This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Carlomagno, F. Articles by Santoro, M. Search for Related Content PubMed PubMed Citation Articles by Carlomagno, F. Articles by Santoro, M.

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)

Istituto di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche c/o Dipartimento di Biologia e Patologia Cellulare e Molecolare "L. Califano" (F.C., D.V., T.G., M.D.C., R.M.M., A.F., M.S.), Università "Federico II", 80131 Naples; and Dipartimento di Oncologia (F.B.), Università degli Studi di Pisa, 56126 Pisa, Italy

Address all correspondence and requests for reprints to: Massimo Santoro, Istituto di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, Facoltà di Medicina e Chirurgia, via S. Pansini 5, 80131 Naples, Italy. E-mail: masantor{at}unina.it.

Abstract

Inappropriate activation of the RET receptor tyrosine kinase causes development of papillary and medullary thyroid cancer. We have previously shown that pyrazolopyrimidine is a potent inhibitor of the RET kinase. Here, we show that 4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) (PP2), another pyrazolopyrimidine, blocks the enzymatic activity of the isolated RET kinase and RET/PTC1 oncoprotein at IC₅₀ in the nanomolar range. PP2 blocked in vivo phosphorylation and signaling of the RET/PTC1 oncoprotein. PP2 prevented serum-independent growth of RET/PTC1-transformed NIH3T3 fibroblasts and of TPC1 and FB2, two human papillary thyroid carcinoma cell lines that carry spontaneous RET/PTC1 rearrangements. Finally, PP2 blocked invasion of type I collagen matrix by TPC1 cells. Thus, pyrazolopirimidines hold promise for the treatment of human cancers sustaining oncogenic activation of RET.

ONCOGENIC ACTIVATION OF the RET kinase is associated to papillary (PTC) and medullary (MTC) thyroid carcinoma (1). In approximately 30% of adult papillary thyroid carcinomas, chromosomal inversions or translocations lead to the recombination of the RET intracellular tyrosine kinase domain with unrelated genes. The resulting chimeric sequences, RET/PTC, exert oncogenic activity (2). RET/PTC1 (the H4-RET fusion; Ref 3) and RET/PTC3 (the RFG-RET fusion; Ref. 4) are the most prevalent variants (5, 6). Germline mutations in RET confer predisposition to multiple endocrine neoplasia type 2 (MEN2A and MEN2B) and familial MTC and sporadic medullary thyroid carcinomas (7, 8, 9, 10, 11).

Upon activation, either induced by ligand-binding or by the above-described mutations, the RET kinase autophosphorylates in a tyrosine residue mapping in its C-domain (Y905). This phosphorylation event stabilizes the kinase in the active conformation and facilitates the autophosphorylation of tyrosines located in the C-tail and comprising Y1015 and Y1062, both essential for the transmission of oncogenic RET signals (12). Y1015 is responsible for coupling to phospholipase C (13), whereas Y1062 is a multiple docking site necessary for activation of Ras/MAPK and PI3K/Akt transduction pathways (1). Tyrosines 1015 and 1062 are phosphorylated in vivo, as phospho-specific antibodies are able to detect RET/MEN2 and RET/PTC oncoproteins in human thyroid cancer cells (14).

A number of tyrosine kinase inhibitors of low molecular weight are now being tested as anticancer agents (15, 16, 17). We have previously shown that a pyrazolopirimidine (PP1) exerts efficacy against RET kinase (18). Here we show that this property is shared by PP2, another compound of the same class.

Materials and Methods

Compounds

PP1 and PP2 were purchased from Alexis (San Diego, CA). Stock solutions (50 mM) were made in 100% dimethylsulfoxide (DMSO) and diluted with culture media or kinase buffer before use. Culture media or kinase buffer containing an equivalent DMSO concentration served as vehicle controls.

Cell culture

Parental NIH3T3 and NIH3T3 cells transfected with RET/PTC1 or v-K-RAS (3) were cultured in DMEM supplemented with 10% calf serum, 2 mM L-glutamine and 100 U/ml penicillin-streptomycin (Life Technologies, Inc., Paisley, Scotland, UK). Human thyroid-carcinoma-derived cell lines, TPC1 (19), FB2 (20), and WRO (21), were cultured in RPMI supplemented with 10% fetal calf serum, 2 mM L-glutamine and 100 U/ml penicillin-streptomycin (Life Technologies, Inc.). HEK293 cells were from American Type Culture Collection (Manassas, VA) and were grown in DMEM supplemented with 10% fetal calf serum. Transient transfections were carried out with the lipofectamine reagent used according to the manufacturer’s instructions (Life Technologies, Inc.).

Generation of phospho-specific anti-RET antibodies

To generate antibodies able to recognize phosphorylated tyrosine 905, the synthetic phosphotyrosine-containing peptide Cys-Val-Tyr-Glu-Glu-Asp-Ser-(pTyr)-Val-Lys-Arg-Ser-Gln and the identical nonphosphate-containing peptide were obtained from Neosystem S. A. (Strasbourg, France). The phosphopeptide was coupled to BSA via m-maleimidobenzoyl-N-Hydroxysuccinimide ester and injected into New Zealand rabbits. The nonphosphorylated peptide was coupled to ovalbumine via succinimidyl 4-N-maleimidomethylcyclohexane-1-carboxylate. Antiserum was affinity-purified by a first passage through a column (Affi-Gel 10, Pierce Chemical Co., Rockford, IL) containing the nonphosphorylated peptide, and then on an Affi-gel 10 column coupled to the phosphopeptide. Bound antibodies were eluted, dialyzed, and used in the present study.

Immunoblotting analysis

Immune complexes were detected with the enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Little Chalfont, UK). Anti-phospho-MAPK and anti-MAPK were from New England Biolabs, Inc. (Beverly, MA). Anti-RET is a polyclonal antibody raised against the tyrosine kinase protein fragment of human RET (9). Anti-pY1062 and anti-pY1015 are polyclonal antibody raised against corresponding phosphorylated peptides. Secondary antibodies coupled to horseradish peroxidase were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

In vitro kinase assays

Subconfluent HEK293 cells were transiently transfected with the appropriate plasmids and solubilized in lysis buffer. Proteins were immunoprecipitated with the required antibodies; immunocomplexes were recovered with protein A Sepharose beads, washed five times with kinase buffer and incubated (20 min at room temperature) in kinase buffer containing 200 µM poly(L-glutamic acid-L-tyrosine) (poly-GT) (Sigma, St. Louis, MO), 2.5 µCi [-³²P]ATP and unlabeled ATP to a final concentration of 20 µM in the presence of the inhibitory compound or vehicle. Samples were spotted on Whatman 3MM paper (Springfield Mill, UK) and washed five times with 1% orthophosphoric acid. ³²P incorporation was measured with a ß-counter scintillator (Beckman Coulter, Inc., Unterschleissheim-Lohhof, Germany). Glutathione-S-transferase (GST)-RET/tyrosine kinase (TK) was purified from HEK293 cell lysates using glutathione Sepharose according to standard procedures (18).

DNA synthesis analysis

For S-phase entry measurements, cells were seeded on glass coverslips. The day after cells were treated with the compound or vehicle alone for 24 h. Bromodeoxyuridine (BrdU; Sigma) was added to the cell culture media at a final concentration of 100 µg/ml for 2 h before harvest. Cells were fixed with paraformaldehyde (4%) and permeabilized with Triton X-100 (0.2%) before staining. Coverslips were incubated with anti-BrdU mouse monoclonal antibody and, then, with a Texas red-conjugated antimouse antibody (Roche Molecular Biochemicals, Mannheim, Germany). All coverslips were counterstained in PBS containing Hoechst 33258 (final concentration, 1 µg/ml; Sigma), rinsed in PBS and mounted in Moviol on glass slides. The fluorescent signal was visualized with an epifluorescent microscope (Axiovert 2, Carl Zeiss, Jena, Germany) interfaced with the image analyzer software KS300 (Carl Zeiss).

Growth curves and collagen gel cell assay

For growth curves, human thyroid carcinoma cells (50,000/dish) were seeded on 60-mm dishes in complete medium. The next day (d 1) 5.0 µM PP2 or vehicle were added to the medium and refreshed every 2 d. Cells were counted every day. For collagen gel cell assay, cells were seeded in tridimensional type I collagen gels as described by Medico et al. (22). Briefly, cells were harvested from cultures using trypsin-EDTA and suspended at a concentration of 10⁵ cells per ml in a solution containing type I collagen (3 mg/ml; Collaborative Biomedical Products, Becton Dickinson and Co. Labware, Franklin Lakes, NJ), 10x DMEM and 0.5 M HEPES (pH 7.4). Aliquots (100 µl) of the cell suspension were dispensed in microtiter 96-well plates and allowed to gel for about 15 min at 37 C before adding 200 µl of complete medium. The medium was changed every 2 d. The morphogenic response was evaluated after 7 d in the presence of the relevant compounds or vehicle alone.

Results

Inhibition of RET/PTC1 autophosphorylation, signaling, and transforming effects by PP2

We used an in vitro phosphorylation assay to determine if PP2 inhibits RET/PTC1-mediated phosphorylation of the synthetic poly-GT peptide. PP1, an inhibitor of RET oncoproteins (18), was used for comparison. As shown in Fig. 1A, PP2 inhibited RET/PTC1 in a dose-dependent fashion. The IC₅₀ of PP2 for both RET/PTC1 and the isolated RET kinase (GST-RET/TK) was in the nanomolar range (100 nM; Fig. 1B), a value similar to that (80 nM) previously reported for PP1 (18). We have previously generated antibodies able to specifically recognize RET/PTC proteins only when phosphorylated on tyrosines 1015 (anti-pY1015) or 1062 (anti-pY1062; Ref. 14). Here, to monitor the earliest step of RET/PTC phosphorylation, we have also developed antibodies selectively recognizing phosphorylated Y905 (anti-pY905). Thus, we evaluated the effects exerted by PP2 on ligand-independent phosphorylation of RET/PTC1 in living cells. Fibroblasts transfected with RET/PTC1 (NIH-RET/PTC1) were serum starved for 24 h and then treated with 5.0 µM PP2 or PP1, as a control, for different time points. Cells were lysed and protein lysates were immunoblotted with the phosphoRET antibodies. As early as 2 h after treatment, PP2, similarly to PP1, abrogated RET/PTC1 phosphorylation in all the three tyrosines (Fig. 2A). Vehicle alone had no effect. Untransfected NIH3T3 cells were used as control for antibody specificity (Fig. 2A). RET signaling leads to the activation of the Ras/MAPK pathway (1). RET/PTC1-expressing NIH3T3 cells, but not parental cells, maintain elevated levels of MAPK phosphorylation upon serum starvation. To determine whether PP2 affected RET/PTC1 signaling, we monitored MAPK activation by immunoblotting with an antibody specific for phosphorylated p42 and p44 MAPK. As controls, we used v-K-RAS transformed NIH3T3 cells (NIH-RAS). Exposure to 5.0 µM PP2 or PP1 caused a dramatic reduction of RET/PTC1-dependent but not Ras-dependent ERK phosphorylation (Fig. 2B). RET/PTC1 expression induces morphological transformation and serum-independent proliferation of NIH3T3 fibroblasts. A 5-µM concentration of PP2 caused a complete morphological reversion of NIH-RET/PTC1 cells, whereas vehicle alone had no effect. Neither parental nor NIH-RAS cells were affected by PP2 (Fig. 2C).

View larger version (14K): [in this window] [in a new window]   Figure 1. Inhibition of RET and RET/PTC1 kinase activity by PP2. A, Protein extracts from HEK293 cells transiently transfected with RET/PTC1 were immunoprecipitated with anti-RET and subjected to a kinase assay on poly-GT substrate in the presence of the indicated compounds. The results are presented as residual phosphorylation levels compared with the control (DMSO). Bars represent the average results of three independent experiments ± SD. B, The IC50 of PP2 for RET/PTC1 or GST-RET/TK was measured with the poly-GT phosphorylation assay. The results of four independent experiments were averaged; deviations were less than 20% of the mean.

View larger version (51K): [in this window] [in a new window]   Figure 2. In vivo inhibition of RET/PTC1 phosphorylation and MAPK activation by PP2. A, NIH3T3 and NIH-RET/PTC3 cell lysates (50 µg) were immunoblotted with anti-RET, or phosphorylation-specific anti-pY905, anti-pY1015 or anti-pY1062 antibodies. Cells were treated as indicated. B, Lysates from serum-starved NIH-RET/PTC1 and NIH-RAS, treated with PP2 or PP1 (5.0 µM) or vehicle for the indicated time points, were immunoblotted with an antibody specific for the p42 and p44 phosphorylated ERKs (pMAPK) or with total anti-ERK (MAPK) for normalization. C, The indicated cell types were treated with PP2 (5.0 µM, 24 h) or vehicle and photographed (magnification, x150). These findings are representative of at least three independent experiments.

The TPC1 and FB2 human thyroid papillary carcinoma cell lines bear the RET/PTC1 rearrangement, whereas the WRO cancer cell line does not. In TPC1 and FB2 cells, PP2 treatment was accompanied by a remarkable reduction of RET/PTC and MAPK phosphorylation levels (Fig. 3A). We measured DNA synthesis after a 24-h treatment with 5.0 µM PP2. The average results of three independent experiments is reported in Fig. 3B, and one representative experiment is shown in Fig. 3C. Treatment with PP2 caused a sharp reduction of BrdU incorporation rate in TPC1 and FB2 cells. Modest effects in RET/PTC-negative WRO cells can be reasonably ascribed to PP2-mediated inhibition of kinases other than RET such as Src-family kinases (23, 24). Then, we measured cell growth upon 1 wk treatment with 5 µM PP2. This treatment completely arrested TPC1 and FB2 cells proliferation, whereas it had only a mild inhibitory effect on WRO cells (Fig. 4A). Growth in type I collagen gels efficiently reflects invasive growth of malignant cells. Whereas untransformed cells distribute in collagen gels as single cells or small regular cell nests, malignant cells proliferate and form irregularly shaped colonies with protruding cells invading surrounding matrix (22). When seeded in collagen, TPC1 cells formed irregularly shaped colonies with protruding cells that rapidly lose contact with the colony of origin and spread in all directions. PP2 and PP1 remarkably arrested this process (Fig. 4B).

View larger version (41K): [in this window] [in a new window]   Figure 3. Effects of PP2 short-term treatment on RET/PTC1-expressing human thyroid carcinoma cells. A, The indicated cell lines were serum starved for 24 h and then treated with 5.0 µM PP2 for 6 h. Cell lysates (100 µg) were immunoblotted with anti-pY905 or pMAPK. Anti-RET and anti-MAPK antibody were used for normalization. These findings are representative of at least four independent experiments. B and C, Serum-independent DNA synthesis was evaluated by BrdU incorporation in TPC1, FB2, and WRO cells upon treatment (5.0 µM, 24 h) with PP2 or vehicle. BrdU was added for 1 h, and cells were fixed and processed for immunofluorescence: anti-BrdU, monitored by a Texas red-conjugated antimouse antibody, was used to detect the fraction of cells in S-phase (red stain). Cell nuclei were counterstained with Hoechst (blue stain). The average results of three independent experiments in which at least 400 cells were counted are reported with SDs in panel B. Representative microscopic fields are shown in C.

View larger version (56K): [in this window] [in a new window]   Figure 4. Effects of PP2 long-term treatment on RET/PTC1 expressing human thyroid carcinoma cells. A, The indicated human cell lines were incubated with vehicle or 5.0 µM PP2 and counted at different time points. Day 1 was the treatment starting day. Data are the mean of two experiments performed in triplicate; SD are indicated. B, TPC1 cells were seeded in collagen gels in the presence or not of 5.0 µM PP2, PP1, or vehicle and photographed after 1 wk.

The RET tyrosine kinase is activated by gene rearrangements in PTCs and by point mutations in sporadic and familial (MEN2) MTC. We have previously reported that the PP1 pyrazolopirimidine exerts potent inhibitory effects on RET kinase (IC₅₀: 80 nM; Ref. 18). Here, we have shown that another pyrazolopyrimidine, PP2, also inhibits RET and RET/PTC1 kinases. PP2 sharply reduced RET/PTC1 phosphotyrosine content. Accordingly, PP2 reduced RET/PTC1-mediated MAPK signaling and cell proliferation. PP2 also inhibited the proliferation and the invasive phenotype of human thyroid carcinoma cells sustaining RET/PTC1 rearrangements. PP2 efficiently inhibits the isolated recombinant RET kinase in vitro; likely, also RET-MEN2A and RET-MEN2B mutants may be sensitive to PP2. We have found that potent kinase inhibitors like tyrphostins (18) and ZD1839 (Carlomagno, F., and M. Santoro, unpublished) do not affect RET, indicating that the effects exerted by PP2 (this paper) and PP1 (18) are specific. However, PP2 is not selective for RET, being also a good inhibitor of c-Src and related kinases (23). Therefore, we cannot exclude additional indirect effects of PP2 mediated in vivo by the inhibition of other kinases and mainly of c-Src, a pivotal downstream RET effector (25). Tumor cells often devise strategies to bypass the effects of antineoplastic agents and selection of therapy-resistant clones is frequently the reason for treatment failure. STI 571 treated chronic myeloid leukemia patients relapse is due to accumulation of very specific point mutations within the catalytic domain of the abl kinase (26). In this frame, to have available a panel of compounds able to disable RET oncoproteins could be an important step to bypass the development of treatment resistance.

Acknowledgments

We are grateful to E. Medico for help in setting-up the collagen assay, to J. A. Fagin for the WRO cells, and to M. Billaud and S. Manie for helpful discussion. We thank F. D’Agnello and Ciotola for the figure artwork.

Footnotes

This study was supported by the Associazione Italiana per la Ricerca sul Cancro, by the Italian Ministero per l’Istruzione, Università e Ricerca Scientifica, and by the Italian Ministero della Salute.

Abbreviations: BrdU, Bromodeoxyuridine; DMSO, dimethylsulfoxide; GST, glutathione-S-transferase; MEN, multiple endocrine neoplasia; MTC, medullary thyroid carcinoma; poly-GT, poly(L-glutamic acid-L-tyrosine; PP1, pyrazolopyrimidine; PP2, 4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine); PTC, papillary thyroid carcinoma; TK, tyrosine kinase.

Received August 13, 2002.

Accepted December 18, 2002.

References

1. Manie S, Santoro M, Fusco A, Billaud M 2001 The RET receptor: function in development and dysfunction in congenital malformation. Trends Genet 17:580–509[CrossRef][Medline]
2. Fagin JA 2002 Perspective: lessons learned from molecular genetic studies of thyroid cancer—insights into pathogenesis and tumor-specific therapeutic targets. Endocrinology 143:2025–2028[Free Full Text]
3. Grieco M, Santoro M, Berlingieri MT, Melillo RM, Donghi R, Bongarzone I, Pierotti MA, Della Porta G, Fusco A, Vecchio G 1990 PTC is a novel rearranged form of the ret proto-oncogene and is frequently detected in vivo in human thyroid papillary carcinomas. Cell 60:557–563[CrossRef][Medline]
4. Santoro M, Dathan NA, Berlingieri MT, Bongarzone I, Paulin C, Grieco M, Pierotti MA, Vecchio G, Fusco A 1994 Molecular characterization of RET/PTC3; a novel rearranged version of the RET proto-oncogene in a human thyroid papillary carcinoma. Oncogene 9:509–516[Medline]
5. Williams D 2002 Science and society: cancer after nuclear fallout: lessons from the Chernobyl accident. Nat Rev Cancer 2:543–549[CrossRef][Medline]
6. Jhiang SM 2000 The RET proto-oncogene in human cancers. Oncogene 19:5590–5597[CrossRef][Medline]
7. Eng C, Clayton D, Schuffenecker I, Lenoir G, Cote G, Gagel RF, van Amstel HK, Lips CJ, Nishisho I, Takai SI, Marsh DJ, Robinson BG, Frank-Raue K, Raue F, Xue F, Noll WW, Romei C, Pacini F, Fink M, Niederle B, Zedenius J, Nordenskjold M, Komminoth P, Hendy GN, Mulligan LM 1996 The relationship between specific RET proto-oncogene mutations and disease phenotype in multiple endocrine neoplasia type 2. International RET mutation consortium analysis. Jama 276:1575–1579[Abstract]
8. Hansford JR, Mulligan LM 2000 Multiple endocrine neoplasia type 2 and RET: from neoplasia to neurogenesis. J Med Genet 37:817–827[Abstract/Free Full Text]
9. Santoro M, Carlomagno F, Romano A, Bottaro DP, Dathan NA, Grieco M, Fusco A, Vecchio G, Matoskova B, Kraus MH, Di Fiore PP 1995 Activation of RET as a dominant transforming gene by germline mutations of MEN2A and MEN2B. Science 267:381–383[Abstract/Free Full Text]
10. Carlomagno F, Salvatore G, Cirafici AM, De Vita G, Melillo RM, de Franciscis V, Billaud M, Fusco A, Santoro M 1997 The different RET-activating capability of mutations of cysteine 620 or cysteine 634 correlates with the multiple endocrine neoplasia type 2 disease phenotype. Cancer Res 57:391–395[Abstract/Free Full Text]
11. Ponder BA 1999 The phenotypes associated with ret mutations in the multiple endocrine neoplasia type 2 syndrome. Cancer Res 59:1736–1741
12. Iwashita T, Asai N, Murakami H, Matsuyama M, Takahashi M 1996 Identification of tyrosine residues that are essential for transforming activity of the ret proto-oncogene with MEN2A or MEN2B mutation. Oncogene 12:481–487[Medline]
13. Borrello MG, Alberti L, Arighi E, Bongarzone I, Battistini C, Bardelli A, Pasini B, Piutti C, Rizzetti MG, Mondellini P, Radice MT, Pierotti MA 1996 The full oncogenic activity of Ret/ptc2 depends on tyrosine 539, a docking site for phospholipase C. Mol Cell Biol 16:2151–2163[Medline]
14. Salvatore D, Barone MV, Salvatore G, Melillo RM, Chiappetta G, Mineo A, Fenzi G, Vecchio G, Fusco A, Santoro M 2000 Tyrosines 1015 and 1062 are in vivo autophosphorylation sites in ret and ret-derived oncoproteins. J Clin Endocrinol Metab 85:3898–3907[Abstract/Free Full Text]
15. Abbott A 2002 On the offensive. Nature 416:470–474[CrossRef][Medline]
16. Zwick E, Bange J, Ullrich A 2002 Receptor tyrosine kinases as targets for anticancer drugs. Trends Mol Med 8:17–23[CrossRef][Medline]
17. Druker BJ, Talpaz M, Resta DJ, Peng B, Buchdunger E, Ford JM, Lydon NB, Kantarjian H, Capdeville R, Ohno-Jones S, Sawyers CL 2001 Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. N Engl J Med 344:1031–1037[Abstract/Free Full Text]
18. Carlomagno F, Vitagliano D, Guida T, Napolitano M, Vecchio G, Fusco A, Gazit A, Levitzki A, Santoro M 2002 The kinase inhibitor PP1 blocks tumorigenesis induced by RET oncogenes. Cancer Res 62:1077–1082[Abstract/Free Full Text]
19. Ishizaka Y, Ushijima T, Sugimura T, Nagao M 1990 cDNA cloning and characterization of ret activated in a human papillary thyroid carcinoma cell line. Biochem Biophys Res Commun 168:402–408[CrossRef][Medline]
20. Basolo F, Giannini R, Toniolo A, Casalone R, Nikiforova M, Pacini F, Elisei R, Miccoli P, Berti P, Faviana P, Fiore L, Monaco C, Pierantoni GM, Fedele M, Nikiforov YE, Santoro M, Fusco A 2002 Establishment of a non-tumorigenic papillary thyroid cell line (FB-2) carrying the RET/PTC1 rearrangement. Int J Cancer 97:608–614[CrossRef][Medline]
21. Fagin JA, Matsuo K, Karmakar A, Chen DL, Tang SH, Koeffler HP 1993 High prevalence of mutations of the p53 gene in poorly differentiated human thyroid carcinomas. J Clin Invest 91:179–184
22. Medico E, Mongiovi AM, Huff J, Jelinek MA, Follenzi A, Gaudino G, Parsons JT, Comoglio PM 1996 The tyrosine kinase receptors Ron and Sea control "scattering" and morphogenesis of liver progenitor cells in vitro. Mol Biol Cell 7:495–504[Abstract]
23. Hanke JH, Gardner JP, Dow RL, Changelian PS, Brissette WH, Weringer EJ, Pollok BA, Connelly PA 1996 Discovery of a novel, potent, and Src family-selective tyrosine kinase inhibitor. Study of Lck- and FynT-dependent T cell activation. J Biol Chem 271:695–701[Abstract/Free Full Text]
24. Fusco A, Berlingieri MT, Di Fiore PP, Portella G, Grieco M, Vecchio G 1987 One- and two-step transformations of rat thyroid epithelial cells by retroviral oncogenes. Mol Cell Biol 7:3365–3370[Abstract/Free Full Text]
25. Melillo RM, Barone MV, Lupoli G, Cirafici AM, Carlomagno F, Visconti R, Matoskova B, Di Fiore PP, Vecchio G, Fusco A, Santoro M 1999 Ret-mediated mitogenesis requires Src kinase activity. Cancer Res 59:1120–1126[Abstract/Free Full Text]
26. Sawyers CL 2002 Disabling Abl-perspectives on Abl kinase regulation and cancer therapeutics. Cancer Cell 1:13–15[CrossRef][Medline]

This article has been cited by other articles:

				M Celano, S Schenone, D Cosco, M Navarra, E Puxeddu, L Racanicchi, C Brullo, E Varano, S Alcaro, E Ferretti, et al. Cytotoxic effects of a novel pyrazolopyrimidine derivative entrapped in liposomes in anaplastic thyroid cancer cells in vitro and in xenograft tumors in vivo Endocr. Relat. Cancer, June 1, 2008; 15(2): 499 - 510. [Abstract] [Full Text] [PDF]

				V. De Falco, V. Guarino, E. Avilla, M. D. Castellone, P. Salerno, G. Salvatore, P. Faviana, F. Basolo, M. Santoro, and R. M. Melillo Biological Role and Potential Therapeutic Targeting of the Chemokine Receptor CXCR4 in Undifferentiated Thyroid Cancer Cancer Res., December 15, 2007; 67(24): 11821 - 11829. [Abstract] [Full Text] [PDF]

				N. Akeno-Stuart, M. Croyle, J. A. Knauf, R. Malaguarnera, D. Vitagliano, M. Santoro, C. Stephan, K. Grosios, M. Wartmann, R. Cozens, et al. The RET Kinase Inhibitor NVP-AST487 Blocks Growth and Calcitonin Gene Expression through Distinct Mechanisms in Medullary Thyroid Cancer Cells Cancer Res., July 15, 2007; 67(14): 6956 - 6964. [Abstract] [Full Text] [PDF]

				R. Ciampi and Y. E. Nikiforov RET/PTC Rearrangements and BRAF Mutations in Thyroid Tumorigenesis Endocrinology, March 1, 2007; 148(3): 936 - 941. [Abstract] [Full Text] [PDF]

				L. Mologni, E. Sala, S. Cazzaniga, R. Rostagno, T. Kuoni, M. Puttini, J. Bain, L. Cleris, S. Redaelli, B. Riva, et al. Inhibition of RET tyrosine kinase by SU5416. J. Mol. Endocrinol., October 1, 2006; 37(2): 199 - 212. [Abstract] [Full Text] [PDF]

				J. W. B. de Groot, T. P. Links, J. T. M. Plukker, C. J. M. Lips, and R. M. W. Hofstra RET as a Diagnostic and Therapeutic Target in Sporadic and Hereditary Endocrine Tumors Endocr. Rev., August 1, 2006; 27(5): 535 - 560. [Abstract] [Full Text] [PDF]

				A. Iervolino, R. Iuliano, F. Trapasso, G. Viglietto, R. M. Melillo, F. Carlomagno, M. Santoro, and A. Fusco The Receptor-Type Protein Tyrosine Phosphatase J Antagonizes the Biochemical and Biological Effects of RET-Derived Oncoproteins. Cancer Res., June 15, 2006; 66(12): 6280 - 6287. [Abstract] [Full Text] [PDF]

				M. C. Zatelli, D. Piccin, F. Tagliati, A. Bottoni, A. Luchin, C. Vignali, A. Margutti, M. Bondanelli, G. C. Pansini, M. R. Pelizzo, et al. Selective Activation of Somatostatin Receptor Subtypes Differentially Modulates Secretion and Viability in Human Medullary Thyroid Carcinoma Primary Cultures: Potential Clinical Perspectives J. Clin. Endocrinol. Metab., June 1, 2006; 91(6): 2218 - 2224. [Abstract] [Full Text] [PDF]

				B. Ouyang, J. A. Knauf, E. P. Smith, L. Zhang, T. Ramsey, N. Yusuff, D. Batt, and J. A. Fagin Inhibitors of Raf Kinase Activity Block Growth of Thyroid Cancer Cells with RET/PTC or BRAF Mutations In vitro and In vivo. Clin. Cancer Res., March 15, 2006; 12(6): 1785 - 1793. [Abstract] [Full Text] [PDF]

				F. Carlomagno, S. Anaganti, T. Guida, G. Salvatore, G. Troncone, S. M. Wilhelm, and M. Santoro BAY 43-9006 inhibition of oncogenic RET mutants. J Natl Cancer Inst, March 1, 2006; 98(5): 326 - 334. [Abstract] [Full Text] [PDF]

				M. C. Zatelli, D. Piccin, F. Tagliati, A. Bottoni, A. Luchin, and E. C. degli Uberti Src Homology-2-Containing Protein Tyrosine Phosphatase-1 Restrains Cell Proliferation in Human Medullary Thyroid Carcinoma Endocrinology, June 1, 2005; 146(6): 2692 - 2698. [Abstract] [Full Text] [PDF]

				M. Santoro, R. M. Melillo, F. Carlomagno, G. Vecchio, and A. Fusco Minireview: RET: Normal and Abnormal Functions Endocrinology, December 1, 2004; 145(12): 5448 - 5451. [Abstract] [Full Text] [PDF]

				J A Fagin How thyroid tumors start and why it matters: kinase mutants as targets for solid cancer pharmacotherapy J. Endocrinol., November 1, 2004; 183(2): 249 - 256. [Abstract] [Full Text] [PDF]

				G. Orgiana, G. Pinna, A. Camedda, V. De Falco, M. Santoro, R. M. Melillo, R. Elisei, C. Romei, S. Lai, C. Carcassi, et al. A New Germline RET Mutation Apparently Devoid of Transforming Activity Serendipitously Discovered in a Patient with Atrophic Autoimmune Thyroiditis and Primary Ovarian Failure J. Clin. Endocrinol. Metab., October 1, 2004; 89(10): 4810 - 4816. [Abstract] [Full Text] [PDF]

				R. M. Melillo, A. M. Cirafici, V. De Falco, M. Bellantoni, G. Chiappetta, A. Fusco, F. Carlomagno, A. Picascia, D. Tramontano, G. Tallini, et al. The Oncogenic Activity of RET Point Mutants for Follicular Thyroid Cells May Account for the Occurrence of Papillary Thyroid Carcinoma in Patients Affected by Familial Medullary Thyroid Carcinoma Am. J. Pathol., August 1, 2004; 165(2): 511 - 521. [Abstract] [Full Text] [PDF]

				G. Cuccuru, C. Lanzi, G. Cassinelli, G. Pratesi, M. Tortoreto, G. Petrangolini, E. Seregni, A. Martinetti, D. Laccabue, C. Zanchi, et al. Cellular Effects and Antitumor Activity of RET Inhibitor RPI-1 on MEN2A-Associated Medullary Thyroid Carcinoma J Natl Cancer Inst, July 7, 2004; 96(13): 1006 - 1014. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Carlomagno, F. Articles by Santoro, M. Search for Related Content PubMed PubMed Citation Articles by Carlomagno, F. Articles by Santoro, M.