Routine Culture Conditions Research Articles

Serum-Free Medium Provides a Clinically Relevant Method to Increase Olfactory Ensheathing Cell Numbers in Olfactory Mucosa Cell Culture.

There is much evidence to suggest that transplanted olfactory ensheathing cells may ameliorate the functional deficits associated with injuries to the nervous system, especially the spinal cord. For clinical implementation of this strategy it will be necessary to derive large numbers of these cells from an accessible and, preferably, autologous source, implying that olfactory mucosa would be ideal. Although olfactory ensheathing cells can be derived from olfactory mucosa, in routine culture conditions the proportion of these cells is unacceptably low for clinical purposes. This study compared the effect of culturing dissociated olfactory bulb and olfactory mucosa in two different media: one containing serum and one serum free. The results indicate that olfactory ensheathing cell proportion, and absolute cell numbers, is greatly increased in serum-free conditions. Further analysis suggests that serum-free medium has a differential effect on contaminating fibronectin-positive and p75-positive cells from olfactory bulb and olfactory mucosa. This study demonstrates that serum-free culture conditions provide a simple and useful means of deriving a sufficient number of olfactory ensheathing cells for transplantation and reveals a difference in biological behavior of the cells contained within olfactory bulb and olfactory mucosa.

The Fatty Acid Transport Protein (FATP) Family: Very Long Chain Acyl-CoA Synthetases or Solute Carriers?

Cellular fatty acids typically derive from uptake from the extracellular milieu and, to a lesser extent, de novo synthesis. Extracellular fatty acids must traverse the plasma membrane, after which they are activated to their CoA thioesters for subsequent metabolism. Both uptake and metabolism are rapid processes, and there has been considerable debate as to whether transport of fatty acids across the lipid bilayer of the plasma membrane proceeds by diffusion or requires transport proteins. One group of proteins proposed to translocate fatty acids is the six-member Fatty Acid Transport Protein (FATP) family. These proteins were designated as such because when overexpressed, host cells exhibited higher rates of accretion of radioactive or fluorescent fatty acids. However, one member of this family, FATP2, is identical to an enzyme with very long-chain acyl-CoA synthetase (ACSVL) activity. This enzyme (ACSVL1 or FATP2), was isolated using classical protein purification techniques. In fact, the six-member ACSVL protein family is identical to the six-member FATP family. We and others have established that all six proteins have acyl-CoA synthetase activity. It remains to be established whether they participate in the physical translocation process, or facilitate transport by trapping, as CoA derivatives, fatty acids that enter cells by diffusion. To characterize the biological functions of the ACSVLs, we are investigating the properties of the overexpressed proteins and the endogenous proteins. We observed that for many ACSVLs, the subcellular location of the overexpressed protein differs from that of the endogenous protein. Using RNA interference (siRNA), we knocked down expression of FATP4 (proposed name: ACSVL5) in Neuro2a cells. Activation of both long-chain (C16:0) and very long-chain fatty acids (C24:0) was decreased when FATP4 was depleted. Despite decreased enzyme activity, initial rates of uptake of [14C]C16:0 were not affected when FATP4 was depleted. In contrast, COS-1 cells overexpressing FATP4 showed enhanced [14C]C16:0 uptake. Neither endogenous (Neuro2a) nor overexpressed (COS-1) FATP4 was localized to plasma membrane under routine cell culture conditions, but rather were found in intracellular membrane compartments. We conclude that, in the cell lines studied, endogenous FATP4 does not function to translocate FA across the plasma membrane.

Hypoxia regulates PGE2 release and EP1 receptor expression in osteoblastic cells

Changes in regional O(2) tension that occur during fracture and skeletal unloading may stimulate local bone cell activity and ultimately regulate bone maintenance and repair. The mechanisms by which bone cells sense and respond to changes in O(2) tension are unclear. In this study we investigated the effects of low O(2) on activation of the hypoxia response element (HRE), prostaglandin E(2) (PGE(2)) production, PGE(2) receptor (EP) expression and proliferation in MC3T3-E1 osteoblastic cells. Cells were cultured for up to 72 h in 2% O(2) (considered hypoxic), 5% O(2) (in the range of normal O(2) tension in vivo) or 21% O(2) (commonly used for cell culture). Cells cultured in 2% O(2) showed activation of the HRE, increased PGE(2) release, increased EP1 expression, and reduced cell proliferation compared to cells grown at 21% O(2). Similarly, cells cultured in 5% O(2) showed increased expression of EP1 and a trend toward a decrease in proliferation, but no activation of the HRE or increase in PGE(2) levels. Expression of EP2, EP3 and EP4 were not affected by O(2) tension. The differences in EP receptor profile observed in cells grown at 5% compared to 21% O(2) suggest that bone cell phenotype may be altered under routine cell culture conditions. Furthermore, our data suggest that hypoxia-dependent PGE(2) production and EP1 expression in bone cells may play a role in bone remodeling and repair in regions of compromised or damaged bone, where O(2) tension is low.

The Early-Immediate Gene EGR-1 Is Induced by Transforming Growth Factor-β and Mediates Stimulation of Collagen Gene Expression

Transforming growth factor-beta (TGF-beta) stimulates collagen synthesis and accumulation, and aberrant TGF-beta signaling is implicated in pathological organ fibrosis. Regulation of type I procollagen gene (COL1A2) transcription by TGF-beta involves the canonical Smad signaling pathway as well as additional protein and lipid kinases, coactivators, and DNA-binding transcription factors that constitute alternate non-Smad pathways. By using Affymetrix microarrays to detect cellular genes whose expression is regulated by Smad3, we identified early growth response factor-1 (EGR-1) as a novel Smad3-inducible gene. Previous studies implicated Egr-1 in cell growth, differentiation, and survival. We found that TGF-beta induced rapid and transient accumulation of Egr-1 protein and mRNA in human skin fibroblasts. In transient transfection assays, TGF-beta stimulated the activity of the Egr-1 gene promoter, as well as that of a minimal Egr-1-responsive reporter construct. Furthermore, TGF-beta enhanced endogenous Egr-1 interaction with a consensus Egr-1-binding site element and with GC-rich DNA sequences of the human COL1A2 promoter in vitro and in vivo. Forced expression of Egr-1 by itself caused dose-dependent up-regulation of COL1A2 promoter activity and further enhanced the stimulation induced by TGF-beta. In contrast, the TGF-beta response was abrogated when the Egr-1-binding sites of the COL1A2 promoter were mutated or deleted. Furthermore, Egr-1-deficient embryonic mouse fibroblasts showed attenuated TGF-beta responses despite intact Smad activation, and forced expression of ectopic EGR-1 in these cells could restore COL1A2 stimulation in a dose-dependent manner. Taken together, these findings identify Egr-1 as a novel intracellular TGF-beta target that is necessary for maximal stimulation of collagen gene expression in fibroblasts. The results therefore implicate Egr-1 in the profibrotic responses elicited by TGF-beta and suggest that Egr-1 may play a new and important role in the pathogenesis of fibrosis.

Interleukin-1β induced vascular permeability is dependent on induction of endothelial Tissue Factor (TF) activity

IL-1β is a pleotropic cytokine that may mediate increased procoagulant activity and permeability in endothelial tissue during inflammatory conditions. The procoagulant effects of IL-1β are mediated through induction of tissue factor (TF) but its alterations on vascular permeability are not well characterized. We found that IL-1β induced a rapid and dose-dependent increase in TF activity in human umbilical vein endothelial cells (ECs) under routine culture conditions. However, IL-1β caused a rapid and marked increase in permeability across confluent EC monolayers using a two-compartment in vitro model only in the presence of factor VIII-deficient plasma that was completely abrogated by neutralizing anti-TF antibody pre-treatment. In vitro permeability was associated with loss of EC surface expression of VE-cadherin and contraction of F-actin cytoskeletal elements that resulted in EC intercellular gap formation. These data demonstrate that IL-1β induces marked changes in permeability across activated endothelium via a TF dependent mechanism and suggest that modulation of TF activity may represent a strategy to treat various acute and chronic inflammatory conditions mediated by this cytokine.

Multinuclear giant cell formation is enhanced by down-regulation of Wnt signaling in gastric cancer cell line, AGS

AGS cells, which were derived from malignant gastric adenocarcinoma tissue, lack E-cadherin-mediated cell adhesion but have a high level of nuclear β-catenin, which suggests altered Wnt signal. In addition, approximately 5% of AGS cells form multinuclear giant cells in the routine culture conditions, while taxol treatment causes most AGS cells to become giant cells. The observation of reduced nuclear β-catenin levels in giant cells induced by taxol treatment prompted us to investigate the relationship between Wnt signaling and giant cell formation. After overnight serum starvation, the shape of AGS cells became flattened, and this morphological change was accompanied by decrease in Myc expression and an increase in the giant cell population. Lithium chloride treatment, which inhibits GSK3β activity, reversed these serum starvation effects, which suggests an inverse relationship between Wnt signaling and giant cell formation. Furthermore, the down-regulation of Wnt signaling caused by the over-expression of ICAT, E-cadherin, and Axin enhanced giant cell formation. Therefore, down-regulation of Wnt signaling may be related to giant cell formation, which is considered to be a survival mechanism against induced cell death.

Molecular typing of Salmonella enterica serovar typhi isolates from various countries in Asia by a multiplex PCR assay on variable-number tandem repeats.

A multiplex PCR method incorporating primers flanking three variable-number tandem repeat (VNTR) loci (arbitrarily labeled TR1, TR2, and TR3) in the CT18 strain of Salmonella enterica serovar Typhi has been developed for molecular typing of S. enterica serovar Typhi clinical isolates from several Asian countries, including Singapore, Indonesia, India, Bangladesh, Malaysia, and Nepal. We have demonstrated that the multiplex PCR could be performed on crude cell lysates and that the VNTR banding profiles produced could be easily analyzed by visual inspection after conventional agarose gel electrophoresis. The assay was highly discriminative in identifying 49 distinct VNTR profiles among 59 individual isolates. A high level of VNTR profile heterogeneity was observed in isolates from within the same country and among countries. These VNTR profiles remained stable after the strains were passaged extensively under routine laboratory culture conditions. In contrast to the S. enterica serovar Typhi isolates, an absence of TR3 amplicons and a lack of length polymorphisms in TR1 and TR2 amplicons were observed for other S. enterica serovars, such as Salmonella enterica serovar Typhimurium, Salmonella enterica serovar Enteritidis, and Salmonella enterica serovar Paratyphi A, B, and C. DNA sequencing of the amplified VNTR regions substantiated these results, suggesting the high stability of the multiplex PCR assay. The multiplex-PCR-based VNTR profiling developed in this study provides a simple, rapid, reproducible, and high-resolution molecular tool for the epidemiological analysis of S. enterica serovar Typhi strains.

Human chorionic gonadotropin and 17-β estradiol regulation of human oviductin/oviduct specific glycoprotein mRNA expression in vitro

Objective To determine whether oviduct mucosal cell culture with exogenous hCG or 17-β estradiol (E 2) supports the continued production of oviductin, a putative embryotrophic protein. Design Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of oviductin mRNA expression after oviduct mucosal cell culture in the presence of hCG or 17-β E 2. Setting University-based Obstetrics and Gynecology Department. Subject(s) Ten women undergoing laparoscopy for tubal sterilization or hysterectomy for uterine fibroids. Intervention(s) The mucosal layer was isolated from the oviduct tissue, subjected to routine culture conditions with the addition of various concentrations of hCG or 17-β E 2 or the equivalent vehicle-only control and semiquantitative RT-PCR performed. Main outcome measure(s) The relationship between exposure to hCG or 17-β E 2 and expression of oviductin mRNA by cultured oviduct mucosal cells. Result(s) There was a significant increase in oviductin mRNA expression after the addition of hCG to the culture medium but only in samples that had maintained a baseline level of oviductin expression. Addition of 17-β E 2 to the culture medium had no significant effect on oviductin mRNA expression. Conclusion(s) Under standard cell culture conditions, baseline human oviductin mRNA expression is increased by the addition of hCG. This effect is likely to be a secondary or synergistic effect as exogenous hCG failed to restore oviductin mRNA expression in samples where expression was lost after culture. E 2 failed to alter oviductin mRNA expression in oviduct mucosal cells cultured under these conditions.

Duration of senescent cell survival in vitro as a characteristic of organism longevity, an additional to the proliferative potential of fibroblasts

More than 40 years have passed since the original publication by Hayflick and Moorhead led to the concept of the ‘Hayflick limit’ of the maximum number of divisions which somatic cells undergo in vitro. This concept is still regarded as a fundamental characteristic of species longevity. Here we want to emphasize another characteristic of somatic cells, namely, the duration of their survival in vitro in the non-dividing state after cessation of proliferation. This is suggested on the basis of results of recent experiments with so-called Japanese accelerated senescent mice. Results of these experiments reveal a good correlation between the longevity of the mice, the number of duplications of their fibroblasts in vitro, and the survival time of these cells in the non-dividing state. In routine culture conditions, cell survival time may be very long, as much as a few years. However, when the cells are grown under conditions of oxidative stress, cellular longevity is markedly shortened. This new test may serve as an additional marker of organismic longevity. The comparative value of both tests, the classical ‘Hayflick limit’ and the new test, is discussed.

Calpain Inhibition Decreases the Growth Rate of Mammalian Cell Colonies

The calpains, a family of calcium-requiring intracellular proteases, are proposed regulators of cell proliferation. However, ablation of the calpain small subunit gene necessary for function of the conventional calpains did not result in decreased rate of proliferative growth of mouse stem cells under routine culture conditions. To address the reasons for this discrepancy, Chinese hamster ovary cell lines were established that overexpress the calpain inhibitor protein, calpastatin, under control of the ecdysone congener, ponasterone A. Overexpression of calpastatin in these cell lines resulted in a decreased growth of isolated colonies adhering to tissue culture plates. However, when cells were plated at higher density, calpastatin overexpression had no influence on proliferative growth rate. Growth of colonies in soft agar was not inhibited by calpastatin overexpression. Cell adhesion, cell de-adhesion, and cell motility all appeared to be normal after calpastatin overexpression. Differential display analysis was initiated to detect possible alteration of gene expression upon calpastatin overexpression. Analysis of approximately 3000 differential display PCR signals resulted in identification of one band that was underexpressed. Northern blot analysis confirmed a decreased amount of approximately 1 kb mRNA in cells overexpressing calpastatin. Sequence analysis identified a putative protein, Csr, containing a region homologous to two ubiquitin transferases and a putative cation channel protein.

Human oviductin mRNA expression is not maintained in oviduct mucosal cell culture

Objective: To determine whether oviduct mucosal cell culture supports the continued production of oviductin, a putative embryotrophic protein. Design: Semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR) analysis of oviductin messenger RNA (mRNA) expression after oviduct mucosal cell culture. Setting: University-based obstetrics and gynecology department. Patient(s): Ten women undergoing laparoscopy for tubal sterilization or hysterectomy for uterine fibroids.. Intervention(s): The mucosal layer was isolated from the oviduct tissue and subjected to routine culture conditions; semiquantitative RT-PCR was performed. Main Outcome Measure(s): The relationship between duration of cell culture and expression of oviductin mRNA. Result(s): There was a significant reduction in oviductin mRNA expression after 3 days in culture, with a complete loss after 6 days in 70% of the samples and after 12 days in the remaining 30%. Conclusion(s): This is the first study to investigate whether oviductin mRNA continues to be expressed in cultured human oviduct mucosal cells. Our results suggest that oviduct mucosal cells lose their ability to produce oviductin after short-term culture. This method of culture does not appear to be appropriate for a coculture system reliant upon oviductal secretion of oviductin.

Cell density mediated pericellular hypoxia leads to induction of HIF-1alpha via nitric oxide and Ras/MAP kinase mediated signaling pathways.

Environmental signals in the cellular milieu such as hypoxia, growth factors, extracellular matrix (ECM), or cell-surface molecules on adjacent cells can activate signaling pathways that communicate the state of the environment to the nucleus. Several groups have evaluated gene expression or signaling pathways in response to increasing cell density as an in vitro surrogate for in vivo cell-cell interactions. These studies have also perhaps assumed that cells grown at various densities in standard in vitro incubator conditions do not have different pericellular oxygen levels. However, pericellular hypoxia can be induced by increasing cell density, which can exert profound influences on the target cell lines and may explain a number of findings previously attributed to normoxic cell-cell interactions. Thus, we first sought to test the hypothesis that cell-cell interactions as evaluated by the surrogate approach of increasing in vitro cell density in routine normoxic culture conditions results in pericellular hypoxia in prostate cancer cells. Second, we sought to evaluate whether such interactions affect transcription mediated by the hypoxia response element (HRE). Thirdly, we sought to elucidate the signal transduction pathways mediating the induction of HRE in response to cell density induced pericellular hypoxia in routine normoxic culture conditions. Our results indicate that paracrine cell interactions can induce nuclear localization of HIF-1a protein and this translocation is associated with strong stimulation of the HRE-reporter activity. We also make the novel observation that cell density-induced activity of the HRE is dependent on nitric oxide production, which acts as a diffusible paracrine factor secreted by densely cultured cells. These results suggest that paracrine cell interactions associated with pericellular hypoxia lead to the physiological induction of HRE activity via the cooperative action of Ras, MEK1, HIF-1a via pericellular diffusion of nitric oxide. In addition, these results highlight the importance of examining pericellular hypoxia as a possible stimulus in experiments involving in vitro cell density manipulation even in routine normoxic culture conditions.

Diversity in a variable-number tandem repeat from Yersinia pestis.

We have identified a tetranucleotide repeat sequence, (CAAA)(N), in the genome of Yersinia pestis, the causative agent of plague. This variable-number tandem repeat (VNTR) region has nine alleles and great diversity (calculated as 1 minus the sum of the squared allele frequencies) (diversity value, 0.82) within a set of 35 diverse Y. pestis strains. In contrast, the nucleotide sequence of the lcrV (low-calcium-response) gene differed only slightly among these strains, having a haplotype diversity value of 0.17. Replicated cultures, phenotypic variants of particular strains, and extensively cultured replicates within strains did not differ in VNTR allele type. Thus, while a high mutation rate must contribute to the great diversity of this locus, alleles appear stable under routine laboratory culture conditions. The classic three plague biovars did not have single identifying alleles, although there were allelic biases within biovar categories. The antiqua biovar was the most diverse, with four alleles observed in 5 strains, while the orientalis and mediaevalis biovars exhibited five alleles in 21 strains and three alleles in 8 strains, respectively. The CAAA VNTR is located immediately adjacent to the transcriptional promoters for flanking open reading frames and may affect their activity. This VNTR marker may provide a high-resolution tool for epidemiological analyses of plague.

Mycobacterium marinum hand infection: case reports and review of literature

Three patients with Mycobacterium marinum infection are described. Key elements in the diagnosis of this infection are a high index of suspicion, taking a history with an emphasis on exposure to tropical fish or other potential sources of M. marinum infection, and tissue biopsy for culture and histology. The microbiologist should be informed about the suspicion of M. marinum infection so that appropriate cultures can be performed. As M. marinum does not grow under routine culture conditions, the diagnosis is easily missed resulting in delayed treatment. The treatment is essentially antimicrobial therapy for the superficial lesions supplemented by an appropriate surgical debridement especially when deep structures are involved.

Expression of transforming growth factor-beta receptors types II and III within various cells in the rat periodontium.

This study reports the immunohistochemical localization of TGF-beta receptor type II (T beta R-II) and type III (T beta R-III) in cells of the forming periodontal ligament (PDL) in rat first molar roots. Mandibular periodontium was obtained from 3, 6 and 12-wk-old rats. This represented tissue from the initial, pre-mature and post-mature stages of root and periodontal development, respectively. Mandibular bone chips and molar roots were used to isolate osteoblasts, fibroblasts and cementoblasts. Cells were obtained using a 2-step trypsinization and explant technique, and cultured in Dulbecco's modification of Eagle's medium (DMEM) under routine cell culture conditions. Cells were cultured on coverslips for the purpose of detecting TGF-beta receptors, and compared with whole tissue sections using the same detection method. Cells which stained positively for T beta R-II and T beta R-III on both paraffin sections and cultured cell slides were counted. Both receptors were expressed in the various periodontal tissue compartments. PDL fibroblasts, cementoblasts and osteoblasts were stained positively for T beta R-II and T beta R-III. Endothelial cells were noted to be positive for T beta R-II only. T beta R-II was more widely distributed in cells than T beta R-III, but T beta R-III was extensively localized in the extracellular matrix. Both receptors were expressed on the cell membrane and also localized in the cytoplasm. The findings for paraffin sections were consistent with the immunohistochemical staining of cultured cells. The percentage of cells which stained positively for T beta R-II was greater (approximately 85%) than that for T beta R-III (approximately 60%) in all major types of the PDL cells on both paraffin sections and cultured cell slides. Extensive location of TGF-beta receptors in both cells and extracellular matrix suggests that several binding sites are available for TGF-beta s to interact with target cells during development and following maturation of the periodontium.

Respiratory metabolism and swimming performance in growth hormone transgenic Atlantic salmon

We show that growth hormone enhanced transgenic salmon have a higher oxygen uptake during routine culture conditions and during forced swimming activity relative to similarly sized control fish. They also have a slightly higher critical oxygen level that limits oxygen uptake. However, they do not differ in regards to critical swimming speed. Growth hormone transgenic Atlantic salmon, Salmo salar, at 12-13°C were an F2 generation using eggs from a transgenic F1 female and milt from a nontransgenic male. They grew two to three times faster than control fish throughout the study period. Under routine culture conditions, both transgenic and control fish exhibited a diurnal cycle in oxygen uptake. However, oxygen uptake of transgenic fish was 1.7 times that of control fish at all times of the day. Oxygen uptake was independent of oxygen concentration above 10 mg/L in both groups of fish; critical oxygen uptake level was 6 mg/L in transgenic fish and 4 mg/L in control fish. During the oxygen decrease, transgenic fish and control fish lost equilibrium at the same low oxygen concentration levels. In the swim tunnel, oxygen uptake of transgenic fish was 1.6 times that of control fish at all swimming speeds. Critical swimming speeds did not differ between transgenic and control fish and were similar to literature values for salmonids.

Production and pre-clinical significance of haematopoietic peptide growth factors (HPGF) in human non-seminomatous germ cell tumour cell lines.

Peptide growth factors involved in the regulation of haematopoiesis (HPGF), for example granulocyte-colony-stimulating factor (G-CSF) and granulocyte/macrophage-colony-stimulating factor (GM-CSF), are of clinical importance in the treatment of testicular germ cell tumour (GCT) patients with modern chemotherapy regimens since they ameliorate chemotherapy-induced neutropenia. Aberrant expression of and/or response to HPGF has been reported in several solid tumour types although no data are available on GCT with the exception of those on stem cell factor (SCF). The aims of this pre-clinical study were twofold: (1) to screen a panel of human non-seminomatous (NS)GCT for the production of HPGF and (2) to test the effects of G-CSF or SCF on the growth of NSGCT cell lines in vitro, and on the growth kinetics of two human NSGCT xenograft models. HPGF concentrations in cell culture supernatant from 11 NSGCT cell lines growing under routine culture conditions were measured by enzyme-linked immunosorbent assay. The growth kinetics of cell lines was quantified in vitro using the sulphorhodamine B assay. The growth kinetics of nude mouse NSGCT xenografts was followed by measuring tumour volumes every 2-3 days over days 1-30, following daily subcutaneous injection of nude mice (days 1-14). The cell lines produced G-CSF (1/11 cell lines), GM-CSF (2/11), SCF (2/11), M-CSF (6/11). and interleukin-6 (9/11). Growth stimulation of cell line H12.1 by SCF was observed in vitro, but no statistically significant differences in NSGCT xenograft tumour volume (VT) or relative VT (r VT) in treated groups were observed on days 14 or 29 compared to the control. The change in rVT of H12.1 xenografts treated with G-CSF alone compared to control (rVT/rVT,c) was 0.96 on day 29. The values for rVT/rVT,c for H12.1 xenografts treated with G-CSF in combination with low- or high-dose SCF were, respectively, 1.67 or 1.7 compared to 1.19 for SCF-treated mice. The results are in agreement with clinical data to date where no observations have been reported of stimulation or inhibition of tumours in patients receiving treatment with G-CSF. Before any clinical trials are initiated in GCT patients treated with G-CSF in combination with SCF, further pre-clinical experiments with this tumour type are recommended to investigate this phenomenon further in a greater number of NSGCT cell lines in vitro and in vivo and with a wider range of SCF/G-CSF schedules. The potential relevance of secretion of HPGF in NSGCT cell lines in vitro to the pathobiology of GCT in patients is also a subject of interest for future research.

Spontaneous immortalisation of ensheathing cells from adult rat olfactory nerve.

In this report, we describe the isolation of a cell line, Rolf B1.T, from cultures of adult rat olfactory nerve cells. Rolf B1.T cells have an antigenic phenotype which closely resembles that of olfactory ensheathing cells. In routine culture conditions, Rolf B1.T cells constitutively express glial fibrillary acidic protein, S1OO, the low-affinity neurotrophin receptor p75 NGF, laminin, tenascin, and the neural cell adhesion molecule (N-CAM); a variable proportion of the cells also express cadherin, which is regulated by local culture conditions and is associated positively with cell proliferation status. We provide evidence that the association may be indirect and linked to a related parameter such as local cell density. Rolf B1.T cells arose from a population of less well-differentiated cells after a spontaneous immortalisation event. The cells retain many characteristics of normal cells, are dependent on serum growth factors for their proliferation, and fail to grow in semi-solid agar. Rolf B1.T cells support the regrowth of neurites from adult retinal ganglion cells in vitro in a heterologous co-culture system and will have potential value in investigations into the mechanisms of glial support for axonal regeneration from adult mammalian central neurons.

Spontaneous immortalisation of ensheathing cells from adult rat olfactory nerve

In this report, we describe the isolation of a cell line, Rolf B1.T, from cultures of adult rat olfactory nerve cells. Rolf B1.T cells have an antigenic phenotype which closely resembles that of olfactory ensheathing cells. In routine culture conditions, Rolf B1.T cells constitutively express glial fibrillary acidic protein, S100, the low-affinity neurotrophin receptor p75 NGF′, laminin, tenascin, and the neural cell adhesion molecule (N-CAM); a variable proportion of the cells also express cadherin, which is regulated by local culture conditions and is associated positively with cell proliferation status. We provide evidence that the association may be indirect and linked to a related parameter such as local cell density. Rolf B1.T cells arose from a population of less well-differentiated cells after a spontaneous immortalisation event. The cells retain many characteristics of normal cells, are dependent on serum growth factors for their proliferation, and fail to grow in semi-solid agar. Rolf B1.T cells support the regrowth of neurites from adult retinal ganglion cells in vitro in a heterologous co-culture system and will have potential value in investigations into the mechanisms of glial support for axonal regeneration from adult mammalian central neurons. © 1996 Wiley-Liss, Inc.

Ligand Activation of Overexpressed Epidermal Growth Factor Receptor Results in Colony Dissociation and Disturbed E-Cadherin Function in HSC-1 Human Cutaneous Squamous Carcinoma Cells

Various types of tumors show aberrant expression and overexpression of epidermal growth factor (EGF) receptor and the degree of receptor expression correlates with a malignant phenotype in many epithelial tumors. However,in vitroevidence supporting the advantageous role of receptor overexpression is deficient. In this study, we compared the effects of exogenous EGF on the cell colony morphology in monolayer and collagen gel culture between HSC-1 human squamous carcinoma cells overexpressing EGF receptor and their revertant subline cells. These cells formed coherent cell colonies under routine culture conditions, but addition of EGF induced dissociation of cell colonies within 24 h in the parent HSC-1 cells, though not in the subline cells. Since the colony dissociation apparently involved loss of cell–cell adhesion, we also studied the effects of EGF on E-cadherin expression and its function. Cell aggregation assays showed that EGF reduced E-cadherin function dose-dependently in the parent cells, but not in the subline cells. However, immunoblotting analysis and ELISA showed the absence of downregulation or degradation of E-cadherin. Instead, EGF tyrosine phosphorylated cadherin/catenin complex components including β-catenin and increased the detergent solubility of E-cadherin in the parent cells. These results suggest that EGF modified the functional association between E-cadherin and actin filament through tyrosine phosphorylation of the cadherin/catenin complex and thereby made the adhesion molecule incompetent. Our results indicate that the ligand activation of overexpressed EGF receptor impairs E-cadherin-mediated cell–cell adhesion and causes dissociation of the squamous carcinoma cell colonies, which facilitates tumor cell invasionin vivo.This might be relevant to the advantageous role of EGF receptor overexpression in malignant phenotype of epithelial tumor cells.