Biochbnica et Biophysica Acta, I 114 (1992) 107-127

107

© 1992 Elsevier Science Publishers B.V. All rights reserved 0304-419X/92/$05.00

BBACAN 87251

An overview of membrane, cytosolic and nuclear proteins associated with the expression of resistance to multiple drugs in vitro Siobhfin M¢Clean and Bridget T. Hill D e p a r t m e n t o f Celhdar Clzemotherapy, bnperial Cancer Research Fund, L o n d o n (UK)

(Received 5 March 1992)

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A, in-vitro drug resistance B, Multiple drug resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C, P-Glycopmlein-mediated drug resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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107 108 108 109

!1.

Resistance-associated membrane proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Overexpression of 300-kDa to 85-kDa proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Multiple membrane-protein alterations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Differential expression of epidermal growth factor receptor . . . . . . . . . . . . . . . . . . . . . . . . . D. Reduced expression of 100-kDa to 70-kDa proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

111 112 113 113 114

111.

Resistance-associated cyto~olic and nuclear proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. 20-22-kDa proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Glutathione S-transfcrases: 23-32-kDa proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Tubulin: 55-60-kDa protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Topoisomerase ll: 170 and 180-kDa proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Heat-shock proteins: 70, 60 and 27 kDa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Transcription factor Spl: 97 kDa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Estrogen receptor and the cytochrome P-450s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

114 115 116 117 117 118 ! 19 ! I')

IV.

Altered regulation of proteins implicated in multiple drug resistance . . . . . . . . . . . . . . . . . . . . . A. Phosphoprotein: 20 kDa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Phosphorylation changes in topoisomerase !! . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Protein-kinase-C-mediated regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

119 120 120 121

V.

Identification of resistance associated proteins by transfection studies . . . . . . . . . . . . . . . . . . . .

121

VI.

Methods used in the detection of alterations in resistance.associated proteins . . . . . . . . . . . . . .

122

VII. What causes protein alterations in MDR cells? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

122

VIIi. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

123

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

! 24

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

124

1. Introduction Correspondence to: B.T. Hill, Department of Cellular Chemotherapy, imperial Cancer Research Fund, P.O. Box 123, London WC2A 3PX, UK.

The development of drug resistance in patients receiving chemotherapy alone, or combined with radio-

therapy, represents a major problem in cancer treatment [1]~ ~though patients may initially respond, a large group will subsequently relap~ and become refractory to further therapy [2]. The underlying mechanisms of this phenomenon, clinical drug resistance, are not fully understood. However, considerable imight has been gained on the subject of in-vitro drug resistance from experimental laboratory studies. A major advance in the treatment of patients with neoplastic disease would be the identification of specific 'markers' predictive for d r u g resistance expressed by tumours, either at presentation or during therapy,

I.A, In.uit~ drug resistance Drug resistance exhibited by mammalian tumour continuous, or as has been examined more re~ntiy, pulsed exposure to antitumour agents in vitro has been associated with changes in expression and/or activity of certain intracellular and plasma membrane proteins, These alterations, which appear numerous and varied, are not restricted only to distinct classes of antitumour drugs since various modified proteins are now considered characteristic features associated with resistance to certain 'alkylating' agents, antimetabolites, antitumour antibiotics, Vinca alkaloids and epipodophyllotoxins, Some 'classical' examples of types of proteins altered in specific drug-resistant cell lines established in the laboratory ar~; listed in Table !. The specific mechanisms involved in some of these cellular protein alterations still remain to be elucidated cells f o l l o w i n g

although amplification of the gene encoding dihydrofolate reductase (DHFR) is one of the known mechanisms responsible for resistance to methotrexate (MTX) [3]. Published data also indicate that resistance to 5-fluorouracil (5-FU) and to cisplatin (CDDP) may be mediated by reduced expression of thymidylate synthase [4] and camptothecin resistance has been associated with resistant forms of its target, topoisomerase I [5]. Furthermore changes in glutathione and its related enzymes have been implicated in drug detoxification mechanisms minimizing the cytotoxicities of CDDP and nitrogen mustards [6,7], while reports of increased expression of metallothioneins in tumour cell lines resistant to CDDP and alkylating agents also suggest a rok for these metal-binding proteins in resistance to these drugs [8,9,10]. in the majority of instances, however, a causal relationship between drug resistance and the specific protein alteration identified remains to be established, in recent years there has been extensive investigation by many laboratories of the phenomenon known as multiple drug resistance (MDR) and from these studies an apparent plethora of protein modifications has been described. This review, therefore will concentrate on those proteins which have been altered as a result of expression of the MDR phenotype. FB, Multiple drag resistm,,ce

MDR is a complex phenotype originally identified in mammalian tumour cells in vitro, involving the expression of cross-resistance to many structurally and func-

TABLE i

Ceil I~ne/typ~

P~ein

~o~,i~in

..................................................... m~mni~ia" '

Drug

..................................~ D R . . . . . . . . . .

Alteration

Re£

ov~r~~i,'e~sed ...................

:'~-27

G l u l a l h ~ Sqr~mst'~ra,~

MCF-7 Human ,~luamous cell ca CHO

MDR CDDP nitrogen mustard

45-fold increase in protein levels 2- to 3-fold increase in activity 40-fold increase in a-class isozyme

II 2 6 7

To~i~r~sc

CHO KB

AMSA VP- 16

resistant form decreased level of protein

126 129

Tubulia

CHO CHO

COL, CMD taxol

altered/~ suhunit altered a subunit

123 124

Met~lk,thh~pacin

Human 'Human pro~e ca Human ovarian ca

alkylating agents ADR CDDP

~werexpressed overcxpressed overexpressed

8 9 10

DHFR

Mammalian

MTX

overexpressed

3

T h v m ~ a ~ ~ynthas~

A2"/t~) ovarian ca

CDDP

2.5-fold increase in activity

4

CIP-.~0

murine lymphoma

CDDP

overexpressed

196

il

t A range of mammalian cell lines, including human. A range o! human tumour cell I;n~s.

109 tionally unrelated antitumour agents [11,12,13]. These drugs include Vinca alkaloids, anthracyclines, and epipodophyllotoxins, while sensitivity is retained towards other drugs such as bleomycin (BLM) and CDDP [12]. Generally, MDR cells appear to have a reduced ability to accumulate drug [14,15], which is often accompanied by overexpression of a 170-kDa ceil-surface glyeoprotein, P-glycoprotein (Pgp)[16,17,18]. This distinct phenotype has been termed 'classical MDR'. In addition~ examples of cell lines have been reported which express a non-Pgp associated drug resistance involving cross resistance to structurally but not necessarily functionally different agents. This group includes cell lines which express the 'atypical' multiple drug resistance phenotype proving cross-resistant to epipodophyllotoxins and anthracyclines but not to Vinca alkaloids [19], This situation cannot be refered to as 'classical MDR', since the drugs involved share a common target, i.e. topoisomerase II, and resistance is generally mediated by modifications in this target en.zymc. However, this type of resistance is discussed in this review since it is conceivable that the both the 'classical' and 'atypical' multiple drug resistance mechanisms could arise in the same cell population. Therefore, for the purpose of this review the term MDR is applied in the broadest sense, and refers to mechanisms by which cells are cross-resistant to any the following classes of antitumour agents: anthracyclines, Vinca alkaloids, epipodophyllotoxins, and other drugs such as colchicine (COL) and actinomycin D (ACTD).

i-C. P.Glycoprotehl-mediated drug resistance Pgp is encoded by a small family of genes [20]. in humans two genes code for MDR1 and MDR3, respectively [21,22], the latter gene is also referred to as MDR2. Elevation in expression of this Pgp, is generally mediated by elevated levels of MDR1 mRNA with or without concomitant amplification of the MDRI gene. Involvement of Pgp in MDR has already been widely reviewed [23-27] and will be discussed only briefly here. Pgp is considered to act as a membrane efflux pump based on the following evidence: the presence of ATP-binding sites within its structure and the striking homology to bacterial transport proteins [28,29,30]. In addition, a pump-like activity has been demonstrated in vesicles prepared from MDR cells [31]. One definitive element of the involvement of Pgp in MDR is that it does confer resistance on drug sensitive cells: for example, Gros et al. [32] transfected the entire coding sequence of the mdrl gene into drug sensitive host cells, which subsequently expressed the M D R phenotype. A feature of the majority of M D R cell lines is that resistance is reversible,at least in part, by the addition of one of a series of agents at non-cytotoxic doses.

These so-called resistance modulators include calcium channel blockers for example, verapamil (VRP), quinidine, nifedipine [33,34], calmodulin inhibitors., such as trifluoroperazine [35] and other unrelated drugs including cyclosporin A [36], dipyridamole [37,38] and tamoxifen [38]. Clinically, VRP has been found to be an effective modulator, since combination chemotherapy including a high dose of VRP produced a response in 13 out of 18 previously relapsed patients with malignant lymphoma [39], although associated normal tissue toxicity clearly limited its usefulness. Pgp can bind drugs, as demonstrated by photoaffinity labelling studies with either an ~2Sl-labelled azido derivative of vinblastine (VBL) [40] or a photoactive analogue of a dihydropyridine calcium channel blocker, ~H-azidopine [41]. Competition studies between these r~hotoactive agents and calcium channel blockers or various MDRassociated drugs have demonstrated that MDR drugs and resistance modulators compete for binding sites on Pgp [41]. Studies carried out by Fojo ct al. [42] have demonstrated Pgp expression at intermediate to high levelsin excretory cells from normal tissue such as colon, small intestine, kidney, liver and adrenal wllich was consistent with Pgp having a transport function. In addition, untreated tumours which arose from these tissueswcrc f'~und consistently to express high levels of M D R I m R N A [43]. Monoclonal antibodies have been raised which recognise different epitopes on Pgp: C219 [44], C494 [44], MRK-16, [45] and JSB-I [46], Hyb 241 [47] and mAb57 [48]. These have enabled the detection of Pgp in human tumour cells and tissues t,sing methods including Western blotting, immunocytochemistry and flow cytometry. The association of the Pgp phenotype with t!le clinical course of disease has also been examined. In a study involving 89 sarcoma patients, turnouts from 15 patients showed elevated Pgp, it was found that no clinical responses were observed in the few patients with Pgp-positive tumours subsequently treated with chemotherapy [49]. A retrospective analysis carried out immunohistochemieally on biopsy samples from 30 children with rhabdomyosarcoma and undifferentiated soft tissue sarcoma using C219 a n d / o r C494 demonstrated the imports.nee of Pgp in the prognosis of childhood soft tissue sarcomas [50]. All nine patients with detectable Pgp in their biopsy samples relapsed after an initial clinical response, however only one patient of the 21 with Pgp-negative tumours relapsed [50], A correlation between high levels of Pgp expression and resistance in patients to a VAD regimen . (vincristine (VCR), Adriamycin (ADR), dexamethasone) was found following a study of Pgp expression in plasma cell myeloma samples using the C219 antibody and flow cytometry [51]. A study of leukaemic cells showed only 23% of Pgp positive turnouts responded

110

clinically to anthracycline therapies, whereas 73% of Pgp negative tumours responded [52]. In addition, the levels of Pgp m R N A have been determined in turnout cells. However, since there is no direct evidence that increased M D R I m R N A levels are nece~arily translated into functional Pgp, these data

need to be interpreted with care. Detectable levels of the MDR1 transcript were found in leukaemic cells of 12 out of 15 adult acute nonlymphocytic leukaemic patients (ANLL) studied [53]. Cons,:stent with the hypothesis that a Pgp-mediated mechanism may confer clinical resistance, lower remission rates and shorter

TABLE il

Altered proteins associated with MDR cells Protein

De~ription

Selecting agent

Implicated in resistance *

Alteration

P,Oly~)protein EOFR

possible el'flux pump 8~vth factor receptor

+ +/ ~

P300 PI~

P70-80 P~5 P~ P36 P ~ =.'),4

membrane protein ATP bi~Inrg protein cell surface protei,~ pho~phoryl~ted cell surface protein membrane glycoprotein cell surfaceprotein cell surface protein membrane protein gl~'oprotein gly¢oprotein gl~co~rotein lab~l~ protein labile protein labile protein cell surface

MDR VCR. ADR, ACTD COL ADR ADR ADR ADR

overexpressed elevated lost elevated increased levt~ls elevated expression increased phosphorylation decreased levels overexpre~sed decreased levels overexpressed decrea~d expression reduced expression decreased expression elevated elevated elevated elevated

8~ 66 88 59 75 85 86 69 69 69, 70 ~9

So~in/Cp22 GSV-~"

calcium binding protein detoxification c,~ymc

elevated hlcreased activity

86, 93-98 112-114

more stable form

124, 126 125 91

PII~, P169, P158 PI~) OPt00

P~q

P?~=gS P75, P72

Tuhulin

microtubule

VBL ADR/VRP

? + ?

ADR VBL COL COL, ADR, VBL ADR ADR ADR ADR

+/ ? ? ? '? '? '?

See Tal)le IV ADrt, VCR COL, X-ra~ C.OL. C M D Taxol ADR

+/-

P~

t~,temblv pha~ I metabolizing e n ~ e (s) phosphoprotein

I~C

protein phosphorylation

See Table V

N~, T o p t ~ i ~ r a ~ II

DNA unknottingenzyme

07towhee P~;0

E~ro~n

÷ ? '?

+

.?

ADR

167-17(1

increased activity decreased activity (cf. Table V)

171-182

ADR, VP-16 VM-26

resistant to VP.16 stimulation,

135.136 136

M,:roid r~c~ptor

ADR

?

heat labile reduced levels of isoforms increased phosphory!ation lost

tran~ription factor

ADR

'?

?

increased expression and DNA binding ability

* Aulho~" ~mclu~ons as to whether the protein alterations have any role to play in multiple drug resistance: + +/-

22=27 72-75 76, 77 60 61 71 ¢)5

increased phosphorylation

~or Spl

less stal)le form reduced expression

Ref.

Role i. resistance likely. Role i. resistance unlikely. ~ role in resistance in certain ceils, Unknown whether protein has any influence on drug resistance.

144,146

140 74 92

111 remission durations were associated with patients expressing higher levels of MDR1 mRNA [53]. More recently, overexpression of the MDR1 transcript was found in 50% of adult acute leukaemias which relapsed after therapy, in comparison to only 19% of untreated leukaemias [54], again contributing to the evidence that expressio, of MDR1 or Pgp is at least in part responsible for drug resistance in human tumours. However, Pgp-mediated resistance does not appear to be the only mechanism operating in these resistant tumour cells, since while all primary breast cancers examined by Sanfilippo et al. [55], which proved sensitive to either ADR or VCR, did not express detectable levels of Pgp by Western blot analysis, only a third of resistant turnouts expressed Pgp, implying other mechanisms of resistance may have been involved. Also, the majority of lung cancer specimens, including small cell and non-small-cell tumours (NCLC), were found to express low levels of the MDRI transcript, since NCLC patients respond poorly to combination chemotherapy, it is clear that non-Pgp mechanisms are involved in the resistance expressed by these tumours [43]. Finally, in a study on a range of human tumour samples, including breast carcinoma, 4 of 6 responsive tumours expressed high levels of Pgp [56] suggesting that high Pgp expression does not alway imply drug resistance. The findings of these latter studies indicate that other mechanisms may play a role in resistance to antitumour agents and clearly argue against the sole use of Pgp as an valuable indicator of the response of a tumour to chemotherapy. In order to predict accurately a patient's response to chemotherapy, the assessment of a panel of 'markers' will undoubtedly prove necessary. It should perhaps be also emphasized that accurate identification of Pgp positivity at either the protein or mRNA level is not a trivial exercise. False negatives may result if the assay used is too insensitive and controls with known lowlevel expression are not used. Similarly, false positives may result from the use of non-specific antisera. Thus reports that tumour biopsies or certain cell lines are Pgp negative need to be evaluated critically. The role of MDR3 in drug resistance remains to be established. MDR3 was undetectable in five human tumour cell lines examined by van der Bliek et al. [22], implying that overexpression of MDR1 was the prime cause of resistance. A recent report demonstrated elevatior, of MDR3 mRNA expression in six out of six prolymphocytic leukaemia samples while only one sample expressed MDRI [57]. The MDR3 expression appeared to correlate with increased accumulation of daunorubucin (DNR) in the presence of cyclosporin A (C'3,cA) indicating the possible function of a drug efflux pump which could be inhibited by CycA [57]. The co-amplification of MDR1 and MDR3 has been observed in a human colon carcinoma cell line using gone specific probes [58]. Both genes were amplified up to

30-fold but the authors were unable to establish whether the MDR3 protein product was expressed [58] and thus could not determine whether the amplification of the MDR3 gone resulted in the acquired MDR phenotype. It was suggested that both MDR genes share common regulatory transcription factors [58]. However, transfection of a human MDR3 eDNA into human BRO melanoma cells did not result in resistance to various MDR-associated drugs including, VCR, COL, ADR, DOX, ACTD [59]. While MDR3 expression may, in time, prove to play a role in resistance in certain tissues, it does not appear to be a general mechanism of drug resistance. Further investigations involving determining specific gene expression in tumour cells and gene transfer studies remain to be carried out to confrm its role, if any, in drug resis. tance. Considering the complexity of the MDR phenotype, it is not surprising that other non-Pgp-mediated alterations have been reported. In this context, the term 'alteration' refers to any change in the level of expres. sion, activity, size or degree of phosphorylation of a protein in drug-resistant cells relative to their drug sensitive parental cell line. This review will outline the many protein alterations which have been observed in tumour cell lines selected for resistance to any of the antitumour agents which are considered 'members' of the various classes of drugs associated with the MDR

phenotype. A considerable number of protein alterations have been associated specifically with only one particular MDR cell line or group of MDR lir~s. The proteins being reviewed here are listed in Table II according to their subcellular location. Membrane protein alterations appear most common among MDR cells although modifications of particular cytosolic and nuclear proteins have also been observed. Among this list are proteins of various function, structure and molecular weight. Their listing does not imply that a causal relationship with resistance has been proven, since it is possible that they may be purely incidental alterations. Each of these proteins will be discussed in turn in an attempt to establish the implication of the changes recorded. In addition, several MDR.associated cell proteins have undergone changes in post-translational regulation, such as phosphorylation levels, and these are outlined in a separate section. Having detailed the various proteins in turn, and briefly discussed the implication of these alterations, their potential relevance to clinical resistance will be summarised.

I!. Resistance-associated membrane proteins Modified cellular proteins which have been linked with the development of resistance to multiple drugs

112 have predominantly been associated with cell membranes. The most frequently reported membrane alteration in MDR cells is Pgp overexpression, discussed briefly above. However, in addition, many other membrane-associated proteins have been identified in dru:g-reshtant cell lines in vitro, although the precise cellular function of the majority of these is unknown to date. These proteins are discussed below in order of their molecular weights. Epidermal growth-factor receptor (EGFR), which is a well characterised protein, has also been shown to be altered in many MDR .cell lines., although its precise role also remains undefined.

IloA. Ot'erexpression of 300°kDa to 85~k~ InvJte#~s IbA~ L 300.k1~,~ prote& The largest of this group of proteins is a 3(8}-kDa membrane protein observed by Tsuruo's group in ADR-sel¢cted human ovarian (A2780), breast (Hattori) and ieukaemia (I~62) cell lines which expressed the MDR phenotypc [60],. However. high levels of this protein were also present in parental human breast carcinoma cells (MCF..7/P) and in human CEM ieukaemic cells [60], demonstrating that the 300.kDa protein did not confer resistance. The function of this protein is unknown, although, studies using the 300kDa protein specific monocional antibody demonstrated a reversible inhibition of growth of MCF-7/P and resistant K562/ADM cells, suggesting the protein may be involved generally in controlling cell proliferation [60]. II-A.! 190.kDa protein A nuclcotid¢.binding protein, immunologicaily distinct from Pgp, was observed in an ADR-selccted human leukaemic cell line, H ~ [61]. These cells, HL-60/ADR, p~ved 80-fold resistant to ADR and showed cross-resistance to VCR, VBL and ACTD [62] and w~r¢ also defective in drug accumulation [63]. in addition, VRP induced a major increase in drug accumulation and enhanced cytotoxicity of ADR in these cells. Although clearly resistant to multiple drugs, Pgp was undetectable in these cells by Western blotting of membrane fractions'using C219 monoclonai antibody while a VCR-sclected HL-60 cell line derived and studied in parallel did ovet~xpress Pgp, Similar to Pgp, this lg0-kDa membrane protein competitively bound ATP and could b¢ selectively labelled with azido~- -~-'P ATP [61], However, in contrast, it could not be labelled with photoaffinity analogues of VBL or calcium channel blockers [61], all of which readily bind to Pgp [40,41]. A later study demonstrated a common recognition site between an antiserum to a short specific sgqu©nc¢ on Pgp and the 190-kDa protein, indicating minor homology between the two proteins [64]. Since these ADR..resistant HL-60 cells were defective in

drug accumulation and it was concluded that this 190kDa protein possibly functions as an energy dependent drug efflux system in these cells [64]. These results provide evidence of a non-Pgp nucleotide-binding protein which may be involved in drug accumulation and which is modulated by VRP.

II-A.3. 150.kDa protein Elevated levels of a 150-kDa cell-surface phosphoprotein have also been identified in an ADR-selected drug-resistant HL-60 line [65]. In-vitro phosphorylation of this protein with .~2p~ facilitated its identification, since the protein was only a minor membrane component. By silver staining isolated 150-kDa protein folleaving ¢leetrophorcsis, the levels of stained protein were comparable in sensitive and resistant cells [65], implying enhanced phophorylation of this protein was responsible for its detection in resistant cells rather than increased expression. 11-,4.4. 95-kDa prote& The expression of yet another protein associated with ADR resistance has been demonstrated by using a novel procedure of simultaneously selecting MCF-7 cells for ADR resistance in the presence of VRP, in an attempt to minimize development of Pgp-related resistance mechanisms [66]. The selected subline, designated MCF-7/ADRVp, overexpressed a membrane protein of 95 kDa, but did not demonstrate elevated Pgp. The level of expression of this 95-kDa protein correlated with levels of ADR resistance. In addition, removal of ADR only from the selection medium resulted in a loss of the protein with a concomitant loss of resistance to ADR. This suggested that the protein played a direct role in ADR resistance in these cells [66], The expression of this qS-kDa protein has also been demonstrated in a number of clinical samples including a malignant pleural effusion of a patient with ADR refractory breast cancer [66], suggesting a possible relationship between this protein and clinical resistance. 11-,4.5. 85-kDa protein Hamada et al. [67] have observed elevated levels of an 85-kDa membrane protein using the monoclonal antibody MRK-20. This 85-kDa protein was overexpressed in ADR-selected ovarian lines but was not detected in other drug~resistant human lines selected with VCR, VBL or colchicine (COL) [67]. The expression of this protein increased in the presence of ADR and diminished when ADR was removed, unlike Pgp expression which remained constant in these lines. Subclones of the K562/ADM line with different levels of this 85-kDa protein expressed similar levels of sensitivity to ADR and to other cytotoxic drugs. Thus, it appears that this protein does not confer resistance

113 although it may play a role in the resistance phenotype [67]. For example, the expression of this protein may be the result of a stress response induced by ADR administration; the possible involvement of stress proteins will be discussed later (cf. Section Ill-E). In a further study a distinct correlation was drawn between expression of this 85-kDa protein and cross resistance to three anthracyclines, to mitozantrone (MX), etoposide (VP-16), BLM and to pepleomycin [68]. Overexpression of the 85-kDa protein was not always associated with Pgp overexpression and it is possible that this 85-kDa protein plays a role in an additional non-Pgp related ADR resistance mechanism [68]. Chen et al. [66] have demonstrated that the 85-kDa was distinct from the 95-kDa protein discussed above: MRK-20 did not cross-react with MCF-7/ADRVp cells and, in addition, a polyclonal antibody to the 95-kDa protein did not detect its specific antigen in human 2780/Ad cells.

II-B. Multiple membmne-lmJtein alterations A series of protein alterations have been identified in a non-Pgp expressing ADR-resistant small cell lung cancer line, H69AR [69]. using monoclonal antibodies, both on the cell surface and on other cellular membranes. Antibodies raised against the non-cell-surface membrane proteins (55, 46 and 36 kDa) of the A69AR cell line were cross-reactive with cell lysates obtained from a multidrug-resistant human fibrosarcoma line, HT 1080/DR4, also selected for resistance with ADR. The cell-surface proteins range in M r from 24.5-34.5 kDa. None of the proteins observed in this study [69] were homologous to any other identified proteins involved in resistance. However, the 36-kDa phosphoprotein (p36) was later identified as lipocortin 11, a family of calcium and phospholipid-binding proteins [70]. The physiological function of p36 remains to be established, however it has been implicated in signal transduction, transformation and differentiation [70]. The expression of this protein in a revertant cell line, however, indi-

cates that this protein is unlikely to be involved in the drug resistance mechanism. In another study [71], this group has demonstrated the presence of proteins of M r 186, 169 and 158 kDa using another monoclonal antibody (mAb 7.4.1) in three ADR-resistant sublines: the fibrocarcinoma cell line, HTI080 DR4, the ovarian carcinoma line, 2780AD, and a MX-se!ected subline of the colon carcinoma line, WiDr. The monoclonal antibody which detected these proteins did not crossreact with Pgp.

II-C. Epidermal growth-factor receptor (EGFR) 170-kDa protein Altered expression of EGFR has been reported in certain MDR cells, (Table liD. However, no consistent paaern of modified expression has emerged. Increased expression of receptor has been demonstrated by two groups [72-75], while loss of receptor has also been observed by others in human tumour cell lines which exhibit MDR [76,77]. Meyers et ai. [72j first examined EGFR expression in murine and Chinese hamster cells while investigating the observed 'normalization' in phenotype of MDR cells and found 1.6- to 10-fold elevations in four MDR lines relative to their drug sensitive parent cells. These lines were selected by continuous exposure to VCR, ADR or ACTD and all overexpressed Pgp. A further study yielded the same trend in human neuroblastoma and neuroepithclioma lines [73]. Although the EGFR and the MDR1 genes are both on chromosome 7 in humans [78,79], the EGFR gene was not amplified in these lines [73], implying that coamplification with the mdrl gene was not the mechanism responsible for EGFR overexpression. Simultaneous amplification of both these genes has been demonstrated, however, in an early passage human lung cancer cell line derived from a patient previously treated with VBL [80]. Although, amplification of both EGFR and MDR1 genes was lost in later passage cells [80]. A human MDR breast carcinoma cell line which was

TABLE Ill

Alterations in EGFR in drug resistant cells Cell line/type

Selecting agent

Alteration

Ref.

murine Chinese hamster lung and bone marrow

VCR, ACTD

increased 1.6- to 10-fold

72

human neuroepithlioma human neuroblastoma

VCR, ACTD

3-30 fold increase in recer~tor levels altered ¢h~,clrophoreticmobility

73

human MCF-7 breast ca.

ADR VCR

increased 1O0-foid increased 10-fold

74 75

human KB epidermoid ca.

COL

37% receptor loss

76

human small cell lung ca.

ADR

3-fold decrease

77

114

~ f o l d ADR resistant (MCF-7/ADR) demonstrated a l ~ f o l d i n c r e ~ in EOFR expression which was associated with a decreased level of receptor respon~ n e s s [74]. In addition, a series of VCR-selected MCF-7 resistant cell lines overexpressed EGFR up to l~fold [751. In contrast, a study of a colchicine-re~tant ~ line showed the reverse situation [76]. While these ~ cells expressed a high level of Pgp, the expression of EGF receptor number by the resistant cells was reduced to 37% of that in the parental cells. More recently, a similar result was obtained in a nonPgp-mediated MDR large cell lung cancer line, L 23/R, in which EOFR exp~ssion was reduced 3-fold [77] and ~ t c h a r d analysis revealed that the h~h affinity recepto~ had been lost, EGFR is up.regulated in several tumours [81,82,83], but appears to have varied expression in drug-resistant tumour ¢¢11 lines. Structural alterations [73] and chanees of receptor affinity [76] have also been report~ in resistant tua~ur cells. It remains uncertain whether EOFR has any definite involvement in the drug resistance mechanism, however, altered TOF alpha s . ~ t i o n by MDR lines has been suggested as a possible explanation for the alterations in EGFR expre~ion [72,73]. The variation in expression of EGFR m y be a phenot~ic change associated with resistance which is cell line- or tissue.dependent. In breast cancer cells taken dir~tly from ~tients' tumours, elevated EGFR tends to be an indicator of poor prognosis [84]. In the event that EGFR overexpression in the MDR breast cancer cell lines is paralleled in breast turnouts in patients, ways of overcoming th~ alt,.ration could be ~ught as a means of modulating resistance,

II1D. Rcduc¢~ ¢xp~ssion of lO0.k~ to 70~kDap~teins most frequently ob~rvod protein alteration as a result of drug resistance have been increased protein oxpr¢~n, hosccvcr, a number of groups have reported a contrasting situation: that is, a loss of expression of membrane proteins in drug-~sistant human tumour cell lines. These proteins appear to form a distinct group, having molecular masseswithin a narrow 25.kDa range from 75 to 100 kDa, The first observation of this type was reported by Beck's group stuclying MDR VBL-selccted CEM I~anphobias' ~id lines which were shown to have reduced ~xp~n of a 75-85-kDa cell-surface glycopt~tein [gS]. Resi~ance develolanent in four resistant KB carcinoma lines selected with COL, ADR or VBL resalted in two consistent changes: (i) Pgp overexprcssion and (ii) a decrease in expression of members of a family of proteins of molecular mass from 70 kDa to 80 kDa [86], This decrease in immunoprccitable proteins associated with a concomitant loss in mRNA for these proteins. A substantial decrease in synthesis of

two membrane glycoproteins of molecular mass 72 and 75 kDa was also reported in COL-selected mu|tidrugresistant KB carcinoma cells [87]. The extent of protein loss correlated with the level of resistance; in the more highly resistant KBC2.5 line these proteins were virtually absent, while both reappeared in a revertant line. Cell fractionation stvdies suggested that the proteins were cell membrane Iocalised. In another series of MDR CEM cells a 90-kDa protein has been reported which underwent progressive reduction in its expression in parallel with acquired resistance levels. This 90-kDa protein was also found on the cell surface [88]. It is unknown at present whether these various proteins (el. Refs. 85-88) are in any way related. As mentioned, protein alterations most frequently reported in drug resistant cells are those involving an increase or overexpression. However, the findings outlined above are distinct in that all involve the decreased expression in proteins of a similar size, which appear to be cell surface or membrane related. It seems plausible therefore, that there is a relationship between these consistent observations; these proteins could be members of the same family of proteins, the loss of which results in or contributes to the resistance mechanism(s). For example, it has been suggested that the 72-75-kDa proteins mediate in drug uptake, implying their loss might therefore impair drug accumulation [87], Whether these proteins play a role in the MDR mechanism or not, the loss of a detectable expression of proteins such as those outlined above may prove a useful marker among a panel of indicators of a refractor~ turnout. Many of the membrane alterations outlined in this section have been associated specifically with ADR selection. ADRoselectod multiple-drug-resistant cell lines often express the MDR phenotype, including Pgp overexpression. However, as described above, additional alterations involving numerous other proteins can arise which may be specific either for ADR resistance itself or to the ADR selection process involved. The fact that many of these proteins are membrane proteins is not surprising since in addition to DNA intercalation, ADR is known also to interact at the cellular membrane [89,90]. While the levels of some of these proteins correlated with resistance to ADR, there are also many reports where the alteration in protein expression is not related to resistance, suggesting that these latter type of proteins are consequences rather than causes of resistance. IlL Resistance-associated cytosolic and nuclear proteins

Compared with the variety of membrane proteins which have been reported as modified in drug-resistant tumour cells, changes in both ~,tosolic and nuclear

I15 proteins are generally fewer and more consistent. Only four major proteins, three cytosolic and one nuclear protein, have been described in more than one model system. However, single reports of alterations in estrogen receptor (ER) [74], cytochrome P-450 (cyt /'-450) [91] and transcription factor, Spl [92] have also been published. I l i A . 20-22-kDa proteins

Sorcin was first observed in 1981 by Meyers and Biedler [84] while studying the possible protein products of gene amplification in VCR-selected drug-resistant Chinese hamster lung cells. This soluble 22-kDa protein with a p l of 5.7 was initially associated only with VCR resistance, However, it has since been shown that a similar 21-kDa protein is overexpressed follow. ing COL [86,94,95] ADR [94,96], ACTD [97], VP-16 [98] or teniposide (VM-26) selection [98] (Table IV). Several laboratories have detected similar acidic proteins all of which may indeed be also identified as sorcin. A 21-kDa protein (pl 5.0) was ove.rexpressed in ACTD- and VCR-selected SEWA mouse cells [97]. Koch et al. [94] have observed a 22-kDa protein (pl 5.3) in two MDR lines, an ADR-selected murine line and a COL-selected hamster line. Djungarian hamster cells exhibited increased levels of a 20°kDa protein following COL-selection [95]. Increased synthesis of a 21-kDa protein was also observed by Shen et al. [86], but only in their COL-selected MDR human KB line and not in either of their VBL or ADR-selected lines. Sorcin was also identified in an ADR-selected K562/ADR line [96]. In all cases mentioned above the detection of 2022-kDa proteins has been associated with some extent of gene amplification [97], exemplified by the presence of homogenously staining regions (HSR) or double

minute chromosomes (DM). In general, the level of protein overproduction correlated with the level of gene amplification, as determined by mean HSR length [98], or the number of DM per ceil [100]. In fact, one

group has demonstrated that loss of DMs following prolonged culture in ACTD of the drug-resistant SEWA cells resulted in the loss of this 21-kDa protein [991. With one exception [89], sorcin was found to be overexpressed only in cells which expressed high levels of Pgp, implying its appearence was the result of coamplification with the mdrl gene. Biedler et al. [101] have reported increased synthesis of sorcin in only 50% of their drug-resistant lines which amplify the mdrl gene, indicating that sorcin may not be a major determinant in the development of drug resistance. Consistent with this, no correlation has been found between the level of sorcin expression and levels of resistance to ADR, VCR and COL in drug resistant murine cells [102]. Sorcin has been found to be encoded by a gene which is one of five linked genes coamplified and overexpressed in the MDR CHO line, CHitC5 [103], One of these genes (class 2) cross hybridized with pCHP1 [104], a Pgp eDNA probe [105]. It seen'is that me class 4 gene (coding for sorcin) was coamplified with the class 2 gene and it is possible that all five classes of genes may be contained on the same amplicon. Evidence for coamplification was outlined by Van der Bleik et al. [103]: following pulsed field gel electrophoresis a large fragment of CH RC5 amplified DNA hybridized with both class 2 and class 4 eDNA probes; both gene classes have been found to be elevated to a similar extent in a range of MDR lines and in metaphas,~ spreads prepared from CHRC5 cells both gene clas.,,es hybridized to the same HSRs. These results may explain the observation of sorcin overexpression in solae MDR lines and not others, since potential for overexpression may be a factor of the proximity of the two gone classes in a particular cell type [101]. However, sorcin may also be amplified independently of Pgp. Overproduction of an acidic 21-kDa protein correlating with the number of DM chromosomes has been observed in SEWA murine cells which did not express detectable levels of Pgp [100]. Should this become a more common finding, the significance of sorcin in MDR may finally be resolved.

TABLE IV 20-22.kDa cytosolicproteins overexpressedin drug resistantcells PROTEIN 21 kDa ~rcin 22 kDa 20 kDa sorein 21 kDa sorcin

pl

5.7 5.3 5.0 acidic

Cell line

Selecting agent

Rcf.

human KB hamster DC3F morine EMt 6/Ca Djungarian hamster human K562 routine SEWA murine LI210

COL VCR ADR, COL COL ADR ACTD, VCR VM-26, VP-1O DOX, ACTD, VCR

86 93 94 95 t)O 97 89

116

Whether sorcin and Pgp are coregulated or not remains to be elucidated conclusively. Sorcin may simply be a passive protein which is coincidentally overexpressed as a result of Pgp overexpression. Alternatively, it may contribute to the MDR phenotype. Studies carried out on the possible biological role for sorcin have revealed that sorcin is a specific calcium-binding protein with a yew high affinity for 45Ca2÷ [94,106]. ~quence analysis has uncovered four calcium-binding sites and a strong homology with the calcium-binding light chain of calpain [103], In addition, the observation of two putative cAMP-dependent protein kinase II recognition sites may imply that activation of ~rein is mediated by cAMP [~], The presence of ~rein, whether neces~r~ or incidental may still modulate resistance in some way. For example, the level of sorein in C H n ~ cells far exceeds the level of free calcium and would be capable of binding 10% of total calcium in the cell [94], Thus it could play a significant role in the regulation of calcium-mediated events such as cell signalling, in resistant cells. Immunocyt~hemistry on 1~62/ADM cells has localized ~rcin to the cytoplasm a~acent to ribosomes, rough endoplasmic reticulum, perinu¢lear membranes and microfilaments [96]. Taken together these data imply that sorcin plays a physiological role, possibly in signal transduction, in these organelles in resistant cells. In summary,, because ~rcin is not overexpressed in all MDR cells it can not be a general cause of resistance. However, when it is overexpressed, it may play some role in the resistance mechanism or influence the patterns of cross-resistance expre~ed to other drugs in a cell line, by having a 'synergistic' effect with PgP. The ability of sorein to sequester Ca ~'* in resistant tumour cells may perturb a variety of Ca ~*-mediated systems, therefore its observed co-amplification with PgP should be carefully examined.

Ill.B. Ghoothion¢ Sotrat~sJ'emses 24-32okl)a proteins The 81utathione S-transferases (GST) are members of a cytoplasmic enzyme family involved in the detoxification of a range of xenobiotics and chemotherapeutic ~ents [107,1~] via conjugation with glutathione [108]. There are a number of GST isoz3nnes, which sub-divide into three classes: ~-e are basic, ~t is neutral and fr and p are acidic [ 109]. Due to the nature of the role of GSTs they might be expected to be altered in some way in cells which become resistant to antitumour drugs, in order to enhan~ the efficiency of the cells' detoxification of such agents. The first MDR cells found to have an elevation in GST activity were derived from a subline of the MCF-7 breast carcinoma cells which was 192-fold resistant to ADR [110]. The~ cells, which expressed comparable levels of resistance to VCR, VBL, ACTD, and VP-16,

expressed a 45-fold increase in GST activity [110]. Western blotting established the elevation in activity to be due to the stable ovcrexpression of the anionic form, ~r [110]. In an ADR-resistant routine P388 leukaemia line GST activity, as measured using either i-chloro-2A,-dinitrob~nzene or ethacrynic acid as substrates, was 1.8and 2.2-fold elevated, respectively [ 111]. Consistent with this increase in activity and also in agreement with Batist etal. [110] a 2.2-fold increase in the ~- isozyme was observed by Western blotting. A two-fold elevation in GST activity has also been observed in two X-ray pro-treated CHO lines which expressed cross resistance to Vinca alkaloids and epipodophyllotoxins, but not to anthracyclines [112], These sublines, designated DXR-101 and DXR-1011 were not radioresistant [112], suuesting the overexpression of GST was not a protection measure against the X-ray pro-treatments but instead, played a role in the drug resistance mechanism, in contrast, no increase in GST activity was detected in two ADR- or VBLoresistant human lines, including a myelogenous leukaemia cell line (K562/ADM), an ovarian carcinoma line (A2780 AD), and a lymphoblastic CCRF-CEM leukaemic line (VLB100) [113]. Only one subline examined in this study, MCF-7/CLI0, expressed an elevation in GST activity. This COL-resistant line had a 7(bfold enhancement in activity, also due to an overexpression of GSTo~" [I 13]. Consistent with this, a series of VCR-resistant MDR MCF-7 cells examined by Whelan etal. [114] demonstrated up to a six-fold increase in GST activity, again with a parallel elevation in the vr isozyme, although these authors reported that this was not an invariable fading in MDR lines since no alterations in GST activities were demonstrated in a series of MDR human teratoma subUnes. The three independent findings of elevated GST activity in differently derived drug-resistant MCF-7 cells suggest that MCF-7 cells may have a higher potential to modify GST in order to overcome the 'insult' of antitumour agents. As two groups [113,114] have suggested the inherently low GST activity level of MCF-7 parental cells may require its upregulation following drug treatment. The increased expression of GST observed in the P388 leukaemia [110] and two CHO sublines [112], however, indicates that GST was not altered solely in drug-resistant MCF-7 cells. Why GST should be involved in MDR in some cell lines and not others is unclear, it is possible that GST elevations may be indicative of a general stress-type response of cells following selection with a cytotoxic agent. However, it does appear significant that when overexpressed it is the Tr isozyme that has been consistently elevated. This is in contrast to studies carried out on cells resistant to aikylating agents in which the GST-a isozyme was generally overexpressed [7,115,116], although as an ex-

117 ception, GST-w overexpression has been demonstrated in melanoma cell lines selected for alkylating agent resistance [117]. Overall, this implies that GST-zr may have a substrate specificity for MDR drugs, including ADR [109], VCR [114] and COL [113]. The finding that two cell lines transfected with GST-rr eDNA proved 1.8- and 3-fold resirtant, respectively, to ADR while no significant resistance was expressed towards alkylating agents, CDDP or radiation [118] is consistent with this suggestion. However, another study involving tranfection of GST-rr expression into MCF-7 cells failed to demonstrate enhanced protection against ADR [119]. Since GST is one of a number of components in the glutathione redox cycle, it is possible that alterations in GST alone cannot confer resistance and so gene transfer studies such as these, may have been inadequate in attempting to establish a direct role for GST in drug resistance. It is unlikely that GST expression alone can cause resistance but its co-modification along with other elements of glutathione metabolism, could mediate the drug resistance mechanism. The ability of inhibitors of GST, such as ethacrynic acid, to circumvent drug resistance in the clinic has yet to be explored. In addition, the ,elev;tnce of GST overexpression to clinical resistance must also be evaluated, to date one study has been carried out where the anionic GST gene was evaluated in tumours from six patients with ANLL and was found to be uniformly expressed at the same level, regardless of anthracycline sensitivity or clinical response [56].

IH-C Tubulin 55-60 kDa Several of the anticancer drugs within the MDR group are antimitotic agents, having microtubules and, more specifically, tubulin as their cellular target. COL, for example, binds to two distinct sites on tubulin [120]. Consequently extensive investigation of tubulin expression in antimitotie-resistant cell lines has revealed alterations in mierotubules in general, [121] or specifically in either the a or ~ subunits of tubulin [122,123,1241. Microtubules of colcemid (CMD)-resistant CHO cells (CM R) studied by immunofluorescence microscopy were found to be resistant to the disruptive action of CMD, requiring a 6- to 7-fold higher concentration to break down the microtubules [121]. It was suggested that the alteration was due to a change in the CMD-binding site of tubulin. Two CHO cell lines, resistant to COL or CMD, when analysed by two dimensional electrophoresis were found to express a mutant ~-tubulin with a more basic isoelectric point, while expression of wild-type tubulin was reduced [122]. By an in-vitro translation assay it was determined that the modified /3-tubulin did not result from post-translational modification. The same

group [123] also obselved an electrophoretic variant of a-tubulin in a taxol°resistant CHO cell line, having a lower isoelectric point. Four species of/3-tubulin were detected by isoelectrio-focusing and gel electrophoresis of cytosols of MDR Chinese hamster lung lines, DC3F/ VCRd-51, compared with a single species in the parental cell eytosols [124]. In agreement with the COL- and CMDresistant CHO cells [122], two of these additional isoforms were considerably more basic than the major isoform common to parental and resistant cells. A review by Cabral and Barlow [125] discussed tubulin mutations in the context of their involvement in the drug resistance mechanism. They considered that a mutation causing COL or CMD resistance might lead to a confirmational change of tubulin subunit resulting in enhancement of stability of the microtubules, while taxol, which promotes tubulin assembly, might cause a mutation which results in a more labile microtubule [125]. This group has since developed an extraction system to determine relative levels of polymerized tubulin in CMD-selected CHO cells. In agreement with their model outlined above, taxol-resistant mutants maintained a lower fraction of tubulin in the assembled form, while CMD-resistant cells had a higher level of cellular tubulin in the polymerized state [126]. However, the buffer used in the assay contained taxol to which some of the cell lines studied were resistant, and to which the CMD-resistant cells would be hypersensitive. Therefore, the enhanced polymerization of tubulin in taxoi hypersensitive cells is not surprising and could perhaps reflect the effect of taxol on these cells rather than a change in the tubulin assembly which correlates with resistance levels. Recent interest in the clinical potential of taxol [127] and the production of a semi-synthetic derivative, Taxotere, for evaluation [128] may provide more knowledge regarding drug mechanisms in this area.

Iii.D. Topoisomerase I1:170 and 180 kDa Topoisomerase I1 (topo 11) is an essential nuclear enzyme which is responsible for the knotting, unknotting, catenation and decatenation of DNA (for reviews see Refs. 129,130). The process involves (i) binding of the enzyme to DNA; (ii) cleavage of the DNA double strand within the enzyme complex; (iii) passage of a second strand through the break; (iv) subsequent resealing of the DNA duplex and (v) release of the duplex from the enzyme. The reaction intermediate involving DNA bound to topo I! may be trapped by detergents and is termed the 'cleavable complex' [131 ]. A number of anticancer agents including the anthracyclines and epipodophylloxins interact with topo II, thereby inducing DNA cleavage (132,133). Therefore it is not surprising that cells which acquire resistance to

118 drugs from either of these classes can express modifications of t o ~ I1. Examples of both quantitative and qualitative changes are discussed below. Drug.resistant cells in vitro having alterations in tope I! express a different cross resistance pattern to the 'classic' MDR phcnotype in that they tend to be cross-resistant to epipodophyllotoxins, anthracyclines, ~hydroxy ellipticine and to MX but not to the various Vinca alkaloids [134]. This drug resistance phenotype has been referred to as 'at-MDR' or altered tope MDR [!9]. A qualitative change in the tope !I e n ~ e of VP16,resistant CHO ceils has been demonstrated [135,136], The~ cells, designated Vpma-5, which expres~d cross resistance to ADR, MX and amascrine (m-AMSA) [137], had comparable levels of tope I! decatenation activity and accumulated the same level of VP-16 as the parental cells, Western blotting conf i n e d comparable leech of expression of tope il in the Vpma-5 cells [1~], However, studies with FPLC purified enzyme revealed it was 30- to 40-fold more resistant to VP-16 stimulation by examination of cleavable complex formation in the presence of ATP [136], In addition, the Vpma-5 enzyme was more heat labile than the wild t ~ e enzyme. Taken together, it was concluded that the tope !1 enzyme itself was modified resulting in a change in interaction between drug and the enzyme-DNA complex, Similarly, human leukaemic CEM cell lines selected with VM-26 expressed a tope 11 enzyme which was resistant to both VM-26-mediatcd inhibition of strand pa~ing and VM-26-induced DNA cleavage and had a reduced rate of catenation up to 30-fold [137], Because the effect of VM-26 on both strand pa~ing and DNA cleavage was inversely proportional to the degree of VM-26 resistance of tile two resistant CEM cell lines studied, it followed that drug resistance w ~ mediated in these cells by an alteration of the tope H protein or its regulation [137], Decreases in the level of tope 11 have been observed in a series of VP-I~resistant KB sublincs which exprtssed resistance levels up to 290-fold [139], As rtsistance to VP-16 incrtasod, the level of tope !! estimated by Western blotting decrea~d, with a concordvnt decrease in tope !! enzyme activity as measured by DNA unknotting activity, Mort recently, two VP-16-rtsistam human tumour cell lines (KB/VP-! and KB/VP.2) were found to have bolh qualitative and quantitative modifications in their tope II enr/me [140], Although VP-16 accumulation and tope 11 activities, as determined by decatenat~n of kinetoplast DNA, were comparable for drug-re. r a n t and -sensitive lines, immunoblotting revealed that levels of tope II were 20-fold less in the two former cell lines [140]. A concordant loss of expression of the 6.2-kb transcript corresponding to tope 11 was ob~rved on Northern blots. However, pbosphorylation

of tope II in both resistant lines was found to be 14- to 18-fold elevated [140]. Since tope !1 activity can be enhanced by protein kinase C (PKC) [141], casein kinase II [142], or a serine kinase [143], it was suggested that phosphorylation of tope 11 in the KB/VP-1 and KB/VP-2 cells maintained the decatenation activity while the reduced levels of the enzyme resulted in the VP- 16 resistance [ 140]. Murine leukaemic cells cross-resistant to m-AMSA, ADR, VM-26 and VBL [134] were found to express two- to thrte-fold less tope II activity. On purification, two active forms of the enzyme were isolated: the 170 isoform and a new form of 180 kDa [145]. Although both isoforms were present in sensitive and in resistant cells, immunoblotting revealed a reduced amount of the 170-kDa isoform in the resistant cells [145], In contrast, cell extracts from a MX-resistant human HL60 human leukaemic line also cross-resistant to several natural product agents contained tile 170-kDa isozym¢, while the 180-kDa form was immunologically undeteetable [ 146]. The value of tope ii mRNA as a clinical marker for ADR response in patients was examined by Kim et al. [56]. Dot blot analysis revealed tope II mRNA expression in 5 of 6 responsive tumours while it was undetectable or detected at only low levels in all nine unresponsive turnouts, It was suggested, therefore, that tope II expression might be a useful tool in predicting the response of a patient to therapy involving ADR [56], A recent report on a study of human ovarian tumours identified a reduced level of tope 11 activity in tumours treated with CDDP and cyclophosphomide, although 16-fold differences in tope II levels were found in the tumours analysed [147], which ,nay be a limiting factor in these types of studies.

Ill.E. Heat-shock proteins: 70, 60 and 27 kDa The heat-shock proteins (HSP) consist of a small number of proteins which undergo enhanced synthesis or accumulation in ceils exposed to increased temperatures for short periods, inducing a state of thermotoleranc¢ in these cells [148]. Evidence linking these pro. teins and other stress-related proteins to MDR have been reported, CHO cells selected for resistance to antimitotic agents have been found to express alterations in two microtubule-associatod proteins, which were identified as a member of the chaperonin family of heat shock proteins (60 kDa) and the constitutive form of the 70-kDa heat shock protein, HSP 70 [149]. Two-dimensional protein acrylamide gel studies on the podophyllotoxin-resistant cells revealed a new 63-kDa protein spot, which peptide mapping studies proved to be a more acidic form of the mammalian homologue of the

119 60-kDa heat-shock protein (HSP 60) [150,151]. In contrast, COL-resistant mutants expressed a more basic form of the 69-kDa protein [141,142] which was later identified as HSP 70 [149,153]. These proteins are thought to be involved in transport of tubulin to mitoehondria and assembly of microtubules [159]. It is possible, therefore, that the mutant form of these HSP molecules may be more efficient in binding or transportation of disassembled tubulin. Chinese hamster lung fibroblast cells transfected with the human estrogen-regulated HSP 27 gene were found to be cross-resistant to VCR, COL, ADR, ACTD and DNR, but not to non-MDR drugs such as 5-FU [154]. These cells did not overexpress Pgp relative to untransfected cells as determined by Western blotting using C219, nor were any accumulation detects de,-eted implying that a Pgp-independent mechanism of resistance was involved [154]. However, since the levels of Pgp were higher than that expressed by drug sensitive CHO cells, AuxBl, which were used as a negative control, this does not rule out the involvement of Pgp completely, since activation of this protein could have occurred in the HSP 27 transfectants. Therefore, the role of HSP 27 in multiple drug resistance needs to be evaluated further. Further reports linking HSPs with drug resistance include the study in which Chinese hamster cells preheated to 43°C for over 50 min prior to ADR treatment, expressed considerable resistance to ADR [155]. A more recent report outlines the induction of MDRI gone expression in human renal carcinoma cells, following a 30-min heat shock up to maximum levels after 2 hours of such heat treatment which correlated with a transient increase in VBL resistance [156]. Other environmental stresses such as glucose starvation and hypoxia may also play some role in the MDR mechanism. Induction of the glucose regulated protein (GRP) system has been linked to both ADR [157] and VP-16 [158] resistance in Chinese hamster ovm3, (CHO) ceils. It was demonstrated that GRP proteins could be induced by two specific conditions, namely, glucose deprivation or anoxia, and by two agents: the calcium ionophore, A23187, and 2-deoxyglucose [157]. Expo. sure of CHO cells to any of these conditions or agents resulted in a significant co-induction of ADR resistance [157]. These findings imply that induction of the 76, 97 and 170-kDa GRP proteins is related to the resistance phenomenon [157]. This effect appears to be due to the observed rapid depletion in topo 11 from nuclei of these cells following any of the treatments [159]. In addition, a correlation has been drawn between ADR resistance and enhanced expression of oxygen-regulated proteins which undergo increased synthesis during hypoxic stress [160]. This close interrelationship between drug resistance, albeit transient, and environmental stress proteins could lead to further

knowledge of the mechanisms involved in development of MDR.

III-F. Transcription factor Spl: 97-kDa protein Eievaiion of expression of a 97-kDa nuclear protein has been noted in ADR-resistant HL-60 cells and had been identified as the transcription factor, Spl [92]. This is known to be. involved in the activation of several mammalian genes [161]. Increased levels of this protein have been found chiefly in the nuclear fraction but also in the membrane and cytosol. Spl could be required for transcriptional activation of genes involved in resistance [92]. The MDRI gone promoter region contains some Spl-binding sites [161] and may play a role in the development of the MDR phenotype. It is also possible that Spl overexpression occurs as a direct result of the MDR phenotype.

ill-G. Eso'ogen receptor and the O,tochrome P.450s An ADR selected MCF-7 cell line expressed a number of alterations when compared to parental MCF-7 cells, including, overexpression of Pgp [163], GST-~r [110] (Table I) and EGFR [74] (discussed earlier). In addition, however, development of resistance in these cells was associated with the loss of detectable levels of estrogen receptor (ER) [74] and reduced expression of the phase I metabolizing enzyme, cytochrome P-450 [91]. This latter modification may, however, be a result of the loss of ER expression rather than a direct result ef either the ADR selection procedure or the MDR phenotype expression [91]. A lack of detectable ER expression was also found in a series of VCR-sclccted resistant MCF-7 ~ublines [74]. Since the presence of ER is an importarg prognostic factor in breast cancer for a reduced risk. of relapse and improved overall survival [164], further investigations into the relationship between ER status and MDR may prove worthwhile.

IV. Altered regulation of proteins implicated in MDR Alterations in protein pho;phorylation including, in certain cells, more specific alterations in PKC activity and increased phosphorylation of particular proteins have been observed in a range of MDR cells by a number of research groups. Pgp is itself phosphorylated, which may have an activating or inhibitory effect on its function depending on the residues which are involved [165]. Meyers [166] reported a significant changes in activities of a range of kinases and phosphatases between drug-resistant hamster lines and their drug sensitive parental lines. Her findings were consistent with an overall increase in protein phosphorylation mediated

120 by elevated levels of one or all of the calcium-dependent kinases, PKC and protein kinase A. However, certain VCR-selected lines expressed an overall decrease in phosphatase activity compared to the parental cells [ 166].

II~A. Plzosphoprote#z: 20 kDa Marsh and Center [167] have identified three proteins which were specifically phosphorylated in vitro in membranes isolated from ADR-resistant Chinese hamster lung cells, A 180-kDa protein and a 220-kDa protein were highly phospho~lated in the presence of Mg** or Mn**and a third protein of 20 kDa was nhosp~rylated only when Mn *+ was added to the incubation mixture. All three proteins were phosphorylated at serin¢ residues, Since the high levels of ph~sphorylation were not ob~rved in drug sensitive or revertant cells, there appeared to I~ a definite relationship between phosphorylation and the drug-resistant phenotype [167], Increased phosphorylation of a 20-kDa protein has al~ been identified in ADR-selected MCF-7 cells (KC/ADRI & KC/ADR 10), in clinical breast biopies and in a series of small cell lung cancer cell lines [168], This group demonstrated a 4-fold increase in phosphorylation of a particulate protein of 20 kDa in the 10(~foid ADR-resistant KC/ADR 10 line [168]. This protein may be the same protein as that found in the CHL lines [168], however, its phosphorylation proved Mg** rather than Mn** dependent, This difference may be real or could be related to the different in-vitro phosphorylation s~tems used in these two studies, Phospho~lation of a protein of 20 kDa has also been demonstrated in a study of four breast tumour biopsies [169], These ~mples were derived from patients treated with, but unresponsive to, combination therapy including ADR and VBL, Biopsy specimens

taken from untreated patients, however, did not show any such in-vitro phosphorylation [169]. A cell line subsequently derived from one turnout of an unresponsive patient expressed resistance to VBL and ADR in clonogenic assays, suggesting a potential role for this protein in a predictive assay of patients' responses [158], This differential phosphorylation was also demonstrated in a study of 17 small cell lung cancer lines [170]. A 1.4-fold increase in phospho~lation of a 20kDa protein was observed in lines obtained from turnouts from six previously untreated patients who subsequently responded to therapy, However, a line derived from a seventh, untreated patient exhibited a 4-fold increase in phosphorylation of a 20-kDa protein and this patient proved unresponsive to chemotherapy [170]. In addition, 10 lines obtained from tumours taken from relapsed or post-chemotherapy patients demonstrated high levels of in-vitro phosphowlation of the 20-kDa protein ranging from 3- to 15.4-fold over control values [ 170]. All lines which had elevated levels of phosphorylation were found to be ADR-resistant in vitro and eight of the 10 lines also expressed resistance to Vinca alkaloids [170]. Although only these two research group~ appear to have reported altered phosphowlation of a small particulate protein, there does appear to be a distinct correlation between its phosphowlation and drug resistance. In addition, this research suggests that this protein could be a potential marker for clinical response in vivo. IV.B, Pho,~ptlorylation changes in topoisomerase H

Tope !! activity can be mediated by phosphorylation [141,142]. As already discussed, Takano et ai. [140] have observed increased serine phosphorylation of tope !1 by immunoprecipitation of .~Zp. labelled cellular proteins from VP-16-resistant KB cells, The phospho.

TAI~LE V

C~II lin~s

Selecting agent

Alteration

Ref.

h.maa MCF-? h.~n ~F-? ~msh, r

ADR ADR VCR ADR ADR

T-fold increase in activity 15-fold increase in activity 1.5-fold increase in activity 1,6 x increase in activity 2 × increase in activity over~xpr~s.sion of y 80-~1~:~ increase overexpre~ion of a reduced rate of degradation 30% decrease in activity 6-fold increase 50% decrease in activity

174 175 175 175 17~ 177 178

hum~m I|L~I

marin~:, h.m~a m d ¢ ~ fibmsarcoma P-~ human MCF-? human MOLT-3 I TMX: t n n ~ t ~ t ¢

ADR VP-16 ADR TMX I

180 181 182 182

121 rylation appeared to result in an increase in decatenation of topo li activity [140]. The enzyme responsible for this elevation in phosphorylation could not be determined, however it was unlikely to be PKC, since phorbol esters, did not result in a further increase in 32P-labelling.

IV-C. Protein-kblase-C-mediated regulation of proteins The biochemical characteristics of phosphorylation patterns in MDR cells suggest a role for PKC. A view reinforced by the observed reversal of resistance by calcium channel blockers which also inhibit PKC [171]. Recent work on staurosporine (STSN), a potent, (but not specific) inhibitor of PKC [172], has provided further indication that phosphorylation of Pgp by PKC plays a role in regulating Pgp function [173]. STSN was found to increase accumulation of VCR in three different human MDR turnout cell lines ahnost to the level of parental cells and inhibited ATP-dependent VCR binding to Pgp by almost 100% in K562/ADM cells [1741. Several investigators have discovered alterations in the levels of PKC activities in drug-resistant lines. However, results are varied and, in some cases, even contradictory (Table V). Fine et al. [174] reported that the basal activity of PKC increased 7-fold in MDR MCF-7 cells (KC/ADRI0). Also in another ADR-resistant MCF-7 line a 15-fold increase in PKC activity was detected, while increases of 1.5- and 1.6-fold in activity of PKC were reported in VCR-resistant hamster lines and in ADR-resistant HL-60 lines respectively [175]. A similar result was obtained by Aquino and collegues [176] studying ADR-se!ected HL.60 cells (HL60/Dox R) which were shown to express a 2,fold elevated level in PKC activity. In particular these cells expressed the PKC isoform-~,, which is absent in the parental sensitive cells [177]. MDR mouse and human tumour cell lines were also found to express 80-90% increases in PKC activity; however, in contrast to Aquino's findings, it was the a isozyme which was overexpressed [178]. A positive correlation between PKC activity and ADR resistance was demonstrated in mouse fibrosareoma cells [179]. The overexpression of PKC in these cells was later reported to be, at least partly, due to a reduced rate of PKC degradation [190]. To counterbalance these reports [174-180], two other groups have identified a significant reduction of PKC in their drug resistant cells. A VP-16-selected P388 cell line expressed a 30% depletion in PKC activity over parental P388 cells [181]. While in a recent publication [182] a 50% reduction in PKC activity in MDR MOLT-3 sublines was described. However, since no absolute correlation between the level of decrease and the degree of MDR was found, these data imply that the PKC alterations observed are merely inciden-

tai alterations and not related to drug resistance in these cells. In this same report [182], a 6-fold increase in PKC activity was demonstrated in the ADR-selected MDR MCF-7 line (MCF-7/DOX R), confirming the observations of Fine et al. [174]. These results, although conflicting, do not rule out an involvement of PKC in drug resistance. However, since the physiological substrates of the various PKC isozymes have not yet been elucidated, it cannot be conclusively established that alterations in PKC activities are responsible for the alterations in substrate labelling. Some authors [177,178,182] have used histones as the substrate in in-vitro labelling assays, but since they are not a specific physiological substrate for PKC it is possible that other kinases present in the extract may also phosphorylate histones. Where possi. hie, attempts should be made to confirm any alterations in PKC octivity as also being reflected in altered levels of protein as judged by Western blotting. Differences in extractability of PKC between sublines should also be minimized in these types of studies. In addi. tion, enhanced .~ap labelling of histone may not be due solely to increased PKC, but other factors such as altered protein phosphatase activities may be responsible for some of the alterations observed. Since increas,:s in PKC activity were observed in ADR-selected drug-resistant cell lines, this increase in enzyme activity may be a feature of ADR selection. PKC activity has been found to increase in Sarcoma 180 cells within l0 minutes of exposure to ADR [183]. Therefore alteration in PKC activity perhaps may relate more to the agent used to select for resistance than to the resistance level itself. V. Identification of resistance-associated proteins by transfeetlon studies Transfection of a full-length mouse eDNA clone encoding Pgp into drug sensitive hamster LR73 cells demonstrated directly that overexpression of a single Pgp gene was sufficient to cause the MDR phenotype [32]. More recently, a mutant MDRI eDNA which resulted in a Gly-185 to Val-185 substitution, was transfected into KB cells and increased the level of COL resistance while it reduced the level of resistance to VBL [184]. When moderately resistant MCF-7 cells, which overexpressed Pgp but exhibited low levels of PKCa isozyme, were transfected with a eDNA for the PKCa protein, resistance to ADR and VBL was greatly increased strengthening the link between PKC and MDR [185]. However, a panel of GST-Tr transfectant cell lines proved only 3-fold resistant to ADR, although Western blot analysis confirmed overexpression of GST-~r, implying GST-~" alone is unlikely to confer resistance [ 118]. Results from other transfection studies have meant

that the list of cellular proteins associated with resistance may be expanded. Mouse cells transfected with a chicken calmodulin gene overexpressed calmodulin and expressed a concomitant increase in sensitivity to Vinca alkaloids, but not to ADR or to BLM [186]. This demonstrates that while caimodulin may not cause resistance to multiple drugs, it may influence the relative cross-resistance patterns expressed to antitumour agents. However. previous studies on drug-resistant mouse, hamster and human lines had failed to reveal any alteration in c a l ~ u l i n expression [187] implying calmodulin alteration is not a general phenomenon. A link has al~ been drawn between drug sensitivity and protein kinaso A ( P ~ ) [188]. This was based on the finding that CHO cells which dominantly expr0ssed a mutant PKA enzyme were more sensitive to a range of drugs, including tht~so associated with the MDR phenotypo, in addition, CHO cells transfected with the mutant P ~ eDNA were found to express sensitivity to multiple dru~s [188]. Vi. Me'dtods used in the detection of alterations in reslstanee.assoelat~ proteins

TABLE VI

Examples of probes used in the detection of proteins altered in MDR tumour cell lines Protein

Probe/Antibody

Ref.

Pgp

C219. C494, C32 MRK-16, MRK-17 Jsb- ! HYB 241 Mab57

44 45 46 47 48

EGFR

mouse monoclonal

197

30(} kDa

MRK-18

60

186. 16g. 158 kDa g$ kDa

7.41 rabbill polyclonai ab t

71 86

85 kDa

MRK-4, MRK-20

67

24.5=34.5 kDa 36 kDa, 55 kDa

2.554 Mab : Mabs 3.5, 3.80, 3.177, 3,186.3,187 Mabs 3.80, 3.177. 3.187

69 69 69 6~)

purified monospecific rabbit ab chicken Imiyclonal ab HOT 104 murine Mab

94 106 96

rabbit polyclonal ab rabbit polyclonal ab

198 148

antiserum against pig brain tubulin Tub 2.1 Mab

123

rabbit polyclonal ab

92

47 kDa Sorcin/Cp22

Topoisomerase !!:

#-isot'orm/plgO

Considerable work involving a variety of model systems and techniques has been carried out aimed at detecting the~ various proteins identified as being modifi~:d in MDR cells. Immune-techniques such as Western blotting, immunoprecipitation, immunocyto. chemistry and flow cytom~y ~,,,inga range of antisera, purified antibodie~ and monoclonal antibodies, to MDR cells have permitted the identification of numerous proteins, Examples of some of the probes used are listed in Table VI, Recently developed antisera against peptide fragments of PgP [189] should also furnish additional useful probes, Enzyme activity a,~mys have allowed the determination of modifications in GST [112,113,114[, t o ~ II [13S,!40] and PKC [174--183] levels and binding activities of receptors such as EGFR [~-77] and ER [74] have been studied using specific receptor-binding assays, While all these techniques may, in theory, be used on biopsy samples, those which are quick, accurate and not requiring large c~ll numbers are preferable, if not essential, for clinical purposes. Therefore, flow c.vtometry and immunocytochemistry are deemed more practical than Western blotting, Merkel et al, [190] have suggested that electrophoretic techniques for the detection of Pgp overexpression are not accurate enough to be predictive in analysing heterogenous human turnout samples, Although many studies of Pgp levels (cf, Section I-C) and some investigations of levels of GST [56[, tope I1 [56,147] and phosphorylation of P-20 [169,1"/0] have been carried out, considerably more work needs to must be done to establish the relevance,

a-i~form/p170 Tubulin

Spl

126

Mab: monoclonal antibody.

if any, of these protein alterations in tumours to clinical resistance. VII. What causes protein alterations in MDR cells?

Whether the altered proteins discussed in this review arise by a process of drug selection or drug induction remains an open question. However, in some cases the reasons behind these alterations of proteins in certain MDR cell lines might be straightforward. Should a protein be directly involved with binding (e.g. Pgp, [40,4!]) or detoxifying (e.g. GST [107,108]) an antitumour drug it could be overexpressed in order to protect the cell-from its cytotoxic effects. Since tope It is a target for some MDR drugs, decreased levels of the enzyme or loss of its DNA-binding function should result in less enzyme being available for or capable of irreversibly binding to DNA, so preventing the cytotoxic action of these potential tope II inhibitors. COL binds tubulin [122] inhibiting tubulin assembly, so that

123 a mutation in tubulin which results in the formation of more stable microtubules would make cells more resistant to the effects of antimitotic agents. Proteins which may be involved in cell signalling such as sorcin [94,106] and PKC [191] may be indirect drug targets involved in regulating the pharmacological response of a cell to antitumour agents. It is difficult to decide how proteins with less well studied functions such as, the list of membrane proteins discussed earlier (cf. Table ll) with a range of molecular weights are altered in an MDR cell. These proteins may interact with antitumour drugs directly "~or, alternatively, could be modified indirectly as a result of another protein change or changes in the cell. Precedent for the latter possibility may be drawn from well studied MDR proteins: for example, the overexpression of sorcin which may be due to co-amplification with the genes which encode for MDR [103] and the loss of expression of cytochrome P.450s in ADRresistant MCF-7 cells could result from the loss of ER in these cells [91]. Many other cascades of events taking place in resistant cells may yet be realised.

VIII, Concluding remarks A variety of proteins have been identified in MDR tumour cells. A considerable number of these must fulfil some function in the cells' response to a cytotoxic agent, whether directly or indirectly. It is possible that the wide range of alterations may be a facet of the different cell lines and different resistance selection procedures used in these various studies. For example, each change may reflect the mechanisms that different cell types utilise to cope with the delivered 'insult' of cytotoxic drugs. If this is the case, attempts to monitor these protein changes in clinical biopsy materials may prove even more difficult to interpret. There may, however, be a number of modifications acting together only some of which are being detected in a given cell type. It is possible that all the proteins mentioned above can be altered in a resistant cell, but in a particular cell line or turnout type only one or a subset of these deviations becomes evident. In the case of clinical resistance, because Pgp has only been detected in tumours taken from a fraction of refractory e~ncer patients, it seems likely that other mechanisms : resistance may play a prominent role and results of efforts to identify these may provide a spectrum of useful 'markers' for predicting clinical response to, or for monitoring the effectiveness of, therapy. Of particular interest are the non-Pgp-associated proteins e.g. HSP-27 [154] and P190 [61]. In view of the variable detectability of Pgp, for example, the possible false positives with C219 monoclonal [191], and large differences in positivity reported using a range of anti-Pgp monoclonals [192,193], not only are a range of anti-Pgp

probes necessary, but also a range of 'markers' need to be investigated if accurate predictions of the response of a patient to therapy is to be attempted, in addition, it is necessary to expand our knowledge of the plethora of changes that may occur in resistant cells in order to fully understand the drug resistance phenomenon. The function of many of these proteins remains to be defined, although a number, including Pgp, clearly are involved in drug efflux and detoxifieation. In only a few defined cases, has a causal relationship between drug resistance and protein overexpression been firmly established, but in many cases the appropriate transfection studies have not yet been carried out. In terms of utilizing the presence or absence of these various proteins as ~mtential 'markers' of clinical drug resistance it is essential to remember that resistance is associated frequemly with modified expression of these molecules, which may also be present in 'normal' tissue carJ),ing out a necessary 'normal' function. For exampie, Pgp has recently been observed to have an ATPdependent, chloride channel activity [194] which may prove to be its physiological function in cells. This qualification therefore means that accurate quantitation of gene or protein expression is necessary and that data are critically evaluated when attempting to detect drug resistant tumour cells. One of the shortcomings of many of the studies outlined in this review is the highly ~elective cell lines which have been examined in terms of resistance and protein levels for example the MCF-7 breast carcinoma, KB epidermoid carcinoma and CHO cell lines. These cell lines 'behave' well in culture and although they have contributed considerably to our knowledge of MDR to date, they may not accurately reflect the events which result in expression of drug resistance. Clearly, future studies should be expanded to include a broader spectrum of turnout cell lines and tissues. Although sometimes difficult to work with, tumour biopsy material obtained from patients in all the various stages of disease should be studied to determine the relevance of protein alterations to clinical drug resistance. In addition, a longitudinal study of cell lines throughout serial drug selection should reveal more information about the sequence of events which may be involved in the induction or selection for MDR and thereby provide information about possible prevention or modulation of these events in the clinic during therapy. The number of different protein changes which have been observed in cells which are resistant to drugs of the MDR phenotype, demonstrates that resistance to multiple drugs can be expressed in many varied ways in individual cells. In addition to Pgp, ma~y other pro° teins can be changed, which may in turn perturb many 'normal' cellular responses, as well as modi~ their responses to cytotoxic agents.

,~knowled~ments We thank Richard Whelan and Louise Hosking for helpful discussions during the preparation of this review and Dr. John Darling for his critical reading of the manuscript,

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