Endogenous thymidine and hypoxanthine are a source of error in evaluating methotrexate cytotoxicity by clonogenic assays using undialyzed fetal bovine serum

None of 13 fresh human tumor samples of various histology cloned in a two-layer agar culture system with 20% undialyzed fetal bovine serum (FBS) showed sensitivity to three antifolates, methotrexate (MTX), trimetrexate and 5,8-dideazaisofolic acid (IAHQ), even after continuous exposure to the highest concentrations (100 microM) for 21 days. In order to investigate this lack of antifolate drug effect, we compared the toxicity of continuous MTX exposure in the human colon carcinoma cell line HCT-8, cloned in a thymidineless medium (RPMI 1640) supplemented with 10% horse serum (HS), 10% fetal bovine serum (FBS), 20% FBS or 20% dialyzed FBS. In the presence of native FBS, when the minimum clone size was set at 30 cells/colony, the survival of HCT-8 cells reached a plateau at approximately 60% of untreated control after exposure to MTX concentrations between 0.1 microM and 100 microM. Only when the minimum clone size was set at 2 X 10(3) cells/colony was the sensitivity of HCT-8 cells to the antimetabolite comparable to that obtained in HS or dialyzed FBS (ED50 values in the range of 0.01 microM). MTX protection experiments indicated that even very small concentrations of thymidine and hypoxanthine together were sufficient to reproduce the pattern of sensitivity to MTX observed under culture conditions with undialyzed FBS. We conclude that for a proper evaluation of MTX cytotoxicity in clonogenic assays, dialyzed FBS and thymidine-less media should be employed; if native FBS is an absolute requirement for growth, only very large colonies (at least 10 cell divisions) should be scored.     

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.     

Stability of DNA/anti-DNA complexes.

Human monocytes in culture release small amounts of prostaglandin E (PGE) into the medium. Addition of Fc fragments of IgG to human monocyte monolayer cultures results in a marked increase in PGE release; Fab fragments, monomeric IgG, and human serum albumin have no effect. An IgG1 myeloma has no effect on PGE levels but addition of the heat aggreagted protein results in a marked increase of PGE secretion. Exposure of the cells to Con A, which binds to a specific monocyte plasma membrane receptor, also results in a large increase in PGE release. The magnitude of the increase in PGE secretion produced by exposure of the monocytes to these ligands greatly exceeds the stimulation observed after the addition of antigen-activated mononuclear cell supernatants, zymosan, Sephadex beads, or endotoxin, to monocyte cultures. Prostaglandin E2 (PGE2) accounts for approximately 70% of the total prostaglandins released by stimulated cells. After addition of Indomethacin to monocyte cultures, the stimulatory effects of the ligands on PGE release are inhibited. Addition of Con A to monocyte cultures results in an increased incorporation of [3H]-arachidonic acid into PGE2. These results suggest that this ligand stimulates synthesis as well as release of this prostaglandin.     

Advances in Rabbit Embryo Culture during Organogenesis

Modified rodent embryo culture techniques were used to maintain 10 1/2 day rabbit embryos in vitro for up to 24 hr. In homologous, immediately centrifuged, heat inactivated serum containing 2 μCi/ml ³H-thymidine, observed status was consistently high at 12 hr but fell thereafter. Liquid scintillation counting and autoradiography revealed rapid uptake of radioactivity during the first 12 hr. The splanchnopleure of the inverted yolk-sac is only partially vascularised in the rabbit and is thus inadequate as a medium of metabolic exchange in extended culture. Nevertheless, this technique will enable a study of the morphology and biochemistry of teratology in vitro in a sensitive species.     

Effect of protein synthesis inhibitors on leukocytic pyrogen-induced in vitro hypothalamic prostaglandin production

In order to study the antipyretic effect of inhibitors of protein synthesis, hypothalamic tissue was incubated in vitro under controlled conditions and the amount of prostaglandin E2 (PGE2) measured in the supernatant medium. Rabbit anterior hypothalamic tissue was incubated with purified human leukocytic pyrogen (LP) and after 60 minutes the supernatant fluid was assayed for PGE2 by radioimmunoassay. Control tissue incubated with Eagle's medium (MEM) released elevated levels of PGE2; however, the addition of polymyxin B (PmxB), a cationic antibiotic which blocks the activities of bacterial endotoxins, significantly reduced PGE2. In addition, endotoxin added to MEM induced from the brain tissue PGE2 production which could be reduced by the addition of PmxB. Thus, commercial culture media such as MEM may contain sufficient amounts of endotoxin to stimulate brain PGE2 production in vitro. Purified human LP incubated with hypothalamic tissue in the presence of PmxB induced PGE2 production in a dose-dependent fashion. This release could be reduced (p less than 0.001) by the presence of either cycloheximide or puromycin during incubation with LP. The addition of these inhibitors to unstimulated hypothalamic tissue incubations did not reduce background levels of PGE2. It is concluded that the antipyretic effect of protein synthesis inhibitors results in a specific decrease in LP-induced levels of PGE2.     

Metabolic dehydration of prostaglandin E2 and cellular uptake of the dehydration product: correlation with prostaglandin E2-induced growth inhibition

When L-1210 murine leukemia cells were incubated with 60 microM PGE2 in culture medium containing fetal calf serum for various time, cell proliferation was inhibited dependent on incubation time. However, when the medium containing PGE2 was changed every 6 h during 24 h exposure to cells, growth inhibition became much weaker. Moreover, when the medium containing PGE2 was aged by preincubating without cells at 37 degrees C, growth inhibitory effect of the medium was enhanced with preincubation time, suggesting that active growth inhibitory compound(s) accumulated during preincubation. In culture medium containing fetal calf serum, PGE2 degraded time-dependently and the major product was identified as PGA2 by HPLC. Furthermore, when cells were incubated with the medium containing 60 microM[3H]PGE2 or the same medium aged by preincubation, we observed that the radioactivity was taken up by the cells time-dependently, and identified the incorporated radioactivity as PGA2. This uptake was closely correlated with decrease in viable cell number during incubation. These results suggested that growth inhibitory effect of PGE2 was due to the metabolic dehydration of PGE2 to PGA2, and PGA2, after taken up by cells, exerted cell growth inhibition.     

Human umbilical cord-derived MSC culture: the replacement of animal sera with human cord blood plasma

Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) hold great potential for their therapeutic use in various clinical diseases. Many publications have reported on human blood-derived alternatives to animal serum for culturing mesenchymal stem cells, such as human serum, allogenic umbilical cord blood serum, and human platelet derivatives. However, it is not clear whether human umbilical cord blood plasma (UCBP), as the surplusage of umbilical cord blood mesenchymal stem cell extraction, could be used. In this study, in order to make the best of umbilical cord blood, the human UCBP was dialyzed to replace fetal bovine serum (FBS) in the culture medium. hUC-MSCs were cultured in the new medium. Cell growth rate, specific biomarkers, and differentiation properties were detected to characterize the cell proliferation and MSC-specific properties. The hUC-MSCs cultured in such derived medium were verified with proliferation rate, cluster differentiation markers, cell cycle, as well as differentiation capabilities. Such dialyzed human UCBP is fully comparable with, if not superior to, FBS in deriving and culturing hUC-MSCs.     

Epidermal growth factor stimulation of prostaglandin E2 biosynthesis in amnion cells. Induction of prostaglandin H2 synthase

Amnion is believed to be a tissue of signal importance, anatomically and functionally, in the maintenance of pregnancy and during the initiation of parturition. Epidermal growth factor (EGF)-like agents cause a striking increase in the secretion of prostaglandin E2 (PGE2) in human amnion cells but only if arachidonic acid is present in the culture medium. To investigate the regulation of arachidonic acid metabolism by EGF-like agents in amnion, we used mEGF and human amnion cells in primary monolayer culture as a model system. The amount of PGE2 secreted into the culture medium was quantified by radioimmunoassay and the rate of conversion of [14C]arachidonic acid to [14C]PGE2 (PGH2 synthase activity) in cell sonicates was determined under optimal in vitro conditions. Treatment of amnion cells with mEGF led to a marked increase in the rate of production of PGE2. The specific activity of PGH2 synthase (viz. the combined activities of prostaglandin endoperoxide (PGH2) synthase and PGH2-PGE isomerase) was increased by 2-5-fold in cells treated with mEGF. Treatment of amnion cells with mEGF for 4 h did not affect the specific activities of phospholipase A2 or phosphatidylinositol-specific phospholipase C. By immunoisolation of newly synthesized, [35S]methionine-labeled PGH2 synthase, we found that mEGF stimulated de novo synthesis of the enzyme. Thus, mEGF acts in human amnion cells in primary monolayer culture to increase the rate of PGE2 biosynthesis by a mechanism that involves induction of PGH2 synthase; the manifestation of EGF action on PGE2 biosynthesis is dependent on the presence of nonesterified arachidonic acid.     

Role of interleukin 1 in promoting human monocyte-mediated tumor cytotoxicity

Human peripheral blood monocytes from normal donors obtained by separation on a Percoll gradient showed considerable cytotoxicity against tumor cells when preincubated in vitro for 24 hr with human monocyte-derived interleukin 1 (IL 1). In contrast, monocytes after pretreatment in medium alone had low cytotoxic activity. All the IL 1 preparations, including IL 1 which was purified by high-performance liquid column chromatography (HPLC), as well as crude culture supernatant from human monocytes promoted monocyte-mediated cytotoxicity in the same dose-dependent manner as the thymocyte growth-promoting activity. There was no endotoxin or interferon (IFN) activity in the highly purified IL 1, suggesting that IL 1 itself was the active moiety. The effect of IL 1 on monocyte-mediated cytotoxicity was partially inhibited by indomethacin, whereas pretreatment of monocytes with prostaglandin (PG) E1 or E2 rather than IL 1 also resulted in substantial monocyte cytotoxicity. Thus, the effect of IL 1 on monocyte-mediated cytotoxicity is presumably mediated by PGE. Since fresh monocytes that were not preincubated exhibited levels of spontaneous cytotoxic activity similar to that of monocytes preincubated with IL 1, it seemed likely that the effect of IL 1 was to maintain the spontaneous level of activity rather than to induce cytotoxic activity. To elucidate this possibility, monocytes were first preincubated in medium alone for a longer period, and after losing their spontaneous activity they were further incubated with or without IL 1. Such "aged" monocytes did not develop cytotoxic activity in response to IL 1 but did in response to other agents known to induce macrophage cytotoxicity, such as endotoxin or lymphokine-containing supernatants. Therefore, the major effect of IL 1 actually seemed to prolong the cytotoxic state of monocytes. These results also suggest that IL 1 released by macrophages or monocytes may play a role in host defense against neoplastic cells by acting on monocytes as an autostimulating factor.     

Inhibition of murine lymphokine-activated killer (LAK) cell activity by adherent cells

The effects of adherent cell depletion, indomethacin, and prostaglandin E2 (PGE2) on murine LAK cell activity were investigated. Removal of plastic adherent cells from splenocyte suspensions either prior to 5-day culture with 1000 U/ml of recombinant human IL-2 (rIL-2) or prior to assay resulted in an enhanced LAK cell cytotoxicity compared to that of whole spleen cell suspensions. Indomethacin enhanced LAK cell cytotoxicity of whole splenocyte suspensions if present during the culture period, but had no effect on whole splenocyte or adherent cell-depleted cell suspensions if added just prior to assay. PGE2 suppressed LAK cell activity of nonadherent splenocyte but not whole splenocyte suspensions when present during the culture period. In vivo treatment of mice with indomethacin enhanced cytotoxicity directed toward both LAK sensitive, natural killer (NK) resistant (P-815) and LAK, NK sensitive (YAC-1) tumor cell targets. Splenocytes from indomethacin-treated mice cultured with additional indomethacin and rIL-2 exhibited highest LAK cell activity. The results from this study indicate that LAK cells are regulated by adherent cells which suppress LAK cell activity. This suppression can be reversed both in vitro and in vivo by indomethacin. This study has important implications for the possible clinical use of indomethacin in the potentiation of in vivo and in vitro LAK cell activity for immunotherapeutic protocols.     

Granulosa cells stimulate in vitro the expansion of isolated mouse cumuli oophori: involvement of prostaglandin E2

Granulosa cells (2 X 10(6) per ml) obtained from pregnant mare's serum gonadotropin (PMSG)-primed mice induce within 24 h the expansion in vitro of cocultured mouse cumuli oophori. Experiments with conditioned media showed that the expansion-promoting action of granulosa cells is due to diffusible factor(s) released into the culture medium. Studies with prostaglandin synthetase inhibitors and direct measurements of prostaglandin E2 (PGE2) released by granulosa cells in the culture medium have also been performed. The results strongly suggest that the cumulus oophorus expansion-promoting action of granulosa cells is mediated by PGE2, and support the hypothesis (Downs and Longo, 1983) that granulosa cells might play a similar role in the mechanism of cumulus expansion in vivo. The suggestion is advanced that coculture with granulosa cells might be of help to allow physiologic expansion in culture of immature cumuli obtained from preovulatory follicles in in vitro fertilization programs.     

The in vitro response of human tumour cells to desferrioxamine is growth medium dependent

Abstract. Iron chelating agents have been demonstrated to inhibit tumour cell growth. However, in vitro and in vivo results using desferrioxamine a hexadentate iron chelating agent, for anti-cancer treatment are not always in agreement. Therefore, we have studied the response of three human tumour cell lines (HL-60 promyelocytic leukaemia, MCF-7 breast cancer and HepG2 hepatoma), grown in culture medium supplemented with either human pooled (HPS) or fet al bovine serum (FBS), to desferrioxamine. Desferrioxamine, at micromolar concentrations, induced severe cytotoxicity in all tumour cell lines grown in FBS medium. When grown in HPS medium, comparable desferrioxamine cytotoxicity was observed in the millimolar range. The addition of 50% saturated human transferrin to FBS medium resulted in protection against desferrioxamine cytotoxicity. HL-60 cells were further studied for iron metabolism characteristics. HL-60 cells, grown in medium with FBS, were found to have an 8.4 fold increase in surface transferrin receptor (TfR) expression ( P < 0.001) as compared with HL-60 cells grown in medium with HPS. However, iron uptake of HPS cultured HL-60 cells, after incubation with saturated human transferrin, was higher, resulting in a higher concentration of iron in HPS cultured HL-60 cells as compared with FBS cultured cells (1.72 ± 0.02 μmol/g protein v. 1.32 ± 0.14 μmol/g protein; P < 0.001). Using desferrioxamine it was shown that TfR expression is dependent on the biological availability of iron in the cell. Consistent with the lower iron content in FBS cultured cells, we conclude that the cytotoxicity of desferrioxamine is dependent on the ability of cells to replenish cellular iron stores from the culture medium. Cells grown in FBS medium lack this ability and are therefore more susceptible to desferrioxamine.     

Interaction of prostaglandins with blood plasma proteins. I. Binding of prostaglandin E 2 to human plasma proteins and its effect on the physiological activity of prostaglandin E 2 in vitro and in vivo

The binding of (PGE2) prostaglandin E2 to human plasma proteins was investigated by DEAE-Sephadex column chromatography and acrylamide gel electrophoresis, and quantitatively assessed by equilibrium dialysis. PGE2 added to human plasma in vitro was found to become mainly bound to plasma albumin. This binding was also demonstrated by adding PGE2 to human serum albumin solutions. The binding of PGE2 to human serum albumin inhibits the contraction-producing effect of PGE2 on the isolated gerbil colon in vitro. The depressor effect of PGE2 on the rat blood pressure was used to assess the in vivo effect of PGE2 albumin interaction. The blood pressure lowering activities of free and albumin-bound PGE2 were found to be the same when administered either intravenously or intraarterially. The significance of these observations with regard to estimation of PG concentration in whole blood or plasma, and their possible effects on PG metabolism is discussed.     

Cytokines produced by blood mononuclear cells stimulated with the streptococcal preparation OK-432: effect on production by supplementing the medium with xenogeneic serum

Summary Human blood MNC were stimulated for 7 days with a streptococcal preparation, OK-432, in a medium supplemented with either 10% autologous serum (autoserum) or 10% FCS. Cytokines, including IL-2, IL-1, IFN, and TNF in the supernatants, and antitumor cytotoxicity of MNC were analyzed. None of the IL-2 was detectable during the culture in medium with autoserum, although significant enhancement of cytotoxicity was observed at day 1. On the other hand, production of IL-2 and higher cytotoxicity were induced in the medium with FCS. Even a control culture of MNC unstimulated with OK-432 in the medium with FCS, showed a slight but significant amount of IL-2 and considerable cytotoxicity. However, such a culture in the medium with autoserum showed no such IL-2 production or cytotoxicity. The cytotoxicity induced in the medium with FCS was significantly inhibited by the addition of anti-IL-2 antibody to the culture as well as by the addition of anti-IFN antibody, whereas the cytotoxicity in the medium with autoserum was not inhibited at all by anti-IL-2 antibody. Therefore, the cytotoxicity induced in the medium with FCS included IL-2-induced killer activity, i.e., LAK activity. Similarly, the levels of IL-1, IFN, and TNF production in the supernatants were variable depending on the serum used to supplement the medium. The nonspecific production of IL-2 and the unexpected induction of cytotoxicity were consistently provoked in the medium using several different lots of FCS, but not in the medium with human allogeneic sera or pooled AB serum nor in that with autoserum. It was revealed that the source of sera used to supplement the medium is an important factor affecting the results of analysis of cytokine production and cytotoxicity of MNC induced by certain stimulants. Abbreviations used: NK, natural killer; IFN, interferon; TNF, tumor necrosis factor; IL-1, interleukin-1; IL-2, interleukin-2; LAK, lymphokine-activated killer; MNC, mononuclear cells; FCS, fetal calf serum; ³H-TdR, ³H-Thymidine     

Stability of antisense DNA oligodeoxynucleotide analogs in cellular extracts and sera

Antisense DNA oligodeoxynucleotides can selectively inhibit the expression of individual (undesirable) genes and thus, have potential in the treatment of cancer and viral diseases. A prerequisite to their use as therapeutic agents is information on the stability of oligodeoxynucleotides, and their structurally modified analogs, in the biological milieu. To this end, degradation of 5' end and internally [32P] labelled unmodified DNA oligodeoxynucleotide (D-oligo) and analogs containing phosphorothioate (S-oligo), methylphosphonate (MP-oligo), and novel alternating methylphosphonate and phosphodiester (Alt-MP-oligo) internucleoside linkages was studied in Hela cell nuclear extract, S100 cytoplasmic extract, normal human serum and calf serum at 37 degrees C. Both 5' end and internally labelled D-oligos showed complete degradation within 30 min incubation in human serum at 37 degrees C. In any given medium, the D-oligo was the least stable oligodeoxynucleotide to nuclease degradation whereas the Alt-MP, MP and S-oligos were generally of comparable stability and all relatively more stable than D-oligo. Interestingly, MP and Alt-MP-oligos also exhibited greater resistance to phosphatases in cellular extracts compared to D and S-oligos. Under the conditions of the experiments, increasing degradation for any given oligonucleotide was observed in the order: S100 cytoplasmic extract less than nuclear extract less than normal human serum less than calf serum. In a study involving alpha-MEM cell culture medium containing 10% heat inactivated fetal calf serum (heated to 56 degrees C for 1 hour), the D-oligo was found to be rapidly degraded (degradation evident within 10 mins) whereas degradation products for the S-oligo were observed within 1 hour. In contrast, the Alt-MP oligo remained stable throughout the 3 hour experiment. These results indicated that in cell culture medium containing heat inactivated serum Alt-MP oligo was more stable than D- and S-oligos.     

Starvation of a clonal osteoblast-like cell line, MOB 3-4-F2, down-regulates prostaglandin E2 receptors but increases cAMP response to prostaglandin E2.

Prostaglandin E2 (PGE2) stimulated cAMP production in the MOB 3-4-F2 cell line, a subclone of the osteoblast-like MOB 3-4 cell line. After being cultured in alpha-minimum essential medium supplemented with 10% heat-inactivated foetal calf serum (HIFCS), cells responded to PGE2 (greater than or equal to 50 ng/ml) with a small, but significant, increase in cAMP production. This response did not vary with duration of culture. In 2% HIFCS-containing medium, despite their lower basal cAMP level, cells responded to PGE2 (greater than or equal to 5 ng/ml) with strikingly increased cAMP production. In addition, prolonged culture in this serum-deficient medium enhanced this response. On the other hand, culture of cells in 2% HIFCS-containing medium decreased the apparent number of PGE2 receptors, which was also enhanced by prolonged culture, without effect on their apparent affinity. Their number in 10% HIFCS-containing medium, more than that in 2% HIFCS-containing medium, was almost constant, independent of the culture period. Starvation of MOB 3-4-F2 cells in serum-deficient medium, therefore, appeared to down-regulate PGE2 receptors but increase the cAMP response to PGE2. Moreover, prolonged starvation of cells appeared to facilitate these phenomena. Our findings suggest that cAMP response to PGE2 does not always reflect the number of available PGE2 receptors in the cells.     

Development of improved media and culture conditions for clonal growth of normal diploid cells

Multiplication of normal diploid cells in culture is controlled by a complex set of interacting extracellular variables. The amount of serum protein needed for colony formation by such cells is affected directly by many of the other variables, including the nature of the culture surface, the type of trypsinization procedure used, and the qualitative and quantitative composition of the culture medium. By a sequential process of adjusting all of these variables to optimum values for cellular multiplication with minimal amounts of serum protein, we have been able to obtain clonal growth of normal human and chicken cells with less than 500 microgram per ml dialyzed serum protein. Precise quantitative adjustment of nutrient concentrations is particularly important. The multiplication-promoting functions of serum can be classified operationally as "replaceable" (those that can be replaced by modifying the medium or the culture conditions) and "nonreplaceable" (those that we have not yet been able to replace). Elimination of the requirement for replaceable functions of serum has improved greatly the specificity and sensitivity of the bioassay for the nonreplaceable functions. The nonreplaceable multiplication-promoting activity from fetal bovine serum for human diploid fibroblasts has been separated from fetuin and serum albumin and purified approximately 15-fold.     

The influence of serum components on the growth and mutation of Chinese hamster cells in medium containing 8-azaguanine

Low concentrations (?20 μg/ml) of 8-azaguanine are 1000 fold more toxic to V79 Chinese hamster cells in medium containing 10% dialyzed fetal calf serum than in medium containing 10% undialyzed serum. Serum enzyme activity that converts AG to nontoxic 8-azaxanthine degrades AG at the same rate, whether or not the serum is dialyzed. However, cytotoxicity results similar to those obtained with US were produced in medium containing DS and 2.5 μg of hypoxanthine (HX)/ml (DSH). Therefore, serum HX is considered to be responsible for the relatively low cytotoxicity of AG in medium containing US. Colonies that arose in medium containing AG were isolated and characterized. Those that remained resistant to AG (40 μg/ml) and sensitive to aminopterin in the presence of HX and thymidine (HAT) were considered mutants; nonmutants were sensitive to AG and resistant to HAT. Colonies isolated from medium containing DSH of US and low concentrations of AG were not mutants, but those from medium containing high concentrations (? μg/l) of AG were mutants. Spontaneous and N-methyl-N′-nitrosoguanidine induced mutants were detectable in medium containing DSH without replating the cells prior to adding AG (?30 μg/ml), but in order to detect MNNG induced mutations in medium containing DS replating was essential. In DS, the mutation frequency increased as an exponential function of the toxicity of MNNG, but remained two orders of magnitude lower than the induced mutation frequencies that occurred in DSH, HX, in DSH or US, produced profound effects, other than interference with AG toxicity, that distort the results of mutagenesis assays. To study mutation using AG resistance as the endpoint, it is essential to use dialyzed serum.     

Clonal growth and serial propagation of rat esophageal epithelial cells

The clonal growth and serial propagation of rat esophageal epithelial cells in low serum-containing medium has been achieved without feeder layers or conditioned medium. To date, a total of four lines have been developed and maintained for as many as 40 passages in culture. Growth of the cells was possible only after modifying the culture medium (PFMR-4) by reducing the calcium concentration from 1 to 0.1 mM, and by adding low levels of dialyzed fetal bovine serum and seven growth factors; i.e. epidermal growth factor, hydrocortisone, ethanolamine, phosphoethanolamine, insulin, transferrin, and cholera toxin. Cell lines have been developed from both explant outgrowths and enzyme dissociated esophagi. The epithelial nature of the cells was confirmed by electron microscopy and immunological methods. Clonal growth studies revealed that optimal cell growth occurred in medium containing 2.4% dialyzed fetal bovine serum and 0.1 mM calcium. Calcium levels of 0.3 mM or higher caused the cells to stratify and undergo terminal differentiation. Coating the culture dishes with collagen, or a combination of collagen, fibronectin, and bovine serum albumin, increased both the cell growth rate and the colony forming efficiency. The successful long term culture of rat esophageal epithelial cells permits their use as models in studies concerned with esophageal differentiation and carcinogenesis.     

The regulation of ionic nickel uptake and cytotoxicity by specific amino acids and serum components

The effects of serum components and amino acids on the uptake and cytotoxicity of NiCl₂ were examined in cultured Chinese hamster ovary (CHO) cells. CHO cells maintained in a minimal salts/glucose medium accumulated 10-fold more⁶³Ni than did cells maintained in complete medium supplemented with 10% fetal bovine serum. Cell-surface binding of⁶³Ni appeared to account for the majority of this increased accumulation of cell-associated nickel observed in the simple maintenance medium since such increases were reduced 70% by trypsin treatment. The addition of the Ni²⁺-binding amino acids cysteine or histidine to the salts/glucose medium markedly decreased⁶³Ni accumulations, an effect not observed following addition of any of several amino acids that do not bind Ni²⁺. Supplementation of the salts/glucose medium with fetal bovine serum decreased in a concentration dependent fashion both the⁶³Ni²⁺ uptake and cell detachment caused by Ni²⁺, while dialyzed (amino acid-free) serum was 3–5-fold less effective than undialyzed serum at reducing⁶³Ni²⁺ uptake and similarly exhibited only a slight protective effect against nickel-induced cytotoxicity. Supplementation of dialyzed serum with cysteine at levels approximating those in whole serum partially restored its inhibitory activity toward nickel uptake by cells and restored completely its inhibition of nickel's cytotoxicity, indicating the predominant role of specific amino acids over serum proteins in regulating the uptake and subsequent cytotoxicity of Ni²⁺. Addition of cysteine to the salts/glucose medium during a 2 h exposure of cells to either 100 μM HgCl₂ or 1 mM NiCl₂ masked the cytotoxic effects of these metal ions. These results demonstrate the importance of extracellular small molecular weight metal ion chelators in altering the biological effects of metal ions at the level of metal uptake.