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Address correspondence to Matthew R. Pincus, M.D., Ph.D., Department of Pathology and Laboratory Medicine, New York Harbor VA Medical Center, 800 Poly Place, Brooklyn, NY 11209, USA; tel 718-630-3688; fax 718 630 2960; e-mail matthew.pincus2{at}med.va.gov.
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(received 30 July 2003; accepted 22 August 2003)
Keywords: glutathione-S-transferase-pi (GST-pi), jun-N-terminal kinase (JNK), jun, GST domain peptides
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionsAcknowledgementsReferences |
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Recently, we found that this enzyme is a powerful and selective inhibitor of the activation of jun by JNK [5]. We found that cell extracts from 3T3/4A cells inhibited JNK-mediated phosphorylation of jun. Purification and analysis of the inhibitory protein resulted in its identification as human GST-pi-1, an isoform of GST-pi [5]. In vitro assays for the effect of purified GST-pi on activation of other kinases (eg, protein kinases A and C, casein kinase II, and MAP kinase) showed that this enzyme blocks only the phosphorylation of jun by JNK [5].
We found that GST-pi strongly blocks oocyte maturation induced by oncogenic p21, but not by insulin-activated wild-type ras-p21 [6,7]. This result is consistent with our finding that oncogenic but not wild-type p21 requires direct activation of JNK.
Because GST-pi is a selective inhibitor of oncogenic ras-p21 through its specific inhibition of JNK-jun, we wished to investigate its mode of action and determine the domains involved in its inhibitory function. A major aim of this work was to synthesize GST-pi peptides corresponding to domains that would inhibit jun activation by JNK and would therefore mimic the inhibitory function of the whole GST-pi protein. Such peptides, if they are specific for blocking direct activation of the JNK-jun system by oncogenic ras-p21, might be potential anti-cancer agents that would not affect normal cell growth.
We found that GST-pi does not bind either to JNK or to jun alone, but only to JNK-jun complexes. It uniquely blocks phosphorylation of jun by JNK but does not interfere with JNK activation [5]. Furthermore, its inhibitory activity is unrelated to its anti-xenobiotic function, since mutant forms of GST-pi that do not interact with substrates like glutathione, still have strong anti-JNK-jun activity [5]. On the other hand, certain inhibitors of GST-pi, like glutathione sulfonate and alkyl derivatives of glutathione, also inactivate this enzyme in its function as an inhibitor of JNK-jun [5].
Based on this finding, we investigated possible regions of GST-pi that might be involved in signal transduction, ie, in inhibiting activation of jun by JNK. Since glutathione sulfonate blocks GST-pi from interacting with the JNK-jun system, in a manner unrelated to its inhibition of enzyme activity, we hypothesized that it might exert this blocking effect by preventing domains of GST from undergoing critical structural changes that would allow them to interact with this system. To infer what these regions might be, using the energy-minimized x-ray crystal structure [8] as the starting point, we performed molecular dynamics calculations on the structures of GST-pi in the absence and presence of the inhibitor, glutathione sulfonate [9].
We then superimposed the average structures of GST-pi in these 2 conditions and found that specific discrete domains change conformation: residues 34–50, 99–121, 165–175, 169–182 and 194–201 [9]. With the exception of the 34–50 domain, none of the other domains is involved in binding to glutathione or in the catalytic mechanism.
We synthesized and tested peptides corresponding to 2 of these domains, 34–50 and 194–201, for their abilities to interfere with the in vitro inhibition of JNK’s activation of jun. We found that the 194–201 peptide strongly blocked this interaction while the 34–50 peptide exhibited only weak activity [5,9]. We concluded that the 194–201 domain is involved in the JNK-jun regulatory function of GST-pi [5,9].
Not addressed in these studies was the effect of these peptides on JNK-jun complexes, in the absence of GST and also intracellularly. We wished to explore the effects of peptides corresponding to the other domains of GST-pi.Therefore, in this paper, we determine the effects of all of the peptides corresponding to the putative effector domains of GST-pi that are involved in regulation of JNK-jun function. Specifically, we examine their effects on JNK-jun activation in the presence or absence of GST-pi in cell-free systems and in cells.
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Materials and Methods |
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Residues 34–50: TIDTWMQGLLKPTCLYG.
Residues 99–121: LRGKYVTLIYTNYENGKNDYVK.
Residues 165–182: LAPGCLDNFPLLSAYVAR.
Residues 165–175: LAPGCLDNFPL.
Residues 169–182: CLDNFPLLSAYVAR.
Residues 194–201: SSPEHVNR.
For experiments in cells, 3 of these peptides (ie, 34–50, 165–175, 194–201) were attached on their carboxyl-terminal end to the penetratin sequence, KKWKMRRNQFWVKVQRG, from Antennapedia, which enables transport of the peptide across cell membranes [10,11]. The negative control peptide, X13, from cytochrome p450, MPFSTGKRIMLGE [3], was synthesized by itself and also attached on its carboxyl-terminal end to the penetratin sequence.
Cells. Two cell lines were employed: NIH 3T3 cells obtained from ATCC (Bethesda, MD) and a human astrocytoma cell line (U 291), donated by Dr. D. Weinstin (GliaMed).
Activated JNK. Quantitative preparation of activated JNK was described previously [1,12]. Briefly, a total of 2 x106 NIH 3T3 cells was incubated in DMEM containing 5% bovine calf serum in the presence of anisomycin (anandamide, Calbiochem, San Diego, CA) (12.5 ug/ml) for 20 min at 37°C. This agent is known to induce the stress-activated protein kinase system (SAP) resulting in JNK activation [5].
After incubation, the cells were washed twice with cold PBS and lysed by adding lysis buffer [5,9] (0.35 M LiCl, 50 mM HEPES, pH 7.6, 1 mM EGTA, 1 mM dithiothreitol (DDT), 2 mM MgCl2, 50 mM NPP, and 1 mM sodium vanadate), and an inhibitor mixture consisting of 1 µg/ml of each of the following protease inhibitors: pepstatin, leupeptin, and aprotinin, plus the following phosphatase inhibitors: 1 mM sodium ortho-vanadate and 5 mM sodium fluoride.
The lysate was centrifuged for 15 min at 17,000 x g at 4°C, and the supernatant was either used directly or was stored at -80°C.
Preparation of a pre-formed JNK-jun complex using c-jun fusion protein. This procedure was described previously [1,2,5,9]. Briefly, 2 µg (20 µl) of c-jun fusion protein beads (Invitrogen, Carlsbad, CA) were incubated with 250 µl of cell lysate (~250 µg total protein) with gentle rocking overnight at 4°C. The mixture was subjected to microcentrifugation for 30 sec at 4°C; the pellet was washed twice with 500 µl of 1x lysis buffer and twice with 500 µl of 1x kinase buffer (25 mM tris, pH 7.5, 5 mM 3-glycerophosphate, 2 mM dithiothreitol, 0.1 mM sodium vanadate, 10 mM MgCl2) [1,2,5] and stored on ice.
Kinase assay. The jun beads complexed with JNK were resuspended in 50 µl of 1x kinase buffer (see above) to which ATP (Sigma, St. Louis, MO) was added to a final concentration of 100 mM and incubated for 30 min at 30°C. Reactions were terminated with 25 µl of 1x SDS sample buffer (62.5 mM tris-HCl, pH 6.8, 2% w/v SDS, 10% glycerol, 50 mM DTT, 0.1% w/v bromphenol blue) [1,2,5]. The resulting solutions were boiled for 5 min, after which they were subjected to microcentrifugation for 2 min. Aliquots of 20 µl were subjected to SDS-PAGE; the gels were transferred onto nitrocellulose membranes (Millipore, Billerica, MA) and incubated with anti-phospho-jun antibodies that included anti-phospho-S63, -T-73, or both, and anti-phospho-JNK antibody (New England Biolab, Beverly, MA).
All antibodies were used at a 1:1000 dilution in blocking buffer (tris-buffered saline, pH 7.6, containing 0.1% Tween-20 with 5% w/v non-fat dry milk [1,2,5]). The secondary antibody was peroxidase-labeled (Pierce, Rockford, IL) and was used in a dilution of 1:100,000 in blocking buffer.
Effects of GST-pi peptides on JNK activation of jun in the presence or absence of GST-pi. To investigate the effect of GST-pi peptides on jun activation by JNK in the presence of GST-pi, we used a protocol described previously [5,9]. To the preformed bead-bound jun-JNK complex was added 0.05 mM GST-pi (Sigma, St. Louis, MO) either alone (control) or in the presence of each GST-pi or negative control X13 peptide (100 µM). Alternatively, the peptides were added to the incubation mixture in the absence of GST-pi. In both cases, the mixture was incubated at room temperature for 30 min, after which ATP was added, and the procedure described for the kinase assay (preceding section) was followed.
Cell experiments. To evaluate the effects of GST-pi peptides on JNK-induced jun activation, we introduced each of 4 different peptides (GST-pi peptides 34–50, 165–175, 194–201, and X13) attached to penetratin on their carboxyl-terminal ends into U 291 astrocytes. In these experiments, 2 x106 U 291 cells were treated with GST peptides attached to penetratin (100 µM) or control X13-penetratin peptide (100 µM) overnight at 37°C in DMEM culture medium, supplemented with 10% fetal bovine serum, after which anisomycin (25 µg/ ml) was added for 45 min to activate JNK [9]. The cells were lysed, and the lysate blotted for phosphorylated JNK and jun as described for NIH 3T3 cells above.
Assay for abilities of peptides to inhibit kinase systems.
Lysates from anisomycin-treated astrocytes (in the absence of peptides) as in the preceding section were used to assay several kinase systems as follows. Incubation mixtures contained 20 µg of cell lysate, 10 µg kinase substrate peptide, and 50 µM final concentration of either negative control (X13) or GST-pi peptide (34–50 and 194–201). Kinase reactions were initiated when 32P-
-ATP (2 µCi) was added to the reaction mixture so the total volume was 10 µl. The kinase substrate peptides were casein kinase substrate peptide (RRKDLHDDEEDEAMSITA); MAP kinase substrate peptide (APRTPGGRR); and JNK substrate jun 5–89 peptide (Upstate Cell Signaling, Charlottesville, VA; Resource International, Camarillo, CA). The mixture was incubated for 30 min at 30°C. The samples were then precipitated with cold TCA and solubilized in SDS protein sample buffer (see above) and electrophoresed in 15% Trichinae PAAG gel (Invitrogen). The radioactivity was detected by a phosphorimager system (Molecular Dynamics, Sunnyvale, CA).
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. As shown in lane 2 of this figure, untreated cells contain low background levels of phosphorylated JNK and no detectable levels of phosphorylated jun. Activated JNK from these anisomycin-treated cells was then incubated with c-jun beads [1,2,5], in the presence of GST-pi alone, or in the presence of peptides from different domains of this protein.
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, lane 1, activated JNK strongly phosphorylates jun peptide in the absence of GST-pi. As shown in lane 2 of Fig. 2, GST-pi completely blocks this activation, as previously reported [5]. Incubation of GST-pi with the JNK-jun peptide system was then carried out in the presence of the peptides from the domains of GST-pi that were found to undergo conformational changes in the molecular dynamics calculations [9].
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, when the JNK-jun-GST system is incubated with the 34–50 domain, that is part of the substrate binding site [8], there is no phosphorylation of jun, indicating that GST inhibition is not relieved by this peptide, as found previously and as also found for the negative control X13 peptide (lane 8, Fig. 2) [5,9]. In contrast, as shown in lane 4 of Fig. 2, the 194–201 peptide from the carboxyl-terminal domain of the protein displaces GST-pi from the complex, allowing phosphorylation of jun, as noted previously [5,9].
We prepared 3 peptides corresponding to 2 neighboring domains, around residues 169–171 and 173–178, that exhibit large differences in average structure when the unbound, active structure is superimposed on that of the inhibitor-inactivated enzyme [9]. Since the conformation of a central residue is most strongly influenced by the 4 neighboring residues on its amino- and carboxyl-terminal ends [13], we prepared peptides corresponding to residues 165–175 and 169–182 that contained the 2 domains. In addition, we prepared a larger peptide that contained both domains, ie, 165–182. Lanes 5,6 and 7 of Fig. 2 show the effects of the 165–182, 165–175, and 169–182, respectively, on GST-pi inhibition. The first 2 peptides are shown to cause some diminished levels of GST-pi inhibition, but at lower levels compared to the 194–201 peptide (lane 4), while the 169–182 sequence has no effect. Since the first 2 peptides both contain residues 165–168, these residues may be important in interacting with the JNK-jun complex.
Interestingly, the 99–121 peptide that, together with the 34–50 domain, exhibits the largest conformational differences between active and inactive forms of GST-pi [9], strongly relieves GST inhibition (lane 9, Fig 2), implicating it as an important domain in interacting with the JNK-jun complex. In addition, it appears actually to enhance phosphorylation in the presence of GST-pi, although this effect is not observed for the peptide in the absence of GST-pi (Fig. 3, lane 8). This result suggests that GST-pi and the GST-pi 99–121 peptide may interact in a cooperative manner to enhance jun phosphorylation by JNK. Overall, of the 6 potential effector domain peptides, two (194–201 and 99–121) appear to interact strongly with the JNK-jun complex, while the 34–50 domain does not bind to the complex in such a way as to compete with GST-pi.
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We therefore examined the effects of each of these peptides on JNK-induced phosphorylation of jun in the absence of GST-pi. As shown in lane 2 of Fig. 3, the 34–50 GST-pi peptide strongly inhibits phosphorylation while, surprisingly, the 194–201 peptide (lane 3) has almost no effect (compare lane 3 with control lanes 1 and 7 to which no peptide [lane 1] or the unrelated X13 peptide [lane 7] was added). In addition, as shown in lane 8 of Fig. 3, the 99–121 peptide, which, from Fig. 2, strongly displaces GST-pi from the JNK-jun complex, causes a low level of inhibition that is comparable to the negative control peptide (lane 7). Furthermore, in Fig. 3, the 3 peptides 165–182 (lane 4), 165–175 (lane 5), and 169–182 (lane 6), the first 2 of which weakly compete with GST-pi (Fig. 2), are seen to block phosphorylation strongly, although this inhibition is weaker for the 165–175 peptide (lane 5). In contrast to the results obtained in Fig. 2, in which the 165–168 segment of the peptides was implicated as important in the interaction of 165–182 domain with JNK-jun, residues 176–182 appear to be important in inhibition of phosphorylation.
Previously, based on the low level of inhibition of GST binding by the 34–50 peptide, we concluded that this peptide might not be part of an effector domain of GST-pi in binding to the JNK-jun complex [9]. In contrast, the results in Fig 3 suggest that this is an effector domain that is involved with phosphorylation. Furthermore, the results in Figs 2 and 3 suggest that the 99–121 and 165–182 domains also constitute effector domains, the former being involved in the binding of GST-pi to the JNK-jun complex, and the latter being involved in phosphorylation and, to a lesser extent, binding.
Overall, from Figs. 2 and 3, it appears that peptides that displace GST from the JNK-jun complex (99–121 and 194–201) have a minimal effect on phosphorylation, while peptides like 34–50 and 165–182 that have a minimal effect on GST binding have a major inhibitory effect on phosphorylation. Evidently, the determinants for binding of GST to the JNK-jun complex are separate from those affecting the phosphorylation of jun by JNK.
Effects of GST-pi peptides in cells.
Since the effects of GST-pi domain peptides have been assayed in vitro in cell-free systems, we have further determined the activities of GST-pi peptide in cells. For this purpose, we synthesized three GST-pi peptides, 34–50, 165–175, and 194–201, attached to a penetratin sequence from Antennapedia that allows them to cross the cell membrane [10,11]. We chose these peptides because each shows a different inhibition pattern in the cell-free system (Figs. 2 and 3). To assay their effects in cells, we chose astrocytes since these cells contain significant amounts of basally activated JNK and show an enhanced response to anisomycin (Adler, V., unpublished observations).
As shown in lane 2 of Fig. 4, activated JNK is present at a significant level in untreated cells, and, as shown in lane 6, anisomycin induces phosphorylation of jun. This phosphorylation is not blocked by the negative control, X13-penetratin (not shown). The jun phosphorylation is almost totally blocked by the 34–50-penetratin peptide (lane 3) while it is not inhibited by the 194–201 peptide (lane 5). The 165–175-penetratin peptide causes modest inhibition (lane 4). These results parallel those observed in the cell-free system, shown in Fig. 3, and therefore suggest that the mechanism of inhibition of jun activation by JNK in cells is similar to that observed in the cell-free system.
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). As shown in Fig. 4, none of the other peptides affects activated JNK levels. Since activated JNK exists in untreated cells (lane 2), the results in lane 3 suggest that 34–50-penetratin peptide may promote JNK inactivation.
Efects of GST-pi peptides on other kinase systems.
We have previously found that GST-pi specifically blocks the JNK-jun system but not other kinase systems such as casein kinase and MAP kinase [5]. Since the GST-pi peptides affect the ability of GST-pi to interact with the JNK-jun system, we have further explored whether these peptides are specific to the JNK-jun system. Cell lysates from anisomycin-treated astrocytes, as described in the preceding section, were incubated with jun substrate peptide (residues 5–89) [1,2,5], casein kinase, or MAP kinase substrate peptides in the presence or absence of either GST peptide 34–50 or 194–201. -32P-ATP was then added to the incubation mixture, and autoradiograms were prepared.
In Fig. 5, lane 3 displays cell lysate incubated with the 3 peptide substrates, all of which are shown to be phosphorylated. Lane 4 shows the results of incubating cell extract with jun and casein kinase substrate peptides in the presence of 50 µM GST peptide 34–50. The casein kinase peptide is phosphorylated as in control lane 3, while the jun peptide is not phosphorylated. Similar results are shown in lane 6, which is the same experiment as in lane 4, except the substrates were jun and MAP kinase peptides. It is evident that the GST 34–50 peptide blocks phosphorylation of jun but not MAP kinase peptide. Lanes 5 and 7 show that the GST-pi 194–201 peptide has no effect on phosphorylation of jun peptide, casein kinase peptide (lane 5), or MAP kinase peptide (lane 7). Thus, it appears that GST-pi 34–50 peptide is a specific inhibitor of JNK-induced phosphorylation of jun.
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Discussion |
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An unexpected finding in the current study is that the 34–50 peptide does not displace GST-pi from the JNK-jun complex and strongly blocks the ability of JNK to activate jun. A similar finding holds for the 165–182 domain peptide although it has some activity in removing inhibition of phosphorylation by GST-pi. Conversely, as found in a previous study [9], the 194–201 domain peptide has strong activity in removing GST-pi inhibition, but has no effect on phosphorylation. The 99–121 domain peptide produces a similar pattern, although it shows weak inhibition of phosphorylation.
Separation of binding vs phosphorylation domains.
These results suggest that peptides that block GST-pi interaction with JNK-jun have minimal effects on phosphorylation, while those that block phosphorylation have minimal effects on GST-pi binding. This implies that sites for strong GST binding to the JNK-jun complex are separate from those that interact with jun phosphorylation sites. Possible overlaps may occur in the 165–182 domain since this peptide interferes minimally with GST-pi binding on its 165–168 end and also strongly blocks jun phosphorylation on its 176–182 end. One mechanism by which the 34–50 peptide blocks phosphorylation of jun by JNK is by either blocking phosphorylation of JNK or by promoting dephosphorylation of activated JNK, since the background levels of activated JNK that occur in astrocytes are not present in astrocytes treated with the 34–50-penetratin peptide (Fig. 4).
Results in cells parallel those in the cell-free system. From prior studies [5], we found that, in resting 3T3/4A cells, JNK is inactivated by binding in complex with jun to monomeric GST-pi. Under conditions of chemical or oxidative stress, GST-pi undergoes multimerization through intermolecular disulfide bond formation that results in its inactivation and allows JNK to activate jun [5]. Since they are presumably unaffected by this process, GST-pi peptides that block phosphorylation of jun by JNK in the cell-free system should likewise block it in cells; conversely, peptides that only affect binding of GST-pi to the JNK-jun complex should not affect JNK-induced phosphorylation of jun, because (a) they do not affect phosphorylation in the cell-free system and (b) GST-pi presumably has dissociated as a multimer from the JNK-jun complex.
As shown in Fig. 4 (lane 3), the 34–50 GST-pi peptide, which completely blocks phosphorylation in the cell-free system (Fig. 3, lane 2), likewise completely blocks phosphorylation in astrocytes. In contrast, the 194–201 peptide that does not block phosphorylation in the cell-free system (Fig. 3, lane 3) likewise does not block it in astrocytes (lane 5, Fig. 4). Furthermore, the 165–175 peptide weakly inhibits phosphorylation (lane 4, Fig. 4) as it likewise was found to do in the cell-free system (lane 5, Fig. 3). Thus, there is close agreement between the results obtained with these GST-pi peptides in the cell-free system and in cells. Since these peptides all contain the penetratin sequence on their carboxyl-terminal ends to enable them to cross the cell membrane, this segment appears to have no effect on their activities. This conclusion is supported by the finding that the negative control X13-penetratin peptide had no effect on the cells.
Specificity of peptide inhibition of the JNK-jun and other kinase systems. Our finding that the GST-pi peptides exert major inhibitory effects on either GST inhibition of phosphorylation or on phosphorylation itself suggests specificity of peptide effects. Thus, for example, the 194–201 peptide blocks GST-pi inhibition of jun phosphorylation by JNK, but the 34–50 peptide does not, while the 34–50 peptide blocks jun phosphorylation by JNK in the absence of GST-pi, while the 194–201 peptide does not. The negative control X13 peptide does not affect either process.
Further evidence regarding peptide specificity is provided by the effects of peptides on different kinase systems. In prior studies [5], we performed in vitro assays of the effect of GST-pi on the JNK-jun system and on other kinase systems, including src, PKA, MAP kinase, and casein kinase, and found that it inhibited only the JNK-jun system. Since oncogenic ras-p21 is selectively blocked by GST-pi [6], we were motivated to find whether one or more peptide domains from GST-pi might also inhibit the JNK-jun system selectively. If so, such peptides would be candidates to inhibit oncogenic ras-p21.
As we found for the whole protein, we find that the 34–50 GST-pi peptide strongly inhibits phosphorylation of jun by JNK (Fig. 3) but has no effect on other kinase systems, such as casein kinase and MAP kinase (Fig. 5). In parallel, the 194–201 peptide has no effect on these kinases, but also has no effect on JNK-induced phosphorylation of jun. Its effect appears uniquely to be to displace GST-pi competitively from its association with JNK-jun.
Despite its selective inhibition of JNK-jun, the GST-pi 34–50 peptide does not competitively displace GST-pi from its complex with JNK-jun, unlike the 165–182 domain peptide that blocks phosphorylation and weakly inhibits GST-pi binding (Figs. 2 and 3). This result suggests that the 34–50 peptide may be well-suited as an anti-oncogenic ras-p21 agent, because (a) it would not displace GST-pi from its inhibitory complex with JNK-jun and (b) it would presumably not be subject to cell stresses that inactivate GST-pi inhibition by causing its dissociation from the complex [5].
Possible structure-function relationships. All of the peptides corresponding to the 6 domains, identified from the molecular dynamics calculations, affect the JNK-jun system and there is a separation of their effects on binding and on kinase activity. These results suggest that GST-pi domains affecting these functions are spatially removed from one another in the 3-dimensional structure of the protein.
Fig. 6 is a color space-filling representation of the molecular dynamics-computed average structure of free (active) GST-pi. In this figure, the main protein chain is colored red while the effector domains are colored differently. As shown in this figure, these domains constitute exposed surfaces that have the potential of interacting with other proteins and are spread over a significant portion of the protein surface, suggesting that the interactions of this protein with JNK-jun are complex, as might be expected since GST-pi interacts with both JNK and jun [5].
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Since the 99–121 domain more strongly affects binding than phosphorylation, while the 165–182 domain affects phosphorylation more strongly than binding, one scenario for the interactions between GST-pi and the JNK-jun complex would be a 4-point attachment in which, as shown in Fig. 6, the left-most (99–121) and right-most (194–201) domains would bind to the JNK-jun complex, while the top-most (165–182) and bottom-most (34–50) domains would interact with the phosphorylation sites. Since the 34–50 peptide appears to affect JNK activation and also to affect JNK-induced phosphorylation of the jun 5–89 peptide (the N-terminal regulatory domain of jun), it may interact both with JNK phosphorylation sites and the N-terminal domain of jun.
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Conclusions |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionsAcknowledgementsReferences |
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Acknowledgements |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionsAcknowledgementsReferences |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionsAcknowledgementsReferences |
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,– a regulator of JNK signaling. EMBO J 1999;18:1321–1334.[Medline]
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