Leukemia Research Vol. 13. No. 5, pp. 351-356, 1989. 01,15-2126/89 $3.00 + .00 Printed in Great Britain. Pergamon Press pie

REVIEW

M E C H A N I S M S O F D R U G R E S I S T A N C E IN A C U T E L E U K A E M I A

ANDREW HALL, ALEX R. CATI'AN and STEPHEN J. PROCTOR

LRF Laboratory, Fourth Floor, Cookson Building, New Medical School, Framlington Place, University of Newcastle, Newcastle upon Tyne, U.K.

(Received 18 January 1989. Accepted 1 February 1989)

Abstract--The development of resistance to chemotherapeutic agents is a frequent cause of treatment failure in acute myeloid and lymphocytic leukaemia. The mechanisms by which resistance develops in these patients are poorly understood, although a framework for their investigation has been provided by a range of studies using animal and human cell lines as model systems. In this review the basic concepts of drug resistance mechanisms are outlined, with special emphasis on studies using cells obtained from patients with resistant forms of leukaemia.

Key words: Leukaemia, cytotoxic, chemotherapy, drug resistance.

MANY PATIENTS diagnosed as suffering from acute leukaemia show an excellent initial response to treat- ment with cytotoxic drugs, only to relapse after a variable period with a recurrence of their disease. Such patients may respond to treatment with the same drugs used to obtain initial remission [1], but relapse with unresponsive disease normally follows. In an attempt to prevent or delay such recurrence many groups have investigated the use of intensive induction and consolidation regimes. This approach has been very successful in the case of children pre- senting with CALLA-positive acute lymphocytic leu- kaemia [2], but has achieved only relatively minor improvements in the outlook of children outside this good prognostic group. Improvements in the long term outlook of adults with acute leukaemia have also been disappointing. In an attempt to suggest rational modifications to existing regimes, more needs to be known about the mechanisms which

Abbreviations: CALLA, common acute lymphoblastic leukaemia antigen; mdr, multi-drug resistance; 6-TG, 6- thioguanine; HGPRT, hypoxanthine-guanine phospho- ribosyltransferase; ARA-C, cytosine arabinoside; Ara- CDP, 1-beta-D-arabinofuranosylcytosine-5'-diphosphate; Ara-CTP, 1-beta-D-arabinofuranosylcytosine-5'-triphos- phate; 6-MPRP, 6-mercaptopurine ribose phosphate; GSH, glutathione; GSTs, glutathione s-transferases; dCTP, deoxycytidine; DHFR, dihydrofolate reductase; AML, acute myeloid leukaemia.

Correspondence to: Dr A. Hall, LRF Laboratory, Fourth Floor, Cookson Building, New Medical School, Fram- lington Place, University of Newcastle, Newcastle upon Tyne, NE2 4HH, U.K.

351

underlie the development of resistance to cytotoxic drugs. By an increased understanding of such mech- anisms it may also be possible to suggest ways in which drug resistance may be overcome.

Due to the problems inherent in establishing pri- mary cell cultures from patients with either fluid or solid tumours, many studies into mechanisms of drug resistance use established cell lines, often of non- human origin. A common approach has been to supplement culture medium with gradually increas- ing doses of cytotoxic drug and to isolate clones which express resistance. Characterization of such sub-lines has yielded a great deal of useful information about the way in which cytotoxic drugs are transported across the cell membrane, their modes of action and their metabolic fate. Extrapolation from such studies to the clinical setting must, however, be guarded. There are two main reasons for caution. Firstly, the behavlour of cells grown in culture, particularly if of non-human origin, cannot be assumed to be the same as that of malignant cells in a patient. Secondly, cells which have undergone malignant transformation, particularly those arising from the bone marrow, often display an abnormal degree of sensitivity to cytotoxic drugs, the factor which underlies the con- cept of a "therapeutic index" in cancer chemotherapy [3]. On relapse these cells lose their abnormal sen- sitivity to treatment and adopt a similar resistance phenotype to their parental cell type. A model system which investigates the phenotype of sensitive rather than resistant sub-lines may, therefore, be closer to the pattern of behaviour of tumours in vivo. So far

352 A. HALL, A. R. CAT'rAN and S. J. PROCTOR

this approach has been adopted in relatively few studies although Robson et al. [3, 4] have successfully isolated and characterised a number of sensitive mutants of Chinese hamster ovary cells in this way.

Although the bulk of research into drug resistance has employed established cell lines, there have been some efforts to characterise cells obtained directly from patients. A major problem with this approach has been the establishment of reliable and repro- ducible methods to assess drug resistance in oitro. Such an estimate is desirable if resistance is to be correlated with phenotypic features of malignant cells, particularly if a number of patients, subjected to a variety of treatment regimes, are to be compared. The use of clinical criteria provides a relatively poor substitute for such assays, as differences in phar- macokinetics may affect response to treatment and be confused with resistance at the cellular level. Particular problems have been encountered in the development of clonogenic, as opposed to cytotoxic assays, as, by definition, these involve the main- tenance of cells in culture which are capable of div- ision. The use of cells in short-term culture has been used to assess the sensitivity of leukaemic cells to cytotoxic drugs [5], but correlation with clinical response has not always been reliable. Care must be taken to establish that cells assessed by such assays arise from the tumour cell population rather than normal precursors. The fact that cells capable of surviving in culture may not be truly representative of the tumour cell population must also be considered.

In an attempt to prevent, or overcome, the devel- opment of resistance in acute leukaemia, a wide range of cytotoxic drugs has been incorporated in a variety of induction, consolidation and maintenance regimes. These drugs differ both in their chemical structure and in their mode of action, although most are believed to exert their main effects by interfering, either directly or indirectly, with the replication or transcription of DNA. As a consequence of this diversity it is perhaps not surprising that differences also exist in the biochemical changes which have been found in resistant cells. The mechanisms which have been identified may be broadly classified into six main groups.

1. D R U G UPTAKE

Due to chemical similarities to naturally occurring metabolites, many drugs are transported across the cell membrane by an active, energy dependent, pro- cess which works against the concentration gradient. Alterations in the structure of the carrier proteins involved in these processes may lead to decreased

drug uptake, and, as a consequence, increased resist- ance.

The transport of methotrexate occurs by an active process which involves a high-affinity transport car- der mechanism shared by aminopterin and 5-methyl- tetrahydrofolate. Initial reports suggested that folic acid is transported by an independent mechanism [6], but this has recently been challenged [7]. A line of L5178Y lymphoblasts resistant to methotrexate has been described which displays a 93% reduction in methotrexate influx, when the extracellular con- centration is 1 Ixm [6]. (Increases in the extracellular level of methotrexate led to a decrease in the dif- ference in uptake between sensitive and resistant cells.) These studies infer either a reduction in the affinity of membrane bound carrier proteins for the drug, or a reduction in the number of carrier proteins expressed.

Observations that the cytotoxic effect of melphalan against murine L1210 leukaemia cells is reduced by the presence of leucine or glutamine in the tissue culture medium led to the characterization of a high-affinity amino acid transport system which is responsible for uptake of the drug at cytotoxic con- centrations [8]. Similar results have been obtained using L5178Y lymphoblasts in a study which estab- lished the presence of at least two carrier systems, capable of generating an eight-fold difference between intracellular and extracellular drug con- centrations [9]. In addition, characterization of a melphalan resistant L1210 cell line has revealed a decrease in intracellular drug concentration associ- ated with diminished uptake. Further support for the hypothesis that alterations in carrier proteins may be responsible for resistance comes from a report by Dantzig et al. [10], who demonstrated that Chinese hamster cells selected on their inability to grow in the absence of high leucine concentrations displayed a 100-fold increase in resistance to treatment with melphalan.

2. D R U G EFFLUX

Many sub-lines selected on the basis of resistance to anthrocyclines have demonstrated cross-resistance to vinca alkaloids, and other unrelated compounds. Recently it has been demonstrated that this "multi- drug resistant" (mdr) phenotype is associated with enhanced efflux of drug across the cell membrane [11], and the increased expression of a specific mem- brane protein with a molecular weight of 170,000 [12] (P-glycoprotein). Drug resistance associated with enhanced P-glycoprotein expression has been demonstrated in both human lymphoblast [13] and myeloma cell lines [14], and in the cells from patients

Mechanisms of resistance in acute leukaemia 353

with relapsed AML [15]. In addition, it has been shown that treatment with verapamil, a calcium chan- nel blocker, causes reversal of resistance in cells with the mdr phenotype, probably by decreasing the rate of drug efflux from the cytoplasm [16]. Limited clini- cal studies have been performed to test the ability of this drug to reverse resistance in vivo. The results of these trials have been generally disappointing, although a recent case report by Durie [17] suggests that this approach may be of value in some cases of refractory multiple myeloma.

3. DECREASED D R U G ACTIVATION

Several cytotoxic drugs are inactive in their admin- istered form, and rely on cellular metabolism to an active form to be effective. A decrease in the activity of the enzymes responsible for this conversion may be responsible for some forms of resistance to cytotoxic effects. The purine analogue, 6-thioguanine (6-TG), is converted to its nucleotide, 6-thioguanylate, by the action of hypoxanthine-guanine phosphoribosyl- transferase (HGPRT) [18]. In-vitro studies have indi- cated that a decrease in the activity of HGPRT may cause resistance to 6-TG [19], although the frequency of this mechanism in vivo has been questioned [20]. Activation of cytosine arabinoside (Ara-C) occurs by conversion into 1-beta-D-arabinofuranosylcytosine- 5'-diphosphate (Ara-CDP) and 1-beta-D-arabino- furanosylcytosine-5'-triphosphate (Ara-CTP). Char- acterization of a cell line resistant to Ara-C has been shown to be due to almost complete absence of an active form of one of the enzymes responsible for this conversion, Ara-C kinase [21]. However, the relevance of this form of resistance in the clinical setting has not yet been established.

4. CELLULAR DETOXIFICATION

Many drugs are detoxified within tumour cells by processes which involve enzymes which are normally engaged in the metabolism of naturally occurring intermediates or xenobiotics. Enhanced expression of protective enzymes has been implicated as a cause for drug resistance, both in model systems and a limited number of clinical studies. Some examples of resistance to 6-mercaptopurine fall within this cat- egory. The active form of the drug, 6-mercaptopurine ribose phosphate (6,MPRP), is degraded to inactive 6-thioinosine by the action of particulate bound alka- line phosphatase. In two reports a significant increase in the activity of alkaline phosphatase has been found in the cells of some patients with resistant forms of acute leukaemia [22, 23]. In one of these [23],

patients were studied both before and after the devel- opment of resistance to thiopurines. Resistance was accompanied by a two-fold, or greater, increase in alkaline phosphatase activity within the blast cells.

Enhanced drug detoxification may also be involved in the development of resistance to the bifunctional alkylating agents, a group which includes chlo- rambucil and melphalan. High performance liquid chromatography studies have indicated that mel- phalan forms a, presumably inactive, conjugate with the tripeptide, glutathione (GSH) [24]. Further evi- dence that this reaction may be important in vivo has been provided by characterization of a melphalan resistant L1210 cell line which was shown to have a significant increase in the intracellular level of GSH [25]. Other studies have indicated that this reaction may be controlled by the action of cytoplasmic glutathione-s-transferases (GSTs) and that elevation of the activity of these enzymes may be associated with resistance to alkylating agents. Evidence comes from work both with cell lines, and tissue biopsies. A Chinese hamster ovary cell variant has been described which shows selective resistance to chlo- rambucil and similar drugs, associated with marked overexpression of a basic form of the enzyme [26]. Inhibition of GST activity using the anti-inflam- matory agent, indomethacin caused partial reversal of the resistance phenotype [27], an observation which may have important therapeutic implications. An attempt has been made to correlate the level of glutathione transferase within a given type of tumour, and the general susceptibility of that tumour type to treatment using chemotherapy [28]. A broad pattern was established in which those tumours generally responsive to cytotoxic drugs, such as those of the head and neck, were found to have low levels of transferase activity, whilst those unresponsive, such as carcinoma of the colon and prostate, had generally high levels. Further studies are needed to document changes in GST expression associated with the devel- opment of resistance to chemotherapy in other forms of malignancy before a final conclusion can be drawn concerning the relative importance of this form of detoxification mechanism.

5. ENHANCED COMPENSATORY METAB- OLISM

In addition to changes in the levels of enzymes directly involved in the metabolism of cytotoxic drugs, resistance may also arise due to increases in the activity of enzymes involved in compensatory biochemical pathways, which bypass the actions of the drugs. Adriamycin is believed to exert some of its cytotoxic actions within target cells by the

354 A. HALL, A. R. CA'I'FAN and S. J. PROCTOR

generation of free hydroxyl radicals, although the relative importance of this mechanism in relation to the interaction with nuclear DNA which also undoubtedly occurs, remains controversial. Recently an adriamycin resistant MCF-7 human breast tumour cell line has been described which demonstrated decreased free hydroxyl radical formation on exposure to the drug, and enhanced levels of glutathione peroxidases, enzymes responsible for the detoxification of these active species [29]. Rapid removal of such potentially toxic products would be expected to reduce the cytotoxic actions of the drug.

Resistance to cytosine arabinoside may arise by a similar process. The drug is normally activated by a series of phosphorylations, the first of which involves the enzyme--deoxycytidine kinase. There is evi- dence that deoxycytidine kinase is inhibited by increases in the level of deoxycytidine 5'triphosphate (dCTP) within the cell, and an inverse relationship between dCTP levels and sensitivity to Ara-C has been established in a range of cell lines [30]. Fur- thermore, concomitant treatment with thymidine, known to reduce cellular dCTP levels, caused an increase in sensitivity to Ara-C of up to 3-fold, and it has been suggested that this may be used as an adjuvant to therapy in resistant cases.

6. BIOCHEMICAL CHANGES IN THE TARGET MOLECULE

Changes may occur in either the level, or the properties, of the target molecules of cytotoxic drugs, with a resulting reduction in the sensitivity of the host cell to damage. A well documented example of this mechanism is believed to be responsible for resistance to methotrexate in some cell lines, and has been implicated in resistance occurring in vioo. Increases in the level of the target for methotrexate, the enzyme dihydrofolate reductase (DHFR), leads to resistance to the effects of the drug, and has been found to be caused by gene amplification [31]. This may occur either within the chromosome, or in smaller pieces of DNA, known as double minute chromosomes [32, 33]. Gene amplification within the chromosome causes a stable form of resistance. In contrast, amplification within double minute chromo- somes causes a form of resistance which is gradually reduced over several cell passages, as these short fragments lack a centromere and are lost by one daughter cell at meiosis [34]. These observations suggest that if methotrexate resistance develops dur- ing treatment, and is found to be associated with the presence of double minute chromosomes, retreat- ment may be successful after several cell cycles. Methotrexate resistance may also be attributed to a

reduction in the affinity of DHFR for the drug, thereby causing an increase in the levels of reduced dihydrofolate in the cells. In a study illustrating this form of resistance a mutant form of DHFR was purified from a methotrexate resistant lympho- blastoid cell line [35]. This protein showed a 50-fold reduction in affinity for methotrexate, accompanied by an 18-fold increase in affinity for dihydrofolate. Interestingly the selective advantage for the cells with this type of enzyme was lost at high drug con- centrations, suggesting the presence of both high, and low-affinity sites of action, and perhaps providing a biochemical rationale for the use of high dose methotrexate in some treatment schedules.

A similar type of mechanism is believed to underlie some forms of resistance to anthrocyclines. An important target for this group of drugs has recently been identified as topoisomerase II, a nuclear enzyme which facilitates controlled breakage of double stranded DNA, and thereby allows the process of decatenation, or unknotting, to occur. During this process the enzyme forms an intermediate "cleavable complex" with the DNA [36]. In the absence of topoisomerase inhibitors this is a short lived species. Anthrocyclines are believed to act by stabilising the cleavable complex, thereby arresting the natural pro- cess of breakage and repair. P388 leukaemia cells have been shown to have two forms of enzyme, with slightly different molecular weights on electro- phoresis. Alterations in the relative proportions of these forms has been associated with resistance to amsacrine [37]. A mutant form of topoisomerase II has also been identified which appears to have reduced affinity for etoposide, suggesting that quali- tative, as well as quantitative, changes may be involved in this form of resistance [38]. Conversely, in a study of cells from patients with chronic lympho- cytic leukaemia, low levels of topoisomerase II, nor- mal in quiescent cells, have been associated with resistance to the action of anthrocyclines, presumably due to a reduction in the number of potential target sites on which the drug can act [39].

7. CONCLUSIONS

As already stated, many of the studies aimed at characterising the biochemical mechanisms under- lying resistance to cytotoxic drugs have relied on the use of cells grown in culture as a model system. Relatively few carefully designed surveys have been undertaken to establish the relative frequency with which these phenotypic modifications occur in relapsed patients. Leukaemia offers a unique oppor- tunity for providing this information, as repeated sampling, and rapid purification of the tumour cell

Mechanisms of resistance in acute leukaemia 355

populat ion can be readily performed. It is important to stress that any one drug may produce a variety of resistance modifications within a blast cell popu- lation. For this reason careful characterization of the biochemical nature of resistant blasts must precede the introduction of therapy a imed at potentiating cytotoxic drugs if the true effect of this form of intervention is to be established.

Acknowledgements--The authors thank Mrs Graham for her expert assistance in preparing the manuscript. Dr A. Hall is funded by the Leukaemia Research Fund.

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