Neoplastic development: paradoxical relation between impaired cell growth at low population density and excessive growth at high density.

The role of heritable, population-wide cell damage in neoplastic development was studied in the 28 L subline of NIH 3T3 cells. These cells differ from the 17(3c) subline used previously for such studies in their lower frequency of "spontaneous" transformation at high population density and their greater capacity to produce large, dense transformed foci. Three cultures of the 28 L subline of NIH 3T3 cells were held under the constraint of confluence for 5 wk (5 wk 1 degree assay) and then assayed twice in succession (2 degrees and 3 degrees assays) for transformed foci and saturation density. After the 2 degrees assay, the cells were also passaged at low density to determine their exponential growth rates and cloned to determine the size and morphological features of the colonies. Concurrent measurements were made in each case with control cells that had been kept only in frequent low-density passages and cells that had been kept at confluence for only 2 wk (2 wk 1 degree). Two of the three cultures transferred from the 2 degrees assay of the 5 wk 1 degree cultures produced light transformed foci, and the third produced dense foci. The light focus-forming cultures grew to twice the control saturation density in their 2 degrees assay and 6-8 times the control density in the 3 degrees assay; saturation densities for the dense focus formers were about 10 times the control values in both assays. All three of the cultures transferred from the 2 degrees assay of the 5 wk 1 degree cultures multiplied at lower rates than controls at low densities, but the dense focus formers multiplied faster than the light focus formers. The reduced rates of multiplication of the light focus formers persisted for > 50 generations of exponential multiplication at low densities. Isolated colonies formed from single cells of the light focus formers were of a lower population density than controls; colonies formed by the dense focus formers were slightly denser than the controls but occupied only half the area. A much higher proportion of the colonies from the 5 wk 1 degree cultures than the controls consisted of giant cells or mixtures of giant and normal-appearing cells. The results reinforce the previous conclusion that the early increases in saturation density and light focus formation are associated with, and perhaps caused by, heritable, population-wide damage to cells that is essentially epigenetic in nature. The more advanced transformation characterized by large increases in saturation density and dense focus formation could have originated from rare genetic changes, such as chromosome rearrangements, known to occur at an elevated frequency in cells destabilized by antecedent cellular damage.

Role of heme regulated eIF2α kinase in pancreatic beta cell function and viability

The eukaryotic translation initiation factor 2 alpha (eIF2α) is part of the initiation complex that drives the initiator amino acid methionine to the ribosome, a crucial step in protein translation. In stress conditions such as virus infection, endoplasmic reticulum (ER) stress, amino acid or heme deficiency eIF2α can be phosphorylated and thereby inhibit global protein synthesis. This adaptive mechanism prevents protein accumulation and consequent cytotoxic effects. Heme-regulated eIF2α kinase (HRI) is a member of the eIF2α kinase family that regulates protein translation in heme deficiency conditions. Although present in all tissues, HRI is predominantly expressed in erythroid cells where it remains inactive in the presence of normal heme concentrations. In response to heme deficiency, HRI is activated and phosphorylates eIF2α decreasing globin synthesis. This mechanism is important to prevent accumulation of heme-free globin chains which cause ER stress and apoptosis. RNA sequencing data from our group showed that in human islets and in primary rat beta cells HRI is the most expressed eIF2α kinase compared to the other family members. Despite its high expression levels, little is known about HRI function in beta cells. The aim of this project is to identify the role of HRI in pancreatic beta cells. This was investigated taking a loss-of-function approach. HRI knock down (KD) by RNA interference induced beta cell apoptosis in basal condition. HRI KD potentiated the apoptotic effects of palmitate or proinflammatory cytokines, two in vitro models for type 2 and type 1 diabetes, respectively. Increased cytokine-induced apoptosis was also observed in HRI-deficient primary rat beta cells. Unexpectedly, we observed a mild increase in eIF2α phosphorylation in HRI-deficient cells. The levels of mRNA or protein expression of C/EBP homologous protein (CHOP) and activating transcription factor 4 (ATF4) were not modified. HRI KD cells have decreased spliced X-box binding protein 1 (XBP1s), an important branch of the ER stress response. However, overexpression of XBP1s by adenovirus in HRI KD cells did not protect from HRI siRNA-induced apoptosis. HRI deficiency decreased phosphorylation of Akt and its downstream targets glycogen synthase kinase 3 (GSK3), forkhead box protein O1 (FOXO1) and Bcl-2-associated death promoter (BAD). Overexpression of a constitutively active form of Akt by adenovirus in HRI-deficient beta cells partially decreased HRI KD-mediated apoptosis. Interestingly, BAD silencing protected from apoptosis caused by HRI deficiency. HRI silencing in beta cells also induced JNK activation. These results suggest an important role of HRI in beta cell survival through modulation of the Akt/BAD pathway. Thus, HRI may be an interesting target to modulate beta cell fate in diabetic conditions.

Modelling oxygen diffusion and cell growth in a porous, vascularising scaffold for soft tissue engineering applications

Soft tissue engineering presents significant challenges compared to other tissue engineering disciplines such as bone, cartilage or skin engineering. The very high cell density in most soft tissues, often combined with large implant dimensions, means that the supply of oxygen is a critical factor in the success or failure of a soft tissue scaffold. A model is presented for oxygen diffusion in a 15-60 mm diameter dome-shaped scaffold fed by a blood vessel loop at its base. This model incorporates simple models for vascular growth, cell migration and the effect of cell density on the effective oxygen diffusivity. The model shows that the dynamic, homogeneous cell seeding method often employed in small-scale applications is not applicable in the case of larger scale scaffolds such as these. Instead, we propose the implantation of a small biopsy of tissue close to a blood supply within the scaffold as a technique more likely to be successful. Crown Copyright (c) 2005 Published by Elsevier Ltd. All rights reserved.

Pioglitazone inhibits cell growth and reduces matrix production in human kidney fibroblasts

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonists are increasingly used in patients with diabetes, and small studies have suggested a beneficial effect on renal function, but their effects on. extracellular matrix (ECM) turnover are unknown. The aims of this study were to investigate the effects of the PPAR-gamma agonist pioglitazone on growth and matrix production in human cortical fibroblasts (CF). Cell growth and ECM production and turnover were measured in human CF in the presence and absence of 1 and 3 muM pioglitazone. Exposure of CF to pioglitazone caused an antiproliferative (P < 0.0001) and hypertrophic (P < 0.0001) effect; reduced type IV collagen secretion (P < 0.01), fibronectin secretion (P < 0.0001), and proline incorporation (P < 0.0001); decreased MMP-9 activity (P < 0.05); and reduced tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2 secretion (P < 0.001 and P < 0.0001, respectively). These effects were independent of TGF-beta1. A reduction in ECM production was similarly observed when CF were exposed to a selective PPAR-gamma agonist (L-805645) in concentrations that caused no toxicity, confirming the antifibrotic effects of pioglitazone were mediated through a PPAR-gamma-dependent mechanism. Exposure of CF to high glucose conditions induced an increase in the expression of collagen IV (P < 0.05), which was reversed both in the presence of pioglitazone (1 and 3 muM) and by L-805645. In summary, exposure of human CIF to pioglitazone causes an antiproliferative effect and reduces ECM production through mechanisms that include reducing TIMP activity, independent of TGF-beta1. These studies suggest that the PPAR-gamma agonists may have a specific role in ameliorating the course of progressive tubulointerstitial fibrosis under both normoglycemic and hyperglycemic states.

Dietary supplementation of vitamin E and -lipoic acid upregulates cell growth and signaling genes in rat myocardium

The efficacy of antioxidant supplementation in the prevention of cardiovascular disease appears equivocal, however the use of more potent antioxidant combinations than those traditionally used may exert a more positive effect. We have shown previously that supplementation of vitamin E and α-lipoic acid increases cardiac performance during post-ischemia reperfusion in older rats and increases Bcl-2 levels in endothelial cells. The purpose of this study was to examine the effects of vitamin E and α-lipoic acid supplementation on myocardial gene expression with a view to determine their mechanism of action. Young male rats received either a control (n=7) or vitamin E and α-lipoic acid supplemented diet (n=8) for 14 weeks. RNA from myocardial tissue was then amplified and samples were pooled within groups and competitively hybridized to 5K oligonucleotide rat microarrays. The relative expression of each gene was then compared to the control sample. Animals that received the antioxidant-supplemented diet exhibited upregulation (>1.5×) of 13 genes in the myocardium with 2 genes downregulated.� �Upregulated genes include those involved in cell growth and maintenance (LynB, Csf1r, Akt2, Tp53), cell signaling (LynB, Csf1r) and signal transduction (Pacsin2, Csf1r). Downregulated genes encode thyroid (Thrsp) and F-actin binding proteins (Nexilin).

The influence of serum, glucose and oxygen on intervertebral disc cell growth in vitro:implications for degenerative disc disease

The avascular nature of the human intervertebral disc (IVD) is thought to play a major role in disc pathophysiology by limiting nutrient supply to resident IVD cells. In the human IVD, the central IVD cells at maturity are normally chondrocytic in phenotype. However, abnormal cell phenotypes have been associated with degenerative disc diseases, including cell proliferation and cluster formation, cell death, stellate morphologies, and cell senescence. Therefore, we have examined the relative influence of possible blood-borne factors on the growth characteristics of IVD cells in vitro.

Regulation of beta-cell viability and gene expression by distinct agonist fragments of adiponectin

Obesity is an established risk factor for type 2 diabetes. Activation of the adiponectin receptors has a clear role in improving insulin resistance although conflicting evidence exists for its effects on pancreatic beta-cells. Previous reports have identified both adiponectin receptors (ADR-1 and ADR-2) in the beta-cell. Recent evidence has suggested that two distinct regions of the adiponectin molecule, the globular domain and a small N-terminal region, have agonist properties. This study investigates the effects of two agonist regions of adiponectin on insulin secretion, gene expression, cell viability and cell signalling in the rat beta-cell line BRIN-BD11, as well as investigating the expression levels of adiponectin receptors (ADRs) in these cells. Cells were treated with globular adiponectin and adiponectin (15-36) +/-leptin to investigate cell viability, expression of key beta-cell genes and ERK1/2 activation. Both globular adiponectin and adiponectin (15-36) caused significant ERK1/2 dependent increases in cell viability. Leptin co-incubation attenuated adiponectin (15-36) but not globular adiponectin induced cell viability. Globular adiponectin, but not adiponectin (15-36), caused a significant 450% increase in PDX-1 expression and a 45% decrease in LPL expression. ADR-1 was expressed at a higher level than ADR-2, and ADR mRNA levels were differentially regulated by non-esterified fatty acids and peroxisome-proliferator-activated receptor agonists. These data provide evidence of roles for two distinct adiponectin agonist domains in the beta-cell and confirm the potentially important role of adiponectin receptor agonism in maintaining beta-cell mass.

The role of adipokines in beta-cell failure of type 2 diabetes

Beta-cell failure coupled with insulin resistance is a key factor in the development of type 2 diabetes. Changes in circulating levels of adipokines, factors released from adipose tissue, form a significant link between excessive adiposity in obesity and both aforementioned factors. In this review we consider the published evidence for the role of individual adipokines on the function, proliferation, death and failure of beta-cells, focusing on those reported to have the most significant effects (leptin, adiponectin, TNFa, resistin, visfatin, DPP-IV and apelin). It is apparent that some adipokines have beneficial effects whereas others have detrimental properties; the overall contribution to beta-cell failure of changed concentrations of adipokines in the blood of obese pre-diabetic subjects will be highly dependent on the balance between these effects and the interactions between the adipokines which act on the beta-cell via a number of intersecting intracellular signalling pathways. We emphasise the importance, and comparative dearth, of studies into the combined effects of adipokines on beta-cells.

Effects of metformin on BRIN-BD11 beta-cell insulin secretory desensitization induced by prolonged exposure to sulphonylureas

Aims: Prolonged exposure of pancreatic beta-cells in vitro to the sulphonylureas tolbutamide and glibenclamide induces subsequent desensitization of insulinotropic pathways. Clinically, the insulin-sensitizing biguanide drug metformin is often administered alongside sulphonylurea as antidiabetic therapy. The present study examines the functional effects of metformin (200 µM) on tolbutamide- and glibenclamide-induced desensitisation. Methods: Acute and prolonged (18 h) effects of exposure to tolbutamide and glibenclamide alone, or in the presence of metformin, were examined in insulin-secreting BRIN-BD11 cells. Results: In acute 20 min incubations at 1.1 mM glucose, metformin increased (1.2-1.7-fold; p <0.001) the insulin-releasing actions of tolbutamide and glibenclamide. At 16.7 mM glucose, metformin significantly enhanced glibenclamide-induced insulin release at all concentrations (50-400 µM) examined, but tolbutamide-stimulated insulin secretion was only augmented at higher concentrations (300-400 µM). Exposure for 18 h to 100 µM tolbutamide or glibenclamide significantly impaired insulin release in response to glucose and a broad range of insulin secretagogues. Concomitant culture with metformin (200 µM) prevented or partially reversed many of the adverse effects on K channel dependent and independent insulinotropic pathways. Beneficial effects of metformin were also observed in cells exposed to glibenclamide for 18 h with significant improvements in the insulin secretory responsiveness to alanine, GLP-1 and sulphonylureas. The decrease of viable cell numbers observed with glibenclamide was reversed by co-culture with metformin, but cellular insulin content was depressed. Conclusions: The results suggest that metformin can prevent the aspects of sulphonylurea-induced beta-cell desensitization. © 2010 Blackwell Publishing Ltd.

Effect of catechol derivatives on cell growth and lipoxygenase activity

Derivatives of salicylic acid have been synthesized as potential lipoxygenase inhibitors. Agents containing a phenolic dihydroxy moiety showed potent (IC 5010 -6-10 -7 M) inhibition of the growth of murine colonic tumour cells in vitro, and were effective inhibitors of 5-, 12- and 15-lipoxygenase in intact cells. The catechols were also potent inhibitors of rabbit reticulocyte 15-lipoxygenase (IC 50 ∼1 μM). © 2003 Elsevier Ltd. All rights reserved.

Topographical guidance of intervertebral disc cell growth in vitro:towards the development of tissue repair strategies for the anulus fibrosus

The anulus fibrosus (AF) of the intervertebral disc consists of concentric sheets of collagenous matrix that is synthesised during embryogenesis by aligned disc cells. This highly organised structure may be severely disrupted during disc degeneration and/or herniation. Cell scaffolds that incorporate topographical cues as contact guidance have been used successfully to promote the healing of injured tendons. Therefore, we have investigated the effects of topography on disc cell growth. We show that disc cells from the AF and nucleus pulposus (NP) behaved differently in monolayer culture on micro-grooved membranes of polycaprolactone (PCL). Both cell types aligned to and migrated along the membrane's micro-grooves and ridges, but AF cells were smaller (or less spread), more bipolar and better aligned to the micro-grooves than NP cells. In addition, AF cells were markedly more immunopositive for type I collagen, but less immunopositive for chondroitin-6-sulphated proteoglycans than NP cells. There was no evidence of extracellular matrix (ECM) deposition. Disc cells cultured on non-grooved PCL did not show any preferential alignment at sub-confluence and did not differ in their pattern of immunopositivity to those on grooved PCL. We conclude that substratum topography is effective in aligning disc cell growth and may be useful in tissue engineering for the AF. However, there is a need to optimise cell sources and/or environmental conditions (e.g. mechanical influences) to promote the synthesis of an aligned ECM.

The association of depression and perceived stress with beta cell function between African and Haitian Americans with and without type 2 diabetes

Background: Diabetes and diabetes-related complications are major causes of morbidity and mortality in the United States. Depressive symptoms and perceived stress have been identified as possible risk factors for beta cell dysfunction and diabetes. The purpose of this study was to assess associations between depression symptoms and perceived stress with beta cell function between African and Haitian Americans with and without type 2 diabetes. Participants and Methods: Informed consent and data were available for 462 participants (231 African Americans and 231 Haitian Americans) for this cross-sectional study. A demographic questionnaire developed by the Primary Investigator was used to collect information regarding age, gender, smoking, and ethnicity. Diabetes status was determined by self-report and confirmed by fasting blood glucose. Anthropometrics (weight, and height and waist circumference) and vital signs (blood pressure) were taken. Blood samples were drawn after 8 10 hours over-night fasting to measure lipid panel, fasting plasma glucose and serum insulin concentrations. The homeostatic model assessment, version 2 (HOMA2) computer model was used to calculate beta cell function. Depression was assessed using the Beck Depression Inventory-II (BDI-II) and stress levels were assessed using the Perceived Stress Scale (PSS). Results: Moderate to severe depressive symptoms were more likely for persons with diabetes (p = 0.030). There were no differences in perceived stress between ethnicity and diabetes status (p = 0.283). General linear models for participants with and without type 2 diabetes using beta cell function as the dependent variable showed no association with depressive symptoms and perceived stress; however, Haitian Americans had significantly lower beta cell function than African Americans both with and without diabetes and adjusting for age, gender, waist circumference and smoking. Further research is needed to compare these risk factors in other race/ethnic groups.