| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Department of Clinical Medicine, Prevention and Biotechnology, University of Milan-Bicocca, Monza, Italy
2 Department of Oncology, McGill University, Montreal, Canada
3 Laboratoire de Chimie Therapeutique, School of Pharmaceutical Sciences, University of Geneva, Geneva, Switzerland
4 MRC Protein Phosphorylation Unit, School of Life Sciences, University of Dundee, Dundee, Scotland, United Kingdom
5 Department of Experimental Oncology, Istituto Nazionale Tumori, Milan, Italy
(Requests for offprints should be addressed to L Mologni at Department of Clinical Medicine and Prevention, DIMEP, University of Milan-Bicocca, via Cadore 48, 20052 Monza, Italy; Email: luca.mologni{at}unimib.it)
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
PTCs are the most frequent cancers of the thyroid gland, accounting for about 80% of all cases (Santoro et al. 2004). About 30–40% of sporadic and up to 85% of radiation-induced PTCs are associated with genomic rearrangements of the RET locus that lead to the expression of constitutively activated RET/PTC fusion tyrosine kinases (Nikiforov et al. 1997, Putzer & Drosten 2004). As rearranged during transfection (RET)/PTC expression is already found in small, indolent nodules, it is believed to be a very early event in thyroid carcinogenesis. Current therapy for PTC is based on surgical removal of the thyroid gland followed by radioiodine therapy and thyroid hormone administration, which can cause a number of undesired effects.
Point mutations of RET are responsible for the onset of both sporadic and familial forms of MTC (Ichihara et al. 2004). All individuals affected by multiple endocrine neoplasia type 2 syndromes (MEN2A and MEN2B) harbor germ line RET mutations. These patients develop MTC in all cases, plus pheochromocy-toma and hyperparathyroidism with variable penetrance, in addition to other symptoms, which are reported in a minority of cases. Similar to PTC, the treatment of choice for MTC relies on surgical intervention. While hereditary MTC is diagnosed at an early stage, sporadic MTC is characterized by a high recurrence rate; more than 50% of patients eventually die of their disease.
Tumor- or gene-specific therapies are thought to be the most promising alternatives to the present chemotherapy or radiotherapy regimes. Different strategies have been devised to achieve selectivity, which includes immunotherapy (Powell et al. 2003), gene therapy (Drosten & Putzer 2003), ribozymes (Parthasarathy et al. 1999), and small-molecule inhibitors. The last option is well represented by the remarkable success of Imatinib in the treatment of chronic myeloid leukemia and gastrointestinal stromal tumors (Kadomatsu et al. 1997, Kantarjian et al. 2002). Given that a certain oncogene is a primary early event and is necessary for tumor cell survival, but could be dispensable for normal cells, blockage of its action is instrumental to a successful therapy. A few small-molecule inhibitors of RET have been identified so far, which demonstrated growth inhibition of cells expressing RET oncogenes (Carlomagno et al. 2002a,b, 2003, Lanzi et al. 2003, Strock et al. 2003).
SU5416 is a 3-substituted indolin-2-one compound that was discovered as a vascular endothelial cell growth factor receptor (VEGFR) inhibitor (Sun et al. 1998) and is currently undergoing clinical evaluation as an anti-angiogenesis drug for a number of solid tumors (Glade-Bender et al. 2003). More recently, SU5416 has been shown to inhibit other tyrosine kinases, such as FLT-3, c-Kit, and c-MET (Krystal et al. 2001, Yee et al. 2002, Wang et al. 2004), while it is inactive against epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), insulin-like growth factor 1 receptor (IGF1R), and platelet-derived growth factor receptor (PDGFR). Phase II clinical trials are ongoing with the compound as a FLT-3 inhibitor in patients with advanced acute myelogenous leukemia (AML) (Fiedler et al. 2003).
In this study, we found that SU5416 blocks RET kinase activity. We show that cell growth and downstream signaling promoted by RET/PTC oncogene are prevented by SU5416.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
All cell culture media were supplemented with 100 U/ml penicillin, 100 µg/ml gentamicin, and 2 mM Gln. Murine pro-B cell line Ba/F3 was maintained in RPMI medium with 10% fetal bovine serum (FBS), plus CHO-conditioned supernatant as a source of IL-3. Three Ba/F3-derived cell lines expressing RET/PTC2 (hereafter referred to as Ba/PTC), NMP/ALK (Ba/NA, Coluccia et al. 2004) and BCR/ABL (Ba/BA, Piazza et al. 2005) were generated by transfection of the corresponding fusion cDNA and selection of stable transfectants with 1 mg/ml G418. These cells were kept in RPMI plus 10% FBS without IL-3. PTC cells, TPC-1, and NPA were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS. Parental NIH-3T3 and NIH-3T3 cells stably transfected with RET/PTC2 (NIH-PTC2) and RETV804M mutant (NIH-V804M) were cultured in DMEM with 10% (parental cells) or 5% FBS. Insect cell line Sf9 was kept at 27 °C in SF900-II medium (Invitrogen) with 10% FBS and 0.1% Pluronic F-68.
Antibodies were used in western blotting according to recommended dilutions. Anti-HisG antibody (Invi-trogen) is directed against the 6xhistidine tag followed by a glycine. Anti-RET (C-19, Santa Cruz Biotechnology, Santa Cruz, CA, USA) recognizes the short isoform of wild-type RET; anti-phospho-RET (recognizing phos-photyrosine 905 of wild-type RET), anti-phospho-JNK1/2 (which detects active diphosphorylated (Thr183/Tyr185) JNK1 and JNK2) and anti-JNK1/2 antibodies were from Cell Signaling Technology (Danvers, MA, USA). Anti-phosphotyrosine (4G10) was purchased from Upstate Biotechnology (Charlottes-ville, VA, USA); anti-ERK1/2 and anti-phospho-ERK1/2 (Thr183/Tyr185) were from Sigma; anti-p21/WAF1 (Ab-1) and anti-p27 (C-19)-G antibodies were bought from Merck Biosciences (Darmstadt, Germany) and Santa Cruz Biotechnology respectively.
SU5416 was purchased from Calbiochem, dissolved in dimethyl sulfoxide (DMSO), aliquoted and stored at –20 °C until used.
Production of recombinant proteins and in vitro kinase assay
Human FLT-3 (amino acids (aa) 589–993, EntrezGene accession number NP_004110
[GenBank]
), ABL (aa 230–517, isoform a, NP_005148
[GenBank]
) and RET (aa 700–1020, NP_066124
[GenBank]
) kinase domain cDNA sequences were cloned in pHisE baculovirus transfer vector (Mologni et al. 2005), in BamHI/SalI, KpnI/XbaI and KpnI/SalI restriction sites respectively. Mutant V804M RET was obtained from wild-type RET plasmid by site-directed mutagenesis using the QuikChange kit (Stratagene, La Jolla, CA, USA), according to manufacturer’s instructions, with the following mutagenic primers: sense, GGCCCGCTCCTCCTCATCATGGAGTACGCCAAA-TACGGC, and antisense, GCCGTATTTGGCGTACTC-CATGATGAGGAGGAGCGGGCC (the nucleotide corresponding to the missense mutation is underlined). ALK sequence was cloned in pBlueBacHis2C vector (Invitrogen) as described (Donella-Deana et al. 2005). The sequence of all inserts was verified by DNA sequencing. Recombinant proteins were expressed in Sf9 cells with an amino terminal hexa-histidine tag to aid in purification, as described (Mologni et al. 2005). Briefly, proteins from total lysate were fractionated in a DEAE-sepharose column and positive fractions were identified by anti-HisG western blotting, loaded on a nickel-chelate affinity column and eluted with imida-zole. The initial anion-exchange step is required to achieve homogeneity in the final preparation. Activity of purified kinase domains was verified by radioactive autophosphorylation assay: a reaction mix containing 0.5 µg recombinant enzyme, 5 µM cold ATP, 1 mM DTT, 10 µCi [
-32P]ATP, 25 mM Hepes pH 7.5, 10 mM MgCl2, and 10 mM MnCl2 was incubated at 30 °C for 15 min. The reaction was stopped by adding Laemmli buffer and heating at 95 °C for 5 min. The samples were resolved on a 10% SDS-PAGE gel and proteins were transferred to an ImmobilonTM-P membrane and visualized by autoradiography.
In vitro kinase assays
ELISA kinase assay was performed as described (Mologni et al. 2005), using approximately 1 pmol recombinant enzyme and 1 nmol peptide-substrate, assuring Michaelis–Menten assumption that the substrate is in vast excess with respect to the enzyme. Peptide substrates used include RET activation loop-derived peptide (SRDVYEEDSYVKRSQGRIPVK, for RET kinase) and ALK activation loop-derived mutant peptide (Donella-Deana et al. 2005) in which Tyr 1282 and 1283 were replaced by phenylalanine (ARDIYR-ASFFRKGGCAMLPVK, used with ALK, ABL, and FLT-3 kinases). For non-radioactive autokinase assay, the recombinant enzyme was incubated for 15 min at 30 °C in kinase buffer (25 mM Hepes, pH 7.0, 1 mM MnCl2, and 5 mM MgCl2) in the presence of 6.5 µM ATP, with or without inhibitors. The reaction was stopped by adding Laemmli buffer and the sample was loaded on SDS-PAGE, transferred to a nitrocellu-lose membrane and detected with anti-phospho-RET or anti-HisG antibodies.
Inhibitor specificity profiling
Radioactive kinase assays were performed at room temperature in the presence of substrate peptides, [-33P]ATP, and 10 µM SU5416, using recombinant kinases obtained from various expression systems. Procedures for purification and assay of 30 of the 52 kinases have been described (Davies et al. 2000, Bain et al. 2003). The other enzymes will be described elsewhere. Kinase assays were performed using ATP concentrations close to the Km value for each kinase. All protein kinases were grouped accordingly into three categories, namely 5, 20, and 50 µM ATP.
Proliferation assay and growth curve
Serial dilutions of kinase inhibitor were prepared in cell culture medium with 1% FBS in 96-well plates. Cells were then resuspended in 1% FBS medium and added to the plate at 104cells/well. Cell proliferation was measured at 72 h using the tritiated-thymidine incorporation assay as described previously (Gambacorti-Passerini et al. 1997). Each data point was done in triplicate.
For growth curves, Ba/F3 and Ba/PTC cells were seeded (105/well) in 24-well plates in triplicate and treated with DMSO or various concentrations of SU5416. Every 2nd day, the cells were counted by Trypan Blue exclusion assay, diluted to keep them in logarithmic growth phase, and fresh inhibitor was added.
Western blot analysis of cell extracts
Ba/PTC cells were grown overnight in 1% FBS and treated with SU5416 or DMSO for 4 h. They were then washed with PBS and lysed in 1 x Laemmli buffer (62.5 mM Tris–HCl, pH 6.8, 2% SDS, 10% glycerol, and 0.3 M ß-mercaptoethanol). Total lysates corresponding to 3 x 105 cells were loaded on SDS-PAGE, transferred to nitrocellulose membrane and probed with anti-RETand anti-phospho-RET antibodies.
TPC-1 cells (6 x 106) were seeded in 100 mm cell culture dishes, grown overnight in 1% FBS and treated with SU5416 or DMSO. Cell lysates were prepared after washing with PBS, by scraping the cells in lysis buffer (25 mM Tris–HCl, pH 7.9, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 2 mM EGTA, 10 mM NaF, 1 mM Na3VO4, 1 mM DTT, 1 mM PMSF, 1 µg/ml leupeptin and aprotinin) and incubating for 40 min on ice. The lysates were then centrifuged for 30 min at 13 000 r.p.m. at 4 °C and total protein content of the supernatants was measured by Bradford assay. Equal amounts (100 µg) of total protein were loaded on SDS-PAGE and analyzed by western blotting using phospho-specific antibodies.
Soft-agar growth assay
NIH-PTC-L cells were seeded in six-well plates at 10 000 cells/well in a medium containing 0.33% low melting agar (type VII, Sigma) and either DMSO or 20 µM SU5416, on a layer of 0.5% agar, as described (Borrello et al. 1996). Fresh inhibitor was added every 3 days to the top layer. The colonies were counted after 15 days.
Cell-cycle analysis
Cells were seeded in six-well plates at a density of 2 x 105/well and treated with inhibitor or vehicle. Cells were harvested at 24, 48, and 72 h after treatment, washed with PBS and fixed in 70% ethanol at –20 °C. The samples were then centrifuged and resuspended in PBS containing 50 µg/ml propidium iodide and 100 µg/ml RNase A, incubated at 37 °C for 30 min and analyzed by FACScan flow cytometer (Beckton Dickinson, Franklin Lakes, NJ, USA).
ATP competition assay
Increasing doses of ATP (0.011–2.4 mM) were incubated with RET in the presence of DMSO, or 0.05, 0.25, and 1.25 µM SU5416, and an ELISA kinase assay was performed as described (Mologni et al. 2005). ATP binding curves were then calculated for each inhibitor concentration and plotted using the Lineweaver–Burke method.
In vivo target modulation
Female CD-1 nu/nu mice (7–9 weeks old) were supplied by Charles River (Calco, Como, Italy) and kept under standard laboratory conditions according to the guidelines of the Istituto Nazionale Tumori (INT), Milan, Italy. Animal studies were approved by the Ethics Committee for Animal Experimentation of INT. Mice were implanted subcutaneously with NIH-PTC cells (106 cells/mouse). When the tumors were measurable, the mice received an acute i.p. dose of 50 mg/kg SU5416 in 60 µl DMSO, or DMSO alone. Mice were sacrificed 6 h after treatment and their tumors resected and snap frozen on liquid nitrogen. Tumor mass was minced in lysis buffer and lysates were then prepared as described earlier. Equal amounts of total protein were loaded on 8% SDS-PAGE and visualized by western blot with anti-RET and anti-phospho-RET antibodies.
Molecular modeling
A model of the tertiary structure of RET tyrosine kinase domain in an active conformation was built by homology modeling, using the phosphorylated active insulin receptor kinase domain (Protein Data Bank entry, 1IR3 [PDB] ) as a template. The amino acid sequence similarity between RETand insulin receptor kinase domains is 57%. The model was refined using AMBER 6 software (Case et al. 1999) for computing molecular dynamic simulations. The model of V804M mutant RET was built by changing Val-804 of wild-type RET model into a methionine; local side chain minimization was then performed with tripos force field and Powell method, with a convergence criterion of 0.05 kcal/mol, using Sybyl 7.0 (Anonymous). The binding mode of SU5416 to wild-type and V804M mutant RET was evaluated by molecular docking, using GOLD algorithm (Tripos Inc., St. Louis, MO, USA) (Jones et al. 1997).
Statistical analysis
Data were always generated in triplicate and mean ± S.D. is reported on graphs. Dose–response curves were normalized over the vehicle control and analyzed by non-linear regression using Graph Pad PRISM 4.0 software. IC50 data are reported as the global fitting of at least three independent experiments, with 95% confidence interval (CI).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
A series of compounds sharing a 2-indolinone core structure was screened for RET inhibition in an in vitro ELISA-based kinase assay, using the recombinant enzyme and an exogenous peptide as a substrate. In a previous work, we reported the activity of two of these compounds (Mologni et al. 2005). SU5416 (Fig. 1
) belongs to the same family of inhibitors and was therefore tested in our assay. SU5416 inhibited RET with an IC50 of 170 nM, using 300 µM ATP (Fig. 2A). In the same experimental conditions, the compound showed a similar degree of inhibition on FLT-3 kinase, a well-known SU5416 target, while it was much less active against ABL and ALK kinases (Table 1; IC50 values from three or more experiments are reported with 95% CI. In order to confirm the data, we performed an autophosphorylation assay of recombinant RET (Fig. 2B). Significant inhibition was already seen at 0.1 µM, supporting the finding that SU5416 is a RET inhibitor. The same membrane was stripped and developed with anti-HisG antibody to show that similar amounts of protein have been loaded.
|
|
|
). A secondary plot of the data from Fig. 2C showed a linear correlation between the slopes of the curves and the inhibitor concentrations (data not shown). These results suggest competition between SU5416 and ATP and are in agreement with virtual docking data showing that SU5416 binds within the ATP-binding pocket (see below). Moreover, dose–response experiments performed at different ATP doses indicated that the IC50 of the compound increased progressively from 0.05 µM (at 11 µM ATP) to over 6 µM (with 3 mM ATP), again indicating an ATP-competitive behavior (data not shown). The calculated Ki of compound SU5416 for RET was 13 nM. Inhibition of RET-mediated transformation
Having established SU5416 as a good inhibitor of RET in cell-free conditions, we sought to determine whether the compound would be toxic to RET oncogene-expressing cells. Murine pro-B cell line Ba/F3 needs exogenous IL-3 for growth and is commonly exploited to study the transforming ability of oncogenes. We selected a stably transfected Ba/F3 clone expressing RET/PTC2 (Ba/PTC). These cells grow independently of IL-3 (Fig. 3A) and show constitutive high levels of tyrosine-phosphorylated proteins (data not shown). Ba/PTC cell growth was blocked by the treatment with 2 µM PP1, a known RET inhibitor (Carlomagno et al. 2002b) (Fig. 3A). Compound SU5416 blocked the proliferation of Ba/PTC cells in a dose-dependent manner, with an IC50 of 7.9 µM (95% CI, 6.8–9.1 µM), while sparing parental Ba/F3 cells (Fig. 3B). The observed proliferation arrest correlated with a strong inhibition of RET/PTC2 phosphorylation, as shown by anti-phospho-RET western blot of total lysates (Fig. 3C). Densitometric analysis of phosphorylated bands revealed an IC50 of approximately 5 µM, consistent with proliferation data. RET expression level was not affected by the treatment, as shown in the lower panel of Fig. 3C. In line with the results obtained in ELISA, the treatment with SU5416 only marginally affected the proliferation of NPM/ALK- and BCR/ABL-transfected Ba/F3 cells, which showed IC50 values between 30 and 40 µM (data not shown). A time-course experiment was carried out by exposing Ba/F3 and Ba/PTC cells to SU5416 for 10 days. The growth of Ba/PTC cells was progressively delayed with increasing SU5416 concentrations, while Ba/F3 cells were not affected (Fig. 3D).
|
). After 48 h of SU5416 treatment, the morphology of cells reverted to a flattened, non-dense appearance (Fig. 4D), similar to parental cells (shown in Fig. 4B), indicating blockage of RET tyrosine kinase signaling. In contrast, DMSO-treated cells maintained a transformed phenotype (Fig. 4C). NIH-PTC2 cells treated with SU5416 were no longer able to grow in soft agar medium (Fig. 4F), or form transformation foci (data not shown). These data suggest that SU5416 is able to abrogate the transforming potential of the RET/PTC2 oncogene.
|
TPC-1 cells are derived from PTC and express the RET/PTC1 fusion kinase. The proliferation of TPC-1 cells was inhibited in the presence of SU5416 (Fig. 5A), with an IC50 of 2.7 µM (95% CI, 2.3–3.2 µM). In contrast, NPA, a RET-negative papillary carcinoma cell line, was much more resistant to the treatment, showing 50% inhibition at 40 µM. The proliferative block of TPC-1 cells correlated at the molecular level with a complete shut off of RET/PTC1 Tyr-905 phosphoryl-ation, after 8 h treatment (Fig. 5B). Tyr-905 (numbering refers to wild-type RET) is a main switch of RET catalytic activity. When phosphorylated, it stabilizes the open conformation of the activation loop, thus allowing substrate entry into the active site. It is therefore considered to be a hallmark of RET kinase activation (Kawamoto et al. 2004). As SU5416 induced inactivation of RET/PTC1 kinase, we investigated whether downstream signaling pathways were affected by the treatment. RET oncogenes have been shown to activate a number of proliferative and anti-apoptotic signals, including the RAS/ERK and the JNK pathways (Chiariello et al. 1998). In TPC-1 cells treated with SU5416 for 36 h, a reduction of both ERK1/2 and JNK1/2 phosphorylation was observed, concomitantly with a block of RET/PTC1 phosphorylation (Fig. 5C). Relative phosphorylation inhibition of p44 and p42 ERK was 90 and 75% respectively, as determined by densitometric analysis of the bands. The phospho-JNK signal in treated cells was reduced by 60%.
|
). No sub-G1 peak was detected, even after 96 h of exposure to the compound (not shown). By contrast, the pan-tyrosine kinase inhibitor, staurosporine, induced massive apoptosis as early as 24 h (data not shown). At the molecular level, SU5416-treated TPC-1 cells showed a remarkable increase of p27/Kip1 expression, while p21/WAF1 was down-regulated (Fig. 6C).
|
In vivo modulation of RET autophosphorylation
To determine whether SU5416 is able to block RET activity in vivo, nude mice were inoculated subcutaneously with NIH-PTC cells. These cells grow as xenograft with high efficiency (Borrello et al. 1996) and formed palpable tumors within 10 days. At day 14 after implantation, tumor-bearing mice were treated with a single dose of inhibitor, or vehicle alone, for 6 h. Western blot analysis of resected tumors showed that RET autophosphorylation on Tyr 905 was inhibited in treated animals, when compared to control mice (Fig. 7). RET protein levels were left unaffected by the treatment. This result indicates that RET catalytic activity was shut down in the tumors and confirms SU5416 as a potent RET inhibitor.
|
Sensitivity of tyrosine kinases to different classes of inhibitors appears to be modulated by a key residue regulating access to the ATP-binding site. For example, replacement of Tyr-315 of ABL by bulkier residues, such as isoleucine, causes resistance to Imatinib (Gorre et al. 2001). Similarly, PP1 is unable to inhibit c-SrcT341M kinase, or oncogenic v-Src, which carries an isoleucine at the corresponding position (Liu et al. 1999). A similar phenomenon was observed for EGFR, FGFR, and PDGFR (Blencke et al. 2004). On the other hand, changing Phe-691 to smaller threonine rendered FLT-3 kinase sensitive to Imatinib (Bohmer et al. 2003). In contrast, three indolinone compounds were found to be insensitive to mutations of this particular residue, suggesting that this class of inhibitors may represent an exception to the rule (Blencke et al. 2004). The corresponding amino acid in wild-type RET is a valine, which is predicted to be small enough to accommodate inhibitors within the ATP pocket. Disease-associated mutations at Val-804 confer resistance to inhibition by PP1, PP2, and ZD6474 (Carlomagno et al. 2004). We expressed RETV804M mutant in Baculovirus and tested its sensitivity to inhibitors. As expected, mutant RET was resistant to PP1 (Fig. 8A). Surprisingly, SU5416 did not inhibit mutant RETV804M, 50% inhibition was not reached at compound concentrations up to 30 µM (Fig. 8B). In contrast, SU4984 (Fig. 1), the compound that inhibited FGFR1V561M mutant (Blencke et al. 2004), blocked RETwt and RETV804M with similar IC50: 1.3 vs 2.6 µM (Fig. 8B). The growth of mutant RETV804M-expressing NIH-V804M cells was not affected by SU5416 up to 10 µM, confirming that SU5416 is incapable of blocking V804M-substituted RET kinase (Fig. 8C). In keeping with this result, phosphorylation of RETV804M was not inhibited in these cells (data not shown).
|
We generated a model of the active RET kinase using the insulin receptor kinase as a template. Molecular docking of SU4984 within the ATP-binding site of RET (Fig. 9A) revealed an orientation of the compound that corresponds very well with the one observed in the crystal structure of the inhibitor in complex with FGFR1 kinase (Mohammadi et al. 1997). The oxindole core is positioned at the same region as the adenine ring of ATP and makes two hydrogen bonds with the backbone of RET hinge region. The carbonyl oxygen of glutamate-805 interacts with the indolinone nitrogen, while the amide nitrogen of Ala-807 is in contact with the oxygen atom of the inhibitor (Fig. 9D). In addition, several hydrophobic residues surround the cavity, contributing to the stabilization of compound binding. The C3 substituent points towards the outside of the pocket. The same binding has been observed for other compounds of the same class (Mohammadi et al. 1997, Moshinsky et al. 2003). This result suggests that our model of RET kinase domain is reliable. Docking of SU5416 showed a similar binding mode, with two hydrogen bonds with the protein backbone (Fig. 9B). When docked on mutant RETV804M, SU5416 could not bind in the same way, because of a clash with Met-804. The software did find a docking solution, but the inhibitor was flipped to 180° with respect to wild-type docking, losing both hydrogen bonds (Fig. 9C). This may explain the resistance to inhibition. The docking of inhibitor SU4984, instead, was only partially affected by the mutation (Fig. 9E). The compound is slightly pushed outwards because of the presence of the bulkier methionine but, while it loses one H-bond (with Glu805), this is compensated by a new interaction between Tyr-806 and the N1 of the piperazine ring. The conservation of one interaction with the hinge region and the establishment of a new interaction by the phenyl–piperazinyl moiety of SU4984 suggest that its activity on RET mutant kinase should not be signi-ficantly reduced.
|
SU5416 was discovered as a VEGFR2 inhibitor, with some activity against PDGFR and no inhibition of EGFR, HER2, and IGF1R (Sun et al. 1998). It was later shown to inhibit FLT-3 and c-Kit tyrosine kinases at nanomolar concentrations (Krystal et al. 2001, Yee et al. 2002), as well as c-Src, MET, FGFR1, and Zap70 at micromolar doses (Sun et al. 2000, Krystal et al. 2001). As for several other kinase inhibitors, initial assumption of selectivity was due to limited experimental testing. Therefore, we sought to enlarge the specificity profile of SU5416 by analyzing its inhibitory activity on a panel of 52 available protein kinases belonging to different classes, at 10 µM inhibitor, a concentration that causes 97% inhibition of RET kinase activity. As reported in Table 2, c-Src was inhibited by approximately 50%, in line with previous reports, while Src-related Lck was 70% inhibited. In addition, we found four serine/threonine kinases that showed less than 10% residual activity in the presence of the inhibitor, phosphorylase kinase (PHK), the checkpoint kinase Chk2 (Bartkova et al. 2004), the centromere-associated kinase Aurora B, which is often over-expressed in cancer cells (Kanda et al. 2005), and mammalian Ste20-like kinase 2 (MST2), a pro-apoptotic factor (O’Neill et al. 2005). Four other kinases (MAPKAP-K1a and 1b, SGK, and p70S6K) were substantially (>80%) but not completely inhibited by 10 µM SU5416.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
In a preliminary screening of potential RET inhibitors, we noticed that a couple of indolinone compounds showed good inhibition in vitro, but poor activity in cell culture, largely due to insolubility. We therefore decided to test SU5416 as it shares the same oxindole scaffold and it has already been demonstrated to possess good properties in vivo. SU5416 inhibited RET in cell-free kinase assays at nanomolar doses, comparable to the IC50 values reported for FLT-3 and c-Kit. Our assay confirmed that SU5416 is a potent inhibitor of FLT-3, while it blocks Alk and Abl kinases only at micromolar concentrations. Previous work showed that this compound is inactive against a number of other kinases, such as EGFR, HER2, IGF1R, and weakly inhibits PDGFR. A related molecule, RPI-1, has anti-tumor activity against thyroid carcinoma in mice. In molecular modeling studies, SU5416 docked in RET active site, in the same position as other indolinone compounds that have been crystallized with the related tyrosine kinase, FGFR1. In view of its potency, safety, its good pharmacokinetics, and the fact that it is amenable to chemical modifications, we suggest that SU5416 may be a good lead structure for new, more selective RET inhibitors.
We show inhibition of RET by SU5416 in various model systems, including IL-3-dependent cells, murine fibroblasts, and human PTC cells. RET/PTC-1-expressing TPC-1 cells were selectively growth inhibited, compared to RET-negative PTC cells NPA, which originate from the same cell type as TPC-1 but carry a B-Raf mutation as the primary oncogenic lesion. This result indicates that growth inhibition induced by SU5416 is indeed mediated by RET. Involvement of the VEGFRs can be excluded because NPA cells express these receptors and are not affected by SU5416. The compound was toxic to NPA control cells only at high concentrations, most likely because of the unspecific inhibition of other proliferative pathways. Also, the use of transfected cell lines, such as NIH and Ba/F3, helps to define the role of RET oncogene in mediating SU5416 biological effects; because parental and transfected cells are genetically identical with the exception of the transgene. Growth arrest observed solely in the latter indicates that RET is the relevant target. Blockage of RET autophosphorylation was observed in TPC-1 cells as early as 8 h and was followed by reduced signaling through the ERK and JNK pathways. RAS/RAF/ERK1/2 pathway is known to be activated by RET both in transfectants and human thyroid carcinoma cell lines, through docking of Shc adaptor proteins to Tyr-1062. JNK1/2 MAPKs are normally involved in stress response and induction of apoptosis. In thyroid tissue, activation of JNKs is instead induced by growth stimuli. Indeed, thyroid carcinoma cells are reported to express high levels of activated JNK, both via Dok-1 phosphorylation and through activation of the small GTPase Cdc42.
Inhibition of RET did not result in apoptosis in TPC-1 cells. This data are in line with previous reports, showing no or little evidence of cell death after block of oncogenic RET activity. Either these cells are somewhat resistant to apoptosis and need longer exposure to drugs in order to induce programmed cell death, or the oncogenic activity of RET/PTC is mainly exerted by growth advantage rather than increased survival. Indeed, a multi-step model of thyroid carcinoma is emerging in which RET is a very early event that needs secondary events (such as loss of p53 expression) to establish full-blown aggressive carcinoma (Powell et al. 2001). Cell-cycle arrest induced by SU5416 coincided with a significant up-regulation of the CDK-inhibitor protein p27, in line with a previous report (Vitagliano et al. 2004). Surprisingly, p21 expression was instead down-modulated. The same result was obtained from two independent experiments, using different antibodies. Although we cannot exclude that this effect may be due to unspecific toxicity of the compound, a few cases have been described where p21 repression, rather than induction, correlates with cell-cycle arrest (Noseda et al. 2004, Gartel & Radhakrishnan 2005).
Activating mutations at Val-804 of RET kinase domain occur both in sporadic and in familial MTC with aggressive behavior and are sometimes associated with a second RET mutation. Importantly, RETV804 mutants show resistance to kinase inhibitors PP1 and ZD6474 (Carlomagno et al. 2004). We studied the possibility that RET mutant V804M is inhibited by SU5416. We could not detect any significant inhibition at concentrations that block almost 100% of wild-type RET activity. This result is in contrast with the proposed property of indolinone compounds entering the kinases by carrying bulky gatekeeper residues (Blencke et al. 2004). In their work, Blencke et al. showed that three such compounds inhibited wild-type and mutant kinases with similar potency, although at rather high doses. In our assay, SU5416 did not alter RETV804M activity even at 30 µM, showing that the rule does not apply to all indolinones. As a control, we tested the compound that showed activity on mutant FGFR1 (SU4984). This inhibitor belongs to the same series of compounds as SU5416, developed by Sun et al.(1998). It has 1-log reduced potency against RET, compared to SU5416, possibly because it exists predominantly as the inactive E isomer (referring to the configuration of the C-3 substituent). It shows broader specificity, as it also inhibits PDGFR and other kinases (Sun et al. 1998). Because of its lower activity and relative lack of specificity, it was not further developed. On the other hand, the compound SU4984 was able to inhibit mutant RET, as it could be predicted by published results (Blencke et al. 2004). This may be explained by our molecular modeling study, showing that, while SU5416 loses hydrogen interactions in the mutant, SU4984 does not. According to our model, the binding of both inhibitors is in fact altered by the V804M substitution, but SU4984 establishes a new H-bond with Tyr-806. However, it should be noted that Tyr-806 is thought to be phosphorylated in fully activated RET (Kawamoto et al. 2004). We do not know whether the enzyme used in our assay is phosphorylated on this particular residue. In this case, the interaction would take place only when the piperazine nitrogen is protonated.
Finally, we described the activity of compound SU5416 on a large panel of protein kinases. New targets of SU5416 were identified, including some serine/threonine kinases, unrelated to known target tyrosine kinases. This is of interest, not only for biochemical and biological studies on the newly identified targets, but also as a caveat for cell-based studies focused on known targets. In this study, biological effects were always correlated to RET inactivation. Although we cannot rule out the possibility that the observations are at least partially due to off-target effects, in vitro data support the notion that RET is a direct target of SU5416.
In conclusion, we have shown that SU5416 is a potent inhibitor of RET catalytic activity in biochemical and cellular assays and may represent a good starting structure in the search of a selective RET inhibitor.
|
Acknowledgements |
|---|
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Bain J, McLauchlan H, Elliott M & Cohen P 2003 The specificities of protein kinase inhibitors: an update. Biochemical Journal 371 199–204.[CrossRef][Web of Science][Medline]
Bartkova J, Guldberg P, Gronbaek K, Koed K, Primdahl H, Moller K, Lukas J, Orntoft TF & Bartek J 2004 Aberrations of the Chk2 tumour suppressor in advanced urinary bladder cancer. Oncogene 23 8545–8551.[CrossRef][Web of Science][Medline]
Blencke S, Zech B, Engkvist O, Greff Z, Orfi L, Horvath Z, Keri G, Ullrich A & Daub H 2004 Characterization of a conserved structural determinant controlling protein kinase sensitivity to selective inhibitors. Chemistry and Biology 11 691–701.[CrossRef][Web of Science][Medline]
Bohmer FD, Karagyozov L, Uecker A, Serve H, Botzki A, Mahboobi S & Dove S 2003 A single amino acid exchange inverts susceptibility of related receptor tyrosine kinases for the ATP site inhibitor STI-571. Journal of Biological Chemistry 278 5148–5155 (Epub 2002 November 5114).
Borrello MG, Alberti L, Arighi E, Bongarzone I, Battistini C, Bardelli A, Pasini B, Piutti C, Rizzetti MG, Mondellini P et al. 1996 The full oncogenic activity of Ret/ptc2 depends on tyrosine 539, a docking site for phospholipase Cgamma. Molecular and Cellular Biology 16 2151–2163.[Web of Science][Medline]
Carlomagno F, Vitagliano D, Guida T, Ciardiello F, Tortora G, Vecchio G, Ryan AJ, Fontanini G, Fusco A & Santoro M 2002a ZD6474, an orally available inhibitor of KDR tyrosine kinase activity, efficiently blocks oncogenic RET kinases. Cancer Research 62 7284–7290.
Carlomagno F, Vitagliano D, Guida T, Napolitano M, Vecchio G, Fusco A, Gazit A, Levitzki A & Santoro M 2002b The kinase inhibitor PP1 blocks tumorigenesis induced by RET oncogenes. Cancer Research 62 1077–1082.
Carlomagno F, Vitagliano D, Guida T, Basolo F, Castellone MD, Melillo RM, Fusco A & Santoro M 2003 Efficient inhibition of RET/papillary thyroid carcinoma oncogenic kinases by 4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Journal of Clinical Endocrinology and Metabolism 88 1897–1902.
Carlomagno F, Guida T, Anaganti S, Vecchio G, Fusco A, Ryan AJ, Billaud M & Santoro M 2004 Disease associated mutations at valine 804 in the RET receptor tyrosine kinase confer resistance to selective kinase inhibitors. Oncogene 23 6056–6063.[CrossRef][Web of Science][Medline]
Case DA, Pearlman DA, Caldwell JW, Cheatham TE, III, Ross WS, Simmerling CL, Darden TA, Merz KM, Stanton RV, Cheng AL et al. 1999 AMBER 6, San Francisco: University of California.
Chiariello M, Visconti R, Carlomagno F, Melillo RM, Bucci C, de Franciscis V, Fox GM, Jing S, Coso OA, Gutkind JS et al. 1998 Signalling of the Ret receptor tyrosine kinase through the c-Jun NH2-terminal protein kinases (JNKS): evidence for a divergence of the ERKs and JNKs pathways induced by Ret. Oncogene 16 2435–2445.[CrossRef][Web of Science][Medline]
Coluccia AM, Perego S, Cleris L, Gunby RH, Passoni L, Marchesi E, Formelli F & Gambacorti-Passerini C 2004 Bcl-XL down-regulation suppresses the tumorigenic potential of NPM/ALK in vitro and in vivo. Blood 103 2787–2794 (Epub 2003 December 2784).
Davies SP, Reddy H, Caivano M & Cohen P 2000 Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochemical Journal 351 95–105.[CrossRef][Web of Science][Medline]
Donella-Deana A, Marin O, Cesaro L, Gunby RH, Ferrarese A, Coluccia AM, Tartari CJ, Mologni L, Scapozza L, Gambacorti-Passerini C et al. 2005 Unique substrate specificity of anaplastic lymphoma kinase (ALK): development of phosphoacceptor peptides for the assay of ALK activity. Biochemistry 44 8533–8542.[CrossRef][Medline]
Drosten M & Putzer BM 2003 Gene therapeutic approaches for medullary thyroid carcinoma treatment. Journal of Molecular Medicine 14 14.
Fiedler W, Mesters R, Tinnefeld H, Loges S, Staib P, Duhrsen U, Flasshove M, Ottmann OG, Jung W, Cavalli F et al. 2003 A phase 2 clinical study of SU5416 in patients with refractory acute myeloid leukemia. Blood 102 2763–2767 (Epub 2003 July 2763).
Gambacorti-Passerini C, le Coutre P, Mologni L, Fanelli M, Bertazzoli C, Marchesi E, Di Nicola M, Biondi A, Corneo GM, Belotti D et al. 1997 Inhibition of the ABL kinase activity blocks the proliferation of BCR/ABL+leukemic cells and induces apoptosis. Blood Cells, Molecules and Diseases 23 380–394.[CrossRef][Web of Science][Medline]
Gartel AL & Radhakrishnan SK 2005 Lost in transcription: p21 repression, mechanisms, and consequences. Cancer Research 65 3980–3985.
Gimm O 2001 Thyroid cancer. Cancer Letters 163 143–156.[CrossRef][Web of Science][Medline]
Glade-Bender J, Kandel JJ & Yamashiro DJ 2003 VEGF blocking therapy in the treatment of cancer. Expert Opinion on Biological Therapy 3 263–276.[CrossRef][Web of Science][Medline]
Gorre ME, Mohammed M, Ellwood K, Hsu N, Paquette R, Rao PN & Sawyers CL 2001 Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification. Science 293 876–880 (Epub 2001 June 2021).
Ichihara M, Murakumo Y & Takahashi M 2004 RET and neuroendo-crine tumors. Cancer Letters 204 197–211.[CrossRef][Web of Science][Medline]
Jones G, Willett P, Glen RC, Leach AR & Taylor R 1997 Development and validation of a genetic algorithm for flexible docking. Journal of Molecular Biology 267 727–748.[CrossRef][Web of Science][Medline]
Kadomatsu K, Hagihara M, Akhter S, Fan QW, Muramatsu H & Muramatsu T 1997 Midkine induces the transformation of NIH3T3 cells. British Journal of Cancer 75 354–359.[Web of Science][Medline]
Kanda A, Kawai H, Suto S, Kitajima S, Sato S, Takata T & Tatsuka M 2005 Aurora-B/AIM-1 kinase activity is involved in Ras-mediated cell transformation. Oncogene 18 18.
Kantarjian H, Sawyers C, Hochhaus A, Guilhot F, Schiffer C, Gambacorti-Passerini C, Niederwieser D, Resta D, Capdeville R, Zoellner U et al. 2002 Hematologic and cytogenetic responses to imatinib mesylate in chronic myelogenous leukemia. New England Journal of Medicine 346 645–652.
Kawamoto Y, Takeda K, Okuno Y, Yamakawa Y, Ito Y, Taguchi R, Kato M, Suzuki H, Takahashi M & Nakashima I 2004 Identification of RET autophosphorylation sites by mass spectrometry. Journal of Biological Chemistry 279 14213–14224 (Epub 12004 January 14206).
Krystal GW, Honsawek S, Kiewlich D, Liang C, Vasile S, Sun L, McMahon G & Lipson KE 2001 Indolinone tyrosine kinase inhibitors block Kit activation and growth of small cell lung cancer cells. Cancer Research 61 3660–3668.
Lanzi C, Cassinelli G, Cuccuru G, Zaffaroni N, Supino R, Vignati S, Zanchi C, Yamamoto M & Zunino F 2003 Inactivation of Ret/Ptc1 oncoprotein and inhibition of papillary thyroid carcinoma cell proliferation by indolinone RPI-1. Cellular and Molecular Life Sciences 60 1449–1459.[CrossRef][Web of Science][Medline]
Liu Y, Bishop A, Witucki L, Kraybill B, Shimizu E, Tsien J, Ubersax J, Blethrow J, Morgan DO & Shokat KM 1999 Structural basis for selective inhibition of Src family kinases by PP1. Chemistry and Biology 6 671–678.[CrossRef][Web of Science][Medline]
Mohammadi M, McMahon G, Sun L, Tang C, Hirth P, Yeh BK, Hubbard SR & Schlessinger J 1997 Structures of the tyrosine kinase domain of fibroblast growth factor receptor in complex with inhibitors. Science 276 955–960.
Mologni L, Sala E, Riva B, Cesaro L, Cazzaniga S, Redaelli S, Marin O, Pasquato N, Donella-Deana A & Gambacorti-Passerini C 2005 Expression, purification, and inhibition of human RET tyrosine kinase. Protein Expression and Purification 41 177–185.[CrossRef][Web of Science][Medline]
Moshinsky DJ, Bellamacina CR, Boisvert DC, Huang P, Hui T, Jancarik J, Kim SH & Rice AG 2003 SU9516: biochemical analysis of cdk inhibition and crystal structure in complex with cdk2. Biochemical and Biophysical Research Communications 310 1026–1031.[CrossRef][Web of Science][Medline]
Nikiforov YE, Rowland JM, Bove KE, Monforte-Munoz H & Fagin JA 1997 Distinct pattern of ret oncogene rearrangements in morphological variants of radiation-induced and sporadic thyroid papillary carcinomas in children. Cancer Research 57 1690–1694.
Noseda M, Chang L, McLean G, Grim JE, Clurman BE, Smith LL & Karsan A 2004 Notch activation induces endothelial cell cycle arrest and participates in contact inhibition: role of p21Cip1 repression. Molecular and Cellular Biology 24 8813–8822.
O’Neill EE, Matallanas D & Kolch W 2005 Mammalian sterile 20-like kinases in tumor suppression: an emerging pathway. Cancer Research 65 5485–5487.
Parthasarathy R, Cote GJ & Gagel RF 1999 Hammerhead ribozyme-mediated inactivation of mutant RET in medullary thyroid carcinoma. Cancer Research 59 3911–3914.
Piazza RG, Magistroni V, Gasser M, Andreoni F, Galietta A, Scapozza L & Gambacorti-Passerini C 2005 Evidence for D276G and L364I Bcr-Abl mutations in Ph+ leukaemic cells obtained from patients resistant to Imatinib. Leukemia 19 132–134.[Web of Science][Medline]
Powell DJ, Jr, Russell JP, Li G, Kuo BA, Fidanza V, Huebner K & Rothstein JL 2001 Altered gene expression in immunogenic poorly differentiated thyroid carcinomas from RET/PTC3p53–/– mice. Oncogene 20 3235–3246.[CrossRef][Web of Science][Medline]
Powell DJ, Jr, Eisenlohr LC & Rothstein JL 2003 A thyroid tumor-specific antigen formed by the fusion of two self proteins. Journal of Immunology 170 861–869.
Putzer BM & Drosten M 2004 The RET proto-oncogene: a potential target for molecular cancer therapy. Trends in Molecular Medicine 10 351–357.[CrossRef][Web of Science][Medline]
Santoro M, Carlomagno F, Melillo RM & Fusco A 2004 Dysfunction of the RET receptor in human cancer. Cellular and Molecular Life Sciences 61 2954–2964.[CrossRef][Web of Science][Medline]
Strock CJ, Park JI, Rosen M, Dionne C, Ruggeri B, Jones-Bolin S, Denmeade SR, Ball DW & Nelkin BD 2003 CEP-701 and CEP-751 inhibit constitutively activated RET tyrosine kinase activity and block medullary thyroid carcinoma cell growth. Cancer Research 63 5559–5563.
Sun L, Tran N, Tang F, App H, Hirth P, McMahon G & Tang C 1998 Synthesis and biological evaluations of 3-substituted indolin-2-ones: a novel class of tyrosine kinase inhibitors that exhibit selectivity toward particular receptor tyrosine kinases. Journal of Medicinal Chemistry 41 2588–2603.[CrossRef][Web of Science][Medline]
Sun L, Tran N, Liang C, Hubbard S, Tang F, Lipson K, Schreck R, Zhou Y, McMahon G & Tang C 2000 Identification of substituted 3-[(4,5,6, 7-tetrahydro-1H-indol-2-yl)methylene]-1,3-dihydroindol-2-ones as growth factor receptor inhibitors for VEGF-R2 (Flk-1/KDR), FGF-R1, and PDGF-Rbeta tyrosine kinases. Journal of Medicinal Chemistry 43 2655–2663.[CrossRef][Web of Science][Medline]
Vitagliano D, Carlomagno F, Motti ML, Viglietto G, Nikiforov YE, Nikiforova MN, Hershman JM, Ryan AJ, Fusco A, Melillo RM et al. 2004 Regulation of p27Kip1 protein levels contributes to mitogenic effects of the RET/PTC kinase in thyroid carcinoma cells. Cancer Research 64 3823–3829.
Wallace A, Laskowski R & Thornton J 1995 LIGPLOT: a program to generate schematic diagrams of protein–ligand interactions. Protein Engineering 8 127–134.
Wang SY, Chen B, Zhan YQ, Xu WX, Li CY, Yang RF, Zheng H, Yue PB, Larsen SH, Sun HB et al. 2004 SU5416 is a potent inhibitor of hepatocyte growth factor receptor (c-Met) and blocks HGF-induced invasiveness of human HepG2 hepatoma cells. Journal of Hepatology 41 267–273.[CrossRef][Web of Science][Medline]
Yee KW, O’Farrell AM, Smolich BD, Cherrington JM, McMahon G, Wait CL, McGreevey LS, Griffith DJ & Heinrich MC 2002 SU5416 and SU5614 inhibit kinase activity of wild-type and mutant FLT3 receptor tyrosine kinase. Blood 100 2941–2949.
Received in final form 25 May 2006
Accepted 6 June 2006
This article has been cited by other articles:
|
|
 |
|
  Y. Arlot-Bonnemains, E. Baldini, B. Martin, J.-G. Delcros, M. Toller, F. Curcio, F. S Ambesi-Impiombato, M. D'Armiento, and S. Ulisse Effects of the Aurora kinase inhibitor VX-680 on anaplastic thyroid cancer-derived cell lines Endocr. Relat. Cancer, June 1, 2008; 15(2): 559 - 568. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Puttini, S. Redaelli, L. Moretti, S. Brussolo, R. H Gunby, L. Mologni, E. Marchesi, L. Cleris, A. Donella-Deana, P. Drueckes, et al. Characterization of compound 584, an Abl kinase inhibitor with lasting effects Haematologica, May 1, 2008; 93(5): 653 - 661. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. V. Sharma and J. Settleman Oncogene addiction: setting the stage for molecularly targeted cancer therapy Genes & Dev., December 15, 2007; 21(24): 3214 - 3231. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Plaza-Menacho, L. Mologni, E. Sala, C. Gambacorti-Passerini, A. I. Magee, T. P. Links, R. M. W. Hofstra, D. Barford, and C. M. Isacke Sorafenib Functions to Potently Suppress RET Tyrosine Kinase Activity by Direct Enzymatic Inhibition and Promoting RET Lysosomal Degradation Independent of Proteasomal Targeting J. Biol. Chem., October 5, 2007; 282(40): 29230 - 29240. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. L. Warner-Schmidt and R. S. Duman VEGF is an essential mediator of the neurogenic and behavioral actions of antidepressants PNAS, March 13, 2007; 104(11): 4647 - 4652. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
