Chromatin binding of epidermal growth factor, nerve growth factor, and platelet-derived growth factor in cells bearing the appropriate surface receptors.

We analyzed the uptake and intracellular distribution of 125I-labeled epidermal growth factor, nerve growth factor, and platelet-derived growth factor in different cell lines that express or do not express the respective surface receptors for these factors. After 1 hr of incubation, all three growth factors were detected in the cytoplasmic fraction and in the nucleus, tightly bound to chromatin. The amount of chromatin-bound growth factors continued to increase during the incubation, and analysis at 48 hr revealed each chromatin-bound labeled growth factor in a nondegraded form. After limited digestion of chromatin with DNase II (10-20% digested sequences), specific release of all three growth factors was detected only after 1 hr of incubation but not after 24 and 48 hr, suggesting that the DNA regions involved in growth factor binding became nuclease-resistant. Binding of labeled epidermal growth factor and nerve growth factor to isolated chromatin was inhibited by monoclonal antibodies specific for the respective growth factor receptor. The data suggest that chromatin binding may represent an important step in the pathway of growth factor action.     

Nerve growth factor binding domain of the nerve growth factor receptor.

A structural analysis of the rat low-affinity nerve growth factor (NGF) receptor was undertaken to define the NGF binding domain. Mutant NGF receptor DNA constructs were expressed in mouse fibroblasts or COS cells, and the ability of the mutant receptor to bind NGF was assayed. In the first mutant, all but 16 amino acid residues of the intracellular domain of the receptor were removed. This receptor bound NGF with a Kd comparable to that of the wild-type receptor. A second mutant contained only the four cysteine-rich sequences from the extracellular portion of the protein. This mutant was expressed in COS cells and the resultant protein was a secreted soluble form of the receptor that was able to bind NGF. Two N-terminal deletions, in which either the first cysteine-rich sequence of the first and part of the second cysteine-rich sequences were removed, bound NGF. However, a mutant lacking all four cysteine-rich sequences was unable to bind NGF. These results show that the four cysteine-rich sequences of the NGF receptor contain the NGF binding domain.     

Differential expression of the insulin-like growth factor binding proteins in spontaneously diabetic rats.

Diabetes-induced growth retardation in the rodent is associated with both reduced circulating insulin-like growth factor-I (IGF-I) and enhanced levels of inhibitors of somatomedin activity. IGF-binding proteins (IGFBPs) are present in the circulation and tissue fluids and are believed to modulate the actions of IGF-I. Since elevated concentrations of the IGFBPs may contribute to the enhanced somatomedin-inhibitor activity observed in serum from diabetic animals, we have examined the amounts of hepatic IGFBP-1, -2, -3 and -4 mRNA in the spontaneously diabetic BioBreeding/Worcester rat. The study used two types of diabetic animal: mildly diabetic animals, which received suboptimal insulin treatment (0.5-1 U/day) and diabetic animals, which received intensive insulin treatment (3-6 U/day). A significant increase in the amount of IGFBP-1 and IGFBP-2 mRNA was seen 1 month and 3 months after the onset of diabetes. Intensive insulin treatment for 3 weeks normalized the amount of IGFBP-1 mRNA in diabetic rats and resulted in a decrease in IGFBP-2 mRNA. In contrast to the increase in IGFBP-1 and IGFBP-2 mRNA, a significant decrease in IGFBP-3 mRNA was seen in diabetic rats (54.6% of control, P less than 0.0005 and 64.6% of control, P less than 0.005 for 1 and 3 months respectively) and intensive insulin treatment for 3 weeks did not restore the IGFBP-3 mRNA level in diabetic rats. No significant difference in IGFBP-4 mRNA levels was seen in diabetic compared with non-diabetic rats. When serum was analysed by ligand blotting the major finding was a reduction in the 39-42 kDa binding protein. No increase in 29-30 kDa IGFBP in the serum was detected in the diabetic rats. From these studies we conclude that the major change in IGFBPs in mildly hyperglycaemic spontaneously diabetic rats is a decrease in IGFBP-3. The changes in hepatic IGFBP-1 and -2 mRNA do not appear to be of sufficient magnitude to result in an increase in serum concentrations of these binding proteins.     

Plasma binding proteins for platelet-derived growth factor that inhibit its binding to cell-surface receptors.

Evidence is presented that the binding of platelet-derived growth factor (PDGF) to plasma constituents inhibits the binding of PDGF to its cell-surface mitogen receptor. Approximately equivalent amounts of PDGF-binding activity were found in plasma from a number of different species known by radioreceptor assay to contain PDGF homologues in their clotted blood. Activation of the coagulation cascade did not significantly alter the PDGF-binding activity of the plasma components. Three molecular weight classes of plasma fractions that inhibit PDGF binding to its cell-surface receptor were defined by gel filtration: approximately equal to 40,000, 150,000, and greater than 500,000. Specific binding of 125I-labeled PDGF to the highest molecular weight plasma fraction could also be demonstrated by gel filtration. The binding of PDGF to these plasma components was reversible under conditions of low pH or with guanidine X HCl, and active PDGF could be recovered from the higher molecular weight fractions. Immunologic and functional evidence is presented that the highest molecular weight plasma fraction may be alpha 2-macroglobulin. A model is proposed in which the activity of PDGF released in vivo may be regulated by association with these plasma binding components and by high-affinity binding to cell-surface PDGF receptors.     

A variant epidermal growth factor receptor exhibits altered type alpha transforming growth factor binding and transmembrane signaling.

Epidermal growth factor (EGF) and type alpha transforming growth factor (TGF-alpha) bind to a specific region in subdomain III of the extracellular portion of the EGF receptor (EGFR). Binding leads to receptor dimerization, auto-and transphosphorylation on intracellular tyrosine residues, and activation of signal transduction pathways. We compared the binding and biological actions of EGF and TGF-alpha in Chinese hamster ovary (CHO) cells expressing either wild-type human EGFR (HER497R) or a variant EGFR that has an arginine-to-lysine substitution in the extracellular domain at codon 497 (HER497K) within subdomain IV of EGFR. Both receptors exhibited two orders of binding sites with radioiodinated EGF (125I-EGF). Similar results were obtained with 125I-TGF-alpha in cells expressing HER497R. In contrast, only one order of low-affinity binding sites was seen with 125I-TGF-alpha in the case of HER497K. Although EGF and TGF-alpha enhanced tyrosine phosphorylation of both receptors, CHO cells expressing HER497K exhibited an attenuated growth response to EGF and TGF-alpha and a reduced induction of the protooncogenes FOS, JUN, and MYC. Moreover, high concentrations of TGF-alpha (5 nM) inhibited growth in these cells but not in cells expressing HER497R. These findings indicate that a region in subdomain IV of EGFR regulates signal transduction across the cell membrane and selectively modulates that binding characteristics of TGF-alpha.     

Insulin-like growth factor binding protein-3 leads to insulin resistance in adipocytes

CONTEXT: Transgenic mice overexpressing IGF binding protein-3 (IGFBP-3) have insulin resistance with reduced uptake of 2-deoxyglucose in muscle and adipose tissue. OBJECTIVE: Our aim was to investigate the effects of IGFBP-3 on glucose uptake in adipocytes. RESULTS: In 3T3-L1 adipocytes, IGFBP-3 reduced insulin-stimulated but not basal glucose uptake. This was independent of IGF binding because IGFBP-2 and IGFBP-1 had no effect, whereas two non-IGF binding mutants of IGFBP-3 were inhibitory. The effect of IGFBP-3 was independent of the blockade of the IGF-I receptor. A mutant form of IGFBP-3 that does not translocate to the nucleus or bind retinoid X receptor-alpha was able to inhibit insulin-stimulated glucose uptake, indicating that nuclear translocation and retinoid X receptor-alpha binding are not essential for this IGFBP-3 action. IGFBP-3 reduced insulin-stimulated glucose transporter-4 translocation to the plasma membrane and reduced threonine phosphorylation of Akt. Collectively, our data indicate that IGFBP-3 impacts on the insulin signaling pathway to inhibit insulin-stimulated glucose uptake independent of IGFs and through nonnuclear mechanisms. Finally, we showed that IGFBP-3 inhibited insulin-stimulated glucose uptake in omental but not s.c. adipose tissue explants. CONCLUSION: IGFBP-3 may contribute to insulin resistance in adipocytes.     

Regulation of insulin-like growth factor (IGF) binding proteins in transgenic mice with altered expression of growth hormone and IGF-I.

The insulin-like growth factors (IGFs) are present in extracellular fluids bound to specific, high affinity IGF binding proteins (IGFBPs). IGFBPs are believed to mediate IGF transport to tissues and to modulate their actions on target cells. To determine whether IGF-I can modulate IGFBP concentrations in blood and to distinguish the effects of IGF-I from those of GH, we assessed serum IGFBP concentrations in four genotypically distinct groups of sibling transgenic (Tg) mice that differed in respect to their expression of IGF-I and GH. This unique physiological situation was created by crossing IGF-I Tg mice to GH-deficient, dwarf mice in whom somatotrophs were genetically ablated by the expression of a diphtheria toxin transgene in the somatotrophs. Because both Tg mouse lines are hemizygous for their respective transgene, progeny of the cross differ genotypically, according to whether or not they carry one or both transgenes, and phenotypically in regard to their relative expression of IGF-I and GH. GH-deficient mice showed a 15.7-fold decrease in serum IGF-I and a 5.5-fold decrease in serum IGFBP-3, but no change in a serum doublet band of 29,000 to 34,000 Mr, as assessed by ligand blotting. When IGF-I was expressed in the GH-deficient mice, serum levels of IGF-I and IGFBP-3 were 69% and 64% of those in normal sera, respectively. The 29,000 to 34,000 Mr doublet bands also increased. The ternary 150 kilodalton IGF-IGFBP complex, however, was not restored, presumably because IGF-I has no influence on the expression of the acid-labile subunit in this complex. In mice with IGF-I overexpression, serum IGFBP-3 was increased 2.1-fold and the sum of the 29,000 to 34,000 doublet bands was increased 2.9-fold. Immunoblotting showed that the changes in the 29,000 to 34,000 Mr forms observed by ligand blotting appeared to be predominantly due to changes in IGFBP-2. The results show that IGF-I can induce IGFBP-3 and IGFBP-2 independently of GH and that IGF-I is a major controller of these binding proteins.     

神经生长因子及其受体在人胰腺导管癌组织中的表达

Objective To investigate nerve growth factor (β-NGF) and its receptors expression in human pancreatic ductal adenocarcinoma. Methods Expression and distribution of β-NGF, tyrosine kinase A (TrKA) and P75NGFR were detected in operation tissue specimens of pancreatic ductal adenocarcinoma with immunohistochemistry and real-time PCR. Relations of β-NGF and its receptors with clinicalpathological characters, especially nerve invasion were analyzed. Results β-NGF and TrKA expression are higher in pancreatic adenocarcinoma than normal pancreas, and the differences are significant (P < 0. 01). Β-NGF and TrKA expression are associated with the differentiation grades(DG), lymphatic node metastasis, nerve invasion and surgical pathological stages. Poorer of DG and later stages, more expression of β-NGF and TrKA. Β-NGF and TrKA expression have positive correlations. Β-NGF, TrKA and P75NGFR mRNA expression have significantly increased 3.84,4. 23 and 2. 41 times than normal tissues by real-time PCR, respectively. Conclusions β-NGF and TrKA might play potential rules in carcinogenesis for pancreatic cancer,have affinity with clinicopathological characters of pancreatic cancer. Β-NGF and TrKA may have mutual effect in signal transduction leading to perineural invasion of pancreatic carcinoma.     

神经生长因子及其受体在人胰腺导管癌组织中的表达

Objective To investigate nerve growth factor (β-NGF) and its receptors expression in human pancreatic ductal adenocarcinoma. Methods Expression and distribution of β-NGF, tyrosine kinase A (TrKA) and P75NGFR were detected in operation tissue specimens of pancreatic ductal adenocarcinoma with immunohistochemistry and real-time PCR. Relations of β-NGF and its receptors with clinicalpathological characters, especially nerve invasion were analyzed. Results β-NGF and TrKA expression are higher in pancreatic adenocarcinoma than normal pancreas, and the differences are significant (P < 0. 01). Β-NGF and TrKA expression are associated with the differentiation grades(DG), lymphatic node metastasis, nerve invasion and surgical pathological stages. Poorer of DG and later stages, more expression of β-NGF and TrKA. Β-NGF and TrKA expression have positive correlations. Β-NGF, TrKA and P75NGFR mRNA expression have significantly increased 3.84,4. 23 and 2. 41 times than normal tissues by real-time PCR, respectively. Conclusions β-NGF and TrKA might play potential rules in carcinogenesis for pancreatic cancer,have affinity with clinicopathological characters of pancreatic cancer. Β-NGF and TrKA may have mutual effect in signal transduction leading to perineural invasion of pancreatic carcinoma.     

Induction of nerve growth factor receptors on cultured human melanocytes.

Normal differentiation and malignant transformation of human melanocytes involve a complex series of interactions during which both genetic and environmental factors play roles. At present, the regulation of these processes is poorly understood. We have induced the expression of nerve growth factor (NGF) receptors on cultured human melanocytes with phorbol 12-tetradecanoate 13-acetate and have correlated this event with the appearance of a more differentiated, dendritic morphology. Criteria for NGF receptor expression included protein accumulation and cell-surface immunofluorescent staining with a monoclonal antibody directed against the human receptor and induction of the messenger RNA species as determined by blot-hybridization studies. The presence of the receptor could also be induced by UV irradiation or growth factor deprivation. The NGF receptor is inducible in cultured human melanocytes, and we suggest that NGF may modulate the behavior of this neural crest-derived cell in the skin.     

神经生长因子及其受体在人胰腺导管癌组织中的表达

Objective To investigate nerve growth factor (β-NGF) and its receptors expression in human pancreatic ductal adenocarcinoma. Methods Expression and distribution of β-NGF, tyrosine kinase A (TrKA) and P75NGFR were detected in operation tissue specimens of pancreatic ductal adenocarcinoma with immunohistochemistry and real-time PCR. Relations of β-NGF and its receptors with clinicalpathological characters, especially nerve invasion were analyzed. Results β-NGF and TrKA expression are higher in pancreatic adenocarcinoma than normal pancreas, and the differences are significant (P < 0. 01). Β-NGF and TrKA expression are associated with the differentiation grades(DG), lymphatic node metastasis, nerve invasion and surgical pathological stages. Poorer of DG and later stages, more expression of β-NGF and TrKA. Β-NGF and TrKA expression have positive correlations. Β-NGF, TrKA and P75NGFR mRNA expression have significantly increased 3.84,4. 23 and 2. 41 times than normal tissues by real-time PCR, respectively. Conclusions β-NGF and TrKA might play potential rules in carcinogenesis for pancreatic cancer,have affinity with clinicopathological characters of pancreatic cancer. Β-NGF and TrKA may have mutual effect in signal transduction leading to perineural invasion of pancreatic carcinoma.     

神经生长因子及其受体在人胰腺导管癌组织中的表达

Objective To investigate nerve growth factor (β-NGF) and its receptors expression in human pancreatic ductal adenocarcinoma. Methods Expression and distribution of β-NGF, tyrosine kinase A (TrKA) and P75NGFR were detected in operation tissue specimens of pancreatic ductal adenocarcinoma with immunohistochemistry and real-time PCR. Relations of β-NGF and its receptors with clinicalpathological characters, especially nerve invasion were analyzed. Results β-NGF and TrKA expression are higher in pancreatic adenocarcinoma than normal pancreas, and the differences are significant (P < 0. 01). Β-NGF and TrKA expression are associated with the differentiation grades(DG), lymphatic node metastasis, nerve invasion and surgical pathological stages. Poorer of DG and later stages, more expression of β-NGF and TrKA. Β-NGF and TrKA expression have positive correlations. Β-NGF, TrKA and P75NGFR mRNA expression have significantly increased 3.84,4. 23 and 2. 41 times than normal tissues by real-time PCR, respectively. Conclusions β-NGF and TrKA might play potential rules in carcinogenesis for pancreatic cancer,have affinity with clinicopathological characters of pancreatic cancer. Β-NGF and TrKA may have mutual effect in signal transduction leading to perineural invasion of pancreatic carcinoma.     

神经生长因子及其受体在人胰腺导管癌组织中的表达

Objective To investigate nerve growth factor (β-NGF) and its receptors expression in human pancreatic ductal adenocarcinoma. Methods Expression and distribution of β-NGF, tyrosine kinase A (TrKA) and P75NGFR were detected in operation tissue specimens of pancreatic ductal adenocarcinoma with immunohistochemistry and real-time PCR. Relations of β-NGF and its receptors with clinicalpathological characters, especially nerve invasion were analyzed. Results β-NGF and TrKA expression are higher in pancreatic adenocarcinoma than normal pancreas, and the differences are significant (P < 0. 01). Β-NGF and TrKA expression are associated with the differentiation grades(DG), lymphatic node metastasis, nerve invasion and surgical pathological stages. Poorer of DG and later stages, more expression of β-NGF and TrKA. Β-NGF and TrKA expression have positive correlations. Β-NGF, TrKA and P75NGFR mRNA expression have significantly increased 3.84,4. 23 and 2. 41 times than normal tissues by real-time PCR, respectively. Conclusions β-NGF and TrKA might play potential rules in carcinogenesis for pancreatic cancer,have affinity with clinicopathological characters of pancreatic cancer. Β-NGF and TrKA may have mutual effect in signal transduction leading to perineural invasion of pancreatic carcinoma.     

神经生长因子及其受体在人胰腺导管癌组织中的表达

Objective To investigate nerve growth factor (β-NGF) and its receptors expression in human pancreatic ductal adenocarcinoma. Methods Expression and distribution of β-NGF, tyrosine kinase A (TrKA) and P75NGFR were detected in operation tissue specimens of pancreatic ductal adenocarcinoma with immunohistochemistry and real-time PCR. Relations of β-NGF and its receptors with clinicalpathological characters, especially nerve invasion were analyzed. Results β-NGF and TrKA expression are higher in pancreatic adenocarcinoma than normal pancreas, and the differences are significant (P < 0. 01). Β-NGF and TrKA expression are associated with the differentiation grades(DG), lymphatic node metastasis, nerve invasion and surgical pathological stages. Poorer of DG and later stages, more expression of β-NGF and TrKA. Β-NGF and TrKA expression have positive correlations. Β-NGF, TrKA and P75NGFR mRNA expression have significantly increased 3.84,4. 23 and 2. 41 times than normal tissues by real-time PCR, respectively. Conclusions β-NGF and TrKA might play potential rules in carcinogenesis for pancreatic cancer,have affinity with clinicopathological characters of pancreatic cancer. Β-NGF and TrKA may have mutual effect in signal transduction leading to perineural invasion of pancreatic carcinoma.     

神经生长因子及其受体在人胰腺导管癌组织中的表达

目的 探讨β神经生长因子(β-NGF)及其受体[酪氨酸激酶A(tyrosine kinase A,TrKA)和P75NGFR]在人胰腺导管癌组织中的表达及意义.方法 选择胰腺导管癌组织标本80例和正常胰腺组织20例,采用免疫组化、荧光定量PCR等方法,定量检测β-NGF及其受体表达,分析β-NGF)及其受体与临床病理学特征,尤其与神经侵袭的关系.结果 随着癌细胞分化程度降低和肿瘤TNM分期增加,β-NGF、TrKA蛋白表达水平增加,且低分化与高分化、中分化组相比显著增加(P<0.01),Ⅲ～Ⅳ期较Ⅰ～Ⅱ期显著增加(P<0.05);β-NGF、TrKA蛋白表达水平在有神经浸润组明显高于无神经浸润组(P<0.01),有淋巴结转移组明显高于无淋巴结转移组(P<0.01),且邻近神经组织的肿瘤细胞β-NGF、TrKA阳性程度高于远离神经组织的癌细胞.P75NGFR表达与肿瘤细胞分化程度关系密切.β-NGF和TrKA表达呈正相关.β-NGF、TrKA和P75NGFR mRNA表达水平在胰腺癌组织中明显高于正常胰腺组织,分别增加3.84、4.23和2.41倍.结论 β-NGF及其受体参与介导胰腺癌的发生,与胰腺癌临床病理学特征关系密切,β-NGF、TrKA高表达和胰腺癌嗜神经性关系密切.     

神经生长因子及其受体在人胰腺导管癌组织中的表达

Objective To investigate nerve growth factor (β-NGF) and its receptors expression in human pancreatic ductal adenocarcinoma. Methods Expression and distribution of β-NGF, tyrosine kinase A (TrKA) and P75NGFR were detected in operation tissue specimens of pancreatic ductal adenocarcinoma with immunohistochemistry and real-time PCR. Relations of β-NGF and its receptors with clinicalpathological characters, especially nerve invasion were analyzed. Results β-NGF and TrKA expression are higher in pancreatic adenocarcinoma than normal pancreas, and the differences are significant (P < 0. 01). Β-NGF and TrKA expression are associated with the differentiation grades(DG), lymphatic node metastasis, nerve invasion and surgical pathological stages. Poorer of DG and later stages, more expression of β-NGF and TrKA. Β-NGF and TrKA expression have positive correlations. Β-NGF, TrKA and P75NGFR mRNA expression have significantly increased 3.84,4. 23 and 2. 41 times than normal tissues by real-time PCR, respectively. Conclusions β-NGF and TrKA might play potential rules in carcinogenesis for pancreatic cancer,have affinity with clinicopathological characters of pancreatic cancer. Β-NGF and TrKA may have mutual effect in signal transduction leading to perineural invasion of pancreatic carcinoma.     

神经生长因子及其受体在人胰腺导管癌组织中的表达

Objective To investigate nerve growth factor (β-NGF) and its receptors expression in human pancreatic ductal adenocarcinoma. Methods Expression and distribution of β-NGF, tyrosine kinase A (TrKA) and P75NGFR were detected in operation tissue specimens of pancreatic ductal adenocarcinoma with immunohistochemistry and real-time PCR. Relations of β-NGF and its receptors with clinicalpathological characters, especially nerve invasion were analyzed. Results β-NGF and TrKA expression are higher in pancreatic adenocarcinoma than normal pancreas, and the differences are significant (P < 0. 01). Β-NGF and TrKA expression are associated with the differentiation grades(DG), lymphatic node metastasis, nerve invasion and surgical pathological stages. Poorer of DG and later stages, more expression of β-NGF and TrKA. Β-NGF and TrKA expression have positive correlations. Β-NGF, TrKA and P75NGFR mRNA expression have significantly increased 3.84,4. 23 and 2. 41 times than normal tissues by real-time PCR, respectively. Conclusions β-NGF and TrKA might play potential rules in carcinogenesis for pancreatic cancer,have affinity with clinicopathological characters of pancreatic cancer. Β-NGF and TrKA may have mutual effect in signal transduction leading to perineural invasion of pancreatic carcinoma.     

神经生长因子及其受体在人胰腺导管癌组织中的表达

Objective To investigate nerve growth factor (β-NGF) and its receptors expression in human pancreatic ductal adenocarcinoma. Methods Expression and distribution of β-NGF, tyrosine kinase A (TrKA) and P75NGFR were detected in operation tissue specimens of pancreatic ductal adenocarcinoma with immunohistochemistry and real-time PCR. Relations of β-NGF and its receptors with clinicalpathological characters, especially nerve invasion were analyzed. Results β-NGF and TrKA expression are higher in pancreatic adenocarcinoma than normal pancreas, and the differences are significant (P < 0. 01). Β-NGF and TrKA expression are associated with the differentiation grades(DG), lymphatic node metastasis, nerve invasion and surgical pathological stages. Poorer of DG and later stages, more expression of β-NGF and TrKA. Β-NGF and TrKA expression have positive correlations. Β-NGF, TrKA and P75NGFR mRNA expression have significantly increased 3.84,4. 23 and 2. 41 times than normal tissues by real-time PCR, respectively. Conclusions β-NGF and TrKA might play potential rules in carcinogenesis for pancreatic cancer,have affinity with clinicopathological characters of pancreatic cancer. Β-NGF and TrKA may have mutual effect in signal transduction leading to perineural invasion of pancreatic carcinoma.