Evidence for efficient phosphorylation of EGFR and rapid endocytosis of phosphorylated EGFR via the early/late endocytic pathway in a gefitinib-sensitive non-small cell lung cancer cell line

Gefitinib (Iressa)–a specific inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase–has been shown to suppress the activation of EGFR signaling required for cell survival and proliferation in non-small cell lung cancer (NSCLC) cell lines. We recently provided novel evidence that gefitinib-sensitive PC9 cells show normal endocytosis of EGFR: internalized EGF-EGFR complexes were transported to late endosomes/lysosomes 15 min after EGF stimulation, and then degraded within the lysosomes. However, gefitinib-resistant QG56 cells showed internalized EGFR accumulation in early endosomes after 60 min of internalization, instead of its trafficking to lysosomes, indicating an aberration in some steps of EGF-EGFR trafficking from the early endosomes to late endosomes/lysosomes. Therefore, we postulate that impairment in some steps of EGF-EGFR trafficking from early endosomes to late endosomes/lysosomes might confer gefitinib-resistance in NSCLC cell lines. To further substantiate the detailed internalization mechanism of gefitinib-sensitive and gefitinib-resistant cells, using confocal immunofluorescence microscopy, we examined the endocytic trafficking of phosphorylated EGFR (pEGFR) in the absence or presence of gefitinib. In PC9 and QG56 cells without EGF stimulation, a large number of pEGFR-positive small vesicular structures not colocalized with late endosomes/lysosomes were spread throughout the cytoplasm, and some pEGFR staining was distributed in the nucleus. This implies a novel intracellular trafficking pathway for pEGFR from cytoplasmic vesicles to the nucleus. Furthermore, an aggregated vesicular structure of early endosomes was observed in the perinuclear region of QG56 cells; it was revealed to be associated with SNX1, originally identified as a protein that interacts with EGFR. Therefore, we confirmed our previous data that an aberration in some steps of EGF-EGFR trafficking from the early endosomes to late endosomes/lysosomes occurs in QG56 cells. Furthermore, in PC9 cells, efficient phosphorylation of EGFR and rapid internalization of pEGFR was observed at 3 min after EGF stimulation; these internalized pEGFR-positive vesicles were trafficked to late endosomes at 15 min, indicating rapid trafficking of EGF-pEGFR complexes from early to late endosomes in PC9 cells. Gefitinib treatment strongly reduced the phosphorylation level of EGFR, and subsequent endocytosis of EGFR was significantly suppressed in PC9 cells. In contrast, in QG56 cells, EGFR trafficking via the early endocytic pathway was basically impaired; therefore, gefitinib appeared to slightly suppress the internalization of pEGFR. Collectively, our data provide novel evidence that extensive impairment in pEGFR endocytosis via the early endocytic pathway might confer gefitinib-resistance in QG56 cells

Crystal growth and its morphology in the mushy zone

In the solidification of multicomponent alloys, a mushy zone appears between the solid and liquid regions and promotes stable solidification by accepting the rejected solute regionally. In this study, the link between heat transfer and microstructures of the mushy zone has been studied experimentally and theoretically. First, the crystal morphology of the mushy zone at a microscale was observed by using succinonitrile–acetone solution and Bi–Sn alloys melts. It was found that the mushy zone consists of a leading front, in which the microstructures originate, and a growing region, where solidification proceeds with the fattening of the crystals. Next, the mechanism of dendritic sidebranch evolution was studied, taking into account the interfacial instability. To summarize these results, a macro–microscopic model is presented, and the change of crystal morphology at the microscale level was analyzed in relation to cooling rate, initial concentration, and distance from a cold wall

Heat transfer and solidification processes of alloy melt with undercooling—Part II: Solidification model

The solidification process of undercooled alloy melts has been clarified experimentally in Part I of this paper. In the present paper, using the experimental evidence, a solidification model linking macroscopic heat transfer and microscopic solidification is presented. The model reflects the microscopic solidification phenomena occurring until the thermodynamically unstable field shifts to equilibrium, consisting of three fundamental processes: (first stage) free growth, (second stage) crystal expansion with relaxation, and (third stage) equilibrium solidification. Based on this model, a numerical simulation is carried out for the temperature change, interface movement, and solute concentration distribution during the solidification of an undercooled Bi-Sn melt. Theoretical predictions of the temperature changes involving the recalescence, terminal time of the relaxation process, and microsegregation for the solidified texture agree quantitatively with experimental observations. © 2005 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved

Bone Regeneration in Artificial Jaw Cleft by Use of Carbonated Hydroxyapatite Particles and Mesenchymal Stem Cells Derived from Iliac Bone

Objectives of the Study. Cleft lip and palate (CLP) is a prevalent congenital anomaly in the orofacial region. Autogenous iliac bone grafting has been frequently employed for the closure of bone defects at the jaw cleft site. Since the related surgical procedures are quite invasive for patients, it is of great importance to develop a new less invasive technique. The aim of this study was to examine bone regeneration with mesenchyme stem cells (MSCs) for the treatment of bone defect in artificially created jaw cleft in dogs. Materials and Methods. A bone defect was prepared bilaterally in the upper incisor regions of beagle dogs. MSCs derived from iliac bone marrow were cultured and transplanted with carbonated hydroxyapatite (CAP) particles into the bone defect area. The bone regeneration was evaluated by standardized occlusal X-ray examination and histological observation. Results. Six months after the transplantation, perfect closure of the jaw cleft was achieved on the experimental side. The X-ray and histological examination revealed that the regenerated bone on the experimental side was almost equivalent to the original bone adjoining the jaw cleft. Conclusion. It was suggested that the application of MSCs with CAP particles can become a new treatment modality for bone regeneration for CLP patients

Heat transfer and solidification processes of alloy melt with undercooling—Part I: Experimental results

The solidification process of Pb-Sn and Bi-Sn alloy melts is discussed to obtain a basic understanding of the essential phenomena of solidification with undercooling. First, from macroscopic observations, it is shown that the solidification process consists of the following three stages: (1) free growth with recalescence dissipation of thermal undercooling, (2) expansion of crystals with the relaxation of constitutional undercooling or with the recovering process of interrupted quasi-steady heat conduction, and (3) equilibrium solidification. The specific features of free growth under non-uniform undercooling are also shown by comparison with the Lipton, Glicksman, and Kurz model. Next, from microscopic observations, the distribution of the solute concentration and the change of crystal morphology in the solidified materials were investigated quantitatively using scanning electron microscopy and energy-dispersive spectroscopy. Finally, the solidification path during the above three fundamental processes is dynamically represented on phase diagrams. © 2005 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved

Inhibition of Rho-associated coiled-coil containing protein kinase enhances the activation of epidermal growth factor receptor in pancreatic cancer cells

<p>Abstract</p> <p>Background</p> <p>Rho-associated coiled-coil containing protein kinase (Rho-kinase/ROCK) is involved in various cellular functions including cell proliferation, and is generally considered to be oncogenic, while some studies show that ROCK functions as a negative regulator of cancer progression. As a result, the precise role of ROCK remains controversial. We have previously reported that Rho-kinase/ROCK negatively regulates epidermal growth factor (EGF)-induced cell proliferation in SW480 colon cancer cells. In the present study, we investigated the role of ROCK in EGF receptor (EGFR) signaling in the pancreatic cancer cell lines, Panc1, KP3 and AsPc1.</p> <p>Results</p> <p>In these cells, Y27632, a specific ROCK inhibitor, enhanced EGF-induced BrdU incorporation. The blockade of EGF stimulation utilizing anti-EGFR-neutralizing antibodies suppressed Panc1 cell proliferation. EGF induced RhoA activity, as well as the phosphorylation of cofilin and myosin light chain (MLC), both targets of ROCK signaling, and Y27632 suppressed both of these processes, indicating that the phosphorylation of cofilin and MLC by EGF occurs through ROCK in Panc1 cells. EGF-induced phosphorylation of EGFR at tyrosine residues was augmented when the cells were pretreated with Y27632 or were subjected to gene silencing using ROCK-siRNA. We also obtained similar results using transforming growth factor-α. In addition, EGF-induced phosphorylation of p44/p42 mitogen-activated protein kinase and Akt were also enhanced by Y27632 or ROCK-siRNA. Moreover, an immunofluorescence microscope study revealed that pretreatment with Y27632 delayed EGF-induced internalization of EGFR. Taken together, these data indicate that ROCK functions to switch off EGFR signaling by promoting the internalization of the EGFR.</p> <p>Conclusions</p> <p>While EGF first stimulates the activation of the EGFR and subsequently increases cancer cell proliferation, EGF concurrently induces the activation of ROCK, which then turns off the activated EGFR pathway via a negative feedback system.</p