胃转流术后远端肠黏膜形态及生长因子表达的变化

目的：探讨胃转流术后远端肠管黏膜的适应性变化及生长因子的表达情况。方法：将8周龄的Wistar大鼠随机分为对照组、假手术组和胃转流术组,术后8周取吻合口远端肠管行常规病理切片检查,测量肠黏膜厚度和绒毛高度,采用免疫荧光法检测肠粘膜中表皮生长因子（EGF）和胰岛素样生长因子-1（IGF—1）的表达。结果：胃转流术组黏膜厚度（672±39与500±31um,P〈0．01）和绒毛高度（445±19与342±15um,P〈0．01）均显著高于假手术组（P〈0．01）,而对照组和假手术组间均无显著差异。与假手术组比较,胃转流术组肠粘膜中EGF和IGF-1的表达显著升高（P〈0．01）,而对照组和假手术组间无显著差异。结论：胃转流术后远端肠管粘膜发生适应性增生,同时伴随着EGF和IGF—1表达水平的升高。     

非小细胞肺癌EGFR 表达与18F-FDG-PET SUVmax 值 的相关性研究

目的:探讨非小细胞肺癌组织中EGFR蛋白在的表达与PET-CT检测的SUVmax的相关性。方法:临床收集30例非小细胞肺癌患者,每一病例首先行PET-CT检查,然后行CT导引下活检穿刺,同一病灶取2个穿刺部位,分别为肿瘤组织内SUVmax值2.5-5区域及SUVmax>5区域,采用免疫组化方法检测检测30例非小细胞肺癌肿瘤内部不同部位EGFR表达。结果:EGFR蛋白表达、SUVmax值在不同年龄、性别、病理类型中表达的差异无统计学意义(P均>0.05)。EGFR蛋白表达、SUVmax值在不同的肿瘤直径、临床分期组表达差异有统计学意义(P均<0.05)。NSCLC肿块SUVmax值与组织EGFR表达呈显著正相关(P<0.05)。结论:NSCLC组织SUV值与组织EGFR表达有显著相关性,可以为非小细胞肺癌的放疗提供指导。     

Simulating the impact of a molecular 'decision-process' on cellular phenotype and multicellular patterns in brain tumors

Experimental evidence indicates that human brain cancer cells proliferate or migrate, yet do not display both phenotypes at the same time. Here, we present a novel computational model simulating this cellular decision-process leading up to either phenotype based on a molecular interaction network of genes and proteins. The model's regulatory network consists of the epidermal growth factor receptor (EGFR), its ligand transforming growth factor-alpha (TGF alpha), the downstream enzyme phospholipaseC-gamma (PLC gamma) and a mitosis-associated response pathway. This network is activated by autocrine TGF alpha secretion, and the EGFR-dependent downstream signaling this step triggers, as well as modulated by an extrinsic nutritive glucose gradient. Employing a framework of mass action kinetics within a multiscale agent-based environment, we analyse both the emergent multicellular behavior of tumor growth and the single-cell molecular profiles that change over time and space. Our results show that one can indeed simulate the dichotomy between cell migration and proliferation based solely on an EGFR decision network. It turns out that these behavioral decisions on the single cell level impact the spatial dynamics of the entire cancerous system. Furthermore, the simulation results yield intriguing experimentally testable hypotheses also on the sub-cellular level such as spatial cytosolic polarization of PLC gamma towards an extrinsic chemotactic gradient. Implications of these results for future works, both on the modeling and experimental side are discussed.     

Growth factors stimulate kidney proximal tubule cell migration independent of augmented tyrosine phosphorylation of focal adhesion kinase

Migration of human proximal tubule cells (HKC-5) was stimulated by epidermal growth factor (EGF), hepatocyte growth factor (HGF), and insulin-like growth factor-1 (IGF-1). Integrin signaling via phosphorylation of focal adhesion kinase (FAK) appears to play a central role in cell migration. Once stimulated, FAK undergoes autophosphorylation at tyrosine (Y) 397, followed by phosphorylation of several sites including Y576/Y577 which increases FAK's kinase activity, as well as at Y407, Y861, and Y925. EGF, HGF, and IGF-1 stimulate FAK phosphorylation in various cells. We showed that endothelin stimulated phosphorylation of Y397 in fibroblasts but not HKC-5 cells. After EGF stimulation, HKC-5 cells showed no change in tyrosine phosphorylation at FAK Y397, 407, 576, 861, or 925. Similarly, HGF and IGF-1 did not stimulate the phosphorylation of FAK Y397 in HKC-5 cells. Further, after inhibition of FAK expression by siRNA, cell migration was similar to cells treated with non-target siRNA and responded to EGF with increased migration. Thus, in proximal tubule cells, stimulation of cell migration by growth factors was independent of augmented FAK tyrosine phosphorylation.     

A basic peptide within the juxtamembrane region is required for EGF receptor dimerization

The epidermal growth factor receptor (EGFR) is fundamental for normal cell growth and organ development, but has also been implicated in various pathologies, notably tumors of epithelial origin. We have previously shown that the initial 13 amino acids (P13) within the intracellular juxtamembrane region (R645-R657) are involved in the interaction with calmodulin, thus indicating an important role for this region in EGFR function. Here we show that P13 is required for proper dimerization of the receptor. We expressed either the intracellular domain of EGFR (TKJM) or the intracellular domain lacking P13 (DeltaTKJM) in COS-7 cells that express endogenous EGFR. Only TKJM was immunoprecipitated with an antibody directed against the extracellular part of EGFR, and only TKJM was tyrosine phosphorylated by endogenous EGFR. Using SK-N-MC cells, which do not express endogenous EGFR, that were stably transfected with either wild-type EGFR or recombinant full-length EGFR lacking P13 demonstrated that P13 is required for appropriate receptor dimerization. Furthermore, mutant EGFR lacking P13 failed to be autophosphorylated. P13 is rich in basic amino acids and in silico modeling of the EGFR in conjunction with our results suggests a novel role for the juxtamembrane domain (JM) of EGFR in mediating intracellular dimerization and thus receptor kinase activation and function.     

Gastrin and interleukin-1beta stimulate growth factor secretion from cultured rabbit gastric parietal cells

The hormone gastrin stimulates proliferation of the gastric mucosa. Inflammation of the stomach is also associated with increased proliferation. The proliferative response is important in the reparative response to injury but can be deleterious by predisposing to the development of cancer. Parietal cells, but not the cells in the proliferative zone of the gastric glands, express the appropriate gastrin receptor. Parietal cells may mediate the trophic effects of gastrin by secreting other growth factors. The role of parietal cells in the proliferative responses has been examined in this study. Rabbit parietal cells were cultured with gastrin or the cytokine interleukin-1beta for 18 hours. The conditioned medium from gastrin or IL-1beta stimulated parietal cells increased proliferation of HeLa cells in an epidermal growth factor-receptor dependant manner. Gastrin and IL-1beta stimulated the secretion of heparin-binding epidermal growth factor and amphiregulin but not transforming growth factor-alpha from parietal cells. Combinations of gastrin and IL-1beta on growth factor secretion were synergistic. The protein kinase C inhibitor staurosporine abolished these stimulatory effects of gastrin and IL-1beta. Divergent effects on histamine-stimulated acid secretion were observed; 18 hours pre-treatment with gastrin enhanced acid secretion by 50% but IL-1beta inhibited acid secretion in both control and gastrin pre-treated parietal cells. The acid-secreting parietal cell plays a central role in the regulation of mucosal proliferation in gastric inflammation. Secretion of paracrine growth factors by parietal cells may be an important point of integration between the endocrine and inflammatory stimuli in determining mucosal responses to injury and inflammation.     

The effect of EGF and the comitogen, norepinephrine, on the proliferative responses of fresh and cryopreserved rat and mouse hepatocytes

The effect of cryopreservation on the proliferative response of fresh and cryopreserved (CP) rat and mouse hepatocytes was studied. Of the parameters measured, incorporation of 3H-thymidine and bromodeoxyuridine (BdrU) incorporation were the most sensitive and LDH content was the least sensitive. The optimal seeding density for epidermal growth factor (EGF)-stimulated proliferative response in fresh rat and mouse hepatocytes was 1.8 x 10(4) cells/cm2 and 2.1 x 10(4) cells/cm2, respectively. 3H-thymidine incorporation by fresh rat and mouse hepatocytes was maximal in cultures treated with 10 and 5 ng/ml EGF, respectively. The cell attachment of fresh rat hepatocytes after 48 h was higher (68%) than CP (42%), therefore, the CP hepatocyte seeding density was increased to 7.1 x 10(4) cells/cm2 so that the cell number after 48 h was the same as fresh hepatocytes. Using the adjusted seeding density, the 3H-thymidine and BdrU incorporation into fresh and CP rat hepatocytes was equivalent. The attachment efficiencies of fresh and CP mouse hepatocytes were the same, therefore, no adjustment was needed. The proliferative response (3H-thymidine incorporation and DNA content) to EGF was the same in fresh and CP mouse hepatocytes. The comitogen, norepinephrine (NE), increased the proliferative response to EGF to the same extent in both fresh and CP rat hepatocytes. In summary, cryopreserved rat and mouse hepatocytes retain their ability to proliferate in culture. Adjustment and monitoring of the seeding density is of high importance, especially with rat hepatocytes, which lose some attachment capacity after cryopreservation. The secondary mitogenic effect of NE is also retained by cryopreserved rat hepatocytes, suggesting that these cells retain alpha1-receptor function.     

Activation of ErbB2 by 2-methyl-1,4-naphthoquinone (menadione) in human keratinocytes: role of EGFR and protein tyrosine phosphatases

Activation of ErbB receptor tyrosine kinases triggers multiple signaling pathways that regulate cellular proliferation and survival. We here demonstrate that ErbB2 is activated via the epidermal growth factor receptor (EGFR) upon exposure of cultured human keratinocytes to 2-methyl-1,4-naphthoquinone (menadione). Both ErbB2 and EGFR are shown to be regulated by protein tyrosine phosphatases that are inhibited by menadione, giving rise to the hypothesis that phosphatase inhibition by menadione may result in a net activation of EGFR and an enhanced ErbB2 phosphorylation. Isolated PTP-1B, a protein tyrosine phosphatase known to be associated with ErbB receptors, is demonstrated to be inhibited by menadione.     

Inhibition of MCF-7 breast cancer cell proliferation by MCF-10A breast epithelial cells in coculture

A coculture system was developed to investigate the interactions between MCF-10A breast epithelial cells and MCF-7 breast cancer cells stably expressing the green fluorescent protein (MCF-7-GFP). Studies with this MCF-10A/MCF-7-GFP coculture system on microtiter plates and on reconstituted basement membrane (Matrigel), revealed paracrine inhibition of MCF-7-GFP cell proliferation. Epidermal growth factor, which in monocultures modestly enhanced MCF-7-GFP and markedly increased MCF-10A cell proliferation, greatly inhibited MCF-7-GFP cell proliferation in MCF-10A/MCF-7-GFP cocultures. 17beta-Estradiol, which stimulated MCF-7-GFP but not MCF-10A cell proliferation in monoculture, inhibited MCF-7-GFP cell proliferation in MCF-10A/MCF-7-GFP cocultures, an effect that was blocked by the antiestrogen, ICI 182,780. On Matrigel, complex MCF-10A/MCF-7-GFP cellular interactions were observed in real time that resulted in the formation of acinus-like structures. These results indicate a role of normal epithelial cells in inhibiting tumor-cell proliferation and demonstrate the utility of this coculture system as a model of early paracrine control of breast cancer.     

Growth hormone modulation of EGF-induced PI3K-Akt pathway in mice liver

The epidermal growth factor (EGF) activates the phosphatidylinositol 3-kinase (PI3K)-Akt cascade among other signaling pathways. This route is involved in cell proliferation and survival, therefore, its dysregulation can promote cancer. Considering the relevance of the PI3K-Akt signaling in cell survival and in the pathogenesis of cancer, and that GH was reported to modulate EGFR expression and signaling, the objective of this study was to analyze the effects of increased GH levels on EGF-induced PI3K-Akt signaling.EGF-induced signaling was evaluated in the liver of GH-overexpressing transgenic mice and in their normal siblings. While Akt expression was increased in GH-overexpressing mice, EGF-induced phosphorylation of Akt, relative to its protein content, was diminished at Ser473 and inhibited at Thr308; consequently, mTOR, which is a substrate of Akt, was not activated by EGF. However, the activation of PDK1, a kinase involved in Akt phosphorylation at Thr308, was not reduced in transgenic mice. Kinetics studies of EGF-induced Akt phosphorylation showed that it is rapidly and transiently induced in GH-overexpressing mice compared with normal siblings. Thus, the expression and activity of phosphatases involved in the termination of the PI3K-Akt signaling were studied. In transgenic mice, neither PTEN nor PP2A were hyperactivated; however, EGF induced the rapid and transient association of SHP-2 to Gab1, which mediates association to EGFR and activation of PI3K. Rapid recruitment of SHP2, which would accelerate the termination of the proliferative signal induced, could be therefore contributing to the diminished EGF-induced activity of Akt in GH-overexpressing mice.