Concentrations Of Fetal Calf Serum Research Articles

In vitro influence of Phaseolus vulgaris, Griffonia simplicifolia, concanavalin A, wheat germ, and peanut agglutinins on HCT-15, LoVo, and SW837 human colorectal cancer cell growth.

Compared with normal colonic mucosa, lectin receptor expression is increased in hyperplastic and neoplastic tissues; some lectins have been shown to influence human colonic epithelial cell proliferation. The aim was to assess further the influence of five lectins (Phaseolus vulgaris (PNA), Griffonia simplicifolia (GSA), concanavalin A (Con A), wheat germ (WGA), and peanut (PHA-L) agglutinins) on cellular growth in three human colorectal cancer cell lines (LoVo, HCT-15 and SW837). Cells were cultured in four lectin concentrations (0.1, 1.0, 10, and 100 micrograms/ml) and growth assessed at days 2, 3, 5, and 7. The experiments were performed in media supplemented with either 1% or 10% fetal calf serum (FCS). Growth was assessed using the MTT (3-(4,5)-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) colorimetric assay. Growth in each cell line was greatly affected by at least two of the lectins tested. There was some variation in the effect of a given lectin on different cell lines. Lectin effects showed a dose-response and the greatest effects generally resulted from the highest concentrations at the longest culture time. WGA and Con A induced large effects in all cell lines; the effects of Con A were partly blocked by the higher concentration of FCS. PNA had modest and uniform stimulatory effects overall. The effects of GSA and PHA-L varied between cell lines. The lectins studied all have the potential to affect colonic cancer growth in vitro. Many dietary lectins are resistant to digestion and may have important effects in vitro but the definition of their role in human colonic cancer biology must take into account the variability in lectin response.

Study of the favorable culture condition to produce granulocyte. Macrophage colony stimulating factor and interleukin-6 by human nasal epithelial cells

Allergic rhinitis and nasal polyposis are upper airway inflammatory conditions characterized by increased numbers of eosinophils and metachromatic cells in the epithelial layer of the nasal mucosa. However, the mechanism by which these cells accumulate still remains obscure in many respects. It is suggested that the granulocyte-macrophage colony stimulating factor (GM-CSF) plays an important role in the nasal mucosa, since the supernatant of cultured epithelial cells induce differentiation of peripheral mononuclear cells into metachromatic cells and eosinophils, and this activity was inhibited by the anti-GM-CSF antibody. In addition, the presence of several cytokines in the supernatant of nasal epithelial cell cultures has been reported. These observations suggest the possibility that GM-CSF and cytokines play an important role in the allergic and inflammatory reactions of the nasal mucosa. In the present study epithelial cells of nasal mucosa were cultured under different conditions to find out the optimal conditions of production for GM-CSF and interleukin-6 (IL-6). When the concentration of fetal calf serum (FCS) in the culture medium was varied, the number of cultured epithelial cells and the concentration of GM-CSF and IL-6 in the supernatant showed an FCS concentration-dependent increase. When nasal mucosal epithelial cells were cultured in 15% FCS, production of both GM-CSF and IL-6 was increased with increasing duration of incubation. There was no significant difference in production of GM-CSF or IL-6 between the patients with allergic rhinitis and those with nasal polyps. These results suggest that neither allergic rhinitis and nor nasal polyposis are associated with the ability of epithelial cells to produce cytokines, but the interplay of various factors in the epithelial layer, as well as of diseases, affects their ability to produce cytokines. When the condition of epithelial cell propagation was rendered favorable or unfavorable by varying the concentration of FCS in the culture medium, using the same culture system, production of GM-CSF and IL-6 was increased in a dose-dependent manner (%) favoring epithelial cell growth. These observations indicate that epithelial cells produce GM-CSF and IL-6 only when conditions are favorable for their proliferation is required.

Cytotoxicity of cholestane 3 beta,5 alpha,6 beta-triol on cultured intestinal epithelial crypt cells (IEC-6).

The effects of cholestane 3 beta,5 alpha,6 beta-triol on intestinal epithelial crypt cells were investigated using the IEC-6 cell line. Cholestane 3 beta,5 alpha,6 beta-triol decreased SH groups (glutathione and protein SH) in the cell, and showed cytotoxicity in a time-dependent manner. Although the concentration of cholestane 3 beta,5 alpha,6 beta-triol used in this study (100 microM) was very high compared with that in plasma of experimental animals, cholestane 3 beta,5 alpha,6 beta-triol did not show any cytotoxicity on IEC-6 cells without fetal calf serum (FCS). The level of cytotoxicity was dependent upon the concentration of FCS in the culture medium. Unknown components in FSC (not VLDL or LDL) were suggested to be associated with the cytotoxicity of cholestane 3 beta,5 alpha,6 beta-triol. Moreover, the fact that even heat-treated FCS (100 degrees C for 30 min) still mediated the cytotoxicity suggested the participation of non-protein components.

Agonist Activation of δ-Opioid Receptor but not μ-Opioid Receptor Potentiates Fetal Calf Serum or Tyrosine Kinase Receptor-Mediated Cell Proliferation in a CellLine-Specific Manner

Activation by opioid receptors of cell proliferation was examined with fibroblast cell lines stably expressing either delta-opioid or mu-opioid receptors. Addition of [D-Ala2, D-Leu5]-enkephalin or [D-Pen2,D-Pen5]-enkephalin to Chinese hamster ovary (CHO) cells transfected with delta-opioid receptor cDNA resulted in an agonist concentration-dependent potentiation of fetal calf serum (FCS)-stimulated cell proliferation. This potentiation by delta-opioid agonists was antagonized by naloxone and was not observed with the kappa-opioid receptor selective agonist U50,488 or the mu-opioid receptor selective agonist [D-Ala2,N-MePhe4, Gly-ol5]-enkephalin. This delta-opioid agonist effect was not observed at FCS concentrations > 0.1% and could be blocked by pretreating cells with pertussis toxin, indicating that Gi/Go were involved in this action. In addition, delta-opioid agonists could potentiate CHO cell proliferation stimulated by those growth factors that are mediated by tyrosine kinase receptors (i.e., insulin, insulin-like growth factor 1, and fibroblast-derived growth factor b). This delta-opioid agonist potentiation of growth apparently was dependent on the level of delta-opioid receptors that were expressed and had cell-line selectivity. Activation of delta-opioid receptors expressed in Rat-1 or NIH3T3 fibroblast did not result in a modulation of the cell growth induced by FCS or by growth factors. Interestingly, in CHO cells transfected with mu-opioid receptor cDNA, activation with agonists did not produce a potentiation of FCS-stimulated proliferation. This lack of mu-opioid receptor effect was not due to the differences among CHO clones. In a CHO cell line transfected with both delta-opioid receptor cDNA and mu-opioid receptor cDNA, activation of delta-but not mu-opioid receptors resulted in a potentiation of growth. These data suggest that delta- and mu-opioid receptors in CHO cells activate similar but divergent second messenger pathways, resulting in the differential regulation of cell growth.

Responses of human MG-63 osteosarcoma cell line and human osteoblast-like cells to pulsed electromagnetic fields

We have studied the effects of low-energy, low-frequency pulsed electromagnetic fields (PEMF) on cell proliferation, in both human osteoblast-like cells obtained from bone specimens and in human MG-63 osteosarcoma cell line. Assessment of osteoblastic phenotype was performed both by immunolabeling with antiosteonectin antibody and by verifying the presence of parathyroid hormone receptors. The cells were placed in multiwell plates and set in a tissue culture incubator between a pair of Helmholtz coils powered by a pulse generator (1.3 ms, 75 Hz) for different periods of time. [3H]Thymidine incorporation was used to evaluate cell proliferation. Since it had previously been observed that the osteoblast proliferative response to PEMF exposure may also be conditioned by the presence of serum in the medium, experiments were carried out at different serum concentrations. [3H]Thymidine incorporation increases in osteoblast-like cells, when they are exposed to PEMF in the presence of 10% fetal calf serum (FCS). The greatest effect is observed after 24 hours of PEMF exposure. No effects on cell proliferation are observed when osteoblast-like cells are exposed to PEMF in the presence of 0.5% FCS or in a serum-free medium. On the other hand, PEMF-exposed MG-63 cells show increased cell proliferation either at 10% FCS, 0.5% FCS and in serum-free medium. Nevertheless, the maximum effect of PEMF exposure on MG-63 cell proliferation depends on the percentage of FCS in the medium. The higher the FCS concentration, the faster the proliferative response to PEMF exposure. Our results show that, although MG-63 cells display some similarity with human bone cells, their responses to PEMF's exposure are quite different from that observed in normal human bone cells.

Responses of human MG‐63 osteosarcoma cell line and human osteoblast‐like cells to pulsed electromagnetic fields

We have studied the effects of low-energy, low-frequency pulsed electromagnetic fields (PEMF) on cell proliferation, in both human osteoblast-like cells obtained from bone specimens and in human MG-63 osteosarcoma cell line. Assessment of osteoblastic phenotype was performed both by immunolabeling with antiosteonectin antibody and by verifying the presence of parathyroid hormone receptors. The cells were placed in multiwell plates and set in a tissue culture incubator between a pair of Helmholtz coils powered by a pulse generator (1.3 ms, 75 Hz) for different periods of time. [3H]-Thymidine incorporation was used to evaluate cell proliferation. Since it had previously been observed that the osteoblast proliferative response to PEMF exposure may also be conditioned by the presence of serum in the medium, experiments were carried out at different serum concentrations. [3H]-thymidine incorporation increases in osteoblast-like cells, when they are exposed to PEMF in the presence of 10% fetal calf serum (FCS). The greatest effect is observed after 24 hours of PEMF exposure. No effects on cell proliferation are observed when osteoblast-like cells are exposed to PEMF in the presence of 0.5% FCS or in a serum-free medium. On the other hand, PEMF-exposed MG-63 cells show increased cell proliferation either at 10% FCS, 0.5% FCS and in serum-free medium. Nevertheless, the maximum effect of PEMF exposure on MG-63 cell proliferation depends on the percentage of FCS in the medium. The higher the FCS concentration, the faster the proliferative response to PEMF exposure. Our results show that, although MG-63 cells display some similarity with human bone cells, their responses to PEMF's exposure are quite different from that observed in normal human bone cells. Bioelectromagnetics 18: 541–547, 1997. © 1997 Wiley-Liss, Inc.

Effects of endotoxin on proliferation of human hematopoietic cell precursors.

In examining the effects of corticosteroids on hematopoiesis in vitro, we observed that results were highly dependent on the lot of commercial fetal calf serum (FCS) utilized. We hypothesized that this variability correlated with the picogram (pg) level of endotoxin contaminating the FCS. Randomly obtained commercial lots of FCS contained 0.39 to 187 pg/ml of lipopolysaccharide (LPS). Standard FCS concentrations in hematopoietic precursor proliferation assays (granulocyte-marcrophage colony forming units [CFU-GM]) resulted in final LPS levels as high as 40 pg/ml. LPS (2-5 pg/ml) added to essentially endotoxin-free cultures, induced human mononuclear cell release of interleukin (IL)-1, IL-6 and granulocyte colony stimulating factor (G-CSF). Lots of FCS induced the release of IL-1, IL-6, and G-CSF from human mononuclear cells and the release of these factors correlated with the level of contaminating LPS. Human bone marrow CFU-GM proliferation, in response to granulocyte-macrophage colony stimulating factor (GM-CSF), positively correlated with the level of LPS contaminating the FCS and the FCS-induced release of IL-6 from mononuclear cells. CFU-GM proliferation of human bone marrow cluster of differentiation (CD) 34+CD14-cells were not affected by the presence of endotoxin. These data suggest that LPS at 2-5 pg/ml may induce bone marrow accessory cell release of hematopoietic growth factors, thus altering proliferative response of hematopoietic precursors and confounding the study of exogenously added cytokines to culture systems.

Cultured gastric parietal cells from the guinea pig: adherence, cell growth and stimulus coupling of Ca 2+ and cyclic AMP

The aim of the study was to establish cell culture conditions for responsive guinea pig parietal cells. Parietal cells were isolated by a pronase/collagenase method, enriched by counterflow elutriation and cultured on plastic culture dishes in minimum essential medium. Precoating with gelatine or collagen increased adherence; optimum fetal calf serum concentration was 10%. Parietal cells were cultured for up to 120 h. Intracellular calcium levels in cells cultured for 48 h were 150 nmol/l and increased to 320 nmol/l after stimulation with carbachol and to 250 nmol/l after histamine stimulation as determined by video imaging microscopy. Intracellular cyclic AMP levels were increased 9-fold by histamine in cells cultured for 24 h and more than 30-fold in cells cultured for 48 h. The results show that guinea pig parietal cells grow in primary culture and are suitable for studying second messenger coupling.

Snow Leopard (Panthera uncia) Spermatozoa Are Sensitive to Alkaline pH, But Motility In Vitro Is Not Influenced by Protein or Energy Supplements

To better understand the biology of snow leopard spermatozoa and to facilitate developing assisted reproduction, a series of studies was conducted to: 1) identify the component(s) of complex culture media responsible for the detrimental effect on sperm survival in vitro, 2) optimize medium for supporting sperm viability, and 3) evaluate sperm capacitation in vitro. Constituents of complex media were added systematically to phosphate-buffered saline (PBS) to isolate the factor(s) influencing snow leopard sperm motility in vitro. Sperm capacitation was also assessed following incubation in PBS with bovine serum albumin (BSA), fetal calf serum (FCS), or heparin. For maintaining sperm motility, there was no benefit (P > or = 0.05) to supplementing PBS with low (5%) or high (20%) concentrations of snow leopard serum (SLS) versus FCS or BSA. Likewise, adding supplemental energy substrates (pyruvate, glucose, lactate, or glutamine) did not enhance or hinder (P > or = 0.05) sperm motility. However, motility rapidly decreased (P < 0.05) with the addition of NaHCO3 to PBS or Ham's F10 nutrient mixture. Surprisingly, Ham's F10 with no buffering component or with both NaHCO3 and N-Z-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) maintained sperm motility at levels similar (P > or = 0.05) to PBS. Although sperm motility in all treatments decreased with time, there was a strong inverse relationship (P < 0.01; r = 0.90) between motility and sample pH at 6 hours. Spermatozoa incubated in PBS containing FCS, BSA, or heparin did not undergo the acrosome reaction when exposed to calcium ionophore. In summary, alkaline pH has a profound detrimental effect on snow leopard sperm motility, and capacitation does not occur under conditions that normally promote this event in other felid species. These results clearly demonstrate a high degree of interspecific variation among felids in fundamental sperm function, and they provide evidence for the necessity of basic research when developing assisted reproduction in little-studied nondomestic species.

Cytotoxic effect of β-carotene in vitro is dependent on serum concentration and source

In a previous study we reported that beta-carotene solubilized in tetrahydrofuran (THF) is toxic for human colonic tumor cells in vitro using media containing 10% fetal calf serum (FCS). Cytotoxicity was evident using beta-carotene concentrations that are achieved in human serum as a result of supplementation with 30 mg beta-carotene/day. In an attempt to determine the mechanism for this toxicity we investigated the effect of beta-carotene when present in human serum as a result of dietary supplementation. This effect was compared to that observed for cells incubated in THF-solubilized beta-carotene. The results indicate that human serum from subjects with a high concentration of beta-carotene is not cytotoxic. Subsequent analysis revealed that in contrast to the results using FCS, THF-solubilized beta-carotene is not cytotoxic in the presence of human serum. In addition, the effect observed with FCS is blunted with increasing FCS concentration from > or = 90% cytotoxicity using 10% FCS to 36% using 100% FCS. The difference in results obtained using FCS and human serum may be due to a serum component that is relatively lacking in FCS as compared to human serum.

Human thyroid epithelial cells cultured in monolayers. II. Influence of serum on thyroglobulin and cAMP production

An in vitro system of secondary cultures of human thyroid follicular epithelial cells in monolayer is described. The 72-h influence of serum and six supplements (thyrotropin, insulin, somatostatin, transferrin, hydrocortisone, glycyl-histidyl-lysine acetate) on growth and function in presence of 3-isobutyl-L-methyl-xanthine (IBMX) was investigated. The function of the cells was evaluated by production of the second messenger adenylate cyclase (cAMP) and the end product thyroglobulin (Tg). Growth was measured as the 3H-thymidine uptake of the cells. Three days of TSH-depletion preceeded the experiments. In presence of IBMX TSH stimulated cAMP production, while stimulation of Tg was only present in some cultures. In absence of IBMX TSH always stimulated the Tg production. The stimulation was independent of the presence of the other five investigated nutritional factors in physiological concentrations. TSH in concentrations from 0.1–10 U/1 stimulated the 72ih 3H-thymidine uptake of the cells. The TSH-stimulated production of Tg and cAMP decreased significantly with increasing concentrations of fetal calf serum (0–10%), (τ = 0.49, P < 0.001, n = 6–29 and τ = 0.75, P < 0.001, n = 6–29, respectively). Thus, serum as a complex, variable and not fully characterized mixture of hormones and growth factors was crucial to the attachment of the cells to the substrate, but inhibited differentiated functions of the human thyroid cells.

Repair Kinetics of Amnion Cells after Microsurgical Injury

There is a paucity of data regarding healing of fetal membranes. We assessed the repairing ability of a microsurgically-injured monolayer of amnion-derived cells compared to an endothelial cell line. Amnion-derived cells (FL cells) were grown to confluence in 10% fetal calf serum (FCS). A microsurgical incision was performed under an inverted microscope. The area of each incision was measured at times 0, 6, 12 and 24 h using MCID M4 image analysis software. The experiments were repeated using 1% FCS. The observations were also made replacing the media at 6 and 12 h. Umbilical-cord-derived endothelial cells were used for comparison. The rate of repair of the incision was measured using three methods: (a) absolute closure rate (ACR): (A0-A1)/T, where A0 is the original incisional area, and A1 is the new area at interval T: (b) relative percentage rate (RPR): (A0-A1)/A0/T x 100, and (c) healing rate (HR): (A0-A1)/A0 x 100. Amnion cells were capable of repairing the microsurgical defect. The ACR decreased over time, and was higher with larger defects. A lower concentration of FCS nor the addition of fresh media altered the repair process significantly. Endothelial cells were significantly faster than amnion cells or amnion-supplemented cells with endothelial media. The RPR remained relatively constant for all groups, suggesting Gompertzian kinetics. Amnion cells are capable of repairing a surgical defect at a relatively constant percent rate, but are slower than an endothelial cell line. The decreased rate of closure over time may be due to relative contact inhibition. Further studies will concentrate on the molecular and cellular mechanisms involved in this process.

Regulation of B cell apoptosis.

Only 1% of thymocytes have hypodiploid nuclei, an index of apoptosis. Yet there is evidence that 99% of thymocytes die by apoptosis, and are destroyed so quickly by phagocytes they are hardly apparent histologically. So when we see 1% of spleen B (or T) cells have hypodiploid nuclei, it tells us little about the apoptotic turnover of these cells. Despite evidence that germinal center B cells enter apoptosis when activated through their antigen receptor in the absence of costimulation1, and that terminal differentiation also leads to apoptosis, life span estimates for the majority of mature B cells run to several months2,3. So we were surprised to find that small dense B cells from the spleens of specific pathogen-free mice spontaneously undergo apoptosis in vitro 4 so fast that about 75% of small dense B cells have hypodiploid nuclei by 64h. The rate is linear with no lag period to indicate an induction process. Varying fetal calf serum concentration from 1 to 10%, varying the B cell (or T cell) concentration from 0.1 to 10 × 106/ml (to rule out autocrine mechanisms), and purifying the cells (adherence to BSA-coated plastic, exposure to anti-thy 1.2 plus complement, centrifugation through Percoll) had no major effect on the spontaneous apoptosis rate5(and our unpublished data). Neither did inclusion of 10% normal mouse serum, or coculture with other spleen cell types5. Cycloheximide (CHX) and other protein synthesis inhibitors accelerated apoptosis in B and T cells4,5 while blocking apoptosis in thymocytes5,6. We appeared to be observing an apoptosis program “hard-wired” into the B cell, very different from that of the thymocyte, requiring no stimulation and no “death protein” synthesis.

Primary cell culture from gill explants of rainbow trout

Primary cultures of gill cells were initiated from gill filament explants of rainbow trout, Oncorhynchus mykiss. The explants were cultured in Leibovitz l‐15 medium with 5, 10 or 20% foetal calf serum (FCS) and l‐glutamine. The attachment efficiency was serum‐dependent though increased FCS concentration did not stimulate further outgrowth of cells. The explants produced cell outgrowth 24 h after attachment as a sheet of cells which exhibited characteristics of gill pavement epithelial cells as indicated by surface microridges revealed by scanning electron micrographs. There was high proliferation for the first 14 days then a stable plateau for 30 days followed by a decline phase from 45 days. Following removal of cells, the explants produced further cell outgrowth which was especially active at the proliferation phase (14 days). Removal of these cells caused the explants to produce a further proliferation of cells reaching confluence in 10–14 days. After the third cell removal cell outgrowth from explants showed migratory activity but did not develop to resemble gill epithelial cells. The use of gill explants to establish primary cultures of fish gill cells has advantages which include longevity of the culture and successive proliferations from explants which could provide a useful tool for the investigation of long‐term processes in cellular biology and reduce the number of culture preparations.

Identification and Conditions for Selective Expression of Megakaryocytic Markers in Friend Erythroleukemia Cells

Friend murine erythroleukemia cells (MELCs) have been reevaluated in terms of their nature and potential pathways of differentiation. MELC induced with 5 mmol/L hexamethylene bisacetamide (HMBA), in addition to expression of known markers of the erythroid phenotype, were also found to exhibit traits of the megakaryocytic lineage. Erythroid differentiation was shown by the typical synthesis and accumulation of hemoglobin (Hb); megakaryoblastoid differentiation of MELCs upon induction was shown by increased specific activity of acetylcholinesterase (AChE). Incubation of MELCs with 5 mmol/L HMBA in RPMI supplemented with 1% fetal calf serum (FCS) (instead of the usual 5%), induced cells to selectively express high levels of AChE (up to approximately 170 mU/mg protein) with little activation of Hb synthesis (less than 5% B+ cells). The increase in AChE levels was a general phenomenon affecting the whole cell population and approached its maximum within 3 days of incubation with the inducer. Subsequently, MELCs become committed to terminal division, undergoing growth arrest and expression of the megakaryocytic phenotype even after the removal of HMBA. There were no appreciable changes of basal AChE levels in MELCs that were either made resistant to HMBA or treated with 0.1 mmol/L hemin that activated differentiated erythroid function without commitment. Phorbol 12-myristate 13-acetate (PMA), known to repress induced Hb synthesis in these cells, did not prevent the full increase in AChE when incubated with MELCs 2 days before HMBA addition. HMBA-induced MELCs always underwent AChE increase that was more or less pronounced depending on the low or high serum content in culture, respectively. Conversely, Hb expression was permitted only when MELCs were transferred in the late phase or at the end of commitment from low to high serum media. Variations of FCS content in culture media proved to be a simple and reliable approach to change the MELC response to inducers and to modulate expression of either megakaryocytic or mixed erythromegakaryocytic phenotype. These findings suggested that MELC might be considered, at least, as a bipotential model of differentiation to be used for studies on regulation of either megakaryocytic or erythroid markers and on competition between the two hematopoietic lineages. In this regard, it was intriguing that AChE levels attained under selective induction (low serum) were always higher than under conditions allowing coexpression of both AChE and Hb (high serum). Moreover, MELCs were also found to bind the specific rat-antimouse platelet monoclonal antibody 4A5.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification and conditions for selective expression of megakaryocytic markers in Friend erythroleukemia cells

Friend murine erythroleukemia cells (MELCs) have been reevaluated in terms of their nature and potential pathways of differentiation. MELC induced with 5 mmol/L hexamethylene bisacetamide (HMBA), in addition to expression of known markers of the erythroid phenotype, were also found to exhibit traits of the megakaryocytic lineage. Erythroid differentiation was shown by the typical synthesis and accumulation of hemoglobin (Hb); megakaryoblastoid differentiation of MELCs upon induction was shown by increased specific activity of acetylcholinesterase (AChE). Incubation of MELCs with 5 mmol/L HMBA in RPMI supplemented with 1% fetal calf serum (FCS) (instead of the usual 5%), induced cells to selectively express high levels of AChE (up to approximately 170 mU/mg protein) with little activation of Hb synthesis (less than 5% B+ cells). The increase in AChE levels was a general phenomenon affecting the whole cell population and approached its maximum within 3 days of incubation with the inducer. Subsequently, MELCs become committed to terminal division, undergoing growth arrest and expression of the megakaryocytic phenotype even after the removal of HMBA. There were no appreciable changes of basal AChE levels in MELCs that were either made resistant to HMBA or treated with 0.1 mmol/L hemin that activated differentiated erythroid function without commitment. Phorbol 12-myristate 13-acetate (PMA), known to repress induced Hb synthesis in these cells, did not prevent the full increase in AChE when incubated with MELCs 2 days before HMBA addition. HMBA-induced MELCs always underwent AChE increase that was more or less pronounced depending on the low or high serum content in culture, respectively. Conversely, Hb expression was permitted only when MELCs were transferred in the late phase or at the end of commitment from low to high serum media. Variations of FCS content in culture media proved to be a simple and reliable approach to change the MELC response to inducers and to modulate expression of either megakaryocytic or mixed erythromegakaryocytic phenotype. These findings suggested that MELC might be considered, at least, as a bipotential model of differentiation to be used for studies on regulation of either megakaryocytic or erythroid markers and on competition between the two hematopoietic lineages. In this regard, it was intriguing that AChE levels attained under selective induction (low serum) were always higher than under conditions allowing coexpression of both AChE and Hb (high serum). Moreover, MELCs were also found to bind the specific rat-antimouse platelet monoclonal antibody 4A5.(ABSTRACT TRUNCATED AT 400 WORDS)

Hepatocyte Growth Factor as a Potent Survival Factor for Rat Pheochromocytoma PC12 Cells

Hepatocyte growth factor (HGF), a natural ligand for the c-met protooncogene product, is a multipotent polypeptide which elicits mitogenic, motogenic, and morphogenic activities for various types of cells. To better understand the biological activity of HGF, as related to neuroectodermal-derived cells, we investigated the effects of HGF on rat pheochromocytoma PC12 cells. HGF increased the number of PC12 cells during long culture, but elicited no direct mitogenic activity, as determined by DNA synthesis. When the cells were cultured in medium containing lower concentrations of fetal calf serum, HGF prolonged the survival of PC12 cells; the number of cells did not decrease during 13 days when the cells were cultured in the presence of HGF, but the cells were completely withdrawn when cultured in the absence of HGF. Nerve growth factor but not HGF induced the differentiation of PC12 cells. High affinity receptor for HGF with Kd values of 20-40 pM was expressed in PC12 cells and other types of cells derived from the central nervous tissue: T98G cells (human glioblastoma), GOTO, and SCCH-26 cells (human neuroblastoma). HGF stimulated motility of T98G cells, while it induced weak mitogenic response in GOTO cells. We suggest that HGF is a potent survival factor for PC12 cells, without exerting any direct mitogenic activity and inducing the cell differentiation, and that this factor may have a distinct biological activity for neuroectoderm-derived cells.

Effects of keratinocyte conditioned medium, amniotic fluid and EGF in reepithelialization of human skin wounds in vitro

Growth factors and combinations of growth promoting substances in amniotic fluid, platelet extracts or factors secreted by cultured human keratinocytes have been shown to stimulate cell proliferation and wound healing. In this report, the possibility of studying reepithelialization of wounds in human skin, using small biopsies maintained in vitro, was examined. The effects of fetal calf serum (FCS), keratinocyte conditioned medium (KCM), amniotic fluid and epidermal growth factor (EGF) on the reepithelialization process was investigated after seven days. KCM alone did not affect healing, but when added to a suboptimal concentration of FCS (2%), KCM induced reepithelialization of the wounds. Amniotic fluid (25%) alone stimulated the formation of a new epidermis, whereas EGF (10 ng/ml) alone or added to 2% FCS did not induce reepithelialization. The model used in this study includes an autologous matrix comprising living fibroblasts and endothelial cells and may thus be used to study aspects of wound healing in human skin.

Platelet-activating factor (PAF) is not an essential component of the cascade leading to smooth muscle cell proliferation following vascular injury

In order to evaluate the relative importance platelet-activating factor (PAF) in the proliferative process leading to restenosis, the effect of SR 27417, a novel highly potent PAF receptor antagonist, on PAF-induced rabbit aortic smooth muscle cell (SMC) proliferation and intimal hyperplasia in rabbit carotid arteries subjected to air-drying endothelial injury was investigated. When added to low concentrations of foetal calf serum, PAF showed a dose-dependent mitogenic effect with regard to rabbit arterial SMC. SR 27417 inhibited PAF-induced SMC growth (IC 50 = 2.4 ± 0.4 nM) but remained without effect on the mitogenic effect of foetal calf serum. A 16 day treatment of SR 27417 (10 mg/kg per day, p.o.) abrogated PAF-induced platelet aggregation ex vivo but did not affect the development of intimal thickening, therefore showing that PAT is not an essential component of the cascade leading to-restenosis following vascular injury.

Human epidermis reconstructed on synthetic membrane: influence of experimental conditions on terminal differentiation.

Cell suspensions of human keratinocytes seeded onto cell culture inserts may undergo terminal differentiation in the absence of fibroblasts. Among the parameters that control these morphogenic events, exposure to air and the composition of the culture medium were investigated. In the latter case, three media were considered DMEM:Ham's F12, MCDB 153, and keratinocyte SFM medium at equivalent calcium (1.5 mM) and fetal calf serum (5%) concentrations. Immunochemical methods and transmission electron microscopy show that cells cultured in DMEM:Ham's F12 medium, and then raised at the air-liquid interface, form a basal layer plus suprabasal cell layers corresponding to the stratum spinosum, stratum granulosum, and stratum corneum. The suprabasal keratinocyte layers show morphologies that resemble intact skin in which cells are connected by desmosomes and contain intermediate filaments and keratohyalin-filaggrin granules. When the cultures are kept submerged, the keratinocytes show occasional keratohyalin granules and are connected by fewer desmosomes. Additionally, no proper stratum corneum is formed. In keratinocyte SFM medium and MCDB 153, cultures raised at the air-liquid interface are not able to form an epithelium of normal architecture and do not express terminal differentiation markers. Differentiation is initiated, however, since desmosomes and bundles of keratin filaments appear; on the other hand, filaggrin is not expressed even after 28 d in culture. Membrane-bound transglutaminase is expressed throughout the entire suprabasal compartment in MCDB153 and DMEM:Ham's F12 media but never appears in keratinocyte SFM medium. These studies show the relative independence of epidermal differentiation program to the composition (including the calcium concentration) of the media contacting the dermis and filling the extracellular space.(ABSTRACT TRUNCATED AT 250 WORDS)