EPR study of the sea anemone cytolysin,equinatoxin II,cytotoxicity on V-79 cells

Electron paramagnetic resonance (EPR) was used to study the effect of equinatoxin II (EqT II), a cytolytic protein isolated from the sea anemone Actinia equina L., on membrane fluidity and cell metabolism of V-79 cells; the reduction of the spin probe incorporated into the cell membranes as well as the oxygen consumption in the cell suspension were measured. The results were compared with the results obtained by the cell viability study. Under the influence of EqT II (less than 37.5 μg/10⁶ cells) no significant changes in cell membrane fluidity were observed, while reduction kinetics of the spin probe and the oxygen consumption decreased when the cells were kept in Tris buffer solution. However, in the presence of 10% fetal calf serum, which prevented cell lysis, the effects of EqT II were diminished. The oxygen consumption corresponds to the cell viability changes but the reduction kinetics alterations indicate that some oxidation-reduction processes other than cell respiration are affected by EqT II in the absence of serum. The effect seems to be indirect, probably due to the formation of pores which are associated with changed permeability of plasmalemma for metabolites and ions.     

Equinatoxin II-induced lysis Of the cultured endothelial cell line ECV-304

Equinatoxin II (EqT II) is a pore-forming actinoporin. Its lethality in rat tissue is due to cardio-respiratory effects. The toxin contracts the vascular smooth muscle only in the presence of intact endothelium. In our study, its effects on the endothelial cell culture ECV-304 were tested. The EqT II effects were dose-dependent and were influenced by calcium ions and sucrose. The obtained results support the conclusion that calcium ions are the intracellular messengers of the EqT II effects on the isolated endothelial cells.     

Doubling of cell mass is not necessary in order to achieve cell division in cultured human cells

When exponentially growing NHIK 3025 cells were shifted from medium containing 30% serum to medium containing 0.03% serum the rate of net protein accumulation was reduced due to both a reduction in the rate of protein synthesis and an increase in the rate of protein degradation. This change in growth conditions increased the protein doubling time from 18 to 140 h. The cell cycle duration of cells synchronized by mitotic selection was, however, only increased from 17 to 26 h by this treatment. Therefore, when the cells divide by the end of the first cell cycle following synchronization, the cells shifted to 0.03% serum contained far less protein than those growing continuously in 30% serum. Hence, the attainment of a critical cell mass is probably not controlling cell division for cells growing in a balanced state.     

Morphological changes of V-79 cells after equinatoxin II treatment.

Morphological observations on the V-79-379 A cells after treatment with equinatoxin II (EqT II), isolated from the sea anemone Actina equina L., and fetal calf serum (FCS) treated toxin were examined by transmission electron microscopy. Our results showed that the cells incubated with FCS treated EqT II were almost ultrastructurally unaltered. When the cells were treated with low concentrations of EqT II alone cell ultrastructure was altered with the evidence of numerous blebs and decreased microvilli number on the cell surface and appearance of numerous vesicles in the Golgi regions. High concentrations of EqT II caused disintegration of plasmalemma and intracellular membranes as well as degradation of cytosol.     

Cutaneous venom of Bombina variegata pachypus (Amphibia, Anura): effects on the growth of the human HL 60 cell line.

The effects of Bombina variegata cutaneous venom (Bvv) on eukaryotic cell growth has been assessed employing the human leukaemic cell line HL 60, by liquid and agar semisolid cultures and 51Cr release assay. HL 60 cells growth is impaired by Bvv in a dose-dependent fashion in both culture systems. The arrest of proliferation requires a contact time lower than 3 min and it is not reversed by washing and culturing the cells in a Bvv-free medium. Similarly, an extremely short exposure time is needed to determine maximum 51Cr release. Neither the agar medium nor the fetal calf serum interact with Bvv effects, which, according to the above findings, must be regarded as cytolytic in nature. In both liquid and the agar-semisolid culture Bvv cytolytic activity half life is about 8 hr. The cytolytic properties of Bvv are thought to be part of the chemical defence system of amphibian skin.     

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Human peripheral blood monocytes were reproducibly shown to lyse a variety of tumor cells in a 3- to 4-hr ⁵¹Cr release assay. Ficoll-Hypaque-purified mononuclear cells were suspended in medium supplemented with either 10% autologous serum or fetal calf serum (PCS). With either serum, highly purified (97–99%) and viable (>99%) monocyte suspensions were obtained by EDTA-reversible adherence to plastic surfaces which had been precoated with autologous serum. When used as effectors in cytotoxicity assays, the monocytes recovered from mononuclear cells suspended in FCS-supplemented medium exhibited higher cytolytic activity and were therefore used for further studies. Using FCS for both coating the plates and supplementing the suspension medium resulted in monocytes with low cytolytic activity. Tumor cell lysis measured by ⁵¹Cr release was detected within 2 hr of incubation and increased gradually with time. The level of lysis was dependent on the effector/target ratio and the tumor target cell employed. The involvement of natural killer lymphocytes in the observed tumoricidal activity was excluded. Detection of cytotoxic activity in a short-term assay will be very helpful in further studies of the mechanism of tumor cell killing by human monocytes since potential complicating effects of long-term in vitro cultivation will be minimized.     

Nicardipine diminished equinatoxin II-induced decrease of coronary flow in isolated rat and pig hearts

Equinatoxin II (EqT II) is a basic, cardiotoxic polypeptide. The vasoconstrictory effect of the toxin on isolated porcine coronary arteries was diminished by nicardipine, an L-type calcium channel antagonist. A comparison was made of the effects of EqT II alone and EqT II in the presence of nicardipine on the coronary flow in porcine and rat hearts isolated according to Langendorff's method. In both models EqT II decreased coronary flow in a dose-dependent manner and there were no statistically significant differences between the two models (p>0.05). However, 1 M nicardipine diminished the effects of EqT II on coronary flow in isolated porcine hearts more than in isolated rat hearts (p<0.05). The results suggest that the activation of L-type calcium channels is one of the mechanisms involved in the lowering of coronary flow induced by EqT II.     

Induction of cytotoxicity of peritoneal exudate cells by agrimoniin,a novel immunomodulatory tannin of Agrimonia pilosa Ledeb

Summary The cytotoxic activities of the PEC after an i.p. injection of agrimoniin, a tannin contained in Agrimonia pilosa Ledeb. were studied. The plastic nonadherent PEC had significantly higher NK cell activity than the untreated control, and the adherent PEC were cytostatic toward MM2 and MH134 cells. The adherent PEC did not cause tumor cell lysis by themselves, but were cytolytic against MM2 cells in the presence of anti-MM2 sera. In the course of these effects of PEC after the i.p. injection of agrimoniin, the augmentation of NK cell activity was the earliest reaction, reaching a peak at 2 days after the injection; then, cytostatic activity increased. The induction of antibody-dependent cell lytic activity was a later reaction, which reached a peak at 6 days after the injection. Abbreviations used: PEC, peritoneal exudate cells; NK cell, natural killer cell; ADCC, antibody-dependent cell-mediated cytotoxicity; PMN, polymorphonuclear leukocytes     

Proliferation- and cell cycle-dependent differences in expression of the 170 kilodalton and 180 kilodalton forms of topoisomerase II in NIH-3T3 cells.

The cellular content of 170kD and 180kD topoisomerase II was studied as a function of the proliferation state and cell cycle position in NIH-3T3 cells. When the cells were synchronized by serum starvation and then stimulated to enter the cell cycle by addition of fresh growth medium, the amount of 170kD topoisomerase II present was undetectable until the cells reached late S phase, peaked in G2-M phase cells, and decreased as the cells completed mitosis. The amount of 180kD topoisomerase II was constant once the cells entered the cell cycle. When exponentially growing cells were induced to enter G0 by serum starvation, the amount of 170kD topoisomerase II decreased in parallel with the loss of cells from the S and G2-M phases of the cell cycle and was undetectable once all of the cells reached G0. In contrast, the 180kD enzyme was still present after all of the cells had entered G0. The tightness of association of the two enzymes with chromatin was measured by determining the concentration of salt required to extract them from isolated nuclei. The 180kD enzyme required a higher concentration of NaCl for extraction than did the 170kD enzyme. The different patterns of expression of the two forms of topoisomerase II suggest that they perform different functions in cells.     

Cytolytic mechanisms of activated macrophages. Tumor necrosis factor and L-arginine-dependent mechanisms act synergistically as the major cytolytic mechanisms of activated macrophages

We examined the cytolytic mechanisms of activated macrophages by using proteose peptone- or thioglycollate broth-induced mouse peritoneal macrophages or mouse macrophage hybridomas as effector cells, L.P3 cells, a clone of L929 cells, and P815 cells as target cells, and IFN-gamma and LPS as activators. It was determined that TNF is the main cytolytic molecule against L.P3 cells from the following results: 1) activated macrophages can produce TNF; 2) TNF shows cytotoxic activity against L.P3 cells; 3) the addition of anti-TNF antibody inhibited most of the cytolytic activity of activated macrophages against L.P3 cells. On the other hand, it was concluded that the main cytolytic mechanism against P815 cells is the production of NO2-/NO3- from L-arginine, from the following results: 1) activated macrophages can produce NO2-; 2) NaNO2 shows high cytotoxic activity against P815 cells; 3) the depletion of L-arginine from the medium inhibited most of the cytolytic activity of activated macrophages against P815 cells and NO2- production by activated macrophages. In this study, however, cytostatic effects of L-arginine-dependent effector mechanism were not studied. Thus, these results show that activated macrophages can express at least two cytolytic mechanisms independently, namely, the one that appears to be mediated by the L-arginine-dependent effector mechanism and the second that appears to be mediated directly by TNF. Furthermore, it was demonstrated that TNF and L-arginine-dependent NO2- production act synergistically as killing mechanisms of activated macrophages. These mechanisms can explain the cytolytic activity of activated macrophages against a variety of target cells.     

Potentiation of the destructive effect of heat (42°C) on synchronized cancer cells in culture by cell specific antiserum

1. 1. A kinetic study was made of the effects of hyperthermia and cell specific antibody on non-synchronized lag phase cultures and synchronized exponentially growing cultures of a malignant cell line. The effects on the degree of cell synchrony were also investigated.

2. 2. Hyperthermic treatment of synchronized SDB monolayer cell populations with maximum replication rate sensitized the cells to subsequent destruction by cell specific antibody.

3. 3. Hyperthermic treatment of SDB monolayer cultures with low replication rate and varying degrees of metabolic activity produced no such sensitization.

4. 4. Hyperthermia was disruptive to a synchrony procedure involving a blockade of DNA synthesis by excess thymidine.

Effect of serum step-down on protein metabolism and proliferation kinetics of NHIK 3025 cells

Human NHIK 3025 cells growing exponentially in 30% or 3% serum had population doubling times of 19.1 and 27.6 hours, respectively. These values were equal to the calculated protein doubling times (17.6 and 26.5 hours, respectively), showing that the cells were in balanced growth at both serum concentrations. Stepdown from 30% to 3% serum reduced the rate of protein synthesis within 1–2 hours, from 5.7% hour to 4.3% hour, while the rate of protein degradation was unchanged (1.7%/hour). In cells synchronized by mitotic selection from an exponentially growing population, the median cell cycle durations in 30% and 3% serum were 17.2 and 23.6 hours, respectively, which were also in good agreement with the protein doubling times. The median G₁ durations were 7.1 and 9.6 hours, respectively. Thus the duration of G₁ relative to the total cell cycle duration was the same in the two cases. Complete removal of serum for a period of 3 hours resulted in a 3-hour prolongation of the cell cycle regardless of the time after mitotic selection at which the serum was removed. For synchronized cells, the rate of entry into both the S phase and into the subsequent cell cycle were reduced in 3% serum as compared to 30% serum, the former rate being significantly greater than the latter at both serum concentrations. Our results thus indicate that these cells are continuously dependent upon serum throughout the entire cell cycle.     

Human interleukin 1 is a cytocidal factor for several tumor cell lines

Highly purified interleukin 1 (IL 1) obtained from stimulated human monocytes appeared to be growth inhibitory and cytocidal for a human melanoma cell line, A375. Although IL 1 did not have an immediate cytolytic effect, with time in culture the growth of the target cells was irreversibly inhibited. The cells eventually lysed and decreased markedly in number; the IL 1 effect can therefore be said to be cytocidal. IL 1 activity could not be separated from the cytocidal activity by a variety of chromatography procedures by using conventional and high-performance liquid chromatography (HPLC). The A375 melanoma cell line was also sensitive to another human cytokine alpha-lymphotoxin (alpha-LT) derived from a human B cell line. IL 1 also appeared to be partially growth inhibitory and cytocidal for a LT-sensitive mouse fibroblast cell line, L929; but not for LT-resistant cells, including a subline of L929; a human epithelial carcinoma cell line, HeLa; a human osteosarcoma cell line, HOS; and a mouse SV40-transformed kidney cell line, TU5. However, the LT-sensitive mouse fibroblast cell line, L-M, was resistant to IL 1. Therefore, the cytocidal activity of IL 1 only partially overlapped the target cell selectivity of alpha-LT. Although natural IFN-alpha and recombinant IFN-beta were appreciably growth inhibitory for the A375 cell line, natural and recombinant IFN-alpha and recombinant IFN-beta and IFN-gamma exhibited little cytocidal activity. Purified IL 1 did not have any antiviral activity, and conversely, IFN and alpha-LT were not co-mitogenic for thymocytes. Furthermore, by ELISA and radioimmunoassays, antibodies against human alpha-LT, tumor necrosis factor, and IFN-gamma did not react with IL 1, indicating that IL 1 is antigenically distinct from these other cytokines. These in vitro results suggest that IL 1 may play a role in host defense against some tumors as a cytocidal factor.     

DNA and cholesterol biosynthesis in synchronized embryonic rat fibroblasts: II. Effects of sterol biosynthesis inhibitors on cell division

The relationships between cholesterogenesis and cell division were studied by using two inhibitors of hydroxymethylglutaryl-CoA reductase activity — 25-hydroxycholesterol and compactin. The effects of both compounds on DNA synthesis were compared in synchronized rat fibroblasts cultured in a cholesterol-containing medium. Compactin did not inhibit DNA synthesis, except after a long time of contact and at high and almost cytotoxic concentrations. 25-Hydroxycholesterol inhibited DNA synthesis (without cytotoxic effects) after only 9–16 h of contact, depending on the phase of the cell cycle at which this compound was added to the culture medium. Sensitivity of cells to 25-hydroxycholesterol was maximal at the end of the S phase/beginning of the G₂M phase. The rapid effect of 25-hydroxycholesterol on DNA synthesis appears to be separate from the inhibitory effect on sterol or non-sterol mevalonate-derived compound synthesis. Indeed, under our experimental conditions, the suppression of cholesterol biosynthesis is compensated by the presence of cholesterol in the culture medium, as demonstrated by the lack of effect of compactin on DNA synthesis; moreover, addition of mevalonolactone to the culture medium did not reverse the effect of 25-hydroxycholesterol. 25-Hydroxycholesterol could inhibit DNA synthesis by a direct action on the nucleus, after transfer by the intermediary of a specific hydroxysterol-binding protein.     

Potentiation of the cytolytic activity of peripheral blood monocytes by lymphokines and interferon

Human peripheral blood monocytes obtained by EDTA-reversible adherence to plastic surfaces precoated with autologous serum can rapidly lyse a variety of tumor cells. That the effector cells in this system are indeed monocytes has been demonstrated (1). Using a short-term (3 to 4 hr) 51Cr-release assay and the single cell conjugate cytotoxic assay, we studied the effects of lymphokine-rich supernatants containing gamma-interferon and partially purified fibroblast interferon on the monocyte cytolytic activity. Overnight incubation of the monocytes in fetal bovine serum-containing medium resulted in a relatively small decrease in cytotoxic activity compared to the one obtained with monocytes incubated in autologous serum. The addition of lymphokines or interferon under both incubation conditions resulted in augmented activity as measured in the 51Cr-release assay. However, the proportions of binding and cytotoxic monocytes, determined in the single cell conjugate assay, did not increase. These results suggest that augmented activity is not due to recruitment of inactive cells. Kinetics studies of tumor cell lysis indicate the increase in killing efficiency is probably due to both an increase in the rate of killing and in the recycling ability of the cytotoxic cells. Using the conjugate/agarose technique, we also demonstrated that excess tumor cells could impair the lytic machinery of freshly isolated monocytes, whereas monocytes treated with lymphokines or interferon partially lost their sensitivity to this inhibitory effect. The ability of tumor cells to impair the lytic machinery of monocytes could be one of the mechanisms by which tumors escape immunosurveillance.     

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.     

Effect of canatoxin on cell cultures

Canatoxin, a toxic protein isolated from Canavalia ensiformis, was shown to inhibit DNA synthesis and to produce a cytolytic effect when added in vitro to various cells, in doses ranging from 50 to 500 nM. In this case no selectivity was found for a certain cell type, as both normal and transformed cells could be affected by the toxic protein. The cytostatic effect was irreversible upon removal of the toxic protein from the medium and could be fully attained after exposing the cells to canatoxin for only 30 min. The use of an in vitro cell culture system may allow for a better insight on the mode of action of canatoxin.     

Fucose-activated killer (FAK) cells: anomalous killers with augmented cytotoxic activity

The effects of monosaccharides on various lymphocyte functions have provided useful probes for the study of cell-cell interactions. In this report, we show that a monosaccharide, alpha-L-fucose, significantly enhances the cytolytic capacity of MLC-induced or preincubated effector cells. The increase in activity was seen against cytotoxic T lymphocyte (CTL) targets (:relevant PHA blasts), natural killer cell (NK) targets (:K562), and natural cytotoxic cell (NC) targets (:MA-160). In addition, traditionally NK-insensitive targets (Raji cells, irrelevant and autologous PHA blasts) were lysed after preincubation of effector cells with fucose. Conversely, ADCC activity was not significantly increased with fucose induction. The addition of fucose directly to assay cultures did not enhance NK or CTL activity, whereas other sugars, such as alpha-methyl-D-mannoside and D-fructose, were inhibitory. The proportion of target-binding cells was not affected by preincubation with fucose, but the percentage of lytic conjugates was doubled. Significant augmentation of NK activity could be observed within 24 hr of incubation with alpha-L-fucose. Conversely, when fucose was added more than 24 hr after initiation of the culture, the increase in cytolytic activity was not observed. Parallel to the increase in cytolytic activity, after preincubation with alpha-L-fucose, an increase in the expression of a newly defined human NC cell marker, HNC-1A3, was observed. The HNC-1A3+ cells were not the major subpopulation responsible for fucose-induced activity, as ascertained by the use of positively sorted cells. The populations expressing antigens defined by the antibodies OKT8 and Leu-7 showed no quantitative change. The treatment of cells with OKM1 and complement (C) before culture eliminated fucose-enhanced killing, whereas similar treatment with OKT8 and C had no significant effect. The induction of fucose-activated killers (FAK) does not result in higher concentrations of interferon (IFN) in culture supernatants, in contrast to poly I:C, which induced both higher cytolytic activity and high titers of IFN. In addition, the induction of FAK was not sensitive to 100 ng/ml of cyclosporin A, suggesting that IL 2 did not play a major role in fucose activation of killing. These results provide strong evidence that alpha-L-fucose is capable of augmenting nonspecific activity by acting on OKM1+ precursors of cytotoxic cells and influencing a postbinding event.     

Recombinant tumor necrosis factor: species specificity for a variety of human and murine transformed cell lines

Tumor necrosis factor (TNF) exhibits cytotoxic or cytostatic activity on a wide range of animal and human transformed cell lines. Using pure, recombinant human and mouse TNF, we examined the degree of species specificity of the in vitro TNF activity on a variety of human and murine transformed cell lines. This species specificity was studied for the TNF activity alone or in synergism with IFN-gamma. Recombinant human and mouse TNF behave remarkably similarly regarding the in vitro cytolytic/cytostatic activity. However, a certain degree of species-specific preference could be revealed as human cell lines needed a higher concentration of recombinant mouse TNF than of recombinant human TNF to attain a similar effect, while on mouse cells the reverse was true. Also, synergism with IFN-gamma seemed more effective when the target cell was treated with homologous TNF.