The effect of transforming growth factor-β on fibroblast cell proliferation in intact connective tissue in vitro

Summary Transforming growth factor-β (TGF-β) has varying effects on cell proliferation, stimulating some cell types while inhibiting others. Its effect on proliferation has mostly been assessed in cell cultures without consideration for the influence of a tissue matrix. In the present investigation we studied the effect of TGF-β on fibroblast cell proliferation in intact connective tissue in vitro using the membranous part of the rat mesentery. Mesenteric membranes were spread over the hole of a cytocentrifuge paper, incubated in vitro, and exposed to various concentrations of TGF-β with or without serum added. At designated times after incubation, the specimens were fixed, spread out on microscope slides, and stained by the Feulgen reaction. Cell proliferation was estimated by counting mitoses in fibroblasts and mesothelial cells and by DNA cytometry of fibroblast nuclei using computer assisted image analyses. Higher concentrations of TGF-β significantly increased proliferation estimated as either the percentage of cells in the S+G₂ phase of the cell cycle or the mitotic index when serum was added. In medium without serum, TGF-β did slightly, but not significantly, increase proliferation. The results show that TGF-β stimulates connective tissue cell proliferation dose-dependently in intact connective tissue in vitro and that addition of serum to the medium is a prerequisite for optimal stimulation.     

Cell density dependent effect of a tumor promoter on proliferation and chondrogenesis of limb bud mesenchymal cells

When limb bud mesenchymal cells are cultured at high density, chondrogenesis takes place in vitro. Treatment of such cultures with the tumor promoter 12-O-tetradecanoyl phorbol 13-acetate (TPA) resulted in complete inhibition of chondrogenesis as indicated from staining the cultures for proteoglycans and from RNA hybridization to cDNA probes specific for four cartilage macromolecules. The effect of TPA varied depending on the initial plating density. At high density, TPA inhibited cell proliferation. At low density, cell proliferation was stimulated by TPA and above a certain cell density, chondrogenesis took place even in the presence of TPA. These results are interpreted to mean that the effect of TPA on chondrogenesis is indirect, possibly through its influence on cell proliferation.     

miR-483 inhibits bovine myoblast cell proliferation and differentiation via IGF1/PI3K/AKT signal pathway

MicroRNAs (miRNAs) have been established to regulate skeletal muscle development in mammals. However, few studies have been conducted on the regulation of proliferation and differentiation of bovine myoblast cells by miRNAs. The aim of our study was to explore the function of miR-483 in cell proliferation and differentiation of bovine myoblast. Here, we found that miR-483 declined in both proliferation and differentiation stages of bovine myoblast cells. During the proliferation phase, the overexpression of miR-483 downregulated the cell cycle–associated genes cyclin-dependent kinase 2 (CDK2), proliferating cell nuclear antigen (PCNA) messenger RNA (mRNA), and the protein levels. At the cellular level, cell cycle, cell counting kit-8, and 5-ethynyl-2´-deoxyuridine results indicated that the overexpression of miR-483 block cell proliferation. During differentiation, the overexpression of miR-483 led to a decrease in the levels of the myogenic marker genes MyoD1 and MyoG mRNA and protein. Furthermore, the immunofluorescence analysis results showed that the number of MyHC-positive myotubes was reduced. In contrast, the opposite experimental results were obtained concerning both proliferation and differentiation after the inhibition of miR-483. Mechanistically, we demonstrated that miR-483 target insulin-like growth factor 1 (IGF1) and downregulated the expression of key proteins in the PI3K/AKT signaling pathway. Altogether, our findings indicate that miR-483 acts as a negative regulator of bovine myoblast cell proliferation and differentiation.     

Effects of 50 Hz pulsed electromagnetic fields on the growth and cell cycle arrest of mesenchymal stem cells: an in vitro study

Mesenchymal stem cells (MSCs) are capable of self-renew and multipotent differatiation which allows them to be sensitive to microenvironment is altered. Pulsed electromagnetic fields (PEMF) can affect cellular physiology of some types of cells. This study was undertaken to investigate the effects of PEMF on the growth and cell cycle arrest of MSCs expanded in vitro. To achieve this, cultured of normal rat MSCs, the treatment groups were respectively irradiated by 50 Hz PEMF at 10 mT of flux densities for 3 or 6 h. The effects of PEMF on cell proliferation, cell cycle arrest, and cell surface antigen phenotype were investigated. Our results showed that exposed MSCs had a significant proliferative capacity (P < 0.05) but the effect of PEMF for 3 and 6 h on cell growth was not different (P>0.05) at an earlier phase after PEMF treatment. Exposure to PEMF had a significant increase the percentage of MSCs in G1 phase compare with the control group, with a higher percentage of cells in G1 phase exposed for 6 h then that for 3 h. At the 16th hour after treatment, PEMF had no significant effect on cell proliferation and cell cycle (P>0.05). These results suggested that PEMF enhanced MSCs proliferation with time-independent and increased the percentage of cells at the G1 phase of the cell cycle in a time-dependent manner, and the effect of PEMF on the cell proliferation and cell cycle arrest of MSCs was temporal after PEMF treatment.     

INSULIN PRODUCES A BIPHASIC RESPONSE INTETRAHYMENA THERMOPHILABY STIMULATING CELL SURVIVAL AND ACTIVATING PROLIFERATION IN TWO SEPARATE CONCENTRATION INTERVALS

Cells ofTetrahymenamay produce autocrine signal molecules with effects on survival and proliferation. Here we have tested the effects of human recombinant and bovine insulin, and the B22–B30 fragment of bovine insulin over a wide range of concentrations (10^?5–10^?18m ) on cell survival and proliferation in a synthetic nutrient medium. The cells were grown in conical flasks at low initial cell densities (40 and 400cells/ml). Insulin prevented rapid cell death and/or promoted cell proliferation over two separate concentration ranges: down to nanomolar levels and again in the low pico- and femtomolar range. At an initial population density of 400cells/ml the cells multiplied at both concentration intervals. At 40 or fewer organisms/ml the cells multiplied in the high concentration interval, whereas in the low interval they survived for about four times longer than those in the control cultures. B22–B30 added to cultures of 40 initial cells/ml produced a stimulation of cell survival in the low pico- and high femtomolar range. In the presence of hemin (50nm ) cells at 400 initial organisms/ml multiplied at insulin concentrations down to about 3nm and again from 300am to 10pm . In some cases, hemin plus insulin activated cell proliferation between the two concentration intervals as well. At 40cells/ml the cells not only survived but proliferated in the femtomolar range. Cells in cultures supplemented with both hemin and B22–B30 multiplied at the low concentration interval (from about 100fm to 10pm ).     

Multifunctional Manganese Ions Trapping and Hydrofluoric Acid Scavenging Separator for Lithium Ion Batteries Based on Poly(ethylene‐alternate‐maleic acid) Dilithium Salt

Manganese dissolution from positive electrodes seriously reduces the life of Li‐ion batteries, due to its detrimental impact on the passivation of negative electrodes. A novel multifunctional separator incorporating inexpensive mass‐produced polymeric materials may dramatically increases the durability of Li‐ion batteries. The separator is made by embedding the poly(ethylene‐alternate‐maleic acid) dilithium salt polymer into a poly(vinylidene fluoride‐hexafluoropropylene) copolymer matrix. LiMn₂O₄‐graphite cells comprising a 1 m LiPF₆ solution in ethylene carbonate plus dimethyl carbonate (1:1 v/v) and the functional separator retain 31% and 100% more capacity than baseline cells with plain commercial separators after 100 cycles at C/5 rate, respectively, at 30 and 55 °C. Analyses of cycled cells indicate greatly reduced Mn contamination of the graphite negative electrodes and almost no irreversible structural change in the LiMn₂O₄ positive electrodes from cells containing the functional separator. The Mn amount in the graphite electrodes from cycled cells with functional separators is ≈80% lower than in the graphite electrodes from cycled baseline cells. Mn ions are found in the functional separators but not in baseline (plain) separators from cycled cells. Finally, it is shown that the reported performance improvements stem from the ability of the novel separator to chelate Mn ions and to scavenge trace HF.     

Structural–Functional Units of Epidermis

The available data suggest that epidermis is organized as a system of discrete structural–functional units (SFUs) that reproduce both in vivo and in vitro. SFUs are formed in the culture of epidermal keratinocytes via self-organization of the developing cellular elements. SFUs are capable of self-maintenance and form a niche for stem cells. At the same time, due to the maintenance of asymmetric proliferation kinetics of the stem cells, SFUs serve as a barrier to their uncontrolled replication.     

Anti‐Androgenic Activity of Fatty Acids

In this study, we show that 5α‐reductase derived from rat fresh liver was inhibited by certain aliphatic free fatty acids. The influences of chain length, unsaturation, oxidation, and esterification on the potency to inhibit 5α‐reductase activity were studied. Among the fatty acids we tested, inhibitory saturated fatty acids had C₁₂–C₁₆ chains, and the presence of a C?C bond enhanced the inhibitory activity. Esterification and hydroxy compounds were totally inactive. Finally, we tested the prostate cancer cell proliferation effect of free fatty acids. In keeping with the results of the 5α‐reductase assay, saturated fatty acids with a C₁₂ chain (lauric acid) and unsaturated fatty acids (oleic acid and α‐linolenic acid) showed a proliferation inhibitory effect on lymph‐node carcinoma of the prostate (LNCaP) cells. At the same time, the testosterone‐induced prostate‐specific antigen (PSA) mRNA expression was down‐regulated. These results suggested that fatty acids with 5α‐reductase inhibitory activity block the conversion of testosterone to 5α‐dihydrotestosterone (DHT) and then inhibit the proliferation of prostate cancer cells.     

Effects of phenytoin and Echinacea purpurea extract on proliferation and apoptosis of mouse embryonic palatal mesenchymal cells

Cleft palate is one of the most common birth defects. Several environment factors are involved in the disorder, such as smoking, vitamin deficiency and teratogens. We investigated the teratogenic agent phenytoin and extract of the immunostimulant Echinacea purpurea in the etiology of cleft palate associated with the proliferation and apoptosis of mouse embryonic palatal mesenchymal (MEPM) cells. We measured the effects of phenytoin, E. purpurea extract, and the mixture of phenytoin and E. purpurea extract on the cell viability of MEPM cells by CCK‐8 assay and on the proliferation and apoptosis of MEPM cells by BrdU labeling assay, flow cytometry, and TUNEL assay. Exposure to phenytoin for 24 h inhibited cell proliferation and increased cell apoptosis of MEPM cells, and E. purpurea extract had the reverse effect. Importantly, treatment with the mixture of phenytoin and E. purpurea extract increased the proliferation and decreased the apoptosis of MEPM cells as compared with treatment with phenytoin alone. The teratogenic effect of phenytoin on cleft palate is associated with the proliferation and apoptosis of MEPM cells, and E. purpurea extract may have a protective effect. J. Cell. Biochem. 112: 1311–1317, 2011. © 2011 Wiley‐Liss, Inc.     

Tanshinone I attenuates proliferation and chemoresistance of cervical cancer in a KRAS‐dependent manner

Chemoresistance is a common occurrence during advanced or recurrent cervical cancer therapy when treated by conventional treatment, platinum‐based chemotherapy. This study aimed to investigate the effect and underlying mechanism of tanshinone I on attenuating proliferation and chemoresistance of cervical cancer cells. In cervical cancer cells, cell proliferation was examined by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT), cell count, and soft‐agar colony‐formation assay. rVista analysis and luciferase reporter assay were used to explore the upstream regulator of KRAS, and the expression levels of key genes were also detected. Western blot analysis showed that tanshinone I significantly suppressed KRAS expression and inhibited AKT phosphorylation. rVista analysis and luciferase reporter assay demonstrated that ELK1 can binds directly to KRAS promoter and positively regulates KRAS expression. MTT assay showed that KRAS or ELK1 overexpression significantly attenuated the suppressive effects of tanshinone I on HeLa cells proliferation. In addition, tanshinone I recovered the cisplatin sensitivity of HeLa CR cells, whereas KRAS or ELK1 overexpression significantly inhibited this phenomenon. Our results suggested that tanshinone I had anticancer effects on cervical cancer cells via inhibiting ELK1 and downregulating KRAS‐AKT axis, which subsequently suppressed the proliferation and cisplatin resistance of cervical cancer cells.     

Knockdown Clock gene suppressing proliferation of 4T1 cells by inhibiting β-catenin expression

Circadian locomotor output cycles kaput (Clock) gene is a core gene in the circadian rhythm system that is involved in cancer cell proliferation. However, the molecular mechanism of Clock gene participate in the cancer cell proliferation is unclear. Previous studies demonstrated that cell proliferation could be regulated by the canonical Wnt pathway (also known as the Wnt/β-catenin pathway), and the Wnt/β-catenin pathway had a relation with the circadian system. To investigate whether the Clock gene affects the proliferation of breast cancer cell by regulating the expression of β-catenin, we knocked down the Clock expression of mouse breast cancer cells (4T1) by RNA interference. Then detected their proliferation rates using CCK8 assay and the expression of the β-catenin gene by real-time PCR and Western blot. The results showed that the proliferation of the Clock knocked down 4T1 cells is slower than the control. The expression level of β-catenin of these 4T1 cells is reduced. Our study showed that Clock gene knocked down inhibiting the proliferation of the 4T1 cells, probably by suppressing the expression of β-catenin.     

The deubiquitinase OTUB1 fosters papillary thyroid carcinoma growth through EYA1 stabilization

Deubiquitinating enzyme OTU domain-containing ubiquitin aldehyde-binding proteins 1 (OTUB1) has been shown to have an essential role in multiple carcinomas. However, the function of OTUB1 in papillary thyroid cancer (PTC) and the underlying mechanisms regulating PTC cells proliferation remain poorly understood. In this study, OTUB1 was significantly upregulated in papillary thyroid carcinoma tissues and cells. Through in vitro and in vivo experiments, knockdown of OTUB1 suppressed PTC cells growth whereas OTUB1 overexpression enhanced the proliferation ability of PTC cells. Moreover, the eyes absent homologue 1 (EYA1) was recognized as a potential target of OTUB1 through mass spectrometry analysis, and we further verified that EYA1 protein level was positively correlated with OTUB1 expression in PTC cells and clinical samples. Mechanistically, OTUB1 could interact with EYA1 directly and deubiquitinate EYA1 to stabilize it. At last, EYA1 was found to play an essential role in OTUB1-derived PTC cells growth. Overall, our investigation reveals that OTUB1 is a previously unrecognized oncogenic factor in PTC cells proliferation and suggests that OTUB1 might be a novel therapeutic target in PTC.     

Attempts at immortalization of crustacean primary cell cultures using human cancer genes

Primary cell cultures from crustacea have been initiated since the 1960s, yet no permanent cell line is available. Primary cells have a limited proliferative capacity in culture due to cellular senescence, which is regulated by a group of dominant senescence genes. The aim of this research was to manipulate cell cycle regulation by transfecting Cherax quadricarinatus primary cells with oncogenes, in an effort to induce a permanent cell line. Human papillomaviruses (HPV) play a critical role in the formation of anogenital cancer. Research has demonstrated that the HPV-expressed E6 and E7 proteins function concomitantly to disrupt the p53 and retinoblastoma (Rb) tumor suppressor genes, regulators of the cell-cycle checkpoints at the first gap (G₁) phase. HPV E6 and E7 genes were transfected into the C. quadricarinatus cells by lipofection. Successful transfection was demonstrated by the presence of oncogene messenger RNA by reverse transciptase polymerase chain reaction. At day 150, transfected cells still remain viable, although cell proliferation was stagnant. It may be that while transfection of the oncogenes was successful, no proliferation of the C. quadricarinatus cells was evident due to a lack of telomere maintenance.     

Promoter methylation status of VEGF receptor genes: A possible epigenetic biomarker to anticipate the efficacy of intracellular-acting VEGF-targeted drugs in cancer cells

We evaluated whether the inhibitory effects of vascular endothelial growth factor (VEGF)-targeted drugs on the proliferation of cancer cells differed according to VEGF receptor (VEGFR) genes, Flt1 and KDR, promoter methylation status. Five hyper-VEGFR-methylation and six no-VEGFR-methylation cancer cells were used for the present study, together with human umbilical endothelial cells (HUVECs) as a control. No-VEGFR-methylation cancer cells showed higher expression of Flt1 and KDR than hyper-VEGFR-methylation cancer cells. Hyper-VEGFR-methylation cancer cells only showed increased expression and protein levels of Flt1 and KDR after treatment with the demethylase 5-aza-2’-deoxycytidine. Two drugs (a VEGF-specific-antibody, bevacizumab, and a KDR-specific-antibody) targeting extracellular VEGF-VEGFR signaling and two VEGF-specific-tyrosine kinase inhibitors (PTK/ZK and sunitinib) targeting intracellular VEGFR signaling were used in the cell proliferation assay. HUVECs showed dose- and time-dependent proliferation decrease with all tested drugs over a 72 h incubation period. No- or hyper-VEGFR-methylation cancer cells showed no significant proliferation differences after treatment with VEGF-specific-antibody or VEGFR2-specific-antibody. After PTK/ZK or sunitinib treatment, no-VEGFR-methylation cancer cells showed dose- or time-dependent decreases in proliferation. Hyper-VEGFR-methylation cancer cells also showed proliferation inhibition by VEGF-specific-tyrosine kinase inhibitors after demethylation of Flt1 and KDR. Proliferation inhibition synergistically increased after combination of demethylation with PTK/ZK in hyper-VEGF-methylation cancer cells. We observed that intracellular targeting of VEGF-VEGFR signaling could be more effective than extracellular targeting of the pathway in the suppression of proliferation of some cancer cells. In particular, the efficacy of intracellular targeting of VEGF-specific-tyrosine kinase inhibitors might be influenced by the epigenetic alteration of VEGFRs.     

Identifying the Impact of Surface Recombination at Electrodes in Organic Solar Cells by Means of Electroluminescence and Modeling

This work reports on combining current‐voltage characteristics, electroluminescence (EL) measurements, and modeling to identify the selectivity of the electrodes in bulk‐heterojunction organic solar cells. Devices with the same photoactive layer but different contact materials are compared and the impact of surface recombination at the contacts on their performance is determined. The open‐circuit voltage, V _OC, depends strongly on the selectivity of the electrodes and it is observed that the EL signal of cells with lower V _OC is dramatically reduced. This is ascribed to an enhanced rate of surface recombination, which is a non‐radiative recombination pathway and does therefore not contribute to the EL yield. In addition, these cells have a lower current in forward direction despite the fact that the surface recombination occurs in addition to the recombination in the bulk. A theoretical model was set up and in the corresponding numerical simulations all three findings (lower V _OC, strongly reduced EL signal and lower forward current) could be clearly reproduced by varying just one single parameter which determines the selectivity of the electrode.     

The Arabidopsis thaliana cDNAs coding for eIF4E and eIF(iso)4E are not functionally equivalent for yeast complementation and are differentially expressed during plant development

Two cDNAs (At.EIF4E1 and At.EIF4E2) encoding, respectively, the eukaryotic initiation factors eIF4E and eIF(iso)4E of Arabidopsis thaliana were isolated by complementation of a Saccharomyces cerevisiae conditional mutant. The deduced amino acid sequences of the proteins are homologous to those from monocotyledonous plants, yeast and mammals. The corresponding genes were identified in YAC clones mapping to chromosome IV (At.EIF4E1) and to chromosome V (At.EIF4E2). The yeast strain complemented by At.EIF4E2 grew poorly compared with an isogenic strain expressing At.EIF4E1. Northern and in situ hybridization analysis show that both Arabidopsis At.EIF4E1 and At.EIF4E2 mRNAs are differentially accumulated in plant tissues. The At.EIF4E1 mRNA is expressed in all tissues except in the cells of the specialization zone of the roots; the At.EIF4E2 mRNA is particularly abundant in floral organs and in young developing tissues. This work further demonstrates an association between a high level of EIF4E mRNAs and cell proliferation and suggests that the plant eIF4E isoforms may have distinct functions in cell development and metabolism.     

GM-CSF accelerates proliferation of endothelial progenitor cells from murine bone marrow mononuclear cells in vitro

Objective: To test whether the GM-CSF accelerates the proliferation of bone marrow endothelial progenitor cells (BM EPCs). Methods: BM EPCs were induced by endothelial cell conditioned medium (EC-CM). The effect of different concentrations of GM-CSF on the proliferation of BM EPCs was evaluated by the formation of EC-cols, MTT assay, and cell cycle assay. The single progenitor cell growth curves were quantified. Results: The data indicated that GM-CSF accelerated the proliferation of BM EPCs both in colony numbers and colony size. MTT confirmed the effect of GM-CSF on accelerating the proliferation of BM EPCs. The single colony experiments showed that EC-cols expressed different proliferation capacity, suggesting that the EC-cols with different proliferation potentials might have been derived from different levels of immature progenitors. The cell cycle assay showed that the rate of cells entering into S phase was 9.3% in the group treated with GM-CSF and 2.1% in the controls. Furthermore, these cells displayed the specific endothelial cell markers and formed capillary-like structures. Conclusions: GM-CSF accelerates proliferation of BM EPCs. The potential beneficial of GM-CSF in the application of treating vascular ischemic patients is promising.