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Departments of Molecular Pharmacology (M.W., C.L.M., M.K.D., P.M.P.) and Pharmaceutical Sciences (A.W., J.S., J.D.S.), St. Jude Children's Research Hospital, Memphis, Tennessee
Received February 23, 2003; accepted April 11, 2003
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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;
Tanizawa et al., 1994). SN-38
is 100- to 1000-fold more cytotoxic than CPT-11. In recent years, CPT-11 has
been approved for the treatment of colon cancer, and it is currently under
investigation for use in a variety of other solid malignancies, in both adults
and children (Conti et al.,
1996; Baker et al.,
1997; Kudoh et al.,
1998; Furman et al.,
1999). Initial studies seem very promising, attesting to the fact
that this is one of the best anticancer agents that has been introduced in the
clinic in the last 30 years.
CPT-11 activation is primarily mediated by carboxylesterases (CEs)
(Satoh et al., 1994; Morton et
al., 1999,
2000;
Khanna et al., 2000). We
recently identified a rabbit liver CE (rCE) that can efficiently activate the
drug (Potter et al., 1998a),
and overexpression of this protein in cells in culture, or when grown as
xenografts in immune-deprived mice, results in enhanced sensitivity to CPT-11
(Danks et al., 1998,
1999; Potter et al.,
1998a,b).
Studies using replication-deficient adenovirus harboring rCE indicate that
tumor cells can be markedly sensitized to the drug after viral transduction
(Wierdl et al., 2001). The
potential application of this reagent to the purging of neuroblastoma cells
from bone marrow has been described previously
(Meck et al., 2001;
Wagner et al., 2002).
Transport proteins mediate the efflux of camptothecin analogs from cells.
However, the ATP-binding cassette (ABC) transporter superfamily members
comprise a large group of functionally diverse proteins whose expression has
been detected in prokaryotes, plants, and mammals. This gene family is defined
by the presence of an approximately 250 amino acid stretch that contains
Walker A and B motifs and a conserved linker sequence termed the "ABC
signature" that is essential for ATP hydrolysis
(Higgins, 2001). Most ABC
transporters are localized on cellular membranes and transport structurally
diverse compounds. They are composed of either one or two membrane-spanning
domains and one or two ATP-binding domains that function as either a single
protein or a multiprotein complex
(Higgins, 2001).
The so-called "half-molecule" transporters within the ABC
superfamily (i.e., containing only one membrane-spanning and one ATP-binding
domain) are typically localized to the membranes of intracellular organelles
(Higgins, 2001). However,
ABCG2 (breast cancer resistance protein, MXR, or ABCP)
(Allikmets et al., 1998;
Doyle et al., 1998;
Bates et al., 2001) is the only
drug-transporting half-molecule transporter currently localized to the plasma
membrane (Rocchi et al., 2000;
Maliepaard et al., 2001) and
has been demonstrated to function as a homodimer
(Ozvegy et al., 2001). ABCG2
is overexpressed in human tumor cell lines selected for resistance to DNA
topoisomerase inhibitors such as topotecan and SN-38
(Van Hattum et al., 2002).
However, drug-selected cells also have increased expression of other ABC
transporters (Litman et al.,
2000) that may facilitate efflux of these camptothecins, thus
leading to potential confusion as to the role of ABCG2 in drug resistance.
To assess the role of ABCG2 in the resistance of cells to camptothecin analogs, we isolated a cDNA encoding the protein and expressed it in an ABCG2- and P-gp—null cell line. These cells were then characterized for transport properties and their ability to confer camptothecin resistance. More importantly, these cells were used to evaluate how ABCG2 interacts with CEs known to activate CPT-11. Because overexpression of ABCG2 would be expected to reduce intracellular concentrations of SN-38 and CPT-11, increased levels of this protein in tumors may result in drug resistance. Therefore the efficacy of enzyme/prodrug therapy approaches such as viral-directed and antibody-directed enzyme prodrug therapy with CPT-11 would depend on the expression of ABCG2. To determine the contribution of this protein in mediating resistance to the camptothecin analogs, we have performed studies on cell lines overexpressing both ABCG2 and rCE. In this series of studies, rCE was used as a tool to create high levels of SN-38 in cells and hence determine the contribution of ABCG2 toward drug resistance. Results demonstrate that reduced intracellular drug concentrations and, consequently, reduced cytotoxicity are observed in cells expressing ABCG2. Additionally and more importantly, enforced overexpression of CEs cannot overcome this drug resistance.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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). CPT-11
and SN-38 were generous gifts from Dr. J. P. McGovren (Pharmacia Upjohn,
Kalamazoo, MI). CPT-11 was dissolved in methanol at 10 mM and stored at
-20°C. Immediately before use, the drug was diluted in serum-free media.
SN-38 was dissolved in DMSO and used in a fashion identical to that of CPT-11.
The fluorescent dye Hoechst 33342 was obtained from Molecular Probes (Eugene,
OR). Reserpine, progesterone, probenecid, prostaglandin A1, and leukotrienes
B4, C4, and D4 were purchased from Sigma Chemical Co. (St. Louis, MO) and
dissolved in DMSO. Antipeptide polyclonal antibodies to ABCG2 were used as
described previously (Zhou et al.,
2002), and an antibody to MRP1 was purchased from Signet
Laboratories (Dedham, MA). Isolation of ABCG2 cDNA. To determine whether an EST clone containing the ABCG2 cDNA existed, the published ABCG2 nucleotide sequence was used to search the EST database. We identified a cDNA that had significant identity (>99%) with the published sequence (dbEST clone number 52176). The clone was purchased from Incyte Systems (Palo Alto, CA), and the cDNA insert was isolated and ligated into pcDNA3 (Invitrogen, Carlsbad, CA). DNA sequence analysis confirmed the identity of the cDNA (GenBank accession number AY017168 [GenBank] ).
Generation of a Nonfunctional ABCG2 Mutant. To render the ABCG2 protein nonfunctional, the highly conserved lysine residue in the Walker A domain (G(X)4GKS) was substituted with a methionine with the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The mutation was confirmed by DNA sequence analysis. This mutant ABCG2 cDNA was transfected into Saos-2 cells as described below.
Cell Transfections. Saos-2 cells were screened by Western analysis
and revealed no immunodetectable MDR1, P-gp, or ABCG2 protein in the
membranes. The cells were transfected with the relevant plasmids (10 µg) by
standard calcium phosphate coprecipitation and cultured in medium containing 1
mg/ml of G418 for at least 14 days, or until colonies were readily visible.
After isolation, membrane fractions were prepared from clones, and ABCG2
expression was determined using a rabbit polyclonal antibody
(Zhou et al., 2002).
Western Analysis. For analysis of MRP1 expression, 100 µg of the membrane-enriched fraction was resolved on 7.5% SDS-polyacrylamide gel electrophoresis, and after transfer to filters, proteins were immunoblotted with an anti-MRP1 antibody at a dilution of 1:300 (Signet). For ABCG2 analysis, the rabbit polyclonal antibody was used at a dilution of 1:500. For both antibodies, specific cross-reactivity was detected using a horseradish peroxidase-conjugated secondary antibody and enhanced chemiluminescence.
Adsorbed ABCG2 antibody (Zhou et al.,
2002) was prepared by incubation with 2 mg/ml of ABCG2 peptide
overnight at 4°C and centrifugation at 10,000g for 5 min. The
supernatant was used for Western analysis at the same dilution as the native
antisera.
Carboxylesterase Assays. CE activity in whole-cell sonicates was
determined spectrophotometrically using o-nitrophenyl acetate as a
substrate (Beaufay et al.,
1974; Potter et al.,
1998a). Data were expressed as micromoles of
o-nitrophenol produced per milligram of protein per minute.
Drug Accumulation and Efflux. Cells were plated at 2 x 105 cells/well in 24-well plates and were allowed to attach overnight at 37°C. After incubation with the dye (for up to 1 h, as indicated in the figure legends), cells were rapidly washed with Hanks' buffer (4°C), and after trypsinization, they were resuspended in PBS buffer (4°C). FACS analysis was performed on a Vantage flow cytometer (BD Biosciences, San Jose, CA). The following excitation and emission wavelengths were used for each dye: Hoechst 33342, 365 nm/400 nm; and mitoxantrone, 647 nm/670 nm. Only intact cells, determined from the appropriate light scatter, were analyzed, and data were collected from at least 5000 cells for each sample.
Cells lacking ABCG2 retain more dye and have greater fluorescence. Therefore, all cells with fluorescence values lower than the ABCG2-null cells were considered to efflux the dye. This was designated the efflux gate ("E" gate) and was used to quantify the proportion of cells effluxing the dye. Inhibitor calculations were made by determining the percentage of the population of cells moving from the low fluorescence "E" gate, to the high-intensity fluorescence observed in cells containing the vector plasmid.
Determination of CPT-11 and SN-38. CPT-11 and SN-38 concentrations
were determined by reverse-phase HPLC as described previously
(Guichard et al., 1998;
Danks et al., 1999). Briefly,
samples were mixed with an equal volume of ice-cold acid methanol, stored at
-80°C for 1 h, and centrifuged at 30,000g to remove particulate
matter. Drug concentrations in the supernatant were determined by separation
on a NovaPak C18 column using 25% acetonitrile/75 mM ammonium acetate, pH 4.0,
as a mobile phase. Detection was achieved using a Jasco 920 fluorescence
detector (Jasco, Tokyo, Japan), and the sensitivity of this system was 20
pg/µl and 1.5 pg/µl for CPT-11 and SN-38, respectively.
Determination of Intracellular CPT-11 and SN-38 Concentrations. Intracellular drug concentrations were determined under the following conditions. Cells (4 x 106) were harvested by trypsinization and resuspended in 1 ml of serum-free media. CPT-11 was added to a final concentration of 1, 10, or 100 µM, and after 1 h of incubation at 37°C, cells were centrifuged, washed rapidly with cold PBS, and finally resuspended in 200 µl of 50 mM HEPES, pH 7.4. After sonication on ice, an equal volume of cold acid methanol was added, and drug concentrations were determined by HPLC as described above.
Viral Transduction of Cell Lines. Cell lines were transduced with
replication-deficient adenovirus expressing rCE (AdCMVrCE), as described
previously (Wierdl et al.,
2001). Routinely, a multiplicity of infection (m.o.i.) of 5 was
used. At this m.o.i., no viral toxicity was observed, and high yields of CE
activity were produced 3 to 4 days after transduction
(Wierdl et al., 2001). Cells
were transduced with virus for 24 h in culture medium, at which point the
reagent was removed and replaced with fresh medium. Cells were then allowed to
grow for 3 to 4 days before growth-inhibition assays or drug-accumulation
studies. When reserpine was used, cells were treated with 5 to 20 µM drug
for 1 h at 37°C.
Growth-Inhibition Assays. Inhibition of cell growth by CPT-11 was assessed by cell counting using a Coulter Z2 multisizer (Beckman Coulter, Inc., Fullerton, CA). Cells were plated at a density of 5 x 104 per well in a six-well plate, and after allowing to adhere overnight, the media were removed and replaced with serum-free media containing CPT-11. After incubation for 2 h, the drug was aspirated, and fresh media were applied. After growth equivalent to three cell doublings (72 h), the cells were counted. Routinely, all data points were repeated in triplicate, and IC50 values were determined using the GraphPad Prism software (GraphPad Software Inc., San Diego, CA).
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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). These resulted in four amino acid changes (A 
V at
position 24, Q E at 166, F S at 208, and A P at 501) that
might reflect polymorphisms in ABCG2. Nevertheless, the predicted protein
sequence of our ABCG2 was 655 amino acids in length, and it was greater than
99% identical to both ABCG2 (Doyle et al.,
1998) and ABCP (Allikmets et
al., 1998). Importantly, this cDNA derived from brain encoded an
arginine at amino acid position 482, unlike the ABCG2 isoform described by
Ross and colleagues, which was isolated from a drug-resistant cell line
(Doyle et al., 1998). In
addition, an analysis of the human genome confirms that Arg-482 is present in
the wild-type ABCG2 sequence. We are currently evaluating the role of the
single amino acid substitutions on the function of ABCG2 (to be reported
elsewhere), but we have evidence that the change to a noncharged amino acid at
position 482 (i.e., from Arg to Thr or Gly) does not occur in the population
and seems to occur only under drug selection
(Zamber et al., 2003).
Characterization of Cell Lines Expressing ABCG2. To eliminate the
possible confounding effects of expression of other transporters or endogenous
ABCG2 in cells, we identified an ABCG2 and P-gp—null cell line by
Western analysis (Saos-2). Additionally, Saos-2 cells have very low levels of
endogenous CE activity (<6 µmol/min/mg) and consequently are relatively
resistant to the cytotoxic effects of CPT-11
(Wierdl et al., 2003). This
line was subsequently transfected with expression plasmids to yield clones
that stably expressed either ABCG2 or ABCG2 containing a mutation in the
Walker A domain (mutABCG2). Membrane fractions were isolated, and Western
analyses were performed on these cell lines
(Fig. 1). Among
Saos-2—transfected clones expressing ABCG2, the rank order of protein
expression was ABCG2#4 
-#6 > -#7
(Fig. 1B). Two clones, ABCG2#4
and ABCG2#7, demonstrated high and low levels of ABCG2 expression,
respectively, and were expanded and used for the subsequent studies described
herein. Transfection with the mutant ABCG2 cDNA yielded several cell lines,
with the clone mutABCG2#10 expanded for further studies. As an additional
control, Saos-2 cells were transfected with pcDNA3, and after clonal selection
with G418, pcDNA#2 cells were isolated. As expected, pcDNA#2 demonstrated no
detectable ABCG2 expression (Fig.
1). All Saos-2 ABCG2 clones demonstrated similar growth rates and
doubling times of 24 h (data not shown).
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Functional Analysis of ABCG2 Cell Lines. To assess the function of ABCG2 in Saos-2 transfected cells, we performed uptake studies using the dye Hoechst 33342 and the chemotherapeutic agent mitoxantrone (Fig. 2). Cells were incubated with either drug for an hour until steady-state levels were achieved, and FACS analysis was performed to determine the levels of intracellular fluorescence. As indicated in Fig. 2A, mitoxantrone was specifically excluded from cells expressing ABCG2. In addition, these studies indicated that mitoxantrone accumulation was inversely proportional to the amount of protein expressed. With Hoechst 33342, although the drug was excluded similar to mitoxantrone, the levels of intracellular fluorescence did not directly correlate with the amounts of ABCG2 protein expressed (Fig. 2B). This discrepancy is probably caused by ABCG2 having different affinities for these drugs.
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To determine whether this was the case, we assessed the uptake of mitoxantrone and Hoechst 33342 as a function of drug concentration (Fig. 3). We observed that ABCG2#4 cells accumulated approximately 50% less mitoxantrone compared with ABCG2#7 cells at each concentration tested and that mutABCG2#10 accumulated twice as much drug as ABCG2#7 (Fig. 3A). In contrast, Hoechst 33342 showed saturation kinetics (Fig. 3B), and the concentrations required to achieve half-maximal intracellular values were estimated to be approximately 5 µM for both ABCG2#4 and ABCG2#7 cells. Nevertheless, the intracellular concentration of Hoechst 33342 in ABCG2#4 cells was approximately 50% of that observed in ABCG2#7 and was much greater than that observed in mutABCG2#10 cells. The difference in drug accumulation between ABCG2#4 and ABCG2#7 was remarkably close to the 3-fold difference in BABCG2 protein levels observed in membranes derived from these cell lines (Fig. 1B).
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Analysis of Hoechst 33342 Efflux in ABCG2-Expressing Cell Lines. To
further characterize cells expressing ABCG2, we measured the rate of Hoechst
influx and efflux in ABCG2#4, ABCG2#7, and mutABCG2#10 cells
(Fig. 3, C and D). As indicated
in Fig. 3C, the steady-state
intracellular dye concentrations are inversely proportional to the level of
expression of functional ABCG2. Hence, ABCG2#4 excludes more dye than ABCG2#7,
which in turn demonstrates approximately 2-fold less Hoechst 33342 than
mutABCG2#10 cells. To determine the rates of dye efflux in these cell lines,
steady-state levels were achieved by incubation in media containing 10, 5, and
1 µM Hoechst 33342 for ABCG2#4, ABCG2#7, and mutABCG2#10 cells,
respectively. The increased concentrations of dye were used to compensate for
the activity of the ABCG2 protein in these cells. After resuspension in fresh
medium, intracellular fluorescence determinations were performed at various
time intervals (Fig. 3D). To
estimate the initial rate of drug efflux, linear regression analysis was
performed on time points up to and including 15 min. It should be noted that
despite the presence of excess Hoechst 33342, the steady-state intracellular
levels of drug in ABCG2#4 only reached approximately one-half that observed in
ABCG2#7 and mutABCG2#10 cells (Fig. 3, C
and D).Taken from the initial slopes, the rate of dye efflux was
50% greater for ABCG2#4 than forABCG2#7. As expected, the efflux of
Hoechst 33342 from mutABCG2#10 was minimal. Overall, these data indicate that
the efflux of Hoechst dye is dependent on the amount of functional ABCG2
protein in cells.
Inhibition of ABCG2-Mediated Transport. To determine whether Hoechst
33342 efflux via ABCG2 could be blocked, we evaluated a variety of compounds
(Table 1) for their ability to
increase dye retention in ABCG2#4 cells. Of the agents tested, reserpine was
found to be the most potent inhibitor, with the MRP1 and P-gp inhibitors MK571
and cyclosporine A1 (CSA), respectively, being much less effective.
The structural analogs of reserpine, yohimbine, and corynanthine were
ineffective at inhibiting ABCG2. Similarly, the MDR1 inhibitor progesterone
(Yang et al., 1990;
Ueda et al., 1992)
(Table 1) was a poor inhibitor
of ABCG2. However, because of the apparent overlap between the inhibition of
MDR1, MRP1, and ABCG2 by reserpine, MK571, and CSA, the relative ability of
these three compounds to inhibit ABCG2 transport was assessed. In these
studies, we also used both ABCG2#4 and ABCG2#7 cells to eliminate the
possibility that the effectiveness of the inhibitor might be dependent on
ABCG2 protein levels.
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Reserpine was a very effective ABCG2 inhibitor, with the concentration
required to inhibit ABCG2 being 2 µM for ABCG2#7 and approximately 5 µM
for ABCG2#4 (Fig. 4A). The MRP1
inhibitor MK571 (Keppler et al.,
1998) was also effective, but less so, requiring concentrations 7-
to 10-fold higher than reserpine for these cells
(Fig. 4B). The least effective
inhibitor, CSA, required concentrations of at least 20 µM to produce 50%
inhibition of dye efflux in ABCG2#7 cells
(Fig. 4C) and 80 µM for
ABCG2#4 (data not shown). These studies demonstrate that ABCG2 transport
inhibition is critically dependent on the amount of protein.
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ABCG2 Transports SN-38. Whereas a number of reports indicate that
ABCG2 transports camptothecin, some confusion exists because ABCG2 contains
two different alleles [either Arg or a noncharged amino acid (e.g., Gly or
Thr) at position 482] that differ in their ability to function as transporters
of certain drugs (Honjo et al.,
2001). Notably, in recently described studies, the expression of
an ABCG2 cDNA containing arginine at position 482 was unable to confer
camptothecin or mitoxantrone resistance in cells
(Komatani et al., 2001). In
contrast, the ABCG2 protein expressed in the Saos-2 clones described in this
article, containing arginine at this position, is capable of transporting and
conferring mitoxantrone resistance but not resistance to either adriamycin or
daunomycin (Fig. 2 and
unpublished data).
Therefore, we assessed intracellular CPT-11 and SN-38 concentrations in the ABCG2#4, ABCG2#7, and pcDNA#2 Saos-2 cell lines after drug treatment. Cells were transduced with adenovirus-expressing rCE, and after 4 days, three identical aliquots were assessed for CE activity and drug accumulation studies, both with and without reserpine. After treatment with 5 µM reserpine for 1 h, concentrations of CPT-11 ranging from 1 to 100 µM were added, and intracellular drug concentrations were determined by HPLC. Table 2 demonstrates the levels of CE and drugs present in extracts of all three cell lines after treatment with 100 µM CPT-11. At all concentrations of CPT-11 tested, the intracellular levels of the parent drug were similar among all cells, even after treatment with reserpine, suggesting that CPT-11 seems to be a poor substrate for ABCG2 (data not shown). However, in ABCG2#4 cells, the levels of SN-38 were approximately 2.4-fold lower than that observed in control cells. In addition, reserpine increased the intracellular concentration of SN-38 by 79%, which is consistent with the hypothesis that this drug is a substrate for ABCG2 and that the transport activity can be inhibited by reserpine.
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ABCG2#7 cells contained much higher levels of CE activity, which resulted in greater intracellular concentrations of SN-38. However, if these values are corrected for the amounts of CE protein present in the extracts, drug concentrations similar to that observed in pcDNA#2 are seen (Table 2). Because lower levels of ABCG2 are expressed in ABCG2#7, these data correlate with the results observed in the Western analyses (Fig. 1). No marked differences in drug concentrations were seen in the presence of reserpine, suggesting that the levels of ABCG2 expression in ABCG2#7 cells were not sufficient to significantly change SN-38 efflux.
ABCG2 Mediates Resistance to CPT-11 in Cultured Cells. Having
determined that reduced intracellular concentrations of SN-38 were present in
cells expressing ABCG2, we sought to determine what contribution this would
have in CPT-11-mediated cytotoxicity. We therefore performed drug survival
assays using cells expressing CE either with or without ABCG2. pcDNA#2 and
ABCG2#4 cells were transduced with AdCMVrCE at an m.o.i. of 5, and after 4
days, cells were harvested and CE activities were determined. Cells were
divided into two aliquots; one was treated with CPT-11, and growth inhibition
was assessed. The second aliquot was incubated with 5 µM reserpine for 1 h
before CPT-11 treatment. Table
3 indicates the CE activity and the IC50 values for
CPT-11 for these cells. As can be seen, ABCG2#4 cells are greater than
160-fold more resistant to CPT-11 compared with the control cell line
(pcDNA#2), and this can partly be overcome by reserpine. Within the same cell
line, reserpine made little difference in the sensitivity of pcDNA#2 cells to
CPT-11. This is presumably because there is no ABCG2 present in these cells.
With ABCG2#4, reserpine reduced the IC50 value by 60 µM,
demonstrating that cytotoxicity could be partially overcome by inhibition of
ABCG2 function.
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To confirm the above studies, we performed growth-inhibition studies with pcDNA#2 and ABCG2#4 cells using SN-38 as the drug. These results are indicated in Fig. 5. Because SN-38 is 100- to 1000-fold more cytotoxic than CPT-11, the IC50 values for the former drug are markedly reduced compared with results for the latter (Table 3). Data in Fig. 5 demonstrate that cells expressing ABCG2 are 18-fold more resistant to SN-38 than are those expressing pcDNA#2. The addition of reserpine at 10 µM and 20 µM reduced the IC50 values from 3.6 to 1.4 and 2.7 µM, respectively, essentially confirming the inhibitory activity of this molecule on the ABCG2 transporter. It should be recalled that at 20 µM reserpine, only 80% inhibition of ABCG2 is observed in ABCG2#4 cells (Fig. 4A), which would probably only result in partial reversal of drug sensitivity to SN-38. Hence, super-imposition of the survival curves for pcDNA#2 and ABCG2#4 cells would not occur at these concentrations of inhibitor.
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Overall, these data indicate three points. First, overexpression of ABCG2 results in modest CPT-11 resistance but marked resistance to SN-38. Second, high-level overexpression of rCE, an enzyme highly efficient at drug activation, cannot overcome this phenotype. Third, reserpine is an inhibitor of the ABCG2 protein.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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;
Meck et al., 2001;
Wierdl et al., 2001). The
studies presented here indicate that in cells which overexpress ABCG2, such an
approach may be unsuccessful. However, it is currently unclear what the levels
of ABCG2 are in both naïve and drug-treated human tumors, and with the
development of selective inhibitors of this protein, improvements in
chemotherapy may be possible.
In ABCG2, the normal allele seems to contain an arginine at amino acid 482,
as determined by DNA sequence analysis of ABCG2 from different ethnic groups
(Zamber et al., 2003). During
drug selection, it has been hypothesized that a loss of charge at this
position is critical for a change in substrate specificity
(Allen et al., 2002). Loss of
arginine at this position changes the substrate specificity in both human and
rodent ABCG2 and further underscores the pivotal role of this amino acid in
substrate selection. Mutations within ABCG2 at codon 482 have been identified
in drug-selected cells during prolonged exposure to high concentrations of
these agents (Bates et al.,
2001; Allen et al.,
2002). However, conflicting data exist in the literature
concerning the true nature of the resistance properties of cells harboring
ABCG2 with either noncharged amino acids (e.g., Gly and Thr) or arginine. For
instance, in a recent publication, the expression of wild-type ABCG2 (arginine
at position 482) was achieved in cells, but drug resistance to topotecan or
mitoxantrone was not observed (Komatani et
al., 2001). This discrepancy could be caused by either intrinsic
properties of the recipient cells, such as the presence of other transporters
known to play a role in transporting these substrates (e.g., P-gp or MRP2), or
by the levels of ABCG2 protein expressed. Thus, because the mutation of amino
acid 482 seems to be present only in cells exposed to selective concentrations
of drug and is not found in the general population
(Zamber et al., 2003), it was
important to determine whether wild-type ABCG2 could transport drugs such as
mitoxantrone and camptothecin analogs.
Because the ABCG2 protein used for the studies described herein contains Arg-482, our results indicate that overexpression of this protein produces resistance to SN-38 and, hence, to CPT-11. Moreover, we provide direct evidence that SN-38 is a substrate for the human ABCG2 transporter. These studies also indicate that expression of the transporter (as observed in ABCG2#4) results in more than a 160-fold increase in the IC50 value for CPT-11. Because drug resistance in vivo is believed to be caused by very small changes in cell sensitivity (as low as a 2-fold change in the IC50 value), potentially, very marked resistance to therapeutic agents might be achieved by small increases in ABCG2 expression.
A number of different inhibitors of ABC transporters have been developed to
inhibit MRP1 and MDR1, with more recent efforts directed toward identifying
ABCG2 inhibitors (Bates et al.,
2001). We found that ABCG2 transport of Hoechst 33342 was
partially inhibited by the MRP1 inhibitor MK571. MK571 is a potent MRP1
inhibitor, and complete inhibition of transport by this protein has been
observed in the presence of 10 µM drug samples
(Renes et al., 2000). However,
at this concentration, MK571 only inhibits 10 and 25% inhibition of Hoechst
33342 efflux in ABCG2#4 and ABCG2#7 cells, respectively
(Fig. 4B). Overall, these
results demonstrate that MK571 is a poor inhibitor of ABCG2.
Cyclosporine A and its potent derivative PSC833 inhibit P-glycoprotein but have marginal effects on ABCG2. Even at 20 µM, CSA was ineffective in inhibiting Hoechst 33342 dye export in ABCG2#4 cells (Fig. 4C). In contrast, 50% inhibition was seen in ABCG2#7 cells. This strongly supports the concept that ABCG2 inhibition is dependent on the amount of protein expressed.
Of all of the compounds tested, reserpine was the most efficacious at inhibiting ABCG2-mediated Hoechst dye transport. However, the analogs yohimbine and corynanthine were ineffective. Because the most prominent difference between the latter compounds and reserpine is the absence of a benzoyl ring, structure-activity relationships can be developed that will assist in the design of potentially novel ABCG2 inhibitors. Thus, exploiting reserpine as a basis for a lead compound in the development of ABCG2 inhibitors seems possible.
Growth-inhibition studies with cells expressing ABCG2 using SN-38 as the
drug indicated that significant decreases in cytotoxicity were observed
compared with control cells. This drug resistance could be partially overcome
by reserpine in a dose-dependent fashion
(Fig. 5). However, even at
concentrations of 20 µM reserpine, ABCG2-expressing cells were still 7-fold
more resistant than cells lacking expression of the transporter. This is in
part because at this concentration of reserpine, only 80% inhibition of
ABCG2 is observed in ABCG2#4 cells. However, a 7-fold increase in drug
resistance would almost certainly result in the development of insensitive
tumor cells in vivo, and it is apparent, therefore, that more potent and
potentially more specific inhibitors of ABCG2 might be necessary. Ultimately,
the contribution of ABCG2 to drug resistance in patients treated with
camptothecin analogs will have to be evaluated to assess whether modulation of
this efflux function could be a viable approach to improving cancer
therapy.
Overall, our studies demonstrate the role of ABCG2 in the resistance to CPT-11, probably via the specific efflux of SN-38, and indicate the potential reversal of this phenotype by reserpine. However, enforced overexpression of rCE, which can efficiently activate CPT-11, could not overcome this drug resistance. Hence, an analysis of expression of ABCG2 in tumors of patients undergoing or about to undergo therapy with topotecan or CPT-11, might indicate the potential for resistance to these agents. Additionally, the development of specific inhibitors of ABCG2 may prove useful in overcoming any observed resistance to topotecan or CPT-11 in vivo.
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Footnotes |
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ABBREVIATIONS: CPT-11, 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin; SN-38, 7-ethyl-10-hydroxycamptothecin; ABC, ATP-binding cassette; CE, carboxylesterase; rCE, rabbit liver carboxylesterase; CSA, cyclosporin A1; HPLC, high-performance liquid chromatography; m.o.i., multiplicity of infection; P-gp, P-glycoprotein; "E" gate, efflux gate; EST, expressed sequence tag; PBS, phosphate-buffered saline; FACS, fluorescence-activated cell sorting; AdCMVrCE, replication-deficient adenovirus expressing rCE; mutABCG2, ABCG2 containing a mutation in the Walker A domain; MK571, 3-([(3-(2-[7-chloro-2-quinolinyl]ethenyl)phenyl)-((3-dimethylamino-3-oxopropyl)-thio)-methyl]thio)propanoic acid; PSC833, 3'-oxo-4-butenyl-4-methyl-(Thr1)-(Val2)-cyclosporin.
Address correspondence to: Dr. Philip M. Potter, Department of Molecular Pharmacology, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105. E-mail: phil.potter{at}stjude.org
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|---|
Allikmets R, Schriml LM, Hutchinson A, Romano Spica V, and Dean M
(1998) A human placenta-specific ATP-binding cassette gene (ABCP)
on chromosome 4q22 that is involved in multidrug resistance. Cancer
Res 58:
5337–5539.
Baker L, Khan R, Lynch T, Savaraj N, Sandler A, Feun L, Schaser R, Hanover C, and Petit R (1997) Phase II study of irinotecan (CPT-11) in advanced non-small cell lung cancer (NSCLC) (Abstract). Proceedings of the American Society for Clinical Oncology 16: 1658.
Bates SE, Robey R, Miyake K, Rao K, Ross DD, and Litman T (2001) The role of half-transporters in multidrug resistance. J Bioenerg Biomembr 33: 503–511.[CrossRef][Medline]
Beaufay H, Amar-Costesec A, Feytmans E, Thines-Sempoux D, Wibo M,
Robbi M, and Berthet J (1974) Analytical study of microsomes and
isolated subcellular membranes from rat liver. I. Biochemical methods.
J Cell Biol 61:
188–200.
Conti JA, Kemeny NE, Saltz LB, Huang Y, Tong WP, Chou TC, Sun M,
Pulliam S, and Gonzalez C (1996) Irinotecan is an active agent in
untreated patients with metastatic colorectal cancer. J Clin
Oncol 14:
709–715.
Danks MK, Morton CL, Krull EJ, Cheshire PJ, Richmond LB, Naeve CW,
Pawlik CA, Houghton PJ, and Potter PM (1999) Comparison of
activation of CPT-11 by rabbit and human carboxylesterases for use in
enzyme/prodrug therapy. Clin Cancer Res
5:
917–924.
Danks MK, Morton CL, Pawlik CA, and Potter PM (1998)
Overexpression of a rabbit liver carboxylesterase sensitizes human tumor cells
to CPT-11. Cancer Res
58:
20–22.
Doyle LA, Yang W, Abruzzo LV, Krogmann T, Gao Y, Rishi AK, and Ross
DD (1998) A multidrug resistance transporter from human MCF-7
breast cancer cells. Proc Natl Acad Sci USA
95:
15665–15670.
Furman WL, Stewart CF, Poquette CA, Pratt CB, Santana VM, Zamboni
WC, Bowman LC, Ma MK, Hoffer FA, Meyer WH, et al. (1999) Direct
translation of a protracted irinotecan schedule from a xenograft model to a
Phase I trial in children. J Clin Oncol
17:
1815–1824.
Guichard S, Morton CL, Krull EJ, Stewart CF, Danks MK, and Potter PM (1998) Conversion of the CPT-11 metabolite APC to SN-38 by rabbit liver carboxylesterase. Clin Cancer Res 4: 3089–3094.[Abstract]
Higgins CF (2001) ABC transporters: physiology, structure and mechanism, an overview. Res Microbiol 152: 205–210.[Medline]
Honjo Y, Hrycyna CA, Yan QW, Medina Perez WY, Robey RW, van de Laar
A, Litman T, Dean M, and Bates SE (2001) Acquired mutations in
the MXR/BCRP/ABCP gene alter substrate specificity in
MXR/BCRP/ABCP-overexpressing cells. Cancer Res
61:
6635–6639.
Keppler D, Jedlitschky G, and Leier I (1998) Transport function and substrate specificity of multidrug resistance protein. Methods Enzymol 292: 607–616.[Medline]
Khanna R, Morton CL, Danks MK, and Potter PM (2000)
Proficient metabolism of CPT-11 by a human intestinal carboxylesterase.
Cancer Res 60:
4725–4728.
Komatani H, Kotani H, Hara Y, Nakagawa R, Matsumoto M, Arakawa H,
and Nishimura S (2001) Identification of breast cancer resistant
protein/mitoxantrone resistance/placenta-specific, ATP-binding cassette
transporter as a transporter of NB-506 and J-107088, topoisomerase I
inhibitors with an indolocarbazole structure. Cancer
Res 61:
2827–2832.
Kudoh S, Fujiwara Y, Takada Y, Yamamoto H, Kinoshita A, Ariyoshi Y, Furuse K, and Fukuoka M (1998) Phase II study of irinotecan combined with cisplatin in patients with previously untreated small-cell lung cancer. West Japan Lung Cancer Group. J Clin Oncol 16: 1068–1074.[Abstract]
Litman T, Brangi M, Hudson E, Fetsch P, Abati A, Ross DD, Miyake K, Resau JH, and Bates SE (2000) The multidrug-resistant phenotype associated with overexpression of the new ABC half-transporter, MXR (ABCG2). J Cell Sci 113: 2011–2021.[Abstract]
Maliepaard M, Scheffer GL, Faneyte IF, van Gastelen MA, Pijnenborg
AC, Schinkel AH, van De Vijver MJ, Scheper RJ, and Schellens JH
(2001) Subcellular localization and distribution of the breast
cancer resistance protein transporter in normal human tissues.
Cancer Res 61:
3458–3464.
Meck M, Wierdl M, Wagner L, Burger R, Harris LC, Potter PM, and
Danks MK (2001) A VDEPT approach to purging neuroblastoma cells
from hematopoeitic cells using adenovirus encoding rabbit carboxylesterase and
CPT-11. Cancer Res 61:
5083–5089.
Morton CL, Wadkins RM, Danks MK, and Potter PM (1999)
CPT-11 is a potent inhibitor of acetylcholinesterase but is rapidly catalyzed
to SN-38 by butyrylcholinesterase. Cancer Res
59:
1458–1463.
Morton CL, Wierdl M, Oliver L, Ma M, Danks MK, Stewart CF, Eiseman
JL, and Potter PM (2000) Activation of CPT-11 in mice:
identification and analysis of a highly effective plasma esterase.
Cancer Res 60:
4206–4210.
Ozvegy C, Litman T, Szakacs G, Nagy Z, Bates S, Varadi A, and Sarkadi B (2001) Functional characterization of the human multidrug transporter, ABCG2, expressed in insect cells. Biochem Biophys Res Commun 285: 111–117.[CrossRef][Medline]
Potter PM, Pawlik CA, Morton CL, Naeve CW, and Danks MK (1998a) Isolation and partial characterization of a cDNA encoding a rabbit liver carboxylesterase that activates the prodrug Irinotecan (CPT-11). Cancer Res 52: 2646–2651.
Potter PM, Wolverton JS, Morton CL, Wierdl M, and Danks MK
(1998b) Cellular localization domains of a rabbit and a human
carboxylesterase: influence on irinotecan (CPT-11) metabolism by the rabbit
enzyme. Cancer Res 58:
3627–3632.
Renes J, de Vries EE, Hooiveld GJ, Krikken I, Jansen PL, and Muller M (2000) Multidrug resistance protein MRP1 protects against the toxicity of the major lipid peroxidation product 4-hydroxynonenal. Biochem J 350: 555–561.
Rocchi E, Khodjakov A, Volk EL, Yang CH, Litman T, Bates SE, and Schneider E (2000) The product of the ABC half-transporter gene ABCG2 (BCRP/MXR/ABCP) is expressed in the plasma membrane. Biochem Biophys Res Commun 271: 42–46.[CrossRef][Medline]
Satoh T, Hosokawa M, Atsumi R, Suzuki W, Hakusui H, and Nagai E (1994) Metabolic activation of CPT-11, 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin, a novel antitumor agent, by carboxylesterase. Biol Pharm Bull 17: 662–664.[Medline]
Tanizawa A, Fujimori A, Fujimori Y, and Pommier Y
(1994) Comparison of topoisomerase I inhibition, DNA damage and
cytotoxicity of camptothecin derivatives presently in clinical trials.
J Natl Cancer Inst 86:
836–842.
Ueda K, Okamura N, Hirai M, Tanigawara Y, Saeki T, Kioka N, Komano
T, and Hori R (1992) Human P-glycoprotein transports cortisol,
aldosterone and dexamethasone, but not progesterone. J Biol
Chem 267:
24248–24252.
Van Hattum AH, Schluper HM, Hausheer FH, Pinedo HM, and Boven E (2002) Novel camptothecin derivative BNP1350 in experimental human ovarian cancer: determination of efficacy and possible mechanisms of resistance. Int J Cancer 100: 22–29.[Medline]
Wagner LM, Guichard SM, Burger RA, Morton CL, Straign CM, Ashmun
RA, Harris LC, Houghton PJ, Potter PM, and Danks MK (2002)
Efficacy and toxicity of a virus-directed enzyme prodrug therapy purging
method: preclinical assessment and application to bone marrow samples from
neuroblastoma patients. Cancer Res
62:
5001–5007.
Wierdl M, Morton CL, Harris LC, Danks MK, Schuetz JD, and Potter PM
(2003) p53-Mediated regulation of expression of a rabbit liver
carboxylesterase confers sensitivity to CPT-11. J Pharmacol Exp
Ther 304:
699–705.
Wierdl M, Morton CL, Weeks JK, Danks MK, Harris LC, and Potter PM
(2001) Sensitization of human tumor cells to CPT-11 via
adenoviral-mediated delivery of a rabbit liver carboxylesterase.
Cancer Res 61:
5078–5082.
Yang CP, Cohen D, Greenberger LM, Hsu SI, and Horwitz SB
(1990) Differential transport properties of two mdr gene products
are distinguished by progesterone. J Biol Chem
265:
10282–10288.
Zamber CP, Lamba JK, Yasuda K, Farnum J, Thummel K, Schuetz JD, and Schuetz EG (2003) Natural allelic variants of breast cancer resistance protein (BCRP) and their relationship to BCRP expression in human intestine. Pharmacogenetics 13: 19–28.[CrossRef][Medline]
Zhou S, Morris JJ, Barnes Y, Lan L, Schuetz JD, and Sorrentino BP
(2002) Bcrp1 gene expression is required for normal numbers of
side population stem cells in mice and confers relative protection to
mitoxantrone in hematopoietic cells in vivo. Proc Natl Acad Sci
USA 99:
12339–12344.
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