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Light microscopy of FLG 29.1 cells. (a) Untreated cells ; (band c) cells treated with 0.1 AM TPA for 72 h (May-Grunwald-Giemsa). Bar, 20 Am .
Source publication
Studies on human osteoclast formation have been hampered by lack of a defined isolated progenitor cell population. We describe here the establishment of a human leukemic cell line (designated FLG 29.1) from bone marrow of a patient with acute monoblastic leukemia. The cultured cells are predominantly undifferentiated leukemic blasts, but addition o...
Contexts in source publication
Context 1
... Cytochemistry and Ultrastructure FLG 29 .1 cells stained with May-Grunwald-Giemsa ap- peared like undifferentiated leukemic blasts, with large round nuclei, prominent nucleoli, dispersed nuclear chro- matin and basophilic cytoplasm . The diameter of the cells ranged from 15 to 20 p,m (Fig. 1 a) . Less than 3 % of the cells were multinucleated, and commensurately larger in dimen- sions . Only a minority of the untreated cells adhered to the plastic surface . After 72 h of treatment with 0.1 p,M TPA, 36% of the cells were attached to the substrate and >45 multinucleated (3 to 12 nuclei) with size ranging from 50 to 100 jm (Fig ...
Context 2
... p,m (Fig. 1 a) . Less than 3 % of the cells were multinucleated, and commensurately larger in dimen- sions . Only a minority of the untreated cells adhered to the plastic surface . After 72 h of treatment with 0.1 p,M TPA, 36% of the cells were attached to the substrate and >45 multinucleated (3 to 12 nuclei) with size ranging from 50 to 100 jm (Fig . 1, b and c) . Multinucleated cells were found attached or in suspension and lacked characteristic periph- eral ruffling. Fluorochrome-conjugated Candida particles were not internalized by both untreated and TPAinduced FLG 29.1 cells . No detectable tPA was found in either the conditioned medium or the cytosol of untreated FLG 29 .1 cells . After ...
Citations
... In adult tissues, CTR is widely expressed, for example in neural networks (Becskei et al., 2004;Sexton, McKenzie & Mendelsohn, 1988), in osteoclasts and osteocytes (Gooi et al., 2010), renal distal epithelium, B and T-cells (Body et al., 1990;Cafforio et al., 2009), testis (Chausmer, Stuart & Stevens, 1980, lung (Fouchereau-Peron et al., 1981) and several other tissues (reviewed in Findlay (2006) and Wookey et al. (2010)). CTR is also expressed by specific cell types in wound healing (Wookey et al., 2010), in cardiovascular diseases (Wookey et al., 2008;Wookey, Zulli & Hare, 2009) and in several types of malignant tissues as breast (Gillespie et al., 1997) and prostate cancer (Thomas et al., 2006), as well as in cell lines derived from neoplasias of the lung (Findlay et al., 1980, Findlay, Michelangeli & Robinson, 1989, breast (Findlay et al., 1981;Gillespie et al., 1997;Kuestner et al., 1994), brain (Wookey et al., 2012a), bone osteoclasts (Gorn et al., 1995;Nicholson et al., 1987), prostate (Thomas, Muralidharan & Shah, 2007), and of lymphoid (Marx et al., 1974) and myeloid tissues (Gattei et al., 1992;Silvestris et al., 2008). ...
Background
Calcitonin expression is a well-established marker for medullary thyroid carcinoma (MTC); yet the role of calcitonin receptor (CTR), its seven-transmembrane G-protein coupled receptor, remains to be established in C-cells derived thyroid tumors. The aim of this work was to investigate CTR expression in MTC and to correlate such expression with clinicopathological features in order to evaluate its possible role as a prognostic indicator of disease aggressiveness and outcome.
Methods
Calcitonin receptor expression was analyzed in a series of 75 MTCs by immunohistochemistry, and by qPCR mRNA quantification in specimens from four patients. Statistical tests were used to evaluate the correlation between CTR expression and the clinicopathological and molecular characteristics of patients and tumors.
Results
Calcitonin receptor expression was detected in 62 out of 75 samples (82.7%), whereas 13 of the 75 samples (17.3%) were completely negative. CTR expression was significantly associated with expression of cytoplasmatic phosphatase and tensin homologue deleted on chromosome 10 and osteopontin, as well as with wild type RET/RAS genes and absence of tumor stroma, suggesting that CTR expression do not associate with clinicopathological signs of worse prognosis.
Discussion
Calcitonin receptor expression appears to be associated in MTC with more differentiated status of the neoplastic cells.
... Th e human preosteoclat FLG29.1 cells, established by Professor Maria Luisa Brandi of University of Florence, were derived from bone marrow cells of a patient aff ected with acute monoblastic leukemia (FAB:M5a), and was characterized as a model of osteoclastic precursor (Gattei et al. 1992). Th e cells were cultured with Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, Grand Island, NY, USA), supplemented with 10% fetal calf serum (Hyclone, Logan, UT, USA), 2 mmol/l L-glutamine (Amresco, Solon, OH, USA), 1 mM sodium pyruvate (Sigma-Aldrich, St Louis, MO, USA), 100 units/ml penicillin (Amresco Inc.) and 0.1 mg/ ml streptomycin (Amresco Inc.). ...
Purpose:
We aimed to investigate the effects of different apparent gravities (μ g, 1 g and 2 g) produced by large gradient high magnetic field (LGHMF) on human preosteoclast FLG29.1 cells.
Materials and methods:
FLG29.1 cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium. Cells were exposed to LGHMF for 72 h. On culture day 1, 2, 3, cell proliferation was detected by 3-(4,5)-dimethylthiahi-azo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) method. On day 3, cell apoptosis and necrosis were assayed by Hoechst and propidium iodide (PI) staining. After cells were exposed to LGHMF for 72 h with the induction of 12-o-tetradecanoylphorbol 13-acetate (TPA), Tartrate-Resistant Acid Phosphatase (TRAP) positive cells and nitric oxide (NO) release were detected by TRAP staining and Griess method, respectively. Intracellular TRAP activity was measured using nitrophenylphosphate (pNPP) as the substrate.
Results:
MTT detection revealed that compared to control, FLG 29.1 cell proliferation in the μ g and 2 g groups were promoted. However, there is no obvious difference between the 1 g and control groups. Hoechst-PI staining showed that LGHMF promoted cell apoptosis and necrosis, especially in the 2 g group. Exposure to LGHMF inhibited the NO concentration of supernatant. Both the TRAP activity and the number of TRAP positive cells were higher in cells of μ g group than those in 2 g group. In the 1 g group, they were decreased significantly compared to control.
Conclusions:
These findings indicate that LGHMF could directly affect human preosteoclast FLG29.1 cells survival and differentiation. High magnetic flux inhibited osteoclasts formation and differentiation while reduced apparent gravity enhanced osteoclastogenesis.
... The development of a maintainable cell line with characteristics of osteoclast precursors would allow further studies of the molecular and cellular biology of osteoclasts. Several cell lines representing osteoclast progenitors have been established from in vivo or in vitro immortalized cells [11][12][13][14], while previously established macrophage cell lines such as human leukemic cell line (FLG 29.1), murine macrophage cell line (BDM-1, RAW 264.7), have also been used to study osteoclastogenesis [9,15,16]. However, an osteoclast precursor cell line recapitulating the features of primary osteoclast differentiation and function has not been established. ...
Osteoclasts are bone-resorbing multinucleated cells differentiated from monocyte/macrophage lineage precursors. A novel osteoclast precursor cell line, 4B12 was established from Mac-1(+)c-Fms(+)RANK(+) cells from calvaria of 14-day-old mouse embryos using immunofluorescence and cell-sorting methods. Like M-CSF-dependent bone marrow macrophages (M-BMMs), M-CSF is required for 4B12 cells to differentiate into TRAP-positive multinucleated cells [TRAP(+) MNCs] in the presence of RANKL. Bone-resorbing osteoclasts differentiated from 4B12 cells on dentine slices possess both a clear zone and ruffled borders and express osteoclast-specific genes. Bone-resorbing activity, but not TRAP, was enhanced in the presence of IL-1alpha. The number of TRAP(+) MNCs and the number of pits formed from 4B12 cells on dentine slices was fourfold higher than that from M-BMMs. 4B12 cells were identified as macrophages with Mac-1 and F4/80, yet lost these markers upon differentiation into osteoclasts as determined by confocal laser scanning microscopy. The 4B12 cells do not have the potential to differentiate into dendritic cells indicating commitment to the osteoclast lineage. 4B12 cells are readily transfectable with siRNA transfection before and after differentiation. These data show that 4B12 cells faithfully replicate the properties of primary cells and are a useful and powerful model for analyzing the molecular and cellular regulatory mechanisms of osteoclastogenesis and osteoclast function.
... [17,18] Brandi and coworkers established osteoclasts-like cells from human leukemic blasts (FLG 29.1), induced by 12-O-tetradecanoylphorbol 13-acetate (TPA). [19] MC3T3-E1 cells, generated from newborn mouse calvaria, have demonstrated to have the capacity of osteoblastogenesis. [20] The cells display a fibroblastic morphology in the active growing phase. ...
... MC3T3-E1, C2C12, Saos-2, and HS-5 were purchased from ATCC, the FLG 29.1 cell line was established in house (M.L. Brandi). [19] Recombinant murine soluble RANK ligand (sRANKL) was purchased from PeproTech (Rocky Hill, NJ). Cell culture media were purchased from Invitrogen (Frederick, MD). ...
... RAW-OCs were purified by serum density gradient fractionation. [28] Human osteoclast precursors, FLG 29.1 cells, were maintained with RPMI-1640 supplemented with 10% FBS and induced with PMA (0.1 × 10 −6 M). [19] Morphological evidence initially appeared after 3 d exposure to PMA and continued to increase. The cells were harvested after one-week induction. ...
Bone‐targeting N ‐(2‐hydroxypropyl)methacrylamide (HPMA) copolymer‐PGE 1 conjugates, containing cathepsin K sensitive spacers, were incubated with induced osteoclasts and osteoblasts, their precursors, and control non‐skeletal cells. The release of PGE 1 was monitored by an HPLC assay. In both murine and human cell lines, osteoclasts appeared to be the most active cells in the cleavage (PGE 1 release). Incubation with osteoblasts also resulted in fast PGE 1 release, whereas precursor and control cells released PGE 1 with a substantially slower rate than bone cells (apparently through ester bond cleavage). Experiments in the presence of inhibitors revealed that other enzymes, in addition to cathepsin K, were participating in the cleavage of the conjugate. Confocal fluorescence studies exposed internalization of the conjugate by endocytosis with ultimate localization in the lysosomal/endosomal compartment.
magnified image
... The FLG 29.1 human cell line was derived and stabilized from a culture of bone marrow cells, collected from a patient affected with acute monoblastic leukemia (FAB: M5a) and then characterized as a model of osteoclastic precursor [23]. The cells were cultured in 250 ml flasks in RPMI 1640 (Sigma-Aldrich, Italy), supplemented with 10% fetal calf serum (FCS) (Sigma-Aldrich, Italy), at 37 • C and 5% CO 2 . ...
The aim of the present work is to determine whether mechanical stress caused by ultrasound (US) exposure affects osteoclastic precursor cells, thus addressing the hypothesis that mechanical strain-induced perturbation of preosteoclastic cell machinery can contribute to the occurrence of bone turnover alterations. Moreover, cell cytoskeleton was studied because of its supposed involvement in cell mechanotransduction.Our experimental model was the FLG 29.1 human cell line, previously characterized as an osteoclastic precursor model. Cell proliferation was quantified by trypan blue exclusion assay. Cell morpho-functional state was monitored by multispectral imaging autofluorescence microscopy. The expression of cytoskeletal components and markers of proliferation (Ki67) and osteoclastic differentiation (RANK) was analysed by immunocytochemistry.The findings demonstrated that US stimulation affects FLG 29.1 cell growth, depresses the expression of cytoskeletal components and markers of proliferation and differentiation, induces cell damage, thus supporting the hypothesis that US exposure inhibits osteoclastogenesis.These results have been compared with those obtained previously by exposure of FLG 29.1 cells to modelled hypogravity conditions. Finally, the possibility to utilize US stimulation for counteracting osteoporosis has been discussed.
... Therefore, in this study, we examined the influence of BVA on osteoblastic cellular responses by using human osteoblastic cells. To evaluate the possible synthesis of estrogen in cells of the osteoclastic lineage, we used the human leukemic cell line FLG 29.1, which is induced to differentiate toward the osteoclastic phenotype by phorbol esters (Gattei et al., 1992) and transforming growth factor-1 (TGF-1) (Fiorelli et al., 1994 ), and also the primary first-passage osteoblastic cells (hOB). The present results clearly demonstrated that the BVA strongly stimulated aromatase activation in FLG19.1 and hOB cells in vitro. ...
The disease preventive and health promotive approach of 'Ayurveda', which takes into consideration the whole body, mind and spirit while dealing with the maintenance of health, promotion of health and treating ailments is holistic and finds increasing acceptability in many regions of the world. Ancient Ayurvedic physicians had developed certain dietary and therapeutic measures to arrest/delay ageing and rejuvenating whole functional dynamics of the body system. This revitalization and rejuvenation is known as the 'Rasayan chikitsa' (rejuvenation therapy). Traditionally, Rasayana drugs are used against a plethora of seemingly diverse disorders with no pathophysiological connections according to modern medicine. Though, this group of plants generally possesses strong antioxidant activity, only a few have been investigated in detail. Over about 100 disorders like rheumatoid arthritis, hemorrhagic shock, CVS disorders, cystic fibrosis, metabolic disorders, neurodegenerative diseases, gastrointestinal ulcerogenesis and AIDS have been reported as reactive oxygen species mediated. In this review, the role of free radicals in these diseases has been briefly reviewed. 'Rasayana' plants with potent antioxidant activity have been reviewed for their traditional uses, and mechanism of antioxidant action. Fifteen such plants have been dealt with in detail and some more plants with less work have also been reviewed briefly.
... Cytogenetic analysis and reverse transcription-PCR to establish the presence of AML-associated fusion genes were performed according to standard methods (22). Primary AML blasts, Kasumi-1 cells (a human AML1/ETOpositive cell line derived from a myeloblastic leukemia; Ref. 23), and the human pre-osteoclastic leukemic cell line FLG 29.1, derived from a monoblastic leukemia (kindly provided by Dr. Bernabei, AOUC Careggi, Firenze, Italy; Ref. 24), were cultured in RPMI 1640 supplemented with 50 units/ml penicillin, 50 g of streptomycin, and 10% FCS at 37°C in a humidified atmosphere containing 5% CO 2 . Kasumi-1 cells (0.4 ϫ 10 6 /ml) and AML blasts (1 ϫ 10 6 /ml) were incubated in the presence of different doses of sodium butyrate (Sigma) or D1 (O-n-butanoil-2,3-O-isopropylidene-␣-D-mannofuranoside; kindly provided by Chiesi Pharmaceuticals, Varese, Italy). ...
Acute myeloid leukemia (AML) is a disease characterized by a block of maturation. Genes coding for core binding factors are rearranged in a considerable subset of AML cases and result in an altered interaction of core binding factor (CBF) subunits with transcriptional coregulators (NCoR/SMRT). Recruitment of histone deacetylase is also altered in AML, and a subsequent transcriptional repression of target genes involved in myeloid maturation is determined. We determined here the effects of two histone deacetylase inhibitors, sodium butyrate and the stable prodrug xylitol butyrate derivative (D1), on a t(8;21)-positive cell line (Kasumi-1) as well as primary AML blasts. Exposure (24-96 h) to butyrates (1 mM) of Kasumi-1 cells induced histone H4 acetylation, whereas H3 acetylation was unchanged. Induction of morphological and immunophenotypic granulocytic maturation (96 h), also confirmed by an increased expression of CAAT/enhancer binding protein alpha, was observed. Inhibition of proliferation and apoptosis via activation of caspase-9 was also observed. In primary AML blasts, butyrates (0.5 mM) increased histone H4 acetylation of 18 of 19 cases tested. Terminal granulocytic maturation was observed in all cases (5 of 5) characterized by chromosomal translocations involving CBF, whereas in non-CBF cases, maturation was incomplete (4 of 8) or absent (4 of 8). Our data indicate the possibility to effectively remove, in CBF AML cases, the maturation block generated by histone deacetylase stable recruitment, contributing to a possible development of molecularly targeted therapies of AML.
... Several haematopoietic cells and cell lines, grown in the presence of osteoblast/stromal cells, growth factors and cytokines, have been used to obtain osteoclast-like cells from their progenitors. These have included mature murine monocytes or macrophages (Udagawa et al. 1990), immortalised murine macrophage-like cells (Miyamoto et al. 1998), avian monocytic cells (Solari et al. 1996), human myelo-monocytic leukaemia cells (Gattei et al. 1992), mononuclear cells from giant cell tumours (James et al. 1996) and human peripheral blood mononuclear cells (Matsuzaki et al. 1998). Fully functional osteoclast-like cells develop from the rodent cultures, whereas multinucleated cells from the avian and the human systems have several osteoclast characteristics, but do not regularly resorb bone. ...
These guidelines review the relevant literature on the way plant phyto-oestrogens act on bone and the responsiveness of different bone cell systems to phyto-oestrogenic compounds. The primary emphasis is on the experimental conditions used, the markers available for assessing osteoblast and osteoclast function, and their expected sensitivity. Finally, we assess the published results to derive some general recommendations for in vitro experiments in this area of research.
... The human SW982 (synovial sarcoma), Hs913T, and HT-1080 (fibrosarcoma), SK-UT-1 (leiomyosarcoma), Jurkat and RAMOS (lymphoblastoid T cell), K562 (myeloid leukemia) cell lines, and the murine NIH3T3 (fibroblast) cells were purchased from ATCC. FLG 29.1 cells have been described previously (23) and were obtained from Dr. V. Gattei (CRO-IRCCS, Aviano, Italy). The murine NQ22 and NQ29 (lymphoblastoid T cells) cell lines have been described (24). ...
EMILIN-1 (Elastin Microfibril Interface Located ProteIN), the prototype of the EMILIN family, consists of a cysteine-rich domain (EMI domain) at the N terminus, an extended region with a high potential coiled-coil structure, a short collagenous stalk, and a self-interacting globular gC1q-l domain. EMILIN-1 is an adhesive extracellular matrix constituent associated with elastic fibers, detected also in the proximity of cell surfaces. To localize the cell attachment site(s), monoclonal antibodies (mAbs) against EMILIN-1 or the gC1q-1 domain were used to inhibit cell attachment to EMILIN-1. Thus, one mAb mapping to the gC1q-1 domain caused complete inhibition of cell attachment. EMILIN-1 and gC1q-1 displayed a comparable dose-dependent ability to promote cell adhesion. Adhesion kinetics was similar to that of fibronectin (FN), reaching the maximum level of attachment at 20 min, but in the absence of cations adhesion was negligible. The relative adhesion strength to detach 50% of the cells was similar for EMILIN-1 and gC1q-1 (250-270 x g) but lower than that for FN (>500). Cell adhesion to EMILIN-1 or gC1q-1 was completely blocked by a function-blocking beta(1) integrin subunit mAb. In contrast, adhesion to the complement C1q component was totally unaffected. Among the various function-blocking mAbs against the alpha integrin subunits only the anti-alpha(4) fully abrogated cell adhesion to gC1q-1 and up to 70% to EMILIN-1. Furthermore, only K562 cells transfected with the alpha(4) integrin chain, but not wild type K562, were able to adhere to EMILIN-1 and were specifically inhibited by anti-alpha(4) function-blocking mAb. Finally, cells attached to EMILIN-1 or gC1q-1, compared with cells plated on FN or vitronectin, which appeared well spread out on the substrate with prominent stress fibers and focal contacts, were much smaller with wide ruffles and a different organization status of the actin cytoskeleton along the cell periphery. This pattern was in accord with the ability of EMILIN-1 to promote cell movement.
... We found that a human leukemia cell line, FLG 29.1 cell, expressed herg, and an HERG current (I HERG ) with a very fast deactivation kinetics, which apparently justifies the low, depolarized value of their V REST (12,20). These cells derive from a patient with an M5a-type leukemia (21) and represent immature preosteoclastic precursors, as revealed by their treatment with phorbol esters (22). They are capable of adhering to bone endothelium, possibly through fibronectin (FN) molecules produced by the endothelial cells (23), and this interaction could influence the maturation of these cells through the osteoclastic pathway. ...
... Cell Culture-FLG 29.1 were obtained in Dr. P. A. Bernabei's laboratory (Hematological Unit, Florence, Italy) as previously reported (22). Cells were routinely cultured in RPMI medium (Hyclone) supplemented with 10% fetal calf serum (Hyclone) (complete medium) and incubated at 37°C in 10% CO 2 . ...
... The effect of FLG 29.1 cell adhesion to FN on cell differentiation was then evaluated: since FLG 29.1 cells show a preosteoclastic phenotype, as revealed by the appearance of osteoclastic markers upon TPA treatment (22), three of these markers, namely the CD 51/␣ V  3 , CtR (30), as well as TRAP (31) were studied. When FLG were left to adhere to FN for 24 h (in complete medium) the appearance of immunoreactivity to CD 51 was first detectable: in fact, this marker cannot be easily detected with this method in control cells ( Fig. 2A), while it appears in FN-seeded cells (Fig. 2B). ...
Integrin receptors have been demonstrated to mediate either "inside-to-out" and "outside-to-in" signals, and by this way are capable of regulating many cellular functions, such as cell growth and differentiation, cell migration, and activation. Among the various integrin-centered signaling pathways discovered so far, we demonstrated that the modulation of the electrical potential of the plasma membrane (V(REST)) is an early integrin-mediated signal, which is related to neurite emission in neuroblastoma cells. This modulation is sustained by the activation of HERG K(+) channels, encoded by the ether-à-go-go-related gene (herg). The involvement of integrin-mediated signaling is being discovered in the hemopoietic system: in particular, osteoclasts are generated as well as induced to differentiate by interaction of osteoclast progenitors with the stromal cells, through the involvement of integrin receptors. We studied the effects of cell interaction with the extracellular matrix protein fibronectin (FN) in a human leukemic preosteoclastic cell line (FLG 29.1 cells), which has been demonstrated to express HERG currents. We report here that FLG 29.1 cells indeed adhere to purified FN through integrin receptors, and that this adhesion induces an osteoclast phenotype in these cells, as evidenced by the appearance of tartrate-resistant acid phosphatase, as well as by the increased expression of CD51/alpha(v)beta(3) integrin and calcitonin receptor. An early activation of HERG current (I(HERG)), without any increase in herg RNA or modifications of HERG protein was also observed in FN-adhering cells. This activation is apparently sustained by the beta(1) integrin subunit activation, through the involvement of a pertussis-toxin sensitive G(i) protein, and appears to be a determinant signal for the up-regulation of alpha(v)beta(3) integrin, as well as for the increased expression of calcitonin receptor.
