Diverse deoxyribonucleotide profiles in cultured human cells with differential sensitivity to thymidine

Intracellular deoxyribonucleotide pools were examined before and after thymidine treatment in highly sensitive T-lymphoid cells, relatively resistant B-lymphoid cells and moderately sensitive melanoma cells. Among the 4 cell lines studied, proportions of the 4 deoxyribonucleotide pools varied appreciably while ribonucleotide profiles were similar. The ratio of dGTP to dCTP increased with sensitivity to thymidine. Increase in dTTP levels with increasing thymidine concentration was dependent on sensitivity of cells to thymidine and was accompanied by reduction in the dCTP pool. dGTP levels increased as did dTTP levels in all cells, while dATP pool expansion correlated with thymidine sensitivity. The results indicate an additional aspect of purine deoxyribonucleotide involvement in the growth inhibitory effects of thymidine.     

Interferon-specific cellular receptors in cultured human cells, differing in sensitivity to alpha-interferon

The interferon-specific cellular receptors in human cells cultures differing in sensitivity to alpha-interferon have been studied. The J-41 cells resistant to alpha-interferon are practically devoid of receptors highly specific to alpha-interferon. The coefficient of equilibrium and the number of receptors analyzed after Scatchard for J-96 culture of cells are 15.6 x 10(11) M-1 and 210 +/- 90, respectively. Evidently, resistance of J-41 cells to alpha-interferon is connected with the absence of interferon receptors and, as a consequence, inability to interferon-receptor interaction.     

Some factors affecting the sensitivity of cultured human cells to high-LET radiation

Summary Comparative effects of decay of DNA-bound¹²⁵I, of-radiation and of tritiated water on survival of the proliferative ability of cultured cells were examined. The results confirm a previous report that cells frozen to -196° C in the presence of 2M glycerol have lost a considerable proportion of their intracellular water. The data also suggest that the fraction of the lethal damage caused by deposition of radiation energy in intracellular water close to the DNA is greater for-radiation than for the decay of DNA-bound¹²⁵I.Inherited differences in the sensitivity of untransformed fibroblasts from individual humans to ionizing radiations and other DNA-damaging agents are being explored. The ratios of the sensitivities of various cell lines to particular agents can vary several-fold. Thus the RBE of various radiations is affected not only by the irradiation conditions and the water content of the cells but also by inherited abnormalities in the DNA repair systems in human cells.Dedicated to Prof. L.E. Feinendegen on the occasion of his 60th birthday     

Altered copper metabolism in cultured cells from human Menkes' syndrome and mottled mouse mutants

Cultured cells of a variety of different types from human Menkes' syndrome patients and brindled mouse mutants exhibit similarly altered responses to changes in extracellular copper concentration. This suggests that the mutations in the mouse and human are very similar and that mutant gene expression is occurring in many different tissues. Intracellular copper levels are markedly elevated in mutant cells in normal medium and in medium containing a hundredfold higher copper. Some cell lines from heterozygotes possess elevated copper levels. Elevated extracellular copper and zinc are significantly more toxic to mutant cells. Mutant cells exhibit normal rates of uptake of copper-64 over a 10-min period but abnormally high accumulation over 24 hr and low rates of efflux. Menkes' fibroblasts become saturated with copper-64 at lower extracellular concentrations than for normal fibroblasts. These data support the idea of enhanced intracellular binding in mutant cells.This work was supported by grants from the Australian National Health and Medical Research Council, the McPherson/Shutt Trust, and the Apex Foundation.     

Electron microscopic evidence for phagocytic properties of human peripheral mononuclear cells cultured with PHA.

The time course of the ultrastructural changes induced in human peripheral mononuclear cells, when cultured with PHA, has been studied. In addition to findings common to many mitotic cells, such as an increase in nuclear and nucleolar size, the presence of free polyribosomes, glycogen and lipid globules in the cytoplasma, and a high number of mitochondria, agglutinating properties due to PHA per se were observed in the first 12 hours. At 72 hours certain cells developed phagocytic-like properties, i.e. they were able to incorporate both material from the extracellular compartment and syngeneic cells. These results are discussed suggesting the possible presence of macrophages in culture or the ability of activated T cells to express it.     

Interferon-gamma reduces the sensitivity of cultured and fresh human tumor cells to lysis by lymphokine-activated killer cells

We have shown that interferon-gamma (IFN-gamma), in pharmacologically achievable doses, can reduce the the sensitivity of human tumor cells to lysis by allogeneic lymphokine-activated killer (LAK) effector cells. Cultured tumor cells showed a consistent reduction in sensitivity to lysis following pretreatment for 18 h with 1-10 units/ml IFN-gamma. Tumor cells cultured up to 7 days in 100 units/ml IFN-gamma remained less sensitive to lysis. Induction of protection from LAK did not appear to correlate with IFN-gamma-induced changes in cell growth or proliferation. Reduced LAK sensitivity also did not correlate with the level of expression of major histocompatibility antigens. Eight of 11 surgically obtained human tumor cell specimens showed a reduction in sensitivity to lysis by allogeneic LAK cells following pretreatment with IFN-gamma. IFN-induced reduction of tumor cell sensitivity to lysis by LAK may play a role in altering the host-tumor relationship, since relatively high concentrations of IFN-gamma may exist in the tumor microenvironment.     

Early sensitivity to colchicine in developing anthers of rye

Plants injected with low concentrations of colchicine at very early stages of premeiotic development showed aneuploidy and slightly reduced chiasmate association at pollen mother cell (PMC) meiosis. With higher concentrations several tetraploid PMCs, reduced pairing, and both univalents and multivalents in diploid cells were found. The results suggest that meiotic chromosome pairing can be affected by colchicine treatment several mitotic cell generations before meiosis.     

Steroid sensitivity of the PHA and PWM responses of fractionated human lymphocytes in vitro

The in vitro PHA and PWM responses of various fractions of human peripheral lymphocytes were tested for sensitivity to water-soluble prednisolone. Removal of cells having the capacity to phagocytize carbonyl iron particles or cells having a tendency to adhere to plastics increased the steroid sensitivity of both the PHA and the PWM responses. T and B cell-rich preparations were obtained by passing cell suspensions, depleted of macrophages-monocytes as described above, through anti-Ig labelled bead columns or by a rosette centrifugation technique. The mitogenic response of cell suspensions enriched for B cells were more steroid resistant than suspensions enriched for T cells.     

High sensitivity of cultured human trophoblasts to ribosome-inactivating proteins.

Many plant proteins possessing abortifacient activities were identified as ribosome-inactivating proteins (RIPs). The effect of several ribosome-inactivating proteins (saporin 6, dianthin 32, pokeweed antiviral protein from seeds, gelonin, bryodin-R, and momordin) on primary cultures of human trophoblasts and human embryonal fibroblasts and on choriocarcinoma (JAR and BeWo) and ovarian carcinoma (TG) cell lines was studied. Protein synthesis of human trophoblasts and BeWo cells was lowered by RIPs more than that of other cells. The trophoblastic receptors for estradiol were not affected by treatment of the cells with momordin. The binding and uptake of saporin 6 and momordin by BeWo and HeLa cells were not correlated to cell toxicity.     

The cytoskeleton of hydatid cyst cultured cells and its sensitivity to inhibitors

Cell cultures obtained from the germinal layer of hydatid cysts of the parasitic tapeworm Echinococcus granulosus were characterized with respect to their microtubule and microfilament systems. These were stained using monospecific antibodies against tubulin from sea urchin spermatozoa or sheep brain and against Dictyostelium discoideum actin as well as rhodamine conjugated phalloidin. The results show that the distribution of microtubules nad actin containing fibres of these cells is remarkably similar to that of mammalian cells both during interphase and mitosis. Hydatid cells, however, could not be stained with a specific antivimentin antibody. Indirect immunofluorescence with antitubulin antibodies of inhibitor treated cells shows that hydatid cell microtubules are sensitive to several antimicrotubular drugs including benzimidazole derivatives, colchicine, vinblastine, and griseofulvin.     

Binding of human thrombin to cultured human endothelial cells.

Binding of thrombin to monolayer cultures of human umbilical vein endothelium is studied. Binding is measured as inhibition by unlabeled ligand of the binding of 125I-thrombin to the cells. Radioactivity bound to cultures at equilibrium is measured after draining but not washing the cells. To correct for unremoved supernatant, 131I-albumin is included as a second label in the medium. Equilibrium between bound and free thrombin is attained within 1 min, and Scatchard analysis indicates a population of approximately 3 x 10(3) sites/cell with a dissociation constant of 10(-10) M, and a larger population with a dissociation constant greater than 10(-8) M. The two populations of sites are also indicated by a biphasic dissociation of bound label. Thrombin inactivated with diisopropyl fluorophosphate binds to the same receptor, with an affinity similar to that of active thrombin. Binding is unaffected by albumin (an acidic protein) and cytochrome c (a basic protein). Cultures of umbilical cord smooth muscle and fibroblasts bind thrombin at least 100 times more weakly than endothelium, and no binding to erythrocytes or a monolayer culture of mouse neuroblastoma is detected.     

Binding of plasminogen to cultured human endothelial cells

Endothelial cells are known to release the two major forms of plasminogen activator, tissue plasminogen activator (TPA) and urokinase. We have previously demonstrated that plasminogen (PLG) immobilized on various surfaces forms a substrate for efficient conversion to plasmin by TPA (Silverstein, R. L., Nachman, R. L., Leung, L. L. K., and Harpel, P. C. (1985) J. Biol. Chem. 260, 10346-10352). We now report the binding of human PLG to cultured human umbilical vein endothelial cell (HUVEC) monolayers, utilizing a newly devised cell monolayer enzyme-linked immunosorbent assay system. PLG binding to HUVEC was concentration dependent and saturable at physiologic PLG concentration (2 microM). Binding of PLG was 70-80% inhibited by 10 mM epsilon-aminocaproic acid, suggesting that it is largely mediated by the lysine-binding sites of PLG. PLG bound at an intermediate level to human fibroblasts, poorly to human smooth muscle cells, and not at all to bovine smooth muscle or bovine endothelial cells; unrelated proteins such as human albumin and IgG failed to bind HUVEC. PLG binding to HUVEC was rapid, reaching a steady state within 20 min, and quickly reversible. 125I-PLG bound to HUVEC with an estimated Kd of 310 +/- 235 nM (S.E.); each cell contained 1,400,000 +/- 1,000,000 (S.E.) binding sites. Functional studies demonstrated that HUVEC-bound PLG is activatable by TPA according to Michaelis-Menten kinetics (Km, 5.9 nM). Importantly, surface-bound PLG was activated with a 12.7-fold greater catalytic efficiency than fluid phase PLG. These results indicate that PLG binds to HUVEC in a specific and functional manner. Binding of PLG to endothelial cells may play a pivotal role in modulating thrombotic events at the vessel surface.     

Decreased sensitivity to diphtheria toxin of Vero cells cultured in serum-free medium.

Vero cell cultures are used in the quality control of Diphtheria vaccines: to estimate vaccine potency and to determine residual toxicity and reversion to toxicity. The impact of replacing foetal calf serum containing medium (SCM) by serum free media (SFM) on the sensitivity of Vero cells to Diphtheria Toxin was studied. Compared to SCM, SFM showed an eight-fold decrease in sensitivity to Diphtheria Toxin. This decrease was almost immediate, indicating that this phenomenon was not caused by a change in membrane structure or protein expression. We investigated the effect of SFM on Diphtheria Toxin in order to determine the cause of the decrease in sensitivity. Our results show that oligopeptides, which are often used in SFM as part of the replacement of foetal calf serum, are the most likely cause.     

Sepiapterin reductase in cultured human cells

Sepiapterin reductase, an enzyme involved in the synthesis of tetrahydrobiopterin (the natural cofactor for phenylalanine, tyrosine and tryptophan hydroxylases), has been assayed in cultured human amniotic fibroblasts and in cultured mononuclear blood cells. In both cases, the Michaelis constants for sepiapterin and NADPH were essentially equal; 20 microM and 6 microM respectively for stimulated mononuclear blood cells and 22 microM and 5 microM respectively for amniotic fibroblasts. The inhibition by N-acetylserotonin was also similar in both cases. The concentration that produced 50% inhibition in stimulated mononuclear blood cells and in amniotic fibroblasts was 2 microM. The results strongly suggest that the same enzyme is acting in both types of cells, at least when grown in culture.