Inhibitory effects of human serum on human fetal skin fibroblast migration: Migration-inhibitory activity and substances in serum, and its age-related changes

Summary In order to clarify the environmental factors modulating cell migration, we investigated the effects of human serum on cell migration, and found that serum from adult donors strongly (by 48%) suppressed the migration of human fetal skin fibroblasts into a denuded area in a cell monolayer. Human serum from old donors inhibited cell migration more strongly than that from adult donors. Next, we investigated the properties of migration-inhibitory activity of human serum and serum proteins in order to identify migration-inhibitory substances. Human serum from adult donors strongly suppressed the migration of human fetal skin fibroblasts, although it stimulated cell proliferation more strongly than fetal bovine serum (FBS), indicating that the inhibitory effects of human serum on cell migration was not due to its toxic effects. The inhibition of cell migration by human serum was concentration dependent. It was demonsstrated that the inhibition did not depend on the inhibitory effects of human serum on collagen synthesis. The migration-inhibitory activity was seen in fractions over 100 kDa, as determined by an ultrafiltration membrane, and no inhibitory activity was observed in fractions under 100 kDa. On the other hand, it was not detected either in fractions over 100 kDa or under 100 kDa in FBS. Among the over 100 kDa human serum proteins examined, γ-globulin, α2-macroglobulin, and low density lipoprotein (LDL) suppressed fibroblast migration in a concentration-dependent manner. However, among the three, cell migration-inhibiting activity of γ-globulin almost disappeared when cell migration was conducted in 10% FBS-supplemented medium. These results indicated that α2-macroglobulin and LDL were candidate substances for cell migration-inhibiting activity in human serum.     

Establishment of an abalone digestive gland cell line secreting various glycosidases in protein-free culture

A cell line designated as ADG was established from an abalone digestive gland using ERDF medium supplemented with 8% fetal bovine serum (FBS), 8% abalone hemolymph, and high concentrations of NaCl, KCl, MgCl₂, MgSO₄, and CaCl₂. ADG cells proliferated better in protein-free medium than in FBS-supplemented medium. Among 9 kinds of media examined, ERDF medium was shown to be optimal for cell growth. ADG cells secreted 13 different kinds of glycosidases in protein-free medium: α-L-fucosidase, β-L-fucosidase, α-D-galactosidase, β-D-galactosidase, N-acetyl-α-D-galactosaminidase, N-acetyl-β-D-galactosaminidase, α-D-glucosidase, β-D-glucosidase, N-acetyl-α-D-glucosaminidase, N-acetyl-β-D-glucosaminidase, α-D-mannosidase, β-D-mannosidase, β-D-xylosidase, and 1-3 xylanase. When ADG cells were cultured in Grace’s insect cell medium, the activity of some secreted glycosidases increased 25-fold to 65-fold per cell as compared with control cells cultured in ERDF medium. ADG - abalone digestive gland; ERDF - enriched RDF; FBS - fetal bovine serum; L-15 - Leibovitz’s L-15 media; DME - Dulbecco’s modified Eagle medium; F-12 - nutrient mixture (Ham); LDF - L-15; DME: F-12 = 10 : 7 : 3; MEM - minimum essential medium; RPMI - RPMI medium 1640; 199 - media 199; GIC - Grace’s insect cell medium; pNP -p -nitrophenol. This revised version was published online in July 2006 with corrections to the Cover Date.     

Purification and Characterization of Serum Serpin from Carp (Cyprinus carpio)

A serine proteinase inhibitor, termed serpin62, was purified to homogeneity from carp serum with an increase in specific inhibitory activity of 6.2-fold and a 3% recovery rate after separation from α₁-antitrypsin. Specific inhibitory activity of serpin62 against bovine pancreatic trypsin was less than half of the specific antitryptic activity of α₁-antitrypsin. Under both reducing and nonreducing conditions, serpin62 was estimated to have a molecular weight (62,000) apparently larger than that of α₁-antitrypsin (55,000). They both consist of single polypeptide chains, but serpin62 differs from serine proteinase inhibitors from muscles of carp and white croaker in molecular weight and structure. Antibody raised against serpin62 immunologically crossreacted with serpin62 and had no crossreactivity with fish serum α₁-antitrypsin and muscular analogues. The antibody was susceptible to both serpin62 and its derivatives, which were widely distributed in carp tissues. Serpin62 is most likely distinct from other fish serine proteinase inhibitors expressing antitryptic activity physicochemically and immunologically. Received June 4, 1998; accepted September 10, 1998.     

Establishment of human parotid pleomorphic adenoma cells in culture: Morphological and biochemical characterization

Summary This study reports the establishment ofα-amylase-producing human parotid pleomorphic adenoma cell lines (2HP and 2HP₁) which have been maintained in culture for over 1 yr. The procedures required preparation of cellular clumps from tumor tissue and plating them on plasma clot or precoated dishes. During the initial phase of growth they required modified MCDB-153 medium without serum. When cells showed signs of degeneration they were changed to MCDB-153 medium containing first 2% and then 10% heat inactivated fetal bovine serum. Although cells grew well in MCDB-153 containing 10% serum, the epithelial cell morphology was not distinct. Therefore, the growth and morphology of cells grown in MCDB-10% serum were compared with those in RPMI growth medium containing 10% fetal bovine serum and F12 containing 10% agammaglobulin newborn bovine serum. Although the growth of cells was a little slower in F12 medium than those in MCDB and RPMI, the epithelial cell morphology was maintained better than in other growth media. The cells of 2HP and 2HP₁ produce low levels ofα-amylase and relatively high levels ofα-amylase mRNAs of 1176 and 702 bp and contain neurofilament-160, a neuronal-specific marker. The cells of 2HP₁ are tumorigenic when tested in athymic mice, but the cells of 2HP are not. The establishment of amylase-producing human parotid adenoma cell lines of different characteristics in culture provides a new opportunity to study the mechanisms of differentiation and transformation, and regulation ofα-amylase in these cells.     

Insulin-like growth factor-I is a serum component stimulating growth of human neuroblastoma

Summary Human non-autocrine neuroblastoma cells SK-N-SH and LF require serum for proliferation in vitro. We wished to determine the role of serum-borne insulin-like growth factor I (IGF-I) as mitogen for these cells. Introduction of the monoclonal antibodyα-IR₃ against human IGF-I receptor reduced proliferation in the presence of fetal bovine serum (FBS). IGF-I (5 nM) was as effective as FBS (10%) in stimulating proliferation. Porcine insulin mimicked the effects of IGF-I, but at a 1000-fold higher concentration. The antibodyα-IR₃ reduced growth stimulated by IGF-I more effectively than growth stimulated by insulin. Thus, proliferation of human non-autocrine neuroblastoma cells can be effectively manipulated by exogenous IGF-I.     

Stimulation of DNA synthesis in primary cultures of adult rat hepatocytes by rat platelet-associated substance(s)

Summary Experiments in whole animals have shown that normally quiescent adult rat hepatocytes are induced to proliferate by blood borne substances, which we are now probing in primary monolayer cultures. Under our conditions, freshly isolated adult hepatocytes do not proliferate actively in a defined medium, but are stimulated to synthesize DNA — an essential first step — by either serum or an EGF-hormone combination. Stimulation of [³H]thymidine incorporation into hepatocyte DNA by addition of dialyzed mouse, human, horse, or bovine (fetal, newborn, or calf) serum, whose activities are all similar, is regularly surpassed by an EGF-insulin mixture without serum. This, in turn, is exceeded by dialyzed normal rat serum, which is several times more potent than the other sera tested. Removal of blood platelets reduces the activity of normal rat serum by over 50%. Heat inactivation (56° C) causes a similar loss, but heat treatment of platelet-poor serum fails to cause further reduction. The activity of mouse and human serum is not reduced by platelet removal. Serum from partially hepatectomized rats is not significantly more stimulatory than normal rat serum, and its activity is depressed in the same way by platelet deprivation and heat inactivation. Lack of enhancement by partial hepatectomy is not consonant with whole animal studies and requires further investigation. The heat-labile portion of the DNA synthesis-stimulating activity of rat serum appears to derive from platelets. This activity differs from the well-characterized heat-stable human PDGF. Its relation to other reported platelet-associated growth factors is still undetermined. This work was supported by USPHS Grants CA-02146 and AM-19435.     

Hydrocortisone stimulation of human mammary epithelial cells

Summary Rapid proliferation of mammary epithelial cells derived from biopsy specimens of human fibroadenomas was observed when medium was supplemented with ten percent fetal bovine serum and hydrocortisone (5 μg per ml⁻¹). Hydrocortisone in combination with FBS also led to a 2.5-fold increase in cell cluster attachment and subsequent colony formation. A similar effect was not observed with human serum. In contrast to fibroblast cell systems, insulin did not significantly alter cell growth. The results show that a mitogenic response to glucocorticoids by mammary epithelium may depend on the presence of factors in sera. Supported in part by NCI Contract CB-33898.     

Molecular cloning, stable expression and cellular localization of human α1-adrenergic receptor subtypes: effect of charcoal/dextran treated serum on expression and localization of α1D -adrenergic receptor

The cDNAs encoding for three subtypes of adrenergic receptors, α_1A-, α_1B- and α_1D-ARs, were cloned and expressed in HEK 293 cells. Expression of α_1A- and α_1B-AR subtypes in HEK 293 cells was stable even with increased passages but that of α_1D-AR was not. Cellular localization studies using immunofluorescence and flow cytometry revealed that expression of α_1A- and α_1B-ARs was primarily localized on the cell membrane whereas expression of α_1D-AR was␣predominantly intracellular. Our studies clearly demonstrated that the culturing of the recombinant cell lines expressing α_1D-AR in charcoal/dextran treated fetal bovine serum (FBS) resulted in targeting of α_1D-AR to the cell membrane and thus, significantly improving its stability and availability for ligand binding studies.Sunil M. Khattar, Roop Singh Bora and Priyanka Priyadarsiny contributed equally to this work.     

Differential release of prostaglandins by organ cultures of human fetal trachea and lung

Summary Human fetal lung at 16–19 weeks gestation has a partially differentiated epithelium, and in organ culture, distal airsacs dilate and the epithelium autodifferentiates to type I and II pneumatocytes, processes regulated by endogenous prostaglandin PGE₂. Human fetal trachea, at the same gestation, has a terminally differentiated mucociliary epithelium but after 4–6 d in organ culture, develops squamous metaplasia. Tracheal cultures restricted to 3 d have normal phase-contrast and light microscopy appearances and immunohistochemical reactivities (epithelium: cytokeratin 7,8,18; glutathione S-transferase pi-isozyme; epithelial membrane antigen and mesenchyme; desmin; vimentin). In human fetal trachea organ cultures, the predominant prostaglandins released are 6-keto-PGF_1α, PGF_2α, and PGE₂, a pattern similar to that previously described for human adult trachea and lung. In fetal lung cultures, 13,14-dihydro-15-keto-PGF_2α is the major prostaglandin released with lesser amounts of 13,14-dihydro-15-keto-PFG_2α, PGF_2α, PGE₂, and 6-keto-PGF_1α. Human fetal lungin vitro has the competence to self-differentiate, as early as 12 weeks gestation and presence of high levels in fetal lung of the inactive metabolite 13,14-dihydro-15-keto-PGE₂ relative to PGE₂ suggests that active prostaglandin catabolism may be one of the mechanisms to retard this stage of maturationin vivo by limiting PGE₂ availability. Surprisingly, the profile of prostaglandins released from fetal lung organ culture does not change to that of a mature lung with terminal differentiation of the epithelium, and this may indicate differences in the expression of key prostaglandin-metabolizing enzymes in developing human fetal lung in culture and within utero ontogeny.     

β-Glucosidase activity in fetal bovine serum renders the plant glucoside,hypoxoside, cytotoxic toward B16-F10-BL-6 mouse melanoma cells

Summary By usingp-nitrophenyl-β-d-glucopyranoside as substrate, β-glucosidase activity was observed in fetal bovine serum (FBS). This activity could be inhibited by heat inactivation of the serum. Gel chromatography of FBS indicated the presence of β-glucosidase activity with an apparent molecular mass of 29 kDa. In McCoy’s 5A medium supplemented with non-heat inactivated FBS, the diglucoside hypoxoside ([E]-1,5-bis[4′β-d-glucopyranosyloxy-3′-hydroxyphenyl]pent-4-en-1-yne) showed cytotoxicity toward B16-F10-BL-6 mouse melanoma cells. In incubations where the media were supplemented with FBS previously heat inactivated at 56° C for 1 h or more, no cytotoxicity was observed in the presence of hypoxoside. The aglucone of hypoxoside, rooperol ([E]-1,5-bis[3′,4′-dihydroxyphenyl]pent-4-en-1-yne), showed cytotoxicity regardless of whether the serum was heat inactivated or not. The kinetics of the heat inactivation of the β-glucosidase activity in FBS coincided with the loss of apparent cytotoxicity of hypoxoside. High performance liquid chromatography analysis showed that rooperol could be generated by incubation of hypoxoside in non-heat inactivated FBS, but that this ability was lost in serum that was heat inactivated for 1 h or longer. Newborn bovine serum did not contain any β-glucosidase activity whereas it was found in three different commercial sources of FBS. This observation is of practical importance because conventional heat inactivation of FBS at 56° C for 30 min was not sufficient to inactivate the β-glucosidase activity completely.     

Heparin inhibits SMC growth in the presence of human and fetal bovine serum

Heparin (HP) has antiproliferative as well as anticoagulant properties, but not all HP preparations are equally antiproliferative. A recent report found that HP lost its total antiproliferative activity when fetal bovine serum (FBS) was replaced with human serum (HS) in culture media. This observation led to the investigation of our most potent antiproliferative Upjohn HP preparation effects on bovine pulmonary artery smooth muscle cells (PASMC) and systemic SMC growth stimulated in the presence of either FBS or HS. Bovine PASMC, human PASMC, and bovine aortic SMC were treated with 10 microg/ml Upjohn HP in either 15% FBS or 15% HS and the cell number was determined by a Coulter counter. We found that Upjohn HP significantly inhibited bovine PASMC and systemic SMC proliferation in both HS and FBS. The antiproliferative activity of the above HP preparation in HS may lead to an effective treatment of pulmonary vascular and systemic remodeling.     

A unique property of fetal bovine serum: High levels of protein-glutathione mixed disulfides

Summary Fetal bovine serum has been reported to delay or inhibit “spontaneous” neoplastic transformation in vitro as compared with all other sera tested. The present results indicate that fetal bovine serum is also unique in containing high levels of protein-glutathione mixed disulfides (3 to 7 μg glutathione as mixed disulfide per ml serum). The level of mixed disulfide appears to vary in accordance with the period of gestation of the fetal calves used to prepare the serum, decreasing below detectable levels (less than 0.2 μg per ml) with nearterm fetal calves. Calf, adult bovine, fetal horse, and swine sera did not contain detectable levels of this type of mixed disulfide. This work was supported by a grant from the National Institutes of Health (CA 08348).     

Detection of squalene in alpha-fetoprotein and fetal serum albumin from bovine

Alpha-fetoprotein and fetal serum albumin have been simultaneously purified from fetal bovine serum by mild procedures utilizing ammonium sulfate, hydrophobic interaction, immobilized metal (nickel) affinity chromatography, and isoelectric focusing. The lipidic extract from each protein was analyzed by gas chromatography and the peak appearing just after the arachidonic acid was identified as squalene by gas chromatography-mass spectrometry. This isoprenoid was not detected formerly in these proteins from human, rat, bovine, and pig. Until recently, in the analysis of the fatty acid composition of the alpha-fetoprotein and serum albumin from mammals, a peak has been assigned in the last part of the chromatographic profile, after arachidonic acid, to docosahexaenoic acid. In the present work, it was found that the peak corresponds to squalene instead of docosahexaenoic acid. Furthermore, we conclude that bovine alpha-fetoprotein and fetal serum albumin carry squalene, but not docosahexaenoic acid. These results agree with others obtained analyzing the same proteins from chick embryo.     

Reversible change in the fibroblast lysosomal enzyme dipeptidyl aminopeptidase-1 (cathepsin c) related to the commercial source of fetal bovine serum in the culture medium

Summary The commercial source of fetal bovine serum used to supplement the growth medium of human skin fibroblasts alters the activity of the lysosomal enzyme dipeptidyl aminopeptidase-1 (DAP-1). Cells grown with one serum were found to have a threefold higher level of DAP-1 than those grown with serum from another source (P<0.001). The effect on DAP-1 activity was specific inasmuch as no differences were found in the activities of a variety of other lysosomal and nonlysosomal hydrolases: DAP-II, DAP-III, DAP-IV, β-glucosidase, β-glucuronidase, andN-acetyl-β-galactosaminidase. The effect is reversible and is observed over a wide range of cell population doublings. Cell growth kinetics were not significantly different with the different sera. This work was supported in part by grants from the National Institutes of Health, Bethesda, MD (NS 16287).     

Putrescine-oxidase activity in adult bovine serum and fetal bovine serum

Summary Putrescine-oxidase activity was found in fetal bovine serum (FBS) with a pH optimum of 8.0 and in adult bovine serum (ABS) with a pH optimum of 9.8. The crude FBS enzyme had a K_M for putrescine of 2.58×10⁻⁶ m and a V_max of 0.53 nmol per hr per 50 μl serum. Aminoguanidine competitively inhibited the enzyme with a K_I of 1.8×10⁻⁸ m. Spermidine and spermine proved competitive inhibitors of putrescine for both the FBS and the crude ABS putrescine oxidases. The V_max for the ABS putrescine oxidase was 2.10 nmol per hr per 50 μl serum, and the K_M for putrescine, 50.3×10⁻⁶ m. The K₁ of the ABS putrescine oxidase for aminoguanidine was 41×10⁻⁶ m. On the basis of both the K_M and K_I values, the adult serum enzyme, at its optimal pH of 9.8, bound spermidine and spermine more avidly than the smaller putrescine and aminoguanidine; whereas the FBS enzyme, at pH 8.0, bound aminoguanidine and putrescine more tightly than the larger polyamines. Each of the enzymes retained over 80% of its activity after heating at 56°C for 30 min. Applications of these data to the study of polyamines in tissue culture and to the purification of diamine oxidases are discussed. This work was supported in part by a grant from the Cystic Fibrosis Foundation.     

Establishment of human osteoblastic cells derived from periosteum in culture

Summary We isolated osteoblastic cells derived from human periosteum and established them in culture. Their growth depended on the presence of ascorbic acid, and the doubling time was 40 to 60 h. The requirement for ascorbic acid was used to high production of collagen. These cells produced mainly type I collagen and only small amounts of type III collagen determined by reducing sodium dodecyl sulfate SDS-polyacrylamide gel electrophoresis. The total collagen yield was about 10 mg from 2×10⁷ cells. The cells could be continuously cultured in α-minimum essential medium supplemented with 10% fetal bovine serum for 18 to 40 population doubling levels, depending on the age of the donated periosteum. These cells have the ability to calcify when incubated with 2 mM α-glycerophosphate-Na₂. Calcification as viewed by the naked eye appeared from Day 15 after treatment. Treatment with the active formed vitamin D₃, 1, 25 dihydroxyvitamin D₃ enhanced calcification significantly and stimulated osteocalcin production. By electron microscopy, cells with many projections on their surfaces showed well-developed rough endoplasmic reticulum and actinlike fibers, and large numbers of lysosomes, mitochondria, and secretion granules. Many matrix vesicles, in which minerals were initially localized, and well-banded collagen fibrils were seen in the intercellular spaces. These observations demonstrate typical osteoblastic morphology. The above results indicate that cultured cells from human periostem are osteoblastic cells that have the capacity to differentiate into osteocytes and to deposit calcified minerals in response to 1,25 dihydroxyvitamin D₃.     

Influence of hormones and growth factors on viability,DNA, and protein content of adult hepatocytes in primary culture

Summary The survival of adult rat hepatocytes in monolayer culture was studied in the presence of different hormones (neurotensin, oxytocin, thyrotropin releasing hormone, luteinizing hormone releasing hormone, cholecalciferol, bradykinin, substance P, aldosterone, melanocyte stimulating hormone, 3,3′,5-triiodo-1-thyronine, corticosterone, human growth hormone, glucagon, insulin, progesterone, testosterone, estradiol, and dexamethasone phosphate) or growth factors (fetal bovine serum). For this purpose trypan blue exclusion, lactate dehydrogenase, and DNA and protein content were measured at 24 and 72 h of culture. 10⁻⁷ M Dexamethasone, a mixture of eight hormones, 10% fetal bovine serum, and a combination of the latter two supplements caused a more than 64% higher DNA content at 72 h when compared to control cultures. A striking agreement of these results with changes of lactate dehydrogenase leakage was observed, whereas trypan blue exclusion gave erratic results. Considerable changes of cell arrangement apparently specific for each supplement were ovserved by low magnification microscopy. It is concluded that glucocorticoids and fetal bovine serum have an outstanding effect on cell viability and that DNA or protein content or both are reliable indicators of cell viability in amitotic cultures.     

Serum leucine-rich alpha-2-glycoprotein-1 binds cytochrome c and inhibits antibody detection of this apoptotic marker in enzyme-linked immunosorbent assay

Cytochrome c (Cyt c) has been implicated as a serum marker for aberrant apoptosis and, thus, has considerable clinical potential. Using a sandwich enzyme-linked immunosorbent assay (ELISA) we found that the sensitivity of Cyt c detection is reduced in the presence of serum. The inhibitory factor responsible was purified from both fetal bovine serum and human serum employing standard chromatography procedures followed by affinity chromatography on Affi-Gel 10-bound Cyt c. In SDS-PAGE, bands at 44 kD and 50 kD were observed for the bovine and human proteins, respectively. Mass spectrometry analysis identified the serum inhibitory factor as leucine-rich alpha-2-glycoprotein-1 (LRα2GP1). This identification may lead to a modified ELISA to quantify total Cyt c in patients’ sera. LRα2GP1 is the first extracellular ligand for Cyt c that has been identified. A physiological function associated with binding is suggested. Supported by: NIH NINDS 5R21-NS45589     

Milk-derived growth factors as serum supplements for the growth of fibroblast and epithelial cells

Summary We have investigated the response of several epithelial and fibroblastic cells to a mitogenic extract of bovine milk. Cation exchange chromatography was used to produce a mitogen-rich fraction from an industrial whey source that, although comprising only 0.5% of total whey protein, contained the bulk of the growth factor activity. This fraction was a source of potent growth promoting activity for all mesodermal-derived cells tested, including human skin and embryonic lung fibroblasts, Balb/c 3T3 fibroblasts, and rat L6 myoblasts. Maximal growth of all these cell types exceeded that observed in 10% fetal bovine serum. Feline kidney and baby hamster fibroblasts and Chinese hamster ovary cells were less responsive, achieving a maximal growth response of 50–75% that observed in 10% fetal bovine serum. Maximal growth achieved in whey-extract-supplemented cultures of Balb/c 3T3 and human skin fibroblasts, and L6 myoblast cultures exceeded that seen in response to recombinant acidic or basic fibroblast growth factor, platelet-derived growth factor, insulin-like growth factor, or epidermal growth factor. Importantly, addition of low concentrations of fetal bovine serum to the whey-derived mitogenic fraction produced an additive response. However, concentrated milk-derived factors were found to be inhibitory to the growth of all epithelial lines tested, including rat intestinal epithelial cells, canine kidney epithelial cells, and mink lung cells. It is concluded that industrial whey extracted in this form constitutes an important source of potent growth-promoting agents for the supplementation of mesodermal-derived cell cultures.     

Effect of normal rat serum injected into the uterus on fetal survival in the rat

We intended to use the rat model to study the effect of autoantibodies on implantation and fetal viability. However, we have since found an effect of normal rat serum on fetal resorption rate and fetal viability. The objective of this study was to determine the consistency of this effect. Wistar strain albino rats were used for injection of 150 μl normal rat serum into the lumen of uterine horn on days L₂–L₆. The other uterine horn, used as a control, was injected with 150 μl normal saline. Percent implantation, fetal resorption rate and fetal viability were determined following the intrauterine injection of normal rat serum as compared with normal saline. A significant increase in fetal resorption rate was observed following the injection of rat serum on days L₄ and L₅ (P = 0.003 and P = 0.001, respectively). A significant decrease in fetal viability was demonstrated following the injection of rat serum on day L₅ (P = 0.01). The rat can provide a suitable animal model for further studies.