Significant Stimulation Of Cell Proliferation Research Articles

Biological interactions of a calcium silicate based cement (Biodentine™) with Stem Cells from Human Exfoliated Deciduous teeth

ObjectiveTo investigate the biological interactions of a calcium silicate based cement (Biodentine™) with Stem Cells from Human Exfoliated Deciduous teeth (SHED), focusing on viability/proliferation, odontogenic differentiation, biomineralization and elemental release/exchange. MethodsBiodentine™ specimens were used directly or for eluate preparation at serial dilutions (1:1–1:64). SHED cultures were established from deciduous teeth of healthy children. Viability/proliferation and morphological characteristics were evaluated by live/dead fluorescent staining, MTT assay and Scanning Electron Microscopy. Odontogenic differentiation by qRT-PCR, biomineralization by Alizarin red S staining, while ion elution by Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES). ResultsSHED effectively attached within the crystalline surface of Biodentine™ specimens acquiring a spindle-shaped phenotype. Statistically significant stimulation of cell proliferation was induced at day 3 by eluates in dilutions from 1:16 to 1:64. Differential, concentration- and time-dependent expression patterns of odontogenic genes were observed under non-inductive and inductive (osteogenic) conditions, with significant up-regulation of DSPP and Runx2 at higher dilutions and a peak in expression of BMP-2, BGLAP and MSX-2 at 1:8 dilution on day 7. Progressive increase in mineralized tissue formation was observed with increasing dilutions of Biodentine™ eluates. ICP-OES indicated that Biodentine™ absorbed Ca, Mg and P ions from culture medium, while releasing Si and Sr ions from its backbone. SignificanceBiodentine™ interacts through elemental release/uptake with the cellular microenvironment, triggering odontogenic differentiation and biomineralization in a concentration-dependent manner. These results reveal a promising strategy for application of the calcium silicate based cement (Biodentine™) for vital pulp therapies of deciduous teeth in Paediatric Dentistry.

Therapeutic-designed electrospun bone scaffolds: Mesoporous bioactive nanocarriers in hollow fiber composites to sequentially deliver dual growth factors

A novel therapeutic design of nanofibrous scaffolds, holding a capacity to load and deliver dual growth factors, that targets bone regeneration is proposed. Mesoporous bioactive glass nanospheres (MBNs) were used as bioactive nanocarriers for long-term delivery of the osteogenic enhancer fibroblast growth factor 18 (FGF18). Furthermore, a core–shell structure of a biopolymer fiber made of polyethylene oxide/polycaprolactone was introduced to load FGF2, another type of cell proliferative and angiogenic growth factor, safely within the core while releasing it more rapidly than FGF18. The prepared MBNs showed enlarged mesopores of about 7nm, with a large surface area and pore volume. The protein-loading capacity of MBNs was as high as 13% when tested using cytochrome C, a model protein. The protein-loaded MBNs were smoothly incorporated within the core of the fiber by electrospinning, while preserving a fibrous morphology. The incorporation of MBNs significantly increased the apatite-forming ability and mechanical properties of the core–shell fibers. The possibility of sequential delivery of two experimental growth factors, FGF2 and FGF18, incorporated either within the core–shell fiber (FGF2) or within MBNs (FGF18), was demonstrated by the use of cytochrome C. In vitro studies using rat mesenchymal stem cells demonstrated the effects of the FGF2–FGF18 loadings: significant stimulation of cell proliferation as well as the induction of alkaline phosphate activity and cellular mineralization. An in vivo study performed on rat calvarium defects for 6weeks demonstrated that FGF2–FGF18-loaded fiber scaffolds had significantly higher bone-forming ability, in terms of bone volume and density. The current design utilizing novel MBN nanocarriers with a core–shell structure aims to release two types of growth factors, FGF2 and FGF18, in a sequential manner, and is considered to provide a promising therapeutic scaffold platform that is effective for bone regeneration.

Leptin accelerates enterocyte turnover during methotrexate-induced intestinal mucositis in a rat model

Gastrointestinal mucositis occurs as a consequence of cytotoxic treatment. In the present study, we tested whether leptin can protect gut epithelial cells from methotrexate (MTX)-induced intestinal damage. Non-pretreated and pretreated with MTX Caco-2 cells were incubated with increasing concentrations of leptin for 24 hours. Cell proliferation and apoptosis were assessed using FACS analysis. Adult rats were divided into three experimental groups: Control rats; MTX-rats were treated with a single dose of MTX, and MTX- LEP rats were also treated with leptin for 3 days. Intestinal mucosal damage (Park's score), mucosal structural changes (bowel and mucosal weight, mucosal DNA and protein content, villus height and crypt depth), enterocyte proliferation, and enterocyte apoptosis were measured at sacrifice. RT-PCR was used to determine the level of bax and bcl-2 mRNA expression. In the vitro experiment, treatment with leptin of Caco-2 cells pre-treated with MTX resulted in a significant stimulation of cell proliferation and inhibition of cell apoptosis in a dose-dependent manner. In the vivo experiment, MTX-LEP rats demonstrated a greater jejunal and ileal bowel and mucosal weight, mucosal DNA and protein, villus height and crypt depth, as well as a greater enterocyte proliferation index compared to MTX-animals. MTX-LEP rats also showed a trend toward an increase in enterocyte apoptosis that was accompanied by an increase in bax mRNA and decrease in bcl-2 mRNA expression. In conclusion, leptin enhances proliferation and decreases apoptosis in Caco-2 cells pretreated with MTX. In a rat model of MTX-induced mucositis, treatment with leptin improves intestinal recovery and enhances enterocyte turnover.

Transforming growth factor-alpha stimulates enterocyte proliferation and accelerates intestinal recovery following methotrexate-induced intestinal mucositis in a rat and a cell culture model

Recent evidence suggests that transforming growth factor-alpha (TGF-alpha) enhances enterocyte proliferation and exerts a gut trophic effect. The purpose of the present study was to evaluate the effect of TGF-alpha on enterocyte proliferation and intestinal recovery following methotrexate (MTX)-induced intestinal mucositis in rats and in Caco-2 cells. Nonpretreated Caco-2 cells and those pretreated with MTX were incubated with increasing concentrations of TGF-alpha. Cell proliferation was determined by FACS cytometry. Adult rats were divided into three groups: control rats treated with vehicle, MTX rats treated with one dose (20 microg/kg) of MTX given intraperitoneally, and MTX-TGF-alpha rats treated with one dose of MTX followed by two doses of TGF-alpha (75 microg/kg a day). Three days after MTX injection, rats were sacrificed. Intestinal mucosal damage (Park's score), mucosal structural changes, and enterocyte proliferation were measured at sacrifice. Western blotting was used to determine the level of extracellular signal-related kinase (ERK) protein, a marker of cell proliferation. A nonparametric Kruskal-Wallis ANOVA test was used for statistical analysis with P value less than 0.05 considered statistically significant. The in vitro experiment demonstrated that treatment with TGF-alpha of Caco-2 cells resulted in a significant stimulation of cell proliferation in a dose-dependent manner. The in vivo experiment showed that treatment with TGF-alpha resulted in a significant increase in bowel and mucosal weight, DNA and protein content in jejunum and ileum, villus height in jejunum and ileum, crypt depth in ileum, and increased cell proliferation in jejunum and ileum compared to the MTX group. MTX-TGF-alpha rats also had a significantly lower intestinal injury score in ileum when compared to MTX animals. The increase in levels of cell proliferation in MTX-TGF-alpha rats corresponded with the increase in ERK protein levels in intestinal mucosa. Treatment with TGF-alpha prevents mucosal injury, enhances ERK-induced enterocyte proliferation, and improves intestinal recovery following MTX-induced intestinal mucositis in rats. These findings correlated with the observation that TGF-alpha also caused a significant stimulation of cell proliferation in a Caco-2 cell culture model treated with MTX. These observations may have significant implications for the treatment of patients on chemotherapy who develop severe mucositis.

Theophylline stimulates cAMP-mediated signaling associated with growth regulation in human cells from pulmonary adenocarcinoma and small airway epithelia

The methylxanthine theophylline is contained in tea and in numerous asthma and cold medications. Theophylline inhibits the enzyme phosphodiesterase, thereby preventing the intracellular break-down of cAMP. The resulting increase in intracellular cAMP reduces smooth muscle tone, thus dilating the airways. Epidemiologic studies on preventive effects of tea on the development of lung cancer have yielded mixed results, with some studies demonstrating a reduction in lung cancer risk whereas others showed evidence for cancer promotion. On the other hand, preclinical studies in mouse models of lung cancer or in vitro systems have consistently demonstrated strong cancer preventive effects of tea and of polyphenols contained in tea. Investigations conducted in our laboratory have recently shown that cell lines derived from human pulmonary adenocarcinomas of Clara cell lineage (PACC) and experimentally induced PACCs in a hamster model are under beta-adrenergic growth control. beta-adrenergic agonists as well as forskolin, which activates cAMP, had strong growth-promoting effects on human PACC cells and on the hamster PACCs. The current project therefore tests the hypothesis that theophylline activates growth-stimulating signaling in human PACC cells and their normal cells of origin, small airway epithelial cells (SAEC). Using assays for the assessment of intracellular cAMP, activated PKA, phosphorylated CREB, ERK1/2 and cell numbers, our data provide evidence for a significant stimulation of cell proliferation in both cell systems via activation of these signaling components.

Biphasic modulation of cell proliferation by quercetin at concentrations physiologically relevant in humans.

Optimal in vitro conditions regarding quercetin solubility and stability were defined. Using these conditions, the effect of quercetin on proliferation of the colon carcinoma cell lines HCT-116 and HT29 and the mammary adenocarcinoma cell line MCF-7 was investigated. For the colon carcinoma cell lines, at relatively high concentrations, a significant decrease in cell proliferation was observed, providing a basis for claims on the anti-carcinogenic activity of quercetin. However, at lower concentrations, a subtle but significant stimulation of cell proliferation was observed for all cell lines tested. These results point at a dualistic influence of quercetin on cell proliferation that may affect present views on its supposed beneficial anti-proliferative effect.

The biphasic stimulation of proliferation of Leydig cells by estrogen exposure.

In this study, we have examined the influence of diethylstilbestrol (DES) and 17 beta-estradiol on the proliferation of TM3 Leydig cells, a normalized mouse cell line. Cells were treated with seven different concentrations (1 pg-1 microg/ml) of DES or 17 beta-estradiol, and cell growth was measured at 24-, 48-, and 72-h periods. DES treatment resulted in a significant (p<0.05) stimulation of cell proliferation. We observed two independent peaks of cell proliferation, one at 1 pg/ml DES (186.87%) and the other at 100 ng/ml DES (248.23%). Cytotoxicity was noted at all time periods with 1 microg/ml DES treatment. 17 beta-estradiol treatment resulted in a significant stimulation of cell proliferation (p<0.05) with a trend similar in response to that of DES treatment, as peak proliferation was noted with 1 pg/ml 17 beta-estradiol (125.27%) and 10 ng/ml 17 beta-estradiol (138.31%). Based on these data, it appears that DES is more mitogenic in these Leydig cells compared to 17 beta-estradiol. Furthermore, for the first time, we detected that both DES and 17 beta-estradiol were able to stimulate proliferation of Leydig cells in a biphasic fashion. Cell cycle kinetic analysis revealed that cell entry into the S-phase was higher in the DES treated cells compared to the controls, and doubling times of DES exposed cells were significantly reduced (p<0.05). Co-administration of tamoxifen at a concentration 1000-fold higher than either DES or 17 beta-estradiol resulted in complete inhibition of cell proliferation. Analysis of expression of ER alpha and ER beta by RT-PCR in untreated Leydig cells, as well as Leydig cells exposed to 1 pg/ml DES, revealed that the transcripts of ER alpha and ER beta were not detectable even after 40 cycles of amplification. A 100-ng/ml dose of DES induced ER alpha expression by 20-fold. These data suggest that estrogen exposure-mediated increases in cell proliferation, coupled with the decrease in cell cycle time, may allow greater accumulation of DNA damage to occur in the testicular target cells compared to untreated cells under normal cell cycle control. In addition, an unidentified estrogen receptor may be responsible for the mitogenic activity of estrogens at low levels.

Dehydroepiandrosterone stimulates proliferation and gene expression in MCF-7 cells after conversion to estradiol

Dehydroepiandrosterone (DHEA) is a mitogen for estrogen-dependent MCF-7 breast cancer cells. Our aims were to determine whether DHEA required conversion to estrogens in order to stimulate cell proliferation and estrogen-dependent gene expression. After incubation of cells with 100 nM DHEA for 4 days, estradiol was present in the medium at a concentration of ∼200 pM. Other compounds identified were testosterone (∼300 pM) and estrone. Significant stimulation of cell proliferation by 1 nM estradiol and 100 nM DHEA was observed after 38 h and 4 days of incubation, respectively, indicating the necessity of DHEA conversion. DHEA doses ≥10 nM induced estrogen-dependent reporter gene expression in MCF-7 cells transfected with a luciferase reporter gene under the control of the estrogen response element. DHEA-dependent stimulation of proliferation and luciferase induction could be inhibited by the anti-estrogens ICI182,780 and tamoxifen, respectively, and by the aromatase inhibitor 4-hydroxyandrostenedione. An androgenic effect of DHEA on proliferation and gene expression of MCF-7 cells was not observed. We conclude that conversion of DHEA to estrogens, particularly estradiol, is required to exert a mitogenic response.

Epidermal growth factor receptor and tyrosine phosphorylation of estrogen receptor.

Activation of estrogen receptor-alpha (ERalpha) by growth factors in the absence of estrogen is a well-documented phenomenon. To study further this process of ligand-independent receptor activation, COS-7 cells without ER were transfected with both ER and epidermal growth factor receptor (EGFR). In the absence of estrogen, epidermal growth factor (EGF) stimulated rapid tyrosine phosphorylation of ER in transfected COS-7 cells. Similarly, in MCF-7 breast cancer cells that have natural expression of ER and EGFR, EGF promoted acute phosphorylation of serine and tyrosine residues in ER, and a direct interaction between ER and EGFR after treatment with EGF was found. In confirmation of a direct interaction between ER and EGFR, activation of affinity-purified EGFR tyrosine kinase in vitro stimulated the phosphorylation of recombinant ER. The cross-communication between EGFR and ER appears to promote significant stimulation of cell proliferation and a reduction in the apoptotic loss of those cells that express both receptor signaling pathways. However, COS-7 cells transfected with both ER and EGFR show minimal stimulation of classical estrogen response element (ERE)-dependent transcriptional activity after stimulation by EGF ligand. This suggests that the proliferative and antiapoptotic activity of EGF-induced ER activation may be dissociated from ERE-dependent transcriptional activity of the ER.

Differential responses of collagen and glycosaminoglycan syntheses and cell proliferation to exogenous transforming growth factor β 1 in the developing mouse skin fibroblasts in culture

Skin fibroblast cultures were established from mouse foetuses at days 14, 15, 16 and 18 of gestation and from newborn mice. Modulations of mitotic and biosynthetic phenotypes of the fibroblasts by transforming growth factor β1 (TGFβ1) were studied. Treatment of the fibroblasts with TGFβ1 at doses of 1 and 10 ng/ml for 48 h resulted in significant stimulation of cell proliferation in the 15-, 16- and 18-day foetal fibroblasts and a slight stimulation in the 14-day foetal fibroblasts. Treatment with TGFβ1 resulted in stimulation of collagen synthesis approximately 2-fold in the 18-day foetal and newborn fibroblasts, but failed to stimulate it in the 14-, 15- and 16-day foetal fibroblasts. TGFβ1 stimulated glycosaminoglycan (GAG) synthesis throughout all developmental stages approximately 1.8–2.6 fold. Histological study demonstrated that skin wounds made at day 16 of gestation were replaced with normal-appearing dermis, but at day 18 the wounds left dermal fibrosis and lack of hair follicles. These results indicate that the modulations of fibroblast phenotypes (proliferation and syntheses of collagen and GAG) in response to TGFβ1 occur at different stages of gestation. Ontogenic transitions of skin wound healing and collagen synthetic phenotype with TGFβ1 treatment in cultured fibroblasts occurred between days 16 and 18 of gestation, suggesting that the unresponsiveness of collagen synthesis to exogenous TGFβ1 in cell culture may be related to the phenomenon of scarless wounds in the foetus.

Platelet-derived growth factor and angiotensin II stimulate the mitogen-activated protein kinase cascade in renal mesangial cells: comparison of hypertrophic and hyperplastic agonists

Exposure of mesangial cells to platelet-derived growth factor (PDGF) BB caused a significant stimulation of cell proliferation and protein synthesis, as measured by [3H]thymidine incorporation and [3H]leucine incorporation respectively. In contrast, cells treated with angiotensin II had no significant increase in [3H]thymidine incorporation, but demonstrated a marked increase in [3H]leucine incorporation. Furthermore, angiotensin II significantly increased total protein content per cell. These data show that, whereas PDGF-BB is a mitogen and stimulates mesangial-cell hyperplasia, angiotensin II causes hypertrophy of the cells without hyperplasia. Treatment of mesangial cells with PDGF and angiotensin II rapidly and dose-dependently stimulated mitogen-activated protein (MAP) kinase activity, as shown by an assay for activity in vitro using myelin basic protein as a substrate, and by immunoprecipitation of 32P-labelled cells with specific antibodies against the 42 kDa and 44 kDa mitogen-activated protein kinases p42mapk and p44mapk, respectively. Whereas stimulation with PDGF-BB caused a potent and sustained (for more than 30 min) phosphorylation and activation of p42mapk and p44mapk, as well as of the upstream activators MAP kinase kinase and c-Raf, the effect of angiotensin II was less potent, reaching a peak at 5-10 min and thereafter declining rapidly. In summary, these results suggest that PDGF-BB and angiotensin II differ in their potency and duration of activation of the MAP kinase cascade, which may explain why PDGF-BB is a potent mitogen for mesangial cells, whereas angiotensin II only triggers mesangial-cell hypertrophy.

The effect of deazaadenosine analogues on the proliferation rate of cultured vero cells

Five adenosine analogues resitant to adenosine deaminase were analyzed for their effects on the proliferation on cultured Vero cells. Cells were exposed for 48 hr to various concentrations of 1-deazaadenosine (c 1Ado), 3-deazaadenosine (c 3Ado), 7-deazaadenosine (c 7Ado), 1,3-dideazaadenosine (c 1,3Ado) or 1,7-dideazaadenosine (c 1,7Ado) and then counted with a hemocytometer. Results indicate that 1) whereas c 7Ado produced a marked decrease in cell growth, c 3Ado was toxic only at the highest concentration tested (30μM), 2) c 1Ado and c 1,7Ado did not affect cell replication and 3) c 1,3Ado produced a significant stimulation of cell proliferation. Further studies have suggested that none of the compounds tested is incorporated in the DNA.