Human Fibroblast-like Cells Research Articles

Cell surface and relative mRNA expression of heat shock protein 70 in human synovial cells

Heat shock proteins (Hsps) have been repeatedly implicated to participate in the pathogenesis of rheumatoid arthritis (RA).Methods: Herein, Hsp70 cell surface and mRNA expression were studied in human fibroblast-like synovial cells, dermal fibroblasts and peripheral blood leukocytes derived from 24 RA patients, who underwent synovectomy by using flow-cytometric analysis and real-time quantitative reverse-transcriptase polymerase chain reaction. For comparison, peripheral blood leukocytes of 17 healthy controls were tested.Results: Significantly higher Hsp70 membrane positivity was found on fibroblast-like synovial cells in RA patients (average 18.3%, median 16.5%) than on autologous and healthy control peripheral blood lymphocytes (RA patients: average 4.7%, median 2.9%, p = 0.002; healthy controls: average 6.0%, median 4.5%, p = 0.002) and/or autologous dermal fibroblasts (average 5.1%, median 4.3%, p < 0.001). Strong Hsp70 cell surface expression was also found on peripheral blood monocytes of RA patients (average 53.0%, median 58.1%) and healthy controls (average 49.4%, median 47.5%, p = 0.52). Peripheral blood granulocytes of healthy controls (average 41.8%, median 41.4%) showed significantly increased Hsp70 expression comparing with RA patients (average 10.7%, median 6.4%, p = 0.005).Significantly higher Hsp70 gene expression was observed in synovial cells of RA patients (average 2.04, median 1.7) when compared with autologous peripheral blood leukocytes (average 0.75, median 0.68; p < 0.001). However, the difference in Hsp70 gene expression between RA-derived synovial cells and healthy control peripheral blood leukocytes (average 1.69, median 1.64) was not observed (p = 0.83). We also found significantly lower relative gene expression in peripheral blood leukocytes of RA patients in comparison with healthy controls (p < 0.001).Interestingly, we found that Hsp70 gene expression in RA non-affected skin dermis gained from the operation wound was 3.7-fold higher in average (average 7.6, median 8.3) when compared to autologous RA-affected synovial tissue (p < 0.001); 10.1-fold higher in average when compared to autologous peripheral blood leukocytes (p < 0.001) and 4.5-fold higher in average comparing to control peripheral blood leukocytes (p < 0.001).Conclusion: Hsp70 gene expression in RA-affected synovial tissue is followed by Hsp70 cell surface expression on fibroblast-like synovial cells growing from RA synovial tissue. Hsp70 may be translocated to the cell surface from the cytosol and/or Hsp70 released from inflamed synovial tissue may be captured onto the membrane of synovial cells from the extracellular space via Hsp receptors.As a physiological response to potentially harmful enviromental stress factors, skin dermis produces higher levels of Hsp70 comparing to the cells of internal organs and tissues.

Ajulemic acid, a synthetic cannabinoid acid, induces an antiinflammatory profile of eicosanoids in human synovial cells

Aims To better understand mechanisms whereby Ajulemic acid (AjA), a synthetic antiinflammatory cannabinoid, promotes resolution of acute and chronic inflammation in animal models, we investigated its influence on cyclooxygenase 2 (COX2) expression and eicosanoid production in human fibroblast-like synovial cells (FLS). Main methods FLS isolated from tissue obtained at joint replacement surgery or cultured from synovial fluid were treated for 60 min with AjA (10–30 μM), then stimulated with tumor necrosis factor α (TNFα). COX2 mRNA was measured by hybridization/colorimetric assay of whole cell lysates collected 4 h after stimulation. To determine effects on arachidonic acid release, FLS were incubated with 14C-arachidonic acid for 20 h then treated with AjA (8–32 μM). Arachidonic acid release was measured by scintillation counting. Prostaglandins (PG) were measured by enzyme linked immunosorbent assay (ELISA) in cell supernatants collected 4 and 24 h after stimulation. Key findings AjA increased the steady state levels of COX2 mRNA in and arachidonic acid release from FLS. Treatment of FLS with AjA increased 15-deoxy-delta 12,14-PGJ 2 (15d-PGJ 2) production in a concentration dependent manner, but did not affect PGE 2 production significantly. Significance The capacity of AjA to increase selectively and markedly 15d-PGJ 2, an eicosanoid which facilitates resolution of inflammation, suggests that AjA may have value as a therapeutic agent for the treatment of rheumatoid arthritis (RA) and other diseases characterized by acute and chronic inflammation.

Effect of estrogen on bone resorption and inflammation in the temporomandibular joint cellular elements

Several epidemiological studies have reported that temporomandibular disorder is more prevalent in women, which suggests the involvement of sex hormones, such as estrogen, in the pathogenesis of this disease. PCR amplification and Western blotting were employed to target the expression of estrogen receptors (ERs) in human fibroblast-like synovial and ATDC5 cells. The effect of estrogen was investigated through the expression of RANKL, osteoprotegerin (OPG), M-CSF/CSF-1 and c-fms. We showed expression of M-CSF/ CSF-1 and c-fms, with time-dependent increase in both after the addition of estrogen. Based on previous studies reporting that M-CSF/CSF-1 regulates the proliferation and differentiation of hemopoietic progenitor cells into mature macrophages, we put forward a new hypothesis based on the increased inflammation and tendency of females to suffer more from temporomandibular disorder (TMD) in the presence of external exacerbating factors. Detection of RANKL and OPG in ATDC5 and expression of both in HFLS was confirmed with complete disappearance of the RANKL band, and marked increase in the expression of OPG after 1 h from the addition of estrogen.

Induced in vitro differentiation of neural-like cells from human amnion-derived fibroblast-like cells

There is growing evidence that the human amnion contains various types of stem cell. As amniotic tissue is readily available, it has the potential to be an important source of material for regenerative medicine. In this study, we evaluated the potential of human amnion-derived fibroblast-like (HADFIL) cells to differentiate into neural cells. Two HADFIL cell populations, derived from two different neonates, were analyzed. The expression of neural cell-specific genes was examined before and after in vitro induction of cellular differentiation. We found that neuron specific enolase, neurofilament-medium, beta-tubulin isotype III, and glial fibrillary acidic protein (GFAP) showed significantly increased expression following the induction of differentiation. In addition, immunostaining demonstrated that neuron specific enolase, GFAP and myelin basic protein (MBP) were present in HADFIL cells following the induction of differentiation, although one of the HADFIL cell populations showed a lower expression of GFAP and MBP. These results indicate that HADFIL cell populations have the potential to differentiate into neural cells. Although further studies are necessary to determine whether such in vitro-differentiated cells can function in vivo as neural cells, these amniotic cell populations might be of value in therapeutic applications that require human neural cells.

W1968 Human Gastric Cancer-Associated Fibroblast-Like Cells Are Derived from the Bone Marrow

W1968 Human Gastric Cancer-Associated Fibroblast-Like Cells Are Derived from the Bone Marrow

Plasmin is involved in inflammation via protease-activated receptor-1 activation in human dental pulp

Plasmin is a proteolytic enzyme produced from plasminogen by plasminogen activators. We investigated the function of plasmin in human dental pulp fibroblast-like cells. Plasmin induced an increase in the intracellular Ca 2+ concentration ([Ca 2+] i) in a concentration-dependent manner. Expression of mRNA for protease-activated receptor-1 (PAR-1) was detected, and the PAR-1 activating peptide SFLLRN induced an increase in [Ca 2+] i in the cells. The plasmin-induced increase in [Ca 2+] i was inhibited in the presence of the PAR-1 antagonist SCH79797. Plasmin stimulated the expression of interleukin-8 (IL-8) mRNA and prostaglandin E 2 release, which are involved in inflammation. These effects of plasmin on expression of IL-8 mRNA and prostaglandin E 2 release were inhibited in the presence of the PAR-1 antagonist SCH79797. These results suggest that plasmin activates PAR-1 and is involved in inflammation in human dental pulp.

Preparation and In Vitro Characterization of Wollastonite Doped Tricalcium Phosphate Bioceramics

In this work a new kind of CaSiO3-doped α-Ca3(PO4)2 ceramic materials, with compositions lying outside the field of the Ca3(PO4)2 solid solution in the system Ca3(PO4)2- CaSiO3, were obtained and some of their properties, relevant for bone repairing, were studied in vitro. Crystalline α-Ca3(PO4)2 solid solution and minor amounts of non-equilibrium residual glass were the only phases in the materials containing 2 and 5 wt% of CaSiO3. α-Ca3(PO4)2, crystalline eutectic-like phase and residual glass were observed for sample containing 15 and 20 wt% of CaSiO3. The mechanical strength improved for all the doped ceramics with regard to un-doped Ca3(PO4)2. The release of ionic Ca and Si in simulated physiological conditions increased with the content of CaSiO3 and favored α-Ca3(PO4)2 surface transformation. The soluble components extracted from the CaSiO3-doped α-Ca3(PO4)2 bioceramics were not cytotoxic to human fibroblastlike cells. Initial cell adhesion onto the surface of the materials seemed to be partially hindered by surface reactivity and remodeling, however those cells adhered to the experimental bioceramics were viable and proliferated normally.

Effects of Levofloxacin and Doxycycline on Interleukin-6 Production of Chlamydia trachomatis-Infected Human Synovial Fibroblasts

Background: Alarge amount (80,000–100,000 pg/ml) of interleukin-6 (IL-6) was detected in cultures of human synoviocytes infected with Chlamydia trachomatis. In this study, we investigated the effect of antibiotics on the IL-6 production of C. trachomatis-infected human fibroblast-like synovial cells (HFLS). Methods: The minimum inhibitory concentrations of levofloxacin and doxycycline against C. trachomatis were determined in either HFLS or HeLa 229 cells. The number of live Chlamydia was also examined. IL-6 in the supernatants of infected cultures was quantified by capture ELISA. Results: The production of IL-6 was suppressed to as low as 1,800 pg/ml in the infected HFLS treated with levofloxacin or doxycycline immediately or early after infection. In HFLS treated with levofloxacin and doxycycline, the IL-6 levels decreased to 37,000 and 21,000 pg/ml, respectively, 48 h after infection, and levofloxacin was thus found to be less effective than doxycycline. In addition, the number of viable C. trachomatis in the infected cultures treated with levofloxacin 24 h after infection was higher than when treated with doxycycline. Conclusions: The early administration and proper selection of antibiotics is important for the suppression of inflammatory cytokine production. These findings indicate that antibiotic therapy will not only work in treating infections but might also be useful in treating reactive arthritis secondary to the infection.

IGF and FGF cooperatively establish the regulatory stem cell niche of pluripotent human cells in vitro

Distinctive properties of stem cells are not autonomously achieved, and recent evidence points to a level of external control from the microenvironment. Here, we demonstrate that self-renewal and pluripotent properties of human embryonic stem (ES) cells depend on a dynamic interplay between human ES cells and autologously derived human ES cell fibroblast-like cells (hdFs). Human ES cells and hdFs are uniquely defined by insulin-like growth factor (IGF)- and fibroblast growth factor (FGF)-dependence. IGF 1 receptor (IGF1R) expression was exclusive to the human ES cells, whereas FGF receptor 1 (FGFR1) expression was restricted to surrounding hdFs. Blocking the IGF-II/IGF1R pathway reduced survival and clonogenicity of human ES cells, whereas inhibition of the FGF pathway indirectly caused differentiation. IGF-II is expressed by hdFs in response to FGF, and alone was sufficient in maintaining human ES cell cultures. Our study demonstrates a direct role of the IGF-II/IGF1R axis on human ES cell physiology and establishes that hdFs produced by human ES cells themselves define the stem cell niche of pluripotent human stem cells.

Human platelet lysate enhances the proliferative activity of cultured human fibroblast-like cells from different tissues

Several studies have shown the presence of fibroblast-like cells in the stromal fraction of different tissues with a high proliferative and differentiation potential. Platelet alpha granules contain growth factors released into the environment during activation. The effects of different supplements for culture medium (human serum, bovine serum and platelet lysate) on cultured human fibroblast-like cells from bone marrow, adipose tissue, trabecular bone and dental pulp have been compared. Expression of typical stromal and hematopoietic markers was analyzed and proliferative rates were determined. Flow cytofluorometry showed a homogenous pattern in serial-passaged cells, with a high level of stromal cell-associated markers (CD13, CD90, CD105). The presence of platelet lysate in culture media increased the number of cell generations obtained regardless of cell source. This effect was serum-dependent. Cell-based therapies can benefit by the use of products from human origin for "ex vivo" expansion of multipotent cells.

Fibroblast-like cells derived from the gonadal ridges and dorsal mesenteries of human embryos as feeder cells for the culture of human embryonic germ cells

The establishment of optimal hEG culture systems is a major challenge for the field of embryonic germ cell research. It is important to find appropriate feeder cells to support the growth of hEG. The clinical application of human embryonic germ cells cultured on mouse-derived feeder cells is restricted, since human embryonic germ cells cultured on mouse-derived feeder cells are at risk of contamination by heterogeneous proteins or pathogens. In order to avoid this limitation, we have isolated and cultured three human embryonic fibroblast-like cell lines derived from the gonadal ridges and dorsal mesenteries of 5- to 10-week old embryos. These cells expressed basic fibroblast growth factor and leukemia inhibitory factor, both essential for the growth of human embryonic germ cells. We then used the mitomycin-inactivited human embryonic fibroblast-like cells as feeder cells to culture human embryonic germ cells derived from the gonadal ridges and dorsal mesenteries of 5- to 10-week old embryos. Of 21 human primordial germ cell cultures initiated, seven were continuously grown and split for 10 passages with normal and stable human karyotypes. These cells expressed markers characteristic of pluripotent stem cells, including alkaline phosphatase, stage-specific embryonic antigens (SSEA)-1, SSEA-3, SSEA-4, tumor related antigens (TRA)-1-60, TRA-1-81, and the POU transcription factor Octamer-4 (Oct-4). Moreover, the cells possessed the capacity to differentiate into all three primary germ layers (ectoderm, mesoderm, and endoderm). Therefore, we have successfully used the human embryonic fibroblast-like cells derived from gonadal ridges and dorsal mesenteries as feeder cells to culture proliferative, undifferentiated and pluripotent human embryonic germ cells.

Differential Activation of ERK 1/2 and JNK in Normal Human Fibroblast-like Cells in Response to UVC Radiation Under Different Oxygen Tensions ¶

The mechanisms by which mitogen-activated protein kinases (MAPK) respond to the input of UV-induced signal transduction pathways and the resulting biological functions are not well understood. We investigated whether the level of oxygen tension of culture was responsible for the differential activation of MAPK and different cellular outcomes in UVC-irradiated cells. The intracellular oxidative level of normal human fibroblast-like cells in a normal atmosphere (normoxic, 20% O2) was increased within 30 min after UVC irradiation. When cells were cultured at lower oxygen tension in the presence of an antioxidant N-acetyl-l-cysteine (NAC) or under physiologically hypoxic (5% O2) conditions, the elevation of the oxidative level by UV-irradiation was significantly reduced. Among MAPK, extracellular-signal related kinase (ERK) 1/2 was activated by UV regardless of the oxidative level, while c-Jun N-terminal kinase (JNK) activation was inhibited in NAC-treated and in hypoxic cultures. In addition, in cultures at lower oxygen tension, there was less apoptosis and cell survival was enhanced. These results suggest that UV-induced oxidative stress was responsible for intracellular signaling through the JNK pathway. Furthermore, the balance between ERK1/2 and JNK activities after UV irradiation under different oxygen tensions possibly modified cellular outcome in response to UV.

Prednisolone phosphate-containing TRX-20 liposomes inhibit cytokine and chemokine production in human fibroblast-like synovial cells: a novel approach to rheumatoid arthritis therapy

To evaluate the potential of using prednisolone phosphate (PSLP)-containing 3,5-dipentadecyloxybenzamidine hydrochloride (TRX-20) liposomes to treat rheumatoid arthritis (RA), we examined their ability to bind human fibroblast-like synovial (HFLS) cells and their effects in these cells. To test for binding, Lissamine rhodamine B-1, 2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (rhodamine)-labelled PSLP-containing TRX-20 liposomes were added to HFLS cells, and the fluorescence intensity of the rhodamine bound to the cells was evaluated. Rhodamine-labelled PSLP-containing liposomes without TRX-20 were used as a negative control. To evaluate the uptake of liposomes by the HFLS cells, we used TRX-20 liposomes containing 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) and p-xylene-bis-pyridinium bromide (DPX), and observed the cells by fluorescence microscopy. The effects of the PSLP in TRX-20 liposomes on HFLS cells were assessed by the inhibition of the production of two inflammatory cytokines (interleukin 6 and granulocyte macrophage colony-stimulating factor) and one inflammatory chemokine (interleukin 8). The interaction of the PSLP-containing TRX-20 liposomes with HFLS cells was approximately 40 times greater than that of PSLP-containing liposomes without TRX-20. PSLP-containing TRX-20 liposomes bound to HFLS cells primarily via chondroitin sulfate. TRX-20 liposomes taken up by the cell were localized to acidic compartments. Furthermore, the PSLP-containing TRX-20 liposomes inhibited the production of the inflammatory cytokines and the chemokine more effectively than did the PSLP-containing liposomes without TRX-20. These results indicate that PSLP-containing TRX-20 liposomes show promise as a novel drug delivery system that could enhance the clinical use of glucocorticoids for treating RA.

Cytofluorometric analysis of phenotypes of human bone marrow and umbilical fibroblast-like cells

Comparative analysis of the expression of some surface markers of human bone marrow mesenchymal stem cells, umbilical fibroblast-like cells, and skin fibroblasts was carried out by the flow cytofluorometry method. Mesenchymal stem cells and umbilical fibroblast-like cells were similar by the levels of expression of the main histocompatibility complex antigens, adhesion molecules, and some growth factor receptors. The profile of skin fibroblast surface antigens was characterized by higher expression of the markers typical of differentiated cells. The results prove the possibility of using umbilical fibroblast-like cells as an alternative source of mesenchymal stem cells for cell replacement therapy.

Suppression of fibroblast metalloproteinases by ajulemic acid, a nonpsychoactive cannabinoid acid

Production of matrix metalloproteinases (MMP) in joint tissue of patients with inflammatory arthritis facilitates cartilage degradation and bone erosion, and leads to joint deformities and crippling. Thus, MMPs are important targets for agents designed to treat inflammatory arthritis. Oral administration of ajulemic acid (AjA), a synthetic, nonpsychoactive cannabinoid acid, prevents joint tissue injury in rats with adjuvant arthritis. AjA binds to and activates PPARgamma directly. Therefore, we investigated the influence of AjA on MMP production in human fibroblast-like synovial cells (FLS), and examined the role of PPARgamma in the mechanism of action of AjA. FLS, treated or not with a PPARgamma antagonist, were treated with AjA then stimulated with TNFalpha or IL-1alpha. Release of MMPs-1, 3, and 9 was measured by ELISA. The influence of AjA on MMP-3 release from stimulated PPARgamma positive (PPAR+/-) and PPARgamma null (PPAR-/-) mouse embryonic fibroblasts (MEF) was also examined. Addition of AjA to FLS suppressed production of MMPs whether or not PPARgamma activation was blocked. Secretion of MMP-3 was also suppressed by AjA in both TNFalpha- and IL-1alpha-stimulated PPARgamma+/- and PPARgamma-/- MEF. Suppression of MMP secretion from FLS by AjA appears to be PPARgamma independent. Prevention by AjA of joint tissue injury and crippling in the rat adjuvant arthritis model may be explained in large part by inhibition of MMPs. These results suggest that AjA may be useful for treatment of patients with rheumatoid arthritis and osteoarthritis.

Mitogen‐activated protein kinase/extracellular signal‐regulated protein kinase activation of cultured human dental pulp cells by low‐power gallium‐aluminium‐arsenic laser irradiation

To examine whether low-power laser irradiation (LPLI) promotes cellular proliferation of human dental pulp-derived fibroblast-like cells (dental pulp cells). Dental pulp cells were obtained by primary culture of human dental pulp tissues from extracted third molar teeth. The phosphorylation of the mitogen-activated protein kinase (MAPK) family after LPLI of these cells was investigated by Western blotting. By using a specific MAPK/ERK kinase (MEK) inhibitor (PD098059), the specific effect of LPLI on the MAPK pathway was also investigated by Western blotting as described above. The incorporation of [3H]thymidine into the cells after LPLI was determined, and statistical analysis was performed by Wilcoxon signed-ranks test. Extracellular signal-regulated protein kinase (ERK) 1/2 was phosphorylated between 5 and 30 min after LPLI. Moreover, PD098059 inhibited LPLI-mediated ERK1/2 activation. LPLI did not affect p38 MAPK or c-Jun N-terminal kinase (JNK) phosphorylation. But LPLI did not stimulate [3H]thymidine incorporation into these cells. These results indicated that LPLI activated MAPK/ERK, a signal for proliferation, differentiation and survival, but did not activate the stress signals p38 MAPK and JNK in human dental pulp cells.

Comparative study of adult human skin fibroblasts and umbilical fibroblast-like cells

Expression of markers, collagens, and HLA-1 by human skin fibroblasts and fibroblast-like cells isolated from the umbilical Wharton's jelly was compared. Skin fibroblasts express collagens (proteins characteristic of differentiated cells of this histogenetic series) and HLA-1, while umbilical cells express, in addition to collagens, juvenile surface markers and almost no HLA-1. This indicates that fibroblast-like cells isolated from different sources are different and can serve as sources for the creation of cell preparations with different characteristics in future.

519. Development of S/MAR Plasmid Vectors for Persistent Expression and Maintenance, In Vivo

Currently used vectors in human gene therapy suffer from a number of limitations with respect to safety and reproducibility. The ideal vector has to be free of these effects and should allowlong-term expression of a transgene in the absence of selection. Recently a novel non-viral episomal expression system which fulfils these criteria, in principle, was reported. This vector, pEPI, replicates episomally in CHO, HeLa, K562, as well as in primary human fibroblast-like cells, and is mitotically stable in the absence of selection for more than 100 generations. Here we show the long term expression of pEPI in a range of cells including cystic fibrosis airway (IB3) cells to be stable for at least 10 weeks. We demonstrate, however, that antibiotic selection pressure, is necessary for the first weeks following transfection. FACS sorting of CHO and IB3 cells, show that expression and maintenance in cells without initial antibiotic selection diminished rapidly, whereas constructs under selection pressure for an initial period of 2 weeks were mitotically stable and expressed transgene for at least 10 weeks, even following the removal of selection pressure. However, initial in vivo results showed no persistance of vector and limited expression of the reporter gene when using viral promoters. We describe the developments we have made to overcome these limitations and the results of the application of an S/MAR plasmid carrying a human promoter and chromosomal destabilising elements to drive the expression and episomal maintenance of these vectors in vivo. Currently used vectors in human gene therapy suffer from a number of limitations with respect to safety and reproducibility. The ideal vector has to be free of these effects and should allowlong-term expression of a transgene in the absence of selection. Recently a novel non-viral episomal expression system which fulfils these criteria, in principle, was reported. This vector, pEPI, replicates episomally in CHO, HeLa, K562, as well as in primary human fibroblast-like cells, and is mitotically stable in the absence of selection for more than 100 generations. Here we show the long term expression of pEPI in a range of cells including cystic fibrosis airway (IB3) cells to be stable for at least 10 weeks. We demonstrate, however, that antibiotic selection pressure, is necessary for the first weeks following transfection. FACS sorting of CHO and IB3 cells, show that expression and maintenance in cells without initial antibiotic selection diminished rapidly, whereas constructs under selection pressure for an initial period of 2 weeks were mitotically stable and expressed transgene for at least 10 weeks, even following the removal of selection pressure. However, initial in vivo results showed no persistance of vector and limited expression of the reporter gene when using viral promoters. We describe the developments we have made to overcome these limitations and the results of the application of an S/MAR plasmid carrying a human promoter and chromosomal destabilising elements to drive the expression and episomal maintenance of these vectors in vivo.

Cellular and molecular effects for mutation induction in normal human cells irradiated with accelerated neon ions

We investigated the linear energy transfer (LET) dependence of mutation induction on the hypoxanthine-guanine phosphoribosyl transferase ( HPRT) locus in normal human fibroblast-like cells irradiated with accelerated neon-ion beams. The cells were irradiated with neon-ion beams at various LETs ranging from 63 to 335 keV/μm. Neon-ion beams were accelerated by the Riken Ring Cyclotron at the Institute of Physical and Chemical Research in Japan. Mutation induction at the HPRT locus was detected to measure 6-thioguanine-resistant clones. The mutation spectrum of the deletion pattern of exons of mutants was analyzed using the multiplex polymerase chain reaction (PCR). The dose–response curves increased steeply up to 0.5 Gy and leveled off or decreased between 0.5 and 1.0 Gy, compared to the response to 137Cs γ-rays. The mutation frequency increased up to 105 keV/μm and then there was a downward trend with increasing LET values. The deletion pattern of exons was non-specific. About 75–100% of the mutants produced using LETs ranging from 63 to 335 keV/μm showed all or partial deletions of exons, while among γ-ray-induced mutants 30% showed no deletions, 30% partial deletions and 40% complete deletions. These results suggested that the dose–response curves of neon-ion-induced mutations were dependent upon LET values, but the deletion pattern of DNA was not.

Differential regulation of anti-inflammatory proteins in human rheumatoid synoviocyte MH7A cell by celecoxib and ibuprofen

Non-steroidal anti-inflammatory drugs are known to be the most widely used drugs to exert their anti-inflammatory activities. It was examined protein expression profiles of human rheumatoid fibroblast-like synoviocyte MH7A cells treated with celecoxib, a selective cyclooxygenase-2 inhibitor, or ibuprofen, a non-selective cyclooxygenase inhibitor, using two-dimensional gel electrophoresis for comparison the mechanism of the drugs. Altered expression pattern in response to celecoxib is significantly different from that of ibuprofen treated cells. When MH7A cells were treated with celecoxib, 28 proteins were affected at their expression levels. Among them, heat shock proteins (Hsp60 and 70), glucose regulated proteins (Hsp75 and 78) were observed to be up-regulated by 1 to 30 μM concentrations of celecoxib but those proteins were not affected in ibuprofen treated cells. On the other hand, the expression of 19 proteins was changed by ibuprofen and the expression of apolipoprotein E, RNA binding motif 4, CTP-phosphocholine cytidylyltransferase, and phospholipase A2 inhibitory protein was only altered by ibuprofen. The expressions of 15 proteins were affected by both celecoxib and ibuprofen. Our results showed that celecoxib and ibuprofen, though they are known to act as cyclooxygenase inhibitors, could exert a different mode of acting mechanisms in anti-inflammatory processes. The chemical proteomic approach will be useful for figuring out the mode of actions of drugs.