Inhibition of cytolytic T lymphocyte maturation with ornithine, arginine, and putrescine

L-Ornithine was shown to inhibit the development of cytolytic T lymphocytes (CTL) in mixed lymphocyte cultures (MLC). Lymphokines were unable to reverse the suppressive effect, and cytotoxic activity was not revealed by coupling ornithine-inhibited MLC cells to target cells with phytohemagglutinin (PHA). If addition of ornithine to MLC were delayed, sensitivity of CTL to inhibition was reduced after 24 hr and lost by 48 hr. Suppression of CTL development was not due to a toxic effect. MLC washed free of ornithine after 3 days produced detectable cytolytic activity within 24 hr of secondary culture, and to the same degree as the uninhibited MLC control within 48 hr. Cytotoxic cells generated in secondary cultures were Lyt-2+, did not kill the natural killer-sensitive YAC-1 cell line, and were shown to be antigen-specific by virtue of the findings that cytolysis and cold target inhibition were observed only with cells carrying the original, inducing H-2 haplotype. Cytolysis of target cells by normal CTL effector cells was not inhibited by L-ornithine. MLC depleted of accessory cells so that CTL activation was dependent upon addition of lymphokines remained susceptible to inhibition by ornithine. Our findings indicate that in the ornithine-inhibited MLC, CTL precursors undergo clonal expansion, but their maturation is arrested at a precytolytic stage. L-Arginine and putrescine also suppressed generation of CTL in primary MLC, and cells recovered from arginine- and putrescine-inhibited MLC developed control levels of CTL within 48 hr of secondary culture. Inhibition by putrescine was observed in tissue culture medium supplemented with human serum but not with fetal calf serum, presumably due to the presence of diamine oxidase activity in fetal calf serum. Similar to ornithine, the suppressive effects of arginine and putrescine on T lymphocytes were apparently selective for CTL because they did not inhibit mitogen activation with concanavalin A or the production of interleukin 2 and interleukin 3. These findings are consistent with a hypothesis that the inhibitory effects of ornithine, arginine, and putrescine are mediated by polyamines, and exerted on the differentiative stage of CTL development.     

Inhibition of cells in culture by polyamines does not depend on the presence of ruminant serum

Using T-lymphocyte (T-LC) and granulocyte colony (GC) assays with truly proliferating cells, the inhibitory dose-response relationships of spermine and spermidine in the presence of selected sera have been examined. In contrast to previous studies which used [3H]thymidine uptake as an index of proliferation, in vitro inhibition by polyamines was shown to require neither foetal calf serum (FCS) nor the addition of any exogenous polyamine oxidase. Cells grown in the absence of FCS were between 5-50% as sensitive to polyamines as in its presence. By using specific inhibitors of polyamine oxidase, it was shown that polyamine-elicited mitotic inhibition in the absence of FCS was still dependent on a polyamine oxidase, and evidence is presented to show that the source of the enzyme is the cells themselves.     

The culture of human skin fibroblasts on microcarriers in the presence of serum substitute: Ultroser G

The effects of the substitution of serum by Ultroser G on human skin fibroblasts cultured on microcarriers were analysed. Cultures could not be established on microcarriers in the presence of Ultroser G. However, microcarrier cultures started in the presence of 10% foetal calf serum, and transferred to 2% Ultroser G after 7 days resulted in high cell densities.     

The culture of human skin fibroblasts on microcarriers in the presence of serum substitute: Ultroser G

The effects of the substitution of serum by Ultroser G on human skin fibroblasts cultured on microcarriers were analysed. Cultures could not be established on microcarriers in the presence of Ultroser G. However, microcarrier cultures started in the presence of 10% foetal calf serum, and transferred to 2% Ultroser G after 7 days resulted in high cell densities.     

Effect of insulin, thyroxine and oestradiol on growth of normal human diploid cells and human heteroploid HeLa cells

Insulin (5 micrograms/ml), thyroxine (1 microgram/ml) and beta-oestradiol (0.1 microgram/ml), if added separately to the medium, did not significantly influence the growth of normal human diploid LEP 19 cells cultivated in Eagle's MEM medium with 1% foetal calf serum. Combined, they did not influence the growth of LEP 19 cells in serum-free MEM medium either. Under the same cultivation conditions, insulin caused 37%, thyroxine 24% and a mixture of insulin, thyroxine and beta-oestradiol 325% (without serum) or 15% (with 1% foetal calf serum) stimulation of the growth of human heteroploid HeLa cells. Isolated beta-oestradiol significantly inhibited the growth of HeLa cells.     

Polyamine-mediated inhibition of in-vitro cell proliferation is not due to acrolein

The influence of acrolein or spermine on the viability and growth of phytohaemagglutinin-stimulated rat thymic lymphocytes in cultures supplemented with foetal calf serum have been investigated. Acrolein (greater than 20 microM) was cytotoxic; spermine had little effect on viability, but inhibited [3H]TdR incorporation at low concentrations (approximately 10 microM). Cells treated with greater than 8 microM acrolein 3 hr before stimulation exhibited irreversible inhibition of protein synthesis, whereas 50 microM spermine had no effect, even when cells were treated for 24 hr before stimulation. However, addition of 25 microM spermine after stimulation did inhibit both [3H]-uridine incorporation and protein synthesis: this was reversible if cells were freed of polyamine within 4 hr, but not if washed after 24 hr. These results show that, contrary to several previous reports, in-vitro inhibition of cell proliferation by spermine is not due to the formation and action of acrolein.     

The effect of polyamines on cell culture cells

Growth of KB cells was inhibited by both spermine and spermidine, but the inhibition is reduced in conditioned medium. The amount of spermine required for 50% inhibition of plating varied according to the type of serum used with medium 199 (calf, fetal bovine, and horse; 0.55, 0.9, and 24 μg/ml respectively). The spermine oxidase activity of the three sera was calf > horse > fetal bovine, which is not the same ordering as was obtained for the inhibition. When the concentration of sera in the media was varied, the inhibition decreased as calf and fetal bovine sera concentration increased, whereas, with horse serum, an increase in serum concentration increased the inhibition. The opposite effects of increasing concentrations of the sera on the inhibition suggest that at least two factors are involved in the inhibition. A scheme which involves three factors (spermine oxidase, another enzyme and its activator) is postulated to account for the inhibitions and reversals observed. Spermine oxidase alone cannot account for the action of polyamine on cells.     

Selective maintenance of cultured epithelial cells from DMBA-induced mammary tumours by bovine colostrum supplement

Growth kinetics in cultures of DMBA-induced mammary tumours supplemented with bovine colostrum were compared with the kinetics of cultures maintained with the conventional supplement of foetal calf serum. Although the latter permitted a greater degree of cell proliferation, a substantial amount of the cell growth was due to the fibroblastic proliferation. In the presence of bovine colostrum, epithelial islands surrounded by a few solitary cells became established. Unlike the foetal calf serum supplemented cultures, these cultures frequently did not become completely confluent within 7 days. The absence of fibroblasts in colostrum supplemented cultures was confirmed by electron microscopy. Results from this study suggest that colostrum may be useful in selective maintenance of primary cultures of epithelial origin.     

Natural killer activity in man under different experimental conditions]

The cytotoxic activity of natural killer (NK) cells against labelled with 3H-uridine target cells of standard human erythromyeloblast line K562 in 63 healthy donors was studied in 14-hr cytotoxic test. The cytotoxic reaction was realized in complete medium supplemented with different types of serum such as foetal calf serum (FCS), autologous or homologous sera in different schemes of incubation. It has been shown that NK cell activity was augmented by 50 per cent (p < 0.05) in the presence of homologous serum added to mononuclear cell suspension for 1 hr at 37 degrees C in comparison with the effect of FCS presence. Thus, the serum of healthy donors contains some factors which not only reflect an individual genotypic information of a donor, but also can be recognized by NK cells and significantly change the cytotoxic NK cell activity. The data obtained exclude possibility of using a homologous serum when positive controls for natural cytotoxic reactions are planned.     

Cytotoxic effect of human monocytes to mouse red cells

Human blood monocytes have been cultivated for various lengths of time. Within a few hours they are well spread on the glass and look very similar to peritoneal macrophages in vitro. After about 10 days of cultivation, they develop the capacity to exert cytotoxic effects against mouse red cells in the presence of foetal calf serum. Red cell ghosts as well as abnormal red cells are attached to the macrophages at this point. In the presence of human serum autologous to the monocytes, the rouse red cells are phagocytosed.     

Separated human breast epithelial and myoepithelial cells have different growth factor requirements in vitro but can reconstitute normal breast lobuloalveolar structure

In order to investigate the specific factors controlling the growth of normal breast cell types, purified populations of human breast epithelial and myoepithelial cells from reduction mammoplasties were grown in primary culture in three defined media and their response to foetal calf serum (FCS), epidermal growth factor (EGF) and basic fibroblast growth factor (FGF2) measured using MTT growth assays. Epithelial and myoepithelial cells differed markedly in their growth requirements. Whereas epithelial cell survival was dependent on the presence of FCS, myoepithelial cell growth was dramatically inhibited by serum. EGF and FGF2 were mitogenic for epithelial cells but not myoepithelial cells, the addition of insulin being the only essential supplement required for myoepithelial cell growth. Heparin inhibited FGF2-stimulated epithelial cell growth but also basal myoepithelial cell proliferation and this inhibition could be overcome by the addition of EGF. Neutralizing antibodies to EGF also inhibited basal myoepithelial cell growth. This suggests the possibility of an autocrine role for a heparin-binding member of the EGF family in the growth of myoepithelial cells. Purified cells combined to form lobuloalveolar structures when incubated in a reconstituted basement membrane matrix (Matrigel) in the presence of EGF and FGF2. J. Cell. Physiol. 171:11–19, 1997. © 1997 Wiley-Liss, Inc.     

The effects of newborn calf serum and foetal bovine serum on the Na+ + K+-activated adenosine triphosphatase activity in 3T3 and SV3T3 cells grown to confluency

The effects of cell density and growth in 10% foetal bovine serum and 10% newborn calf serum on the activity of the enzyme (Na+ + K+)-ATPase were studied in 3T3 and SV3T3 cells. The enzyme activity decreases in 3T3 cells grown in foetal bovine serum as the cells approach confluency while in those grown in newborn calf serum the enzyme activity increases. The (Na+ + K+)-ATPase activity does not change with increase in cell density in SV3T3 cells grown in foetal bovine serum while the enzyme activity in those grown in newborn calf serum increases with increase in cells density up to about 1.35 x 10(5) cells/sq. cm. and then decreases with further increase in cell number. At confluency it was found that the enzyme activity is higher in the SV3T3 as compared to the 3T3 cells when the cells were grown in 10% foetal bovine serum, whereas in those grown in 10% newborn calf serum the enzyme activity is higher in the 3T3 as compared to the SV3T3 cells.     

Isolation and culture techniques of foetal calf chondrocytes.

Large quantities of differentiated mammalian chondrocytes from normal hyaline cartilage were isolated after digestion of foetal bovine tracheas with collagenase. Incubation of the newly isolated cells for 1 day in the presence of dextran sulphate inhibited formation of cell aggregates during subsequent subculture in the absence of dextran sulphate. After incubation with dextran sulphate, the cells were plated in Ham's F12 medium with or without foetal calf serum on hydrophilic or hydrophobic Petri dishes. Chondrocytes cultured on hydrophilic substrates in the presence of serum attached to the substrate and showed cytoplasmic spreading. The cells did not attach to hydrophobic substrates in the presence of serum, but remained in suspension as single cells. In the absence of serum the chondrocytes attached to either substrate, but did not show any cytoplasmic spreading. By using labelling with [35S]sulphate and [3H]-thymidine it was shown that glycosaminoglycan synthesis did not require the presence of serum, whereas DNA synthesis required serum factors. Extracellular glycosaminoglycans were recovered in two pools: the medium pool and the pericellular pool, the latter being isolated by proteolytic digestion. The kinetics of these pools differed, depending on the presence or absence of serum and the type of substrate used. The turnover of the pericellular pool was studied in a pulse-chase experiment. At the end of the chase (72 h), only 60% of the material in the pericellular pool had been metabolized.     

1,25-Dihydroxyvitamin D3 inhibitory effect on the growth of two human breast cancer cell lines (MCF-7, BT-20)

1,25 (OH)2 D3 has been shown to be able to reduce the growth of several human cell lines. The effect of 1,25 (OH)2 D3 on the growth of breast cancer cell lines in relation to their oestrogen (ER) and progesterone (PGR) receptor content has been investigated. The growth inhibition of BT-20 and MCF-7 cell lines is related to the dose of 1,25 (OH)2 D3. It is dependent on the foetal calf serum concentration in the culture medium. At low concentration of 1,25 (OH)2 D3 the inhibitory effect is not detectable in the presence of 10% FCS. The rescue of cells from inhibition by serum was less effective when 1,25 (OH)2 D3 was present in the medium. The results of [3H]thymidine incorporation experiments and DNA measurements are in agreement with the reduction of cell number. Analysis in flow cytometry indicated a reduced number of cells in S phase. These data indicate that 1,25 (OH)2 D3 is able to modulate the growth of human breast adenocarcinoma cells regardless of their sex steroid dependency.     

Reduced frequency of sister chromatid exchanges in human lymphocytes cultured with autologous serum

Summary The incidence of sister chromatid exchange (SCE) was determined in human lymphocytes cultured with fetal calf, human AB, and autologous serum. In each individual studied, cells grown in medium supplemented with fetal calf and human AB serums showed higher yields of SCE than those cultured with autologous serum. Increased concentration of fetal calf and human AB serum in the tissue culture medium resulted in elevated frequency of SCE. No such elevation in SCE frequency was observed with increased concentration of autologous serum. The results indicate the presence of extraneous SCE-inducing factors in fetal calf and human AB serum, the nature of which is not precisely known.Aided by C.S.I.R. Grant No. 7/45 (1052/77) EMR I     

The preparation of pyrogen-free foetal calf serum for use in cell and tissue cultured

A method is described for the preparation of foetal calf serum from blood drawn by heart puncture from calf foetuses obtained from a local municipal abattoir. This method differs from that previously described in that the Seitz Supra EK filter pads used in preliminary purification steps have been abandoned. These pads contain significant amounts of pyrogenic bacterial lipopolysaccharides and attempts to depyrogenate them with sodium hypochlorite, hydrogen peroxide and 1% foetal calf serum were unsuccessful. The modified method uses pyrogen-free glass fibre and cellulose fibre filters and provides an entirely satisfactory serum as proved by negative Limulus amoebocyte lysate tests and the excellent growth of cells.     

Carotenoids inhibit DNA synthesis in human aortic smooth muscle cells

Quiescent, serum-starved human aortic smooth muscle cells were restimulated with 20% foetal calf serum in Dulbecco's modified Eagle medium, in the presence and absence of beta-carotene, canthaxanthin, zeaxanthin, lycopene, lutein or beta-cryptoxanthin, at final concentrations up to 23 microM. Concentration-dependent inhibition of DNA synthesis, measured by [methyl-3H]thymidine incorporation, was observed for the carotenoids, except for canthaxanthin and lutein which had no effect. Lycopene was the most potent of the carotenoids tested. The results suggest that antiproliferative effects of dietary carotenoids might be of significance in vivo.     

Oesophagostomum radiatum: inhibition of in vitro development by newborn calf serum

Oesophagostomum radiatum developed to fourth stage larvae after 14 days in vitro culture. However, development was totally inhibited if the standard 50% foetal calf serum in the medium was replaced by newborn calf serum. Inhibition did not occur with serum from cattle immune to O. radiatum through natural infection or experimental vaccination irrespective of the titre of specific antibodies to O. radiatum in each serum. The inhibitory action of NCS could be abolished by heat treatment at 56 degrees C for 1 h but not by dialysis or repeated freeze-thawing. The inhibition was not consistent with observed differences in the activity of 19 enzymes in the various sera or the absence of various thiol-containing stimulants of worm development.     

Establishment of internal-image anti-idiotype monoclonal antibodies to a human antibody to lung cancer

 Internal-image anti-idiotype antibodies are expected to enhance anticancer effector mechanisms in vivo. The objective of this study was to establish hybridomas producing anti-idiotype monoclonal antibodies against a human monoclonal antibody (hmAb) 4G12 that reacts strongly with lung squamous cell carcinomas. BALB/c female mice 6 weeks old were immunized with 4G12. Splenocytes were hybridized with P3U1 cells and hybrid cells secreting anti-4G12 hmAb were cloned. Two clones reacted with 4G12 hmAb but not with 3H12 IgM hmAb, human IgM, human serum or fetal calf serum. These two Ab2 antibodies (IgG1κ) 2B12 and 2H1 demonstrated 91.5% and 90.3% inhibition in their reactivity with radiolabelled 4G12 on PC10 cells, indicating that 2B12 and 2H1 antibodies were of the Ab2β type. In criss-cross inhibition assays, the binding of 2B12 or 2H1 to 4G12 was not inhibited by 2H1 or 2B12. Thus 2B12 and 2H1 were thought to recognize the different epitopes on the antigen-binding sites. Antisera against 2B12 and 2H1 demonstrated specific reactivity to PC10 cells. The two Ab2β antibodies, 2B12 and 2H1, express internal images of lung squamous cell carcinoma recognized by the 4G12 antibody and may be useful for cancer immunotherapy. Received: 20 September 1996 / Accepted: 2 January 1997     

Diadenosine 5',5'-P1,P4-tetraphosphate (AP4A) levels under various proliferative and cytotoxic conditions in several mammalian cell types

Diadenosine 5',5'-P1,P4-tetraphosphate (Ap4A) has been proposed as an intracellular signal for growth. In order to test this hypothesis Ap4A levels were followed in several cell types under various conditions. Quiescent dog thyroid cells in a primary culture were induced to proliferate by addition of a mixture of epidermal growth factor, thyrotropin and foetal calf serum; V79 cells were synchronized by serum depletion then stimulated to proliferate by addition of foetal calf serum. Protein and DNA synthesis increased in both cases, although no significant changes in Ap4A levels per cell could be demonstrated. HeLa D98/AH2 and L929 cells were treated with human recombinant tumour necrosis factor alpha which caused marked cell death. This was measured by a decrease in DNA content and a release into extracellular medium of incorporated radioactive precursor. No concomitant variations in Ap4A concentrations could be observed under these conditions. The data from these various systems do not support the hypothesis that changes in Ap4A levels regulate cellular proliferation.