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- Materials and methods
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.
Multidrug resistant (MDR) cancers are refractory to a wide range of anti-neoplastic drugs and the difficulty in treating such tumours has inspired extensive efforts to clarify the underlying causes. The best studied mediator of this phenomenon is the product of the MDR1 gene, P-glycoprotein, which is a 170 kDa transporter situated in the plasma membrane of MDR cells. It uses energy from ATP hydrolysis to export a diverse range of substrates (Gottesman & Pastan, 1993) including anti-cancer drugs such as vinca alkaloids, anthracyclines and taxoids (Gottesman & Pastan, 1993) and fluorescent dyes such as rhodamine 123, calcein acetoxymethylester (AM) and fluo-3 AM (Neyfakh, 1988; Homolya et al, 1993).
The roles of P-glycoprotein have also received attention outside cancer research, as P-glycoprotein is expressed in organs of detoxification such as the intestine and blood–brain barrier where it mediates the extrusion of natural toxins and protects the organism from toxicity (Schinkel et al, 1994; Sparreboom et al, 1997; Schinkel, 1998). However, other cell types that express P-glycoprotein, e.g. the haematopoietic lineage, do not fulfil detoxification functions and the role of P-glycoprotein in these cells is not yet clear (Chaudhary & Roninson, 1991; Drach et al, 1992).
The natural killer (NK) cell features the strongest P-glycoprotein expression within the haematopoietic compartment. NK-cell P-glycoprotein has been detected at the protein and mRNA levels (Chaudhary et al, 1992; Drach et al, 1992; Klimecki et al, 1994, 1995), and its transport competence is suggested by the ability of NK cells to export rhodamine 123 (Chong et al, 1993; Kobayashi et al, 1994). Several putative functions have been attributed to P-glycoprotein in NK cells, with roles being hypothesized in cell-mediated cytotoxicity and cytokine secretion (Chong et al, 1993; Markham et al, 1993; Kobayashi et al, 1994; Klimecki et al, 1995). Little is known of the specific properties of P-glycoprotein and its regulation in NK cells, rather NK-cell P-glycoprotein is assumed to be identical to its better known counterpart expressed in MDR cell lines (Robey et al, 1999). We describe deviations in the behaviour of P-glycoprotein in NK cells compared with the classic P-glycoprotein of MDR models.
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- Materials and methods
Functional assays provided the first indication that NK-cell P-glycoprotein was distinct from classic P-glycoprotein. Despite efficiently transporting rhodamine 123, fluo-3 AM, BODIPY FL verapamil and ST BODIPY dihydropyridine, NK cells did not export daunorubicin and calcein AM, each of which was transported by CEMVLB. Furthermore, differential antibody reactivity was established using three antibodies that bind to internal regions of P-glycoprotein. CEMVLB reacted with each of these antibodies, however, NK cells demonstrated reactivity with C494 and JSB-1 but did not react appreciably with C219. The use of MM4·17, an antibody recognizing an external epitope, also highlighted differences between the two cell types. Unfixed CEMVLB demonstrated strong reactivity with MM4·17, however, unfixed NK cells did not react with MM4·17 and reactivity was only evident after fixation and permeabilization of the NK cells. Confocal microscopy demonstrated substantial P-glycoprotein expression within the plasma membrane of NK cells, indicating that this fixation requirement could not be entirely explained by a cytoplasmic location of P-glycoprotein in NK cells. Collectively, these data suggest that the P-glycoprotein product of NK cells is distinct from classic P-glycoprotein.
Western blot analysis provided the greatest insight into the unique nature of NK-cell P-glycoprotein. In CEMVLB lysates, classic P-glycoprotein migrated as a broad band at 170 kDa and above, reactive with the antibodies C494, JSB-1 and C219. Notably, this high-molecular-weight band was not detected in NK-cell lysates, suggesting negligible expression of classic, 170 kDa P-glycoprotein. Instead, NK cells featured expression of 70 and 80 kDa proteins, herein referred to as ‘mini P-glycoproteins’. In addition to differences in molecular weight, these products were distinguished by their differential antibody reactivity. The 70 kDa form was reactive with both C494 and JSB-1, whereas the 80 kDa form reacted only with C494. Neither product reacted with C219. Corresponding mini P-glycoproteins were also detected in CEMVLB, however, at significantly lower levels than classic P-glycoprotein. It is highly improbable that these small-molecular-weight proteins are breakdown products resulting from the proteolytic cleavage of full-length P-glycoprotein, as our evidence for the absence of 170 kDa P-glycoprotein in NK cells is not confined to Western analysis, but is supported by our flow cytometric data. Thus, the negligible C219 reactivity in NK cells, the requirement for fixation in order to liberate MM4·17 reactivity despite the apparent plasma membrane expression of the protein and the atypical dye efflux profile all argue against the expression of classic P-glycoprotein in NK cells.
The antibodies reactive with NK-cell P-glycoprotein all bind to the C-terminal half of P-glycoprotein. The C494 epitope has been mapped to aa 1027–1032 (Georges et al, 1990), the JSB-1 epitope is believed to overlap this site (Rao et al, 1995) and the predominant MM4·17 epitope occurs at aa 740–747 (Cianfriglia et al, 1994). In contrast, C219 binds to two sites within the molecule, at aa 567–572 in the N-terminal half and aa 1213–1218 in the C-terminal half (Georges et al, 1990). Therefore, an attractive explanation accounting for NK mini P-glycoproteins is that they are truncated MDR1 products comprising the C-terminal region between the C219 epitopes. Such a truncation would yield an approximately 640 aa protein corresponding to an area between aa 573 and 1213 of the MDR1 sequence, consistent with the size of the proteins reported herein and the pattern of antibody reactivity observed.
Alternative splicing of the MDR1 gene could account for smaller-molecular-weight P-glycoproteins and examples of alternative splicing of mdr genes have been reported previously (Barg et al, 1999). Alternatively, NK mini P-glycoproteins may be encoded by distinct genes with high structural homology to the C-terminal half of MDR1. Regardless of their origins, the fact that mini P-glycoproteins are constitutively expressed in primary human NK cells warrants an exploration of their physiological function in this cell type. The discovery of small-molecular-weight P-glycoproteins in NK cells adds to a growing list of mini P-glycoproteins reported in different species (Kawai et al, 1994; Thévenod et al, 1996; Barg et al, 1999; Ma et al, 1999).
The existence of smaller-molecular-weight P-glycoproteins in NK cells warrants reappraisal of the previous studies that guided the general acceptance of classic P-glycoprotein expression in this cell type. With regard to the numerous reports of PCR-based MDR1 detection, it is prudent to note that the primers chosen for NK-cell studies amplify regions located exclusively in the C-terminal half of the molecule (Drach et al, 1992; Gupta et al, 1992; Markham et al, 1993; Wilisch et al, 1993; Kobayashi et al, 1994; Yamashiro et al, 1998; Egashira et al, 1999). We suggest that these studies may have detected mini P-glycoproteins rather than full-length MDR1. With regard to immunological detection of P-glycoprotein via flow cytometry, the majority of studies in NK cells have been performed using the monoclonal antibody MRK16. The epitope recognized by MRK16 is a conformational one, encompassing regions of both the N- and C-termini, within the first and fourth external loops respectively (Georges et al, 1993). It is interesting to note, however, that a synthetic fourth loop epitope demonstrates strong and independent reactivity with MRK16 (Georges et al, 1993). On the basis of this precedent, the possibility that MRK16 reacts with hypothetical C-terminal mini P-glycoproteins is therefore feasible.
The dye efflux observed in NK cells is not compatible with the transport properties of multidrug resistance-associated protein (Davey et al, 1996; Hollo et al, 1996; Germann et al, 1997), sister of P-glycoprotein (Lecureur et al, 2000) or the mitoxantrone resistance protein (Litman et al, 2000). In contrast, C-terminal mini P-glycoproteins are predicted to contain drug-binding sites in transmembrane domain 12 (Ambudkar et al, 1999) and, therefore, deserve further scrutiny as possible mediators of the P-glycoprotein-like dye transport executed by NK cells.
The present study raises important issues concerning the diagnostic detection of P-glycoprotein and the chemotherapeutic strategies employed to treat P-glycoprotein-positive malignancy. Evidence of P-glycoprotein expression and activity cannot be automatically extrapolated to assume expression of the fully functional P-glycoprotein observed in MDR cell lines. The differential handling of P-glycoprotein substrates by NK cells suggests the potential for a restricted MDR phenotype, which has ramifications for the spectrum of drugs and inhibitors employed in its treatment. Consequently, maximizing therapeutic utility will require discrimination between these different phenotypes.