SEARCH

SEARCH BY CITATION

Keywords:

  • multidrug resistance protein;
  • pancreatic carcinoma;
  • ABCC family;
  • MRP

Abstract

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Pancreatic ductal adenocarcinoma is among the top 10 causes of death from cancer in industrialized countries. In comparison with other gastrointestinal malignancies, pancreatic cancer is one of the tumors most resistant to chemotherapy. An important mechanism of tumor multidrug resistance is increased drug efflux mediated by several transporters of the ABC superfamily. Especially BCRP (ABCG2), MDR1 P-glycoprotein (ABCB1) and members of the MRP (ABCC) family are important in mediating drug resistance. The MRP family consists of 9 members (MRP1–MRP9) with MRP1–MRP6 being best characterized with respect to protein localization and substrate selectivity. Here, we quantified the mRNA expression of BCRP and of all MRP family members in normal human pancreas and pancreatic carcinoma and analyzed the mRNA level of the transporters most abundantly expressed in pancreatic tissue, BCRP, MRP1, MRP3, MRP4 and MRP5, in 37 tissue samples. In addition, we determined the localization of the 4 MRP proteins in normal human pancreas and in pancreatic carcinoma. The expression of BCRP, MRP1 and MRP4 mRNA did not correlate with tumor stage or grading. On the other hand, the expression of MRP3 mRNA was upregulated in pancreatic carcinoma samples and was correlated with tumor grading. The MRP5 mRNA level was significantly higher in pancreatic carcinoma tissue compared to normal pancreatic tissue. These data suggest that MRP3 and MRP5 are involved in drug resistance of pancreatic tumors and that quantitative analysis of their expression may contribute to predict the benefit of chemotherapy in patients with pancreatic cancer. © 2005 Wiley-Liss, Inc.

Pancreatic cancer is the 4th to 5th leading cause of cancer-related death in most Western industrialized countries1 with an average survival after diagnosis of 3 to 6 months. In Europe, it is the 8th most common cancer with approximately 74,000 newly diagnosed cases per year.2 In spite of impressive advances in the field of diagnostic imaging of the pancreas, the availability of numerous tumor markers and an aggressive therapeutic approach, the prognosis of pancreatic carcinoma continues to be poor, with less than 5% surviving beyond 5 years. Surgical resection is possible in up to 40% of the patients with localized disease, but even in this group of patients, prognosis is relatively poor.3, 4 Most treatment failures are due to local recurrence, hepatic metastases or both and occur within 1 to 2 years after surgery.5, 6 Adjuvant therapy may improve long-term survival7, 8, 9, 10 but its routine use is not universal9 because the results of randomized trials have been inconclusive.8 In case of nonresectable pancreatic carcinomas infiltrating the retroperitoneal plexi or the superior mesenteric artery, chemotherapy might be the option of choice for the treatment. Until now, much impact on survival has not been achieved regarding the different chemotherapies, with maximum median survival times lying between 4 and 9 months and relatively low response rates.11, 12 Therefore, in comparison with other gastrointestinal malignancies, pancreatic cancer seems to be one of the most resistant tumors to chemotherapy. This fact underscores the urgency to find novel therapeutic strategies to understand the mechanism of drug resistance in pancreatic carcinoma in order to develop more effective drugs in the future.

Drug resistance is attributable to several processes taking place in neoplastic cells. One of these processes is the decreased accumulation of drugs within cancer cells because of increased drug efflux. Proteins mediating this drug efflux mostly belong to the large superfamily of ABC transporters. Especially members of the ABCB family including MDR1 P-glycoprotein13 and members of the ABCC family14, 15, 16 have been shown to be responsible for mediating multidrug resistance. In addition, the BCRP (ABCG2) protein, a member of the ABCG family was shown to mediate resistance against several anticancer drugs.17 Substrates for BCRP include mitoxantrone, methotrexate and topoisomerase I inhibitors.17 The human ATP-binding cassette transporter family C (symbol ABCC) consists of 12 members, 9 of which comprise the group of multidrug resistance proteins (MRP1–MRP9; ABCC1–ABCC6 and ABCC10–ABCC12).14, 15, 16 MRPs are integral membrane proteins mediating the ATP-dependent export of organic anions out of cells. So far, the family members MRP1–MRP6 are the best characterized paralogs with respect to their substrate spectrum. MRP1, MRP2, MRP3 and MRP6 transport lipophilic compounds conjugated with glutathione, glucuronate or sulfate.14, 15, 16, 18 Substrates for MRP4 and MRP5 include cyclic nucleotides and nucleotide analogs.19, 20, 21 Furthermore, MRP4 has been identified as a cotransporter for reduced glutathione with bile salts22 and as a transporter for prostaglandins23 and the steroid dehydroepiandrosterone-3-sulfate (DHEAS).24 In addition to endogenous compounds, MRP family members are able to export a variety of organic anions of toxicological relevance and are important in conferring resistance to cytotoxic and antiviral drugs.19, 20, 23 Whereas the expression of MDR1 P-glycoprotein has been analyzed in detail in pancreatic carcinoma,25, 26 the knowledge on the expression and localization of MRP family members and of BCRP is very limited. In pancreas the expression of MRP3 (ABCC3) has been analyzed by Northern blotting and RT-PCR.27, 28, 29, 30 In addition, the MRP3 protein has been detected in normal pancreatic tissue and in pancreatic adenocarcinoma.30 Furthermore, the protein expression of MRP1 (ABCC1) and MRP2 (ABCC2) has been analyzed in rat and human tissue samples with chronic pancreatitis.31

In our study, we analyzed the mRNA expression of all 9 human MRP (ABCC) family members and of BCRP (ABCG2) in normal pancreatic tissue and pancreatic carcinoma. Based on these data, we subsequently quantified the mRNA expression of BCRP (ABCG2) and of the 4 most abundant MRP family members MRP1 (ABCC1), MRP3 (ABCC3), MRP4 (ABCC4) and MRP5 (ABCC5) by semiquantitative real-time RT-PCR in 31 pancreatic carcinoma samples and in 6 samples from healthy tissue donors and correlated these mRNA expression data to clinical parameters. In addition, we studied the localization of the 4 MRP family members by immunofluorescence analysis. These expression and localization studies contribute to the understanding of the role of drug transporters in patients with normal pancreatic tissue and in pancreatic carcinoma thus supporting the understanding of the impact of these transporters in causing intrinsic multidrug resistance.

Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Patients and tissue collection

Normal human pancreatic tissue was obtained through an organ donor program from 6 individuals who were free of any pancreatic disease. The median age of the organ donors was 44 years (percentiles 35/50). Pancreatic cancer tissue samples were obtained from 31 patients (14 women and 17 men) undergoing a pylorus-preserving Whipple resection for ductal adenocarcinoma of the pancreas. The median age of the patients with pancreatic cancer was 63 years (percentiles 58/71). According to the international classification of the UICC, there were 8 stage I, 5 stage II, 10 stage III and 8 stage IV pancreatic cancers. Tumor grading was well differentiated in 7 cases, moderately differentiated in 17 cases and undifferentiated in 7 cases. The median survival time of all patients together regardless of their tumor stage was 12 months (percentiles 5.8/25.3). None of the patients received chemotherapy before surgery. In pancreatic carcinomas with early tumor stages (I + II) the median survival was 20 months (percentiles 10.5/31.5), in late tumor stages (III + IV) the median survival was only 10 months (percentiles 5.0/21.8).

Freshly removed tissue samples were cut in the operating room and randomly divided for histologic analysis (immediately fixed in paraformaldehyde solution for 12–24 hr and paraffin-embedded for immunohistochemistry) or were snap-frozen in liquid nitrogen and maintained at −80°C until further analysis. Studies were approved by the Human Ethics Committee of the Universities of Bern and Heidelberg.

Antibodies

The polyclonal antisera EAG5,32 FDS,28 SNG22 and AMF19 were raised in rabbits against the carboxy-terminal sequences of human MRP2, MRP3, MRP4 and MRP5, respectively. The EAG5, SNG and AMF antisera were affinity-purified as described33 using MRP2-,34 MRP4-22 and MRP5-expressing19 cells. The monoclonal mouse antibodies QCRL1 against MRP1, M2III-6 against MRP2 and M3II-9 against MRP3 were from Alexis Biochemicals (Günzburg, Germany). The monoclonal rat M6II-31 antibody against MRP6 was a kind gift of Dr. G. Scheffer (Free University Medical Center, Amsterdam, The Netherlands) and is now commercially available from Alexis. Alexa Fluor488-conjugated goat anti-rabbit, anti-mouse or anti-rat IgG were from Molecular Probes (Eugene, OR).

Immunofluorescence microscopy

Cryosections (4–5 μm) were prepared with a cryotome (Leica, Bensheim, Germany), air-dried for at least 2 hr and fixed in precooled acetone (−20°C for 10 min). Immunofluorescence staining was carried out as described35 with antibodies or antisera diluted in phosphate-buffered saline (PBS). Antisera FDS and AMF were diluted 1:50 and the fluorochrome-conjugated antibodies 1:300. The monoclonal antibodies and the affinity-purified antibodies were used at the following final concentrations: QCRL1 1–1.5 μg/ml, M2III-6 5 μg/ml, M3II-9 10–13 μg/ml, M6II-31 0.2 mg/ml, affinity-purified EAG5 0.5–1 mg/ml, affinity-purified SNG 0.3–0.5 mg/ml and affinity-purified AMF 0.5–1 mg/ml. Pictures were taken on a confocal laser scanning microscope (LSM510, Carl Zeiss, Jena, Germany). Some cryosections were stained with hematoxylin/eosin and photographed with a digital video camera (Hamamatsu, Hamamatsu, Japan) on an Axiovert S100TV (Carl Zeiss). Pictures were analyzed using Openlab imaging software (Improvision, Coventry, UK).

Real-time semiquantitative RT-PCR

Total RNA was isolated by the single-step guanidinium method. Total RNA (1 μg) was reverse-transcribed using 2.5 μg oligo(dT)18 primer as described.28 Synthesized single-stranded DNAs were purified using Microcon 100 (Millipore, Billerica, MA) filter units. BCRP (ABCG2) and MRP1–MRP9 (ABCC1–ABCC6 and ABCC10–ABCC12) mRNA levels in relation to β-actin mRNA levels were determined using the LightCycler™ System and the FastStart DNA Master SYBR Green I kit (both from Roche, Mannheim, Germany). PCRs were performed according to the manufacturer's instructions with 0.5 μM of the respective sense and 0.5 μM of the respective antisense primers, 4 mM MgCl2, and 1-fold LightCycler-FastStart DNA Master SYBR Green I mix in a total volume of 20 μl including 1 μl of the synthesized sscDNA. Cycling conditions were as follows: 10 min denaturation at 94°C, followed by 45 cycles of 10 sec denaturation at 94°C, 15 sec primer annealing at 64°C and 30 sec of fragment elongation at 72°C. Sequence and position of MRP-specific primers used in this analysis and the amplified fragment lengths are summarized in Table I. For β-actin amplification, the sense primer oActin.for (5′-TGACGGGGTCACCCACACTGTGCCCATCTA-3′) and the antisense primer oActin.rev (5′-CTAGAAGCATTTGCGGTGGAC-GATGGAGGG-3′) was used. The amount of β-actin and MRP1 to MRP9 single-stranded cDNAs was determined as described35 using a serial plasmid dilution (human MRP5 cDNA in pcDNA3.1/Hygro19); from 1 × 106 to 1 × 103 fg as amplification standard. The β-actin mRNA concentration, calculated in relation to the standard curve, was set to 100% and the respective BCRP and MRP mRNA value is given as a percentage of β-actin amplification.

Table I. Primer Sequences, Positions and Length of the Amplified Fragment used for Semiquantitative Real-Time RT-PCR
 Sense primerPositionAntisense primerPositionFragment length (bp)
MRP1 (ABCC1)5′-CTGACAAGCTAGACCATGAATGT-3′bp 4244 – bp 42695′-TCACACCAAGCCGGCGTCTTT-3′bp 4596 – bp 4576353
MRP2 (ABCC2)5′-CTTCGGAAATCCAAGATCCTGG-3′bp 4354 – bp 43755′-TAGAATTTTGTGCTGTTCACATTCT-3′bp 4637 – bp 4613284
MRP3 (ABCC3)5′-GGACCCTGCGCATGAACCTG-3′bp 4133 – bp 41525′-AGGCAAGTCCAGCATCTCTGG-3′bp 4582 – bp 4562450
MRP4 (ABCC4)5′-GGATCCAAGAACTGATGAGTTAAT-3′bp 3621 – bp 36445′-TCACAGTGCTGTCTCGAAAATAG-3′bp 3978 – bp 3956358
MRP5 (ABCC5)5′-GCTGTTCAGTGGCACTGTCAG-3′bp 3834 – bp 38545′-TCAGCCCTTGACAGCGACCTT-3′bp 4314 – bp 4294481
MRP6 (ABCC6)5′-CACTGCGCTCCAGGATCAGC-3′bp 4010 – bp 40295′-CAGACCAGGCCTGACTCCTG-3′bp 4511 – bp 4492502
MRP7 (ABCC10)5′-AGGACAGGGCCTTGTGGCAG-3′bp 4043 – bp 40625′-TCAGGGACCTCCGAGTGAGG-3′bp 4479 – bp 4460437
MRP8 (ABCC11)5′-GAAGTCCTCCTTGGGCATGGC-3′bp 3540 – bp 35605′-TTATCTCAGTGAAGAAGTGGCTGT-3′bp 4149 – bp 4126610
MRP9 (ABCC12)5′-AGAGACACAATAATGAAACTCCCA-3′bp 3706 – bp 37295′-CTACAATCTGACTTCTGCTGCTA-3′bp 4080 – bp 4058375
BCRP (ABCG2)5′-CAAAGGCAGATGCCTTCTTCG-3′bp 1502 – bp 15225′-CATACTGAATTAAGGGGAAATTTAA-3′bp 1989 – bp 1965488

Statistical analysis

The age and survival of all patients are reported as median together with the percentiles. Furthermore, we analyzed the median of MRP3 and MRP5 mRNA levels according to the 4 tumor stages (UICC), and according to the different tumor grade (I, II and III).

Differences between groups were analyzed using the Kruskal-Wallis and Mann-Whitney tests for non-parametric data with p < 0.05 considered statistically significant. For survival analysis related to MRP3 and MRP5 expression the log-rank test was used. The correlation of MRP3 and MRP5 mRNA levels with tumor grading and tumor stage was analyzed using the Pearson test.

Results

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Expression of BCRP and MRP family member mRNAs in human pancreas and pancreatic carcinoma

The first aim was to investigate the mRNA expression of BCRP and of all 9 human MRP family members in normal human pancreas and pancreatic carcinoma. This expression analysis was performed using the LightCycler™ system and primer pairs directed against BCRP and each of the MRP paralogs. These primer pairs (Table I) were tested for their specificity and affinity against the respective cDNA template to ensure that this experimental setup can be used not only for detecting a specific BCRP or MRP mRNA but also for quantifying the amount of the respective mRNA in a given sample. Using these primer pairs and RNA from normal pancreatic tissue and 4 different pancreatic carcinoma samples cDNA fragments of the expected sizes were amplified for BCRP, for MRP1 to MRP5 and MRP7 (Fig. 1). For MRP6 mRNA, only very weak amplification products of the expected size were detected (Fig. 1). The MRP7 mRNA level was low in all samples investigated (<<0.1% β-actin mRNA), whereas no amplification product was observed for MRP8 mRNA. MRP9 mRNA was detected only in normal human pancreas as indicated by a weak amplification product (Fig. 1) with a mRNA level below 0.1% of β-actin mRNA. Because the MRP family members MRP1 to MRP6 are best characterized with respect to protein localization and transport properties, with MRP1 to MRP5 being expressed in human pancreas, and because MRP7 and MRP9 were only weakly amplified, we further quantified the mRNA levels of the MRP family members MRP1 to MRP5 in 37 tissue samples from normal pancreas and pancreatic carcinoma.

thumbnail image

Figure 1. Agarose gel electrophoresis analysis of BCRP and MRP family member mRNA expression in normal human pancreas and pancreatic carcinoma. RT-PCR was performed using the LightCycler™ system as described and the primer pairs specific for BCRP and each MRP family member as presented in Table I. Tissue samples were obtained from normal human pancreas (Pa normal) and from 4 different pancreatic carcinoma samples ranging from tumor stage I to stage IV (Pa-Ca S1–Pa-Ca S4).

Download figure to PowerPoint

For BCRP, the mRNA values varied between lower than 0.1% and 18.7% of the respective β-actin value, with most values being lower than 1% of the β-actin mRNA (Table II). The values of MRP1 mRNA levels were between 0.1% and 3.0% of the quantity of the β-actin mRNA levels of the respective sample (Table II). Amplified MRP2 cDNA fragments were detected in 32 of 37 samples but in all cases the amount of the MRP2 mRNA in relation to β-actin was below 0.1% confirming the weak MRP2 amplification products in the first expression analysis (Fig. 1). Therefore, we did not include the data on MRP2 mRNA quantification into Table II. The quantity of MRP3 mRNA was moderate to high in all samples and varied between 0.3% and 44.0% of the respective β-actin mRNA value. The quantities of MRP4 mRNA varied between 0.2% and 1.6% of the quantity of β-actin mRNA with 1 sample being below 0.1% β-actin mRNA level. MRP5 mRNA was detectable in all 37 samples, with values between 0.2% and 5% of the respective β-actin mRNA level, only in 2 pancreatic carcinoma samples the values in relation to β actin were below 0.1%.

Table II. Clinical Data of Patients with Ductal Pancreatic Carcinoma and Quantification of MRP1, MRP3, MRP4, MRP5 and BCRP mRNA Expression1
Patient numberGenderAgeGStage(UICC)PTPNPMSurvival(months)mRNA expression (%)
MRP1MRP3MRP4MRP5BCRP
  • 1

    G = tumor grading; PT = tumor state; PN = lymph node state; PM = metastasis state; quantification of MRP1, MRP3, MRP4, MRP5 and BCRP mRNA expression (%) is presented as percentage of β-actin mRNA expression.

  • 2

    Normal pancreatic tissue.

12Male50      0.60.70.20.20.2
22Female38      1.22.90.30.20.1
32Male43      0.92.90.20.30.1
42Male25      0.53.60.30.2<0.1
52Female45      1.22.71.00.50.3
62Male50      0.61.40.30.30.3
7Male55II100450.33.90.10.80.6
8Female71II100331.81.81.21.30.2
9Male56II100561.44.70.81.41.1
10Male60III300121.82.81.30.70.4
11Female52IIII300670.70.31.62.40.5
12Female64II400210.31.50.20.30.1
13Female61IIV111 0.31.5<0.10.10.6
14Male62III200 0.616.60.10.80.3
15Male62III100 0.316.90.10.34.9
16Female52IIII300290.94.50.22.89.9
17Female81III200120.27.40.83.05.6
18Male75IIII300 0.66.20.31.60.3
19Female71IIII300 0.49.20.311.10.3
20Male68IIII300301.43.10.51.75.8
21Male65IIIII31021.22.91.00.70.3
22Male79IIIII31050.72.90.10.40.1
23Male76IIIII210690.35.90.32.018.7
24Female77IIIII41020.86.50.32.00.8
25Female64IIIII41031.77.60.41.91.3
26Male61IIIV401213.03.41.04.21.2
27Male38III20032.310.70.41.60.3
28Male63IIIV21151.15.40.71.40.2
29Female71IIIV211 0.55.60.70.80.9
30Male60IIIV31190.14.90.11.07.5
31Female73IIIIII310150.70.40.30.40.5
32Male46IIIIII310891.19.30.50.70.3
33Female60IIIIII21092.044.00.4<0.10.1
34Male70IIIIII31061.832.70.35.01.6
35Female52IIIIV31151.210.50.5<0.12.0
36Female68IIIIV21110.820.40.21.31.4
37Male58IIIIV311 0.815.20.32.00.6

Immunolocalization of MRP family members in normal pancreas and pancreatic carcinoma

Immunofluorescence signals were detected for MRP1, MRP3, MRP4 and MRP5 in normal pancreas and pancreatic carcinoma tissue (Figs. 2, 3). However, no MRP2-positive staining was observed in normal pancreas and pancreatic carcinoma when using the affinity-purified EAG5 antibody (Fig. 2a) or the M2II-6 antibody (not shown), which is in agreement with a study by Sandusky et al.36 Normal pancreas and pancreatic carcinoma were also negative when stained with the M6II-31 antibody, whereas this antibody readily detected MRP6 on liver sections being used as positive control. Similar negative staining results for MRP6 in normal human pancreas were obtained by Scheffer et al.37 In contrast, MRP3 was readily detectable with the FDS antibody (Figs. 2, 3) or the M3II-9 antibody (not shown). MRP3 was predominantly localized in the basolateral membrane of epithelial cells of ducts in normal pancreas and in the cells of ductal pancreatic carcinoma (Fig. 2b, 3b), which is in accordance with a study by Scheffer et al.30 In addition, MRP3 was also present in the plasma membrane of acinar cells in normal pancreas. Similar localizations in duct cells, acinar cells and pancreatic cancer cells were observed for MRP4 and MRP5 (Figs. 2c,d, 3c,d). MRP1 mRNA was detectable in normal pancreas and pancreatic carcinoma (Table II), the source of the MRP1 mRNA probably being fibroblasts of the connective tissue, which showed strong staining (Fig. 3f), rather than acinar cells or pancreatic carcinoma cells, which lacked MRP1 staining (Fig. 3e,f). Because the affinities of the antibodies towards their respective antigen differ from one antibody to the other, the staining intensities do not reflect the exact relative amounts of the MRP proteins to each other. The stainings rather give a qualitative assessment of the localization of different MRP isoforms. However, when normal pancreatic tissue and pancreatic carcinoma samples with high MRP3 mRNA expression levels were analyzed exemplarily with identical settings on a confocal laser scanning microscope (Fig. 3g,h), staining for MRP3 with the FDS antibody was more intense in the carcinoma sample than in normal human pancreas.

thumbnail image

Figure 2. Representative confocal laser scanning micrographs of cryosections of normal human pancreas stained for MRP2, MRP3, MRP4 or MRP5. (a) Cryosections of normal human pancreas were incubated with the affinity-purified EAG5 antibody to detect MRP2, which is apparently not present in normal human pancreas. (a*) Positive control staining for MRP2 was observed in cryosections of human liver with the affinity-purified EAG5 antibody. (b) cryosections of normal human pancreas were incubated with the FDS antibody to detect MRP3, which is present in the basolateral membrane of epithelial cells of ducts (box) and in the acinar cells. (c,d) MRP4 and MRP5 were detected with the affinity-purified SNG and AMF antibodies, respectively, and localized in the plasma membrane of acinar cells and in the basolateral membrane of ductular epithelial cells (boxes). Because the antibodies differ in their affinities towards their respective antigen, the staining intensities do not reflect the relative amounts of one MRP isoform to the other. Bars = 20 μm.

Download figure to PowerPoint

thumbnail image

Figure 3. Immunolocalization of MRP1, MRP3, MRP4 and MRP5 in cryosections of human pancreatic ductal adenocarcinoma. (a) Hematoxylin/eosin-stained section with a ductal adenocarcinoma. (be) Confocal laser scanning micrographs of the same area as in (a), stained with the FDS, SNG, AMF or QCRL1 antibody to detect MRP3, MRP4, MRP5 or MRP1, respectively. MRP3, MRP4 and MRP5 were detectable in ductal adenocarcinoma (green), whereas MRP1 was not. (f) Strong fluorescence signals (green) were however obtained for MRP1 in fibroblasts of the connective tissue. (g) Confocal laser scanning micrograph of normal pancreatic tissue. (h) Micrograph of a pancreatic carcinoma sample with high MRP3 mRNA expression level. Pictures were taken with identical settings demonstrating increased amount of MRP3 protein in the pancreatic carcinoma sample. Nuclei are stained in red with propidium iodide. Bar (identical magnification in af) = 50 μm.

Download figure to PowerPoint

Relationship of MRP3 and MRP5 mRNA expression levels to histopathologic and clinical parameters

The statistical analysis of MRP1, MRP4 and BCRP mRNA in relation to histopathologic and clinical parameters demonstrated that there was no correlation of the mRNA expression level to carcinoma stages or grades. Therefore, these 3 transporters were not included into the statistical analysis. The MRP3 mRNA expression level in pancreatic carcinoma tissue samples was, irrespective of the tumor stage (tumor stage classified by UICC with UICC I = T1-2N0M0, UICC II = T3N0M0 and T1-3N1M0, UICC III = T4N0-1M0, and UICC IV = T1-4N0-1M1 with T = tumor, N = lymph node, and M = metastasis) significantly higher than the MRP3 mRNA level in normal pancreatic tissue (Mann-Whitney test p = 0.02 for UICC stage I and stage II and p = 0.03 for UICC stage II and IV). With regard to the correlation between the MRP3 mRNA expression in lower tumor stages (I+II) with higher stages (III+IV), the mRNA expression did not significantly increase in the higher tumor stages (p = 0.7). The comparison of the histopathological data (tumor grade of differentiation with grade I = highly differentiated, grade II = less-differentiated and grade III = low-differentiated) and the MRP3 mRNA expression data revealed that tumor differentiation was related to MRP3 mRNA expression levels. The Mann-Whitney test showed significant differences of MRP3 mRNA expression levels between grade 1 vs. 2 (p = 0.001), between grade 1 vs. 3 (p = 0.017) and between grade 2 vs. 3 (p = 0.028). Between normal pancreatic tissue and tissue samples with tumor grade 1, there was no significant relationship (p = 1). However, significant differences in MRP3 mRNA expression levels between normal pancreatic tissue and pancreatic carcinoma grade 2 and grade 3 were observed (Mann-Whitney test p = 0.01 or p = 0.035) (Fig. 4). The Kaplan-Meier graph (Fig. 5) shows a difference in survival between patients (n = 8) with a high MRP3 mRNA expression level (>10% β-actin mRNA expression level) and patients (n = 23) with moderate to low MRP3 mRNA expression levels (<10% β-actin mRNA expression level; p = 0.0005). A lower MRP3 mRNA expression was associated with a better survival prognosis.

thumbnail image

Figure 4. Quantification and correlation of MRP3 and MRP5 mRNA expression levels in normal human pancreas (normal) and pancreatic carcinoma tissue samples from different tumor gradings (GI–GIII). Values for the respective mRNAs are given in Table II. p < 0.05.

Download figure to PowerPoint

thumbnail image

Figure 5. Kaplan-Meier curve showing a different survival of patients with a high MRP3 mRNA level (> 10% β-actin mRNA) and patients with a moderate to low MRP3 mRNA level (< 10% β-actin mRNA).

Download figure to PowerPoint

In contrast to the MRP3 mRNA levels, MRP5 mRNA expression levels were not related to tumor grade (Fig. 4) or tumor stage. A correlation in the sense of rising expression of MRP5 mRNA level together with the rising tumor stage could not be shown like for MRP3 mRNA expression levels (Mann-Whitney test p = 0.52). MRP5 mRNA expression levels in pancreatic carcinoma tissue samples were significantly higher than in normal pancreatic tissue samples for the tumor stages I, II and III (UICC stage I: p = 0.005, UICC stage II: p = 0.009, UICC stage III: 0.02). In UICC stage IV, however, there was no significant difference of the MRP5 mRNA expression level in comparison to normal pancreatic tissue (p = 0.1). MRP5 mRNA expression levels in tumors with grading 1 and 3 were similar, and the mRNA level in grade 2 tumors was not significantly different (Fig. 4). Furthermore, the Mann-Whitney test did not show significant differences of MRP5 mRNA expression levels between grade 1 vs. 2 (p = 0.087), between grade 1 vs. 3 (p = 0.8) or between 2 vs. 3 (p = 0.187). In contrast, MRP5 mRNA expression levels in normal pancreatic tissue samples were significantly lower in comparison to pancreatic carcinoma samples.

Discussion

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Increased export of cytotoxic drugs from cells is one major mechanism of drug resistance. Proteins involved in this increased drug efflux mostly belong to the large family of ABC transporters. In our study, we investigated the expression of BCRP, a member of the ABCG family and the expression and localization of members of the MRP (ABCC) family of export pumps in normal human pancreatic tissue and pancreatic carcinoma. Semiquantitative real-time RT-PCR demonstrated the expression of BCRP (ABCG2), MRP1 (ABCC1), MRP3 (ABCC3), MRP4 (ABCC4) and MRP5 (ABCC5) mRNA in normal pancreatic tissue and in pancreatic carcinoma samples with different tumor gradings. In addition, cDNA fragments of the expected length were amplified for MRP2 (ABCC2), MRP6 (ABCC6) and MRP7 (ABCC10), but for these the quantification of the mRNA levels relative to the respective β-actin mRNA levels demonstrated mRNA levels below 0.1% (for MRP6 below 0.01%) and, therefore, may reflect amplification products originating from nonpancreatic cells within the tissue samples used for RNA isolation. MRP9 (ABCC12) was amplified only in normal pancreatic tissue, whereas MRP8 (ABCC11) was neither amplified in normal nor in the pancreatic carcinoma samples (Fig. 1). Based on the semiquantitative RT-PCR data, we subsequently analyzed the expression levels of BCRP, MRP1, MRP3, MRP4 and MRP5 mRNA in 31 different pancreatic carcinoma tissue samples and in 6 samples from normal human pancreas. In addition, the localization of the 4 MRP family members was analyzed.

The quantification of the BCRP and the MRP family member mRNA levels demonstrated that BCRP, MRP1 and MRP4 mRNA levels did not change significantly between normal pancreatic tissue and pancreatic carcinoma samples with different tumor gradings. Differences between samples may thus reflect interindividual variations rather than adaptive mechanisms during tumor development. Furthermore, MRP1 protein was localized only in fibroblasts and not in acinar cells or pancreatic carcinoma cells. Expression of MRP1 protein in human38 and mouse39 fibroblasts has been demonstrated earlier and supports the importance of this export pump in protecting cells of the connective tissue. In contrast, MRP3 mRNA expression was significantly lower in normal pancreatic tissue compared to pancreatic cancer tissue (Fig. 4). The MRP3 protein mediates the efflux of bile salts and several other organic anions out of cells,40, 41, 42 and confers resistance to several anticancer drugs including vincristine, etoposide, teniposide and methotrexate.29, 43, 44, 45 Several of these substances have been used in clinical studies for the treatment of pancreatic cancer.46, 47, 48 Interestingly, in most cases, the beneficial effect was very low. Especially methotrexate treatment had a marginal antitumor activity against pancreatic carcinoma,46, 48 suggesting that upregulation of MRP3 in pancreatic carcinoma may play a role in the resistance of these tumors. The upregulation of MRP3 in pancreatic carcinoma without prior treatment of patients with anticancer drugs could be explained by the fact that MRP3 was found to be an inducible transporter. Studies on the regulation of MRP3 protein and mRNA expression have demonstrated that MRP3 is upregulated in obstructive cholestasis30, 49 and in regenerating liver.50 Liver regeneration is accompanied with dedifferentiation of hepatocytes and a change in cellular polarity. The fact that MRP3 mRNA expression correlated with tumor grading in pancreatic carcinoma may demonstrate this inducible expression during dedifferentiation. Furthermore, a correlation of the MRP3 mRNA level with survival of patients after resection has been observed. Patients with a MRP3 mRNA level below 10% β-actin mRNA expression level have a longer survival period than patients with a higher MRP3 mRNA expression level. Although this correlation between MRP3 mRNA level and survival of patients is demonstrated only in a small group of patients, MRP3 expression may be an important factor for prognosis.

The missing correlation of MRP3 mRNA levels with the tumor stage could be explained by the fact that chemoresistance is supposed to correlate less with tumor extension, reflected by the tumor stage, than with the differentiation of the tumor. Therefore, the inducible nature of MRP3 together with its ability to confer drug resistance suggests that MRP3 is important in mediating resistance and could be a potential marker for chemoresistance in pancreatic adenocarcinoma.

MRP5 mRNA expression levels in pancreatic carcinoma were neither related to tumor grade nor to tumor stage. Therefore, determination of MRP5 mRNA expression may be less predictive for prognosis and chemoresistance. Higher MRP5 mRNA expression levels in pancreatic carcinoma samples compared to normal pancreatic tissue may indicate that MRP5 could account, at least in part, for the intrinsic drug resistance of ductal pancreatic carcinoma. Many chemotherapeutic agents have been tested in the treatment of pancreatic cancer, but only 5-fluorouracil and mitomycin C have been shown to have beneficial effects.51, 52 Therefore, several other cytotoxic agents have been actively investigated in the treatment of pancreatic carcinoma and from these, gemcitabine has become increasingly the chemotherapeutic drug of choice.8, 53, 54 Gemcitabine is a deoxycytidine analog that competes for the incorporation into DNA.55 Nucleoside analogs are an important class of drugs used in the treatment of cancer and viral infections. Several studies have shown that resistance against these drugs can be mediated by a change in uptake transporters and metabolizing enzymes.56, 57 However recent studies suggest that also efflux pumps are important modulators of resistance against nucleoside-based analogs. Thus, it has been shown that nucleotide analogues and cyclic nucleotides are substrates for MRP423, 58, 59 and MRP5.19, 21, 59, 60 Overexpression of one of these export pumps may cause resistance against these anticancer and antiviral agents. Although MRP5-mediated transport of gemcitabine phosphates using inside out-oriented membrane vesicles has not yet been described and MRP5-mediated resistance against gemcitabine is controversally discussed,21, 61 one study demonstrated that HEK293 cells overexpressing the human MRP5 protein are 2-fold more resistant to gemcitabine compared to vector control-transfected cells.61 Therefore, increased MRP5 expression as observed in pancreatic carcinoma samples may influence the chemotherapy following curative resection by mediating resistance against this cytotoxic agent.

In conclusion, we have shown that the ABCG2 family member BCRP and the MRP family members MRP1, MRP3, MRP4 and MRP5 are expressed in human pancreas and pancreatic carcinoma and that the MRP3, MRP4 and MRP5 proteins were localized to the plasma membranes of ductular and acinar cells. The quantitative mRNA expression analysis demonstrated that MRP3 and MRP5 mRNA levels change during tumor development, whereas BCRP, MRP1 and MRP4 mRNA levels remained stable, when comparing the mRNA levels in normal human pancreatic tissue and pancreatic carcinoma. In addition, MRP3 mRNA levels significantly correlated with tumor grade and prognosis of the patients.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We thank E. Böhm, M. Brom, J. Longin and M. Meinhardt for their excellent technical help during our study, H. Spring for his expert help in obtaining confocal laser scanning micrographs and I. Esposito for her support in the analysis of the histopathological samples.

References

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  • 1
    Fernandez E, La Vecchia C, Porta M, Negri E, Lucchini F, Levi F. Trends in pancreatic cancer mortality in Europe, 1955–1989. Int J Cancer 1994; 57: 78692.
  • 2
    Bray F, Sankila R, Ferlay J, Parkin DM. Estimates of cancer incidence and mortality in Europe in 1995. Eur J Cancer 2002; 38: 99166.
  • 3
    Gudjonsson B. Cancer of the pancreas: 50 years of surgery. Cancer 1987; 60: 2284303.
  • 4
    Manabe T, Ohshio G, Baba N, Miyashita T, Asano N, Tamura K, Yamaki K, Nonaka A, Tobe T. Radical pancreatectomy for ductal cell carcinoma of the head of the pancreas. Cancer 1989; 64: 11327.
  • 5
    Griffin JF, Smalley SR, Jewell W, Paradelo JC, Reymond RD, Hassanein RE, Evans RG. Patterns of failure after curative resection of pancreatic carcinoma. Cancer 1990; 66: 5661.
  • 6
    Sperti C, Pasquali C, Piccoli A, Pedrazzoli S. Recurrence after resection for ductal adenocarcinoma of the pancreas. World J Surg 1997; 21: 195200.
  • 7
    Birkmeyer JD, Warshaw AL, Finlayson SR, Grove MR, Tosteson AN. Relationship between hospital volume and late survival after pancreaticoduodenectomy. Surgery 1999; 126: 17883.
  • 8
    Neoptolemos JP, Cunningham D, Friess H, Bassi C, Stocken DD, Tait DM, Dunn JA, Dervenis C, Lacaine F, Hickey H, Raraty MG, Ghaneh P. Adjuvant therapy in pancreatic cancer: historical and current perspectives. Ann Oncol 2003; 14: 67592.
  • 9
    Sener SF, Fremgen A, Menck HR, Winchester DP. Pancreatic cancer: a report of treatment and survival trends for 100,313 patients diagnosed from 1985–1995, using the National Cancer Database. J Am Coll Surg 1999; 189: 17.
  • 10
    Sohn TA, Yeo CJ, Cameron JL, Koniaris L, Kaushal S, Abrams RA, Sauter PK. Resected adenocarcinoma of the pancreas-616 patients: results, outcomes, and prognostic indicators. J Gastrointest Surg 2000; 4: 56779.
  • 11
    Landis SH, Murray T, Bolden S, Wingo PA. Cancer statistics, 1998. CA Cancer J Clin 1998; 48: 629.
  • 12
    Günzburg WH, Lohr M, Salmons B. Novel treatments and therapies in development for pancreatic cancer. Expert Opin Investig Drugs 2002; 11: 76986.
  • 13
    Ambudkar SV, Kimchi-Sarfaty C, Sauna ZE, Gottesman MM. P-glycoprotein: from genomics to mechanism. Oncogene 2003; 22: 746885.
  • 14
    Borst P, Evers R, Kool M, Wijnholds J. A family of drug transporters: the multidrug resistance-associated proteins. J Natl Cancer Inst 2000; 92: 1295302.
  • 15
    König J, Nies AT, Cui Y, Leier I, Keppler D. Conjugate export pumps of the multidrug resistance protein (MRP) family: localization, substrate specificity, and MRP2-mediated drug resistance. Biochim Biophys Acta 1999; 1461: 37794.
  • 16
    Kruh GD, Belinsky MG. The MRP family of drug efflux pumps. Oncogene 2003; 22: 753752.
  • 17
    Doyle LA, Ross DD. Multidrug resistance mediated by the breast cancer resistance protein BCRP (ABCG2). Oncogene 2003; 22: 734058.
  • 18
    Haimeur A, Conseil G, Deeley RG, Cole SP. The MRP-related and BCRP/ABCG2 multidrug resistance proteins: biology, substrate specificity and regulation. Curr Drug Metab 2004; 5: 2153.
  • 19
    Jedlitschky G, Burchell B, Keppler D. The multidrug resistance protein 5 functions as an ATP-dependent export pump for cyclic nucleotides. J Biol Chem 2000; 275: 3006974.
  • 20
    Adachi M, Reid G, Schuetz JD. Therapeutic and biological importance of getting nucleotides out of cells: a case for the ABC transporters, MRP4 and 5. Adv Drug Deliv Rev 2002; 54: 133342.
  • 21
    Reid G, Wielinga P, Zelcer N, De Haas M, Van Deemter L, Wijnholds J, Balzarini J, Borst P. Characterization of the transport of nucleoside analog drugs by the human multidrug resistance proteins MRP4 and MRP5. Mol Pharmacol 2003; 63: 1094103.
  • 22
    Rius M, Nies AT, Hummel-Eisenbeiss J, Jedlitschky G, Keppler D. Cotransport of reduced glutathione with bile salts by MRP4 (ABCC4) localized to the basolateral hepatocyte membrane. Hepatology 2003; 38: 37484.
  • 23
    Reid G, Wielinga P, Zelcer N, van der Heijden I, Kuil A, de Haas M, Wijnholds J, Borst P. The human multidrug resistance protein MRP4 functions as a prostaglandin efflux transporter and is inhibited by nonsteroidal antiinflammatory drugs. Proc Natl Acad Sci U S A 2003; 100: 92449.
  • 24
    Zelcer N, Reid G, Wielinga P, Kuil A, van der Heijden I, Schuetz JD, Borst P. Steroid and bile acid conjugates are substrates of human multidrug-resistance protein (MRP) 4 (ATP-binding cassette C4). Biochem J 2003; 371: 3617.
  • 25
    Suwa H, Ohshio G, Arao S, Imamura T, Yamaki K, Manabe T, Imamura M, Hiai H, Fukumoto M. Immunohistochemical localization of P-glycoprotein and expression of the multidrug resistance-1 gene in human pancreatic cancer: relevance to indicator of better prognosis. Jpn J Cancer Res 1996; 87: 6419.
  • 26
    Lu Z, Kleeff J, Shrikhande S, Zimmermann T, Korc M, Friess H, Büchler MW. Expression of the multidrug-resistance 1 (MDR1) gene and prognosis in human pancreatic cancer. Pancreas 2000; 21: 2407.
  • 27
    Kiuchi Y, Suzuki H, Hirohashi T, Tyson CA, Sugiyama Y. cDNA cloning and inducible expression of human multidrug resistance associated protein 3 (MRP3). FEBS Lett 1998; 433: 14952.
  • 28
    König J, Rost D, Cui Y, Keppler D. Characterization of the human multidrug resistance protein isoform MRP3 localized to the basolateral hepatocyte membrane. Hepatology 1999; 29: 115663.
  • 29
    Kool M, van der Linden M, de Haas M, Scheffer GL, de Vree JM, Smith AJ, Jansen G, Peters GJ, Ponne N, Scheper RJ, Elferink RP, Baas F, et al. MRP3, an organic anion transporter able to transport anti-cancer drugs. Proc Natl Acad Sci U S A 1999; 96: 69149.
  • 30
    Scheffer GL, Kool M, de Haas M, de Vree JM, Pijnenborg AC, Bosman DK, Elferink RP, van der Valk P, Borst P, Scheper RJ. Tissue distribution and induction of human multidrug resistant protein 3. Lab Invest 2002; 82: 193201.
  • 31
    Schaarschmidt T, Merkord J, Adam U, Schroeder E, Kunert-Keil C, Sperker B, Drewelow B, Wacke R. Expression of multidrug resistance proteins in rat and human chronic pancreatitis. Pancreas 2004; 28: 4552.
  • 32
    Büchler M, König J, Brom M, Kartenbeck J, Spring H, Horie T, Keppler D. cDNA cloning of the hepatocyte canalicular isoform of the multidrug resistance protein, cMrp, reveals a novel conjugate export pump deficient in hyperbilirubinemic mutant rats. J Biol Chem 1996; 271: 150918.
  • 33
    Mayer R, Kartenbeck J, Büchler M, Jedlitschky G, Leier I, Keppler D. Expression of the MRP gene-encoded conjugate export pump in liver and its selective absence from the canalicular membrane in transport-deficient mutant hepatocytes. J Cell Biol 1995; 131: 13750.
  • 34
    Cui Y, König J, Buchholz U, Spring H, Leier I, Keppler D. Drug resistance and ATP-dependent conjugate transport mediated by the apical multidrug resistance protein, MRP2, permanently expressed in human and canine cells. Mol Pharmacol 1999; 55: 92937.
  • 35
    Nies AT, König J, Pfannschmidt M, Klar E, Hofmann WJ, Keppler D. Expression of the multidrug resistance proteins MRP2 and MRP3 in human hepatocellular carcinoma. Int J Cancer 2001; 94: 4929.
  • 36
    Sandusky GE, Mintze KS, Pratt SE, Dantzig AH. Expression of multidrug resistance-associated protein 2 (MRP2) in normal human tissues and carcinomas using tissue microarrays. Histopathology 2002; 41: 6574.
  • 37
    Scheffer GL, Hu X, Pijnenborg AC, Wijnholds J, Bergen AA, Scheper RJ. MRP6 (ABCC6) detection in normal human tissues and tumors. Lab Invest 2002; 82: 5158.
  • 38
    Wijnholds J. Drug resistance caused by multidrug resistance-associated proteins. Novartis Found Symp 2002; 243: 6979; discussion 80-2, 180–5.
  • 39
    Hayashi A, Suzuki H, Itoh K, Yamamoto M, Sugiyama Y. Transcription factor Nrf2 is required for the constitutive and inducible expression of multidrug resistance-associated protein 1 in mouse embryo fibroblasts. Biochem Biophys Res Commun 2003; 310: 8249.
  • 40
    Hirohashi T, Suzuki H, Sugiyama Y. Characterization of the transport properties of cloned rat multidrug resistance-associated protein 3 (MRP3). J Biol Chem 1999; 274: 151815.
  • 41
    Zeng H, Liu G, Rea PA, Kruh GD. Transport of amphipathic anions by human multidrug resistance protein 3. Cancer Res 2000; 60: 477984.
  • 42
    Zelcer N, Saeki T, Bot I, Kuil A, Borst P. Transport of bile acids in multidrug-resistance-protein 3-overexpressing cells co-transfected with the ileal Na+-dependent bile-acid transporter. Biochem J 2003; 369: 2330.
  • 43
    Zeng H, Bain LJ, Belinsky MG, Kruh GD. Expression of multidrug resistance protein-3 (multispecific organic anion transporter-D) in human embryonic kidney 293 cells confers resistance to anticancer agents. Cancer Res 1999; 59: 59647.
  • 44
    Zelcer N, Saeki T, Reid G, Beijnen JH, Borst P. Characterization of drug transport by the human multidrug resistance protein 3 (ABCC3). J Biol Chem 2001; 276: 464007.
  • 45
    Zeng H, Chen ZS, Belinsky MG, Rea PA, Kruh GD. Transport of methotrexate (MTX) and folates by multidrug resistance protein (MRP) 3 and MRP1: effect of polyglutamylation on MTX transport. Cancer Res 2001; 61: 722532.
  • 46
    Di Costanzo F, Canaletti R, Sdrobolini A, Olmeo N, Luppi G, Pucci F, Cavicchi F, Gasperoni S, Rodino C, Zironi S, Angiona S, Contu A. Modulation of fluorouracil by methotrexate, leucovorin, and cisplatin (M-FLP) in the treatment of advanced pancreatic cancer: a phase II study of the Italian Oncology Group for Clinical Research (GOIRC). Am J Clin Oncol 2000; 23: 3148.
  • 47
    Fjallskog ML, Granberg DP, Welin SL, Eriksson C, Oberg KE, Janson ET, Eriksson BK. Treatment with cisplatin and etoposide in patients with neuroendocrine tumors. Cancer 2001; 92: 11017.
  • 48
    Ikeda M, Okada S, Ueno H, Okusaka T, Tanaka N, Kuriyama H, Yoshimori M. A phase II study of sequential methotrexate and 5-fluorouracil in metastatic pancreatic cancer. Hepatogastroenterology 2000; 47: 8625.
  • 49
    Donner MG, Keppler D. Up-regulation of basolateral multidrug resistance protein 3 (Mrp3) in cholestatic rat liver. Hepatology 2001; 34: 3519.
  • 50
    Chang TH, Hakamada K, Toyoki Y, Tsuchida S, Sasaki M. Expression of MRP2 and MRP3 during liver regeneration after 90% partial hepatectomy in rats. Transplantation 2004; 77: 227.
  • 51
    Haycox A, Lombard M, Neoptolemos J, Walley T. Review article: current treatment and optimal patient management in pancreatic cancer. Aliment Pharmacol Ther 1998; 12: 94964.
  • 52
    Oster MW, Gray R, Panasci L, Perry MC. Chemotherapy for advanced pancreatic cancer. A comparison of 5-fluorouracil, adriamycin, and mitomycin (FAM) with 5-fluorouracil, streptozotocin, and mitomycin (FSM). Cancer 1986; 57: 2933.
  • 53
    Berlin JD, Catalano P, Thomas JP, Kugler JW, Haller DG, Benson AB,3rd. Phase III study of gemcitabine in combination with fluorouracil versus gemcitabine alone in patients with advanced pancreatic carcinoma: Eastern Cooperative Oncology Group Trial E2297. J Clin Oncol 2002; 20: 32705.
  • 54
    Louvet C, Andre T, Lledo G, Hammel P, Bleiberg H, Bouleuc C, Gamelin E, Flesch M, Cvitkovic E, de Gramont A. Gemcitabine combined with oxaliplatin in advanced pancreatic adenocarcinoma: final results of a GERCOR multicenter phase II study. J Clin Oncol 2002; 20: 15128.
  • 55
    Huang P, Chubb S, Hertel LW, Grindey GB, Plunkett W. Action of 2′,2′-difluorodeoxycytidine on DNA synthesis. Cancer Res 1991; 51: 61107.
  • 56
    Obata T, Endo Y, Murata D, Sakamoto K, Sasaki T. The molecular targets of antitumor 2′-deoxycytidine analogues. Curr Drug Targets 2003; 4: 30513.
  • 57
    Weckbecker G. Biochemical pharmacology and analysis of fluoropyrimidines alone and in combination with modulators. Pharmacol Ther 1991; 50: 367424.
  • 58
    Lai L, Tan TM. Role of glutathione in the multidrug resistance protein 4 (MRP4/ABCC4)-mediated efflux of cAMP and resistance to purine analogues. Biochem J 2002; 361: 497503.
  • 59
    Wielinga PR, van der Heijden I, Reid G, Beijnen JH, Wijnholds J, Borst P. Characterization of the MRP4- and MRP5-mediated transport of cyclic nucleotides from intact cells. J Biol Chem 2003; 278: 1766471.
  • 60
    Wijnholds J, Mol CA, van Deemter L, de Haas M, Scheffer GL, Baas F, Beijnen JH, Scheper RJ, Hatse S, De Clercq E, Balzarini J, Borst P. Multidrug-resistance protein 5 is a multispecific organic anion transporter able to transport nucleotide analogs. Proc Natl Acad Sci U S A 2000; 97: 747681.
  • 61
    Davidson JD, Ma L, Iverson PW, Lesoon A, Jin S, Horwitz L, Gallery M, Slapak CA. Human multi-drug resistance protein 5 (MRP5) confers resistance to gemcitabine. Proc Am Assoc Cancer Res 2002; 43: 3868.