| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Experimental Therapeutics |
Divisions of Experimental Therapy [J. D. A., R. F. B., A. H. S.] and Molecular Biology [L. v. D., J. W.], The Netherlands Cancer Institute, 1066CX Amsterdam, The Netherlands
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
A reasonable criticism of this approach is that high levels of drug resistance are atypical of clinical tumors. Chemotherapy is usually a marginal proposition in the sense that the maximum dose tolerated by the patient is often barely sufficient to kill a useful percentage of the tumor cells. Relatively small increases in drug resistance in tumor cells are thus sufficient to render the drug ineffective. Such small increases may be achieved by a much wider variety of mechanisms than those able to confer high levels of drug resistance. The relevance of the (elevated) drug transporter expression seen in drug-selected cell lines to clinical drug resistance cannot, therefore, be taken for granted.
A growing body of evidence links P-gp expression in untreated tumors to poor prognosis for chemotherapy with substrate drugs (for instance, see Refs. 8 , 9 ). The connection is firm for several hematological cancers but, thus far, remains tentative for most other malignancies. Part of the reason is that reliable quantitation of gene expression in heterogeneous patient material is technically problematic (e.g., see the meta-analysis of P-gp expression in breast cancer in Ref. 10 ). Data concerning the contribution of MRP1 to clinical drug resistance are still sparse (8 , 9 , 11) , and the issue is complicated by the widespread expression of MRP1 in normal tissues. In general, it is still unclear whether the (often low) basal levels of multidrug transporter expression significantly affect the innate drug sensitivity of most types of tumors.
We have therefore tested the effects of eliminating normal (i.e., not drug-selected) P-gp and Mrp1 expression on drug sensitivity by comparing mouse cell lines carrying either functional or targeted null alleles of the genes encoding these transporters. This model circumvents issues surrounding the heterogeneity of gene expression in tumor cells and permits a direct assessment of the contribution of multidrug transporters to basal drug sensitivity at levels of expression that can be more readily related to those in normal tissues or untreated tumors. We found that these contributions can be very substantial for drugs that are transporter substrates, implying that basal expression of multidrug transporters could substantially affect innate drug sensitivity of clinical tumors and that even small increases (in absolute terms) in their expression may well mediate acquired resistance. The remarkable sensitivity of cell lines lacking both P-gp and Mrp1 to antineoplastic drugs also makes them valuable in other contexts as tools for analysis of drug resistance mechanisms.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
P-gp and Mrp1 Protein Levels.
Total tissue and cell lysates for immunoblots were prepared by
homogenizing and sonicating cells at 107/ml or
tissues at 50 mg wet weight/ml in 10 mM Tris-HCl (pH 7.4),
10 mM KCl, 1.5 mM MgCl2,
1% w/v SDS, 1% v/v 2-mercaptoethanol, 1 mM
phenylmethylsulfonyl fluoride and Complete protease inhibitor mixture
(Boehringer Mannheim, Mannheim, Germany). Samples were mixed with
SDS-PAGE loading buffer and heated to 65°C for 5 min, this being
adequate to dissociate immunoglobulins in the tissue samples without
causing significant aggregation of either P-gp or Mrp1. Samples were
analyzed on 8% SDS-PAGE gels and electroblotted to nitrocellulose.
Blots were divided at the 97-kDa marker. The high molecular weight
halves were probed for either P-gp or Mrp1 using the C219 (Centocor,
Leiden, The Netherlands) or MRPr1 (16)
monoclonal
antibodies, respectively. These antibodies were raised against the
human and Chinese hamster proteins but cross-react with the mouse
homologues. The low molecular weight halves of the blots were probed
with the anti-
-tubulin monoclonal YL 1/2 (17)
as a
loading control. Target proteins were visualized with horseradish
peroxidase-conjugated secondary antibodies and enhanced
chemiluminescence (ECL). Replicate blots of total RNA from the cell
lines and mouse tissues were hybridized with
32P-labeled antisense RNA probes for
Bcrp1 (18)
or Mdr2 (19)
or random-primed DNA probes for Spgp (IMAGE clone
313236, GenBank accession number W11894) or Cyp3a16 (a
complete coding sequence amplified by PCR from liver cDNA, kindly
provided by J. W. Smit, The Netherlands Cancer Institute).
Hybridization was performed in Ultrahyb buffer (Ambion, Austin, TX)
under the recommended conditions, except for Cyp3a16, for
which the hybridization and wash temperatures were reduced by 5°C to
ensure detection of other homologous Cyp3a mRNAs. One blot
was rehybridized with a cDNA probe for the mouse 18S rRNA as a loading
control.
Drugs.
Paclitaxel, vinblastine sulfate, doxorubicin (Adriamycin), cytarabine,
melphalan, and sodium arsenite were obtained from Sigma-Aldrich
(Zwijndrecht, The Netherlands). Formulations of vincristine sulfate,
cisplatin, daunorubicin, mitoxantrone, etoposide, and teniposide were
obtained from local pharmaceutical suppliers; the topotecan
formulation was from SmithKline Beecham (Brentford, Middlesex,
England); and Taxotere (docetaxel) and SN-38 were from
Rhône-Poulenc Rorer (Vitry-Alfortville, France).
Drug Sensitivity Assays.
Growth inhibition (IC50) assays were performed by
seeding 200 cells per well in 96-well plates in complete medium and,
after cell attachment, applying drugs in a dilution series of 2-fold
concentration steps, with each concentration in quadruplicate wells.
After 4–4.5 days, when control wells were still subconfluent, cells
were lysed in situ, and nucleic acids were stained with a
proprietary dye (Cyquant; Molecular Probes, Eugene, OR) and quantified
by fluorescence (485 nm excitation, 530 nm emission).
Descriptive and Inferential Statistics.
The SDs shown in Table 2
represent variability in absolute
IC50 determinations across independent
experiments. Sensitivity factors in Table 3
are computed directly from
the means shown in Table 2
. Because distributions of drug resistance
data are, by nature, positively skewed, IC50
values were transformed to logarithms for the purpose of statistical
comparisons (20)
. All tests were two-tailed t
tests assuming unequal variances.
|
|
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
. Although growth properties might be expected to affect drug
sensitivity, in practice no systematic effects from this source were
observed.
|
. P-gp levels in the wild-type cell lines
were comparable with or lower than those seen in normal mouse tissues.
The C219 antibody cross-reacts with Mdr2 P-gp (homologue of human MDR3)
and Spgp, but their expression is mostly confined to liver (21
, 22) . Two observations exclude significant levels of either of
these proteins in the fibroblast cell lines: Mdr2 and
Spgp mRNAs were undetectable by Northern analysis of the
cell lines but readily detectable in mouse liver (Fig. 2,A and B)
, and the C219 antibody detected no Mdr2
or Spgp protein in the six fibroblast lines null for
Mdr1a/1b (Fig. 1A)
. Thus, the C219 signal in the
wild-type cell lines reliably reflects their expression of P-gp.
|
|
. Mrp1 levels in the cell lines were somewhat higher than in most tissues examined. This result is adequate for the present purpose, given that Mrp1 tends to be concentrated in epithelial layers having substantially higher protein levels than the tissue taken as a whole (23, 24, 25) . This also holds true for P-gp (26) .
Sensitivity of the Lines to Antineoplastic Drugs.
The relative sensitivities of the cell lines to an extensive panel of
antineoplastic drugs were determined by growth inhibition assay (Table 2)
. The drug panel contained representatives from the major families of
clinically important antineoplastic agents that are known substrates
for P-gp or Mrp1, including taxanes, Vinca alkaloids,
anthracyclines, epipodophyllotoxins, and camptothecin derivatives, as
well as an arsenical. Control drugs were also included from several
classes that are poor or insignificant substrates of these proteins but
for which acquired resistance is also a significant clinical problem:
cisplatin, cytarabine (a nucleoside analogue), and melphalan (an
alkylating agent). Mitoxantrone was included because it is an
excellent substrate for the recently identified multidrug transporter
BCRP/Bcrp1 (5
, 6
, 18)
.
The data show that basal expression of P-gp and Mrp1 can have a major
impact on innate resistance to substrate drugs. Table 2
presents the
mean IC50 values, and Table 3
, to aid comparisons, shows the sensitivity factors plus results of
statistical tests of the differences between genotypes. The
Mdr1a-/-1b-/-
cell lines were substantially more sensitive than the wild-type lines
to all drugs tested that are good P-gp substrates—the taxanes,
anthracyclines, and Vinca alkaloids. The differences were
surprisingly large given the relatively low levels of P-gp in the
wild-type cell lines; the averages were 16-fold for paclitaxel,
4–5-fold for the anthracyclines, and 
3-fold for Vinca
alkaloids. It may be noted that the cell lines lacking P-gp expressed
somewhat higher levels of Mrp1 than the wild-type lines (Fig. 1)
. It is
not clear whether this is a coincidental or a compensatory effect.
Either way, it might partially mask the effect of inactivating P-gp for
drugs that are substrates of both transporters, Vinca
alkaloids, epipodophyllotoxins, and possibly anthracyclines (see
below).
The
Mdr1a-/-1b-/-Mrp1-/-
cell lines were markedly more sensitive than the
Mdr1a-/-1b-/-
cell lines to several Mrp1 substrate drugs, most notably vincristine
(>10-fold), sodium arsenite (7-fold), and the epipodophyllotoxins
etoposide (7-fold) and teniposide (4-fold). Qualitatively, these
differences in drug sensitivity are in agreement with those seen in
previous studies of mouse embryonic stem cell lines and bone marrow
mast cells in which only the Mrp1 gene had been inactivated
by gene targeting (27
, 28)
. However, the differences we
observed are generally greater due, at least in part, to the absence of
masking P-gp activity on drugs that are common substrates. For such
drugs, the combined effect of eliminating the function of two
transporters can sometimes be very striking, as exemplified by
vincristine, for which the difference in sensitivity between
Mdr1a-/-1b-/-Mrp1-/-
and wild-type lines averaged 28-fold.
Human MRP1 transports anthracyclines effectively and can confer
resistance to these drugs, but it is unclear to what extent mouse Mrp1
does likewise (28, 29, 30, 31, 32)
. We found that mouse fibroblast
lines lacking both Mrp1 and P-gp were only slightly more sensitive to
doxorubicin and daunorubicin than lines lacking only P-gp
(1.5-fold), suggesting that these drugs are relatively poor
substrates for mouse Mrp1.
It is interesting that the Mdr1a-/-1b-/-Mrp1-/- cell lines were 2–3-fold more sensitive to the camptothecin derivatives topotecan and SN-38 than the Mdr1a-/-1b-/- lines. This result suggests that mouse Mrp1 can transport these drugs, in line with a recent report (33) that elevated expression of human MRP1 in drug-selected or MRP1-transfected cells mediates resistance to SN-38 and CPT-11 (another camptothecin derivative), which is associated with ATP-dependent drug efflux and reversed by MRP1 inhibitors.
Overexpression of P-gp is usually associated with only modest
resistance to mitoxantrone (e.g., Refs. 34
, 35
), implying that this drug is a relatively poor substrate.
Increases in resistance to mitoxantrone have also been observed in
MRP1-transfected cells (11)
. In line with these
results, we did observe moderately increased sensitivity to
mitoxantrone in
Mdr1a-/-1b-/-
and
Mdr1a-/-1b-/-Mrp1-/-
fibroblast lines of 2.5- and 4.3-fold, respectively, compared with
wild-type lines, but only the latter difference was statistically
significant (P = 0.041, two tailed). This is
in part due to the considerable variability in resistance to
mitoxantrone between different cell lines within each genotype.
Mitoxantrone is a good substrate of the BCRP/Bcrp1 transporter
(5
, 6
, 18)
, and there is indeed a qualitative
correlation between Bcrp1 mRNA levels (Fig. 2D)
and resistance to mitoxantrone. However, the
relationship is not so good that Bcrp1 expression might
account for all of the differences in sensitivity to this drug.
Within genotypes, substantial systematic differences in drug
sensitivity were evident only in the wild-type group of cell lines; the
WT1.2 and especially the NIH/3T3 lines were frequently more sensitive
than the 2ac.1 and 2ac.2 lines. This pattern was most pronounced for
P-gp substrate drugs and was not observed consistently for drugs that
are neither P-gp nor Mrp1 substrates (cisplatin, cytarabine, and
melphalan). The systematic differences are thus probably at least
partly the result of the lower P-gp expression observed in the WT1.2
and NIH/3T3 lines (Fig. 1)
. The different mouse strain origin of the
NIH/3T3 line might also contribute to the differences observed.
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Although many other mechanisms may intrude to alter drug resistance, there can be little doubt that the increased sensitivities seen in the knockout fibroblast lines are due to the loss of P-gp and Mrp1. Multiple independently derived lines of each genotype were examined, with similar origins and genetic background. Results for the cell lines of each genotype are in good agreement, and the pattern of drug sensitivities observed matches the known substrate specificities of the two transporter proteins.
These observations strongly support the position that the normal levels of expression of MDR transporters in cancer (or normal) cells can be important for innate resistance to antineoplastic drugs. They also imply that low absolute levels of expression of drug transporters in tumors can still be significant for acquired resistance, in that a change to low expression from very low (or no) initial expression could produce a difference in drug sensitivity of the same scale as observed in the cell line model. Although our results concern P-gp and Mrp1, there is no obvious reason to suspect that other drug transporters, such as Bcrp1, will differ in this respect. In "Introduction" we alluded to attempts to predict the responses of primary tumors to specific chemotherapeutic regimens on the basis of P-gp expression, which have been generally disappointing. Our data suggest that such failures are not reason to conclude that P-gp (or Mrp1) is irrelevant to clinical drug resistance but rather that the task of assessing expression of these (and other) drug transporters, although difficult, is worthwhile. It seems clear that simultaneous assessment of expression of multiple transporters of a given drug will be beneficial.
The relevance of basal expression of MDR transporters to chemotherapy will be increased with the adoption of potent inhibitors of P-gp such as Valspodar (and, ultimately, inhibitors of other transporters) for coadministration during chemotherapy, now being tested in Phase III clinical trials (e.g., see Ref. 9 ). Our results lend credence to the idea that inhibitors of MDR transporters may be useful not only to reverse or prevent acquired drug resistance but also to sensitize drug-naive, untreated tumors to substrate drugs, even when expression of the relevant transporter(s) is low. A caveat to these approaches is that coadministration of inhibitors with antineoplastic drugs might increase the risk of toxic side effects in normal cells that are otherwise protected by drug transporter expression (14 , 25 , 36) . The importance of such side effects can only be decided in the clinical trials.
It must be reiterated that we are not suggesting that P-gp, Mrp1, or other drug transporters are the only important influences on basal resistance in our cell lines, let alone in clinical tumors. The influence of other, unknown factors is evident in the data, for example, in the marked sensitivity of NIH/3T3 cells to arsenite and the variability in mitoxantrone sensitivity, only part of which is attributable to Bcrp1 expression. This is not surprising, given that drug sensitivity may be influenced by many factors. (In fact, the cell lines lacking P-gp and Mrp1 function will be useful tools for investigating these other factors.) Other cell types, including tumor cells, may have a more extensive repertoire of drug resistance mechanisms than fibroblasts. If so, the relative contribution of P-gp and Mrp1 to drug resistance in tumors or normal cells thus may (or may not) be less than is suggested by our results. Nevertheless, acknowledging the limitations of the cell line model, our data indicate that this contribution can be very marked.
|
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 This work was supported by Dutch Cancer Society
Grants NKI 95-963 and NKI 97-1433. 
2 To whom requests for reprints should be
addressed, at Division of Experimental Therapy, The Netherlands Cancer
Institute, Plesmanlaan 121, 1066CX Amsterdam, The Netherlands. Phone:
(31) 20-512-2046; Fax: (31) 20-512-2050; E-mail: alfred{at}nki.nl
3 The abbreviations used are: P-gp,
P-glycoprotein; BCRP, breast cancer resistance protein; MDR, multidrug
resistance; MRP, multidrug resistance-associated protein; MEF, mouse
embryo fibroblast; SN-38, (4S)-11-ethylcamptothecin-ol;
Spgp, sister of P-gp.
Received 2/21/00. Accepted 8/17/00.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  C.-P. Wu, S. Shukla, A. M. Calcagno, M. D. Hall, M. M. Gottesman, and S. V. Ambudkar Evidence for dual mode of action of a thiosemicarbazone, NSC73306: a potent substrate of the multidrug resistance linked ABCG2 transporter Mol. Cancer Ther., December 1, 2007; 6(12): 3287 - 3296. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. R. Gerstner and R. L. Fine Increased Permeability of the Blood-Brain Barrier to Chemotherapy in Metastatic Brain Tumors: Establishing a Treatment Paradigm J. Clin. Oncol., June 1, 2007; 25(16): 2306 - 2312. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Haber, J. Smith, S. B. Bordow, C. Flemming, S. L. Cohn, W. B. London, G. M. Marshall, and M. D. Norris Association of High-Level MRP1 Expression With Poor Clinical Outcome in a Large Prospective Study of Primary Neuroblastoma J. Clin. Oncol., April 1, 2006; 24(10): 1546 - 1553. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Green, P. Soderkvist, P. Rosenberg, G. Horvath, and C. Peterson mdr-1 Single Nucleotide Polymorphisms in Ovarian Cancer Tissue: G2677T/A Correlates with Response to Paclitaxel Chemotherapy Clin. Cancer Res., February 1, 2006; 12(3): 854 - 859. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. J. Dilda, A. S. Don, K. M. Tanabe, V. J. Higgins, J. D. Allen, I. W. Dawes, and P. J. Hogg Mechanism of Selectivity of an Angiogenesis Inhibitor From Screening a Genome-Wide Set of Saccharomyces cerevisiae Deletion Strains J Natl Cancer Inst, October 19, 2005; 97(20): 1539 - 1547. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Pavek, G. Merino, E. Wagenaar, E. Bolscher, M. Novotna, J. W. Jonker, and A. H. Schinkel Human Breast Cancer Resistance Protein: Interactions with Steroid Drugs, Hormones, the Dietary Carcinogen 2-Amino-1-methyl-6-phenylimidazo(4,5-b)pyridine, and Transport of Cimetidine J. Pharmacol. Exp. Ther., January 1, 2005; 312(1): 144 - 152. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Kalin, J. Fernandes, S. Hrafnsdottir, and G. van Meer Natural Phosphatidylcholine Is Actively Translocated across the Plasma Membrane to the Surface of Mammalian Cells J. Biol. Chem., August 6, 2004; 279(32): 33228 - 33236. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Cisternino, C. Mercier, F. Bourasset, F. Roux, and J.-M. Scherrmann Expression, Up-Regulation, and Transport Activity of the Multidrug-Resistance Protein Abcg2 at the Mouse Blood-Brain Barrier Cancer Res., May 1, 2004; 64(9): 3296 - 3301. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. D. Allen, S. C. van Dort, M. Buitelaar, O. van Tellingen, and A. H. Schinkel Mouse Breast Cancer Resistance Protein (Bcrp1/Abcg2) Mediates Etoposide Resistance and Transport, but Etoposide Oral Availability Is Limited Primarily by P-glycoprotein Cancer Res., March 15, 2003; 63(6): 1339 - 1344. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. P. Lin, D. R. Johnson, R. A. Finch, M. G. Belinsky, G. D. Kruh, and A. C. Sartorelli Comparative Study of the Importance of Multidrug Resistance-associated Protein 1 and P-Glycoprotein to Drug Sensitivity in Immortalized Mouse Embryonic Fibroblasts Mol. Cancer Ther., October 1, 2002; 1(12): 1105 - 1114. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. D. Allen, S. C. Jackson, and A. H. Schinkel A Mutation Hot Spot in the Bcrp1 (Abcg2) Multidrug Transporter in Mouse Cell Lines Selected for Doxorubicin Resistance Cancer Res., April 1, 2002; 62(8): 2294 - 2299. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. D. Allen, A. van Loevezijn, J. M. Lakhai, M. van der Valk, O. van Tellingen, G. Reid, J. H. M. Schellens, G.-J. Koomen, and A. H. Schinkel Potent and Specific Inhibition of the Breast Cancer Resistance Protein Multidrug Transporter in Vitro and in Mouse Intestine by a Novel Analogue of Fumitremorgin C Mol. Cancer Ther., April 1, 2002; 1(6): 417 - 425. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. D. Allen and A. H. Schinkel Multidrug Resistance and Pharmacological Protection Mediated by the Breast Cancer Resistance Protein (BCRP/ABCG2) Mol. Cancer Ther., April 1, 2002; 1(6): 427 - 434. [Full Text] [PDF]
|
|
|
|
|
|
N. Zelcer, T. Saeki, G. Reid, J. H. Beijnen, and P. Borst Characterization of Drug Transport by the Human Multidrug Resistance Protein 3 (ABCC3) J. Biol. Chem., November 30, 2001; 276(49): 46400 - 46407. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Duesberg, R. Stindl, and R. Hehlmann Origin of multidrug resistance in cells with and without multidrug resistance genes: Chromosome reassortments catalyzed by aneuploidy PNAS, September 5, 2001; (2001) 201398998. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Liu, H. Chen, D. S. Miller, J. E. Saavedra, L. K. Keefer, D. R. Johnson, C. D. Klaassen, and M. P. Waalkes Overexpression of Glutathione S-Transferase II and Multidrug Resistance Transport Proteins Is Associated with Acquired Tolerance to Inorganic Arsenic Mol. Pharmacol., August 1, 2001; 60(2): 302 - 309. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. R. Johnson, R. A. Finch, Z. P. Lin, C. J. Zeiss, and A. C. Sartorelli The Pharmacological Phenotype of Combined Multidrug-Resistance mdr1a/1b- and mrp1-deficient Mice Cancer Res., February 1, 2001; 61(4): 1469 - 1476. [Abstract] [Full Text]
|
|
|
|
|
|
P. Duesberg, R. Stindl, and R. Hehlmann Origin of multidrug resistance in cells with and without multidrug resistance genes: Chromosome reassortments catalyzed by aneuploidy PNAS, September 25, 2001; 98(20): 11283 - 11288. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
