BBA - Molecular Cell Research (v.1538, #1)

Glioblastoma cells express a mutant EGF receptor (EGFRvIII) that has constitutive tyrosine kinase activity and enhances their tumorigenicity. Here we show that EGFRvIII promotes constitutive phosphorylation of extracellular regulated kinases (ERKs) in glioblastoma cells in the absence of EGF. EGFRvIII also promoted constitutive activation of phosphoinositide 3-kinase in these cells, as assessed by phosphorylation of protein kinase B/akt. As expected, phosphorylation of protein kinase B/akt was blocked by the phosphoinositide 3-kinase inhibitors wortmannin and LY294002. Less expectedly, we found that this treatment also blocked EGFRvIII-induced phosphorylation of ERKs. In contrast, ERK phosphorylation induced by EGF-activated normal EGF receptor in the same cells was largely unaffected by treatment with phosphoinositide 3-kinase inhibitors. This difference in behavior between the normal receptor and EGFRvIII was not due to differences in the levels of activated EGFRvIII and wild-type EGF receptor, as the two types of receptor were tyrosine phosphorylated to a similar extent under the experimental conditions used. EGFRvIII activation of ERKs was also sensitive to the phospholipase C inhibitor U73122, whereas ERK activation by normal EGF receptor was not. These results show that EGFRvIII and wild-type EGF receptor preferentially use different signaling pathways to induce ERK phosphorylation. The different mechanisms of ERK activation used by normal and mutant EGF receptors may be important in understanding the potent tumorigenic activity of EGFRvIII.
Keywords: EGF receptor; EGFRvIII; Extracellular-regulated kinase; Phosphoinositide 3-kinase; Phospholipase C; Glioblastoma;

Dynamin inhibits phosphatidylinositol 3-kinase in hematopoietic cells by Duygu Harrison-Findik; Suniti Misra; Suresh K. Jain; Marilyn L. Keeler; Kate A. Powell; Chandra S. Malladi; Lyuba Varticovski; Phillip J. Robinson (10-19).
Phosphatidylinositol 3-kinase (PI 3-kinase) plays a role in late stages of endocytosis as well as in cellular proliferation and transformation. The SH3 domain of its regulatory p85 subunit stimulates the GTPase activity of dynamin in vitro. Dynamin is a GTPase enzyme required for endocytosis of activated growth factor receptors. An interaction between these proteins has not been demonstrated in vivo. Here, we report that dynamin associates with PI 3-kinase in hematopoietic cells. We detected both p85 and PI 3-kinase activity in dynamin immune complexes from IL-3-dependent BaF3 cells. However, this association was significantly reduced in BaF3 cells transformed with the BCR/abl oncogene. After transformation only a 4-fold increase in PI 3-kinase activity was detected in dynamin immune complexes, whereas grb2 associated activity was elevated 20-fold. Furthermore, dynamin inhibited the activity of both purified recombinant and immunoprecipitated PI 3-kinase. In BaF3 cells expressing a temperature-sensitive mutant of BCR/abl, a significant decrease in p85 and dynamin association was observed 4 h after the induction of BCR/abl activity. In contrast, in IL-3-stimulated parental BaF3 cells, this association was increased. Our results demonstrate an in vivo association of PI 3-kinase with dynamin and this interaction regulates the activity of PI 3-kinase.
Keywords: Phosphatidylinositol 3-kinase; Dynamin; Endocytosis; Hematopoietic cell; Cell signaling; Transformation;

Osmotically induced cytosolic free Ca2+ changes in human neutrophils by M.Rachel Morris; Iolo J.M Doull; Maurice B Hallett (20-27).
Cytosolic free Ca2+ concentration in neutrophils was measured by ratiometric fluorometry of intracellular fura2. Increasing the extracellular osmolarity, by either NaCl (300–600 mM) or sucrose (600–1200 mM), caused a rise in cytosolic free Ca2+max≅600 nM). This was not due to cell lysis as the cytosolic free Ca2+ concentration was reversed by restoration of isotonicity and a second rise in cytosolic free Ca2+ could be provoked by repeating the change in extracellular osmolarity. Furthermore, the rise in cytosolic free Ca2+ concentration occurred in the absence of extracellular Ca2+, demonstrating that release of intracellular fura2 into the external medium did not occur. The osmotically-induced rise in cytosolic free Ca2+ was not inhibited by either the phospholipase C-inhibitor U73122, or the microfilament inhibitor cytochalasin B, suggesting that neither signalling via inositol tris-phosphate or the cytoskeletal system were involved. However, the rise in cytosolic free Ca2+ may have resulted from a reduction in neutrophil water volume in hyperosmotic conditions. As these rises in cytosolic Ca2+max≅600 nM) were large enough to provoke changes in neutrophil activity, we propose that conditions which removes cell water may similarly elevate cytosolic free Ca2+ to physiologically important levels.
Keywords: Neutrophil; Cytosolic free Ca2+ concentration; Osmolarity; Cystic fibrosis; Signalling; Imaging; Cell shape; (Lung damage); (Inflammation);

Electrical stimulation influences mineral formation of osteoblast-like cells in vitro by Hans-Peter Wiesmann; Mareke Hartig; Udo Stratmann; Ulrich Meyer; Ulrich Joos (28-37).
The aim of the present study was to assess the structure of newly formed mineral crystals after electrical stimulation of osteoblast-like cells in vitro. Pulsed electrical stimulation was coupled capacitively or semi-capacitively to primary osteoblast-like cells derived from bovine metacarpals. Computer calculations revealed that the chosen input signal (saw-tooth, 100 V, 63 ms width, 16 Hz repetition rate) generated a short pulsed voltage drop of 100 μV (capacitive coupled mode) and of 350 μV (semi-capacitive coupled mode) across the cell-matrix layer. Stimulated cultures showed an enhanced mineral formation compared to the non stimulated controls. In cultures exposed to capacitively coupled electric fields and in control cultures nodules and mineralized globules were found. Nodules with a diameter of less than 200 nm covered the cell surface, whereas mineral globules with a diameter of up to 700 nm formed characteristic mineral deposits in the vicinity of the cells similar to biomineral formations occurring in mineralizing tissues. In contrast, large rod-shaped crystals were found in cultures stimulated by semi-capacitive coupled electric fields, indicating a non-physiological precipitation process. In conclusion, osteoblasts in culture are sensitive to electrical stimulation resulting in an enhancement of the biomineralization process.
Keywords: Apatite; Mineralization; Electrical stimulation; Cell culture;

Modulation of the stress response during apoptosis and necrosis induction in cadmium-treated U-937 human promonocytic cells by Alba Galán; Alfonso Troyano; Nuria E. Vilaboa; Carlos Fernández; Elena de Blas; Patricio Aller (38-46).
Treatment for 2 h with 200 μM cadmium chloride, followed by recovery, caused apoptosis and induced heat-shock protein 70 (HSP70) expression in U-937 promonocytic cells. However, pre-incubation with the GSH depleting agent l-buthionine-[S,R]-sulfoximine (BSO, 1 mM for 24 h) caused necrosis instead of apoptosis and failed to induce HSP70 expression. This failure was a consequence of necrosis instead of GSH depletion, since BSO allowed or even potentiated HSP70 induction when used in combination with heat shock (2 h at 42.5°C) or with 50 μM cadmium, which caused apoptosis. The administration of N-acetyl-l-cysteine (NAC) at the beginning of recovery after BSO/200 μM cadmium treatment prevented the execution of necrosis and restored the execution of apoptosis, but did not restore HSP70 induction, indicating that the inhibition by BSO of HSP70 expression is an early regulated event. This contrasted with the capacity of NAC to prevent the alterations caused by BSO/200 μM cadmium in other proteins, namely the suppression of Bax expression and the increase in Bcl-2 and HSP-60 expression. Finally, it was observed that treatment with 200 μM cadmium rapidly increased the HSP70 mRNA level and stimulated heat-shock factor 1 (HSF1) trimerization and binding, and that these effects were prevented by pre-incubation with BSO. Taken together, these results indicate that the stress response is compatible with apoptosis but not with necrosis in cadmium-treated promonocytic cells. The suppression of the stress response is specifically due to the early inhibition of HSF1 activation.
Keywords: Cadmium; Apoptosis; Necrosis; Stress response; U-937 promonocytic cell;

Distinct patterns of insulin-like growth factor binding protein (IGFBP)-2 and IGFBP-3 expression in oxidant exposed lung epithelial cells by Valerie Besnard; Sophie Corroyer; Germain Trugnan; Katarina Chadelat; Elodie Nabeyrat; Véronique Cazals; Annick Clement (47-58).
Oxygen (O2) species are involved in a large variety of pulmonary diseases. Among the various cell types that compose the lung, the epithelial cells of the alveolar structure appear to be a major target for oxidant injury. Despite their importance in the repair processes, the mechanisms which regulate the replication of the stem cells of the alveolar epithelium, the type 2 cells, remain poorly understood. Based on the results of several studies which have documented the involvement of the insulin-like growth factor (IGF) system in lung epithelial cell replication, and which have also suggested a role for IGF binding proteins (IGFBPs) in the control of cell proliferation, the aim of the present work was to determine whether IGFBPs could be involved in the modulation of growth of human lung epithelial cells exposed to oxidants. Experiments were performed using a human lung adenocarcinoma cell line (A549) which was exposed for various durations to hyperoxia (95% O2). We observed a rapid and reversible growth arrest of the cells after only 24 h of O2 exposure. When oxidant injury was prolonged, growth arrest was followed by induction of apoptosis with activation of the Fas pathway. These effects were associated with an increased expression of IGFBP-2 and IGFBP-3. In addition, study of localization of these proteins revealed distinct patterns of distribution. IGFBP-3 was mainly present in the extracellular compartment. In comparison, the fraction of IGFBP-2 secreted was less abundant whereas the IGFBP-2 fraction in the intracellular compartment appeared stronger. In addition, analysis of the subcellular localization provided data indicating the presence of IGFBP-2 in the nucleus. Taken together these data support a role for IGFBP-2 and IGFBP-3 in the processes of growth arrest and apoptosis in lung epithelial cells upon oxidant exposure. They also suggest that distinct mechanisms may link IGFBP-2 and IGFBP-3 to the key regulators of the cell cycle.
Keywords: Lung epithelial cell; Oxidant; Insulin-like growth factor; Cell proliferation;

HCV core protein modulates Rb pathway through pRb down-regulation and E2F-1 up-regulation by Jae-We Cho; Won-Ki Baek; Se-Hwan Yang; Jun Chang; Young Chul Sung; Min-Ho Suh (59-66).
It has been recognized that the HCV (hepatitis C virus) core protein plays an important role in hepatocarcinogenesis. The functional inactivation of the Rb pathway appears to be a major event for multi-step cancer carcinogenesis. To elucidate the role of the HCV core protein in hepatocarcinogenesis, we investigated the effect of the HCV core protein on the Rb pathway in both Rat-1 cell lines, stably expressing the HCV core protein and the doxycycline-regulated cell lines. The HCV core stable transfectants showed a dramatic decrease in the pRb levels and E2F-1 up-regulation. In the doxycycline-regulated cell lines, the pRb levels were significantly decreased which are followed by E2F-1 up-regulation. HCV core stable transfectants showed higher cell growth rates and were sensitize to apoptosis. Thus, our results first indicate that the HCV core protein decreases the expression of pRb, thereby allowing E2F-1 to be constitutively active, which is thought to result in rapid cell proliferation or sensitizing to apoptosis.
Keywords: Hepatitis C virus; Core protein; Hepatocarcinogenesis; Rb pathway;

Replication protein A is the major single strand DNA binding protein of human cells, composed of three subunits with molecular weights of 70, 32, and 14 kDa. Most of the DNA binding activity of RPA has been mapped to the largest subunit that contains two OB-fold DNA binding domains and a third, OB-like structure in the carboxyterminal domain (CTD). This third domain resembles an OB-fold with a zinc binding domain inserted in the middle of the structure, and has recently been shown to carry a coordinated Zn(II) ion. The bound metal ion is essential for the tertiary structure of the RPA70-CTD, and appears to modulate its DNA binding activity when tested with synthetic oligonucleotides. We show here that zinc strongly affects the conformation of nucleoprotein filaments formed between RPA and long natural DNA molecules. In these experiments, the CTD is dispensable for DNA binding and the unwinding of long double stranded DNA molecules. However, using band shift assays and electron microscopy, we found that RPA–DNA complexes contract at zinc concentrations that do not affect the conformations of complexes formed between DNA and a RPA70 deletion construct lacking the CTD. Our data suggest that nucleoprotein complexes with RPA in its natural, zinc-bearing form may have a compact rather than an extended conformation.
Keywords: Replication protein A; Zinc; Conformation; Electron microscopy;

Influence of proteasome and redox state on heat shock-induced activation of stress kinases, AP-1 and HSF by Lorenza Tacchini; Paola Dansi; Emanuela Matteucci; Aldo Bernelli-Zazzera; Maria Alfonsina Desiderio (76-89).
We studied the pattern of activation of stress kinases and of transcription factors activator protein-1 (AP-1) and heat shock factor (HSF) in FAO cells by combining two treatments, i.e. heating (42°C for 1 h) and proteasome inhibition, each known to cause cellular heat shock response. The co-treatment heat shock (HS) and proteasome inhibitor (a peptidyl aldehyde or lactacystin) showed cumulative effects on the intensity and duration of activation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) at the end of the HS period and during recovery. Similarly, the thiol-reducing agents N-(2-mercaptoethyl)-1,3-diaminopropane and dithiothreitol strongly activated both JNK and p38 MAPK in cells undergoing HS. AP-1 DNA binding activity in response to proteasome inhibitors was so strong that it shadowed the stimulatory effect of HS in the combined treatment, but lactacystin, which is the most potent and specific proteasome inhibitor, decreased the binding late during recovery from HS. Thiol-reducing agents prevented AP-1 DNA binding induced by HS. The combined HS/proteasome inhibitors or HS/thiol-reducing agents treatments cooperatively activated HSF DNA binding. Expression of collagenase I and hsp 70 mRNAs reflects the different behavior of AP-1 and HSF transcription factors in cells exposed to HS and proteasome inhibition. The data seem to indicate that JNK and p38 MAPK activations are not necessarily coupled to DNA binding of AP-1, which can be either increased or inhibited when these kinases are activated. AP-1 and HSF show opposite patterns of response to HS in the presence of proteasome inhibitors or reducing agents.
Keywords: Heat shock; Stress kinase; Proteasome; Redox state; Activator protein-1; Heat shock factor;

Exposure to high concentrations of oxygen has previously been shown to cause growth arrest in A549 cells, a distal lung epithelial cell line. We found that when A549 cells were exposed to 95% oxygen they underwent substantial growth inhibition. This was associated with induction of p21Waf1/Cip1/Sdi1 protein and a decrease in cyclin B1 protein. Flow cytometry revealed that A549 cells exposed to hyperoxia had a significant decrease in the percentage of cells in G1 and a modest but significant increase in the percentage of cells in S phase and G2/M, consistent with cells entering S phase. A549 cells in room air and hyperoxia were then treated with nocodazole, a mitotic inhibitor. Room air A549 cells treated with nocodazole showed a marked increase in G2/M consistent with mitotic arrest. In contrast, hyperoxic treated cells had a modest but significant decrease in G1 but only a minimal increase in G2/M consistent with partial G1/S arrest and growth inhibition in S phase. To further investigate the role of p21Waf1/Cip1/Sdi1 as a checkpoint regulator during hyperoxic growth inhibition, HCT116 cells with wild-type and null p21Waf1/Cip1/Sdi1 were exposed to hyperoxia. Both wild-type p21(+/+) cells and null p21(−/−) cells underwent growth inhibition when exposed to hyperoxia. At 48 h the hyperoxic treated HCT116 p21(+/+) had a similar cell cycle distribution as the hyperoxic treated HCT116 p21(−/−) cells, suggesting that p21Waf1/Cip1/Sdi1 may not be essential for growth arrest during hyperoxia. These findings suggest that hyperoxia causes partial growth arrest at different phases of the cell cycle but primarily in S phase, that hyperoxic growth arrest is associated with a decrease in cyclin B1 protein and that p21 induction may not be essential for hyperoxic growth arrest.
Keywords: Hyperoxia; A549 cell; p21Waf1/Cip1/Sdi1;