Microtubule inhibition causes epidermal growth factor receptor inactivation in oesophageal cancer cells
- Authors:
- Published online on: November 20, 2012 https://doi.org/10.3892/ijo.2012.1710
- Pages: 297-304
Abstract
Introduction
The cytoskeleton is comprised of actin filaments, intermediate filaments and microtubules. Microtubules are dynamic structures that are important for a range of cellular functions, such as intracellular trafficking, cell movement and division, where they are involved in chromosome segregation. α- and β-tubulin heterodimers polymerise into a hollow tube denoted microtubule (1,2). The stability of the microtubule is regulated through GTP-hydrolysis of β-tubulin; binding of GTP allows polymerisation, but within the microtubule GTP can be hydrolysed to GDP (1,2). Microtubules constantly polymerise and depolymerise, a process termed dynamic instability. Since micro-tubules play an essential role in chromosome segregation, drugs that interfere with microtubule function prevent the cell from mitosis. A number of microtubule targeting drugs are used in the clinic to treat human cancers, including oesophageal cancer. Examples are the vinca alkaloids that destabilise microtubules by binding close to the GTP-binding region in β-tubulin (3), and the taxanes which bind to polymerised microtubules and stabilise the GDP-bound form of β-tubulin by forcing them into a configuration resembling the GTP-bound state (4).
Growth factors and their receptors have been shown to be of pivotal significance for the occurrence and development of cancer. The human epidermal growth factor receptor (HER) family is comprised of four members, i.e. EGFR (HER1, ErbB1), HER2 (ErbB2, Neu), HER3 (ErbB3) and HER4 (ErbB4) (5). These receptors are tyrosine kinases that are activated by ligand-induced dimerisation. There are several ligands for the receptors in the HER family, and these have different binding specificities, resulting in formation of homo- or heterodimeric receptor complexes. HER family members are commonly activated in human cancer cells by different mechanisms including autocrine stimulation, mutations or overexpression (6). Dysregulated and improper receptor activation leads to induction of signals that promote proliferation, survival, migration and angiogenesis, events that are all central for tumour development and progression. Over-activated EGFR is recognised as an important mechanism in several types of cancer, including colorectal cancer (7), head and neck cancer (8), and non-small cell lung cancer (9) and has become a target of interest in the treatment of these tumours.
There are data indicating possible interactions between EGFR and the microtubule system. Gao et al demonstrated that histone deacetylase 6 (HDAC6), a microtubule-associated deacetylase, associates with the endosomal compartments and controls EGFR trafficking and degradation (10). This is consistent with data from Deribe et al showing that HDAC6 negatively regulates EGFR endocytosis and degradation by controlling the acetylation status of α-tubulin and subsequently receptor trafficking along microtubules (11).
Oesophageal carcinoma is the seventh most common cause of cancer-related death in the Western world (12). The standard treatment for localised oesophageal carcinoma includes a combination of radiation and chemotherapy, sometimes followed by surgery. Preoperative chemoradiotherapy or chemotherapy (13) has been demonstrated to give a significant survival benefit. However, patients with advanced metastatic disease that are treated with palliative chemotherapy have a poor prognosis with a median survival time of less than one year. The 5-year survival rate of all diagnosed patients is only around 15%. Thus, there is an urgent need to improve current therapies. In oesophageal carcinoma patients, EGFR has been reported to be commonly overexpressed (14) and the overexpression is correlated to lymph node metastasis, vascular invasion and shorter survival (15–17). The EGF and TGF ligands function as mitogens for oesophageal tumour cells (18) and activation of EGFR signalling has been implicated in metastasis via modulation of cell adhesion, angiogenesis, invasion and migration.
In the current study we have investigated the possible interaction of anti-microtubule drugs and the EGFR signalling system in human oesophageal cancer cells. Treatment with the drugs led to inhibition of proliferation of the cells. Additionally, microtubule destabilising agents were also shown to inhibit EGFR phosphorylation. These effects could be inhibited by simultaneous addition of the protein tyrosine phosphatase inhibitor sodium orthovanadate, suggesting that disruption of the microtubule network leads to release or activation of a tyrosine phosphatase. This study shows that microtubule targeting drugs have other effects beyond interfering with the mitotic spindle.
Materials and methods
Cell culturing and counting
The human ESCC (oesophageal squamous cell carcinoma) cell lines Kyse30, Kyse70, Kyse140, Kyse150, Kyse180, Kyse410, Kyse450, Kyse510 and Kyse520 (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany) were cultivated in RPMI-1640 medium, supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 50 units of penicillin and 50 μg streptomycin/ml (Sigma-Aldrich, St. Louis, MO, USA). Cells were split twice a week by incubation in 13–20 μl/cm2 5X Trypsin-EDTA Solution (Sigma-Aldrich) in 37°C until detachment from the surface. Cells were used for further experiments when 70–90% confluent. Cell counting was performed using a Coulter® Z2 Particle count and size analyser.
Cell growth analysis using resazurin assay
Kyse70 and Kyse140 cells were seeded in 96-well plates at a concentration of 5,000 cells per well and were allowed to grow overnight. Docetaxel, podophyllotoxin (PPT) and vincristine are from Sigma-Aldrich. Five or seven different concentrations of docetaxel, podo phyllotoxin (PPT) or vincristine were then added to the cell medium. After 24 and 48 h, resazurin (Alamar Blue, Sigma-Aldrich) was added at a concentration of 440 μM to each well followed by incubation in the dark for 1 h at room temperature. Wells containing only culture medium served as blank. The analysis of fluorescence (560EX nm/590EM nm) using a Wallac Victor Multilabel Counter (Wallac, Turku, Finland) was followed by calculations of relative numbers of viable cells expressed as percentages of untreated cells. Resazurin detects cell viability by converting the reagent to a fluorescent indicator in response to metabolically active cells (19,20). The resazurin assay is quantitative with respect to time and dose, and separate experiments showed a linear correlation between the number of viable cells and the emitted light (data not shown). Each experiment was performed three times.
Microtubule staining
Kyse70 and Kyse140 cells were grown on coverslips overnight and then treated with 5 μM of either docetaxel, PPT or vincristine for 24 h. Cells were fixed in 2% formaldehyde and permeabilised with 0.2% Triton X-100. Coverslips were blocked with 10 mM glycine at room temperature for 1 h, incubated with primary mouse anti-α-tubulin antibody (Sigma-Aldrich), followed by incubation with a secondary polyclonal goat anti-mouse antibody labelled with FITC (Dako, Glostrup, Denmark). The nuclei were stained with DAPI. Coverslips were mounted on object slides using Fluoromount-G (Southern Biotechnology Associates, Birmingham, AL, USA). Microtubule staining was visualised using a Zeiss immunofluorescence microscope at x40 magnification.
Immunoprecipitation and western blot analysis
Subconfluent Kyse70 and Kyse140 cells were treated with different concentrations (0.5, 5 and 10 μM) of PPT, vincristine or docetaxel for 24 and 48 h in starvation medium containing 0.1% FBS. Subsequently, cells were stimulated with 100 ng/ml EGF (Chemicon, Temecula, CA, USA) or IGF-1 (R&D Systems, Minneapolis, MN, USA) for 5 min and washed with ice-cold phosphate-buffered saline before lysis. Cell lysates were prepared according to Lennartsson et al(21). Briefly, total protein concentration was determined using the BCA Protein Assay Kit (Pierce, Rockford, IL, USA). Total cell lysate (TCL) were submitted to SDS-polyacrylamide gel electrophoresis (SDS-PAGE). For immunoprecipitation, antibodies against IGF-1Rβ were added to each lysate at a concentration of 1 μg/ml. Protein A-Sepharose was added in order to collect immunocomplexes. After washing of the beads, samples were boiled in reducing sample buffer and subjected to SDS-PAGE. Separated proteins were electrotransferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA), membranes blocked using 5% BSA, and then incubated with primary antibody overnight at 4°C. Antibodies used were anti-EGFR, anti-IGF-1Rβ and anti-Akt1/2 rabbit polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phosphotyrosine mouse monoclonal antibodies PY99 (Santa Cruz Biotechnology), phospho-specific anti-Erk and phospho-specific anti-Akt antibodies (Cell Signalling Technology, Beverly, MA, USA), anti-PTP1B mouse antibodies (BD Biosciences, San Jose, CA, USA), anti-β-actin mouse monoclonal antibodies (Sigma-Aldrich) and anti-Erk2 rabbit serum (Ludwig Institute for Cancer Research, Uppsala, Sweden). Anti-PTPε rabbit serum was from Dr A. Elson (The Weizmann Institute of Science, Israel). EGF was from Chemicon and IGF-1 from R&D Systems. After washing, membranes were incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG antibodies (Amersham Biosciences, Uppsala, Sweden) and proteins visualised using ECL western blotting detection systems (Roche Applied Science, Indianapolis, IN, USA) on a cooled charge-coupled device (CCD) camera (Bio-Rad Life Science, Hercules, CA, USA). The images were analysed using the software Quantity One.
siRNA interference
siRNA was employed to knockdown PTPε and PTP1B expression. Anti-PTPε siRNA targets sequence: GCGAACAGGUACAUUCAUA; anti-PTP1B siRNA targets sequence: GGAGAAAGGUUCGUUAAAA; non-targeting siRNA was used as a control (target sequence CGTACGCGGA ATACTTCGA). To downregulate PTP1B and PTPε expression, different concentrations of siRNA for anti-PTP1B and anti-PTPε were incubated with Kyse70 and Kyse140 cells for 48 h. Levels of knockdown were tested after measuring protein levels by immunoblotting. Meanwhile, different concentrations of microtubule targeting drugs were added to the cell cultures for 24 h.
Sodium orthovanadate treatment
Subconfluent Kyse70 and Kyse140 cells were treated with 10 μM PPT, vincristine or docetaxel for 24 h. Before cell lysis, sodium orthovanadate (Na3VO4) was added in the medium at a concentration of 1 mM for 1 h, followed by stimulation with 100 ng/ml EGF for 5 min. Total cell lysates were used for immunoblotting analysis.
Results
Podophyllotoxin (PPT), vincristine and docetaxel interact with the microtubule network and affect survival of ESCC cells
After 24-h treatment, the microtubule destabilising drugs PPT (5 μM) and vincristine (5 μM) disrupted the microtubule network while the microtubule stabilising drug docetaxel (5 μM) stabilised the network (Fig. 1) in oesophageal cancer cells. After 24-h drug treatment, we did not observe nuclear pyknosis and massive apoptotic bodies in either Kyse70 or Kyse140 cells under microscope following DAPI staining. Treatment with PPT, docetaxel and vincristine in Kyse70 and Kyse140 cells resulted in time-dependent plateau-shaped dose-response curves in a proliferation assay, consistent with tubulin interaction and cell cycle arrest. Maximal inhibition (>80%) of Kyse70 (Fig. 2A) was achieved at low concentrations (0.1 μM PPT, 0.1 μM docetaxel and 0.05 μM vincristine at 48 h), while Kyse140 cells appeared slightly less sensitive (Fig. 2B).
Microtubule destabilising drugs decrease EGFR phosphorylation and downstream signalling in ESCC cells
It has been reported that EGFR is commonly overexpressed in oesophageal carcinoma (14). Variable but detectable levels of EGFR were observed in all 9 oesophageal carcinoma cell lines used in this study (Fig. 3). The possible interactions between EGFR and microtubules were investigated by treatment of Kyse70 and Kyse140 cells with increasing concentrations of micro tubule targeting drugs for 24 or 48 h, followed by 5 min EGF stimulation. The concentrations used in this study are comparable to or slightly higher than the clinically achievable peak plasma concentrations in patients (22,23). The microtubule destabilising agents PPT (Fig. 4A) and vincristine (Fig. 4B) inhibited EGFR tyrosine phosphorylation after 24 h treatment at all concentration where we did not observe the inhibition of EGFR expression. However, the microtubule stabilising agent docetaxel had a minor inhibitory effect on EGFR phospho rylation after 24 h treatment (Fig. 4C). After 48 h of drug treatment with PPT, vincristine and docetaxel, a substantial inhibition of EGFR expression and phosphorylation could be observed, indicating a possible indirect effect due to the cytotoxicity of microtubule targeting drugs (Fig. 4). Dephosphorylation was observed already after 24 h of treatment whereas EGFR downregulation was most pronounced after 48 h, indicating that inhibition of phosphorylation and receptor downregulation are two distinct events.
To investigate if decreased phospho-EGFR also resulted in decreased signalling downstream of EGFR, cells were treated with microtubule targeting drugs for 24 h thus avoiding EGFR degradation. Docetaxel, PPT and vincristine downregulated phosphorylation as well as protein levels of Akt (Fig. 5) in both Kyse70 and Kyse140 cells. Moreover, the microtubule destabilising agents PPT and vincristine inhibited phosphorylation of Erk but did not cause downregulation of total Erk protein levels. Docetaxel had no effect on the activation of Erk in either cell line.
Microtubule targeting drugs demonstrated no inhibition of phosphorylation or expression of IGF-1R in ESCC cells
IGF-1R, which is becoming an important target in the treatment of cancer, has been found to be significantly overexpressed in oesophageal squamous cell carcinoma tissue compared with adjacent normal tissue (24). We found that IGF-1R was expressed in all nine ESCC cell lines (Fig. 3). To test whether microtubule targeting drugs can affect IGF-1R, we treated cells with increasing concentrations of microtubule targeting drugs (0.5, 5 and 10 μM) for 24 or 48 h, followed by 5 min of IGF-1 stimulation. We were not able to observe any effect either on ligand-induced IGF-1R phosphorylation or on IGF-1R expression (Fig. 6). Thus, it appears that disruption of the microtubule system selectively inhibits EGFR function over that of IGF-1R, indicating that EGFR downregulation is not caused by general drug cytotoxicity.
Reduced EGFR phosphorylation induced by microtubule destabilising agents can be reversed by treatment with a tyrosine phosphatase inhibitor
EGFR dephosphorylation caused by microtubule destabilising agents was not correlated with protein downregulation for all drugs and cell lines. In addition, the EGFR dephosphorylation occurred before substantial receptor downregulation could be seen, suggesting that these effects are independent of each other (Fig. 4). To further explore the mechanism of EGFR dephosphorylation we treated cells for 24 h, to avoid major EGFR downregulation, with microtubule targeting drugs in the presence or absence of the tyrosine phosphatase inhibitor sodium orthovanadate (Na3VO4). As can be seen in Fig. 7, sodium orthovanadate treatment to a large extent abolished EGFR dephosphorylation. Thus, one possibility is that disruption of the microtubule network releases or activates a tyrosine phosphatase that can dephosphorylate EGFR but not IGF-1R.
PTP1B or PTPε downregulation can not reverse EGFR dephosphorylation induced by microtubule disrupting agents
The tyrosine phosphatase PTPε co-localises with microtubules in cells and its binding to tubulin can inhibit its activity; conversely disrupting microtubule structures increased PTPε activity (25). PTP1B has been reported to interact with endocytosed EGFR and promote its dephosphorylation, and this complex is disrupted by sodium othovanadate (26,27). To further explore which protein tyrosine phosphatase is involved in EGFR dephosphorylation induced by microtubule disruption, RNAi was employed to downregulate PTP1B and PTPε expression in Kyse70 and Kyse140 cells as shown in Fig. 8A and B. Neither PTP1B nor PTPε downregulation could reverse the effect of the microtubule disrupting drugs on EGFR dephosphorylation (Fig. 8C and D). Furthermore, we tested if PTPε downregulation could affect the EGFR dephosphorylation induced by micro-tubule targeting agents in the A431 cell line which is originated from epidermoid carcinoma and known to overexpress EGFR. PPT dephosphorylated EGFR in both A431 moc (cytPTPε negative) and A431 (cytPTPε overexpressed) cells (Fig. 8E), proving also in other cells than ESCC cells that PTPε downregulation can not reverse EGFR dephosphorylation induced by micro-tubule targeting agents.
Discussion
Microtubules are involved in various cellular functions, including cell adhesion, movement, replication and division. Microtubule inhibition can interfere with chromosome segregation during mitosis and disrupt cell signalling, hence promoting cell cycle arrest and cell death (28,29). In the present study, we have investigated the cytotoxic effects of the microtubule targeting drugs docetaxel, vincristine, and PPT in oesophageal carcinoma cell lines. As expected, microtubule targeting drugs disrupted the microtubule network and inhibited cell survival in oesophageal carcinoma cells. Surprisingly, we also found that disruption of the microtubule network was associated with dephosphorylation of EGFR and subsequent reduced activation of Akt and Erk. Co-treatment with a tyrosine phosphatase inhibitor diminished this effect, suggesting that disruption of the microtubule network leads to exposure of EGFR to an active tyrosine phosphatase. Neither 24 nor 48 h of drug treatment had any effect on IGF-1R phosphorylation or stability, suggesting some degree of receptor selectivity and that the EGFR downregulation is not due to general toxicity.
In both Kyse70 and Kyse140 cells, phosphorylation of Akt was inhibited by docetaxel, vincristine and PPT. However, the degradation of total Akt protein after drug treatment may partially explain the dephosphorylation of Akt. Compared to Akt, Erk protein levels were not affected by drug treatment, while phosphorylation of Erk was inhibited by vincristine and PPT but not docetaxel in both Kyse70 and Kyse140 cells. This suggests that Akt protein levels may be more easily affected by a general drug response and that microtubule destabilisation, but not stabilisation, affects downstream signalling of EGFR. The observed dephospho rylation of EGFR could be reversed by a tyrosine phosphatase inhibitor, suggesting that a tyrosine phosphatase is activated following microtubule disruption. It is possible that microtubule destabilising agents activate a phosphatase that is selective for EGFR over IGF-1R in ESCC cells. Protein tyrosine phosphatases (PTPs) strictly control receptor tyrosine kinase (RTK) phosphorylation and downstream signalling. Several PTPs have been reported to dephosphorylate tyrosine residues of EGFR and regulate signalling, including T-cell PTP (TCPTP), Src homology phosphotyrosine phosphatase 1 and 2 (SHP1 and 2), PTP1B, PTPN9, density-enhanced phosphatase-1 (DEP-1), RPTPσ and RPTPκ (30–35). So far only PTPε and PTP1B have been reported to interact with the microtubule system (25–27). However, using siRNA to downregulate PTP1B and PTPε expression in ESCC cells, we found no evidence supporting that PTP1B or PTPε downregulation could influence the effect of the microtubule disrupting drugs on EGFR dephosphorylation (Fig. 8C and D). Elucidating which PTP(s) is important for regulation of EGFR phospho rylation in ESCC cells following disruption of the microtubule network is subject for future studies.
The additional mechanism of action of tubulin inhibitors on EGFR signalling suggested in the present work may have clinical impact on the selection of drug combinations for the treatment of oesophageal cancer as well as other cancer types. Several clinical studies involving EGFR targeted therapies in oesophageal cancer have been performed, including the antibody cetuximab as well as the tyrosine kinase inhibitors erlotinib (Tarceva®) and gefitinib (Iressa®). Although not yet a standard of care, the results from these studies suggest that treatment with EGFR targeted therapies, alone or in combination with chemotherapy and/or radiotherapy, is feasible with promising clinical activity (36,37). Ongoing and future clinical trials involving EGFR targeted therapies and anti-tubulin acting chemotherapy in combination is recommended to consider the potential interactions between these treatments, both with respect to clinical efficacy of the treatment and to the selection of appropriate biomarkers.
We demonstrated that microtubule targeting drugs inhibited the survival of oesophageal cancer cells involving a reduction of tyrosine phosphorylation and activation of EGFR, and that this effect is reversible by inhibition of tyrosine phosphatases using sodium orthovanadate. Thus, we propose that in addition to the previously described mechanisms of action for microtubule disrupting chemotherapeutics, these drugs may also lead to EGFR dephosphorylation and downregulation of EGFR-induced signalling. These findings may have a clinical impact on the selection of chemotherapeutic drug combinations for the treatment of oesophageal cancer as well as other cancer types.
Acknowledgements
The authors are grateful to Dr Markus Dagnell at Cancer Center, Karolinska Institutet, for providing A431 moc (cytPTPε negative) and A431 (cytPTPε overexpressed) cells. We also thank Dr Ari Elson from Weizmann Institute of Science for providing anti-PTPε antibody.
References
Li H, DeRosier DJ, Nicholson WV, Nogales E and Downing KH: Microtubule structure at 8 Å resolution. Structure. 10:1317–1328. 2002. | |
Mitchison T and Kirschner M: Dynamic instability of microtubule growth. Nature. 312:237–242. 1984. View Article : Google Scholar : PubMed/NCBI | |
Lobert S, Vulevic B and Correia JJ: Interaction of vinca alkaloids with tubulin: a comparison of vinblastine, vincristine, and vinorelbine. Biochemistry. 35:6806–6814. 1996. View Article : Google Scholar : PubMed/NCBI | |
Burkhart CA, Berman JW, Swindell CS and Horwitz SB: Relationship between the structure of taxol and other taxanes on induction of tumor necrosis factor-alpha gene expression and cytotoxicity. Cancer Res. 54:5779–5782. 1994.PubMed/NCBI | |
Bazley LA and Gullick WJ: The epidermal growth factor receptor family. Endocr Relat Cancer. 12:S17–S27. 2005. View Article : Google Scholar : PubMed/NCBI | |
Normanno N, Bianco C, Strizzi L, et al: The ErbB receptors and their ligands in cancer: an overview. Curr Drug Targets. 6:243–257. 2005. View Article : Google Scholar : PubMed/NCBI | |
Arnold D and Seufferlein T: Targeted treatments in colorectal cancer: state of the art and future perspectives. Gut. 59:838–858. 2001. View Article : Google Scholar : PubMed/NCBI | |
Bonner JA, Harari PM, Giralt J, et al: Radiotherapy plus cetuximab for squamous-cell carcinoma of the head and neck. N Engl J Med. 354:567–578. 2006. View Article : Google Scholar : PubMed/NCBI | |
Pao W and Chmielecki J: Rational, biologically based treatment of EGFR-mutant non-small-cell lung cancer. Nat Rev Cancer. 10:760–774. 2010. View Article : Google Scholar : PubMed/NCBI | |
Gao YS, Hubbert CC and Yao TP: The microtubule-associated histone deacetylase 6 (HDAC6) regulates epidermal growth factor receptor (EGFR) endocytic trafficking and degradation. J Biol Chem. 285:11219–11226. 2010. View Article : Google Scholar : PubMed/NCBI | |
Deribe YL, Wild P, Chandrashaker A, et al: Regulation of epidermal growth factor receptor trafficking by lysine deacetylase HDAC6. Sci Signal. 2:ra842009.PubMed/NCBI | |
Devesa SS, Blot WJ and Fraumeni JF Jr: Changing patterns in the incidence of esophageal and gastric carcinoma in the United States. Cancer. 83:2049–2053. 1998. View Article : Google Scholar : PubMed/NCBI | |
Gebski V, Burmeister B, Smithers BM, Foo K, Zalcberg J and Simes J: Survival benefits from neoadjuvant chemoradiotherapy or chemotherapy in oesophageal carcinoma: a meta-analysis. Lancet Oncol. 8:226–234. 2007. View Article : Google Scholar : PubMed/NCBI | |
Jankowski J HD, Hopwood D and Wormsley KG: Expression of epidermal growth factor, transforming growth factor alpha and their receptor in gastro-oesophageal diseases. Dig Dis. 11:1–11. 1993. | |
Gibault L, Metges JP, Conan-Charlet V, et al: Diffuse EGFR staining is associated with reduced overall survival in locally advanced oesophageal squamous cell cancer. Br J Cancer. 93:107–115. 2005. View Article : Google Scholar : PubMed/NCBI | |
Kim LW, Tsung-Teh W, In Seon C, et al: Expression of epidermal growth factor receptor in esophageal and esophagogastric junction adenocarcinomas. Cancer. 109:658–667. 2007. View Article : Google Scholar : PubMed/NCBI | |
Wei Q, Chen L, Sheng L, Nordgren H, Wester K and Carlsson J: EGFR, HER2 and HER3 expression in esophageal primary tumours and corresponding metastases. Int J Oncol. 31:493–499. 2007.PubMed/NCBI | |
Oku K, Tanaka A, Yamanishi H, et al: Effects of various growth factors on growth of a cloned human esophageal squamous cancer cell line in a protein-free medium. Anticancer Res. 11:1591–1595. 1991.PubMed/NCBI | |
Ahmed SA, Gogal RM Jr and Walsh JE: A new rapid and simple non-radioactive assay to monitor and determine the proliferation of lymphocytes: an alternative to [3H]thymidine incorporation assay. J Immunol Methods. 170:211–224. 1994.PubMed/NCBI | |
Nociari MM, Shalev A, Benias P and Russo C: A novel one-step, highly sensitive fluorometric assay to evaluate cell-mediated cytotoxicity. J Immunol Methods. 213:157–167. 1998. View Article : Google Scholar : PubMed/NCBI | |
Lennartsson J, Wardega P, Engstrom U, Hellman U and Heldin CH: Alix facilitates the interaction between c-Cbl and platelet-derived growth factor beta-receptor and thereby modulates receptor down-regulation. J Biol Chem. 281:39152–39158. 2006. View Article : Google Scholar : PubMed/NCBI | |
McLeod HL, Kearns CM, Kuhn JG and Bruno R: Evaluation of the linearity of docetaxel pharmacokinetics. Cancer Chemother Pharmacol. 42:155–159. 1998. View Article : Google Scholar : PubMed/NCBI | |
Sethi VS, Jackson DV Jr, White DR, et al: Pharmacokinetics of vincristine sulfate in adult cancer patients. Cancer Res. 41:3551–3555. 1981.PubMed/NCBI | |
Nemoto T, Ohashi K, Akashi T, Johnson JD and Hirokawa K: Overexpression of protein tyrosine kinases in human esophageal cancer. Pathobiology. 65:195–203. 1997. View Article : Google Scholar : PubMed/NCBI | |
Sines T, Granot-Attas S, Weisman-Welcher S and Elson A: Association of tyrosine phosphatase epsilon with microtubules inhibits phosphatase activity and is regulated by the epidermal growth factor receptor. Mol Cell Biol. 27:7102–7112. 2007. View Article : Google Scholar | |
Flint AJ, Tiganis T, Barford D and Tonks NK: Development of ‘substrate-trapping’ mutants to identify physiological substrates of protein tyrosine phosphatases. Proc Natl Acad Sci USA. 94:1680–1685. 1997. | |
Eden ER, White IJ, Tsapara A and Futter CE: Membrane contacts between endosomes and ER provide sites for PTP1B-epidermal growth factor receptor interaction. Nat Cell Biol. 12:267–272 | |
Poruchynsky MS, Wang EE, Rudin CM, Blagosklonny MV and Fojo T: Bcl-xL is phosphorylated in malignant cells following microtubule disruption. Cancer Res. 58:3331–3338. 1998.PubMed/NCBI | |
Blagosklonny MV, Giannakakou P, el-Deiry WS, et al: Raf-1/bcl-2 phosphorylation: a step from microtubule damage to cell death. Cancer Res. 57:130–135. 1997.PubMed/NCBI | |
Tarcic G, Boguslavsky SK, Wakim J, et al: An unbiased screen identifies DEP-1 tumor suppressor as a phosphatase controlling EGFR endocytosis. Curr Biol. 19:1788–1798. 2009. View Article : Google Scholar : PubMed/NCBI | |
Xu Y, Tan L-J, Grachtchouk V, Voorhees JJ and Fisher GJ: Receptor-type protein-tyrosine phosphatase-kappa regulates epidermal growth factor receptor function. J Biol Chem. 280:42694–42700. 2005. View Article : Google Scholar : PubMed/NCBI | |
Suarez Pestana E, Tenev T, Gross S, Stoyanov B, Ogata M and Bohmer FD: The transmembrane protein tyrosine phosphatase RPTPsigma modulates signaling of the epidermal growth factor receptor in A431 cells. Oncogene. 18:4069–4079. 1999.PubMed/NCBI | |
Keilhack H, Tenev T, Nyakatura E, et al: Phosphotyrosine 1173 mediates binding of the protein-tyrosine phosphatase SHP-1 to the epidermal growth factor receptor and attenuation of receptor signaling. J Biol Chem. 273:24839–24846. 1998. View Article : Google Scholar : PubMed/NCBI | |
Agazie YM and Hayman MJ: Development of an efficient ‘substrate-trapping’ mutant of Src homology phosphotyrosine phosphatase 2 and identification of the epidermal growth factor receptor, Gab1, and three other proteins as target substrates. J Biol Chem. 278:13952–13958. 2003. | |
Yuan T, Wang Y, Zhao ZJ and Gu H: Protein-tyrosine phosphatase PTPN9 negatively regulates ErbB2 and epidermal growth factor receptor signaling in breast cancer cells. J Biol Chem. 285:14861–14870. 2010. View Article : Google Scholar : PubMed/NCBI | |
Ku GY and Ilson DH: Esophagogastric cancer: targeted agents. Cancer Treat Rev. 36:235–248. 2010. View Article : Google Scholar : PubMed/NCBI | |
Ekman S, Dreilich M, Lennartsson J, et al: Esophageal cancer: current and emerging therapy modalities. Expert Rev Anticancer Ther. 8:1433–1448. 2008. View Article : Google Scholar : PubMed/NCBI |