Voacamine, an alkaloid extracted from Peschiera fuchsiaefolia, inhibits P-glycoprotein action in multidrug-resistant tumor cells.

Multidrug resistance (MDR) in tumor cells is generally associated with increased efflux of the cytotoxic compounds, due to the activation of mechanisms of intracellular transport and to the overexpression of surface proteins, such as P-glycoprotein (Pgp), which act as ATP-dependent molecular pumps. In a previous study, voacamine, a bisindolic alkaloid from Peschiera fuchsiaefolia, was examined for its possible capability of enhancing the cytotoxic effect of doxorubicin (DOX) on resistant human osteosarcoma cells. The effects of voacamine on the cell survival and on accumulation of DOX were investigated on both the parental cell line, U-2 OS-WT, and its resistant counterpart, U-2 OS-R. A differential effect between sensitive and resistant cells on the intracellular DOX concentration and distribution was revealed. In particular, voacamine induced a significant increase of drug retention and intranuclear location in resistant cells. Moreover, the cell survival analysis and the electron microscopic observations revealed an enhancement of the cytotoxic effect of DOX induced by the plant extract. In the present study, a panel of monoclonal antibodies (MAbs), recognizing different and specific structural and functional state of Pgp, was used. By flow cytometry and immunofluorescence confocal microscopy, a dose-dependent increase of the reactivity of Pgp with MAb UIC2, which specifically recognizes an epitope of the drug transporter in its functional conformation, was detected in voacamine-treated U-2 OS-R cells. Conversely, the expression of the epitope recognized by MAb MC57 was downregulated while MAb MM4.17 did not change its binding level to treated and untreated MDR cells. These data suggest that the plant extract reacts with Pgp producing conformational changes with consequent epitope modulation. Taken together, our observations seem to demonstrate that voacamine is a substrate for Pgp and, therefore, interferes with the Pgp-mediated drug export, acting as a competitive antagonist of cytotoxic agents.

P-glycoprotein expression affects the intracellular concentration and antiviral activity of the protease inhibitor saquinavir in a T cell line.

A number of ATP-binding cassette proteins, which function as cellular efflux pumps, are known to be expressed on the membranes of human cells, including CD4-positive lymphocytes. It has also been shown recently that most anti-HIV protease inhibitors (PIs) are first-rate substrates of one of these membrane transporters, P-glycoprotein (Pgp). These findings raise the question of whether Pgp expression could influence HIV replication and/or affect the action of PIs. To gain new insight into this, initially unexpected, phenomenon, a study was undertaken with the aims of investigating whether treatment with saquinavir (SQV) induces Pgp expression in primary or transformed human T cell lines and, primarily, establishing whether Pgp expression could modify both the uptake of SQV and its antiviral action. Pgp expression, mainly measured by reverse transcription-PCR, was found to be variably detectable in healthy individuals, and short or prolonged SQV treatment was unable to induce or increase the expression of Pgp in a lymphoblastoid cell line or in primary lymphocytes derived from these individuals. However, further experiments, performed in a cell line with high Pgp expression (CEM(VBL100) cells) and its parental cell line (CEM cells), demonstrated that over-expression of Pgp reduces the uptake of SQV This result is consistent with the finding that CEM(VBL100) cells are less sensitive to the antiviral activity of SQV, the ID50 value (100 microM) being significantly higher than the corresponding value in parental CEM cells (4 microM).

Saquinavir induces stable and functional expression of the multidrug transporter P-glycoprotein in human CD4 T-lymphoblastoid CEMrev cells.

BACKGROUND: The multidrug transporter P-glycoprotein (P-gp) is expressed in HIV-1 target cells, in a range of pharmacological barriers and in AIDS-associated tumours. P-gp substrates include HIV-1 protease inhibitors (PIs) and anticancer drugs, which are efficiently effluxed from multidrug-resistant (MDR) cells. OBJECTIVES: The aim of this study was to investigate the effect on human CD4 T-lymphoblastoid CEMrev cells of saquinavir and other PIs in terms of P-gp expression and to characterize the functional and biochemical patterns of PI-induced P-gp molecules. METHODS: CEMrev cells no longer expressing detectable amounts of P-gp were cultured for a prolonged period in the presence of 10 microg/mL saquinavir (CEMsaq10) and tested for P-gp expression and function. Subsequently, CEMsaq10 cells were transferred into medium containing 15 microg/mL saquinavir (CEMsaq15) and cultured for several months. These cell lines were continuously monitored for P-gp expression, function and immunochemical patterns. A similar strategy was adopted to determine whether other PIs, such as ritonavir and indinavir, were able to induce P-gp expression in CEMrev cells. RESULTS: Compared with the drug-diluent control, the exposure of CEMrev cells to 10 microg/mL saquinavir induced, in a consistent fraction of cells (45-50%), de novo expression of functioning P-gp molecules. The transfer of CEMsaq10 cells to 15 microg/mL saquinavir was associated with a dramatic increase in P-gp expression and function (85-90% of CEMsaq15 cells expressed P-gp and effluxed P-gp dye substrates). These saquinavir-induced P-gp molecules included 75-kDa molecules as well as the classical 170-kDa form of P-gp, suggesting induction of a particular isoform of P-gp termed mini-P-glycoprotein. Conversely, ritonavir and indinavir induced transient P-gp expression in a small percentage of the CEMrev cells. CONCLUSIONS: Treatment of human CD4 T-lymphoblastoid CEMrev cells with saquinavir caused over-expression of functioning P-gp molecules. This de novo acquired MDR phenotype, which differed from that induced by other PIs, was stable, as expression and activity of P-gp were observed in CEMsaq10 and CEMsaq15 cells during prolonged in vitro culturing, even in drug-free conditions.

Subcellular detection and localization of the drug transporter P-glycoprotein in cultured tumor cells.

In vitro studies on the cellular location of P-glycoprotein (Pgp) are reported with the aim to clarify the relationship between its intracellular expression and the multidrug resistance (MDR) level of tumor cells. Pgp was found abnormally expressed on the plasma membrane of tumor cells with "classical" MDR phenotype. However, Pgp was also often detected on the nuclear envelope and on the membrane of cytoplasmic organelles. The hypothesis that this drug pump maintains a transport function when located in these compartments, is still under debating. Our results, together with those obtained by other researchers, demonstrate that cytoplasmic Pgp regulates the intracellular traffic of drugs so that they are no more able to reach their cellular targets. In particular, we revealed that in MDR breast cancer cells (MCF-7) a significant level of Pgp was expressed in the Golgi apparatus. A similar result was found in human melanoma cell lines, which never undergone cytotoxic drug treatment and did not express the transporter molecule on the plasma membrane. A strict relationship between intracellular Pgp and intrinsic resistance was demonstrated in a human colon carcinoma (LoVo) clone, which did not express the drug transporter on the plasma membrane. Finally, a structural and functional association between Pgp and ERM proteins has been discovered in drug-resistant human T- lymphobastoid cells (CEM-VBL 100). Our findings strongly suggest a pivotal role of the intracytoplasmic Pgp in the transport of drugs into cytoplasmic vesicles, thus actively contributing to their sequestration and transport outwards the cells. Thus, intracellular Pgp seems to represent a complementary protective mechanism of tumor cells against cytotoxic agents.

Monoclonal antibodies as a tool for structure-function studies of the MDR1-P-glycoprotein.

P-glycoprotein is considered one of the most important member of the rapidly growing superfamily of integral proteins known as the ATP-binding cassette (ABC) which in human also include several other multidrug resistance membrane proteins (i.e., MRP), the product of the cystic fibrosis gene, the TAP-1/TAP2 peptide transporters encoded by the major histocompatibility complex genes and the gene encoding for breast cancer resistance protein (BCRP) also known as MXR1 (mitoxantrone resistance protein). Many monoclonal antibodies (MAbs) reacting with distinct P-glycoprotein domains have been isolated and used to study the molecular organization and cellular functions of this ABC protein. MAbs have been used for multidrug resistance (mdr) gene cloning, delineation of the secondary and tertiary structure of P-glycoprotein and molecular analysis of the mechanisms involved in substrate recognition and transport. The immunodetection of the distinct products of the mdr gene family in normal and malignant cells and tissues has greatly contributed to the understanding of the physiological role of P-glycoprotein and its possible involvement in the refractory of tumors to chemotherapy. The present article deals with the immunological methods used for the structure-function studies of the P-glycoprotein. After introducing the basic structural features of this ABC transporter, the antibody based-approach is discussed with aiming to furnishing methodological perspectives for further investigations of the physiological role of P-glycoprotein and the multidrug resistance phenomenon.

What is the relationship between P-glycoprotein and adhesion molecule expression in melanoma cells?

A number of studies have reported that increased P-glycoprotein expression in drug-resistant tumour cells may be associated with decreased expression of a family of surface glycoproteins. However, despite its potential biological and clinical relevance, this phenomenon has not been extensively studied. In this study the phenotypic alterations that are associated with the acquisition of the multidrug-resistant phenotype in tumour cells, together with drug transporter overexpression, were investigated in human melanoma cells. The expression of cell adhesion molecules was analysed in a panel of multidrug-resistant melanoma cell lines (M14Dx) showing different degrees of resistance to doxorubicin and different levels of the expression of the drug transporter P-glycoprotein. In particular, expression of intercellular adhesion molecule-1 (ICAM-1), CD44, very late activation antigen (VLA)-5 and VLA-2 was determined by flow cytometry in the different resistant cell lines. A progressive downregulation of all the adhesion molecules examined was revealed in M14Dx cells, in parallel with an increasing level of expression of the drug transporter P-glycoprotein. The results obtained raise the question of the role of P-glycoprotein in the invasive and metastatic behaviour of tumour cells.

Intracellular P-glycoprotein in multidrug resistant tumor cells.

The overexpression of the drug-efflux molecular pump P-glycoprotein (P-gp) may confer to tumor cells the multidrug resistant (MDR) phenotype, which is one of the causes of cancer chemotherapy failure. By investigating several in vitro models of human tumor cells, we observed that P-gp, in addition to its localization on the plasma membrane, can also be found intracellularly. In particular, by using immunocytochemical and cytofluorimetric methods, we revealed that in MDR breast cancer cells (MCF-7) a significant level of P-gp was expressed in the Golgi apparatus, which is the major site of accumulation of the antitumoral compound doxorubicin. Moreover, we demonstrated the intracellular location of P-gp in three stabilized human melanoma cell lines which had never undergone cytotoxic drug treatment and did not express the transporter molecule on the plasma membrane. Double immunofluorescence labelling and immunoelectron microscopy revealed, also in this tumor cell type, the location of P-gp in the Golgi apparatus where it seems to play a pivotal role in intracellular drug transport. Finally, we analyzed the expression, localization and function of drug transport proteins in human colon carcinoma lines (LoVo) exhibiting different degrees of intrinsic or drug-induced resistance. We found that only MDR LoVo cells expressed P-gp on the plasma membrane while both low-level drug resistant clonal LoVo cells and MDR LoVo cells appeared to be positive for intracellular P-gp. Our findings suggest a functional role of the intracytoplasmic P-gp in the transport and sequestration of drugs. This represents a complementary protective mechanism of tumor cells against cytotoxic agents.

Immunogold localisation of P-glycoprotein in supported lipid bilayers by transmission electron microscopy and atomic force microscopy.

In this study, purified P-glycoprotein molecules, a membrane drug pump responsible for the multidrug resistance phenomenon, were incorporated in model membranes deposited onto solid supports, according to the method described by Puu and Gustafson (1997). The insertion of proteins into planar supported model membranes is of interest, as the films are fundamental in biosensor applications and for the investigation of how proteins conform and aggregate in a lipid environment. In our investigation, two model membranes were prepared by transferring liposomes containing P-glycoprotein to different hydrophobic supports: (a) thin amorphous carbon films; (b) Langmuir-Blodgett lipid monolayers on mica. After the labelling of P-glycoprotein with two well-characterised monoclonal antibodies, MM4.17 and MRK-16, samples (a) were observed by transmission electron microscopy (TEM) and samples (b) by atomic force microscopy (AFM). The comparative analysis performed by TEM and AFM allowed us to demonstrate the successful insertion of P-glycoprotein in the model membranes and their stability under different environmental conditions (vacuum, air and water). P-glycoprotein appeared to maintain, after purification and insertion in lipid bilayers, a good part of its conformational features as shown by the P-glycoprotein segments bearing the specific monoclonal antibody epitopes.

Evidence that natural killer cells express mini P-glycoproteins but not classic 170 kDa P-glycoprotein.

Several lines of evidence including reverse transcription polymerase chain reaction, immunoreactivity and their ability to efflux rhodamine 123 have implied the existence of P-glycoprotein in natural killer (NK) cells. It has been a natural tendency to assume that NK-cell P-glycoprotein is identical to the P-glycoprotein of multidrug resistant (MDR) cell lines, however, the present study uncovered major differences. Functionally, NK cells demonstrated a restricted substrate profile, being unable to transport daunorubicin and calcein acetoxymethylester while efficiently transporting other P-glycoprotein substrates. Furthermore, physical differences in NK-cell P-glycoprotein were established by differential reactivity with P-glycoprotein antibodies. NK cells demonstrated strong reactivity with C494 and JSB-1, but did not react appreciably with C219. In addition, NK cells were unable to bind to the antibody MM4.17 unless they had been fixed and permeabilized, yet this antibody normally recognizes an extracellular epitope of P-glycoprotein. These differences culminated in the demonstration using Western analysis that NK cells did not express detectable levels of 170 kDa P-glycoprotein. Instead, NK cells expressed small-molecular-weight 'mini P-glycoprotein' products, of approximately 70 and 80 kDa. Collectively, these data indicate that the predominant P-glycoprotein species of NK cells are novel mini P-glycoproteins and not the classic P-glycoprotein of MDR models.

P-Glycoprotein conformational changes detected by antibody competition.

Conformational changes accompanying P-glycoprotein (Pgp) mediated drug transport are reflected by changes in the avidity of certain monoclonal antibodies (mAbs). More of the UIC2 mAb binds to Pgp-expressing cells in the presence of substrates or modulators [Mechetner, E.B., Schott, B., Morse, S.B., Stein, W., Druley, T., Dvis, K.A., Tsuruo, T. & Roninson, I.B. (1997) Proc. Natl Acad. Sci. USA 94, 12908-12913], while the binding of other mAbs (e.g. MM12.10, MRK16, 4E3) is not conformation sensitive. Pre-staining of Pgp+ cells with UIC2 decreased the subsequent binding of MM12.10 mAb by about 30-40%, suggesting that there are Pgp molecules available for both UIC2 and MM12.10, and others accessible only for MM12.10. In the presence of certain substrates/modulators such as vinblastin, cyclosporin A or valinomycin, the MM12.10 reactivity was completely abolished by preincubation with UIC2. However, verapamil, Tween-80 and nifedipine did not influence the ratio of bound mAbs significantly. This is the first assay to our knowledge, sharply distinguishing two classes of modulators. The conformational changes accompanying the mAb competition phenomenon appear to be closely related, though not identical to those accompanying the UIC2-shift, as suggested by the simultaneous assessment of the two phenomena.

Identification of a LFA-1 region involved in the HIV-1-induced syncytia formation through phage-display technology.

We have identified a peptide region on CD18 molecule (the beta subunit of the LFA-1 molecule) involved in syncytia formation of HIV-1-infected lymphocytes. Several phage clones mimicking an epitope of the CD18 cell-surface determinant were isolated from two 9-mer random peptide phage-displayed libraries via their binding to the CD18-specific monoclonal antibody (mAb) MHM23, which in in vitro assay inhibits syncytia formation in HIV-1-infected cells. The peptide sequences displayed on phages that blocked immunolabeling of this mAb on LFA-1-expressing cells were used to identify the epitope recognized by mAb MHM23 by sequence comparison. On the basis of this analysis, two peptides which inhibited syncytia formation in HIV-1-infected cells in vitro were synthesized, thus confirming that they mimic a CD18 domain that is involved in this phenomenon. The results here presented highlight the potential of phage-display technology for the study of biological processes at the basis of virus infection, but also suggest new approaches for the therapy of AIDS.

Induction of P-glycoprotein expression on the plasma membrane of human melanoma cells.

Melanoma cells exhibit, both in vivo and in vitro, intrinsic drug resistance to various chemotherapeutic agents. Cultured human melanoma cells (M14) intrinsically express significant amounts of multidrug resistance-related protein (MRP1) and P-glycoprotein (P-gp) in the Golgi apparatus, but do not express these drug transporters on the plasma membrane. A panel of multidrug resistant (MDR) melanoma cell lines (M14Dx), showing different degrees of resistance to doxorubicin (DOX), were isolated. In M14Dx lines, the appearance of surface P-gp, but not of MRP1 or lung resistance related protein (LRP), occurred in cells grown in the presence of DOX concentrations higher than 60 nM. Furthermore, P-gp levels appeared to be dose-dependent. Flow cytometry, laser scanning confocal microscopy and cytotoxicity studies demonstrated that the activity of the drug extrusion system was related to both surface P-gp expression and resistance to DOX. In conclusion, P-gp, but not MRP1 or LRP, might play a pivotal role in the pharmacologically-induced MDR phenotype of melanoma cells.

LC determination of indinavir in biological matrices with electrochemical detection.

A high performance liquid chromatographic (HPLC) method with electrochemical detection for the quantification of Indinavir in cell culture is described. The sample pre-treatment involved a protein precipitation procedure using acetonitrile. Chromatography was carried out on a base-deactivated reversed-phase column with an isocratic mobile phase. The method was validated with regard to specificity, linearity, limits of detection and quantitation, precision and accuracy, recovery and ruggedness. The proposed HPLC assay was utilised to directly evaluate the capability of P-glycoprotein expressing multidrug resistant cells in mediating the transport and efflux of protease inhibitor (PI) Indinavir, a basic compound in AIDS care.

Detection of P-glycoprotein in the nuclear envelope of multidrug resistant cells.

P-glycoprotein is a plasma membrane efflux pump which is responsible for multidrug resistance of many cancer cell lines. A number of studies have demonstrated the presence of P-glycoprotein molecules, besides on the plasma membrane, also in intracellular sites, such as the Golgi apparatus and the nucleus. In this study, the presence and function of P-glycoprotein in the nuclear membranes of human breast cancer cells (MCF-7 WT) and their multidrug resistant variants (MCF-7 DX) were investigated. Electron and confocal microscopy immunolabelling experiments demonstrated the presence of P-glycoprotein molecules in the nuclear membranes of MCF-7 DX cells. Moreover, the labelling pattern was strongly dependent on pH values of the incubation buffer. At physiological pH (7.2), a strong labelling was detected in the cytoplasm and the nuclear matrix in both sensitive and resistant MCF-7 cells. By raising the pH to 8.0, the P-glycoprotein molecules were easily detected in the cytoplasm (transport vesicles and Golgi apparatus), plasma and nuclear membranes exclusively in MCF-7 DX cells. Furthermore, drug uptake and efflux studies, performed by flow cytometry on isolated nuclei in the presence of the P-glycoprotein inhibitor cyclosporin A, suggested the presence of a functional P-glycoprotein in the nuclear membrane, but not in the nuclear matrix, of drug resistant cells. Therefore, P-glycoprotein in the nuclear envelope seems to represent a further defense mechanism developed by resistant cells against antineoplastic agents.

Multidrug resistance and retroviral transduction potential in human small cell lung cancer cell lines.

Multidrug resistance (MDR) remains a major problem in the successful treatment of small cell lung cancer (SCLC). New treatment strategies are needed, such as gene therapy specifically targeting the MDR cells in the tumor. Retroviral LacZ gene-containing vectors that were either pseudotyped for the gibbon ape leukemia virus (GALV-1) receptor or had specificity for the amphotropic murine leukemia virus (MLV-A) receptor were used for transduction of five SCLC cell lines differing by a range of MDR mechanisms. Transduction efficiencies in these cell lines were compared by calculating the percentage of blue colonies after X-Gal staining of the cells grown in soft agar. All examined SCLC cell lines were transducible with either vector. Transduction efficiencies varied from 5.7% to 33.5% independent of the presence of MDR. These results indicate that MDR does not severely impair transduction of SCLC cells, and that MLV-A as well as GALV-1 retroviral vectors are suitable for further development of gene therapy in SCLC.

FCE 24517-resistant MCF-7 human breast cancer cell line: selection and characterization.

We have developed a stable line of the human breast carcinoma cell line MCF-7 by in vitro continuous exposure to increasing concentrations of the antitumoral alkylating agent FCE 24517 (tallimustine). The selected line, MCF-7/24517(1), was resistant to the selecting agent (RI=10) and to a lesser degree to melphalan, MEN 10710 (a related dystamycin analog), doxorubicin and etoposide, but not to m-AMSA. MCF-7/24517(1) cells did not express the multidrug-resistant phenotype, evaluated in terms of mRNA for mdr-1 and gp170 glycoprotein. A significant, albeit modest, increase in the cellular content of glutathione was measured and therefore other resistance mechanism(s) should be operative. We conclude that the MCF-7/24517(1) line is a valuable model to investigate the mechanisms of resistance of FCE 24517 and its derivatives.

Interferon-gamma up-regulates expression and activity of P-glycoprotein in human peripheral blood monocyte-derived macrophages.

P-glycoprotein (Pgp), the product of the multidrug resistance (MDR1) gene, is expressed in a variety of normal tissues but very little is known about its expression and function in cells of the immune system. In this study, we investigated the effect of interferon-gamma (IFN-gamma) on the expression and activity of Pgp in human peripheral blood monocyte-derived macrophages (MDM). We report that IFN-gamma up-regulated Pgp expression in a dose- and time-dependent manner. We show that IFN-gamma slightly increased the accumulation of MDR1 mRNA and induced a polarized redistribution of Pgp, as well as of some cytoskeletal proteins (ie, ezrin, actin, and alpha-actinin) on cell pseudopodia. Notably, confocal microscopy studies showed that Pgp and ezrin colocalized in these cellular structures. The IFN-gamma-induced Pgp up-modulation was a specific response of primary macrophages, as IFN-gamma treatment of primary lymphocytes and monocytic cell lines did not result in any increase of Pgp expression. Finally, IFN-gamma stimulated the Pgp transport activity in MDM, as rhodamine 123-efflux increased in treated cells as compared with control cultures. These results indicate that Pgp expression and activity can be up-regulated in human MDM in response to IFN-gamma. We suggest that IFN-gamma may be involved in the induction of multidrug resistance in macrophages.

Epitope mapping of the monoclonal antibody MM12.10 to external MDR1 P-glycoprotein domain by synthetic peptide scanning and phage display technologies.

Epitope mapping of MDR1-P-glycoprotein using specific monoclonal antibodies (mAbs) may help in delineating P-glycoprotein topology and hence in elucidating the relationship between its structural organization and drug-efflux pump function. In this work, by using synthetic peptide scanning and phage display technologies, the binding sites of the mAb MM12.10, a novel antibody to intact human multidrug resistant (MDR) cells, were studied. The results we obtained confirm that two regions localized on the predicted fourth and sixth loops are indeed external and that MDR1 peptides covering the inner domain of the current 12 transmembrane segment (TMs) model of P-glycoprotein could form part of the MM12.10 epitope.

Intracellular expression of P-170 glycoprotein in peripheral blood mononuclear cell subsets from healthy donors and HIV-infected patients.

BACKGROUND AND OBJECTIVE: P-glycoprotein (P-gp) is a transmembrane efflux pump that actively extrude a variety of unrelated drugs from cancer cells, leading to the so-called multidrug resistance (MDR) phenomenon. However, P-gp has also been found in normal bone marrow and peripheral blood mononuclear cells (PBMC). Recently, the presence of P-glycoprotein in PBMC from human immunodeficiency virus (HIV)-infected patients has also been investigated and a phenotype-associated P-gp expression has been detected. DESIGN AND METHODS: A total of thirty-eight HIV-1 positive patients with a mean age of 34 years (range, 24-41 years) were studied after an informed consent. Peripheral blood mononuclear cells (PBMC) were isolated by centrifugation on a Ficoll/Hypaque and P-glycoprotein expression was investigated on lymphocyte population by single and double-color immunofluorescence techniques. We investigated: i) both surface and intracellular expression of the P-gp molecule in different PBMC subsets, ii) P-gp expression modifications occurring during HIV infection, and iii) the effect of HIV-gp120 on the expression of P-gp by T lymphocyte subsets from healthy donors. RESULTS: Our experimental findings indicate that: a) P-gp glycoprotein can be detected on an intracellular level in different PBMC subpopulations (mainly CD8+ T lymphocytes, CD16+ NK cells and CD14+ monocytes); b) this intracellular expression is decreased in specific PBMC subsets (i.e. T-CD8+ and NK-CD16+) from HIV-infected patients and c) a rearrangement was obtained when CD4+ and CD8+ lymphocytes from healthy donors were exposed in vitro to the HIV-binding glycoprotein gp120. INTERPRETATION AND CONCLUSIONS: Our results indicate that P-gp glycoprotein can also be expressed intracellularly and can be rearranged in PBMC subsets from HIV-infected patients.

Detection of P-glycoprotein in the Golgi apparatus of drug-untreated human melanoma cells.

The intracellular location of the MDR1 gene product, known as P-glycoprotein (P-gp), has been detected by flow cytometry in 3 stabilized human melanoma cell lines which had never undergone cytotoxic drug treatment and did not express P-gp on the plasma membrane. In addition, MDR1 mRNA expression was revealed by RT-PCR in the same cell lines. Immunofluorescence microscopy, performed by using the same 2 monoclonal antibodies (MM4.17 and MRK-16) as employed in the flow-cytometric analysis, revealed the presence of P-gp intracytoplasmically, in a well-defined perinuclear region. Double immunofluorescence labelling and immunoelectron microscopy strongly suggested the location of the transporter molecule in the Golgi apparatus. The same observations have been obtained on a primary culture from a metastasis of human melanoma. Analysis of the expression of another membrane transport protein, the multidrug-resistance-related protein (MRP1), showed that it was present in the cytoplasm of all the melanoma cell lines examined. MRP1 also showed Golgi-like localization. The study by laser scanning confocal microscopy on the intracellular localization of the anti-tumoral agent doxorubicin (DOX) during the drug-uptake and -efflux phases, indicated the Golgi apparatus as a preferential accumulation site for the anthracyclinic antibiotic. P-gp function modulators (verapamil and cyclosporin A) were able to modify DOX intracytoplasmic distribution and to increase drug intracellular concentration and cytotoxic effect in melanoma cells. On the contrary, MRP1 modulators (probenecid and genistein) did not significantly influence either DOX efflux and distribution or the sensitivity of melanoma cells to the cytotoxic drug.