effector domain itself, although it also inter-acts with other proteins (RIP) which may recruit cas-pases by other pathways).

Unlike Fas, other members of the TNF receptorfamily can activate the transcription factor NF-kB,via the TRAF protein (TNF receptor-associated fac-tor), thus, in certain cellular/metabolic situations-re-sulting in an overall survival or growth signal. Thismay represent a safety cut-off mechanism to preventinappropriate induction of apoptosis. Bcl-2 and Bcl-XL can inhibit Fas-induced apoptosis [67], but not inall cell systems [68,69]; for further discussion relevantto this, see Section 4.1 and Section 5.1.

Production of Fas L (in the absence of Fas orelements of its transduction pathway) could rendertumours resistant to immune attack by Fas positivelymphocytes. For example, many human lung cancersexpress Fas ligand [70].

Tumours such as prostatic carcinomas [71] havefunctional Fas-mediated cell death pathways. Treat-ment of a number of tumour cell types includingcolon carcinoma [72] and leukaemia [73] with clini-

cally relevant concentrations of anti-cancer drugs in-creases levels of Fas, which could be relevant in vivoby making cells more sensitive to cell killing, e.g. byFas L from immune lymphocytes. There is also recentevidence that drug treatment can induce expression ofFas L [74]. The importance of these observations inchemotherapy (and resistance mechanisms) remains tobe established by further investigations.

3. Bcl proteins in the regulation of apoptosis

3.1. Function of Bcl-2/Bcl-XL, Bax and relatedproteins and their mechanism of action in antagonisingapoptosis

It appears that members of the CED-3 subfamilyof caspases (caspases 2, 3, 9 and 10) and caspase 8(FLICE) are among the most important mediators ofthe proteolytic events determining commitment of acell to apoptosis [75,76]. For example, when U937cells are treated with anticancer agents, caspase 3 isconverted from an inactive 32 kDa precursor to anactive 17 kDa fragment resulting in cleavage of spe-cific cellular substrates including actin [35] and polyADP-ribose polymerase [77]. Selective inhibitors ofcaspase activity (Z-Asp-CH2-DCB) completely blockchemotherapeutic drug-induced apoptosis in cell linessuch as U937 and Jurkat, even under conditionswhere significant DNA damage occurs [78–80]. Itshould be noted, however, that in certain systems,caspase inhibition does not block apoptosis [81]. Re-lease of cytochrome c from mitochondria into the cy-tosol appears to be one of the key steps in caspaseactivation; cytochrome c has been designated asApaf-2 (apoptosis activating factor 2) based on activ-ity in cell-free extracts. Another possibly importantfactor released from mitochondria is AIF (apoptosis-inducing factor) which is a caspase-inhibitor-sensitiveprotease released following mitochondrial depolarisa-tion and permeability transition; this release can beinhibited by bcl-2 [82,83]. Bcl-2 or Bcl-XL inhibit re-lease of cytochrome c from mitochondria followingapoptosis-inducing stimuli, and Kharbanda et al. [84]have demonstrated direct interaction between bcl-XL

and cytochrome c. Zou et al. [85] have recently re-ported the cloning of Apaf-1, a 130 kD protein,which may be the mammalian homologue of CED-4.Apaf-1 binds to cytochrome c and to procaspase 9,and this complex may be responsible for activation ofcaspase 3 [86].

Caspase 3 may activate DNA fragmentation factor(DFF) which initiates DNA fragmentation, long con-sidered a key event in apoptosis [87].

Reed [88] has recently reviewed the mechanisms bywhich Bcl-2 and Bcl-XL may antagonise apoptosis.


These proteins are localised in the outer mitochon-drial membrane, endoplasmic reticulum and nuclearenvelope, and may have cellular functions in additionto modulation of apoptosis. Most emphasis in theliterature to date, however, has been on mitochon-drial activity relating to apoptosis. Bcl-2/bcl-XL createion channels in membranes; Bax may also be able todo this, and possibly by heterodimerization with Bcl-2or Bcl-XL it may alter the properties of channels inthe mitochondrial membrane. Mitochondrial mem-brane depolarisation, with formation of megaporesand calcium efflux, is generally observed during apop-tosis, but there is disagreement on whether this is aconsequence of caspase activation, or a trigger forrelease of cytochrome c and AIF. Hacker and Vaux[89] and Reed [88] have reviewed the actions of pro-apoptotic (Bax, Bcl-XS, Bad, Bak, Bik, Bok) andanti-apoptotic (bcl-XL, bcl-2, Bag-1, Bfl-1, Mcl-1) bclfamily members.

The actual mechanism by which bcl-2/bcl-XL andBax, possibly as homodimers, may form channelswhich facilitate or block apoptosis is not fully under-stood. In one model, heterodimerisation would inhibitformation of functional channels. This hypothesiswould explain some surprising results where overex-pression of Bcl-2 sometimes promotes cell death, andBax or Bak can in some cellular backgrounds unex-pectedly protect against apoptosis. Another model en-visages Bax channels facilitating cytochrome ctransport across the outer mitochondrial membrane,with bcl-2/bcl-XL interfering with this activity [88,90].

Bcl family members (whether pro- or anti-apop-totic) contain several (or all) of 4 bcl-2 homologydomains (BH1 to 4) and in most cases a C-terminaltransmembrane anchor (TM). The different domainsmay correspond to different functions, including inter-action with other proteins. Immunoprecipitation stud-ies and use of yeast-two-hybrid systems haveidentified many proteins which can bind to bcl-2 orbcl-XL (reviewed by Reed [88]). These include theprotein kinase Raf-1, the protein phosphatase cal-cineurin, the GTPases R-Ras and H-Ras, the p53-binding protein p53-BP2, the prion protein, caspasessuch as FLICE (caspase 8) and ICE (caspase 1) anda number of other proteins including CED-4, BAC-1,Nip-1, Nip-2 and Nip-3. Bax does not appear to in-teract with these proteins. These interactions may in-volve sequestering proteins from the cytosol, so thatit is suggested that bcl-2/bcl-XL have a dual function,(membrane channel component and adaptor/dockingprotein). Recently, evidence has been presented to in-dicate that caspase cleavage may convert bcl-2 to aform which has Bax-like apoptosis-promoting func-tions; this could contribute to the rapid collapse ofcells in the latter stages of apoptosis [91]. Other re-cent results indicate that regulation of mitochondrial

mediator release is not the only anti-apoptotic rolewhich bcl-XL and related proteins can play (Section5.5). The various cellular locations of bcl-2/bcl-XL

may be relevant in this regard.Reactive oxygen species (ROS) may be involved in

terminal stages of apoptosis in some situations; forexample, in apoptosis of neuronal cells followingNGF withdrawal, cell death can be prevented byROS traps and antioxidants; ROS production can beblocked by actinomycin D or cycloheximide (whichcan also block apoptosis induction in this system), orby caspase inhibitors [92].

3.2. Western blot analysis of Bcl family members

Krajewska et al. [93] used anti-peptide rabbit anti-seram to examine expression of bcl family membersin tissue from human colorectal tumours. For Bcl-2,the main band was at 26 kDa, with a second band(possibly the phosphorylated inactive form) at 30 kD,in some samples. Bcl-XL migrated as a doublet band(28–30 kD) and a 19 kDa bcl-XS band was also seenin most samples. Bcl-XL levels were elevated in tu-mour versus adjacent normal tissues, but no differ-ence was apparent in bcl-Xs levels. The doubletbcl-XL had also been noted by Krajewski et al. [94],in normal human tissues; they suggested proteolyticprocessing, covalent modifications, initiation of trans-lation from a downstream AUG or CUG in the bcl-XL open reading frame, or additional alternativelyspliced forms of bcl-X.

Bax-a gave a single 21 kDa M.W. band, and Bak asingle 25 kDa band.

Many antibodies available commercially for detec-tion of apoptosis-related proteins give variable resultsand show multiple unexplained bands.

3.3. Phosphorylation of Bcl-2/Bcl-XL and Bad

It is unclear whether or not Bcl-XL activity is regu-lated by phosphorylation, although the doublet bandobserved for the dimeric form on Western blots sug-gests that this is a possibility, and the general similar-ity with Bcl-2 is a further indication that this mayoccur, since phosphorylation of bcl-2 is well docu-mented. Haldar et al. [95], using the phosphatase in-hibitor okadaic acid, demonstrated that Bcl-2 can bephosphorylated in lymphoid cells, and that phospho-rylation inhibits the ability of Bcl-2 to bind to Baxand to modulate apoptosis. Haldar et al. [96,97] fur-ther showed that taxotere and to a lesser extent taxol,cause induction of Bcl-2 phosphorylation in tumourcell types including breast, prostate, leukaemia andlymphoma (possibly as a result of effects on micro-tubules). Phosphorylation of Bad is discussed in Sec-tion 5.2.


3.4. Bcl-X gene structure and alternati6e splicing

The promoter region and genomic organisation ofthe mouse bcl-X gene have been analysed in detail[98]. Two major alternatively spliced mRNAs, withopposing actions on cell death, bcl-XL and bcl-XS,have been described. Bcl-XS lacks the BH1 and BH2domains of bcl-2 and as a negative regulator of apop-tosis it is dominant over bcl-2 and bcl-XL, possiblyby heterodimersing with them. Two additional formsof bcl-X mRNA, bcl-Xb and bcl-XDTM have been de-scribed in rodents, but their function is unknown.Shiraiwa et al. [99] have suggested that rat bcl-Xb (anunspliced form of bcl-X mRNA) may, like bcl-XS,promote apoptosis. A fifth novel alternatively splicedform containing a unique 47 amino acid C-terminus,and termed bcl-X gamma, appears to play a role ininhibiting activation-induced apoptosis in T-cells. Ithas also been found in thymocytes, where interactionof the T-cell receptor with host MHC molecules mayinvolve a cellular pathway affected by bcl-X gamma[100].

A number of alternatively spliced Bax transcriptshave also been described [101].

Pecci et al. [102] found that in a rat endometrialcell line which expresses functional progesterone re-ceptors, cell survival/prevention of apoptosis is depen-dent on the presence of progesterone (underconditions of low serum and absence of glucocorti-coids). Apoptosis could be induced by progesteroneantagonists. In this cell line, progesterone administra-tion increased overall bcl-X mRNA levels, and alsoshifted the ratio of spliced transcripts in favour ofbcl-XL as opposed to bcl-XS. Administration ofprogesterone antagonists increased the relativeamount of bcl-XS, without altering overall bcl-XmRNA levels. The authors speculate that modulationof the bcl-XL: bcl-XS ratio may be involved in degen-eration of the endometrial wall lining at the end ofthe menstrual cycle, when progesterone levels (fromthe ovary) drop.

Tilly et al. [103] found that, in equine systems, cho-rionic gonadotrophin-mediated inhibition of apoptosisin ovarian granulosa cells may be linked to a shift inthe ratio between bcl-2/bcl-XL and bax/bcl-XS mRNAlevels, although in this system it appeared that alter-ation in bax levels was the most important determi-nant.

Chang et al. [104] reported that treatment of a hu-man gastric cancer line with translation/transcriptioninhibitors upregulated bcl-XS and induced apoptosis;this could be inhibited by dexamethasone treatmentwhich suppressed the chemically-induced upregulationof bcl-XS mRNA, and also increased the bcl-XL

mRNA level as well as enhancing overall bcl-XmRNA stability.

4. Bcl proteins in the regulation of cancerchemotherapeutic drug-induced apoptosis

4.1. Bcl-XL, like bcl-2, can protect cancer cells in 6itroagainst chemotherapeutic drug-induced apoptosis; thepresence of antagonistic proteins (Bax) is a crucialfactor

Transfection with bcl-XL cDNA has been shown toprotect several cell types in vitro against apoptosisinduced by a wide range of chemotherapeutic drugs.Minn et al. [105] transfected human bcl-XL cDNA intothe murine IL-3-dependent prolymphocytic cell lineFL5.12; they found increased resistance to the anti-cancer drugs bleomycin, cisplatin, etoposide and vin-cristine, as well as the protein synthesis inhibitorhygromycin B and mycophenolic acid (which inhibitsguanine biosynthesis). Reversible cell cycle arrest wasnot prevented by bcl-XL overexpression; the protectionappeared, instead, to involve direct protection againstundergoing apoptosis. Vincristine-treated bcl-XL over-expressing cells became polyploid after drug removal,and Minn et al. [105] suggest a role for bcl-XL protec-tion against apoptosis in accumulation of chromosomalabnormalities in tumours.

Dole et al. [106] examined a range of neuroblastomacell lines with different levels of bcl-2 and bcl-XL ex-pression; they also transfected bcl-XL cDNA into oneof the lines which expressed very low endogenous levelsof bcl-XL. The transfected lines displayed enhancedresistance to apoptosis induced by cisplatinum or by anactivated form of cyclophosphamide (4-hydroperoxy-cyclophosphamide); apoptosis induced by the topoiso-merase II inhibitor VP-16 was, however, slightlydelayed, but not prevented.

Transfection with bcl-2 or bcl-XL cDNA conferredresistance to taxol in human leukaemia cell lines HL-60(acute myeloid leukaemia) and 697 (pre-B humanleukaemia), [107,108]. Bcl-XL cDNA transfection alsoconferred resistance to cycloheximide-induced apopto-sis in mouse myeloma cells [109].

In vitro experiments to downregulate bcl-2 expres-sion using antisense oligodeoxynucleotides in small-celllung cancer cells [110] and a hammerhead ribozyme inprostatic carcinoma cells [111] have been successful,and represent a possible prelude to gene therapy appli-cations. Kitada et al. [31] found enhanced sensitivity tomethotrexate and ara C following bcl-2 antisense treat-ment of non-Hodgkins leukaemia cells. Webb et al.[112] described the first trial of bcl-2 antisense oligos inhumans. Nine patients who had bcl-2 positive relapsednon-Hodgkin’s lymphoma received an 18-base phos-phothioated sequence over a 2-week period. No side-ef-fects were noted, and tumour response was recorded.

Bcl-2 and bcl-XL appear to act similarly in antagonis-ing apoptosis induced by cancer chemotherapeutic


drugs and other chemicals. Ibrado et al. [113] foundthat overexpression of bcl-2 or bcl-XL in the humanacute myelogenous leukaemia line HL-60 inhibited acti-vation of one of the caspases believed to be involved inapoptosis (Caspase 3/CPP32/Yama/apopain); in thiscase, the apoptosis-inducing stimulus was cytosine ara-binoside. This group later [114] reported that the in-hibitory effect of bcl-XL may involve blockingcytochrome c release from mitochondria, and thus pre-venting caspase 3 activation. Similarly, Decaudin et al.[115] found that, in human leukaemia cell lines, overex-pression of bcl-2 or bcl-XL protected against chemi-cally-(VP-16, adriamycin, cytosine arabinoside,ceramide) induced disruption of mitochondrialtransmembrane potential (which may be a key step inprocaspase activation via release of mitochondrial me-diators such as cytochrome c). Decaudin et al., alsofound that while overexpression of the protease in-hibitor cytokine response modifier A, blocked Fas/Apo-1/CD95-mediated effects on mitochondrialtransmembrane potential and nuclear apoptosis, it hadno effect on the corresponding chemotherapy-inducedevents.

Simonian et al. [116] generated clones of FL5.12murine lymphoid cells with equivalent levels of bcl-2and bcl-XL expression (using constructs coding for theflag epitope, to facilitate monitoring of equal expressionlevels). Their assays involved continuous exposure totoxic agents, followed by viability assessment usingtrypan blue exclusion (which would, of course, identifycells only at a late stage in cell damage, when theplasma membrane becomes generally permeable). Withthis assay, their results indicated that bcl-XL providedsomewhat better protection than did bcl-2, againstchemotherapeutic agents that these authors classified asbeing specific for M phase (vincristine, vinblastine,VP-16, VM-26) and for S-phase (methotrexate, 5-FU,hydroxyurea) or which are non-phase specific (cis-platin). Bcl-XL and bcl-2 provided similar levels ofprotection against irradiation-induced apoptosis. (Irra-diation, like cisplatin, was classified as not cell cyclephase specific). On the other hand, Tu et al. [117] foundthat drug treatment of myeloma cells caused up-regula-tion of bcl-2 but not of bcl-X.

Huang et al. [68] have presented evidence that bcl-2,bcl-XL and adenovirus protein EIBI9 kDa are function-ally equivalent, can interact with Bax and Bak, and canblock cell death induced by many cytotoxic agents; inthe systems they examined, however, these proteinswere ineffective against death signals from the Fas orTNF receptors although in other systems Fas-inducedapoptosis can be blocked by bcl-2 [30]. Susin et al. [69]have shown that caspases, (which could be activateddirectly rather than via mitochondrial disruption inFas-activated pathways in certain situations or celltypes) can cause permeability transition and AIF re-

lease in isolated mitochondria. This is not inhibited bybcl-2, in contrast to permeability transition induced byceramide or peroxidants, and provides a partial modelfor understanding bcl-2-independent Fas-induced apop-tosis (but for other possibilities, see Section 5.1).

4.2. Enhancement of chemosensiti6ity in 6itro and ofcancer cell apoptosis in 6i6o by o6erexpression ofbcl-XS

Bcl-XS is the translation product of the short (alter-natively spliced) form of bcl-X mRNA. It exerts aneffect on apoptosis which is antagonistic to bcl-XL andbcl-2, serving as a dominant negative inhibitor of theiractivity. Bcl-XS cDNA transfectants of the humanbreast carcinoma line MCF-7 (which overexpresses bcl-XL) show a 5–10-fold increase to apoptosis induced invitro by VP-16 or taxol [118] Bcl- XS cDNA transfec-tion also enhanced sensitivity to adriamycin, vincristine,VP-16 and 5-FU in a low-level adriamycin pulse-se-lected resistant variant of the human lung carcinomacell line DLKP (R. NicAmhlaoibh and M. Clynes, inpreparation).

Transient adenovirus-vector mediated bcl-XS cDNAinfection induced apoptosis of MCF-7 cells in vitro,and also when injected intratumorally into MCF-7derived tumours in nude mice [119].

4.3. Role of bcl-2 in modulation of telomerase acti6ity

Telomerase activity is involved in maintaining telom-eres at the ends of chromosomes; loss of telomeres mayplay an important role in cellular senescence/differenti-ation, and telomerase activity is high in reproductivecells and most cancer cells. Mandal and Kumar [120]found that over-expression of Bcl-2 in human cervicalcarcinoma HeLa cells and in the human colorectalcarcinoma line DiFi led to a significant increase intelomerase activity. They also showed that IL-2 depri-vation of the cytotoxic T-cell line, CTLL-2, causeddown-regulation of both Bcl-2 and of telomerase activ-ity.

5. Other proteins which may modulate apoptosis

5.1. Bag-1 co-operates with Bcl-2 in promoting cellsur6i6al and also acti6ates Raf-1

Takayama et al. [121] used an interesting approach tosearching for new proteins which might interact withBcl-2. They screened a mouse embryo phage expressionlibrary with recombinant bcl-2, and then screened witha human-specific anti-Bcl-2 monoclonal antibody. Thisresulted in discovery of Bag-1 (Bcl-2-associated athano-gene 1, apparently named from the Greek


athanos, anti-death). Bag-1 is not homologous to theBcl-2 family, but co-transfection of Bag-1 and Bcl-2into the human lymphoid line, Jurkat, enhanced protec-tion against apoptosis induced by staurosporine, anti-Fas antibody and cytolytic T-cells (in comparison tocells transfected with either cDNA alone). Takayama etal. [121] suggested that some pathways previously de-scribed as bcl-2/XL independent may not be indepen-dent, but may require co-expression of Bag-1 (Section4.1). Investigations using yeast two-hybrid systems andco-immunoprecipitation indicate that Bag-1 also inter-acts with Raf-1 (a protein kinase), although it shares nohomology with the previously-described Raf-1 activat-ing proteins Ras and 14-3-3 [122]. Overexpression ofeither Bcl-2 or Bag-1 (or, more potently, both together)blocks apoptosis caused by NGF withdrawal in PC12cells [92]. Bag-1 can occur, in human cells, in 36 and 50kDa isoforms (32 and 50 kDa in mice), due to alterna-tive transcription initiation sites. The lower M.W. formseems to be cytosolic, with the 50 kD form beinglocated in nuclei and mitochondria. Bag-1 is virtuallyidentical to RAP46, which interacts with activatedsteroid hormone receptors; it also binds to hepatocytegrowth factor receptors and PDGF receptor, enhancingtheir anti-apoptotic activity [123].

5.2. Bad can heterodimerise with Bcl-XL displacingBax and promoting cell death. Bad antagonises Bcl-XL

rather than Bcl-2 acti6ity

Bad [124] was discovered by using yeast two-hybridsystems and by expression library cloning. Bad antago-nises cell death inhibition by bcl-XL. It contains theBH1 and BH2 domains, but lacks the hydrophobicC-terminal signal anchor sequence which other familymembers use for membrane integration. Yang et al.[124] suggest that Bad in itself has little effect on theapoptotic pathway, but that it promotes cell death bydisplacing Bax from Bcl-XL: Bax heterodimers (whichpromote cell survival). They cite evidence that Bcl-2and bcl-XL which are mutated so that they no longerinteract with Bax, also lose their ability to block celldeath. They also suggest an analogy with the Myc,Max, Mad regulatory network [125], in which, forexample, while Myc:Max heterodimers are transcrip-tional activators, Max:Max or Mad:Max are believedto be transcriptional repressors [126].

Bcl-2 can sequester the protein kinase Raf-1 fromcytosol to the mitochondrial membrane, resulting inphosphorylation of BAD (which can in its unphospho-rylated form heterodimerise with Bcl-2 or Bcl-XL); thephosphorylated BAD dissociates from bcl-2 or bcl-XL

and moves into the cytosol in a complex with theRaf-1-interacting protein 14-3-3, and no longer inter-feres with bcl-2/bcl-XL activity [88]. BAD is also phos-phorylated by active Akt protein kinase in a PI

3-kinase-dependent, IL-3 stimulated pathway [59], asmentioned in Section 2.3.

5.3. Galectin

Galectin 3 is a beta-galactoside binding proteinwhich has recently been shown [127] to inhibit cisplatininduced apoptosis, without altering expression of Bcl-2,Bcl-XL or Bax. This protein contains the highly con-served NWGR amino acid sequence in the BH1 domainand mutations in this region abolish its ability to pro-tect against apoptosis.

5.4. MCL-1, BFL-1, sur6i6in, IAP-like protein

Mcl-1 [128,129], and bfl-1 [130] are bcl-2 homologueswhich suppress apoptosis. Their importance remains tobe established. A human gene, termed survivin, withhomology to the baculovirus IAP gene has been clonedrecently by [131]; it is widely expressed in tumours andin foetal tissues, but not in terminally differentiatedcells. It can (on transfection) antagonise IL-3-depriva-tion-induced apoptosis in lymphocytes, and may havean anti-apoptotic role in vivo. Another human IAP-likeprotein (hILP) may inhibit apoptosis at a step down-stream of mitochondrial events [132].

5.5. Bcl-XL/Bcl-2-interacting proapoptotic proteins

Recently-discovered proteins which may be impor-tant in bcl-XL activity include:� MRIT, (a caspase homologue-named from the San-

skrit word for death, which is one up on the Greekscholars-whose C-terminal lacks the caspase catalyticconcensus sequence, but which contains a DED,Section 2.4); MRIT, like CED-4, interacts with Bcl-XL and with caspases including FLICE and possiblycaspase 3 [133];

� p28 Bap 31, an integral endoplasmic reticulumprotein which interacts with Bcl-2, Bcl-XL and pro-caspase 8 (PRO-FLICE), this interaction beingblocked by Bax. Bcl-2/bcl-XL may inhibit a caspase-activation function of this protein [134];

� Harakiri, which interacts with Bcl-2 or Bcl-XL (butnot with Bax or Bak), and promotes apoptosis in abcl-2/bcl-XL inhibitable fashion [135];

� Bim is a pro-apoptotic protein, containing the BH3domain common to most of the bcl family; it existsin three isoforms (possibly due to alternative splic-ing) and its pro-apoptotic activity is antagonised bybinding of bcl-2, bcl-XL or bcl-w [136].The discovery of these proteins illustrates the com-

plexity of the cell’s apoptotic machinery, and indicatesthat release of mitochondrial mediators is only part ofthe overall picture; furthermore, the results of Li et al.[137] support an anti-apoptotic role of bcl-XL indepen-dent of cytochrome c release.


6. Bcl-X expression in vivo

6.1. Expression of bcl-X in 6arious tissues, andphysiology of bcl-x, bcl-2- and bax-deficient mice

Krajewski et al. [94] used an anti-peptide polyclonalantiserum which detects both Bcl-XL and Bcl-XS toexamine, by immunohistochemistry, expression of Bcl-X in normal tissues in vivo. High level expression wasobserved in specific subsets of the haemopoietic system(including cortical lymphocytes, activated lymphocytes,and several bone marrow cell types), reproductive tis-sues, and a variety of epithelial cells. In general, expres-sion patterns of Bcl-X were different from that of Bcl-2,indicating that they may play different, non-redundantbut possibly similar roles in different tissues and atdifferent stages of differentiation; the differences infunction are not, however, clear at the moment.

Bcl-X-deficient mice [138] died around embryonicday 13, and widespread apoptosis was observed inpostmitotic immature neurons of developing brain,spinal cord and dorsal root ganglia. The lifespan ofimmature lymphocytes was also shortened. Bcl-2-knockout mice (lacking both Bcl-2a and Bcl-2b

proteins) were less severely affected; they had appar-ently normal embryonic development, but tended to dieprematurely and showed (to a variable degree) growthretardation, and exhibited abnormalities in kidney,spleen, thymus, lymphocytes, hair pigmentation andsmall intestine [139].

Bax-deficient mice were viable, displaying hyperplasiain thymocytes, B cells and ovaries, but abnormalities ofmale reproductive organs and associated sterility; theabnormalities in testes included hypoplasia and in-creased apoptosis. Obviously, the functions of genessuch as bax in the whole animal cannot be reduced tosimple positive or negative effects on apoptosis rates[140].

6.2. Expression of bcl-2, bcl-X and bax in tumours

As already discussed, bcl-XL appears to be widelyexpressed in normal tissues, [94]. Any cancer therapydirected against bcl-XL must take account of possibleside-effects on normal tissues, unless it can be targetedto the tumour tissue. Previous experience with modula-tors of P-170 indicates the types of problem which canarise from inhibition of a target tumour protein whichis also expressed in normal tissue [141–143].

There is little information available in the literatureabout expression of bcl-XL in tumours, and little clearevidence on correlation of bcl-XL expression with resis-tance to chemotherapy. This is in contrast with a fairlyextensive literature on bcl-2 (which of course was dis-covered much earlier). There is some evidence thatbcl-XL expression may be correlated with chemoresis-

tance in multiple myeloma [144] and that bcl-2, bcl-XL

and Bax may not be relevant to nitrogen mustard drugresistance in B-CLL [145].

Bcl-2 may be overexpressed in more than half of allhuman cancers (see [146] for review) and may be associ-ated with poorer prognosis in some lymphomas andleukaemias, and in prostate adenocarcinoma. Increasedexpression of bcl-2 may be associated with drug resis-tance in squamous cell lung carcinoma [147] and bcl-2is widely expressed in breast cancer [148,149]. Reed’sgroup [93] used polyclonal antibodies to specific peptidesequences to examine expression of bcl-XL and relatedproteins in primary colorectal adenocarcinomas andalso in adenomatous colonic polyps. Their results sug-gested a tendency for Bcl-XL expression to increase,and for Bcl-2 and Mcl-1 to decrease as differentiationstate decreased. Expression of the anti-apoptoticprotein Bak was also reduced in both adenomas andcarcinomas, while Bax expression did not show majorchanges.

The ratio between Bcl-XL (and related anti-apoptoticproteins) and Bax (or similar pro-apoptotic proteins)seems to be important in determining sensitivity tochemotherapy. Reduced expression of Bax (by im-munocytochemistry) is associated with poor responserates to chemotherapy in breast cancer [150]. The bcl-2/bax ratio showed good correlation with chemoresis-tance in acute myeloid leukaemia patients [151]. HighBax expression [152] relative to Bcl-2 may contribute tothe inherent chemosensitivity of testicular tumours.

It should be realised that resistance is often multi-functional [153], that tumour cell populations can bevery heterogenous with respect to resistance level andmechanism [154] and that some resistance mechanismsmay be dominant over others; therefore measurementof a single biochemical determinant, even one appar-ently so central as bcl-2/bcl-XL, may be unable topredict sensitivity to chemotherapy-induced apoptosisin patients. For example, bcl-2 expression per se wasnot a useful predictive marker of chemosensitivity innon-Hodgkins lymphoma [155] or chronic lymphocyticleukaemia [156,157] and bcl-2 was surprisingly reportedto be a good predictor of response in breast carcinomawhen analysed on its own [158,159].

7. Recent developments in understanding the role ofMRP in drug-resistance

7.1. MRP expression in cell lines and in humantumours

Since its initial discovery in the drug-resistant smallcell lung cancer cell line H69AR [160], MRP has beenshown to be present in multidrug resistant cell linesfrom a variety of tumor types. These tumors


include leukaemias [161,162], fibrosarcoma [163], smallcell and non-small cell lung carcinomas [164–166],breast carcinoma [167,168], melanoma [169] and bladdercarcinoma [170,171]. There have also been reports ofco-expression of P-glycoprotein (Pgp) and MRP in anumber of drug-selected cell lines [172,173]. Cell linesoverexpressing MRP are typically cross-resistant to an-thracyclines, epipodophyllotoxins and some vinca alka-loids [174–176]. The resistance profiles of MRP and Pgpoverexpressing cell lines exhibit similarities, but theseprofiles differ, notably with regard to Taxol resistance[177] (see Section 7.4). The explanation for a cell devel-oping MRP rather than Pgp-mediated resistance duringdrug exposure remains unclear, but it is believed thatoverexpression of MRP may confer initial low levelresistance, while Pgp overexpression develops as higherlevels of resistance are required for survival [165,172].

The role of MRP expression in drug-resistance inhuman cancers in vivo remains uncertain, althoughpreliminary evidence suggests a correlation in neuroblas-toma [178] retinoblastoma [179] primary breast car-cinoma [167] and non-small cell lung cancer [180]. MRPis present in many normal tissues, but Flens et al. [181]reported that immunostaining was predominantly cyto-plasmic in normal cells, but predominantly plasmamembrane-located in malignant tissue. Althoughderived from data with cell lines rather than tumourtissue, the data presented by Berger et al. [169] stronglysupports a role for MRP in the intrinsic drug resistanceof melanoma.

7.2. MRP gene structure

The MRP gene has been localised to band p13.13-13.12 on chromosome 16 [160]. Sequence analysis per-formed by Zhu et al. [182] of a 2.2 kb 5%-flankingsequence of genomic MRP from HL60-ADR cells sug-gested the presence of regulatory elements in this region.These elements included AP-1, AP-2, SP-1, ERE (estro-gen response element), GRE (glucocorticoid responseelement) and CRE (cyclic AMP response element) sites.

Zhu and Center [182] discovered that the most signifi-cant region of promoter activity may be present in ahighly GC-rich region of 194 nucleotides. This regiondoes not contain a TATA box for facilitating site-spe-cific initiation of transcription. It contains multiple startsites for transcription, as is found in the case of Pgp[183]. The MRP gene has been shown to be amplified ina number of drug- selected cell lines [184,185]. In othercases MRP mRNA levels have been shown to be in-creased in the absence of gene amplification[186,187,160] and this is believed to be due to alterationsin transcriptional control of the MRP gene. The MRPgene encodes a message of 6.5 kb which has the potentialfor synthesis of the 1531 amino acid MRP protein.Transfection studies using full length MRP cDNA have

shown that MRP can confer vincristine and VP-16resistance but not cisplatin resistance to drug sensitiveHeLa cells [176] (see Section 7.4).

7.3. MRP protein structure

A number of models have been proposed [170] for thesecondary structure of the MRP protein. Hipfner et al.[188] demonstrated that MRP has an extracytosolic NH2

terminus and provided evidence to suggest that in theNH2 terminal domain, the membrane-spanning domainof MRP (MSD-1) contains five transmembrane (TM)segments. MSD-2 was predicted as possessing six TMsegments and MSD-3 was predicted as possessing eitherfour or six TM regions. Kast and Gros [189] alsoprovided evidence to suggest that MRP possesses 5 and6 TM regions in MSD-1 and MSD-2, respectively.Bakos et al. [190] presented a membrane topology modelof MRP which predicted that MSD-1 contained four orfive TM segments and that both MSD-2 and MSD-3each contained six TM segments. Other members of theABC (ATP-binding cassette) transporter family, such asthe pancreatic b-cell sulfonylurea receptor (SUR), theyeast cadmium resistance factor YCF1 and the MOAT(multispecific organic anion transporter) protein mayalso have topologies similar to MRP [191]. MRP may belocalised in the endoplasmic reticulum [192], post-Golgicytoplasmic vesicles and in the plasma membrane ofresistant cells [193].

The amino acid sequence of MRP contains a numberof sites known to be relevant for ATP binding andpost-translational modification [188,190,170]. It hasbeen demonstrated that the unmodified MRP proteinhas an apparent mass of 170 kDa and is processed intoa mature 190 kDa form by the addition of N-linkedoligosaccharides [191,193]. Human MRP is now be-lieved to contain 14 potential sites for N-linked glycosy-lation. Only three of these sites (Asn19, Asn23 andAsn1006) are believed to be glycosylated [188]. The effectsof glycosylation on MRP activity are not fully known.It has been demonstrated that tunicamycin-induced inhi-bition of glycosylation has little effect on the cellulardrug accumulation characteristics of MRP-transfectedcells [193,192].

The specific location and functional role of the phos-phorylated residues on MRP are currently unclear. InCFTR (cystic fibrosis transmembrane conductance reg-ulator) and Pgp (P-glycoprotein), phosphorylation oc-curs predominately in the linker region [194,195]. Thisis also believed to be the case for phosphorylation ofthe MRP protein, but comparison of the linker regionamino acid sequences of human and murine MRP haveshown that there is only a low level of conservation ofpotential PKC phosphorylation sites between these twohomologs. Human and murine MRP proteins share avery high proportion of conserved sequences


[174,191,196]. MRP is phosphorylated on serineresidues only and the phosphate groups are containedin nine tryptic peptides [197]. The phosphorylation ofthese amino acids was shown to be blocked by com-pounds such as staurosporine and this inhibition ofphosphorylation may result in an increase in drugaccumulation in MRP overexpressing cells [197]. Theprotein kinases involved in the phosphorylation ofMRP have not been conclusively identified. There arepotential sites for phosphorylation of MRP by proteinkinase C, cyclic AMP/cyclic GMP-dependent proteinkinases and tyrosine kinases [193].

7.4. MRP transport acti6ity

The MRP transporter has the ability to transportcysteinyl leukotrienes such as leukotriene C4 (LtC4) andglutathione conjugates such as glutathione disulphide(GSSG), [198–200]. A wide variety of organic anionsincluding anionic conjugates of bile salts and steroidhormones have the potential to serve as physiologicalsubstrates of MRP [201]. Transport of heavy metaloxyanions by MRP was demonstrated by Cole et al.[177].

A relationship exists between MRP protein expres-sion and drug resistance in various MRP expressing celllines but the mechanism by which MRP confers resis-tance to chemotherapeutic drugs is currently unclear.MRP has the ability to transport glutathione conju-gates and so if a cytotoxic drug became conjugated toglutathione then this drug conjugate could serve as apotential substrate for transport by MRP. Glutathioneconjugation is not known to occur for natural productdrugs [202]. In addition, MRP transfectants do notexhibit increased resistance to alkylating agents, a classof drugs for which glutathione conjugation is known tooccur [175,177]. Although MRP has been indirectlyshown to reduce cellular accumulation of drugs such asadriamycin and daunorubicin [203,166], only a limitednumber of reports have described the direct transportof cytotoxic drugs by MRP. Studies carried out by Paulet al. [204] demonstrated that MRP isolated fromMRP-transfected NIH/3T3 fibroblasts could serve asan ATP-dependent pump for a variety of naturalproduct, unconjugated cytotoxic drugs including an-thracyclines, vincristine and etoposide. Paul et al. [205]demonstrated that MRP isolated from the HL60-ADRcell line actively transported daunorubicin, VP-16 andvincristine but not vinblastine or Taxol. Stride et al.[174] and Loe et al. [206] demonstrated that transportof vincristine by MRP occurred only in the presence ofglutathione.

The analysis of drug resistance profiles of MRP-over-expressing, drug-selected cell lines has generated infor-mation on the potential drug transporting abilities ofMRP [207,208]. This information is complicated by the

fact that many of these cell lines were obtained by astepwise selection in drug, which facilitates the develop-ment of multiple collateral resistance mechanisms. Thismay result in the simultaneous expression of numerousdrug resistance mechanisms which ultimately results indifficulties in interpreting the contribution of any indi-vidual mechanism to drug resistance levels [172,173].Studies carried out with MRP-transfected cell lines mayserve as more useful models for definition of the profileof drugs potentially transported by MRP. A studycarried out by Breuninger et al. [175] using MRPcDNA-transfected NIH/3T3 fibroblasts demonstratedthat these transfectants displayed increased resistanceto several lipophilic drugs including adriamycin,daunorubicin, VP-16, actinomycin D and vincristine,but negligible levels of resistance to vinblastine andTaxol, but were more sensitive to cisplatin (Table 1).The absence of high level resistance to Taxol in MRP-transfected cells is significant as this drug is a highaffinity substrate for transport by Pgp. In a similarstudy carried out by Cole et al. [177] it was found thatMRP-transfected cell populations possessed a moderatelevel resistance to adriamycin, daunorubicin, epirubicin,vincristine and VP-16. These cells exhibited only lowlevel resistance to Taxol, vinblastine and colchicine. Inaddition, these transfectants were not resistant to mi-toxantrone or cisplatin. The MRP-transfected cellswere also resistant to a selection of heavy metal anionsincluding arsenite, arsenate and trivalent and pentava-lent antimonials, but were not resistant to cadmiumchloride [177].

7.5. Modulation of MRP mediated drug resistance

The chemosensitisers verapamil and cyclosporin Aare significantly less effective at reversing drug resis-tance in MRP overexpressing cell lines, than is the casein Pgp overexpressing cell lines, [175,209,166]. It was

Table 1Summary of drug resistances of two MRP transfected cell lines

Fold resistanceaCytotoxic drug Fold resistanceb

5.6 9.4Adriamycin5.1Daunorubicin 7.8

Epirubicin N.D.c9.0Mitoxantrone 1.2 1.5

3.8Vincristine 8.44.3Vinblastine 1.2

11.411.6VP-16N.D.c1.1Colchicine

2.0Taxol 1.30.9 0.9cisplatin

2.3N.DcActinomycin D

a IC50 of MRP transfectant/IC50 of control transfectant ([177]).b IC50 of MRP transfectant/IC50 of control transfectant ([175]).c N.D.=Not done.


shown by McGrath et al. [210] that MRP was notlabelled with a photoaffinity analogue of verapamil inthe MRP-overexpressing HL60-ADR cell line. Thissuggests that verapamil does not directly interact withMRP. In the case of Pgp, verapamil is known to binddirectly to the protein and interfere with transport inthis manner [211]. A number of compounds have beenidentified as having the ability to efficiently modulateMRP activity. The use of these compounds as modula-tors of MRP has been reviewed by Twentyman andVersantvoort [212]. The leukotriene D4 receptor antag-onist MK571 has been extensively characterised byGekeler et al. [213] in selected Pgp and MRP expressingcell lines. A complete reversal of vincristine resistancewas achieved by co-incubation with a non-toxic level ofMK571 in an MRP-expressing cell line. This compoundwas found to have no effect on the level of toxicityachieved in a Pgp-overexpressing cell line. Gekeler et al.[214] showed that the specific bisindolylmaleimideprotein kinase C inhibitor GF109203X totally reversedvincristine resistance in one MRP-overexpressing cellline. This compound only partially reversed adriamycinand vincristine resistance in a second MRP-overex-pressing cell line. The effect of this compound on drugresistance of a Pgp-overexpressing cell line was notexamined in this study [214]. Duffy et al. [215] demon-strated that a sub-set of NSAIDs including in-domethacin, sulindac and tolmetin enhanced drugtoxicity (in MRP-expressing cell lines) of drugs such asadriamycin, vincristine and VP-16 which are MRP sub-strates (but not of non-MRP-substrate drugs), probablyas a result of interference with MRP activity. Draper etal. [216] reported similar effects with indomethacin. Invivo efficacy of MRP-substrate drug /NSAID combina-tions has also been demonstrated in transplantablemurine tumours and human lung tumour xenografts(R. O’Connor, E. Moran and M. Clynes, unpublished).Consideration must obviously be given also to possiblesensitisation of normal tissues to anticancer drugs, ifMRP is inhibited; the studies of Wijnholds et al. [217]showing hypersensitivity to VP-16 in mrp−/− miceand of Lorico et al. [218] showing increased drugsensitivity in double-MRP-knockout embryonic stemcells, are relevant in this regard.

7.6. MRP as a member of the ATP-binding cassettesuperfamily

Members of the ATP-binding cassette (ABC) super-family contain at least one hydrophobic transmem-brane region and a cytoplasmic nucleotide bindingdomain (NBD). These proteins are involved in energydependent transport of a variety of substrates acrossmembranes. The ATP binding NBDs contain conservedresidues (Walker A and B motifs) spaced by 90–120amino acids (Walker C motif), [219]. A total of 21 new

genes from the human ABC superfamily were relativelyrecently identified, bringing the total number of charac-terised human ABC genes to 33 [220].

A number of human ABC proteins have been wellcharacterised and these include proteins involved indrug transport (Pgp and MRP) and peptide transport(TAP), as well as proteins such as CFTR (cystic fibrosistransmembrane conductance regulator) and SUR (sul-fonylurea receptor), [221–223]. MRP exhibits signifi-cant sequence homology to CFTR (19% amino acididentity), rat SUR (29% amino acid identity), YORI(33% amino acid identity), a yeast protein with theability to confer oligomycin resistance, ItpgpA (32%amino acid identity), a Leishmania protein involved inresistance to oxyanions and YCF1 (43% amino acididentity), a yeast protein involved in cadmium resis-tance [170].

As already described, MRP has the ability to trans-port a wide variety of hydrophobic anionic compounds.The multispecific organic anion transporter (MOAT) inliver canicular membranes and other tissues has similartransport abilities [224–226]. This suggested that theMRP-1 gene may have been responsible for cMOATprotein synthesis. The extremely low expression ofMRP-1 protein in the liver [227,160] and the predomi-nantly basolateral localisation of MRP-1 in the hepato-cyte membrane [228], indicated that the cMOATprotein was not a product of the MRP-1 gene.Paulusma et al. [229] provided evidence that MRP-1and cMOAT are encoded by two different genesnamely MRP-1 and MRP-2.

It has been shown that increased levels of GST(glutathione S-transferase) are involved in resistance toalkylating agents [230]. Ishikawa and Ali-Osman [231]demonstrated that GST-mediated cisplatin resistanceinvolves two steps. The initial step involves the forma-tion of a glutathione (GSH) S-conjugate and this isthen followed by the subsequent removal of the toxicconjugate from the cell by a glutathione conjugatepump (GS-X pump). Conjugation of cisplatin andGSH can occur non-enzymatically [231] but exportfrom the cell requires a GS-X pump [232].

The activity of a GS-X pump was shown by Ishikawaet al. [233] to be enhanced in cisplatin-resistant humanpromyelocyte leukemia HL60/R-CP cells. Muller et al.[200] found no increased GS-conjugate transport activ-ity in a cisplatin resistant lung cancer cell line, butfound a substantial increase in GS-conjugate transportactivity in the MRP-overexpressing GLC4/ADR cellline relative to the sensitive parental cells. Muller et al.[200] and Leier et al. [234] demonstrated that overex-pression of the MRP protein in cells resulted in in-creased ATP dependent GS-conjugate transport. Thecisplatin resistance conferred by the GS-X pump inHL60/R-CP cells was dependent on intracellular levelsof GSH in this cell line. The sensitivity of the vast


majority of MRP overexpressing cell lines to cisplatinwas explained by Ishikawa et al. [187] as being a resultof insufficient intracellular levels of GSH in these cells.Indeed, not only the function of MRP-1, but alsoregulation of its expression may be closely correlatedwith glutathione metabolism. Gamma-glutamylcysteinesynthetase is coordinately upregulated with MRP-1 fol-lowing exposure to heavy metals, cisplatin or the bi-functional nitrosourea, ACNU; post-transcriptionalregulation may be involved [235,236]. In colorectalcarcinoma, coordinated overexpression of MRP-1 andgamma-glutamylcysteine synthetase is frequently ob-served [237].

Database screening identified three new homologuesof the MRP-1 and MRP-2 genes. The MRP-3 protein,like cMOAT is predominantly expressed in the liver,with MRP-4 expressed at very low levels in a numberof different tissues and MRP-5 expressed in almost alltissues analysed [207]. Preliminary investigations havealso been carried out to investigate the role of theseMRP homologues in drug resistance. MRP-4 was notover-expressed in any of the drug resistant cell linestested by Kool et al. [207]. The MRP-3 and MRP-5transporters were over-expressed in a limited number ofcell lines. Kool et al. [207] demonstrated that cMOATwas over-expressed in a number of drug resistant celllines and that mRNA levels correlated with cisplatinbut not adriamycin resistance. HepG2 cells transfectedwith cMOAT antisense cDNA were shown by Koike etal. [238] to exhibit increased sensitivity to cisplatin,vincristine and adriamycin. Further investigations arenecessary in order to investigate the exact contributionof each of the homologues of MRP to drug resistancein MRP expressing cells.

8. Clinical studies on reversal of multi-drug resistancein cancer

8.1. Introduction

The resistance of tumour cells to cytotoxicchemotherapy may be intrinsic or acquired. Tumoursare termed intrinsically resistant when they fail to re-spond to the initial chemotherapy schedule. Tumoursmay also acquire resistance to subsequent cytotoxictreatment presumably by mutation and/or gene induc-tion, and/or due to the survival of a resistant tumourfraction from the initially responsive tumour [239].Certain cancers are generally associated with acquiredor intrinsic resistance; for example, leukaemias havebeen shown to display acquired P-170-mediated resis-tance, whereas others, e.g. of colorectal origin, typicallyexpress P-170 and/or MRP constitutively without anyprior therapy. Attempts have been made to use currentknowledge of drug-resistance mechanisms to circum-

vent resistance and thereby extend patient survival.Agents which inhibit cancer resistance mechanisms aretermed resistance modulators, chemosensitising agents,resistance circumvention agents. As described below,the experience to date of clinical trials using inhibitorsof P-glycoprotein underlines the need for new pharma-cological agents and new cellular targets such as thosedescribed earlier in this review.

8.2. Problems associated with chemosensitisation

Resistance mechanisms seen in cancer cells are de-pendent on the ability of tumour cells to use innateprotective or adaptive responses which may also beexpressed by normal cells. Therefore, any inhibition ofthese mechanisms in resistant cancer may augment thetoxicity of cytotoxic therapy in normal tissues [240]. Todate, there are no significant reports of agents withparticular affinity for cancer cells versus normal tissue.Many modulators interfere with the normal pharma-cokinetic variables of standard chemotherapeuticagents [241]. Commonly, the peak plasma level (Cmax),area under curve (AUC) and elimination half life (t1/2)are all elevated, thus increasing the toxic burden on therest of the body and necessitating dose rescheduling ormore intensive patient monitoring [240,242]. For exam-ple Lum et al. [243] showed that cyclosporin signifi-cantly increased the AUC, and decreased the CL (totalrate of clearance) values of etoposide when co-adminis-tered. Additionally, many of the earlier clinical investi-gations specifically in the area of P-gp modulation usedsensitisers with relatively weak inhibitory efficiency.

8.3. Circum6ention of P-170 o6erexpression

One of the most studied mechanisms of resistance isthe 170 kDa protein pump termed P-glycoprotein (P-gp). This pump utilises the energy of ATP to activelyefflux a variety of agents including a broad spectrum ofcancer chemotherapeutic drugs [244]. The net effect ofthis efflux mechanism is to reduce the time a cell isexposed to the agent and to reduce its maximum intra-cellular concentration. Since most cytotoxic cancertherapies are given at or near the maximum tolerabledose, and have little selectivity for cancer cells, the dosegiven cannot be increased to compensate without be-coming toxic for the patient. P-gp can pump a widevariety of structurally dissimilar agents including manyof the more effective, first-line anti-cancer drugs suchas adriamycin and vinca alkaloids [240]. If cells alreadyexpress a high level of P-gp activity they will be intrin-sically resistant to the cytotoxic effects of these agents.Likewise, if P-gp expression is induced in a cancer cellby initial cytotoxic exposure, all of the other substrateagents will be less effective if given subsequently [240].Because P-gp was the earliest resistance


Table 2Selected reports of clinical trials involving P-gp inhibition

Authors Toxicity findingsClinical protocol drugs and tumour type

[247] 24 Patients, Cyclosporin A and epidoxorubicin, Colorectal Increased incidence of leucocytopenia; no significant effect onoutcomecancer

[276] High incidence of serious cardiotoxicity; no objective responsesEight patients; verapamil and adriamycin; ovarian cancerSeven paediatric patients; verapamil, etoposide and vin-[248] Cardiac and haematological toxicty evident; partial responses

but no improvement on overall outcomeblastine; various refractory malagnancies20 Paediatric patients; verapamil and etoposide; various re-[276] Lower toxicity; six partial responsesfractory malagnancies16 Female patients; trifluoperazine and vinblastine; refractory[251] Significant CNS toxicity; similar response rate to controlbreast cancer

[252] 59 Patients; verapamil and VAD (vincristine, doxorubicin and No increased toxicity; no beneficial effect versus controldexamethasone); resistant melanoma18 Patients; verapamil and CVAD (cyclophosphamide vin- Significant but manageable toxicity; high (72%) response rate[277]cristine adriamycin and dexamethasone); drug refractorylymphoma16 Patients; cyclosporine A and teniposide; metastatic renal[278] Haematopoietic toxicity; one minor responsecell carcinoma

mechanism studied, it is also the first mechanism to betargeted by clinical strategies aimed at returning normaldrug sensitivity to the cancer cell. More recently, avariety of other cellular pump resistance mechanismshave been described. These include the multiple drugresistance protein (MRP), which is an ATP dependentpump with a similar, but not identical, substrate menuto that of Pgp (Section 7).

The intense investigation of P-gp has led to theidentification of a broad range of chemicals that modu-late its activity. They have been classed into sevenbroad categories: (1) calcium channel blockers; (2) Im-munosupressive agents; (3) vinca alkaloid analogues;(4) steroidal agents; (5) cyclosporin analogues; (6)calmodulin antagonists; and (7) a miscellaneous collec-tion of hydrophobic agents [245,246]. In many cases theinhibitory action of these agents is more an experimen-tal curiosity than a useful effect, since the concentra-tions at which MDR modulation is evident are greaterthan the obtainable therapeutic levels. Many of theagents also have significant therapeutic effect or toxicityin their own right that precludes their clinical use asP-gp modulators.

Several clinical studies of P-gp modulators in combi-nation with chemotherapeutic regimes have been re-ported or are underway [247–252] (Table 2). There arelimited claims for measurable increases in the anti-cancer effect using P-gp modulation in a chemothera-peutic combination to treat some haematologicalmalignancies. However, overall the results have beendisappointing and the studies have not mirrored thesuccess seen with MDR modulation in vitro. The rea-sons for this poor therapeutic activity are not simplydue to problems with the direct inhibition of drugefflux; pharmacokinetic and other considerations alsolimit therapeutic use.

As one might anticipate from the broad spectrum ofagents that affect P-gp activity, several forms of modu-lation are thought to occur. Most agents appear toblock the P-gp pump competitively [253]. However,differences are seen in the pump affinity for varyingcytotoxic agents under the modulation of particularsensitisers. This suggests the possibility of distinctbinding sites for certain P-gp substrates [254–256]. Theamount of drug required for P-gp modulation variesvery significantly among the list of agents. Some in-hibitors such as the antibiotics erythromycin and tetra-cycline are active only under in vitro experimentalconditions as the drug concentrations required aregreatly in excess of those attainable in the body [246].The ability to obtain physiologically relevant levels ofthe modulator significantly limits the number of agentsavailable. It has been suggested that this problem canbe overcome by combining modulators with non-over-lapping toxicities [254], but this does not seem to havebeen investigated clinically. These concentrations mustalso be sustainable for a suitable length of time so theP-gp-mediated drug efflux can be blocked over theduration of chemotherapy.

Historically, the screening for P-gp modulators hastended to use existing pharmacological agents. As aresult, most of these agents have strong pharmacologi-cal actions distinct from their activity on the effluxpump. In many cases these actions reduce the overalldose or the duration of that dose which can be givenwithout very significant side effects. The two mostcommonly used modulators, verapamil and cyclosporingenerally cause the side effects of cardiac abnormali-ties, circulatory problems, constipation, headache [249]and potent immunosupression, nephrotoxicity, muscu-loskeletal pain, nausea and vomiting, respectively [247].


As with any pharmaceutical, P-gp modulators canhave toxic effects. The presence of additional toxicchemotherapeutic agents increases the susceptibility tothese toxic effects. P-gp is expressed in normal tissuesincluding the liver, kidney and brain capillary endothe-lial cells (the blood brain barrier) and has an activerole in protecting the body from the effects of xenobi-otics [257,258]. The non-selective body-wide nature ofthe P-gp inhibition caused by these agents renders thebody more sensitive to the direct toxicity of the anti-cancer drugs being administered. Additional cytotoxicdose refinement may be required due to inhibited drugelimination mechanisms and direct modification of theusual pharmacological profile of the anti-cancer agent.

Several studies have shown that the correlation be-tween P-gp and tumour response is quite variable be-tween patients and different forms of cancer [259]. Thisfinding may be partly explained by the observationthat the relative activity of P-gp can vary betweentumour cells [260,261].

Additionally, since several resistance mechanismsmay be operational at the same time in a given tu-mour, the ideal approach would be to identify thepresence or absence of the P-gp protein and to quan-tify the level of its impact on the resistance of atumour before deciding on a particular chemothera-peutic regime [262].

Agents such as verapamil and cyclosporin which arepotent in vitro and which can reach sufficientchemosensitising concentrations in vivo have markedpharmacological effects as already described. There-fore, more recent approaches have utilised analogueswith similar or greater potency while largely lackingthe additional pharmacological actions regarded as sideeffects in this application of therapy. Several differentanalogues of verapamil have been produced and testedin vitro. Most notably the R-isomer of verapamil hasbeen brought to the stage of clinical testing as it largelylacks the calcium channel blocking capacity of theS-isomer while still efficiently blocking P-gp efflux[263]. A more potent P-gp modulating cyclosporinderivative, PSC 833, with reduced immunotoxicity, isalso now in clinical trial stage [264,240]. There is noclear evidence so far that either of these drugs willmake a very significant contribution to treating pa-tients with resistant tumours.

8.4. Resistance due to DNA repair mechanisms

There are a few reports of attempts to overcomesome forms of resistance due to the overexpression ofDNA repair mechanisms. Willson et al. [265], reportedthe results of a clinical trial using streptozotocin todeplete the level of the resistance enzyme O6-alkylgua-nine-DNA alkyltransferase (AT) in resistant colon andrectal cancer. However, although the dose of modula-

tor administered depleted AT levels, increased toxicitywas evident without significant increases in therapeuticresponse.

8.5. New strategies to o6ercome drug resistance

8.5.1. RibozymesA paper by Holm et al. [266], indicated that cultures

of resistant cells could be rendered more sensitive bytransfecting with an MDR-1-specific ribozyme, whichselectively destroyed the mRNA for P-gp in cancercells in vitro. Other authors (including our own group)have shown similar results; however, clinical use of thistechnology still awaits a suitable delivery system for invivo use [267]. The use of antisense agents and ri-bozymes for MDR modulators has been reviewed re-cently by Byrne et al. [268].

8.5.2. CytokinesRecent in vitro data indicates that certain cytokines

may also have activity as resistance modulating agentsboth directly inhibiting the mechanisms and indirectlyby affecting the expression of certain genes leading tothe emergence of resistance to chemotherapy [269,270].However, clinical application of this finding is likely tobe difficult due to the inherent biological actions ofthese agents in a variety of other cellular systems in thebody.

8.6. Conclusion

Based on the clinical findings with MDR modulationto date we can draft a set of specifications for an agentthat would be therapeutically most useful to modulateMDR. The ideal agent should inhibit the resistancemechanism at a low, and readily physiologicallyachievable, concentration. It should display some levelof selectivity for cancerous tissue over normal tissue tomaintain the protective mechanisms in non-cancerouscells while producing a more toxic effect in the tumour.If selectivity is achievable, the ideal agent should po-tently and irreversibly inhibit the resistance mechanismin the tumour cell for the duration of chemotherapy orlonger. The agent should be able to permeate throughall cells in a tumour at sufficient concentration to beeffective in modulating resistance. The agent shouldhave no significant effect on the pharmacokinetics ofthe anti-cancer drugs either directly or through inter-fering with the normal cytotoxic elimination processes.On its own this novel chemosensitiser should be rela-tively non-toxic and free from other pharmaceuticalactivity. It should be available as a pure chemical withwell-described, consistent pharmacokinetics. The toxic-ity profile of this agent should be distinct from that ofthe chemotherapeutic partner so there would not be a


need to reduce the dose of anti-cancer agent. It shouldalso not increase the toxicity of the chemotherapy inthe rest of the body, allowing the maximum chemother-apeutic dose to be used. In particular liver, kidney,cardiac, nervous and immune functions should not berendered more sensitive. Combination therapy withsuch an ideal agent should be considered as a first linetreatment to stem the emergence of acquired resistanceafter the initial dose of chemotherapy.

The initial trials of chemosensitising agents werelargely unsuccessful due to a complex set of factors.These included the use of modulators with low potency,drug-induced alterations in the pharmacokinetics of thecancer therapy, overt toxicity and other pharmacologi-cal effects of the modulators and the multifunctionalnature of cancer resistance. However, research in thisfield continues with clinical trials of more potent P-gpmodulating agents and a collection of promising invitro ideas which await extrapolation into the patient.

9. Conclusion/prospects

The inherent genetic instability of cancer cells conferson them the ability to generate variants exhibiting atremendous variety of resistance mechanisms to toxicagents [27]. Furthermore, the mechanisms by whichnormal cells protect themselves against toxins can berapidly recruited by cancer cells to protect them againstdrugs. In spite of these problems, chemotherapy curesmany cancers, and it is reasonable to hope that as weunderstand in more detail the mechanisms of resistance,we should be able to devise strategies so that more andmore cancer patients can be cured.

In spite of the significant expansion in our knowledgeof the P-170 efflux pump, clinical trials involving use ofP-170 modulators have produced disappointing results,due at least in part to enhanced toxicity to normalP-170-expressing tissues [272]. Many unanswered ques-tions remain-the circumventing agents may have af-fected other cellular activities; perhaps more specificP-170 inhibitors will be better? Could modulators bedirected to the tumour cells, and not to normal tissues?Or, must we use drugs not affected by P-170, in Pgp-positive tumours?

It is possible that inhibitors of other pumps (e.g.MRP [273,274,275]) may have fewer side-effects, butdata is as yet insufficient to reach any conclusions. Thefrequency of MRP-positive tumours (and in which P-170 does not play a significant role) remains unclear.The role of more recently-discovered proteins (such asLRP) in MDR is still not established; they could con-ceivably be future targets for modulation.

If it turns out to be the case that many/all anticancerdrugs converge on a (few) final death/apoptosis path-way(s), steps in these pathways will be promising

targets for new chemotherapeutic agents. These stepscould, unfortunately, also represent mechanisms forcancer cells to develop resistance to a very wide rangeof drugs; this may already be part of the clinicalproblem, where the multidrug resistance observed inpatients often seems to be wider than can be explainedby known MDR mechanisms (P-170, MRP, Topoiso-merase II). On the positive side, alternative redundantpathways may represent an opportunity for new combi-nation chemotherapy approaches to overcome resis-tance and the closer the targets are to the final celldeath committment steps (caspase activation?) thefewer downstream chances exist for additional resis-tance mechanisms. Obviously, once again, if ways couldbe found to selectively stimulate apoptosis in tumourcells versus normal tissue (either by physical targettingor by exploiting any differentially expressed pathwaysor proteins), the prospects for cure could be enhancedsignificantly.

There may also be a subset of tumours for whichmolecular approaches alone will not overcome resis-tance, and in which therapies must address drug pene-tration/vascularisation/ oxygenation.

In principle, based on our knowledge to date, atumour could exhibit multiple drug resistance due to acombination of all of the following:1. Poor drug supply/oxygenation2. Over-expression of drug efflux and sequestration

mechanisms (P-170, MRP)3. Altered multidrug targets (e.g. topoisomerase)4. Resistance to apoptosis (e.g. by overexpression of

anti-apoptotic proteins such as bcl-2 or bcl-XL)5. Enhanced degradation or impaired activation of

drugs6. Altered cell cycle properties

Tackling such a worst-case scenario could be virtu-ally impossible, but if chemotherapy can be made moreeffective at the first phase, the tumour should not havetime to co-express these multiple mechanisms; manyrounds of different types of chemo- and radio-therapymight encourage development of such super-resistanttumours.

The realistic target of MDR research may thereforebe to identify the range of mechanisms operating indifferent kinds of pre-treatment tumours; to developreliable diagnostic and therapeutic strategies associatedwith these mechanisms; and to start optimized treat-ment, based on the resistance mechanisms expressed,immediately. The disappointing progress to date inclinical application of MDR research reflects the inher-ent complexity of the problem, and should not discour-age researchers from pursuing the valuable andachievable goal of rational mechanism-based therapy ofmulti-drug resistant cancer.


Acknowledgements

We are grateful to Yvonne Reilly for patientlyretyping the many versions of this manuscript.

Reviewer

This paper was reviewed by Toshihisa Ishikawa,Ph.D., Manager, Medicinal Biology Laboratory, PfizerInc. Central Research.

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Biography

Martin Clynes is Professor of Biotechnology atDublin City University, and Director of BioResearchIreland’s National Cell and Tissue Culture Centre(NCTCC). A major focus of the research programmesat NCTCC is to discover how cancer cells becomeresistant to chemotherapy, and how this clinical prob-lem could be overcome. Carmel Daly, Roisin Nic Amh-laoibh, Robert O’Connor, Cathal Elliott and ToniO’Doherty (Research Scientists) and Deirdre Croninand Lisa Connolly (Postgraduate Students) are workingin this research programme. Kevin Scanlon has pio-neered studies on oncogene cDNA transfection andribozyme technology to investigate drug resistance incancer cells, and is currently Head of Cancer Researchat Berlex Biosciences; Anthony Howlett is Senior Scien-tist in the cancer Research Department at Berlex..

Documents

Recent developments in drug resistance and apoptosis research