The effect of chelating agents on iron mobilization in Chang cell cultures.
The investigation of chelating agents with potential therapeutic value in patients with transfusional iron overload has been facilitated by the use of Chang cell cultures. These cells have been incubated with [59Fe]transferrin for 22 hr, following which most of the intracellular radioiron is found in the cytosol, distributed between a ferritin and a nonferritin form. Iron release from the cells depends on transferrin saturation in the medium, but when transferrin is 100% saturated, which normally does not allow iron release, desferrioxamine, 2,3-dihydroxybenzoic acid, rhodotorulic acid, cholythydroxamic acid, and tropolone all promote the mobilization of ferritin iron and its release from cells. They are effective to an approximately equal degree. The incubation of [59Fe]transferrin with tropolone in vitro at a molar ratio of 1:500 results in the transfer of most of the labeled iron to the chelator, reflecting the exceptionally high binding constant of this compound. How far these phenomena relate to therapeutic potentially remains to be seen. (+info
In vitro effects of 2,4-dichlorophenoxy acetic acid (2,4-D) on bovine cells.
Bovine fetal muscle cells were exposed to culture media containing 2 mg and 20 mg per liter of 2,4-dichlorophenoxy acetic acid (2,4-D) for varying intervals to determine the in vitro response of mammalian cells to this compound. The concentrations of 2,4-D used were comparable to those used in spray programmes although the residues normally found in pasture are much lower since 2,4-D is rapidly degraded under field conditions. Untreated and treated cultures were analyzed for total cell count, mitotic index and the percentages of differentiating and degenerating cells. The response of cultures to treatment was similar irrespective of the concentrations of 2,4-D used although in higher concentrations there was an initial drop in mitotic index. Other changes noted in treated cultures included an increase in differentiating and degenerating cells compared to those in control. The mitotic cells in treated cultures exhibited unipolar and tripolar spindles and a variety of other abnormalities including malorientation of the mitotic apparatus in relation to the axis of the cell. Myoblasts in initial stages of myogenesis were noted to be in mitosis in treated cultures suggesting that 2,4-D may have a stimulatory effect on myoblasts which in normal myogenesis are in post mitotic stage. (+info
Differences in benzo(a)pyrene metabolism between rodent liver microsomes and embryonic cells.
Differences in benzo(a)pyrene metabolite pattern have been shown by rodent liver microsomes (Sprague-Dawley) and rodent embryo cells from Syrian hamsters and NIH Swiss mice. Rodent liver induced by methylcholanthrene shows marked quantitative variation between species. Additional pattern changes were found in mouse and hamster embryo secondary cultures with a reduction of the K-region metabolites and a marked increase in 9-hydroxybenzo(a)-pyrene. These results are indicative of a region-specific attack on the carcinogen by the cell monooxygenases which is distinct from the liver attack of microsomal enzymes on benzo(a)pyrene. These results suggest that activation and detoxification of benzo(a)pyrene may be species and tissue variable, and susceptibility and resistence to malignant transformation may be predicted on induction of a fortuitous combination of intermediate metabolic steps. (+info
Glycopeptides from the surgace of human neuroblastoma cells.
Glycopeptides suggesting a complex oligosaccharide composition are present on the surface of cells from human neuroblastoma tumors and several cell lines derived from the tumors. The glycopeptides, labeled with radioactive L-fucose, were removed from the cell surface with trypsin, digested with Pronase, and examined by chromatography on Sephadex G-50. Human skin fibroblasts, brain cells, and a fibroblast line derived from neuroblastoma tumor tissue show less complex glycopeptides. Although some differences exist between the cell lines and the primary tumor cells, the similarities between these human tumors and animal tumors examined previously are striking. (+info
Stimulation of thymidine uptake and cell proliferation in mouse embryo fibroblasts by conditioned medium from mammary cells in culture.
Undialyzed conditioned medium from several cell culture sources did not stimulate thymidine incorporation or cell overgrowth in quiescent, density-inhibited mouse embryo fibroblast cells. However, dialyzed conditioned medium (DCM) from clonal mouse mammary cell lines MCG-V14, MCG-T14, MCG-T10; HeLa cells; primary mouse adenocarcinoma cells; and BALB/c normal mouse mammary epithelial cells promoted growth in quiescent fibroblasts. The amount of growth-promoting activity produced per cell varied from 24% (HeLa) to 213% (MCG-V14) of the activity produced by primary tumor cells. The production of growth-promoting activity was not unique to tumor-derived cells or cells of high tumorigenicity. The amount of growth-promoting activity produced per cell in the active cultures was not correlated with any of the following: tumorigenicity, growth rat, cell density achieved at saturation, cell type, or species of cell origin. It is concluded that transformed and non-transformed cells of diverse origin, cell type, and tumorigenicity can produce growth factors in culture. The growth-promoting potential of the active media from primary tumor cultures accumulated with time of contact with cells and was too great to be accounted for entirely by the removal of low-molecular-weight inhibitors by dialysis. The results are consistent with the hypothesis that conditioned medium from the active cultures contained a dialyzable, growth-promoting activity. Different cell lines exhibited differential sensitivity to tumor cell DCM and fetal bovine serum. Furthermore, quiescent fibroblasts were stimulated by primary tumor cell DCM in the presence of saturating concentrations of fetal bovine serum. These observations support the notion that the active growth-promoting principle in primary tumor cell DCM may not be a serum factor(s). (+info
Diphtheria toxin effects on human cells in tissue culture.
HeLa cells exposed to a single sublethal concentration of diphtheria toxin were found to have diminished sensitivity when subsequently reexposed to the toxin. Three cells strains exhibiting toxin resistance were developed. In the cells that had previously been exposed to toxin at 0.015 mug/ml, 50% inhibition of protein synthesis required a toxin concentration of 0.3 mug/ml, which is more than 10 times that required in normal HeLa cells. There appears to be a threshold level of diphtheria toxin action. Concentrations of toxin greater than that required for 50% inhibition of protein synthesis (0.01 mug/ml) are associated with cytotoxicity, whereas those below this concentration may not be lethal. Several established human cell lines of both normal and neoplastic origin were tested for their sensitivity to the effects of the toxin. No special sensitivity was observed with the cells of tumor origin. Fifty % inhibition of protein synthesis of HeLa cells was achieved with diphtheria toxin (0.01 mug/ml) as compared to the normal human cell lines tested (0.03 and 0.5 mug/ml) and a cell line derived from a human pancreatic adenocarcinoma (0.2 mug/ml). A human breast carcinoma cell line showed a maximum of 45% inhibition of protein synthesis. This required a diphtheria toxin concentration of 5 mug/ml. These results suggest that different human cell lines show wide variation in their sensitivity to the toxin. (+info
The effects of estrogens and antiestrogens on hormone-responsive human breast cancer in long-term tissue culture.
We have established or characterized six lines of human breast cancer maintained in long-term tissue culture for at least 1 year and have examined these lines for estrogen responsiveness. One of these cell lines, MCF-7, shows marked stimulation of macromolecular synthesis and cell division with physiological concentrations of estradiol. Antiestrogens are strongly inhibitory, and at concentrations greater than 3 X 10(-7) M they kill cells. Antiestrogen effects are prevented by simultaneous treatment with estradiol or reversed by addition of estradiol to cells incubated in antiestrogen. Responsive cell lines contain high-affinity specific estradiol receptors. Antiestrogens compete with estradiol for these receptors but have a lower apparent affinity for the receptor than estrogens. Stimulation of cells by estrogens is biphasic, with inhibition and cell death at concentrations of 17beta-estradiol or diethylstilbestrol exceeding 10(-7) M. Killing by high concentrations of estrogen is probably a nonspecific effect in that we observe this response with 17alpha-estradiol at equivalent concentrations and in the otherwise unresponsive cells that contain no estrogen receptor sites. (+info
The effects of glucocorticoids and progesterone on hormone-responsive human breast cancer in long-term tissue culture.
Glucocorticoids, at physiological concentration, inhibit cell division and thymidine incorporation in three lines of human breast cancer maintained in long-term tissue culture. At steroid concentrations sufficient to inhibit thymidine incorporation 50%, little or no effect is seen on protein synthesis 48 hr after hormone addition. All three of these lines are shown to have glucocorticoid receptors demonstrable by competitive protein binding assays. Receptors are extensively characterized in one line by sucrose density gradient analysis and binding specificity studies. Good correlation between receptor-binding specificity and biological activity is found except for progesterone, which binds to glucocorticoid receptor but is noninhibitory. Cross-competition and quantification studies demonstrate a separate receptor for progesterone. This receptor has limited binding specificities restricted largely to progestational agents, whereas the glucocorticoid receptor bound both glucocorticoids and progesterone. Two other human breast cancer lines neither contain glucocorticoid receptor nor are inhibited by glucocorticoids. It is concluded that in some cases glucocorticoids can directly limit growth in human breast cancer in vitro without requiring alterations in other trophic hormones. (+info