Multidrug resistance in human tumors—molecular diagnosis and clinical significance

Molecular Diagnosis Vol. 4 No. 2 1999

Review Multidrug Resistance in Human Tumors Molecular Diagnosis and Clinical Significance CHEPPAIL RAMACHANDRAN,

P h D , S T E V E N J. M E L N I C K , P h D , M D

Miami, Florida

Background: Multidrug resistance (MDR) of human tumors is one of the major reasons for the failure of chemotherapy in refractory cancer patients. MDR can be intrinsic or acquired, depending on the time of its occurrence, either at diagnosis or during chemotherapy. Molecular investigations in MDR during the last two decades have resulted in the isolation and characterization of genes coding for Pglycoprotein, multidrug resistance-associated protein, lung resistance-related protein, drug resistance-associated protein, breast cancer resistance protein, and adenosine triphosphate-binding cassette protein. Several molecular probes, primer pairs, and monoclonal antibodies have been developed over these years to quantify the regulation and expression of these drug resistance markers in tumor cells. Methodologies have also been standardized to estimate the gene amplification, mRNA and protein expression, and functionality of drug resistance proteins in clinical specimens from cancer patients. Methods and Results: This review describes these drug resistance genes and techniques for detection and quantification of their expression and function. Conclusions: Because these markers have clinical significance and usefulness, currently available technology warrants the application of these markers in clinical oncology. Key words: multidrug resistance, chemotherapy, molecular diagnosis, gene amplification, gene expression, functional assay.

Cellular drug resistance is o n e of the major limiting factors for the success of c h e m o t h e r a p y in refractory cancer patients. W h e n t u m o r cells develop drug resistance, they b e c o m e resistant not only to the treated drug, but also to a variety of structurally and functionally unrelated drugs of natural origin. T h e r e f o r e , the t u m o r cell resistance is often

r e f e r r e d to as multidrug resistance ( M D R ) . M D R can be intrinsic or acquired, the latter usually occurring after a brief remission. Even t h o u g h Farber et al. [1] initially noticed this p h e n o m e n o n in y o u n g leukemia patients in 1948, m a j o r progress in M D R research has occ u r r e d during the last three decades. The M D R p h e n o t y p e was first described in 1970 by Biedler and R i e h m [2] in Chinese h a m s t e r lung cells and P388 m u r i n e leukemia cells. Later, Ling's group in T o r o n t o d e m o n s t r a t e d that M D R is related to decreased intracellular drug accumulation [3] and c o r r e l a t e d with the p r e s e n c e of a 170 k D a plasma m e m b r a n e glycoprotein (P-glycoprotein) [4]. Mo-

From the Department of Pathology, Miami Children's Hospital, Research Institute, Miami, Florida 33155.

Reprint requests: Cheppail Ramachandran, PhD, Department of Pathology, Miami Children's Hospital, Research Institute, 3100 SW 62nd Avenue, Miami, FL 33155. Copyright © 1999 by Churchill LivingstonM ~

1084-8592/99/0402-000258.00/0

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lecular investigations since then have concluded that M D R is a multifactorial problem involving several genes, either acting alone or cumulatively. Basic research in this area has identified several drug transporter genes coding for P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), lung resistance-related protein (LRP), drug resistance-associated protein (DRP), breast cancer resistance protein (BCRP), and adenosine triphosphate-binding cassette protein (ABCP).

Mechanism of MDR P-glycoprotein The best-characterized mechanism of M D R involves P-gp. P-gp is a 170 kDa transmembrane protein encoded by the human MDR1 gene that is associated with the transport of natural anticancer drugs. P-gp overexpression in tumor cells results in broad resistance to a variety of anticancer drugs with different chemical structures and mechanism of action [5-8]. The MDR1 gene belongs to a small ATP-binding cassette (ABC) gene family that includes two members (MDR1, MDR3) in humans. Transfection studies with full-length functional cDNAs for the MDR1 gene has been shown to confer a full M D R phenotype in drug-sensitive cells [912]. Even though the P-gp-dependent efflux pump results in decreased drug accumulation and diminished cytotoxicity, the exact mechanism by which P-gp regulates intracellular drug levels is not completely understood. Drug transport experiments with multidrug-resistant cells suggest that decreased drug influx or increased drug efflux or both could be responsible for maintaining drug at low level. However, the kinetic analysis of drug uptake and efflux data supports the hypothesis that drugs are directly removed from the plasma membrane [13]. A growing body of evidence indicates that MDR1 is a key determinant of chemotherapeutic response of some forms of cancer. Gene expression studies with human tumor samples have suggested a correlation between MDR1 gene expression and both intrinsic and acquired resistance. Fojo et al. [14] found increased MDR1 m R N A levels in several tumor types that had not been previously exposed to chemotherapy. These tumors include colon carcinoma, renal cell carcinoma, hepatocellular carcinoma, adenocortical carcinoma, pheochromocyoma, pancreatic islet cell carcinoma, chronic

leukemia, and non-small-cell lung carcinoma. These tumors are considered to intrinsically express the MDR1 gene and characteristically display poor durable response rates to chemotherapy. Cancers that do not usually express MDR1 at diagnosis include many tumor types generally sensitive to chemotherapy such as acute lymphoblastic leukemia of childhood; lymphoma; neuroblastoma; and ovarian, breast, and small-cell lung carcinomas [15-18]. In several types of cancer such as acute myeloid leukemia (at the time of diagnosis), pediatric soft tissue sarcoma, pediatric neuroblastoma, and regionally advanced breast cancer, overexpression of MDR1/P-gp has been found to correlate with poor treatment outcome in patients with chemotherapy [19-26]. These data have been interpreted as an indication of P-gp-mediated drug resistance induced by treatment in P-gp-positive tumors. However, several clinical studies have suggested that P-gp positivity is associated with the aggressive tumor behavior. In colon cancer, P-gp was expressed predominantly in the tumor cells at the invading edge of primary tumors, and P-gp positivity in primary tumors was associated with a higher incidence of lymph node metastases [27]. In renal cell carcinoma, P-gp positivity was found more frequently in invasive than in noninvasive tumors [28]. In primary breast cancer, the expression of MDR1/P-gp seems to be more common in advanced regional disease than in small tumors [29-31]. A significant correlation has been reported between P-gp positivity and lower probability of event-free survival in patients with osteosarcoma treated with preoperative and postoperative chemotherapy [26], Multivariate analysis showed that P-gp status is a more significant variable than the extent of tumor necrosis. Hence, P-gp positivity was a strong predictor for poor treatment outcome in this study, but was not correlated with tumor response to preoperative chemotherapy as assessed by histochemical examination. These data seem to be the strongest evidence so far that P-gp indeed might be a marker for more aggressive tumor behavior and thus poor treatment outcome, independent of its effect on chemosensitivity [31]. In contrast to several reports describing the prognostic significance of P-gp, some studies have indicated the absence of any apparent relationship between MDR1 mRNA, P-gp expression, and response to therapy [32,33]. Most of these studies measured either MDR1 m R N A or P-gp expression

Molecular Diagnosis of MDR

in tumor cell. None of these studies analyzed P-gp function as an efflux pump. Several recent studies have suggested that it may not be sufficient to measure MDR1 m R N A and/or P-gp expression alone without determining the P-gp pump function and its role in reducing drug accumulation and chemosensitivity [34]. Modulation of multidrug transport function of P-gp has been documented with a variety of noncytotoxic compounds that competitively inhibit P-gpmediated antineoplastic drug efflux. A wide variety of these chemical compounds have shown reversal of M D R in cell culture systems [35,36]. Among these compounds, clinical trials have been performed with efflux blockers such as calcium channel blockers (verapamil, nifedipine), antipsychotic agents (phenothiazines), calamodulin inhibitors (trifluoperazine), antihormonal agents (tamoxifen), immunosuppressants (cyclosporin A, FK 506) and antiarrhythmic agents (quinoline) [37 41]. However, these clinical trials have not resulted in any recommendations to date, and most of these trials have thus far been disappointing. The major reasons for failure of these trials are as follows: 1. Inability to achieve adequate plasma concentrations of potential modulating agents because of toxicities (e.g., hypotcnsion and heart block with verapamil). . Presence of multiple mechanisms of drug resistance in tumor cells in addition to MDR1/P-gp. 3. The bioavailability of modulating agent in vivo may be affected by serum proteins (e.g., amiodarone, dipyridamole) and the lack of P-gp binding specificity. The classes of drugs lirst investigated as inhibitors of P-gp function were already in clinical use such as calcium channel blockers, ealamodulin antagonists, antihormonal agents, or immunosuppressive agents and hence were not targeted specifically against P-gp. [42, 43]. 4. P-gp modulators alter the pharmacokinetics of anticancer drugs, which can increase toxicity if the dose of anticancer drug is not appropriately reduced. P-gp modulators also inhibit the normal functioning of P-gp expressed in a variety of normal human tissues including liver, kidney, and intestines [44]. Additionally P-gp is present in the central nervous system in the bloodbrain barrier. In some tissues such as liver, kidney, and intestine, P-gp has an excretory function, whereas in other tissues such as brain,

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testis, and placenta, it provides a barrier function against toxic substances. Clinical trials with a nonimmunotoxic analog of cyclosporin A, PSC833, is progressing, and reports are encouraging [45]. In a phase I trial, the combination of vincristine, doxorubicin, and dexamethasone plus PSC833 has been shown to cause measurable reduction in the number of P-gp plasma cells in multiple myeloma patients [46]. However, it is too early to predict the outcome of clinical trials with PSC833. Recently List et al. [47] presented the results of a phase Ill Southwest Oncology Group (SWOG) study involving 226 relapsed or refractory acute myeloid leukemia patients treated with ara C (induction therapy) and daunorubicin or ara C, daunorubicin, and cyclosporin A. In this study, no correlation between P-gp and LRP expression or P-gp function and treatment outcome was observed among the treatment arms. However, cyclosporin A reduced daunorubicin resistance and improved remission duration and survival in patients with acute myeloid leukemia independent of systemic daunorubicin exposure.

Multidrug Resistance-Associated Protein The Canadian group led by Cole et al. [48] discovered the MRP and cloned the gene from a nonP-gp MDR cell line, H69 AR. However, as early as 1990, Marquardt et al. [49] described a 190 kDa phosphoprotein, which later was identified as MRR MRP belongs to the A B e transporter family, having only 15% amino acid sequence similarity with P-gp. Nevertheless, MRP and P-gp confer resistance to a similar profile of chemotherapeutic agents [5(I-52]. Even though the drug selection procedure for deriving cell lines that overexpress MRP or P-gp is similar, it is not clear what determines the preferential overexpression of either MRP or P-gp. Also, the overexpression of one protein does not preclude the other [53-55]. Since its discovery in the human small-cell lung cancer cell line H69AR, MRP has been identified in non-P-gp MDR leukemia, fibrosarcoma and non-small-cell lung carcinoma, other small-cell lung carcinoma, and in breast, cervical, prostate, and bladder carcinomas. Reduced drug accumulation and drug efflux are usually observed in drug selected cells that overexpress MRP [56] as is the case with P-gp overexpress-

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ing cells. Altered intracellular distribution of anthracyclines has been detected in several drugselected MRP overexpressing cell lines, and in some cases the process was energy dependent [54, 57,58]. This has led to the speculation that MRP may participate in sequestration of drugs away from their cellular target [59]. Alterations in the subcellular distribution of MRP have been detected in drug-selected and MRP transfected cell lines. However, the mechanism underlying this apparent difference in subcellular localization is not clearly understood [59-61]. Compounds reported to increase drug accumulation, alter drug distribution, and/or modulate resistance in MRP-overexpressing cells to varying degrees include verapamil, nicardipine, oubain, quinoline, genistein, and PSC833. However, the problems encountered with P-gp modulation are also seen in the modulation of MRP with these blockers. This has limited the use of MRP modulators in clinical trials. The mechanism by which MRP confers resistance to multiple drugs is not well understood. It has not been possible to phototabel MRP with affinity analogs of vinblastine or doxorubicin, and ATP-dependent transport of unmodified drug by itself has not been shown [51,62]. However, MRP transport of glutathione-drug conjugates has been shown in cell lines [63,64]. High levels of MRP m R N A have been detected in some tumor cell lines that characteristically respond poorly to chemotherapy. Examples include non-small-cell lung cancer [65], thyroid carcinoma [66], glioma [67], and neuroblastoma [68]. The stepwise selection of drug-resistant cell lines often exhibit the overexpression of MRP in low-level resistant cells, followed by P-gp [53-55]. Therefore, it is suggested that elevated MRP expression may be more likely to occur at clinically relevant levels of drug resistance. However, the evaluation of the importance of MRP expression as a prognostic factor and as a target for chemotherapeutic agents is being studied in several laboratories. Efforts in this area are also facilitated by the availability of reagents and methods for the sensitive and specific detection of MRP m R N A and protein. However, the absence of a rapid, reliable, and specific functional assay for MRP is still hindering the studies on prognostic evaluation of MRP in human tumors. Current and future studies are aimed at understanding the MRP-mediated M D R and designing or identifying specific molecules that could

reverse it, so that they could be used in functional assays and for clinical modulation.

Lung Resistance-Related Protein Overexpression of LRP was identified in a n o n P-gp M D R lung cancer cell line [69]. Later it was confirmed that LRP is a human vault protein [70]. Since its characterization, LRP has been found to be overexpressed in a large number of P-gp-negative drug-selected M D R cell lines such as lung carcinoma, fibrosarcoma, breast carcinoma, and melanoma [69,71,72]. The concomitant overexpression of LRP and MRP appears to be a frequent event in non-P-gp M D R cell lines [73,74]. LRP gene codes for a 110 kDa vault protein, which is the most abundant component of vaults [70,75,76]. Because LRP-associated drug resistance phenotype appears to be broad and includes drugs that are not substrates for P-gp or MRP [77], LRP has shown great value as a marker of in vitro resistance to both MDR-related drugs (doxorubicin, vincristine) and to some nonclassical M D R drugs (cisplatin, carboplatin, and melphalan). The presence of vaults in human tumors as revealed by LRP expression and its correlation with clinical parameters have been investigated. A n initial screening of 174 tumor specimens comprising 27 tumor types showed LRP expression in 63 % of the case [78]. In childhood acute lymphoblastic leukemia, the expression of LRP but not P-gp was significantly associated with an increased in vitro resistance of fresh leukemia cells to daunorubicin. Schadendorf et al. [79] studied the expression of P-gp, MRP and LRP in 21 primary and 37 metastatic melanomas. P-gp was expressed in only one case, whereas MRP and LRP were expressed in 50% and 62% of the tumors, respectively, with no difference between primary and metastatic tumors. In metastatic melanomas, a significant correlation was reported between prior chemotherapy treatment and increased LRP expression, whereas no relationship was evident for MRP expression. In 42 cases of lung tumors, LRP positivity was reported in 83% of squamous cell carcinomas, 59% of adenocarcinomas, and 36% of large-cell undifferentiated carcinomas [80]. The independent prognostic significance of LRP has also been reported in acute myeloid leukemia, as poor response to induction chemotherapy, remission achievement, and as a predictor of overall survival [81]. Similarly, in

Molecular Diagnosis of MDR

ovarian carcinomas, tumors with LRP positivity had a significantly inferior response to chemotherapy, shorter interval until tumor progression, and shorter overall patient survival [82]. Transfection studies with LRP gene have failed to confer M D R phenotype to sensitive cells. However, it has been suggested that complete vault particles formed by at least four separate proteins including LRP might be required to show the functional activity of LRP in transfected cells [70,83].

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ATP-Binding Cassette Protein More recently, another ATP-binding cassette gene (ABCP) located on human chromosome 4@2 involved in M D R has been described [88]. ABCP is expressed abundantly in the human placenta. The predicted protein is closely related to the Drosophila white and yeastADPI genes and is a m e m b e r of a superfamily that includes several multidrug transporters. ABCP expression in different types of human cancers and its clinical relevance in M D R are being investigated.

New MDR Markers Drug Resistance-Associated Protein We have identified a novel D R P from an intrinsically multidrug resistant melanoma cell line (FCCM-9) established from a patient [84,85]. The full-length 1.3 kb D R P cDNA codes for a 50 kDa protein. Analysis of the deduced protein sequence showed that D R P has sites for ATP-binding, ceasin kinase and protein kinase C phosphorylation, Nlinked glycosylation, N-myristoylation, and plasma membrane attachment. Additionally, DRP has sequence similarity with cyclosporine binding cyclophilin proteins at the 3'-end. DRP transfection into drug-sensitive NIH3T3 and CEM cells have conferred clinical level (9- to 10-fold) doxorubicin resistance compared with parental-sensitive cell lines. DRP is overexpressed in drug-resistant leukemia and breast cancer cell lines. Immunoperoxidase staining with DRP81 polyclonal antibody (DRP81) showed an intracellular localization of D R P protein. Further studies on the mechanism and expression of DRP in pediatric tumors are in progress at the Miami Children's Hospital.

Breast Cancer Resistance Protein Recently Ross et al. [86,87] discovered the breast cancer resistance protein (BCRP), a novel member of the ATP-binding cassette superfamily of transport proteins, from the atypical multidrug resistant human breast cancer cell line (MCF-7/AdrVp). Transfection of BCRP into drug-sensitive MCF-7 cells confers the drug resistance phenotype of MCF/ AdrVp including resistance to anthracyclines and mitoxantrone. A novel chemosensitizing agent fumitremorgin C at noncytotoxic concentrations inhibits BCRP transporter and sensitizes resistant cells to mitoxantrone.

Molecular Techniques to Study MDR Gene Amplification Drug-resistance genes such as M D R and MRP were identified and cloned based on D N A amplification observed in in vitro drug-selected M D R cell lines [48,89,90]. Even though amplification of drugresistance genes (MDR, MRR and LRP) is a common event in cell lines, it is quite uncommon in clinical specimen [91]. Several investigators have used Southern and Slot blot hybridization of labeled gene probes to genomic DNA to determine genc amplification [92-94]. Although measurements of amplification of drug-resistance genes are of interest to researchers, they appear to be of limited value as a practical clinical test. Although the presence of common amplified sequences in several human and rodent cell lines selected for resistance to different cytotoxic drugs provides strong evidence for the importance of these regions in M D R [92], the role of gene amplification in clinical drug resistance must await a more comprehensive assessment of amplification of newly cloned drug resistance genes such as DRR BCRR and ABCR

Gene Expression mRNA expression Quantification of m R N A expression of drugresistance genes is quite critical in the clinical setting as a measure of drug resistance. However, an important limitation for this approach is the low level of m R N A expression of M D R genes that is likely to be seen in patient material. When stable

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M D R cell lines are selected in tissue culture by drug exposures, they often show high levels of resistance easily detectable by the numerous applied detection techniques. In the patient material showing a low level of resistance, m R N A expression is usually low and extremely sensitive technique must be used to detect it. Investigators have used various methods of analysis for measuring m R N A expression such as Northern blot hybridization, slot blot hybridization, RNase protection analysis, in situ hybridization, and reverse transcription-polymerase chain reaction (RT-PCR). Northern and Slot blot hybridization techniques are relatively sensitive methodologies and offer assurance of specificity, but they are difficult to apply in clinical samples because the assays are time consuming and require relatively large samples [95,96]. In situ hybridization and in situ RT-PCR detect m R N A expression in individual cells, and allow for the detection of the heterogeneity of gene expression in a tumor. All other methods provide m R N A estimates on a "pooled" basis. In situ hybridization provides morphologic confirmation of the presence of m R N A in tumor cells, and can determine spatial expression of genes [97-100]. This m e t h o d has been performed with MDR1 and M R P genes [99-101]. However, unless the expression of m R N A in individual cells is high, the results of in situ hybridization may be difficult to interpret. Furthermore, in situ hybridization is laborious and requires high level of technical expertise. Application of the in situ RT-PCR assay to amplify the low level of transcripts may facilitate their easy detection and quantification [102]. The RT-PCR assay is more sensitive for gene expression studies with the MDR1 gene. This technique, which uses a set of gene specific primers, is a reliable semiquantitative method for very low levels of m R N A expression. Some of the limitations imposed by small tissue samples can be overcome by the RT-PCR method. However, an important limitation of this method is the inability to determine the heterogeneity of m R N A expression in a tissue specimen [103-105]. This limitation occurs mainly because the RT-PCR assay detects m R N A species on a "pooled" basis in the total R N A or total m R N A extracted from clinical specimens and cannot differentiate between normal and tumor cells. That all drug resistance genes cloned to date are expressed at varying levels in different normal h u m a n tissues supports this observation. For ex-

ample, P-gp is expressed at high levels in human adrenal cortical cells, brush border of renal proximal cells, biliary canaliculi of the liver, small and large intestinal mucosal cells, and pancreatic cells. P-gp is also expressed at lower levels in epithelial cells of brain, stomach, and CD34+ bone marrow stem cells [106,107]. Similarly M R P m R N A is expressed at high levels in testis, skeletal muscle, heart, kidney, and lung [48,108]. L R P is also widely distributed in normal h u m a n tissues [78]. We have seen that D R P m R N A is expressed at high levels in normal human kidney, liver, and lung tissues and low levels in stomach, colon, heart, small intestine, adrenal gland, and appendix tissues (Ramachandran et al. unpublished results). Protein Expression. Protein expression of drugresistance genes can be determined by Western blotting, immunocytochemistry, and flow cytometry. Western blotting can facilitate the estimation of the molecular weight of the protein. However, it requires a large n u m b e r of cells and is unable to discriminate between tumor and n o n t u m o r cells [109,110]. Immunohistochemistry is able to detect the heterogeneity of protein expression among tumor cells on one hand and between tumor and nontumor cells on the other. Flow cytometry is a more rapid technique, even though one has to make cells in suspension before the assay. Although this method can give a semiquantitative estimate of protein expression, heterogeneity cannot be ascertained well, especially in solid tumors. Sometimes flow cytometry is reported to be equally effective as immunohistochemistry in measuring heterogeneity of P-gp expression in hematologic malignancies, although such conclusion is hard to justify in solid tumors because of the absence of markers to identify different cell types. In the multicenter trials performed in the United States [111] and subsequently in Europe [112] in patients with acute myeloid leukemia, flow cytometry was clearly superior to immunohistochemistry in distinguishing specimens with low or intermediate levels of P-gp expression using MRK16 antibody. In contrast, identical results were obtained with C494, C219, and JSB-1 antibodies both by flow cytometry and immunohistochemistry. A variety of monoclonal antibodies that recognize internal (C219, C494, JSB-1) or external epitopes (HYB-612, MRK16, MRK17, 4E3, UIC2) have been developed for P-gp [113-116]. Similarly, antibodies recognizing M R P (MRPm6, MRPrl) and

Molecular Diagnosis of MDR

LRP (LRP56, 3B6) have been developed and are commercially available [69,72,117]. Sensitivity and specificity are critical parameters in evaluating individual antibodies and detection techniques. In the case of P-gp, immunohistochemical methods have been reported to be as sensitive as the ribonuclease protection assay and more sensitive than Western blot hybridization [118]. Immunohistochemical detection techniques have intrinsic advantages for detection of P-gp expression in leukemia and solid tumors that include the delineation of histologic details, recognition of tumor cell heterogeneity, delineation of admixed normal cells bearing M D R proteins, and opportunity to correlate histology and phenotype. Subjectivity is the primary disadvantage. Because some commercially available anti-P-gp antibodies recognize epitopes in other molecules [90,94], at least two antibodies recognizing separate epitopes (one surface, one cytoplasmic) should be used. It is also critical to use both positive and negative cell lines that reflect the physiologic and pathophysiologic level and pattern of site-specific P-gp expression in vivo. In flow cytometric analysis, antibodies recognizing external epitope are always better than those recognizing internal epitopes.

Functional Assay Most of the MDR-rclated proteins undergo posttranslational modifications for activation of drug transport function [119,120]. Several investigators seeking to correlate P-gp expression with clinical outcome have reported the absence of any apparent relationship between MDR1 mRNA, P-gp expression, and response to therapy [21-23, 121].Therefore, it may not be sufficient to measure mRNA or P-gp expression alone without determining the P-gp pump function and its role in reducing cellular drug accumulation and chemosensitivity. Recently, we have reported that P-gp in a breast cancer cell line HTB-123 is nonphosphorylated and nonfunctional. Treatment of drug sensitive HTB-123 cells with phorbol ester activates P-gp phosphorylation and function. This quick induction of drug resistance can also be reversed by verapamil or dipyridamole [23]. Therefore, it is always desirable to analyze the tumor specimen for the level of gene expression (mRNA and protein), as well as protein function (drug accumulation and distribution) and correlate with clinical data.

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The earliest assays to measure P-gp function used radiolabeled anticancer drugs (adriamycin, vincristine) for studying their influx, accumulation, and efflux from cultured cell lines and were used primarily for research purposes [122-125]. They are laborious techniques and require handling of radioactive materials that are not suitable for routine testing of clinical samples. Subsequently, rapid and easier methods have been developed that involve the use of fluorochromes in flow cytometric tests and/or cell imaging and analysis systems. The first assay to detect the function of P-gp with fluorochromes made use of the inherent fluorescent properties of antitumor drugs daunorubicin and doxorubicin. These agents are well-known substrates of multidrug transporters and are actively transported out of the cells. Several dyes such as rhodamine 123, fluo-3, and hydroethidine [126129] were also reported to be good substrates of P-gp. These agents can be used to discriminate between drug-sensitive and MDR cell lines using a flow cytometer. Flow cytometry can be applied in routine clinical screening, as it is available in many hospitals; and the assay is relatively inexpensive, fast, reproducible, and easy to perform. Measuring dye or drug efflux in the presence or absence of a P-gp modulator (cyclosporin A, dipyridamole, or verapamil) is preferable to measuring dye or drug accumulation only. Dyes such as rhodamine 123, or fluorescent cancer drugs, such as daunorubicin or doxorubicin, may be used to assess P-gp function [130-132]. However, rhodamine 123 efflux assay is reported to yield better functional estimate [130] probably because of more favorable uptake/efflux kinetics. Several investigators have suggested that calcein-acetoxymethylester (calcein-AM) is a better substrate for MRP than rhodamine 123 because it displays a larger and more rapid differential accumulation efflux [133-135]. However, calcein-AM is also a substrate for P-gp, and similar efflux kinetics have been observed in cells overexpressing P-gp [136], which limits the specificity of calcein-AM based functional assay. A major drawback to the use of flow cytometry in solid tumors is the need for a cell suspension, a problem not found in hematologic malignancies. Even though cell suspension can be prepared from solid tumor samples by mechanical and/or enzymatic techniques, cell damage, loss of membrane integrity, and tissue architecture may cause

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problems in deriving reproducible data. Furthermore, solid tumors are heterogeneous by definition, which may make it difficult to distinguish among different cell populations. However, with suitable control cell lines and reproducible protocols, P-gp function in solid tumors can be analyzed [137]. Cell imaging is another method to differentiate between drug-sensitive and -resistant cells in a clinical specimen. Light microscopic cellular fluorescence can be measured using a photometer and computer-assisted image analysis systems. Several investigators have shown that the anthracyclines such as doxorubicin and daunorubicin are accumulated in the nuclei of living cells, and to a lesser extent in the plasma membrane, cytoplasm, in the organelles of the Golgi region, and in lysosomes [138, 139]. In M D R cell lines, drugs are localized in a distinct pattern within the cytoplasm corresponding to intralysosomal distribution. Most of the fluorescence is localized first to the Golgi apparatus and then is gradually shifted to the lysosomes and/or mitochondria. This may enhance the ability of the cells to remove drugs and may reduce the accessibility of the anthracyclines to intracellular targets impairing their ability to exert cytotoxic effect [140]. The addition of inhibitors of P-gp function (verapamil) redistributes the fluorescence to the more diffuse pattern of nuclear and cytoplasmic compartments seen in drugsensitive cells. By using high-resolution techniques (scanning laser and confocal microscopy), several investigators reported a significant decrease in the doxorubicin fluorescence in the nucleus compared with cytoplasm of M D R cells [57,141,142]. One of the important advantages of this quantitative approach is the low number of cells (n = 30) required to obtain reliable results and to give significant differences between sensitive cells and cells with low levels of resistance. Recently, Piwnica-Worms et al. [143] reported that [99mTc]SESTAMIBI, a lipophilic cationic radiopharmaceutical, is actively transported by P-gp. Exploiting the favorable ,/-emission properties of 99mTc,functional expression of P-gp was successfully imaged in human tumor xenografts in nude mice with pharmacologically inert tracer quantities of [99mTc]SESTAMIBI. Further improvements in this technique may provide a novel technique to rapidly characterize P-gp function in human tumors in vivo.

Conclusion M D R has been one of the more intensely investigated fields of cancer research during the last three decades, mainly because of its close association with the failure of chemotherapy leading to increased morbidity and mortality in cancer patients. Molecular investigations in M D R have identified many drug transporter genes and established the role of these marker genes in the refractory status of cancer patients. These investigations have used reagents to detect the expression and function of these drug resistance genes such as monoclonal antibodies, cDNA probes, and primer pairs; and, techniques to detect and quantify expression and function of drug resistance genes have been standardized. Investigators have organized conferences and workshops to discuss the variations in the methodologies and results and to translate M D R laboratory research into clinical set up [111,112,144,145]. In these meetings, several areas of consensus have been reached regarding the use of molecular diagnostic assays of M D R in cancer patients. However, additional research including clinical trials are needed before molecular diagnostic assays of M D R are translated to clinical oncology.

Acknowledgment The authors are thankful for the developmental grants from Miami Children's Hospital Foundation and American Cancer Society Florida Division.

Received February 25, 1999. Received in revised form April 1, 1999. Accepted April 9, 1999.

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