• plasma cell myeloma;
  • multidrug resistance;
  • P-glycoprotein;
  • flow cytometry;
  • protein function


  1. Top of page
  2. Abstract
  7. Supporting Information


Multidrug resistance (MDR) mediated by P-glycoprotein (P-gp) can compromise the successful treatment of many malignancies including plasma cell myeloma (PCM). However, methods do not yet exist that can accurately determine P-gp activity in PCM patient samples.


In this study, we have utilized new advances in flow cytometric methods to determine the activity of P-gp in PCM tumor cells. Furthermore, we have used several PCR-based approaches to perform a pilot study determining the functional impact of ABCB1 SNPs in patients with PCM.


No associations were seen between P-gp activity or expression and subgroups of PCM. Similarly, no association was seen between P-gp expression and SNPs within ABCB1 although a nonsignificant reduction in activity was demonstrated for rs1045642 (P = 0.121).


We have described a new method for the determination of P-gp and MRP activity suitable for use in clinical studies and have optimized this method to include a gating strategy, allowing routine use on PCM bone marrow aspirate samples. This is the first patient study to consider the full impact of SNPs within ABCB1 all the way from the genome to the proteome in PCM. The methods described here could also be utilized for future studies of “stem cell like” side populations in PCM that are considered to be drug resistant. Furthermore, minor amendments to these methods will facilitate studies of P-gp, MRP, and BCRP activity in other haematological malignancies. © 2012 International Clinical Cytometry Society

Multidrug resistance (MDR) is a significant factor compromising the successful treatment of a range of malignancies. A prominent factor effecting MDR is the adenosine tri-phosphate binding cassette (ABC) transporter protein, P-glycoprotein (P-gp). This protein, encoded by the gene ABCB1, plays a vital role in the protection of tissues from dietary xenobiotic agents (1).

However, expression of P-gp in tumor cells has been associated with the MDR phenotype where it is thought to play a direct role in the exclusion of drugs from the malignant cells (2). Indeed, study of P-gp expression in lymphoid malignancies has demonstrated that patients with increased levels of P-gp, either at diagnosis or upon relapse, have a poorer prognosis than those that do not (3–7). Furthermore, there have been studies which suggest alternative modes of action for P-gp, other than substrate efflux, contributing to the MDR effect. These include P-gp involvement in intramembranal substrate transport suggesting a potential role as an antiapoptotic protein. Conversely, a recent report on a clinical trial of Zosuquidar, a novel and highly selective inhibitor of Pgp, concluded that it did not significantly alter the outcome in acute myeloid leukaemia (AML) patients (8). Thus, the role of P-gp in MDR remains to be fully elucidated.

The work of Grogan et al. has shown that higher dosages of chemotherapy used to treat patients with plasma cell myeloma (PCM) can correlate with increases in the amount of P-gp expression upon relapse (9). The hypothesis suggested by Grogan et al. was that P-gp was upregulated in direct response to the increasing doses of cytotoxic drugs. However, an alternative hypothesis was postulated for the Grogan et al. study, namely that the increased P-gp expression is due to “selective pressure” within the initial clone (10).

Several factors are pertinent in the study of P-gp in PCM. First, the majority of studies to date comment on P-gp expression for mononuclear cells or bone marrow aspirate cells, rather than positively selected tumor cells which would provide results more relevant to tumor cell drug efflux. Also, P-gp expression is normally described as either positive or negative using western blot or immuno-precipitation methodologies. This “total P-gp” reading fails to provide a fully quantitative result which would facilitate the statistical analysis required for a patient population study of P-gp and does not take cellular location of protein into account. Additionally, PCM is considered a “patchy” disease within bone marrow (BM) and hence BM aspirate samples obtained for diagnosis and used for clinical research often contain relatively low levels of the tumor cells. This compromises the use of western blot methodologies which require large numbers of tumor cells to obtain sufficient protein for a reliable result. A more focused approach using flow cytometric methodologies offers the potential of a solution to this problem.

Also, it remains to be determined if the amount of P-gp protein can be correlated with the expression of the ABCB1 gene as discrepancies between these readings would suggest the involvement of post translational modifications in the upregulation of P-gp within PCM. Furthermore, it was suggested recently that it may be more appropriate in clinical investigations to study the activity of surface expressed P-gp as it is this expression that facilitates the extrusion of therapeutics from tumor cell cytoplasm (11).

Previous research has highlighted a single nucleotide polymorphism within ABCB1, rs1045642, which may mediate P-gp activity and could be a useful predictor for overall survival in PCM (12–16). rs1045642 has also demonstrated a degree of linkage disequilibrium (LD) with two further SNPs (rs2032582 and rs1128503) raising the possibility that rs1045642 is simply “tagging” for the effects of another SNP (17). It is also still undetermined if rs1045642 generates a reduction in P-gp function in PCM. Indeed, a suitable method to determine P-gp function in PCM tumor cells to allow investigations of this nature had not been developed in advance of this study.

This study was designed to develop flow cytometric methods to facilitate clinical screening of P-gp protein expression and P-gp function in the tumor cells of PCM patients. A pilot study in PCM patients (premalignant, newly diagnosed malignant and relapsed) of the effects of the ABCB1 SNPs (rs1045642, rs2032582, and rs1128503) on P-gp activity was also performed using these novel methods to determine if P-gp expression and function were altered in their presence.


  1. Top of page
  2. Abstract
  7. Supporting Information

Patient Recruitment

This study had ethical approval from the Office for Research Ethics Committees Northern Ireland (ORECNI). Informed consent was obtained before samples collection, in accordance with the Declaration of Helsinki. Bone marrow aspirates, and 5 ml EDTA anticoagulated peripheral blood (PB) were collected at the time of diagnosis from patients with a suspected diagnosis of plasma cell dyscrasia (PCD), or at relapse in patients with a previously confirmed diagnosis of PCM.

Peripheral Blood Mononuclear Cell (PBMC) Selection

PBMC were separated by density gradient centrifugation using Ficoll-Paque™ (Cat no.17-1440-02: Amersham Pharmacia Biotech, UK) at 1,600 rpm (800xG) for 30 min. Genomic DNA was then extracted from these cells using the Flexigene™ DNA isolation kit (Cat no. 51204: Qiagen, UK).

CD138+ Cell Selection

To obtain a purified sample of patient tumor cells, mononuclear cells (MNC) were separated from BM aspirates by density gradient centrifugation using Ficoll-Paque™ (Amersham Pharmacia Biotech) with the MNC layer harvested for subsequent CD138+ cell selection using anti-CD138 magnetic microbeads and an autoMACS instrument (Cat no. 130-051-301: Miltenyi Biotech, Germany). Tumor specific RNA was then extracted from these CD138+ cells using the recognized Trizol method (Cat no. 15596-026: Invitrogen, UK).

Real Time Quantitative (RQ) PCR and Melting Curves Analysis for rs1045642, rs2032582, and rs1182503 Genotyping

Genotyping for all three SNPs was performed using real time PCR and melting curves analysis on the LightCycler 1.2 instrument (Roche Diagnostics, UK) with predesigned fluorescence resonance energy transfer (FRET) hybridization probe and primer sets designed for rs1045642, rs1128503, and rs2032582 detection (Custom order: TIB MOLBIOL, Germany). PCR was performed using a 10 μl reaction volume in LightCycler capillaries (Cat no. 04929292001: Roche Diagnostics, UK) with primers and probes at the concentrations shown in Table 1, and ∼ 40 ng of genomic DNA. The PCR protocol utilized a preliminary denaturation at 95°C for 10 min, followed by a number of cycles of denaturation, annealing, and extension (Table 1). Melting curve analysis directly followed the amplification with a denaturation at 95°C for 0 s and annealing (Table 1) for 2 min using a 10°C/s ramp rate. The temperature was then increased to 75°C at 0.1°C/s, with fluorescence monitored continuously on the FL-2 channel. Genotype discrimination was determined as the difference in melting temperatures of the sensor probe from the target strand with peaks at distinct temperatures demonstrating the C-T, G-T-A, and C-T alleles (Table 1). This method was previously validated by using sequencing performed on four selected samples representing each genotype (18).

Table 1. PCR Conditions and Melting Temperatures for ABCB1 SNP Determination
GenotypePrimer/probe concentrations (μmol/l)PCR protocol (°C)(s)Melting temperatures (°C)
F.PrimerR. PrimerSensorAnchorCyclesDenatureAnnealingExtensionCGTA
rs10456420. (0)58 (4)72 (4)6255
rs11285030. (0)58 (4)72 (4)5763
rs20325820. (0)58 (10)72 (15)484252

Generation of cDNA from Patient RNA

PCM patient RNA (concentration adjusted to 100 ng) was set up for a standard reverse transcription PCR reaction with a two-step PCR protocol using the random primers method (Cat no. N8080127: Invitrogen, UK). cDNA generation was performed on a thermalcycler instrument (Applied Biosystems, UK).

Relative Quantification of ABCB1

Determination of ABCB1 gene expression was achieved by qRT-PCR using the Roche Lightcycler 1.2 instrument and a validated Taqman assay, (Hs00184491_m1: Applied Biosystems, UK). The relative quantification approach was chosen using the housekeeper gene GUSB (Hs03929099_m1: Applied Biosystems, UK) as was used for our previous work (18).

Flow Cytometric Surface and Intracellular Staining for P-gp Expression in PCM Tumor Cells

BM aspirate samples were washed once in prewarmed phosphate buffered saline (PBS). A total of 100 μl of washed BM cells were then incubated at room temperature (RT) for 15 min with 5 μl of the antibodies anti-CD138-APC (Clone MI15, Cat no. 347216: BD Biosciences, UK), anti-CD38-PE-Cy7 (Clone HIT2, Cat no. 560677: BD Biosciences, UK), and anti-CD45-APC-H7 (Clone 2D1, Cat no. 560178: BD Biosciences, UK) and 10 μl of an antibody raised against an external epitope of P-gp (17F9) and conjugated to phycoerythryn (PE) fluorochrome (Cat no. 557003: BD Biosciences, UK). Two millilitres of FACSlyse™ red cell lysing reagent were then added for 10 min at RT and the sample was washed twice using PBS and centrifugation at 2,000 rpm for 5 min. A total of 500 μl of Cellfix™ cell fixation reagent were added after the final wash to stabilize the cells for further analysis. A negative control was included, which was a sample also processed as above but without P-gp antibody. Samples were also set up for intracellular P-gp detection using the method above, with unconjugated 17F9 antibody to block surface sites and then employing the BD IntraSure kit™ (Cat no. 641776: BD Biosciences) method with our PE-17F9 antibody to stain for intracellular P-gp expression.

A Becton Dickinson (BD) FACSCanto II flow cytometer was used for all analyses and was calibrated with Cytometer setup and tracking beads to correct for any day to day variability in mean channel fluorescence (MCF) results. Quantibrite beads (Cat no. 340495: BD Biosciences) were also analyzed under the same constant voltage settings as for patient samples to standardize the fluorescence readings as antibody binding capacity (ABC). One thousand tumor cell events were acquired for each patient with a tumor cell event defined as cells which were positive for CD38 and CD138 but negative for CD45, in line with current guidelines (19) (Fig. 1).

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Figure 1. Flow cytometric detection of P-gp expression in bone marrow aspirate from patient diagnosed with plasma cell myeloma and associated plasma cell gating strategy based on positivity for CD138 and CD38 and negativity for CD45. Figure demonstrates sample unstained for P-gp which serves as a negative control. [Color figure can be viewed in the online issue, which is available at]

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Quantitative Evaluation of P-gp Function in PCM Tumor Cells

PCM patient BM aspirates were processed using a recently introduced kit for the detection of MDR activity (Cat no. 168MDQKit: SOLVO Biotechnology, Hungary), incorporating surface staining for the cellular antigens CD138, CD38, and CD45 to enable PCM tumor cell detection. Briefly, patient BM aspirate samples were separated by density gradient centrifugation using 5 ml of Ficoll Paque™ (Amersham Pharmacia Biotech) and centrifugation at 1,600 rpm for 30 min to obtain the mononuclear cell fraction. These cells were washed once in a specialized wash buffer at 1,600 rpm for 10 min and adjusted to a concentration of 2–6 × 106 cells in 8 ml of wash buffer. The cells were then split across three replicate sets of three Falcon flow cytometry test-tubes with 800 μl included in each tube; one set containing 5 μl of an inhibitor specific to both MRP-1 and P-gp (I1), one set containing 5 μl of an inhibitor specific to MRP-1 (I2) and one containing no inhibitors (B). These tubes were then incubated in the dark at 37°C for 5 min after which 200 μl of calcein AM were added for a further 10 min. The tubes were then immediately centrifuged at 2,000 rpm for 5 min and the supernatant discarded to stop the main reaction. Following this, 5 μl of the antibodies anti-CD138-APC, anti-CD38-PE-cy7, and anti-CD45-APC-H7 were incubated in each tube for 15 min. The cells were then washed once in wash buffer and reconstituted in 500 μl of wash buffer for further analysis.

Analysis was performed as per the above directions for P-gp expression, with calcein AM expression detected and presented as MCF on the FITC channel for the sample groups I1, I2, and B. The following equations were then employed to determine the functional activity of the proteins MRP1 and P-gp expressed as the multidrug resistance activity factor (MAF) for each: MAFtotal = [(I1B)/I1] × 100, MAFmrp = [(I2B)/I1] × 100 and MAFpgp = MAFtotal − MAFmrp. Results for this study could then be presented as a MAF specific to P-gp (Fig. 2).

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Figure 2. Flow cytometric determination of P-gp activity using detection of Calcein AM accumulation within cell cytoplasm. Groups I1(d), I2(e), B(f) denote the use of inhibitors to both MRP-1 and Pgp-1 (d) and specifically to MRP-1 (e) to determine the activity of Pgp-1 specifically in a bone marrow aspirate from a patient diagnosed with plasma cell myeloma. A plasma cell gating strategy was employed to ensure tumor specific results (ac). [Color figure can be viewed in the online issue, which is available at]

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Analysis of P-gp expression and activity was also performed on the DLKP-A cell line, a cell line engineered to upregulate P-gp and which demonstrates high levels of drug resistance. This cell line was used to optimize and validate flow cytometric methodologies and to provide a positive baseline for comparison with patient samples assayed (Supporting Information Fig. 1).


  1. Top of page
  2. Abstract
  7. Supporting Information

Patients and Genotyping

Thirty two patients, with a suspected or confirmed diagnosis of PCD were enrolled on the study. These patients were subsequently confirmed as having either monoclonal gammopathy of undetermined significance (MGUS) (n = 10) or PCM (n = 17), with a further five PCM patients enrolled at relapse. Genotyping for the three SNPs within the possible ABCB1 common haplotype was successful in 27 of these patients. The rarer A allele at rs2032582 was not demonstrated within any individuals included in this study. Most of the 27 patients were included in each part of the study; however, samples from some did not provide sufficient material for all analyses.

Effect of SNPs on ABCB1 Gene Expression

The expression of ABCB1 mRNA was measured in 20 patients to confirm the expression of P-gp at the RNA level and to determine if genotype at the three loci generated an alteration to ABCB1 expression. ANOVA analysis was performed using SPSS to determine if a significant difference in ABCB1 mRNA levels between genotype at the three loci could be detected. No association was seen between ABCB1 mRNA levels and genotype at any of the three loci under study (P > 0.05) (Supporting Information Fig. 2). However, the expression of ABCB1 mRNA was detected in all patients included in this analysis.

Correlations Analysis of ABCB1 Gene and P-gp Expression and Activity

We next investigated whether ABCB1 mRNA levels could be correlated with either surface or intracellular P-gp protein expression levels and if these protein levels could be correlated with the P-gp specific MDR activity of PCM tumor cells (n = 14). Correlations were determined using Spearman correlation analysis in SPSS. No significant associations were found between ABCB1 gene expression, surface membrane and intracellular P-gp levels and P-gp specific MDR activity (Table 2). However, a negative correlation between P-gp surface and intracellular levels was demonstrated which was approaching significance (P = 0.069).

Table 2. Matrix of Correlation for ABCB1 Gene Expression and P-gp Protein Expression and Activity (n = 14)
 Gene expressionProtein expression (surface)Protein expression (intracellular)Protein activity
  • a

    Correlation demonstrated borderline significance (P = 0.062).

Gene expression1.00   
Protein expression (surface)0.3081.00  
Protein expression (intracellular)−0.152−0.510a1.00 
Protein activity0.1670.407−0.3731.00

P-gp Surface and Intracellular Expression in PCM

The surface and intracellular expression of P-gp in the PCM patients was then investigated. As shown in Figure 3, surface and intracellular levels of P-gp are highly variable between patients. Also, such expression could be demonstrated within the cytoplasm but not necessarily on the membrane of the tumor cells. No association was seen between the level of P-gp expression and the subgroup of PCM in our preliminary cohort.

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Figure 3. P-gp membrane and intracellular specific expression (a+b) and P-gp activity (c+d) demonstrates a high degree of intrapatient variability but is not shown to be associated with diagnostic stage in PCD. (a) Shows P-gp membrane staining specific to plasma cells in patient bone marrow biopsies (n = 32). (b) Demonstration of intracellular specific levels of P-gp in the same 32 patients presented in (a). An inverse correlation between surface and intracellular levels of P-gp was demonstrated in a subset of these patients, albeit without quite reaching statistical significance (P = 0.069). (c+d) Results shown are of MAF values indicative of P-gp activity on a quantifiable scale of 1–100 (c). To ensure results of activity were reported in relation to the membrane expression of P-gp, we applied a correction factor, dividing P-gp activity by the surface expression to normalize the MAF values and provide a result for corrected P-gp activity (d).

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We next investigated whether the three ABCB1 SNPs were correlated with P-gp expression, both on the surface of the cell and within the cytoplasm (n = 25). As P-gp expression was determined quantitatively, ANOVA analysis was employed on each genotype in turn and as no difference in expression was seen for the three groups studied (MGUS, PCM, and RELAPSE) these patients were combined for this analysis. There was no significant association demonstrated between genotype at any of the three loci and P-gp expression either on the surface of the cell or within the cytoplasm (P > 0.05) (data not shown). Interestingly, surface expression of P-gp was low and demonstrated a mean ABC of just 30.48. Both of these levels were substantially lower than those demonstrated in the DLKPA cell line which was also assayed (Table 3).

Table 3. Mean P-gp Expression and Activity for PCM Patients and DLKP-A Cell Line
 P-gp expression (surface)P-gp expression (intracellular)P-gp activity
PCM Patients0.481326.076.2

P-gp Activity in PCM

To fully assess the effect of P-gp in PCM, we performed an investigation of its functional activity using a newly introduced flow cytometric assay based on Calcein accumulation in the presence and absence of P-gp inhibitors, with results reported quantitatively as MAF values (n = 21). Figure 3 demonstrates that P-gp activity can have considerable variation on an individual patient basis and that, as with expression, this activity does not appear to be correlated with the subcategory of PCD. To ensure our activity results were reported in relation to the membrane expression of P-gp, we applied a correction factor to normalize the MAF values by dividing them by the amount of membrane expressed P-gp in the patient tumor cells. These corrected MAF values (Fig. 3d) were also not seen to be associated with the subcategory of PCD (MGUS, PCM at diagnosis, or PCM at relapse).

We then investigated whether the three SNPs were correlated with activity in this preliminary cohort of patients (n = 18). P-gp activity was not significantly associated with genotype at any of the three loci studied (Fig. 4) (P > 0.05). However, when the analysis was repeated using the corrected activity values there appeared to be an association between this activity and genotype at rs1045642 with the CC genotype patients having increased activity in comparison to the TT genotype patients (Fig. 4b). However, this finding failed to reach statistical significance (P = 0.121). As with the P-gp expression results, mean uncorrected MAF values were low in comparison to the DLKP-A cell line used for validation (Table 3).

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Figure 4. SNPs in ABCB1 are not associated with (a) P-gp activity in PCD (rs1045642 (P = 0.864), rs2032583 (P = 0.934), rs1128503 (P = 0.798)) but may be associated with (b) P-gp activity when normalized against surface expression (rs1045642 (P = 0.121), rs2032583 (P = 0.818), and rs1128503 (P = 0.771)). Significance was determined by ANOVA analysis on each genotype in turn although results are shown here only for rs1045642 which was the only finding approaching statistical significance.

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  1. Top of page
  2. Abstract
  7. Supporting Information

ABC-transporter-mediated MDR remains a complex issue in the treatment of haematological malignancies, with the MDR phenotype being associated with several different proteins (1, 9, 18, 20, 21), and particularly since SNPs within these genes are now under scrutiny as further potential modifiers of the MDR proteins and hence phenotype (11, 12, 14). Genotype at rs1045642 within the ABCB1 gene has been shown previously to affect overall survival (OS) within PCM (14) but it was as yet undetermined if it affected P-gp expression and/or activity in PCM. Additionally, given increasing awareness of the need for more personalized treatment strategies for cancer patients (22–24), methods to assess P-gp expression and activity in tumor cells were urgently required (25).

In this preliminary study, ABCB1 gene expression was found in all patients; however, no associations were demonstrated between genotype at rs1045642, rs2032583, or rs1128503 and ABCB1 mRNA levels. This finding would suggest that these SNPs, if functional, may exert their effect on the P-gp protein post translation, in keeping with our current hypotheses (18).

We next determined that ABCB1 mRNA expression does not correlate significantly with either P-gp expression or P-gp functional activity. Additionally, neither surface nor intracellular P-gp expression correlates with the P-gp-mediated drug efflux potential of the tumor cells. This suggests that P-gp expression and activity in PCM tumor cells may be mediated by both post transcriptional and post translational factors, some of which have been identified in other studies (25–27). Furthermore, this finding demonstrates that studies of MDR which rely on gene expression based approaches may not fully appreciate the importance of the MDR phenotype in individual patients where post translational modifications may result in a more active MDR protein. Interestingly, P-gp surface expression showed an inverse correlation with the intracellular protein levels, suggesting that immature P-gp is actively shuttled from this cytoplasmic location and inserted into the cellular membrane in order to exert the noted drug efflux in malignancy (28–30).

Our flow cytometric results have demonstrated that none of the three SNPs investigated is significantly associated with either surface or intracellular levels of P-gp. This would suggest that none of these SNPs affects P-gp protein expression levels in PCM and that if any effect is mediated by these SNPs it is a more subtle interaction, possibly affecting only protein function in line with our current evidence (14, 18). A large degree of intrapatient variability was seen for P-gp expression in this preliminary study and emphasizes both the need for study of larger patient cohorts and also the need to assess tumor cell P-gp expression in individual patients at diagnosis and during treatment in PCM (22).

To complete our investigation of the ABCB1 SNPs, we studied their possible effect on the activity of P-gp by utilizing a newly developed assay based on Calcein accumulation as opposed to the traditional approach of rhodamine 123 efflux (16, 31, 32). This approach was preferred as rhodamine 123 fluorescence levels are known to fluctuate between experiments making it unsuitable for a longitudinal patient study. Our initial analysis demonstrated no association between any of these SNPs, subcategory of PCD and the functional activity of P-gp. This was an unexpected finding given that a previous in vitro study has confirmed a reduction in P-gp activity in the presence of the variant SNP at rs1045642 (15).

As the above finding was unexpected, a correction factor was applied to compensate for individual differences in P-gp protein expression. When the data was “normalized” in this manner a possible association between rs1045642 and P-gp activity was observed, with the CC genotyped patients having higher P-gp activity and TT genotyped patients having lower P-gp activity. Although this did not reach statistical significance, it is in concordance with effects seen in other malignancies (16) and we again highlight the small patient numbers in this preliminary study. Furthermore, this trend was not seen in conjunction with the other two SNPs studied, providing evidence that it is indeed rs1045642, alone, which mediates the observed effects on P-gp activity (15).

It should be noted that, in this study, P-gp expression was not significantly different in patients when grouped by the different subcategories of PCD (MGUS, PCM at diagnosis, and PCM at relapse). However, this patient cohort was small and a further larger study is required to provide more robust statistical analyses to confirm our preliminary findings. The flow cytometric methods described here will be highly suited to such a study, enabling scrutiny of PCM tumor cells at the single cell level. Furthermore, since P-gp and other ABC transporters have been implicated in resistance to treatment, or relapse, of other haematological malignancies such as chronic myeloid leukaemia (32), this method can be adapted to study these malignancies, with some simple alterations to the antibodies used for an appropriate tumor cell gating strategy.

A very recent investigation (33) has also shown that a stem cell like plasma cell sub population can be identified in PCM patients that has a noted increase in drug resistance and shows increased expression of either P-gp or BCRP, another ABC transporter (ABCG2) (11, 18). Interestingly, increased expression of ABC transport proteins in such a flow side population would account for the ability of these cells to evade cytotoxic treatment and sustain the main tumor population upon relapse. No such plasma cell side populations were investigated by Hoechst 33342 efflux in this study. If such side populations were identified in further primary cases of PCM, however, the methods outlined here would be highly suited for the characterization of the multidrug resistance activity of these cells, potentially revealing the mechanism by which these cells are able to survive following treatment. Furthermore, the assay kit used for this study has since been updated to include reagents and inhibitors that will allow specific determination of BCRP activity in addition to MRP and P-gp (34).

To the best of our knowledge, this is the first study to utilize methods capable of accurately evaluating the expression and function of P-gp specific to tumor cells in PCM patients. This is also the first patient study to consider the full impact of SNPs within ABCB1 all the way from the genome to the proteome in PCM. Our results corroborate previous work suggesting that rs1045642 may affect the activity of P-gp in vivo. Moreover, we have confirmed that this SNP may affect P-gp activity without altering the protein expression and that this impact can be attributed solely to the actions of rs1045642, and not rs2032582 or rs1128503 which can form a haplotype with it. Further investigations of P-gp, MRP, and indeed BCRP in patient samples should utilize the methods outlined in this study as they provide the most accurate evaluation of P-gp expression and activity currently available.


  1. Top of page
  2. Abstract
  7. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  7. Supporting Information

Additional Supporting Information may be found in the online version of this article.

CYTO_21018_sm_SuppFig1.pdf6751KSupplementary Figure 1. Validation of methodologies developed for assessment of P-gp activity and expression in PCM using DLKP-A cell line. (a) Demonstration of high P-gp membrane expression in DLKP-A cell line with similarly high levels shown for intracellular specific staining (data not shown). (b) Demonstration of high P-gp activity in DLKP-A cell line which shows large amount of Calcein accumulation following inhibition of P-gp function and loss of this retention when P-gp function is not inhibited.
CYTO_21018_sm_SuppFig2.pdf2962KSupplementary Figure 2. ABCB1 gene expression in PCM is not associated with rs1045642, rs2032582 or rs1128503 in plasma cell myeloma although expression of ABCB1 mRNA is noted for all samples (n=20). Significance was determined by ANOVA analysis on each genotype in turn; (a)rs1045642 (p=0.959), (b)rs2032583 (p=0.945) and (c)rs1128503 (p=0.875).

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