Breast CancerResearch and Treatment,4, 89-94 (1984) © 1984, MartinusNijhoffPublishers. Boston. Printed in the Netherlands

6th Annual San Antonio Breast Cancer Symposium

Multidrug resistance V. Ling, J. Gerlach, and N. Kartner Department of Medical Biophysics, University of Toronto and the Ontario Cancer Institute, 500 Sherbourne Street, Toronto, Ontario, Canada M4X1K9 Keywords: collateral sensitivity, combination chemotherapy, cross-resistance, mutation, P-glycoprotein

Summary Mutations resulting in a complex phenotype of cross-resistance and collateral-sensitivity are frequently observed in mammalian cell lines. A cell surface 170 000 dalton glycoprotein (P-glycoprotein) has been identified to be intimately associated with this multidrug resistance phenotype. We speculate that such mutations also occur in advanced cancers and play a major role in contributing to the non-response to combination chemotherapy. In this context, P-glycoprotein may be a useful molecular marker for diagnostic and therapeutic applications.

Introduction Failure in chemotherapeutic management of advanced metastatic cancers is a common and frustrating clinical problem. This non-response is often wide-ranging, extending to unrelated drugs, or combination of drugs, with which the patient had not been treated previously. The recent discovery that mutations resulting in multidrug resistance are common in tumor cell lines provides new insights into this problem (1-4). This finding supports the currently held view that tumor cells acquiring drug resistance mutations could play a major role in the development of a non-responsive disease (see Refs. 1, 5-7 for reviews). In addition it provides a rational explanation for the non-response to combination chemotherapy so frequently encountered in cancer therapy. Thus it is postulated that multidrug resistance mutations occur spontaneously in the malignant stem cell population. These variant cells, initially present in low numbers, would have a selective growth advantage during chemo-

therapeutic treatment and would eventually dominate, contributing to a therapy-resistant disease. The objective of the present article is to review aspects of the multidrug resistance phenotype, and to speculate on implications of this phenotype for cancer chemotherapy.

The multidrug resistance phenotype The multidrug resistance phenotype has been studied most extensively in tumor cell lines in culture (1). In all cases, a pleiotropic expression of crossresistance to structurally and functionally unrelated drugs is observed, and in some instances increased sensitivity (collateral sensitivity) is also found. Several features of this phenotype are illustrated in Table 1. 1. As mentioned above, there are no obvious structural or functional relationships among the drugs to which the mutant cells are resistant. Correla-

90 Table 1. Relative drug resistance.a

Drugs

Colchicine Daunorubicin Vinblastine Vincristine Adriamycin Puromycin Emetine 1-Dehydrotestosterone Acronycine Triton X:I00

Cell lines CHaC5

DNRR51

CEM/ VLB100

180 76 30 NA 25 - 100 29

25 41 22 NA - 30 38 11

19 19 70 - 500 NA 39 9

~

~ -

0.1 < 0.06 0.3

1 0.6 NA

1 1 NA

a Relative resistance was determined by the concentration of drug required to inhibit growth in the drug-resistant line divided by that required for the parental line (9). Cross-resistance is denoted by a value greater than i and collateral sensitivity by a value less than 1. N A - not analyzed. The drug-sensitive parent of CHaC5 and DNRa51 is the C H O cell line AuxBl (22,3) and the drug-sensitive parent of CEM/VLB100 is the h u m a n lymphoid line C C R F - C E M (25). CHRC5 was selected for resistance to colchicine, DNRR51 to daunorubicin, and CEM/VLB~00 to vinblastine. It should be noted that the CEM/VLB100 line used in this study is a subclone derived from the original resistant population (25). This may account for the overall lower relative resistance values recorded here compared to those reported by Beck (10).

tions of cellular resistance with molecular size (8) or hydrophobicity (9) of the drugs have been observed, but these correlations are rather limited. Therefore, it is not possible at this point to predict a priori what classes of drugs might or might not be involved in the multidrug resistance of a particular cell line. 2. The cross-resistance pattern could be quite different among resistant isolates selected with different drugs. For example, the CHO cell lines CHgC5 and DNRR51 were isolated from the same drug-sensitive parental line but with colchicine and daunorubicin, respectively. As seen in Table 1, CHRC5 is most resistant to colchicine and daunorubicin ranked third, among the drugs used, while DNRR51 is most resistant to daunorubicin and colchicine ranked fourth. In general, it appears that the mutant line is most

resistant to the selecting drug, although exceptions are frequently observed. This is illustrated by the human line CEM/VLB100 selected for resistance to vinblastine (Table 1). In this case, CEM/VLB100 is far more resistant to vincristine, a drug to which it had not been exposed previously, than to vinblastine. . Collateral sensitivity to some agents is observed. The basis for this response is not fully understood and what compounds might be involved cannot be predicted. The complexity of this response is illustrated when the line CHRC5 is tested against a series of closely related detergents of the Triton X family. Degrees of crossresistance or collateral sensitivity are observed depending on the number of ethylene oxide residues in the side chain of the detergent molecule involved (9). Thus, CHRC5 is cross-resistant to Triton X-165 with 16 ethylene oxide residues but is collaterally sensitive to Triton X-102 with 12-13 residues. The picture that emerges from consideration of the properties of the multidrug resistance phenotype is that it is highly complex and enigmatic. On the one hand, resistance to a very wide range of structurally different drugs is observed and on the other hand, very different responses (cross-resistance and collateral sensitivity) are observed to two very closely related detergents. Mutations resulting in multidrug resistance are frequently observed in lines isolated for resistance to a single agent and with a single-step selection procedure. In the Chinese hamster ovary (CHO) tissue culture cell system, multidrug resistance is observed in clonal lines isolated for stable resistance to drugs such as colchicine, daunorubicin, adriamycin, taxol, puromycin, vinblastine, podophyllotoxin, cytochalasin B, actinomycin D and others. They occur at frequencies ranging from 10`5 to 10-7. Similar findings have been described in a variety of cell lines (1, 4, 8, 10). The question of whether or not multidrug resistance mutations occur in human cancers is obviously an important one. Malignant cells carrying such a mutation could be refractile to a combination of unrelated drugs. This would not be the case

91 if the cells were resistant to single agents. A sobering consideration is that if the frequency of multidrug resistance mutations observed in tumor cell lines is representative of that in human cancers, then most human cancers would contain some multidrug-resistant cells by the time they are clinically detectable.

Genetic studies

Genetic analyses were undertaken to determine if aspects of the multidrug resistance phenotype can be delineated. Findings from such studies in the CHO cell system indicate that many features of the multidrug resistance phenotype are co-expressed under a variety of conditions (1, 11-14). For example, extensive cross-resistance is observed in independent, single-step, clonal isolates obtained in the absence of mutagen treatment, in cell:cell hybrids made between sensitive and resistant lines, and in mouse cells transfected with genomic D N A from multidrug-resistant hamster lines. In revertant lines, isolation for sensitivity to one compound results in the reversion of the whole spectrum of drugs involved. These studies indicate that multidrug resistance has a genetic basis, and that the multiplicity of response to different drugs is likely due to a single genetic event. The fact that multidrug resistance is dominantly expressed in cell hybrids further implies that such mutations can be expressed in a variety of cells, including polyploid malignant cells. Clues as to the nature of the genetic alteration(s) associated with multidrug resistance were obtained from karyotypic analysis. In a number of cases, karyotypic abnormalities such as double-minute chromosomes (DMs) or chromosomes with homogeneously staining regions (HSRs) were observed in mutant lines expressing multidrug resistance (15-19). These karyotypic markers are usually associated with gene amplification (20, 21). Recently we have demonstrated in a mouse cell system that increased number of DMs was associated with increased multidrug resistance, and that in revertant cells, the number of DMs was concordantly reduced (16). Thus it appears that gene

amplification is a likely genetic basis for the multidrug resistance phenotype.

Molecular studies Previous studies indicated that the uptake of radioactively labeled drugs was reduced in the multidrug-resistant lines and that the degree of reduced uptake correlated with the increased resistance (3, 22). Therefore it appears that the molecular alteration(s) responsible for multidrug resistance is likely to be localized at the plasma membrane. A membrane mutation resulting in multidrug resistance is compatible with the concept of the dynamic nature of mammalian cell plasma membranes (11). The pleiotropy observed in the multidrug resistance phenotype presumably could reflect a perturbation in the multimolecular interactions required for many membrane functions. Comparison of plasma membranes prepared from drug-sensitive and drug-resistant cells indicated that the resistant cells contain an increased amount of a 170 000 dalton glycoprotein (3, 14, 23, 24). We have called this the P-glycoprotein, as it is associated with the pleiotropy of the multidrug resistance phenotype. Antisera raised against membranes from drug-resistant cells were used to further confirm that muttidrug-resistant cells possess an altered surface membrane. Such an antiserum absorbed with drug-sensitive cells stains the surface of drug-resistant cells specifically (Fig. 1). Moreover the intensity of staining correlates with the degree of resistance in the colchicine-resistant CHO cell lines (data not shown). Using the same antiserum in an immunoblot (Western blot) procedure, an increased staining of the 170 000 dalton P-glycoprotein component is observed in the mutant line (Fig. 2A). This staining is not significantly diminished by extensive absorption of the antiserum with membrane proteins from drug-sensitive cells (Fig. 2B); moreover, this absorbed antiserum is almost monospecific for P-glycoprotein. Use of this absorbed antiserum to examine membrane components in a variety of cell lines expressing the multidrug resistance phenotype revealed that, in each case, an increased staining of a compo-

92

Fig. 1. Immunofluorescentdetectionof multidrug-resistantCHO cells. (A) Mixtureof sensitive(AuxB1) and resistant (CHRC5)CHO cells photographedusingphase contrastmicroscopy.(B) The same fieldusingfluorescencemicroscopyrevealingresistantcellsheavily labeled with an antibody specificfor multidrug-resistantCHO cells. The antibody was produced by injecting rabbits with plasma membranes from CHRC5cells. It was then extensivelyabsorbed with glutaraldehyde-fixed,sensitive(AuxB1) cells. Bindingto azidepoisoned cells was detected with a fluorescein-labeled,swine antibodyto rabbit immunoglobulins.

nent in the 170 000 dalton region was observed (4). The identical molecular weight and immunological cross-reactivity observed in mammalian cells from different species indicated that P-glycoprotein is a highly conserved membrane component. In addition, it demonstrates that increased expression of P-glycoprotein is a good indicator of the multidrug resistance phenotype. The intimate association of P-glycoprotein with multidrug resistance is also demonstrated by other studies. The level of P-glycoprotein expression correlates with the degree of drug resistance (1-4). P-glycoprotein expression is greatly reduced in revertant cells (4, 24) and is expressed in cells transfected with DNA from multidrug-resistant cells (12). Furthermore, increased expression of P-glycoprotein correlates with increased numbers of DMs and increased multidrug resistance in a mouse line (16). We speculate, therefore, that gene amplification results in over-expression of P-glycoprotein and expression of the multidrug resistance phenotype. Whether or not over-expression of P-glycopro-

tein alone is sufficient to mediate the multidrug resistance phenotype is still not known. Molecular cloning of the P-glycoprotein gene and transfer of the purified gene into drug-sensitive cells to test for function may provide a definitive answer to this question. Different changes associated with multidrug resistance have been reported (10, 15, 19). How these changes relate to over-expression of P-glycoprotein is not known. At present, P-glycoprotein is the best molecular indicator of the multidrug resistance phenotype.

Concluding remarks The questions of whether or not multidrug resistance mutations do in fact occur in human cancers, and whether they play a role in the development of therapy-resistant disease are important ones with wide implications. For example, if chemotherapy with the current repertoire of antineoplastic agents is indeed limited by the occurrence of multidrug resistance mutations, then new drugs will be re-

93 quired. A s n o t e d above, the multidrug resistance p h e n o t y p e is wide-ranging. Its particular p a t t e r n of resistance m a y be affected by a variety of factors and c a n n o t be predicted a priori. Clinically useful drugs not affected by this p h e n o t y p e or those in the category of being collaterally sensitive will n e e d to be identified. A t present, this has b e e n explored by empirical m e a n s in defined m o d e l systems. E x t e n sion of this w o r k to h u m a n cancers poses an important challenge. Despite the complexity of the multidrug resistance p h e n o t y p e it is gratifying that a constant feature of this p h e n o t y p e is the expression of a

highly-conserved cell-surface antigen, the P-glycoprotein. It is likely that this m a r k e r will facilitate investigation of the role of multidrug resistance in a d v a n c e d malignancies. R e c e n t results o b t a i n e d in our l a b o r a t o r y are relevant. W e o b s e r v e d by imm u n o b l o t analysis, that cells o b t a i n e d directly f r o m ascites fluid of some non-responsive ovarian patients contained significant a m o u n t s of P-glycoprotein (Bell, Gerlach, Kartner, E v e r n d e n - P o r elle, Buick, and Ling, unpublished observation). T h e implications of this finding will n e e d to be further determined, h o w e v e r , it indicates the potential of P-glycoprotein as a m a r k e r that m a y be exploited for diagnostic and therapeutic applications.

Acknowledgements This w o r k was s u p p o r t e d by the National C a n c e r Institute of C a n a d a , the Medical R e s e a r c h Council of C a n a d a , and the O n t a r i o C a n c e r T r e a t m e n t and Research Foundation.

References

Fig. 2. Immunoblots (Western blots) of plasma membranes. Membrane proteins (50 gg per lane) from colchicine-resistant CHO cells, CHRC5 (lane a) or from drug-sensitive parental line AuxB1 (lane b) were fractionated by gel electrophoresis and replica electroblotted onto nitrocellulose paper. The 'Western' blots were overlaid with rabbit antiserum (raised against CHRC5plasma membrane), washed, probed with I25I-labeledS. aureus protein A, washed, and exposed to X-ray film as previously described (3). In blot A, unabsorbed antiserum was used, and in blot B, antiserum preabsorbed with membrane proteins from AuxB1 cells was used (3). Preimmune rabbit serum yielded no labeled components under the same conditions. The P-glycoprotein region and approximate molecular sizes in kilodaltons are indicated.

1. Ling V: Genetic basis of drug resistance in mammalian cells. In Bruchovsky N, Goldie JH (eds), Drug and Hormone Resistance in Neoplasia. CRC Press, Miami, Vol I, pp 1-19, 1982 2. Ling V, Kartner N, Sudo T, Siminovitch L, Riordan JR: Multidrug resistance phenotype in Chinese hamster ovary cells. Cancer Treat Rep 67: 869-874, 1983 3. Kartner N, Shales M, Riordan JR, Ling V: Daunorubicinresistant Chinese hamster ovary cells expressing multidrug resistance and a cell surface P-glycoprotein. Cancer Res 43: 4413-4419, 1983 4. Kartner N, Riordan JR, Ling V: Cell surface P-glycoprotein associated with multidrug resistance in mammalian cell lines. Science 221: 1285-1288, 1983 5. Goldie JH, Coldman AJ: Clinical implications of the phenomenon of drug resistance. In Bruchovsky N, Goldie JH (eds), Drug and Hormone Resistance in Neoplasia. CRC Press, Florida, 1982, Vol II, pp 111-127 6. De Vita, Jr V: The relationship between tumor mass and resistance to chemotherapy. Cancer 51: 1209-1220, 1983 7. Schabel, Jr FM, Skipper HE, Trader MW, Laster Jr WR, Griswold Jr DP, Corbett TH: Establishment of cross-resistance profiles for new agents. Cancer Treat Rep 67: 905922, 1983

94 8. Biedler JL, Peterson RHF: Altered plasma membrane glycoconjugates of Chinese hamster cells with acquired resistance to actinomycin D, daunorubicin, and vincrisfine. In Sartorelli AC, Lazo JS, Bertino JR (eds), Molecular Actions and Targets for Cancer Chemotherapeutic Agents. Academic Press, New York, 1981, pp 453-450 9. Bech-Hansen NT, Till JE, Ling V: Pleiotropic phenotype of colchicine-resistant CHO cells: cross-resistance and collateral sensitivity. J Cell Physiol 88: 23-31, 1976 10. Beck WT: Vinca alkaloid-resistant phenotype in cultured human leukemic lymphoblasts. Cancer Treat Rep 67: 875882, 1983 11. Baker RM, Ling V: Membrane mutants of mammalian cells in culture. In Korn ED (ed), Vol 9, Methods in Membrane Biology. Plenum Publishing, New York, 1978, pp 337-384 12. Debenham PG, Kartner N, Siminovitch L, Riordan JR, Ling V: DNA-mediated transfer of multiple drug resistance and plasma membrane P-glycoprotein expression. Mol Cell Biol 2: 881-889, 1982 13. Ling V: Drug resistance and membrane alteration in mutants of mammalian cells. Can J Genet Cytol 17: 503515, 1975 14. Ling V, Baker RM: Dominance of colchicine resistance in hybrid CHO cells. Somatic Cell Genet 4: 193-200, 1978 15. Biedler JL, Chang T, Meyers MB, Peterson RHF, Spengler BA: Drug resistance in Chinese hamster lung and mouse tumor cells. Cancer Treat Rep 67: 859-867, 1983 16. Robertson SM, Ling V, Stanners CP: Co-amplification of double minute chromosomes, multidrug-resistance, and cell surface P-glycoprotein in DNA-mediated transfor-

mants of mouse cells. Mol Cell Biol (in press) 17. Baskin F~ Rosenberg RN, Dev V: Correlation of doubleminute chromosomes with unstable cross-resistance in uptake mutants of neuroblastoma cells. Proc Natl Acad Sci USA 78: 3654-3658, 1981 18. Kopnin BP: Specific karyotypic alterations in colchicineresistant cells. Cytogenet Cell Genet 30: 11-14, 1981 19. Kuo T, Pathak S, Ramagli L, Rodiguez L, Hsu TC: Vincristine-resistant Chinese hamster ovary ce}ls. In Schimke RT (ed), Gene Amplification. Cold Spring Harbor Laboratory, CSH, New York, 1982, pp 53-57 20. Cowell JK: Double minutes and homogeneously staining regions: Gene amplification in mammalian cells. Ann Rev Genet 16: 21-59, 1982 21. Schimke RT (ed): Gene Amplification. Cold Spring Harbor, New York, 1982 22. Ling V, Thompson LH: Reduced permeability in CHO cells as a mechanism of resistance to colchicine. J Cell Physiol 83: 103-116, 1974 23. Juliano RL, Ling V: A surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants. Biochim Biophys Acta 455: 152-162, 1976 24. Riordan JR, Ling V: Purification of P-glycoprotein from plasma membrane vesicles of Chinese hamster ovary cell mutants with reduced colchicine permeability. J Biol Chem 254: 12701-12705, 1979 25. Beck WT, Mueller TJ, Tanzer LR: Altered surface membrane glycoproteins in Vinca alkaloid-resistant human leukemic lymphoblasts. Cancer Res 39: 2070-2076, 1979