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*Adrenal Gland Cancer
Endocrinology Vol. 142, No. 10 4282-4287
Copyright © 2001 by The Endocrine Society

ARTICLES

The Regulation of MAPKs in Y1 Mouse Adrenocortical Tumor Cells

Thuyanh Le and Bernard P. Schimmer

Banting and Best Department of Medical Research and Department of Pharmacology, University of Toronto, Toronto, Ontario, Canada M5G 1L6

Address all correspondence and requests for reprints to: Bernard P. Schimmer, Ph.D., Professor, Banting and Best Department of Medical Research, University of Toronto, 112 College Street, Toronto, Ontario M5G 1L6, Canada. E-mail: bernard.schimmer{at}utoronto.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The regulation of the MAPKs, Erk1 and Erk2, and the MAPK kinase, Mek, were examined in the Y1 mouse adrenocortical tumor cell line and in the protein kinase A-defective mutant, Kin-8. ACTH and basic fibroblast growth factor each increased Mek phosphorylation and stimulated Mek activity in both cell lines and also activated the Erks at concentrations that paralleled their effects on Mek. The specific Mek inhibitor, PD98059, blocked the activation of the Erks by ACTH and basic fibroblast growth factor, indicating that Mek is the upstream activator of Erk. PD98059 did not block the phosphorylation of Mek, as might have been expected from previous studies; instead PD98059 inhibited the activity of the activated enzyme. In ACTH-stimulated, mutant Kin-8 cells, PD98059 paradoxically increased the amount of phosphorylated Mek, while preventing the activation of Erk. These results are interpreted as reflecting the loss of a protein kinase A-mediated inhibitory influence on Mek phosphorylation and activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE MAPK CASCADE is an important regulatory pathway in cell cycle progression and has been used as a biochemical marker to evaluate the mitogenic potential of a variety of hormones and growth factors. The MAPK pathway is involved not only in the mitogenic effects of growth factors acting through receptor tyrosine kinases but also in the mitogenic effects of many hormones acting through G protein-coupled receptors that lack intrinsic tyrosine kinase activity (1). The signals that link G protein-coupled receptors to the MAPK pathway are complex and may involve the Ras pathway as well as other convergent protein kinase cascades (1).

We previously examined the actions of ACTH on MAPK and its linkage to mitogenesis in Y1 mouse adrenocortical tumor cells. The Y1 cell line is an ACTH-responsive, steroid-secreting cell line that behaves, in many respects, like fasciculata cells from the normal adrenal cortex and is used widely as a model system to study the regulation of adrenocortical function (2, 3, 4). We found that ACTH stimulated the phosphorylation of the p44 MAPK (Erk1) and the p42 MAPK (Erk2), and consequently increased MAPK activity in the Y1 cell line. These observations led to the discovery of a mitogenic action of ACTH in the cell line that long had been missed because of an overriding growth-inhibitory effect of the hormone (5). Though it was clear that cAMP and PKA mediated the growth inhibiting effects of ACTH (6, 7), cAMP and PKA were not responsible for the activation of MAPK or for the mitogenic effects of the hormone (5). Little else is known about the pathways leading to the activation of MAPK in adrenocortical cells; most studies published to date have focused on the regulation of MAPK in cells derived from the adrenal zona glomerulosa (8, 9, 10). In particular, the role of MAPK kinase (Mek) as an upstream regulator of MAPK in these cells has been inferred only through the use of the Mek inhibitor, PD98059.

In this study, we have compared the effects of ACTH and fibroblast growth factor (FGF) on the activation of Mek and Erk in Y1 cells and investigated the actions of the Mek inhibitor PD98059. We show that ACTH and FGF both use Mek as an upstream regulator of MAPK and present evidence for a negative influence of PKA on Mek activation. Interestingly, PD98059 inhibits the activity of Mek without preventing its phosphorylation, contrary to earlier findings (11).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and cell culture
The cells used in this study were the Y1 mouse adrenocortical tumor cell line (12) and Kin-8, a cAMP-resistant mutant, of the Y1 cell line (7). Cells routinely were cultured as monolayers at 6.5 C, under a humidified atmosphere of 95% air-5% CO2, in Nutrient Mixture F10 supplemented with 15% heat-inactivated horse serum, 2.5% heat-inactivated FBS, and antibiotics. Cells were arrested early in the G1 phase of the cell cycle by transferring cells in the logarithmic phase of growth to serum-free {alpha} MEM for 72 h (5).

Detection of phosphorylated Erks and Meks
Cell lysates were prepared in radioimmunoprecipitation assay buffer in the presence of protease and phosphatase inhibitors as described previously (5). Equivalent amounts of the lysates (10–25 µg protein) were electrophoresed on 10% polyacrylamide-SDS gels and assayed for phosphorylated Erks and Meks by Western blot analysis on nitrocellulose membranes (Schleicher & Schuell, Inc., Keene, NH). The phosphorylated forms of Erk1 and Erk2 were detected using a primary rabbit antibody that specifically recognizes the tyrosine-204 phosphorylated forms of Erk1 and Erk2. The phosphorylated forms of Mek were detected using an antibody that specifically recognizes Mek phosphorylated at both Ser-217 and Ser-221. Antibody interactions with Erk and Mek were detected by chemiluminescence using a secondary horseradish peroxidase-conjugated antirabbit antibody.

Mek kinase activity
Mek kinase activity was assayed using the protocol from a MEK kinase assay kit provided by New England Biolabs, Inc. (Mississauga, Ontario, Canada). Cells were scraped into a nondenaturing lysis buffer containing protease and phosphatase inhibitors [20 mM Tris.HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% NP40, 0.1% (wt/vol) SDS, 2 mM dithiothreitol, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 mM phenylmethylsulfonylfluoride, 1 mM sodium pyrophosphate, 50 mM NaF, and 1 mM sodium orthovanadate]. The scraped cells were sonicated in an ice-water bath and clarified by microcentrifugation for 10 min at 4 C. Phosphorylated Mek was immunoprecipitated from the supernatant fraction using the phospho-Mek specific antibody described above preabsorbed onto protein A-Sepharose beads. Cell extracts (100 µg protein) were incubated with the antibody-bead complex in a total vol of 200 µl nondenaturing lysis buffer at 4 C for 3 h with gentle rocking. The beads were isolated by centrifugation, washed twice with nondenaturing lysis buffer and twice with kinase assay buffer [25 mM Tris.HCl (pH 7.5), 5 mM ß-glycerophosphate, 2 mM dithiothreitol, 0.1 mM sodium orthovanadate, and 10 mM MgCl2], and resuspended in 50 µl kinase assay buffer containing 200 µM ATP and 2 µg inactive Erk2. The kinase reaction was carried out for 30 min at 30 C, chilled, and centrifuged for 1 min at 4 C to remove the beads and terminate the reaction. When measuring the activity of purified, activated Mek1 or purified, activated Erk2, the enzyme was added directly to the kinase assay buffer; Erk2 was assayed using 2 µg Elk-1 fusion protein as substrate. Samples were solubilized in SDS sample buffer and processed for Western blot analysis of phosphorylated Erk2 as described above. Phosphorylated Elk-1 was detected by Western blot analysis using a phospho-specific Elk-1 primary antibody and a horseradish peroxidase-conjugated antirabbit secondary antibody.

Reagents and chemicals
Tissue culture media and sera were obtained from Canadian Life Technologies, Inc. (Burlington, Ontario, Canada). Protein A-Sepharose beads were from Amersham Pharmacia Biotech (Baie d’Urfé, Canada); antibodies for the phosphorylated forms of Erk, Mek, and Elk-1 were obtained from New England Biolabs, Inc., as were recombinant Mek-activated Erk2, inactive (Lys52Arg) Erk2, and Elk-1 fusion protein. Recombinant, Raf-activated Mek1 was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Chemiluminescence was performed with Rennaissance Chemiluminescence Reagent Plus Detection Kit (NEN Life Science Products, Boston, MA), and reactions were visualized using X-Omat Blue XB-1 film (Eastman Kodak Co., Rochester, NY). ACTH (Acthar) was from Rh\|[ocirc ]\|ne-Poulenc Rorer Canada, Inc. (Mississauga, Ontario, Canada); FGF (basic) was from Sigma (St. Louis, MO); PD98059 was from Calbiochem (San Diego, CA).

Quantitation of results and statistical analyses
Signals from immunoblots were quantitated by densitometric analysis using the NIH 1.52 Image gel plotting and quantitation program. The signals obtained were within the linear range, with respect to protein concentration for the phosphorylated forms of Erk and Mek. Data were analyzed for statistical significance using Peritz’s F test (a parametric, multiple comparison test for all differences among group means) (13).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of ACTH and FGF on the phosphorylation of Erk and Mek in Y1 cells
Y1 cells were placed in serum-free medium for 72 h to arrest cells early in the G1 phase of the cell cycle. Cells were pulsed with ACTH at concentrations ranging from 1.5 x 10-10 to 1.5 x 10-8 M; extracts were then prepared and assayed for the phosphorylated forms of Erk and Mek by Western blot analysis. As shown in Fig. 1Go, the effects of ACTH on Erk phosphorylation were observed only in cells that were first growth-arrested by serum deprivation. In cells growing logarithmically in serum-supplemented medium, the levels of phosphorylated Erks and Erk2 were high and not affected by the hormone (Fig. 1aGo). This finding is consistent with the overexpression of c-Ki-ras in this cell line (14) and with the observation that approximately 60% of logarithmically growing Y1 cells are distributed in the G1 phase of the cell cycle (15). In contrast, the levels of phosphorylated Erk1 and Erk2 were considerably lower in cells arrested early in G1 by serum deprivation (Fig. 1bGo). Treatment of these growth-arrested cells with ACTH increased the levels of phosphorylated Erk1 and Erk2 in a dose-dependent manner; with increases detectable at ACTH concentrations as low as 1.5 x 10-10 M (Fig. 1bGo). The increased levels of phosphorylated Erks reflect activation of MAPK activity, as we demonstrated previously (5). As shown in Fig. 1cGo, ACTH also stimulated the accumulation of phosphorylated Mek over a concentration range that paralleled the effects on Erk phosphorylation. Both Mek1 and Mek2 are detected by the antibody, but the isoforms are poorly resolved on the electrophoretic gels.



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  		Figure 1. Effects of ACTH on Erk and Mek phosphorylation in Y1 cells. Logarithmically growing cells (a) or cells growth-arrested by serum starvation (b and c) were treated with varying concentrations of ACTH for 5 min. Cell extracts then were immunoblotted for phosphorylated Erks (a and b) or Meks (c) as described in Materials and Methods. The results are representative of three separate experiments.

 
The effects of FGF on Erk and Mek phosphorylation were determined using the experimental paradigm described for ACTH above. Like ACTH, FGF also caused parallel increases in Erk and Mek phosphorylation (Fig. 2Go, a and b), with effects detectable at FGF concentrations as low as 0.3 ng/ml (approximately 2 x 10-11 M). Although serum-deprived Y1 cells had very low levels of phosphorylated Erk and Mek before stimulation (Figs. 1Go and 2Go), the levels of total Erk and Mek under basal conditions were appreciable and did not change upon stimulation with FGF (Fig. 2Go, c and d). Thus, the changes in amounts of phosphorylated Erk and Mek reflected increases in protein phosphorylation rather than increases in protein synthesis.



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  		Figure 2. Effects of FGF on Erk and Mek Phosphorylation in Y1 cells. Cells were growth-arrested by serum starvation and treated for 5 min with varying concentrations of FGF as indicated. Cell extracts were immunoblotted for phosphorylated Erks (a), phosphorylated Meks (b), total Erks (c), or total Meks (d) as described in Materials and Methods. The results in A and B are representative of four separate experiments, whereas the results in c and d are representative of two separate experiments.

 
Effects of the Mek inhibitor, PD98059, on Erk and Mek phosphorylation
PD98059 is a Mek-specific inhibitor commonly used to study Mek-dependent signal transduction. This inhibitor blocks the Mek-dependent activation of MAPK activity and other Mek-dependent downstream events in a variety of cell lines (e.g. Ref. 16) and has been reported to act by blocking Mek phosphorylation (11). We thus examined the effects of PD98059 on Mek phosphorylation and activity and the consequent effects on Erk phosphorylation in Y1 cells.

As shown in Fig. 3Go, PD98059 inhibited the ACTH-induced phosphorylation of Erk1 and Erk2 in a dose-dependent manner, with an apparent ED50 of 5.4 ± 1.0 µM. Results from a representative Western blot are shown in Fig. 3aGo; results compiled from eight experiments are shown in Fig. 3bGo. Surprisingly, PD98059 did not inhibit Mek phosphorylation (Fig. 3Go), indicating that the inhibitor blocked the phosphorylation and activation of the Erks without affecting the phosphorylation of Mek.



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  		Figure 3. Effects of PD98059 on ACTH-stimulated Erk and Mek phosphorylation in Y1 cells. Y1 cells were growth-arrested by serum starvation, incubated for 30 min in the presence of varying concentrations of PD98059, and then stimulated with ACTH (1.5 nM) for 5 min in the continued presence of the inhibitor. Cell extracts were immunoblotted for phospho-Erk and phospho-Mek as described in Materials and Methods. A representative Western blot is shown in a. Chemiluminescent signals for phospho- Erk1, phospho-Erk2, and phospho-Mek (Mek1 + Mek2) were quantitated by densitometry after ensuring that they were linear with respect to protein concentration. Results were compiled from eight experiments and expressed as a percentage of the signal obtained in the absence of inhibitor. Asterisks denote statistically significant inhibition of phosphorylation (P < 0.05).

 
PD98059 also inhibited FGF-stimulated Erk phosphorylation without affecting FGF-stimulated Mek phosphorylation (Fig. 4Go). The inhibitor, however, seemed to be less effective in blocking FGF action than in blocking ACTH action. As seen from a representative Western blot, PD98059 inhibited the phosphorylation of Erk1 and Erk2 to the same extent (Fig. 4Go). The concentrations of PD98059 that were required to inhibit Erk phosphorylation in response to FGF (1 ng/ml) were approximately 6-fold higher than those required to inhibit ACTH-stimulated Erk phosphorylation (Fig. 4bGo). Furthermore, the inhibitory effects of PD98059 could be overcome by raising FGF concentrations to 10 ng/ml (data not shown), as reported previously for mouse fibroblasts (11).



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  		Figure 4. Effects of PD98059 on FGF-stimulated Erk and Mek phosphorylation in Y1 cells. Phospho-Erk and phospho-Mek were analyzed as described in the legend to Fig. 3Go, except that cells were stimulated with FGF (1 ng/ml) instead of with ACTH. A representative Western blot is shown in a. Results compiled from quantitative densitometric analysis of at least six experiments are shown in b and are expressed as a percentage of the signals obtained for Erk1 + Erk2 (open bars) and Mek (filled bars) in the absence of inhibitor. Asterisks denote statistically significant inhibition of phosphorylation (P < 0.05).

 
The finding that PD98059 did not inhibit ACTH- or FGF-stimulated Mek phosphorylation prompted us to examine the effects of the inhibitor on Mek activity. For these experiments, growth-arrested Y1 cells were treated with ACTH (1.5 x 10-9 M) or FGF (1 ng/ml) for 5 min; phosphorylated Mek then was immunoprecipitated and assayed for its ability to phosphorylate Erk2. As shown in Fig. 5aGo, Y1 cells treated with either FGF or ACTH exhibited increased Mek activity (lanes 3, 4, and 6) when compared with unstimulated controls (lane 1). The signals obtained in the unstimulated controls reflected cross-reactivity of the antibody with the unphosphorylated Erk2 substrate rather than a residual basal Mek activity (data not shown). Adding PD98059 (60 µM) to intact cells before treatment with ACTH inhibited Mek activity (lane 8). PD98059 also inhibited Mek activity when added directly to assays containing phosphorylated, activated Mek immunoprecipitated from FGF- or ACTH-stimulated cells (lane 5 vs. lane 4, and lane 7 vs. lane 6, respectively).



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  		Figure 5. Effects of PD98059 on Mek activity in Y1 cells. a, Growth-arrested Y1 cells, either unstimulated (lane 1), treated with 1 ng/ml FGF (lanes 2–5), or treated with 1.5 nM ACTH (lanes 6–8), were tested for Mek activity by measuring the phosphorylation of an inactive Erk2 substrate (arrow) as described in Materials and Methods. Lane 2 shows the activity obtained in the absence of the exogenously added Erk2 substrate. For some samples, 60 µM PD98059 (lanes 5 and 7) or diluent (3% DMSO, lane 4) was added to cell extracts during the assay for Mek activity. In lane 8, PD98059 (60 µM) was added to growth-arrested cells 30 min before the addition of ACTH. The results are representative of three independent experiments. b, Activated recombinant Mek1 (50–500 fmol) was assayed for protein kinase activity, in the absence (-) or presence (+) of PD98059 (60 µM), by measuring the phosphorylation of Erk2. Activated recombinant Erk2 (24–240 fmol) was assayed for activity, in the absence or presence of PD98059, by monitoring the phosphorylation of Elk-1. The change in mobility of phosphorylated Elk-1, with increasing concentrations of Erk2, likely reflects progressive phosphorylation of the substrate. Results are representative of three independent experiments.

 
The ability of PD98059 to inhibit the kinase activity of activated Mek was further tested using purified, preactivated enzymes from commercial sources. As shown in Fig. 5BGo, PD98059 (60 µM) markedly inhibited the protein kinase activity of Raf-activated Mek1. In contrast, PD98059 had no effect on the activity of a purified, Mek2-activated Erk2, confirming the specificity of PD98059 for Mek.

Effects of PD98059 on Mek and Erk phosphorylation in the PKA mutant, Kin-8
To assess the involvement of cAMP and PKA in the actions of ACTH, Erk and Mek regulation were investigated in the PKA-defective mutant, Kin-8 (6). ACTH increased Erk phosphorylation in the mutant over the same concentration range as in parent Y1 cells and also increased Mek phosphorylation, though only at higher concentrations of the hormone (Fig. 6Go). The basis for this difference in sensitivity of the Erks and Meks to ACTH in the Kin-8 mutant is not known; however, the finding that PD98059 inhibited ACTH-induced phosphorylation of the Erks in Kin-8 (Fig. 7Go) indicates that the phosphorylation of Erk is Mek-dependent. It is possible that at the lowest concentrations tested, ACTH induced only small changes in phosphorylated Mek that escaped detection. PD98059 seemed to be a much more effective inhibitor of ACTH-stimulated Erk phosphorylation in the Kin-8 mutant (ED50 = 0.9 ± 0.2 µM, n = 4) than in parent Y1 cells. Concentrations of PD98059 as low as 5 µM maximally inhibited the ACTH-stimulated accumulation of Erk1 and Erk2 (P < 0.05), reducing the levels to values that were below the basal level. Intriguingly, PD98059 increased the levels of phosphorylated Mek in ACTH-treated Kin-8 cells despite the persistent inhibition of Erk phosphorylation (Fig. 7Go).



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  		Figure 6. Effects of ACTH on Erk and Mek phosphorylation in the PKA mutant, Kin-8. Kin-8 cells were growth-arrested by serum starvation and treated with varying concentrations of ACTH for 5 min. Cell extracts were prepared and immunoblotted for phosphorylated Erks (a) or Meks (b) as described in Materials and Methods. The results are representative of an experiment carried out in triplicate.

 


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  		Figure 7. Effects of PD98059 on Erk and Mek phosphorylation in the Kin-8 mutant. Kin-8 cells were growth-arrested by serum starvation, incubated for 30 min in the presence of varying concentrations of PD98059, and then stimulated with ACTH (1.5 nM) for 5 min in the continued presence of the inhibitor. Cell extracts were immunoblotted for phospho-Erk1, phospho-Erk2, and phospho-Mek as described in Materials and Methods. A representative Western blot is shown in a. Results in b were compiled from three separate experiments and expressed as percentages (mean ± SE) of the corresponding signals obtained in the absence of PD98059.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The Erks are MAPK isoforms that have been shown to be critical molecules for the transduction of proliferative signals from hormones and growth factors. Erk activation depends on phosphorylation at specific Thr and Tyr residues by Mek isozymes. The Meks, in turn, are activated by phosphorylation at specific Ser residues by upstream Mek kinases. Although the Meks are the only known activators of the Erks so far (17), Mek-independent pathways for the activation of Erks have been proposed (18). The results presented here demonstrate the essential role of Mek in the activation of the MAPK cascade in the Y1 mouse adrenocortical cell line. FGF (a receptor tyrosine kinase agonist) and ACTH (a G protein-coupled receptor agonist) both rapidly increased the phosphorylation of Mek and Erk isoforms and increased Mek activity in Y1 cells. On a molar basis, FGF was approximately 10-times more potent than was ACTH; however, in each case, the concentrations of agonist required to activate the Meks paralleled those required to activate the Erks. Furthermore, the specific Mek inhibitor, PD98059 (11, 16), inhibited both FGF-stimulated and ACTH-stimulated Erk phosphorylation.

PD98059 did not block the phosphorylation of Mek by FGF or by ACTH; a finding that was unanticipated. Alessi et al. (11) previously reported that PD98059 failed to inhibit bacterially-expressed Mek that was phosphorylated and activated by c-Raf in vitro but did prevent the in vitro phosphorylation and activation of Mek by c-Raf or by Mek kinase. On the basis of these findings, they concluded that PD98059 inhibited Mek by preventing its phosphorylation. Our results, however, show that PD98059 did not prevent the phosphorylation of Mek in Y1 adrenal cells; rather, the inhibitor blocked the activity of the phosphorylated, activated enzyme. PD98059 had no effect on the kinase activity of activated Erk2, confirming the specificity of PD98059 for Mek and establishing that PD98059 inhibits the MAPK cascade via inhibition of Mek. The basis for the difference in findings presented here and in the work by Alessi et al. is not clear.

The ability of ACTH to activate the MAPK pathway was not disrupted in the PKA-defective Kin-8 mutant (5) and was not mimicked by forskolin, a general activator of adenylyl cyclase (data not shown), indicating that cAMP and PKA did not mediate this action of ACTH. Nevertheless, the activation of Erk phosphorylation was considerably more sensitive to inhibition by PD98059 in the Kin-8 mutant than in parent Y1 cells. We have shown previously that PKA mutations disrupt the expression of the P-glycoprotein drug efflux pump, an important determinant of drug sensitivity and resistance (19). Although we do not know the contribution of the P-glycoprotein drug efflux pump to the intracellular accumulation of PD98059, it is interesting to speculate that the increased sensitivity of the Kin-8 mutant to the Mek inhibitor resulted from the decreased expression of the multidrug resistance gene. Strikingly, PD98059 increased the levels of phosphorylated Mek in the Kin mutant after ACTH treatment without producing a significant effect on Mek phosphorylation in ACTH-treated parent Y1 cells. The regulation of Mek is complex and not only involves its phosphorylation and activation by upstream protein kinases such as the Rafs but also involves negative feedback regulation by the downstream Erks (20). In addition, PKA has been reported to inhibit the MAPK cascade by phosphorylating upstream Rafs, thereby preventing Ras-GTP binding to Raf and Raf activation (21). Thus, the levels of phosphorylated Mek (at Ser-217 and Ser-221) observed after ACTH stimulation are the net result of activation by ACTH, feedback inhibition by Erk, and PKA-mediated inhibition of upstream Rafs. We suggest that the PD98059-dependent increase in Mek phosphorylation seen in the Kin-8 mutant after ACTH treatment may reflect the combined effects of PD98059 (which relieves Erk-dependent feedback inhibition) and the PKA mutation (which relieves the PKA-mediated inhibition of upstream rafs).


   Acknowledgments
 
We thank Jennivine Tsao for excellent technical assistance.


   Footnotes
 
This work was supported by a research grant from the National Cancer Institute of Canada with funds provided by the Canadian Cancer Society.

Abbreviations: FGF, Fibroblast Growth factor; Mek, MAPK kinase.

Received April 11, 2001.

Accepted for publication June 25, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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  3. Schimmer BP 1989 Cyclic AMP and other effectors of cyclic AMP-dependent pathways. In: Gupta RS, ed. Drug resistance in mammalian cells. Boca Raton: CRC Press, Inc.; vol 1:185–210
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