Adhesion of different cell cycle human hepatoma cells to endothelial cells and roles of integrin β1

AIM: To investigate the adhesive mechanical properties of different cell cycle human hepatoma cells (SMMC-7721) to human umbilical vein endothelial cells (ECV-304), expression of adhesive molecule integrinβ1 in SMMC-7721 cells and its contribution to this adhesive course. METHODS: Adhesive force of SMMC-7721 cells to endothelial cells was measured using micropipette aspiration technique. Synchronous G1 and S phase SMMC-7721 cells were achieved by thymine-2-deoxyriboside and colchicines sequential blockage method and double thymine-2-deoxyriboside blockage method, respectively. Synchronous rates of SMMC-7721 cells and expression of integrinβ1 in SMMC-7721 cells were detected by flow cytometer. RESULTS: The percentage of cell cycle phases of general SMMC-7721 cells was 11.01% in G2/M phases, 53.51% in G0/G1 phase, and 35.48% in S phase. The synchronous rates of G1 and S phase SMMC-7721 cells amounted to 74.09% and 98.29%, respectively. The adhesive force of SMMC-7721 cells to endothelial cells changed with the variations of adhesive time and presented behavior characteristics of adhesion and de-adhesion. S phase SMMC-7721 cells had higher adhesive forces than G1 phase cells [(307.65±92.10)×10^-10N vs (195.42±60.72)×10^-10N, P<0.01]. The expressive fluorescent intensity of integrinβ1 in G1 phase SMMC-7721 cells was depressed more significantly than the values of S phase and general SMMC-7721cells. The contribution of adhesive integrinβ1 was about 53% in this adhesive course. CONCLUSION: SMMC-7721 cells can be synchronized preferably in G1 and S phases with thymine-2-deoxyriboside and colchicines. The adhesive molecule integrinβ1 expresses a high level in SMMC-7721 cells and shows differences in various cell cycles, suggesting integrin β1 plays an important role in adhesion to endothelial cells. The change of adhesive forces in different cell cycle SMMC-7721 cells indicates that S phase cells play predominant roles possibly while they interact with endothelial cells.     

Nesfatin-1 exerts a direct, glucose-dependent insulinotropic action on mouse islet β- and MIN6 cells

Nesfatin-1 is a recently discovered multifunctional metabolic hormone abundantly expressed in the pancreatic islets. The main objective of this study is to characterize the direct effects of nesfatin-1 on insulin secretion in vitro using MIN6 cells and islets isolated from C57BL/6 mice. We also examined the expression of the nesfatin-1 precursor protein, nucleobindin 2 (NUCB2) mRNA, and nesfatin-1 immunoreactivity (ir) in the islets of normal mice and in the islets from mice with streptozotocin-induced type 1 diabetes and diet-induced obese (DIO) mice with type 2 diabetes. Nesfatin-1 stimulated glucose-induced insulin release in vitro from mouse islets and MIN6 cells in a dose-dependent manner. No such stimulation in insulin secretion was found when MIN6 cells/islets were incubated with nesfatin-1 in low glucose. In addition, a fourfold increase in nesfatin-1 release from MIN6 cells was observed following incubation in high glucose (16.7 mM) compared to low glucose (2 mM). Furthermore, we observed a significant reduction in both NUCB2 mRNA expression and nesfatin-1-ir in the pancreatic islets of mice with type 1 diabetes, while a significant increase was observed in the islets of DIO mice. Together, our findings indicate that nesfatin-1 is a novel insulinotropic peptide and that the endogenous pancreatic islet NUCB2/nesfatin is altered in diabetes and diet-induced obesity.     

Designed β-sheet peptides that inhibit proliferation and induce apoptosis in endothelial cells

Novel β-sheet-forming peptide 33mers, βpep peptides, have been designed by using a combination approach employing basic folding principles and incorporating short sequences or proposed key residues from the β-sheet domains of interleukin-8 (IL-8), platelet factor-4 (PF4) and bactericidal/permeability increasing protein (B/PI). Since PF4 and B/PI are anti-angiogenic and IL-8 is angiogenic, the library of 30 βpep peptides was assayed for the ability to affect the growth of endothelial cells. Results indicate that five βpep peptides (βpep-2, 7, 8, 21 and 25) demonstrate greater than 50% anti-proliferative activity at 30 μg/ml, and one of those (βpep-25) is similarly active at 10 μg/ml. Insight into the mechanism of action was probed in an apoptosis assay. Anti-proliferative activity was found to be correlated with the induction of apoptosis. For example, at 100 μg/ml βpep-25 induces 85% of endothelial cells to undergo apoptosis within 2 days. These effects from βpep peptides appear to be selective for endothelial cell (EC) because normal cells (fibroblasts and leukocytes) and various tumor cells are not significantly affected at peptide concentrations used in this study. Comparison of active and inactive βpep sequences allows structure–function relationships to be deduced. Five hydrophobic residues and two lysines appear to be crucial to activity. This research contributes to the development of novel anti-angiogenic peptides. This revised version was published online in June 2006 with corrections to the Cover Date.     

Adhesion of different cell cycle human hepatoma cells to endothelial cells and roles of integrin β_1

AIM: To investigate the adhesive mechanical properties of different cell cycle human hepatoma cells (SMMC-7721) to human umbilical vein endothelial cells (ECV-304), expression of adhesive molecule integrinβ1 in SMMC-7721 cells and its contribution to this adhesive course. METHODS: Adhesive force of SMMC-7721 cells to endothelial cells was measured using micropipette aspiration technique. Synchronous G1 and S phase SMMC-7721 cells were achieved by thymine-2-deoxyriboside and colchicines sequential blockage method and double thymine-2-deoxyriboside blockage method, respectively. Synchronous rates of SMMC-7721 cells and expression of integrinβ1 in SMMC-7721 cells were detected by flow cytometer. RESULTS: The percentage of cell cycle phases of general SMMC-7721 cells was 11.01% in G2/M phases, 53.51% in G0/G1 phase, and 35.48% in S phase. The synchronous rates of G1 and S phase SMMC-7721 cells amounted to 74.09% and 98.29%, respectively. The adhesive force of SMMC-7721 cells to endothelial cells changed with the variations of adhesive time and presented behavior characteristics of adhesion and de-adhesion. S phase SMMC-7721 cells had higher adhesive forces than d phase cells [(307.65±92.10)×10-10N vs(195.42±60.72)×10-10N, P<0.01]. The expressive fluorescent intensity of integrinβ1 in G1 phase SMMC-7721 cells was depressed more significantly than the values of S phase and general SMMC-7721cells. The contribution of adhesive integrinβ1 was about 53% in this adhesive course. CONCLUSION: SMMC-7721 cells can be synchronized preferably in d and S phases with thymine-2-deoxyriboside and colchicines. The adhesive molecule integrinβ1 expresses a high level in SMMC-7721 cells and shows differences in various cell cycles, suggesting integrin β1 plays an important role in adhesion to endothelial cells. The change of adhesive forces in different cell cycle SMMC-7721 cells indicates that S phase cells play predominant roles possibly while they interact with endothelial cells.     

Characterisation and expression of β1-, β2- and β3-adrenergic receptors in the fathead minnow (Pimephales promelas)

Complimentary DNAs for three beta-adrenergic receptors (βARs) were isolated and characterised in the fathead minnow. The encoded proteins of 402 (β₁AR), 397 (β₂AR) and 434 (β₃AR) amino acids were homologous to other vertebrate βARs, and displayed the characteristic seven transmembrane helices of G Protein-coupled receptors. Motifs and amino acids shown to be important for ligand binding were conserved in the fathead minnow receptors. Quantitative RT-PCR revealed the expression of all receptors to be highest in the heart and lowest in the ovary. However, the β₁AR was the predominant subtype in the heart (70%), and β₃AR the predominant subtype in the ovary (53%). In the brain, β₁AR expression was about 200-fold higher than that of β₂- and β₃AR, whereas in the liver, β₂AR expression was about 20-fold and 100-fold higher than β₃- and β₁AR expression, respectively. Receptor gene expression was modulated by exposure to propranolol (0.001-1 mg/L) for 21 days, but not in a consistent, concentration-related manner. These results show that the fathead minnow has a beta-adrenergic receptor repertoire similar to that of mammals, with the molecular signatures required for ligand binding. An exogenous ligand, the beta-blocker propranolol, is able to alter the expression profile of these receptors, although the functional relevance of such changes remains to be determined. Characterisation of the molecular targets for beta-blockers in fish will aid informed environmental risk assessments of these drugs, which are known to be present in the aquatic environment.     

HIT cells secrete β-cell mitogenic factors

We studied the growth characteristics of the insulin-producing HIT cells. Although successful in many cell lines such as βTC1, growth arrest could not be obtained with HIT cells left for 3 days without serum. Cytofluorometric analysis showed that about 24% of the cells continuously exposed to serum peaked in the S phase. A similar proportion was found for cells cultured for 1 or 2 days in serum-free medium. A treatment with suramin, disrupting the binding of ligands from their receptors, was associated with a rapid and transient increase in c-fos and c-jun gene expression after suramin removal, in the absence of serum. In addition, HIT cells secrete mitogenic factors, different from IGF-I or IGF-II, acting on insulin-secreting βTC1 cells and on BP-A31 fibroblasts. Chromatography of the medium conditioned by the HIT cells on gel filtration gave two major mitogenic fractions, of hydrodynamic characteristics 33 000 and 3000-10 000. The activity was heat stable and bound to heparin. Comparative studies of the self-regulatory HIT cells, with the βTC1 cells requiring external growth factors, should contribute significantly to our understanding of the regulation of β cell growth.     

Longevity of human islet α- and β-cells

Pancreatic islet cell regeneration is considered to be important in the onset and progression of diabetes and as a potential cell therapy. Current hypotheses, largely based on rodent studies, indicate continuous turnover and plasticity of α- and β-cells in adults; cell populations in rodents respond to increased secretory demand in obesity (30-fold β-cell increase) and pregnancy. Turnover and plasticity of islet cells decrease in mice within >1 year. In man, morphometric observations on postmortem pancreas have indicated that the cellular expansion is much smaller than the increased insulin secretion that accompanies obesity. Longevity of β-cells in humans >20-30 years has been shown by (14) C measurements and bromo-deoxyuridine (BrdU) incorporation and there is an age-related decline in the expression of proteins associated with cell division and regeneration including cyclin D3 and PDX-1. Quantitative estimation and mathematical modelling of the longevity marker, cellular lipofuscin body content, in islets of subjects aged 1-84 years indicated an age-related increase and that 97% of the human β-cell population was established by the age of 20. New data show that human α-cell lipofuscin content is less than that seen in β-cells, but the age-related accumulation is similar; lipofuscin-positive (aged) cells form ≥ 95% of the population after 20 years. Increased turnover of cellular organelles such as mitochondria and endoplasmic reticulum could contribute to lipofuscin accumulation with age in long-lived cells. Induction of regeneration of human islet cells will require understanding of the mechanisms associated with age-related senescence.     

Induction of glycogenolysis in cultured Ewing's sarcoma cells by dopamine and β-adrenergic agonists

Summary This study describes hormonal regulation of glycogen metabolism in Ewing's sarcoma cells. ³H-Glycogen synthesized in cultured Ewing's sarcoma WE-68 cells from ³H-glucose was hydrolyzed in a concentration-dependent manner by various catecholamines. The order of potency for the glycogenolytic effects of catecholamines was isoproterenol dopamine > norepinephrine > epinephrine. The concentrations giving half-maximal effectiveness (EC₅₀) were about 2×10^-8 M, 3×10^-8 M, 8×10^-8 M, and 5×10^-7 M for isoproterenol, dopamine, norepinephrine, and epinephrine, respectively. Higher concentrations of each of the catecholamines were necessary to elicit EC₅₀ stimulation of cyclic AMP production in Ewing's sarcoma cells. Glycogenolysis induced by dopamine was blocked by chlorpromazine, a dopamine D₁-receptor antagonist, but not by haloperidol, a dopamine D₂-receptor antagonist. The glycogenolytic action of norepinephrine was markedly reduced by propranolol, a -adrenoreceptor antagonist, and was not affected by yohimbine, an -adrenoreceptor antagonist. In addition, chlorpromazine also antagonized the glycogenolytic response to norepinephrine. Dibutyryl cyclic AMP, 3-isobutyl-1-methylxanthine, and the diterpene forskolin were also found to induce ³H-glycogen hydrolysis. Our data indicate that cate-cholamines exert their glycogenolytic effects in Ewing's sarcoma cells by stimulation of cyclic AMP formation via -adrenergic recepetors and dopamine D₁-receptors.     

CCR5 expression in inflammatory bowel disease and its correlation with inflammatory cells and β-arrestin2 expression

Objective: To elucidate the correlation of expression of CC chemokine receptor 5 (CCR5) with degrees of inflammatory cells infiltration and expression of β-arrestin2 in biopsic intestinal mucosa of the patients with inflammatory bowel disease (IBD).

Methods: Paraffin sections were derived from 53 patients with active IBD, 26 patients with remissive IBD and 30 healthy people. Immunohistochemical envision two-step method was used to test the expression of CCR5 and β-arrestin2 in biopsic intestinal mucosa. HE and toluidine blue staining were used to detect the pathological cytological analysis and classification in lamina propria of colonic mucosa.

Results: The positive rate, strong positive rate and immunohistochemical score of CCR5 expression in active IBD were significantly higher than that in normal controls and remissive IBD (p?<?.05). CCR5 expression had no obvious correlation with clinical severity, lesion distribution and endoscopic classification of active IBD. Neutrophils, eosinophils and lymphocytes in active IBD were significantly higher than that in normal controls and remissive IBD (p?<?.05), while the lymphocyte grade had a positive correlation with CCR5 expression (p?=?.042, r?=?.286). Mastocytes in active IBD, remissive IBD and normal controls had no obvious difference (p?>?.05). β-arrestin2 expression was significantly lower in active IBD than that in remissive IBD and normal controls, and it had a negative correlation with CCR5 expression (p?=?.01, r?=??.247).

Conclusions: CCR5 is highly expressed in active IBD, and it has positive correlation with lymphocyte grade and negative correlation with expression of β-arrestin2.     

The role of α-, δ- and F cells in insulin secretion and action

Nowadays, most of the basic pancreatic islet research is focused on the beta and alpha cells. Nonetheless, the pancreatic islets are complete organs with five, up to now, different cell types and dedicated vasculature and neural supply. Every cell contributes in the integration of the islet function and deserves a closer lookup in the saga of the glucose homeostasis. In this brief report we describe the current concepts about the contribution of the non-beta cells in the overall islet contribution to glucose metabolism.     

Human β-mannosidase deficiency: Biochemical findings in plasma,fibroblasts, white cells and urine

Summary Marked deficiencies of -mannosidase activity were demonstrated in plasma, leukocytes, fibroblasts and urine of a patient with -mannosidosis, similar deficiencies were observed in the proband's sibling. All other lysosomal enzymes measured, including sulphamidase, exhibited normal activity. Both parents showed reduced plasma and leukocyte -mannosidase activity. Urinary glycosaminoglycan excretion was normal but TLC of urinary oligosaccharides revealed an abnormal band with the mobility of a disaccharide. This finding was confirmed by Bio-Gel P2 column chromatography. Further purification of this compound revealed two disaccharides, both of which yielded mannose and glucosamine following acid hydrolysis and mannose andN-acetylglucosamine following enzymic digestion. These two compounds are thought to be structural isomers of the disaccharide Man-GlcNAc.     

Sonic hedgehog maintains survival and growth of chronic myeloid leukemia progenitor cells through β-catenin signaling

Sonic hedgehog (Shh) signaling plays an important role in many human cancers and cancer stem cells. Here we investigate the activity and functional role of Shh signaling in chronic myeloid leukemia (CML) and leukemia progenitor cells. Differential activation of Shh signaling was found in about 50% CML chronic phase samples, about 70% of CML accelerated phase samples, and >80% CML blast crisis phase samples. Deregulated activation of Shh signaling was observed in CD34(+) and c-kit(+) leukemia progenitor cells. Stimulation of Shh signaling with exogenous Shh peptide induced expansion of CD34(+) and c-kit(+) progenitor cells (p < 0.05), inversely, blocking the pathway with signal inhibitor induced cell apoptosis (p < 0.05). Low level of Shh protein was observed in CML bone marrow stromal cells, and CD34(+) progenitor cells are less sensitive to exogenous Shh peptide and more sensitive to cyclopamine than CD34(-) cells (p < 0.05), implying cell-autonomous activation of Shh signaling play a predominant role in progenitor cells. Coactivation of Shh and β-catenin signaling was found in CD34(+) and c-kit(+) progenitor cells. Administration of Shh-neutralizing antibody or Wnt3a-neutralizing antibody in c-kit(+) progenitor cells induced cell apoptosis; however, Wnt3a peptide could salvage cell apoptosis, while Shh peptide failed to revert anti-Wnt3a-induced cell apoptosis. C-MYC, GLI1, BCL-2, and P21 were also found to be downstream targets of Shh signaling, mediating apoptosis or G(2)/M cell cycle arrest of progenitor cells. Our results demonstrate that autoactivated Shh signaling provides survival and proliferative cues in CML progenitor cells through downstream β-catenin signaling, suggesting a novel therapeutic approach in CML.     

Expression of E-selectin, integrin β1 and immunoglobulin superfamily member in human gastric carcinoma cells and its clinicopathologic significance

AIM: To study the expression levels of E- selectin, integrin beta1 and immunoglobulin superfamily member-intercellular adhesion molecule-1 (ICAM-1) in human gastric carcinoma cells, and to explore the relationship between these three kinds of cell adhesion molecules and gastric carcinoma. METHODS: The serum contents of E-selectin, integrin beta1 and ICAM-1 were detected by enzyme-linked immunosorbent assay (ELISA), in 47 healthy individuals (control group) and in 57 patients with gastric carcinoma (gastric carcinoma group) respectively prior to operation and 7 d after operation.RESULTS: The serum E-selectin, ECAM-1 and integrin beta1 were found to be expressed in both control and gastric carcinoma groups. However, they were highly expressed in patients with gastric carcinoma patients before operation or with unresectable tumours. The expression levels of ICAM-1 and integrin beta1 were significantly higher in gastric carcinoma patients than in controls (P < 0.01). A comparison of the E-selectin levels between the two groups showed statistically insignificant difference (P = 0.64). In addition, the expression levels were all decreased substantially in the postoperative patients subjected to radical resection of the tumours, indicating that the high level expressions of these compounds might be the important factor for predicting the prognosis of these patients. CONCLUSION: Serum E-selectin, ICAM-1 and integrin beta1 expression levels are probably related to the metastasis and relapse of gastric cancer.     

Peroxisome-proliferator-activated receptors γ and β/δ mediate vascular endothelial growth factor production in colorectal tumor cells

Background  

Peroxisome-proliferator-activated receptors (PPARs) are nuclear receptors for fatty acids and their derivatives. PPAR subtypes PPARγ and PPARβ/δ are suspected to modulate cancer development in the colon, but their exact role is still discussed controversially.