British Journal of Rheumatology 1996;35:43(M35

EXPRESSION OF THE MULTIDRUG RESISTANCE GLYCOPROTEIN 170 IN THE PERIPHERAL BLOOD LYMPHOCYTES OF RHEUMATOID ARTHRITIS PATIENTS. THE PERCENTAGE OF LYMPHOCYTES EXPRESSING GLYCOPROTEIN 170 IS INCREASED IN PATIENTS TREATED WITH PREDNISOLONE J. F. MAELLEFERT, M. MAYNADDE,* J. G. TEBEB,t S. AHO,J P. WALKER,§ C. CHATARD, V. DULIEU, M. BOUVIER,t P. M. CARLI* and C. TAVERNIER Service de Rhumatologie and *Laboratoire d'Hematologie, CHU Dijon, ^Service de Rhumatologie, Centre Hospitalier Lyon-Sud, %Service d' Epidimiologie et Hygiine Hospitaliere, CHU Dijon and %Laboratoire de Biophysique, Faculti de Midecine, Dijon, France SUMMARY The objective was to evaluate the expression of the multidrug resistance P-glycoprotein (P-gp) in peripheral blood lymphocytes (PBL) of patients with rheumatoid arthritis (RA). PBL from 68 RA patients and 44 controls were evaluated. RA patients had a mean disease duration of 10.7 yr, with a mean number of past resistances to DMARDs of 0.82, and were treated with NSAIDs (n = 34), DMARDs (n = 25) and prednisolone (n = 40). Fluorescence flow cytometry was used to assess P-gp membrane expression on PBL. In the RA group, the percentage of PBL expressing P-gp was Higher in patients treated with prednisolone than in other patients [mean ± s.D.: 10.7 ± 15.8% vs 3.3 ± 7.6%, P < 0.03, Student] and was not related to other therapies, age, sex, RA duration, number of past resistances to DMARDs, activity, ESR, CRP. The percentage of PBL expressing P-gp did not differ in RA and control groups, but was higher in the prednisolone-treated RA patients than in controls. Prednisolone could induce a rise in the percentage of PBL expressing P-gp. On the contrary, patients with a high percentage of PBL expressing P-gp could be more resistant to DMARDs and need prednisolone earlier. Further studies are needed to address this question and to evaluate the potential implication of P-gp in drug resistance in RA. KBY WORDS:

Rheumatoid arthritis, Treatment, MDR, Drug, Resistance, Glucocorticoids.

efflux pump that exports MDR-related drugs out of the cells, lowering their intracellular concentration [13]. The MDR-related drugs can enhance expression of the mdrl gene and P-gp [14]. Reversal of drug resistance has been obtained by different agents which usually act as competitive inhibitors. These so-called resistancemodifying agents include verapamil, quinidine and cyclosporin [15-17]. P-gp is absent in most normal tissues, but is present in adrenal cortex, kidney, intestine, liver and pancreas [18]. The physiological function of P-gp in normal tissues is still not fully understood. P-gp could be involved in cellular detoxication and excretion of cellular metabolites [18]. A number of disease-modifying anti-rheumatic drugs (DMARDs) have been shown to be more effective than placebo in the management of rheumatoid arthritis (RA). However, most courses of DMARDs, except methotrexate, are discontinued after 2 or 3 yr, because of toxicity, lack of efficacy or escape from control [19-21]. Recently, Jorgensen et al. [22] have reported that the mdrl gene and P-gp were expressed in the synovia] tissue of patients with RA. These authors suggested that the MDR phenomenon could be implicated in the acquired resistance to DMARDs in RA. In order to investigate this hypothesis, we have evaluated in a preliminary study the percentage of peripheral blood lymphocytes (PBL) expressing P-gp in RA patients, and have searched for correlations between this percentage and different variables,

drug resistance remains one of the most important clinical obstacles in the treatment of certain cancers. One particular mechanism is that characterized by the multidrug resistance phenotype (MDR). MDR is a phenomenon in which cells develop cross-resistance to many agents, such as anthracyclines, vinca alkaloids and colchicine [1-3]. The MDR-related drugs have little similarity in their chemical structure or mechanism of action, but they are all lipophilic compounds derived from various natural products [1-3]. MDR is thought to mostly result from an overexpression of the mdrl gene [4, 5]. This gene is a constituent of a small gene family in mammals. Three mdr genes in rodents [6] have been isolated. In humans, two mdr genes have been identified [7], but functional analysis demonstrated that only one {mdrl) can confer the MDR phenotype to drug-sensitive cells [4]. Moreover, this gene family exists throughout the evolutionary paradigm, since MDR-like sequences have been identified in bacteria [6], mosquito parasites [8], plants [9] and Drosophila [10]. In humans, the mdrl gene encodes a 170 kDa transmembrane glycoprotein (P-gp) composed of 1280 amino acids with 12 transmembrane domains [11, 12]. P-gp acts as an energy-dependent unidirectional drug ACQUIRED

Submitted 4 July 1995; revised version accepted 9 November 1995. Correspondence to: J. F. Maillefert, Service de Rhumatologie, Hdpital General, 3 rue du Fb Raines, 21 000 Dijon, France.

© 1996 British Society for Rheumatology 430

MAILLEFERT ET AL.\ EXPRESSION OF P-GLYCOPROTEIN IN RA including clinical activity, number of past resistances to DMARDs and current therapy. PATIENTS AND METHODS Patients Sixty-eight patients hospitalized for RA were included. All patients fulfilled the ARA revised criteria for diagnosis of RA [23]. There were 48 females and 20 males, of mean age 62.8 ± 12.9 (s.D.) yr. The mean ± S.D. disease duration was 10.7 ± 10.6 yr. The mean ± s.D. number of previous resistances to DMARDs was 0.82 ±1.1. Forty-six patients were seropositive for rheumatoid factors, 29 had active RA (active disease was defined by at least three of the following: > 9 painful or tender joints, ^ 6 swollen joints, ^45min of morning stiffness, St 28 mm/h ESR). The mean ± S.D. ESR and CRP were, respectively, 41.1 ± 31.2 mm/h and 39.4 ± 44 mg/1. All patients were treated with analgesics (paracetamol and/or dextropropoxyphen and/or codeine). In addition, the therapy included non-steroidal antiinflammatory drugs (NSAIDs) (n = 34), prednisolone (n = 40; mean ± s.D. daily dose = 14.7 ± 9.7 mg; mean ± S.D. duration of therapy = 62.7 ± 77.6 months), DMARDs (n = 25), including methotrexate (n = 11; mean ± S.D. weekly dose = 8.41 ± 3 mg; mean ± S.D. duration of therapy = 30.8 ± 24.9 months), tiopronin (n = 4), hydroxychloroquine (n = 4), gold salts (n = 4), sulphasalazine (n = 1) and D-penicillamine (n = 1). The combinations of these are summarized in Table I. As controls, we have studied PBL of 44 patients hospitalized for sciatica, acute back pain or osteoarthritis. They were 30 females and 14 males, of mean age 61.2 ± 18yr. They were not treated with conventional RA therapies, except for NSAID and analgesics (paracetamol and/or dextropropoxyphen and/or codeine). Determination of the percentage of PBL expressing P-gp The percentage of PBL expressing P-gp was determined using indirect immunofluorescence. We used monoclonal antibody MRK16 (Immunotech, France) which recognizes an extracellular epitope of P-gp. Mononuclear cells were separated from peripheral blood by density-gradient centrifugation

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(d = 1.077) (Eurobio, France) and washed once in Hank's buffer (Sigma) and twice in Hank's buffer containing 1% (w/v) bovine serum albumin (BSA) (Sigma). Cells were fixed for 30min at 4°C in phosphate-buffered saline without Ca2+ and Mg2* (PBS) (Eurobio) containing 1% (w/v) paraformaldehyde. They were then washed in PBS containing 1% (w/v) BSA (PBS-1% BSA) and incubated for 30 min at 4°C with 2.5 ng of MRK16/500 000 cells in 200/il of PBS-1% BSA. After two washes in PBS-1% BSA, the cells were incubated with a goat anti-mouse fluoresceinconjugated polyclonal F(ab)j fraction (Silenus, Australia). Cells were then washed twice in PBS-1% BSA, resuspended in PBS and stored at 4°C in the dark until flow cytometric analysis. A non-specific fluorescence control was performed using purified mouse monoclonal IgG2a (Coulter, France). Flow cytometry analysis was performed on a FACScan flow cytometer (Becton Dickinson, Montain View, CA, USA). The excitation source was an argon ion laser emitting a 488 nm beam at 15 mW. The red and the greenfluorescenceswere collected, respectively, through a 585/42 and 530/30 nm band-pass filters. Five thousand events were analysed with Lysis I software (Becton Dickinson, Montain View, CA, USA). The fluorescences were measured on a four decade logarithmic scale. A 1 % fluorescent cell cut-off was determined on the isotypic control and used for the MRK16 assay (Fig. 1). The results were expressed as the percentage of fluorescent PBL. Statistical analysis The mean values were compared using the Student test if normality and homoscedasticity were met, and by Wilcoxon test if not. The homoscedasticity was assessed by the Bartlett test. The qualitative variables were compared using the %2 test. The Pearson r was used to test linear associations between two quantitative variables. In the RA group, the Student test was used to test the relationships between the percentage of PBL P-gp-I- and gender, seropositivity for rheumatoid factors, treatment with NSAID, treatment with prednisolone, and to compare age, number of past resistances to DMARDs and CRP between patients treated and not treated with prednisolone. The

TABLE I Current combination of therapies in RA patients* Current therapy Prednijolone Prednisolone Prednisolone Prednisolone Prednisolone Prednisolone Prednisolone Prednisolone

+ + + + + + +

NSAID NSAID + MTX MTX tiopronin hydroxychloroquine gold salts NSAID + D-penicillamine

•All patients treated with analgesics.

Patients (no.) 21 6 4 3 2 2 1 1

Current therapy Tiopronin NSAID MTX NSAID + MTX NSAID + tiopronin NSAID + hydroxychloroquine NSAID + gold salts NSAID + sulphasalazine Analgesics alone

Patients (no.) 1 14 2 2 1 2 3 1 2

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8

(A)

ISOTYPIC NEGATIVE CONTROL Toui- seee X ToUl- 1.11

(B)

16 LABELLING

(C)

-ES-

s* seee 4359 ToUl- 87.18

-Si 38

ScatUr

|

FK3- 1 •—Example offlowcytometric data. (A) Representation of PBL after separation by density gradient centrifugation on the FSC/SCC double scattergram. (B) Histogram of fluorescence of the non-specific negative control. (Q Histogram of fluorescence of PBL labelled with MRK16 monoclonal antibody. In this example, 87% of PBL express P-gp.

Wilcoxon test was used to test the relationships between the percentage of PBL P-gp-t- and RA activity, treatment with methotrexate, treatment with other DMARDs, and to compare ESR and the duration of RA between patients treated and not treated with prednisolone. The Pearson r test was used to test the relationships between the percentage of PBL P-gp+ and age, duration of RA, number of past resistances to DMARDs, ESR and CRP. Additionally, the Pearson r test was used in the subgroup of patients treated with prednisolone to test the relationships between the percentage of PBL P-gp+ and the duration and daily dosage of steroids. Finally, the x2 test was used to compare gender and RA activity between patients treated and not treated with prednisolone. In the control group, the Pearson r test was used to test the relationships between the percentage of PBL Pgp + and age, and the x2 test was used to test the relationships between the percentage of PBL P-gp+ and gender. The comparison between the RA and the control group used the Wilcoxon test (age and percentage of PBL P-gp+) and the x2 test (gender). The comparison between the percentage of PBL P-gp+ in control and RA patients treated with prednisolone used the Wilcoxon test. A multivariate analysis using a multiregression analysis was performed in the RA group. The

dependent variable was the rate of PBL expressing the P-gp. The independent variables were all those that were related to the dependent variable with P < 0.1 in univariate analysis. The qualitative variables were coded 0-1 (dummy variables). Statistical significance was defined as P < 0.05. RESULTS RA group Age and gender. The percentage of PBL expressing P-gp (P-gp+) was not related to gender (mean ± s.D. = 12.1 ± 18.6% in men; 5.8 ± 10.5% in women; P < 0.09) and age (P < 0.8). RA history and current assessment. The percentage of PBL P-gp+ was not related to the duration of RA (P = 0.11). The percentage of PBL P-gp+ was higher in the prednisolone-treated RA patients group than in the control group (mean ± S.D. = 10.7 ± 15.8% in the

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prednisolone-treated RA patients vs 4.91 ± 9.9% in controls; P < 0.02). DISCUSSION We have found a higher percentage of PBL P-gp + in RA patients treated with prednisolone than in other RA patients. This high percentage does not seem to be a part of a generalized abnormality in phenotype in RA since RA patients and controls did not differ in the percentage of PBL P-gp+, neither prednisolone non-treated RA patients and controls. Although these results need to be confirmed by an evaluation of mdrl gene expression, and of P-gp functional activity, several hypotheses can already be put forward to explain them. First, prednisolone could have induced an enhancement of the percentage of PBL P-gp -I-. Several observations suggest interactions between P-gp and steroids, and thus support this hypothesis. P-gp is expressed in normal human adrenal cortex [24], where high levels of steroids are synthesized. In the pregnant mouse, P-gp and mdrlb gene mRNAs are found at high levels in the uterus [25]. Various steroids, such as hydrocortisone, dexamethasone, deoxycorticosterone, corticosterone, testosterone and especially progesterone, inhibit [3H]azidopine labelling of P-gp in MDR cells and in endometrium of gravid uterus [26]. Deoxycorticosterone, corticosterone, testosterone, and especially progesterone, enhance vinblastine accumulation in MDR cells [26]. Progesterone has a high affinity for human P-gp, but is not transported by this pump, whereas cortisol and dexamethasone have a lower affinity for P-gp, but appear to be substrates for transport by P-gp [27]. Accumulation of hydrocortisone is reduced in MDR pituitary tumour cells and is restored after blockade of P-gp [28]. Steroid accumulation and sensitivity are reduced in variants of murine thymoma W7 cells which highly express P-gp. Intracellular accumulation and sensitivity of the drugs can be restored by P-gp blockade with verapamil [29]. A putative glucocorticoid-responsive element has been identified in the promoter of the mouse mdrlb gene [30]. The functional MDR genes (mouse mdrla and mdrlb and human mdrl) can be transcriptionally activated by dexamethasone in cells of hepatoma origin in a time- and concentration-dependent manner [31]. In mouse Yl adrenal cells, the adrenocorticotrophininduced increase in steroid secretion enhances the mdrlb mRNA levels. However, the ability of P-gp to export daunomycin and vinblastine decreases when the cells are engaged in steroid secretion [32]. All these observations suggest that some steroids could be physiological substrates for P-gp, and that steroids interfere with the mdr system, modulating mdr gene expression and acting as competitive inhibitors for P-gp drug transport function. Another explanation for the higher percentage of PBL P-gp+ in RA patients treated by prednisolone is that patients with a high percentage of PBL P-gp + could be more resistant to DMARDs and need prednisolone earlier. Our results do not support this

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hypothesis, since we did not find a correlation between the percentage of PBL P-gp+ and past resistances to DMARDs or current treatment with such therapies. On the contrary, the percentage of PBL P-gp + tended to be lower in patients treated with DMARDs. However, our population was heterogeneous and included a small number of patients treated with each DMARD, so we could not test them separately, except for methotrexate. Such resistances could be observed only in subgroups of patients, and could concern only certain DMARDs. As a consequence, our population did not allow us to correctly study the relationship between DMARDs and the percentage of PBL P-gp+. Interestingly, Jorgensen et al. [33] have recently shown that sulphasalazine, but not methotrexate, increases P-gp membrane expression on PBL in vitro. We did not find any correlation between methotrexate therapy and P-gp expression in our patients. These results, and the fact that methotrexate (a hydrophilic drug) is usually not considered to interact with the mdr system in cancerology [2], suggest that this drug does not interact with P-gp in RA patients. Further prospective studies are needed to confirm this and to study the relationship between other DMARDs and P-gp. Finally, a confounding variable might explain our results. RA patients treated with prednisolone were older and tended to have a longer duration of disease than the other RA patients. However, the percentage of PBL P-gp-t- was not related to age in the RA population or in the control group. Similarly, no correlation was found in the RA group between the percentage of PBL P-gp + and the duration of disease. Elsewhere, the activation of PBL could be of importance in the percentage of PBL P-gp + , but this percentage was not correlated with the activity of the disease, the ESR and the CRP. Moreover, these variables were not related to therapy with prednisolone. Potential therapeutic implications The discovery of the mdr system and its potential role in multidrug resistance in human cancer has led to therapeutic trials using resistance-modifying agents (RMA) in conjunction with cytotoxic drugs. Numerous protocols have been studied in various types of neoplasia, including haematological malignancies in which promising results have been obtained [34-36]. The observation of a higher percentage of PBL P-gp -I- in RA patients treated with prednisolone raises some interesting questions. In the hypothesis that P-gp extrudes steroids out of the cell, the use of RM As could diminish the cellular efflux and enhance the intracellular accumulation of corticosteroids. This phenomenon might not be ubiquitous and concern only some tissues. As a consequence, the use of RMAs could permit one to reduce the daily dosage and some of the systemic side-effects of corticosteroids without modification of the clinical efficacy. The relationship between the mdr system and DMARDs needs to be explored. If this system is

implicated in some resistances to DMARDs, a use of RMAs could reverse some of these. Finally, the interactions between corticosteroids, DMARDs and P-gp have to be explored. Steroids could decrease the efficiency of some DMARDs by reducing their intracellular accumulation through an enhancement of the expression of P-gp. On the contrary, steroids could enhance the intracellular accumulation of some DMARDs by acting as competitive inhibitors of P-gp. On the other hand, some DMARDs could modify the cellular accumulation of corticosteroids through similar phenomena. All these interesting hypotheses have to be explored in further studies. Exploration of the relationship between the mdr system and the therapeutic regimens in RA could lead to a new approach in the management of DMARDs and corticosteroids. REFERENCES

1. McClean S, Hill BT. An overview of membrane, cytosolic and nuclear proteins associated with the expression of resistance to multiple drugs in vitro. Biochim Biophys Ada 1992;1114:107-27. 2. Nooter K., Herweijer H. Multidrug resistance (mdr) genes in human cancer. Br J Cancer 1991;63:663-9. 3. Weinstein RS, Kuszak JR, Kluskens LJ, Coon JS. P-glycoprotein in pathology: the multidrug resistance gene family in humans. Human Pathol 1990;21:34-48. 4. Ueda K, Cardarelli C, Gottesman MM, Pastan I. Expression of a full-length cDNA for the human 'MDR1' gene confers resistance to colchicine, doxorubicin, and vinblastine. Proc Natl Acad Sci USA 1987;84:30O4-8. 5. Lincke CR, Van der Bliek A, Schuurhuis GJ, Van der Velde-Koerts T, Smit J, Borst P. The multidrug resistance phenotype of human BRO melanoma cells transfected with a wild-type human mdrl cDNA. Cancer Res \990;SO:\ 779-85. 6. Gros P, Croop J, Housman D. Mammalian multidrug resistance gene: complete cDNA sequence indicates strong homology to bacterial transport proteins. Cell 1986;47:371-80. 7. Chin JE, Soffir R, Noonan KE et al. Structure and expression of the human MDR (P-glycoprotein) gene family. Mol Cell Biol 1989;9:3808-20. 8. Wilson CM, Scrranom AE, Wasley A, Bogenschutz MP, Shankar AH, Wirth DF. Amplification of a gene related to mammalian mdr genes in drug-resistant Plasmodium falciparum. Science 1989;244:1184-6. 9. Dudler R, Hertig C. Structure of an mdr-like gene from Arabidopsis thaliana. J Biol Chem 1992;267:5882-8. 10. Wu CT, Budding M, Griffin MS, Croop JM. Isolation and characterization of Drosophila multidrug resistance gene homologs. Mol Cell Biol 1991;ll:3940-8. 11. Ueda K, Cornwell MM, Gottesman MM et al. The mdrl gene, responsible for multidrug resistance, codes for P-glycoprotein. Biochem Biophys Res Commun 1986;141: 956-62. 12. Endicott JA, Ling V. The biochemistry of P-glycoprotein mediated multidrug resistance. Annu Rev Biochem 1989;58:137-71. 13. Horio M, Gottesman MM, Pastan I. ATP-dependent transport of vinblastine in vesicles from human multidrug-resistant cells. Proc Natl Acad Sci USA 1988;85:3580-4.

MAILLEFERT ET AL.: EXPRESSION OF P-GLYCOPROTEIN IN RA 14. Kohno K, Sato S, Takano H, Matsuo K, Kuwano M. The direct activation of human multidrug resistance gene (MDR1) by anticancer agents. Biochem Biophys Res Commun 1989;165:1415-21. 15. Tsuoro T, Ida H, Tsukagoshi S, Sakuri Y. Overcoming of vincristine resistance in P388 leukemia in vivo and in vitro through enhanced cytotoxicity of vincristine and vinblastine by verapamil. Cancer Res 1981;41:1967-72. 16. Tsuoro T, Ida H, Kitatani Y, Yokota K, Tsukagoshi S, Sakuri Y. Effects of quinidine and related compounds on cytotoxicity and cellular accumulation of vcr and adriamycin in drug resistant tumor cells. Cancer Res 1984;44:4403-12. 17. Slater LM, Sweet P, Stupcky M, Gupta S. Cyclosporin A reverses vincristine and Daunorubicin resistance in acute lymphatic leukemia in vitro. J Clin Invest 1986;77:1405-8. 18. Borst P, Schinkel AH, Smit JJM et al. Classical and novel forms of multidrugresistanceand the physiological functions of P-glycoproteins in mammals. Pharmacol Ther 1993;6O:289-99. 19. Situnayake RD, Grindulis KA, McConkey B. Long term treatment of rheumatoid arthritis with sulphasalazine, gold or penicillamine: a comparison using life-table methods. Ann Rheum Dis 1987;46:177-83. 20. Wolfe F, Hawley DJ, Cathey MA. Termination of slow acting antirheumatic therapy in rheumatoid arthritis: a 14-year prospective evaluation of 1017 consecutive starts. J Rheumatol 1990;17:994-1002. 21. Buchbinder R, Hall S, Sambrook PN et al. Methotrexate therapy in rheumatoid arthritis: a life tablereviewof 587 patients treated in a community practice. J Rheumatol 1993;2(k639-44. 22. Jorgensen C, Sun R, Rossi JF et al. Multidrug resistance genes (mdrl) RNA in rheumatoid synovium. Arthritis Rheum 1993;36(suppl. 9):S266. 23. Arnett FC, Edworthy SM, Bloch DA et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988;31:315-24. 24. Sugawara I, Nakahara M, Hamada H, Tsuruo T, Mori S. Apparent stronger expression in the human adrenal cortex than in the human adrenal medulla of Mr 170 000-180 000 P-glycoprotein. Cancer Res 1988;48: 4611-4.

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25. Arceri RJ, Croop JM, Horwitz SB, Housman D. The gene encoding multidrug resistance is induced and expressed at-high levels during pregnancy in the secretory epithelium of the uterus. Proc Natl Acad Sci USA 1988;85:4350-4. 26. Huang Yang CP, God DePinho S, Greenberger LM, Arceri RJ, Horwitz SB. Progesterone interacts with P-glycoprotein in multidrug-resistant cells and in gravid uterus. / Biol Biochem 1989^64:782-8. 27. Ueda K, Okamura N, Hirai M et al. Human P-glycoprotein transports cortisol, aldosterone, and dexamethasone, but not progesterone. J Biol Chem 1992;267:24248-52. 28. Nelson EJ, Hinkle P. Characterization of multidrug-resistance pituitary tumor cells. Endocrinology 1992;130: 3246-56. 29. Bourgeois S, Gruol DJ, Newby RF, Rajah FM. Expression of an mdr gene is associated with a new form of resistance to dexamethasone-induced apoptosis. Mol Endocrinol 1993;7:840-51. 30. Cohen D, Piekarz R, Hong Hsu SI, DePinho RA, Carrasco N, Horwitz SB. Structural and functional analysis of the mouse mdrl b gene promoter. J Biol Chem 1991;266:2239-44. 31. Zhao JY, Ikeguchi M, Eckersberg T, Kuo MT. Modulation of multidrug resistance gene expression by dexamethasone in cultured hepatoma cells. Endocrinology 1993;133:521-8. 32. Altuvia S, Stein WD, Goldenberg S, Kane SE, Pastan I, Gottesman MM. Targeted disruption of the mouse mdrlb gene reveals that steroid hormone enhance mdr gene expression. / Biol Chem 1993;268:27127-32. 33. Jorgensen C, Bologna C, Sany J. In vitro induction of multidrug resistance genes (mdrl) on lymphocytes by DMARDs. Arthritis Rheum 1994;37(suppl. 9):S255. 34. Dalton WS, Grogan TM, Meltzer PS et al. Drug resistance in multiple myeloma and non-Hodgkin's lymphoma: detection of P-glycoprotein and potential circumvention by addition of verapamil to chemotherapy. J Clin Oncol 1989;7:415-9. 35. Sonneveld P, Nooter K. Reversal of drug-resistance by cyclosporin A in a patient with acute myeloid myeloma. Br J Haematol 1990;75:208-l 1. 36. Sonneveld P, Durie BGM, Lokhorst HM et al. Modulation of multidrug-resistance multiple myeloma by cyclosporine. Lancet 1992;340:255-9.