8

Multidrug resistance in leukaemia

PAUL BAINESPETER CUMBERROSE ANN PADUA

A major problem in the treatment of leukaemia is the cross-resistance ofsome primary and many recurrent tumours to chemotherapeutic agents.Tumours can evade chemotherapy in several ways. At the population level,residual surviving cells may adapt to the toxicity, or resistant cells may havealready existed. Frequently the malignant population, as a whole, appearsinsensitive even in the absence of prior exposure to cytotoxic drugs (Sato etal, 1990a).

The individual cell can use several mechanisms when faced with a toxicinsult. These include decreased drug accumulation, increased intracellulardetoxification, amplification of genes coding for detoxifying enzymes,increased repair of DNA and activation of oncogenes . Recently, however,attention has focused on the roles of efflux pumps and topoisomerasesbecause these mechanisms confer the phenomenon of multidrug resistance,where cells become insensitive to a broad spectrum of dissimilar chemo­therapeutic agents (Biedler and Riehm, 1970).

MDR P·GLYCOPROTEINS

The M DR1 gene which codes for a 170kDa transmembrane P-glycoprotein(P.170) has been extensively investigated in association with multidrugresistance (Hamada and Tsuruo, 1986, 1988;Willingham et al, 1987). P-170is an energy-dependent efflux pump, and increased levels of the MDR1transcript and protein have been detected in drug-resistant cells (Juliano andLing, 1976; Beck et ai, 1979;Bradley et ai, 1988). The MDR1 gene has beenshown to confer drug resistance when introduced into sensitive mammaliancells in vitro (Gras et al, 1986; Veda et ai, 1987a; Choi et al, 1988; Pastan etai, 1988) and in vivo (Mickisch et ai, 1991).

The MDR1 p.l70 is a member of the ATP-binding cassette family oftransporters which are responsible for the uptake of metabolites in bacteria(Chen et al, 1986; Gras et ai, 1986), the secretion of pheromone in yeast(McGrath and Varshavsky, 1989) and pigment uptake into retinal cells inDrosophila (Dreesen et al, 1988). The protein includes 12 membrane­spanning hydrophobic regions and two ATP-binding sites (Figure 1). P-170

Bailli~re's ClinicalHaematology- 943Vol.5. No.4. October 1992 Copyright© 1992.byBailliereTindallISBN0-7020-1691-8 All rightsofreproduction in anyformreserved

944

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IN

P. BAINES ET AL

COOH

Figure 1. Schematicdiagram of P-glycoprotein (P-170) in the cell membrane. The moleculecomprisesa short, hydrophilic N-terminal region,a longhydrophobic region,which contains12segmentslikelyto be closely associatedwiththe membrane,and a long,hydrophilic C·terminalregion. The nucleotide-binding sites (marked N) are the probable sitesof ATP binding.

is also closely related to the cystic fibrosis transmembrane regulator and issimilar in its function as an ATP-dependent chloride channel (Valverde etaI, 1992). If the chloride and drug export transport mechanisms are linked.they do not appear to form part of a chloride-drug countertransportmechanism.

Much of our knowledge of P·170 comes from cell lines selected forresistance by gradually increasing exposure to cytotoxic drugs in vitro.Characteristically, P-170confers resistance to drugs other than the selectingagent. This phenomenon of multidrug resistance is limited in vitro to certaincategories of drugs such as the vinca alkaloids (mitotic spindle poisons).epipodophyllotoxins (topoisomerase II inhibitors) and anthracyclines(DNA-interacting agents), and does not extend to the alkylating agents(chlorambucil, cyclophosphamide) or the antimetabolites (cytarabine,methotrexate, 5-fluorouracil), all of which are important in the treatment ofleukaemia.

It seems unlikely that P·170 can have a high affinity for all the diversesubstrates with which it can evidently interact and which are selected fortheir affinity to their intracellular target molecules. This has led to theproposal that P-170 must intercept the drug before it enters the cell (Gros etaI, 1986) (Figure 2). If this is true, the ability of P-170 to mediate resistanceagainst broad categories of some drugs, but not others, may reflect the wayin which a drug enters the cell. This would explain why multidrug resistantcells are resistant to trimetrexate, which enters the cell via passive diffusion.but not to methotrexate, which enters via the folate receptor (Klohs et al,1986).

Despite their undoubted contribution to our understanding of multidrugresistance, the mechanisms employed by drug-selected cell lines may notalwaysbe those used by malignant populations in vivo. This may well be truefor mutations in the MDRI gene at codon 185 which give rise to specificpatterns of cross-resistance (Choi et aI, 1988), and for gene amplification(Veda et aI, 1987a). Both these abnormalities may be artefacts of the drugselection conditions imposed on emergent resistant lines and are unlikely to

MULTIDRUG RESISTANCE IN LEUKAEMIA

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MRK16P-glycoprotein •

945

Figure 2. Schematic diagram of the P-glycoprotein (P·170) resistance mechanism and levels ofcontrol. The dark oval represents P·170. which is encoded by the MDRI gene and can bedetected using the MRK16 antibody against an extraceUular epitope. P-170can use ATP as anenergy source from within the cell to pump cytotoxics out (thick arrows) , which reduces theamount of drug reaching the nucleus (thin arrow) . F1TC, fluorescein isothiocyanate.

prevail in vivo, as some resistant tumours have had no exposure to drugs atall and have elevated levels of MDR1 expression. Much of the gene amplifi­cation occurs on unstable double minutes and homogeneously stainingregions (Baskin et al, 1981; Biedler et ai, 1983;Tsuruo et ai, 1986), featuresrarely seen in fresh tumours. Amplification of the MDR1 gene has rarelybeen observed in clinical haematological samples (Holmes et al, 1989; Ito etal, 1989) and does not correlate with resistance (Sugimoto et ai, 1987).Altered patterns of cross-resistance have been found associated with par­ticular point mutations at codon 185of the MDR1 gene in human carcinomacell lines (KB) selected in vinblastine or colchicine (Choi et al, 1988). Cellsselected in, and with a preferential resistance to colchicine, had a two-basepair change at nu~leotide pos~tio? 554 (g~anine.to thyt;nine)an~ position 555(adenine to thymine], resulting In a glycine (vinblastine-specific sequence)to a valine (colchicine-specific) change at codon 185. Cells transfected withmutant MDR1 cDNA clones had increased relative resistance to colchicinecompared with wild-type transfectants. It has been proposed that position185 is the site of drug binding, and mutations at this position may alter theaffinity of the P-170 !or .different ~rugs, allow.ing cells to become drugresistant due to a quahta tive change in the protem. However, mutations atamino acid position 185 have not been found in DNA from leukaemic celllines and acute lymphoblastic leukaemia (ALL) patients (Gekeler et aI,

946 P. BAINES ET AL

1991), and in chronic lymphocytic leukaemia (CLL) and acute myelogenousleukaemia (AML) demonstrating a range of MDR1 mRNA expression(Holmes et ai, 1992).

A further divergence between in vitro and in vivo models was highlightedby the data of Siapak et al (1990). Cell lines exposed to increasing toxic stressbecame partially resistant before a noticeable increase in drug efflux wasdiscernible. At higher doses, increased drug efflux preceded the upregu­lation of MDR1, which was increased only at the highest drug concen­trations despite being one of the first mechanisms recruited in de novomalignancy.

MDR1 mRNA levels (4.5 kb transcript) are raised in resistant acutenon-lymphoblastic leukaemia (ANLL) (Goldstein et ai, 1989; Holmes et ai,1989; Sato et aI, 1990a; Herweijer et aI, 1990; Nooter et aI, 1990a), but arelow in sensitive ALL and ANLL (Fojo et ai, 1987a; Goldstein et aI, 1989).Consequently, cell lines selected for lower levels of resistance, without geneamplification but with increased MDR1 mRNA expression, may be bettermodels for the in vivo situation (Shen et al, 1986).

Increased levels of MDR1 mRNA and protein have been observed inB-cell CLL (Groulx et ai, 1988; Perri et al, 1989; Herweijer et al, 1990;Holmes et ai, 1990a; Cumber et ai, 1991) and in relapsed chronic myelo­genous leukaemia (CML) (Kuwazuru et ai, 1990). In CML, overexpressionof MDR1 has been detected in both chronic and blast crisis phases of thedisease (Goldstein et ai, 1989; Herweijer et al, 1990; Weide et al, 1990). Incontrast, P-170 has been reported to be present on CML blasts but absent onchronic phase cells (Kuwazuru et al, 1990;Sato et al, 1990b). No correlationwith clinical resistance was made in all these studies.

P-170-positive cell numbers correlated with in vitro resistance to doxo­rubicin in a study of myeloma, lymphoma and breast cancer (Salmon et al,1989), but again no clinical data were available for these patients. Highlevels of P-170 and increased daunorubicin efflux were reported in blastsfrom a case of resistant ALL (Redner et ai, 1990). Increased MDR1 mRNAand P-170 levels were found in relapsed and refractory AML patients(Holmes et ai, 1989; P.M. Cumber, H. Limaye, T. Hoy, A. Al Sabah, J.Whittaker and R.A. Padua, unpublished data) and in relapsed ALL(Rothenberg et ai, 1989).

There is little published data on MDR1 mRNA levels in the same patientbefore and after chemotherapy, although the incidence of raised MDR1mRNA is higher in secondary AML patients following previous exposure tocytotoxic drugs, in myelodysplastic marrow (Ma et ai, 1987; Holmes et al,1989) and in B-cell CLL following chlorambucil or cyclophosphamidetreatment (Holmes et al, 1990a). The mechanism by which MDR1 mRNAtranscription might be increased is uncertain, but it is possible that promoterusage may playa role (Veda et ai, 1987b). Sequential analyses of AML,ALL and B-cell CLL patients did not reveal consistent increases of eitherMDR1 transcripts or protein (Ito et al, 1989;Shustik et aI, 1991). A prelimi­nary study of P-170 in over 200 AML patients did not find a correlationbetween protein levels and prognosis (Ball et al, 1990). In contrast, a recentcomprehensive study clearly demonstrated that complete remission rates

MULTIDRUG RESISTANCE IN LEUKAEMIA 947

were significantly lower in P-17D-positive ANLL than in P-17D-negativeANLL (Campos et al, 1992).

Transcription of MDR3, which shows close homology to MDR1 and issimilarly located on chromosome 7 (Chen et al, 1986; Roninson et aI, 1986;Van der Bliek et aI, 1987, 1988), is increased in prolymphocytic leukaemia,and there is evidence that this may also be associated with an efflux pump(Nooter et aI, 1990b). Surprisingly, unlike MDR1, MDR3 does not conferdrug resistance to sensitive cells in in vitro transfection experiments (Vander Bliek et al, 1988) and its role in conferring drug resistance is unclear. Inmice, there is a third member of this gene family, M DR2 (Croop et al, 1989).However, there is no human homologue of this gene (Van der Bliek et aI,1988).

MDRI IN NORMAL TISSUES

Cells of some normal tissues (adrenal, kidney) contain as much or moreMDR1 mRNA as cell lines several hundred-fold resistant to drugs (Shen etal, 1986; Fojo et al, 1987a). This distribution is probably determined by thenormal chloride channel function of the MDRI P-17D in the apical regions ofthe secretory epithelia common to these regions (Valverde et al, 1992).Colon, rectum, liver and kidney also exhibit elevated levels of P-17D. It hasbeen suggested that the normal physiological role of P-17D is to export toxinsfrom these organs.

Primitive haematopoietic stem cells also possess high levels of P-170, andit was in this situation that the common association of P-170, the presence ofthe CD34 sialomucin and drug resistance were first noted (Chaudhary andRoninson, 1991; Geller et aI, 1991; List et aI, 1991; Campos et ai, 1992).Marrow lymphocytes also exhibit elevated P-170. If this is a general featureof the lymphoid lineage, this would explain the resistance of peripheralblood lymphocytes to cytotoxic drugs (Kaspers et al, 1991). However,Holmes et al (1990a) reported that MDR1 mRNA levels were low in thesecells, whereas total peripheral blood RNA from these normal individualshad high levels of transcripts, presumably from the myeloid compartment.Why cells of some lineages should express more P-170 than others is far fromclear. This may be related to cell cycle state and previous exposure to toxicagents, but the answer may well lie in the requirement for a chloride channelnetwork by the cell.

DETECTION OF MULTIDRUG RESISTANCE

Apart from the measureme~t of intracellular mRNA levels by Northern/slotblotting and RNAse protection (Bradley et ai, 1988), P-170 positive cells canbe detected using the MRK16 antibody to an extracellular epitope (Hamadaand Tsuruo, 1986) by indirect immunofluorescence. However abnormalglycosylation may mask the epitope re~ognized by MRK16, anda neurami­nidase incubation should precede antibody labelling (Cumber et aI, 1990;

948 P. BAINES ET AL

1991), this also means that considerable care needs to be exercised wheninterpreting the results of earlier studies.

Another parameter of multidrug resistance is the reduction in intracellu­lar accumulation of drug. The reduced autofluorescence of daunorubicindue to increased efflux in multidrug resistant cells can be detected at 488 nm(Herweijer et al, 1989; Cumber et al, 1991) using a fluorescence-activatedcell sorter.

Methods for determining the sensitivity of cell populations to cytotoxicdrugs in vitro have been recently reviewed by Veerman and Pieters (1990).Clonogenic assays have the advantage of measuring the effects of drugs onleukaemic stem cells, but the effect on resting clonogenic cells may bemissed and, in any case, many samples fail to grow. The differential stainingcytotoxicity assay, or DISC assay (Weisenthal et ai, 1983), has the advan­tages of being rapid (2-4 days), of being applicable to most haematologicaltumours, and of detecting the morphology of viable and dead cells, which isuseful when non-tumour cells contaminate the sample. A modification ofthe MIT assay (Hansen et al, 1989), in which a soluble tetrazolium salt, 3-4,5-dimethylthiazol-2-5-diphenyl tetrazolium bromide, is added to cells fol­lowing 2-4 days of culture in decreasing drug concentrations, can also beused (Santini et al, 1989; Sargent and Taylor, 1989; Pieters et al, 1991).Viable cells convert the salt into a purple formazan precipitate, which can bedissolved with a dimethylformamide/sodium dodecyl sulphate (SDS) solu­tion and the extinction read at 570nm on a microplate reader. The dose ofdrug required to kill 50% of cells can be calculated from the resulting curve.Unlike the DISC assay, the MIT technique only gives an overall result forthe entire population, so contaminating maturing cells may influence the

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Daunorublcln cone • JJg'm1

Figure 3. Decreasing cell viability, measured by decreasing reduction of MIT. in resistant VLB(0 and .) and sensitive CEM (0 and .) human leukaemic T-cell lines, with increasingconcentrationsofdaunorubicin alone (open symbols) orwith both daunorubicin and1.25l'-glmlof the 'modifier' cyclosporin A (closed symbols) added over 48 h of culture.

MULTIDRUG RESISTANCE IN LEUKAEMIA 949

results. Nevertheless, this is a quick procedure not open to subjectiveinterpretation and is highly reproducible when tested on multidrug resistantcell lines (Figure 3). Furthermore, resistance, as determined by MIT, candistinguish between responders and non-responders in de novo AML(Santini et al, 1989; Sargent and Taylor, 1989) and has been found to bepredictive of remission duration induced by thioguanine, daunorubicin andprednisolone in childhood ALL (Pieters et ai, 1991).

REVERSAL OF MULTIDRUG RESISTANCE

A typical feature of P-170-mediated multidrug resistance is that it can bereversed by a variety of 'modifiers' which also inhibit its chloride channelactivity (Valverde et al, 1992). These are diverse and have been recentlyreviewed by Ford and Heit (1990), but include calcium channel blockers(verapamil), cyclosporins, vinca alkaloid and anthracycline analogues,steroids, tamoxifen and metabolic poisons. The analogues of vinca alkaloidsand anthracyclines seem likely to compete for P-170 binding, and photo­active analogues of verapamil show that this drug can also bind P-170directly (Safa, 1988). Cyclosporins may also act in this way. The mechanismsthough are far from clear, with some workers (Hamada and Tsuruo, 1988)claiming that the ATPase associated with P-170 is the target of verapamil, inwhich case phosphorylation of P-170 could affect drug binding via a confor­mational change (Hamada et aI, 1987). Cyclosporins can also bind calmo­dulin and cyclophilins, which in turn inhibits the translocation oftranscription factors to the nucleus (Flanagan et al, 1991; Liu et al, 1991);this could restrict MDRl expression.

Consistent with the reversal of resistance, verapamil (Tsuruo et ai, 1981,1982) increases intracellular vincristine or doxorubicin levels in multidrugresistant cells by inhibiting their efflux, and this results in an increase in thesensitivity of these cells to these drugs. Verapamil can increase daunorubicinaccumulation in AML blasts in vitro (Maruyama et al, 1989), and cyclo­sporin A can restore daunorubicin accumulation in AML and B-cell CLL(Nooter et al, 1990a; Cumber et al, 1991). The change in drug accumulationmediated by cyclosporin A treatment appears to be correlated with levels ofP-170 (Cumber et al, 1991). Cyclosporin A has already been used to increasethe sensitivity of resistant AML to daunorubicin therapy (Sonneveld andNooter, 1990).

Multidrug resistant cells are quite often more sensitive than their parentlines to the modifier alone. Verapamil, alone, is particularly toxic to severalmultidrug resistant lines (Twentyman et aI, 1986; Warr et al, 1986; Cano­Gauci and Riordan, 1987), but this effect is also seen for less-specific agentssuch as detergents (Bech-Hansen et al, 1976), shear forces (Riordan andLing, 1979) and osmotic lysis'.These less-specific effects may reflect lipidchanges in the mem~rane, ~hlCh are not regarded as instrumental in thedevelopment of.multldrug reslsta!1ce (Montaudan et al, 1986) but which maymediate the action of some modifiers.

Modifiers may do more than reverse P-170-mediated resistance.

950 P. BAINES ET AL

Doxorubicin-selected HL60 lines did not express P-170 but showeddecreased drug accumulation, reversible with verapamil (McGrath andCenter, 1987; McGrath et aI, 1989). This suggests other efflux pumps mayexist which can be blocked by modifiers.

TOPOISOMERASES

Although increased P-170-mediated drug efflux is a common response ofcells to toxic insult, some drugs such as VP-16, VM-26 and mitoxantroneselect lines which exhibit non-P-170 forms of multidrug resistance (Odaimiet aI, 1986; Beran and Andersson, 1987; Yalowich et al, 1987; Dalton et al,1988; Harker et aI, 1989). In addition, these ceIl lines are cross-resistant tomost of the anticancer drugs but, unusually, not to the vinca alkaloids (Becket aI, 1987; Danks et aI, 1987)-a situation termed 'atypical multidrugresistance'. Many of these drugs, in fact, appear to exert their pharmaco­logical activity via interaction with DNA topoisomerase II (Topo II) (Lockand Ross, 1987; Sullivan et al, 1987; D'Arpa and Liu, 1989). Together withits family member, topoisomerase I, Topo II is an enzyme responsible forthe breakage and religation of coiled DNA (see Wang 1985, 1989 forreviews). Both of these enzymes are associated with highly transcribedgenes (Fleischman et aI, 1984; Riou et aI, 1989). A number of drugs appearto stabilize the complex between Topo-II and DNA (Osheroff, 1989),increasing single- and double-strand breaks (Ross et al, 1984; Gewirtz,1991). This can lead to G2 arrest and inhibition of p34 cdc2 kinase activity(Dive and Hickman, 1991).

Atypical multidrug resistance is associated with a reduced level of Topo IIactivity (Danks et aI, 1988; Cole et aI, 1991; Sinha and Eliot, 1991). TheTopo II gene has been cloned and localized to chromosome 17 (Tsai­Pflugfelder et aI, 1988). Undetectable levels have been observed in thelymphocytes of CLL patients, whereas detectable levels of expression wereobserved in ALL and non-Hodgkin's lymphoma (Potmesil et al, 1988).These data have not been correlated with clinical drug resistance. Pointmutations in Topo II have been observed in drug-resistant cell lines (Bugg etaI, 1991; Hinds et aI, 1991), but such mutations have not yet been detected invivo.

Intercalating agents can also stabilize Topo II-DNA complexes (Tewey etaI, 1984; Fox and Smith, 1990), which would explain why this category ofdrugs can select ceIl lines exhibiting both P-170-mediated and atypicalmultidrug resistance (Caprinico et aI, 1986; Zijlstra et aI, 1987; Sinha et al,1988; Friche et aI, 1991). These two forms of multidrug resistance mayinteract within resistant cells. Modifiers, such as verapamil, potentiateetoposide-induced single-strand breaks associated with increased etoposideaccumulation (Yalowich and Ross, 1984), which probably results fromverapamil inhibition of P-170 activity. Even greater complexity arises inlines where verapamil appears to inhibit repair of single-strand breaksdirectly (Harker et al, 1986). Immunological detection of Topo II is nowpossible (Smith and Makinson, 1989) and assays for its activity and DNA

MULTIDRUG RESISTANCE IN LEUKAEMIA 951

cleavage are becoming available (Andrea et al, 1991), but the number ofstrand breaks induced by some drugs (anthracyclines) does not alwayscorrelate well with toxicity (Gerwirtz, 1991). This may mean that damage tospecific genes is more important or it may reflect the variable presence ofother resistance mechanisms.

GLUTATHIONE-S-TRANSFERASES

The glutathione-S-transferases (GSTs) are a family of enzymes involved indrug detoxification (Hayes and Wolf, 1988). The cytosolic GST isoenzymesconjugate electrophilic drugs, toxins and carcinogens to reduced glutathione(GSH) before elimination from the body. The GST a isoenzymes haveglutathione peroxidase activity and prevent oxidative damage (Ketterer etai, 1986). GST .... reduces peroxidized DNA, which may have a role in DNArepair (Tan et al, 1988). Neither GST a nor I-L seem to be expressed inhaematopoietic cells (Hall et al, 1990a,b). Increased expression of GST 1Thas been demonstrated in cell lines resistant to alkylating agents (Wang andTew, 1985; Batist et al, 1986; Kramer et al, 1988) and doxorubicin (Robsonet ai, 1986; Samuels et ai, 1991). In chemically induced hepatocarcinogene­sis, and in non-small cell lung carcinoma, both GST1T and MDRI P-170 areoverexpressed (Kitahara et ai, 1984; Thorgeirsson et ai, 1987; Volm et al,1991).

The expression of GST 1T is increased in a number of haematologicalmalignancies (McQuaid et al, 1989; Holmes et al, 1990b; Schisselbauer et al,1990) and may be higher on relapse (Moscow et al, 1989a). However,sequential studies have not found modulation of GST 1T expression in CLLpatients treated with chlorambucil (Holmes et ai, 1990b). In AML patientssampled at presentation and subsequent relapse, the levels were reduced orremained constant following chemotherapy. There was no coordinateexpression of GST 1T and MDRI. While GST is an important detoxifyingenzyme in the liver and expression of GST 1T or a in yeast confers drugresistance (Black et ai, 1990), the evidence to support a role for the GSHredox system in the resistance expressed by haematological neoplasms isuncertain (Holmes et ai, 1990b; Begleiter et ai, 1991). Furthermore, intro­ducing GST 1T into MCF-7 breast cancer cells by transfection did not confer amultidrug resistant phenotype (Moscow et ai, 1989b).

OTHER DRUG RESISTANCE MECHANISMS

Little work has been carried out on other known detoxification pathwayssuch as cytochrome P450 (Porter and Coon, 1991) and carbonyl reductase(Forrest et ai, 1991).

Oncogene activation has been linked to drug resistance since trans­formation with c-MYC, c-RAS, v-ras or v-rafcan increase P-170 and GSTIGSH levels in NIH 31'3 cells (S~lar, 1988; Niimi et ai, 1990) and in rat liverepithelial cells transformed with v-Ha-RAS or v-raf (Burt et ai, 1988).

952 P. BAINES ET AL

Oncogene levels increased following etoposide, but this was concluded toreflect a first step in cell death (Rubin et aI, 1991).

Finally, there are a number of ill-defined proteins whose levels areelevated in resistant lines (Bhalla et aI, 1985; Danks et aI, 1985;Tsuruoet al,1986).

THERAPEUTIC OPTIONS

Although tumour drug resistance in vitro, and its parameters, do not takeaccount of the overall pharmacology of cytotoxic drugs in vivo, these assayscan be of diagnostic and prognostic value. For example, in de novo AML,responders showed lower in vitro resistance than non-responders (Santini etaI, 1989;Sargent and Taylor, 1989). In childhood ALL, in vitro resistance tothioguanine, daunorubicin and prednisolone was associated with pooreroutlook (Pieters et al, 1991). Poor remission rates correlated with highMDRI expression in a varietyofleukaemias (Marie et aI, 1991) and in AML(Sato et aI, 1990a). Remission rates in ANLL were significantly lower inleukaemias with high numbers of P-170-positive cells (Campos et al, 1992),and residual P-170-positive cells during remission may underlie early relapse(Musto et aI, 1991).

Strategies for the reversal of drug resistance which have emerged fromresearch into drug resistance mechanisms include inhibition of the promoterof the dihydrofolate receptor gene by rnithramycin, which restores theefficacy of methotrexate in breast carcinoma lines with multiple copies of thedihydrofolate reductase gene. Such a molecular approach might beextended to MDRI transcription, perhaps via anti-sense oligonucleotidetherapy. More immediate attention has focused on the reversal of multidrugresistance by modifiers. Verapamil has been studied in most detail and initialresults in clinical trials have been encouraging. Dalton et al (1989) studiedpatients with resistant multiple myeloma and non-Hodgkin's lymphoma.Three out of seven patients with P·170-positive tumours, previously refrac­tory to vincristine, doxorubicin and dexamethazone, showed improvementupon the addition of verapamil during their chemotherapy regimens. Thedose-limiting factor was cardiotoxicity with hypotension, first degreeatrioventricular block and junctional rhythms. The use of the racemic formD-verapamil to decrease cardiotoxicity has been disappointing (Dalton et aI,1989). Cyclosporin A has been used to treat a patient with refractory AML,with a transient elimination of an MDRI positive clone and a short-livedclinical response (Sonneveld and Nooter, 1990). The major toxicities associ­ated with cyclosporin A are renal impairment and immunosuppression. Thedevelopment of cyclosporin A analogues which are far less immunosup­pressive and nephrotoxic may circumvent these problems. These agentshave shown promise in vitro and are shortly to be used in clinical trials(Twentyman, 1988; Gaveriaux et aI, 1989; Boesch et al, 1991). Quinine,which has been shown to increase anthracycline accumulation in vitro, is lesscardiotoxic than verapamil and may be useful (Chauffert et al, 1990).Tamoxifen has also been shown to be effective in the reversal of multidrug

MULTIDRUG RESISTANCE IN LEUKAEMIA 953

resistance in cell lines which appear to be using non-P-170 mechanisms(Chatterjee and Harris, 1990).

The development of these rational, therapeutic options from a betterunderstanding of the mechanisms involved in clinical drug resistance isencouraging and can be seen as an important step towards specific therapydesigned for the individual patient.

SUMMARY

Multidrug resistance hampers successful chemotherapy in many haernato­logical neoplasms and is mediated by several cellular proteins. In somecases, the genes encoding these proteins have been shown to conferresistance on transfer to drug-sensitive cell lines. This is true for the effluxpump product of the MDRI gene, P-170. Upregulation of enzymes such asGST has been observed, although the contribution of this enzyme in drugresistance expressed by malignant haematopoietic cells is still uncertain.Cells also appear to be able to downregulate enzymes which are drugtargets. Examples include the decrease in Topo II which accompanies theresistance shown by cells to VP·16 and VM-26.

Although many reports include both presentation and relapsed patients,there are few data on samples drawn from the same patients before and afterchemotherapy. While P-170 and GST appear to be raised more often in cellsfrom resistant and relapsed disease, it is quite clear that such mechanismscan be active in de novo malignancy and do not necessarily emerge as aconsequence of prior chemotherapy.

Methods of detecting drug resistance are reviewed here; these include invitro cellular assays for drug toxicity, and molecular, immunological andfunctional detection of P-170 or Topo II. The clinical evaluation of suchassays is only just beginning and some of the data are contradictory. To someextent, this may reflect the complex way in which the various resistancemechanisms may interact. Nevertheless, there are some encouraging earlysigns that the application of these assays to clinical material will yieldvaluable data on the relative contributions of these mechanisms and on waysin which they may be overcome. At present, much attention has focused onthe potential of agents which prevent the P-170 efflux pump from exportingcytotoxics from the cell. This is likely to be only the first of new therapiesarising from an improved understanding of multidrug resistance. Moreimmediately, assays for multidrug resistance and its parameters may findtheir place as routine diagnostic and prognostic tools in the laboratory.

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