Proliferation Of Normal Human Fibroblasts Research Articles

Inhibitory effect of Porphyromonas gingivalis-derived phosphoethanolamine dihydroceramide on acid ceramidase expression in oral squamous cells.

The maintenance of diminished acid ceramidase (ASAH1) gene expression leading to the accumulation of antiproliferative intracellular ceramides in oral squamous cell carcinoma (OSCC) has emerged as a prospective oral cancer therapeutic regimen. Our published study demonstrated that the key periodontal pathogen Porphyromonas gingivalis downregulates the expression patterns of ASAH1 mRNA in normal epithelial cells in vitro. Therefore, P. gingivalis may also beneficially diminish the expression of ASAH1 in OSCC. Because a uniquely structured P.gingivalis-derived phosphoethanolamine dihydroceramide (PEDHC) inhibits the proliferation of normal human fibroblasts, this study aimed to test the effect of PEDHC on the survival of human oral squamous OECM-1 cells in vitro. We demonstrated that the P. gingivalis dihydroceramide-null (ΔPG1780) strain upregulates the expression of ASAH1 mRNA and promotes aggressive proliferation and migration of OECM-1 cells compared to the parent P. gingivalis-W83 strain. In addition, the intracellular concentration of ceramides was dramatically elevated in OECM-1 cells exposed to PEDHC in vitro. Furthermore, PEDHC inhibited expression patterns of ASAH1 mRNA as well as some genes associated with degradation of the basement membranes and extracellular matrix, for example, MMP-2, ADAM-17 and IL-6, in OECM-1 cells. Altogether, these data indicated that PEDHC produced by P. gingivalis inhibits acid ceramidase expression, promotes intracellular ceramide accumulation and suppresses the survival and migration of OSCC cells in vitro. Further studies are needed to determine molecular mechanisms of PEDHC-mediated inhibitory effect(s) on OSCC using in vivo models of oral cancer.

Cytotoxicity of 3D Printed Materials for Potential Dental Applications: An In Vitro Study

Aim of the Study: This study aims to evaluate the cytotoxicity of different stereolithographic 3D printed materials and the polyetheretherketone for varying time intervals using MTT assay and by application of these materials for intra-oral usage. Materials and Methods: Three groups of disc specimens (5 mm diameter, 2 mm thick) were manufactured by either selective laser melting (PA6 nylon, fiber reinforced PA6 nylon) or milling (PEEK). The cytotoxicity of these materials was tested by culturing the samples on human fibroblast cell lines prior to MTT assays (3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole). MTT assays were completed at 3, 6, and 10 days. Cell grown in normal medium without experimental material served as control. The total cell number and viability of cells pre-incubated with PA6 nylon, fiber-reinforced PA6 nylon, and PEEK were comparable to the control samples. Differences between the growth inhibitory effects of the samples in the MTT assay were below 0.05%. Results: Both nylon and fiber-reinforced nylon reduced the proliferation of normal human fibroblasts up to 6 days of treatment. PEEK had better biocompatibility than PA6 nylon and fiber-reinforced nylon. Both PA6 nylon and fiber-reinforced are 3DPrinted materials that showed cytotoxicity at 10 days. However, soaking the nylon materials for 6 days made them safe on normal human cells. Conclusion: PEEK material can be considered for intraoral usage as the material is biocompatible. Both PA6 nylon and fiber-reinforced PA6 nylon materials showed increased in-vitro cell death at 10 days, suggesting that they are non-biocompatible for intraoral usage beyond 6 days.

Fabrication of biodegradable textile scaffold based on hydrophobized hyaluronic acid

In this work, we report on the preparation of a novel biodegradable textile scaffold made of palmitoyl-hyaluronan (palHA). Monofilament fibres of palHA with a diameter of 120μm were prepared by wet spinning. The wet-spun fibres were subsequently processed into a warp-knitted textile. To find a compromise between swelling in water and degradability of the final textile scaffold, a series of palHA derivatives with different degrees of substitution of the palmitoyl chain was synthesized. Freeze-drying not only provided shape fixation, but also speeded up scaffold degradation in vitro. Fibronectin, fibrinogen, laminin and collagen IV were physically adsorbed on the textile surface to enhance cell adhesion on the material. The highest amount of adsorbed cell-adhesive proteins was achieved with fibronectin (89%), followed by fibrinogen (81%). Finally, textiles modified with fibronectin or fibrinogen both supported the adhesion and proliferation of normal human fibroblasts in vitro, proving to be a useful cellular scaffold for tissue engineering.

Anticancer properties of peptide fragments of hair proteins.

The primary function of hair and fur covering mammalian skin is to provide mechanical and thermal protection for the body. The proteins that constitute hair are extremely resistant to degradation by environmental factors. However, even durable materials can be slowly broken down by mechanical stresses, biodegradation mediated by endogenous enzymes in the skin or host microbes. We hypothesised that the biodegradation products of hair may possess bioprotective properties, which supplement their physical protective properties. Although evolutionary processes have led to a reduction in the amount of hair on the human body, it is possible that the bioprotective properties of hair biodegradation products have persisted. The human skin is exposed to various environmental carcinogenic factors. Therefore, we hypothesised that the potential bioprotective mechanisms of hair degradation products affect melanoma growth. We used pepsin to partially digest hair enzymatically, and this process produced a water-soluble lysate containing a mixture of peptides, including fragments of keratin and keratin-associated proteins. We found out that the mixtures of soluble peptides obtained from human hair inhibited the proliferation of human melanoma cells in vitro. Moreover, the hair-derived peptide mixtures also inhibited the proliferation of B lymphoma cells and urinary bladder cancer cells. Normal human cells varied in their susceptibility to the effects of the lysate; the hair-derived peptide mixtures modulated the proliferation of normal human fibroblasts but did not inhibit the proliferation of human mesenchymal cells derived from umbilical cord stromal cells. These results suggest that hair-derived peptides may represent a new class of anti-proliferative factors derived from basically structural proteins. Identification of active regulatory compounds and recognition of the mechanism of their action might pave the way to elaboration of new anticancer drugs.

Natural rubber latex LbL films: Characterization and growth of fibroblasts

AbstractThe recent biomedical applications of natural rubber (NR) latex, mostly in dry membranes, have motivated research into novel, more noble uses of this low‐cost biomaterial. In this article, we provide the first report on the fabrication of layer‐by‐layer (LbL) films of NR alternated with the polyelectrolytes polyethylenimine (PEI) and polyallylamine hydrochloride (PAH). Stable (PAH/NR)n and (PEI/NR)n LbL films displayed similar physicochemical properties, but differed in terms of film morphology according to atomic force microscopy (AFM) and scanning electron microscopy (SEM) data. Most significantly, (PEI/NR)5 LbL films were made of smaller and flattened particles, which were not efficient for the growth and proliferation of normal human fibroblasts (NHF). In contrast, efficient NHF proliferation could be obtained with (PAH/NR)n LbL films, with the fibroblasts exhibiting the expected elongated morphology. Furthermore, cell growth did not occur for cast films of NR, thus demonstrating the suitability of the LbL method for this biologically related application. The differences between the two polyelectrolytes illustrate the importance of the film architecture and morphology, which open the way for exploiting the molecular control inherent in the LbL technique for further applications of NR‐containing films. © 2012 Wiley Periodicals, Inc. J Appl Polym Sci, 2012

Overexpression of the Pituitary Tumor Transforming Gene Induces p53-dependent Senescence through Activating DNA Damage Response Pathway in Normal Human Fibroblasts

Pituitary tumor transforming gene (PTTG1, securin) is involved in cell-cycle control through inhibition of sister-chromatid separation. Elevated levels of PTTG1 were found to be associated with many different tumor types that might be involved in late stage tumor progression. However, the role of PTTG1 in early stage of tumorigenesis is unclear. Here we utilized the adenovirus expression system to deliver PTTG1 into normal human fibroblasts to evaluate the role of PTTG1 in tumorigenesis. Expressing PTTG1 in normal human fibroblasts inhibited cell proliferation. Several senescence-associated (SA) phenotypes including increased SA-beta-galactosidase activities, decreased bromodeoxyuridine incorporation, and increased SA-heterochromatin foci formation were also observed in PTTG1-expressing cells, indicating that PTTG1 overexpression induced a senescent phenotype in normal cells. Significantly, the PTTG1-induced senescence is p53-dependent and telomerase-independent, which is distinctively different from that of replicative senescence. The mechanism of PTTG1-induced senescence was also analyzed. Consistent with its role in regulating sister-chromatid separation, overexpression of PTTG1 inhibited the activation of separase. Consequently, the numbers of cells with abnormal nuclei morphologies and chromosome separations were increased, which resulted in activation of the DNA damage response. Thus, we concluded that PTTG1 overexpression in normal human fibroblasts caused chromosome instability, which subsequently induced p53-dependent senescence through activation of DNA-damage response pathway.

Heat Shock-Induced Three-Dimensional-Like Proliferation of Normal Human Fibroblasts Mediated by Pressed Silk

The aim of this study was to determine the optimal heat treatment conditions for enhancement of pressed silk-mediated 3D-like proliferation of normal human dermal fibroblasts, as well as to determine the responses to heat shock of cells and intracellular signaling pathways. The beginning of 3D-like pattern formation of cells was observed in the second week after the start of the experiment. The mean rates of beginning of 3D-like pattern formation by cells heat-treated at 40 ºC and 43 ºC for 10 min were significantly higher (3.2- and 8.6-fold, respectively) than that of untreated cells. We found that apoptosis had occurred in 7.5% and 50.0% of the cells at one week after heat treatment for 10 min at 43 ºC and 45 ºC, respectively. Western blot analysis demonstrated that phosphorylation of p38 MAPK and that of Hsp27 were markedly increased by heat treatment at 43 ºC for 10 min. The results of an experiment using a p38 MAPK inhibitor and Hsp27 inhibitor suggest that activation of p38 MAPK by heat shock is associated with 3D-like cell proliferation and that Hsp27 contributes to the inhibition of apoptosis. The results of this study should be useful for further studies aimed at elucidation of the physiologic mechanisms underlying thermotherapy.

Roles of focal adhesions and fibronectin‐mediated cohesion in proliferation of confluent fibroblasts

Multilayered fibroblast sheets have applications as cell transplants for tissue engineering. One way to increase their therapeutic efficacy is to increase cell numbers in a graft, but the factors influencing multilayered growth remain poorly understood. In this study, we investigated the roles of focal adhesion (FA) assembly and intercellular cohesion through fibronectin (FN) in the proliferation of normal human fibroblasts at confluence. Density-dependent growth-arrested fibroblasts resumed DNA synthesis when cultured in multilayer formation medium (MFM) containing transforming growth factor-beta1, ascorbic acid, and serum. This proliferation depended on alpha 5 beta 1-integrin-mediated cell-FN-cell interactions because blocking them with antibodies inhibited DNA synthesis. However, cell-FN-cell cohesion operated well regardless of exposure to MFM, judging from several parameters, including FN matrix deposition, activated beta1 integrin expression, and stress fiber development. Density-arrested cells formed few FAs at the cell center. Exposure of the cells to MFM induced the formation of vinculin-, paxillin-, and phosphotyrosine-containing FAs throughout the ventral cell-surface, indicating ROCK-mediated actomyosin contractile force generation. When the assembly of FAs was inhibited with either the ROCK inhibitor Y-27632 or the myosin II inhibitor blebbistatin, the up-regulation of DNA synthesis by MFM was suppressed. The drugs did not impair FN matrix deposition, activated beta1 integrin expression, and stress fiber development. Thus, these results indicate that the formation of FAs promotes the proliferation of confluent fibroblasts with the support of alpha 5 beta 1-integrin-mediated cell-FN-cell cohesion. The present findings provide insights into the rational design of high-density fibroblast transplants.

Small-Molecule Targeting of Heat Shock Protein 90 Chaperone Function: Rational Identification of a New Anticancer Lead

Heat shock protein 90 (Hsp90) is a significant target in the development of rational cancer therapy due to its role at the crossroads of multiple signaling pathways associated with cell proliferation and cell viability. Here we present a combined structure- and dynamics-based computational design strategy, taking the flexibility of the receptor and of a lead peptidic antagonist into account explicitly, to identify the nonpeptidic small molecule 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) as a structurally novel inhibitor of Hsp90. The compound is selected to bind the Hsp90 N-terminal domain, mimicking the chemical and conformational properties of the recently described peptidic antagonist of the survivin-Hsp90 complex, shepherdin [Plescia et al. Cancer Cell 2005, 7, 457-468]. Experimental tests show that AICAR binds the Hsp90 N-domain, destabilizes multiple Hsp90 client proteins in vivo, including survivin, and exhibits antiproliferative and proapoptotic activity in multiple tumor cell lines, while not affecting proliferation of normal human fibroblasts. We propose that AICAR represents a viable lead for further development of anticancer drugs with wide therapeutic opportunities.

TRAIL-mediated enhancement of collagen production by human lung fibroblasts

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family that induces apoptosis in a variety of transformed cell lines and in normal human hepatocytes in vitro. The expression of TRAIL was found in CD8 T cells that had undergone oligoclonal expansion in the lungs of patients with systemic sclerosis (scleroderma) and were able to stimulate collagen production in lung fibroblasts in vitro. Among the family members, TRAIL displays highest homology to CD95 ligand, a receptor of which may not only mediate apoptosis of T cells, but also mediate the proliferation of normal human fibroblasts. Considering structural and functional similarities between TRAIL and CD95 ligand, we examined the effects of soluble TRAIL on normal human lung fibroblasts. Collagen α2(I) mRNA expression was assessed by real-time RT-PCR, with ribosomal protein S9 as an internal standard. Total soluble collagen was measured in culture supernatants using the Sircol Biocolor Assay. Both normalized α2(I) collagen mRNA expression and total soluble collagen secretion were increased upon TRAIL stimulation, with peak response (>5-fold increase) at 10 ng/ml TRAIL. DNA microarray hybridization revealed 78 genes involved in signal transduction, DNA transcription, and tissue remodeling, whose expression level increased, and 8 genes whose expression level decreased >2.5-fold in comparison with quiescent fibroblasts. Augmented expression of a number of genes involved in the TGFβ pathway suggested that TRAIL might induce TGFβ-mediated autocrine and/or paracrine stimulation of fibroblasts. This was confirmed by the ability of anti-TGFβ antibody to inhibit the effects of TRAIL on collagen production by fibroblasts. DNA mobility shift assay also revealed TRAIL-induced increase in protein binding to the collagen promoter that was substantially inhibited by consensus oligonucleotide for Smad3/4, mediators of TGFβ signaling. These data suggest that TRAIL can enhance extracellular matrix synthesis in fibroblasts by triggering TGFβ production, which acts in autocrine manner.

Caffeine does not cause override of the G2/M block induced by UVc or gamma radiation in normal human skin fibroblasts.

Caffeine has for many years been known to be involved in the sensitization of DNA to damage. One potential mechanism recently put forward is an override of the G2/M block induced by irradiation, which would leave the cells less time for DNA repair prior to mitosis. However, different cell types display a variety of responses and no clear pathway has yet emerged, especially as little is known about the capacity of this agent to enhance DNA damage in normal, untransformed cells. Continuous exposure to commonly used caffeine concentrations (1–5 mM) inhibited the proliferation of normal human fibroblasts (NHFs) in a dose-dependent manner to up to 80% at 5 mM. Exposure of exponentially growing NHFs to UVc radiation (20 J m–2) or γ radiation (2.5–8 Gy) led to a 45–60% inhibition of proliferation and protracted accumulation of cells in the G2/M phase. Addition of 2 mM caffeine after irradiation induced slowing of the S phase passage, with a resultant delay in G2/M accumulation mimicking a G2/M block override. These results were confirmed by stathmokinetic studies, which showed delayed entry of the cells into mitosis in the presence of caffeine. Our data demonstrate that caffeine primarily inhibits replicative DNA synthesis and suggest that, at least in normal cells, caffeine potentiates the cytotoxicity of radiation by intervening in DNA repair rather than by overriding the G2/M block. © 2000 Cancer Research Campaign

The proliferation of normal human fibroblasts is dependent upon negative regulation of p53 function by mdm2

Loss of function of the tumour suppressor gene p53 is a key event in most human cancers. Although usually occurring through mutation, in some tumour types this appears to be achieved via an indirect mechanism involving inappropriate expression of a functional inhibitor, mdm2, which binds to the transactivation domain of p53. This interaction offers an ideal potential target for novel cancer therapies. However, therapeutic specificity may depend on the extent to which this p53-inhibitory action of mdm2 is also required by normal cells. Transgenic data have already established that mdm2 is needed to prevent embryonic lethality, but the situation in adult cells is still unclear. Here we show that micro-injection of normal human fibroblasts with an antibody directed against the p53-binding domain of mdm2 induces expression of p53-responsive genes, and furthermore results in p53-dependent growth arrest. We conclude that normal cell proliferation can be dependent on negative regulation of p53 by mdm2, a finding which raises an important note of caution for mdm2-directed cancer therapies.

Investigation of calmodulin and basic fibroblast growth factor (bFGF) in idiopathic myelofibrosis: evidence for a role of extracellular calmodulin in fibroblast proliferation.

The urinary concentration of calmodulin and basic fibroblast growth factor (bFGF) was determined in a total of 53 patients with various chronic myeloproliferative disorders (CMPD), including 22 patients with idiopathic myelofibrosis (IMF). Calmodulin excretion was significantly elevated in IMF (0.29 +/- 0.04 microgram/mmol creatinine) (P < 0.001), when compared to polycythaemia vera (PV) (0.14 +/- 0.02), essential thrombocythaemia (ET) (0.13 +/- 0.04), chronic myeloid leukaemia (CML) (0.16 +/- 0.02), unclassified myeloproliferative disorders (UMPD) (0.11 +/- 0.02) and age-matched controls (0.1 +/- 0.02) (P < 0.001). In contrast, bFGF was slightly elevated in all CMPD conditions when compared to age-matched controls. A neutralizing antibody to calmodulin was demonstrated to significantly influence the in vitro proliferation of normal human fibroblasts, an effect dependent on both cell density and the presence of fetal calf serum (FCS). Essentially, the antibody reduced FCS-induced proliferation of low-density fibroblasts but had little or no inhibitory effect on high-density fibroblasts in the absence of FCS. In addition, extracellular calmodulin was shown not to interact with known fibroblast mitogens, namely, IFG-1, EGF, bDGF and PDGF. We conclude that extracellular calmodulin should be considered, in addition to PDGF, TFG-beta and EGF, as a potential mitogen involved in the stromal reaction of idiopathic myelofibrosis.

SIMPLE AND EFFICIENT CHEMOSENSITIVITY ASSAY FOR HUMAN PRIMARY TUMOR-CELLS USING CONTACT-SENSITIVE MICROTITER PLATES

A simple and efficient chemosensitivity assay for human primary tumors has been developed using 96-well microtiter plates covered totally with lethally irradiated 3T3 monolayers termed cell mats, on which the proliferation of normal human fibroblasts is preferentially inhibited. Two days after the inoculation of tumor cells into the microtiter cell mat plates, the cells were treated with various anticancer drugs, and the cell numbers were assessed by radioactivity using a 96-well automatic scintillation counter after subsequent radiolabeling with H-3-deoxyuridine for 24h. In this manner, complete dose response curves of many anticancer drugs were available from one plate within 5 days. Drugs tested in triplicate were adriamycin, mitomycin C, cisplatin, etoposide, and 5-fluorouracil at 0.01, 0.1, and Ix the peak tolerated drug concentrations in serum. Clinically, of the 39 primary tumor specimens of different types received, 4 were contaminated. The remaining 35 samples were successfully cultured, with 5 cultures being abandoned due to an insufficient cpm. As a result, cell survival curves were obtained from 30 (77%) specimens. This high evaluable rate might be due to the feeder effect brought by cell mats. Although optimization of the assay system has yet to be determined, the ability to have multiple drug sensitivity per plate with short-term duration would make this system an efficient assay for chemosensitivity test of human primary tumor cells.

Effects of Human and Ovine Pancreatic Secretory Trypsin Inhibitors on the Proliferation of Normal Human Fibroblasts

Human pancreatic secretory trypsin inhibitor inhibited cell-surface proteolytic activity in human fibroblasts. In the range of concentrations which caused proteinase inhibition, fibroblast proliferation was also inhibited by this reagent and by the ovine equivalent. At lower concentrations, there was some evidence for a mitogenic effect, and this was confirmed by obvious stimulation of DNA synthesis at these concentrations. Human alpha 1-proteinase inhibitor, previously demonstrated to be an inhibitor of fibroblast proliferation, was also mitogenic at concentrations lower than those which inhibited proteolytic activity and cell proliferation. Human pancreatic secretory trypsin inhibitor and epidermal growth factor apparently work through independent mechanisms, since their mitogenic effects are additive.

Differential expression of mRNA coding for heparin-binding growth factor type 2 in human cells.

The proliferation of normal human fibroblasts, keratinocytes, and melanocytes in vitro can be controlled by purified polypeptide growth factors and serum. We have studied the cellular expression of the heparin-binding growth factor type 2/basic fibroblast growth factor (HBGF-2/bFGF) gene to determine whether these cell types synthesize mRNA for this mitogen. Our results indicate that normal human fibroblasts synthesize four distinct mRNAs of 7.0, 3.7, 2.2, and 1.5 kilobases, which hybridize to a specific HBGF-2/bFGF cDNA probe. In fibroblasts, the level of all four of these transcripts increases dramatically (more than tenfold) within 4 hours of treatment of quiescent cells with fresh fetal bovine serum. Of the purified growth factors tested, transforming growth factor type-beta also increased HBGF-2/bFGF mRNA abundance, but not to the levels attained by serum treatment. Treatment of fibroblasts with cycloheximide before and during serum treatment blocked the ability of serum to induce the expression of the HBGF-2/bFGF gene. The gene is expressed at low levels in human fibroblasts rapidly growing in serum-free medium and at higher levels in cells rapidly growing in serum-containing medium. In contrast to fibroblasts, mRNA coding for HBGF-2/bFGF is undetectable in proliferating normal human keratinocytes, melanocytes, or mammary epithelial cells. Because keratinocytes and melanocytes proliferate in response to purified HBGF-2/bFGF, our results suggest that HBGF-2/bFGF may mediate the proliferation of epidermal cells through paracrine mechanisms involving stromal fibroblasts. Moreover, we have shown that a human squamous cell carcinoma cell line (SCC-25) expresses mRNA coding for HBGF-2/bFGF, suggesting that the gene may become activated in some carcinomas.