Circumvention of P-glycoprotein-mediated Multiple Drug Resistance by Phosphorylation Modulators Is Independent of Protein Kinases*

Expression of P-glycoprotein by tumor cells confers resistance to multiple natural product drugs because of its ability to export these compounds. This transporter is a substrate for several protein kinases; however, the functional significance of its phosphorylation is not de- fined. We examined the effects of many activators and inhibitors of protein kinases on the activity of P-glyco- protein in drug-resistant human breast carcinoma cells (MCF-7/ADR). Several phorbol esters sensitized these cells to P-glycoprotein substrate drugs; however, there was no correlation with activation of protein kinase C. The 4 (cid:97) - and 4 (cid:98) -isomers of phorbol 12-myristate 13-ace-tate were equally potent in sensitizing the cells to acti- nomycin D and daunomycin and in increasing the intracellular accumulation of [ 3 H]vinblastine. These effects of 4 (cid:98) -phorbol myristate acetate required much higher concentrations than were needed to increase P-glyco- protein phosphorylation and were not antagonized by staurosporine. Similar to verapamil, the phorbol esters did not sensitize MCF-7/ADR cells to cisplatin, nor parental MCF-7 cells to any of the anticancer drugs. Mezerein, K-252a, and H-89 sensitized MCF-7/ADR cells, increased intracellular accumulation of [ 3 H]vinblastine, and antagonized photolabeling of P-glycoprotein by [ 3 H]azidopine. Therefore, phosphorylation does not appear to play a significant role in regulating P-glycopro-

The term multiple drug resistance (MDR) 1 refers to the phenomenon in which tumor cells which survive an initial round of chemotherapy subsequently demonstrate decreased sensitivity to both the original therapeutic agent and other seemingly unrelated drugs (reviewed in Refs. [1][2][3][4][5]. This is commonly mediated by overexpression of Pgp, a transmembrane protein (M r ϭ ϳ170,000) which acts as an energy-dependent drug efflux pump. This transporter actively removes a variety of structurally diverse compounds, including anthracyclines, Vinca alkaloids, epipodophyllotoxins, actinomycin D, and paclitaxel. Enhanced efflux of these compounds reduces their intracellular accumulation and so reduces their cytotoxicity. In contrast, Pgp does not export small hydrophilic drugs such as cisplatin, 5-fluorouracil and melphalan. Certain agents, such as verapamil, have been shown to reverse MDR by competing with the cytotoxic drugs for binding to Pgp, thereby promoting drug accumulation and cytotoxicity (reviewed in Ref. 6). Because of their potential usefulness as anticancer agents, the identification of compounds with this ability to reverse MDR is an area of research receiving high priority in both academic and pharmaceutical settings.
Another approach to the development of MDR reversing compounds involves characterization of the molecular mechanisms which regulate Pgp function and expression. Many cellular activities are regulated by the phosphorylation and dephosphorylation of specific proteins. Shortly after its discovery, Pgp was shown to be phosphorylated on serine residues in resting cells (7)(8)(9)(10)(11), most likely at consensus recognition sites for PKC and PKA (12) present in the deduced amino acid sequence of mdr genes. Recently, convincing data that Pgp is a substrate for both PKC and PKA in vitro and in intact cells has been provided (13)(14)(15). These kinases phosphorylate serine residues (669 and 681, respectively) in the linker region of Pgp (13,16). Additional studies demonstrated that treatment of MDR cells with PKC-activating phorbol esters enhances Pgp phosphorylation (9,17,18), while the nonspecific protein kinase inhibitor staurosporine reduces Pgp labeling (19,20). However, the effects of phosphorylation on Pgp activity remain controversial since both stimulation (17,(21)(22)(23) and inhibition (9,24) of drug transport have been reported. Additionally, phorbol esters have been found to decrease the drug sensitivities of cells which do not express Pgp (25,26), casting doubt on a specific role of Pgp in these responses The combined information indicates that multiple protein kinases are able to phosphorylate Pgp; however, the significance of these reactions in regulating Pgp activity remains undefined. To address this ambiguity, we have characterized the effects of a battery of pharmacological inhibitors and activators of several protein kinases on Pgp activity in human breast carcinoma cells which overexpress Pgp (MCF-7/ADR). In these cells, reduction of Pgp activity is manifested as increased cytotoxicity of substrate drugs such as actinomycin D, daunomycin, and vinblastine (27)(28)(29). If phosphorylation is important in regulating the function of Pgp, phosphorylation modulators would be expected to demonstrate patterns of reversal of MDR consistent with their effects on specific types of protein kinases EXPERIMENTAL PROCEDURES Materials-4␣-PMA and 4␣-PDBu were purchased from LC Laboratories (Woburn, MA). 4␣-and 4␤-Phorbol, 4␣-and 4␤-PDD, and K-252a were from Calbiochem-Novabiochem, while all other drugs and reagents were purchased from the Sigma. These compounds were dissolved in absolute ethanol and stored at Ϫ20°C. To test the effects of drugs on growth, cells were seeded in 96-well tissue culture dishes (Corning Glass Works, Corning, NY) at approximately 10% confluency and were allowed to attach and recover for at least 24 h. Varying concentrations of drugs alone or combined with the test compound were then added to each well, and the plates were incubated for an additional 48 h. The number of surviving cells was then determined by staining with sulforhodamine B as described by Skehan et al. (31). The percentage of cells killed was calculated as the percentage decrease in sulforhodamine B binding compared with control cultures. Control cultures included equivalent amounts of ethanol, which does not modulate the growth or drug sensitivity of these cells at doses utilized in these studies. In figures in which the cytotoxicity of the drug is plotted, values represent the percentage of cells killed by the combination of modulator and drug with normalization to the effect of the modulator alone. Reversal of MDR is defined as the ability of the compound to potentiate the cytotoxicity of Pgp-transported drugs (i.e. daunomycin, vinblastine, and actinomycin D).
[ Phosphorylation of Pgp-MCF-7/ADR cells were plated into 6-well tissue culture plates as described above for the drug accumulation assay. When cultures reached approximately 75% of confluency, the growth medium was removed, replaced with PO 4 -free minimum essential medium (Sigma M3786) for 1 h, and then supplemented with 200 Ci of 32 PO 4 per well. Cells were incubated for 2 h at 37°C, at which time the labeled medium was removed and replaced with unlabeled growth medium (to mimic drug exposure conditions in the cytotoxicity assay). Cells were treated with 0 or 200 nM staurosporine for 15 min and then exposed to either ␣or ␤-phorbol ester for 30 min. Reactions were terminated by lysing the cells in ice-cold PBS containing 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 10 mM NaF, and 100 M phenylmethylsulfonyl fluoride. After 5 min on ice, cell debris was pelleted by centrifugation at 12,000 ϫ g for 1 min. The resulting supernatants were precleared by incubation with rabbit IgG and protein Aagarose (Sigma) for 1 h at 4°C and centrifugation at 12,000 ϫ g for 1 min. Pgp was immunoprecipitated by repeating the incubation with anti-Pgp antibody, mdr(Ab-1) from Oncogene Science (Manhasset, NY, 1 g/sample). The immunoprecipitate was washed twice with cold lysis buffer, dissolved in sample buffer, and subjected to SDS-PAGE on 7.5% acrylamide gels. The gels were fixed with isopropyl alcohol:water:acetic acid (25:65:10, by volume) for 30 min, dried, and then exposed to Kodak X-AR film for 3-5 days. The amounts of 32 P incorporated into Pgp were determined by image analysis of the autoradiograms using the program NIH-Image. Additionally, bands were excised from the gels, and radioactivity was measured by liquid scintillation counting. Essentially identical results were obtained with these two techniques.
Photoaffinity Labeling of Pgp-Membranes (ϳ25 g of protein per sample) were prepared from MCF-7/ADR cells, incubated with compounds of interest and 0.75 M [ 3 H]azidopine (ϳ1 Ci) as described previously (27,28). Samples were then exposed to 200,000 J of UV light in a Stratagene UV Stratalinker at room temperature and analyzed by SDS-PAGE and fluorography using Amplify and Hyperfilm-MP from Amersham. An approximately 170-kDa protein which reacts with anti-Pgp antibodies is photolabeled using membranes from MCF-7/ADR cells, while no such protein is present in membranes isolated from MCF-7 cells (27,28).
Other Methods-Protein concentrations were determined by the method of Bradford (32) using reagents from Bio-Rad and bovine serum albumin (Sigma) as the standard. Molecular modeling of certain com-pounds was conducted following the Polak-Ribiere minimum energy optimization routines using the HyperChem for Windows (Release 3) program from Autodesk, Inc. (Sausalito, CA). Estimations of physical properties were calculated with ChemPlus extensions from Hypercube, Inc. (Waterloo, Ontario, Canada).

Cytotoxicities of Phosphorylation-modulating Compounds to-
ward Drug-sensitive and -resistant Cells-To assess the effects of potential modulators on drug sensitivity, it is necessary to first determine the intrinsic cytotoxicity of the compounds. Therefore, we have determined IC 50 values (drug concentrations which reduce cell proliferation by 50%) for a battery of protein phosphorylation modulators toward MCF-7 and MCF-7/ADR cells. Compounds which are subject to transport by Pgp usually demonstrate significant resistance factors 2 in the MCF-7/ADR (versus MCF-7) cells (29). As indicated in Fig. 1 (for several phorbol esters) and Table I (for other phosphorylation modulators), many of these compounds are very weak cytotoxins, i.e. IC 50 Ͼ 100 M. Of the compounds tested, only staurosporine and calyculin A demonstrated IC 50 values below 1 M for either MCF-7 or MCF-7/ADR cells. Interestingly, the 4␣-isomers of PDBu and PDD were more cytotoxic than the 4␤ compounds. Resistance factors for MCF-7/ADR cells were generally between 0.5 and 2, indicating that expression of Pgp confers neither resistance nor sensitivity to most of these compounds. However, these cells were 7-and 6-fold resistant to calyculin A and A23187, respectively, suggesting that these compounds may be transported by Pgp.
Effects of Phosphorylation-modulating Compounds on Drug Resistance-Reversal of MDR is manifested by the ability of compound to increase cell killing by daunomycin and/or actinomycin D without modulating the cytotoxicity of cisplatin (27,28). As summarized in Table I, most compounds did not reverse MDR at 100 M or doses which approached the IC 50 for that compound. For example, sn-1,2-dioctanoylglycerol was ineffective at 100 M, and staurosporine did not increase the cytotoxicities of the drugs at its IC 50 dose of 0.04 M. Several phosphorylation modulators (e.g. calphostin C, K-252a, chlorpromazine, and trifluoperazine) did significantly sensitize MCF-7/ADR cells to the natural product drugs, although these effects were observed only at doses which substantially inhibited cell proliferation. A measure of the potential usefulness of a compound as an MDR reversing agent is the "efficacy index," calculated as the IC 50 for cell growth/the minimum dose for reversal of MDR (29). This index for the above modulators is below 3 suggesting a narrow therapeutic window; however, much larger efficacy indices were observed for the selective PKA inhibitor, H-89 (Ͼ20 , Table I)  Several phorbol esters were tested to explore structure-function relationships among these compounds for reversal of MDR. Dose-response curves for the inhibition of cell proliferation by actinomycin D (Fig. 2A) indicate that the 80-fold resistance of the MCF-7/ADR cells can be fully eliminated by 10 M verapamil. Similarly, resistance was completely reversed by 5 M mezerein and slightly reversed by 0.5 M mezerein. The resistance of MCF-7/ADR cells was reduced 30-fold by 10 M of either 4␣or 4␤-PMA (Fig. 2B), while a 100 nM concentration of either compound was ineffective. High concentrations (50 M) of 4␣and 4␤-PDBu reduced the resistance factor of MCF-7/ADR cells by 20-and 80-fold, respectively, whereas 500 nM doses had no effects on the IC 50 for actinomycin D (Fig. 2C). Similar studies with vinblastine demonstrated that high, but not low, doses of PMA and PDBu sensitized MCF-7/ADR cells to this drug regardless of the stereochemistry at the 4-position of the phorbol ester (data not shown). None of the compounds tested, i.e. verapamil, mezerein, or the 4␣and 4␤-isomers of PMA and PDBu, affected the sensitivity of parental MCF-7 cells to either actinomycin D or vinblastine (data not shown).
The effects of multiple concentrations of mezerein and 11 phorbol compounds on the cytotoxicities of daunomycin, actinomycin D, and cisplatin toward MCF-7/ADR cells were examined. As indicated in Fig. 3, 5 M mezerein strongly enhanced

TABLE I
Cytotoxicities and MDR-reversal activities of non-phorbol phosphorylation modulators MCF-7 and MCF-7/ADR cells were exposed to multiple concentrations of the indicated compounds for 48 h. Cell survival was determined using the sulforhodamine B assay, and IC 50 concentrations for each drug were calculated. Values represent the means for at least 2 experiments. The resistance factor (RF) is calculated as the IC 50 for MCF-7/ADR cells/the IC 50 for MCF-7 cells. Reversal of MDR is scored as the ability to enhance the cytotoxicity of daunomycin and/or actinomycin D, without modulating the sensitivity of the cells to cisplatin using the following qualitative scale: ϩϩϩ, cell kill is at least 75% (approximately as high as that observed in the presence of 10 M verapamil); ϩϩ, cell kill is between 25 and 75%; ϩ, cell kill is between 15 and 25%; Ϫ, no significant increase in cell kill over that induced by the drug alone. In scoring for effects on [ 3 H]vinblastine accumulation: ϩϩϩϩ, greater than response to 20 M verapamil (Ͼ6-fold); ϩϩϩ, 4-to 6-fold increase; ϩϩ, 2-to 4-fold increase; ϩ, Ͻ2-fold, but significant; Ϫ, no significant increase over control. Numbers in parentheses indicate the maximum micromolar concentrations tested.
the abilities of daunomycin and actinomycin D to kill these cells, but did not modulate the cytotoxicity of cisplatin. Similar studies indicated that neither the 4␣nor the 4␤-isomers of phorbol or PDD, at doses up to 100 M, modulated the toxicities of actinomycin D (Fig. 4A). 4␤-PDBu was slightly more potent than 4␣-PDBu in enhancing the cytotoxicities of actinomycin D (Fig. 4B), while the monobutyrated phorbol was ineffective. 4␤-PMA increased the cytotoxicities of actinomycin D at 10 M (Fig. 4C); however, its IC 50 of ϳ20 M precluded analysis of its effects on MDR at higher doses. The 4␣-isomer of PMA and 4␤-phorbol 12-myristate were substantially less effective at reversing MDR, while 4␤-phorbol 13-acetate was essentially inactive even at 100 M. Virtually identical results were obtained when these phorbol compounds were tested in combina-tion with daunomycin, whereas none of these compounds modulated the cytotoxicity of cisplatin (data not shown). Staurosporine, which inhibits PKC with a K i of approximately 1 nM (33), was tested for its effects on reversal of MDR by mezerein and phorbol esters. As demonstrated in Fig. 5A, pretreatment of MCF-7/ADR cells with 50 nM staurosporine did not reduce cell killing by combinations of daunomycin and mezerein, 4␤-PDBu, 4␣-PMA, or 4␤-PMA. Similarly, staurosporine did not antagonize the effects of these compounds on the cytotoxicity of actinomycin D (Fig. 5B).
Effects of Phosphorylation-modulating Compounds on Pgp Activity-Levels of intracellular drug accumulation in Pgpoverexpressing cells can be used as a convenient measure of Pgp activity. For example, "classical" Pgp antagonists such as verapamil promote 5-to 8-fold increases in the accumulation of [ 3 H]vinblastine and [ 3 H]taxol by MCF-7/ADR cells (34). To assess the effects of the phosphorylation modulators on the intracellular accumulation of [ 3 H]vinblastine, MCF-7/ADR cells were treated with doses of the modulators up to either their IC 50 or to 100 M. The results for non-phorbol compounds are summarized in Table I. Several compounds demonstrated good ability to enhance [ 3 H]vinblastine accumulation. For example, mezerein, K-252a, H-89, and trifluoperazine demonstrated efficacies at least as great as that of verapamil. Conversely, many kinase activators and inhibitors, as well as two phosphoprotein phosphatase inhibitors, had no effect on Pgp activity.
The dose dependences for modulation of the intracellular accumulation of [ 3 H]vinblastine by mezerein and 5 phorbol esters were tested. None of these compounds significantly altered the accumulation of reduce the abilities of mezerein or the phorbol esters to increase intracellular accumulation of [ 3 H]vinblastine (Fig. 7). Staurosporine, K-252a, and calphostin C promoted modest dosedependent increases in [ 3 H]vinblastine accumulation (Fig. 8); however, responses were seen only at concentrations of these kinase inhibitors which were very close to their IC 50 values. In contrast, the PKA inhibitor H-89 caused very marked accumulation of [ 3 H]vinblastine at doses much lower than its IC 50 for either MCF-7 or MCF-7/ADR cells.
Effects of Phosphorylation-modulating Compounds on Pgp Phosphorylation and Photolabeling-To ensure that PKC-activating phorbol esters increase the phosphorylation state of Pgp, MCF-7/ADR cells were labeled with 32 PO 4 and then exposed to 4␣or 4␤-PMA with or without pretreatment with staurosporine. Immunoprecipitation of Pgp (Fig. 9) demonstrated that Pgp is phosphorylated to a modest extent in unstimulated cells. Exposure to either 100 nM or 10 M 4␣-PMA did not significantly enhance the phosphorylation state of Pgp (Fig. 9, lanes 2-5). In contrast, 100 nM and 10 M 4␤-PMA increased Pgp phosphorylation by approximately 3-fold (lanes 6 and 8). Pretreatment of the cells with 200 nM staurosporine blocked the ability of either dose of 4␤-PMA to increase the level of phosphorylation of Pgp (lanes 7 and 9), indicating that PKC is strongly inhibited by this compound. P-glycoprotein in membranes isolated from 4␤-PMA-treated cells, with or without pretreatment with 200 nM staurosporine, was equivalently photolabeled with [ 3 H]azidopine, indicating that phosphorylation does not strongly affect the drug binding affinity of Pgp (data not shown).
Interaction of a compound with the drug binding site(s) of Pgp can be inferred if the compound antagonizes the ability of Pgp to bind and become photolabeled by [ 3 H]azidopine (27,28,35). As demonstrated in Fig. 10, lanes 1, [ 3 H]azidopine can be cross-linked to Pgp in membranes isolated from MCF-7/ADR cells, whereas no such protein in membranes from MCF-7 cells is photolabeled by [ 3 H]azidopine (data not shown). As expected, photolabeling of Pgp by [ 3 H]azidopine was substantially reduced by including verapamil in the binding buffer (A, lane 2). Photolabeling was very strongly suppressed by H-89 and A23187 (lanes 6 and 7), while calyculin A, dibutyryl-cAMP, dibutyryl-cGMP, W-7, and genestein caused more modest decreases in [ 3 H]azidopine binding. The effects of PKC modulators were also tested (Fig. 10B)

DISCUSSION
Despite several years of effort, there have been no definitive demonstrations of reversal of MDR due to phosphorylation or dephosphorylation of Pgp. We sought to assess the roles of protein kinases in regulating Pgp function using a pharmacological approach. To this end, a panel of compounds which activate or inhibit protein kinases were tested for their effects on Pgp activity in MCF-7/ADR cells. These compounds included both general protein kinase inhibitors, such as staurosporine, and quite selective protein kinase inhibitors and activators, e.g. calphostin C, H-89, and phorbol esters.
Consideration of the effects of this large panel of phosphorylation modulators indicated that there were no patterns of differential cytotoxicity of inhibitors or activators of any particular class of protein kinase or phosphatase toward the two cell lines. Only calyculin A and A23187 demonstrated significantly different toxicities for the two cell lines. Therefore, PKC, PKA, PKG, and Ca 2ϩ /calmodulin-dependent protein kinases appear to be similarly regulated and functional in the two cell types, even though their actual levels may be different (15,25). Cell cycle times and cell phase distributions were similar in wild-type MCF-7 and drug-resistant MCF-7/ADR cells (data not shown), indicating that pathways involved in regulating cell proliferation have not been grossly altered in the selected cells.
Recently, we have identified several novel natural products which overcome MDR by acting as antagonists for Pgp (e.g. 27,28). Using the same methods, we have assessed the ability of the phosphorylation modulators to overcome Pgp-mediated MDR in MCF-7/ADR cells. While certain of these compounds inhibited Pgp activity, there are no correlations with the activation or inhibition of any class of protein kinase. Similarly, phosphoprotein phosphatase inhibitors, which have more global effects on protein phosphorylation, did not modulate MDR at subcytotoxic doses.
Because of the interest in the potential role of PKC in regulating MDR, we conducted an extensive characterization of the effects of activators and inhibitors of this family of kinases. Overall, our data argues against a substantial role of PKC in regulating Pgp activity for the following reasons. Previous studies have shown that calphostin C increases drug accumulation in MDR cells (36,37), but this effect is not mediated by inhibition of PKC (37).
2. Drug resistance was substantially reversed by micromolar concentrations of several phorbol esters, including both the ␣and ␤-isomers of PMA and PDBu. The high doses required and the lack of specificity for the ␤-isomers suggest that these effects do not involve protein kinase C. Furthermore, 1,2-dioctanoylglycerol, another PKC activator, did not enhance the cytotoxicities of the drugs. 4␤-PDBu was particularly interesting since it demonstrated MDR reversing activity at doses at least 12-fold lower than its IC 50 . This efficacy index of 12 is at least as good as the index for well-characterized Pgp antagonists, e.g. verapamil.
3. The phosphorylation state of Pgp was significantly increased by 4␤-PMA, but was unaffected by 4␣-PMA, even at 10 M, indicating that the effects of the latter compound are not mediated by phosphorylation.
4. The abilities of mezerein and phorbol esters to reverse MDR and to potentiate intracellular drug accumulation were not altered by pretreatment of the cells with staurosporine, even though this compound very effectively blocked PKC-mediated phosphorylation of Pgp. 5. All of the "PKC modulators" which reversed MDR inhibited the photolabeling of Pgp by [ 3 H]azidopine. Therefore, it is very likely that the reversing effects of these compounds reflect their abilities to interact directly with Pgp rather than alteration of Pgp phosphorylation by PKC.
Similar conclusions can be drawn from data on PKA modulators. For example, H-89, which is ϳ650-fold more active toward PKA than PKC (38), effectively inhibited drug transport by Pgp and reversed MDR. However, this was associated with high efficiency of inhibiting Pgp photolabeling by [ 3 H]azidopine, indicating that the activity of H-89 is due to direct interaction with Pgp rather than through alteration of transport activity via the phosphorylation state of Pgp. Notably, the efficacy index of H-89 (i.e. 10 -20) is superior to many previously described Pgp antagonists, suggesting that this compound may be therapeutically useful for reversal of MDR. A structurally related PKA inhibitor, H-87, has been shown to partially reverse resistance in MDR cells (39).
Antagonism of Pgp by staurosporine (40 -42), H-7 (43), chlorpromazine (44), and trifluoperazine (45) have been described previously, and these compounds demonstrated moderate-togood inhibition of Pgp in our model system. However, these agents simultaneously inhibit more than one protein kinase, making it difficult to assess the involvement of individual kinases. With the exception of staurosporine, all of these agents markedly inhibited Pgp photolabeling by [ 3 H]azidopine (Fig.  10), again suggesting that they act by direct antagonism of the drug binding site of Pgp rather than by altering its phosphorylation state.
The phorbol ester family represents a new class of Pgp antagonists which demonstrate interaction at low micromolar concentrations. These compounds are of moderate size and are composed of multiple ring systems, similar to several other Pgp antagonists (6); however, their lack of ionizable functional groups is somewhat unusual. The hydrophobicity of these compounds appears to determine interaction with Pgp such that log P values of Ͼ2, but Ͻ7, effectively block drug transport. While PMA and PDBu are able to bind to Pgp, our results indicate that they are not transported out of the cells by this protein. In this regard, they resemble estramustine (32,34).
In certain model systems, reversal of MDR is correlated with down-regulation of the expression of Pgp (24, 46 -48). Additional studies have suggested that PKA activity may be necessary for the expression of the mdr1 gene (39,46,49). However, the relevance of study of the regulation of expression of highly amplified genes, e.g. mdr1 in MCF-7/ADR cells, is open to debate. Therefore, we have not shown the effects of phosphorylation modulators on levels of Pgp in these cells. We have recently found that A-498 kidney adenocarcinoma cells have significant levels of Pgp and are moderately resistant to Pgp-  8 and 9). Pgp was then immunoprecipitated using the Oncogene Science antibody, subjected to SDS-PAGE, and dried gels were analyzed by autoradiography and scintillation counting as indicated under "Experimental Procedures." The positions of the following prestained molecular mass markers are indicated: myosin, 217 kDa, and ␤-galactosidase, 130 kDa.  1-9, respectively). B, samples contained ethanol, mezerein, ␣-PMA, ␤-PMA, ␣-PDBu, ␤-PDBu, staurosporine, K252-a, or H-7 (lanes 1-9, respectively). The positions of prestained molecular mass markers are indicated. substrate drugs. 3 Thus, it would seem appropriate to analyze mdr1 gene regulation in this nonamplified system as well.
In conclusion, it is apparent that several compounds which are commonly used as phosphorylation modulators, i.e. to study the roles of specific protein kinases in cellular regulation, are able to interact directly with Pgp. This results in inhibition of drug transport and reversal of the MDR phenotype independent of the involvement of protein kinases. There is little doubt that several protein kinases, including PKC and PKA, are able to phosphorylate Pgp, but this does not appear to significantly alter its activity in MCF-7/ADR cells. Protein phosphorylation may play a role in regulating Pgp expression; however, additional studies are needed to further explore such possibilities.