Primary culture of mouse mammary tumor epithelial cells embedded in collagen gels

Summary Mammary tumor epithelial cells from BALB/cfC3H mice were dispersely embedded inside the collagen gels in Ham's F-12 medium containing horse serum. A sustained cell growth leading to a 5- to 10-fold increase in cell number over initial level was observed in less than 2 weeks. The extent of this growth was found to be dependent on serum concentration. However, addition of various protein and steroid hormones, both singly and in combination, to low-serum-containing medium failed to achieve a comparable level of growth to that promoted by higher serum concentration. Mammary tumor cells can now be consistently propagated in primary culture. This investigation was supported by Grants CA05388 and CA09041 awarded by the National Cancer Institute, Department of Health, Education and Welfare, and by cancer research funds of the University of California.     

Synchronization of 9L rat brain tumor cells by centrifugal elutriation

Asynchronous 9L cells were separated into relatively homogeneously-sized populations using centrifugal elutriation with both a conventional collection method and a long collection method. A substantial increase in the homogeneity of the volume distributions and in the degree of synchrony of the separated fractions was obtained using the long collection method. Autoradiographic data indicated that fractions containing ≥97% G₁ cells, ≥80% S cells, and 70–75% G₂ cells could be routinely recovered with this procedure. Recovery in these fractions varied from 5 to 8% of the total number of cells elutriated. The colony forming efficiency (CFE) of cells from fractions representing each phase of the cell cycle was a constant 60–70%, which was comparable to the 60–80% usually found for asynchronous 9L cells. The percentage of cells in the G₁, S, and G₂ phases in the elutriated fractions was more accurately determined from the volume distribution than from computer fits of the DNA histogram obtained from flow cytometry. In general, the degree of synchrony was related to the coefficient of variation (CV) of the volume distributions of the elutriated fractions. The CV was about 14% for all elutriated fractions. When the ≥97% G₁ population was allowed to progress to S and G₂, the CVs were about 17 and 20.2%, respectively. Thus, the best nonperturbing method for obtaining synchronous 9L cells in the S or G₂ phases was direct elutriation with the long collection method.     

Evaluation of immobilized cytochrome P-448 from Saccharomyces cerevisiae using permeabilized whole cell, microsomal fraction and highly purified reconstituted forms, with benzopyrene-3-monooxygenase activity

Cytochrome P-448 from Saccharomyces cerevisiae in permeabilized whole cell, microsomal fraction and in a highly purified reconstituted benzopyrene-3-monooxygenase (EC 1.14.14.1) system have been immobilized on various supports. Calcium alginate was found to be especially useful and the kinetics of hydroxylation were close to that of the free enzyme system with all three forms of enzyme, even with permeabilized whole yeast cells (V _max of 664 pmol 3-hydroxybenzo(a)pyrene produced per h per nmol cytochrome P-448 compared with 1000 for free highly purified reconstituted enzyme system). Only the highly purified reconstituted form was successfully immobilized by BrCN-activated Sepharose-4B or by acrylamide. Both of these supports stabilized the highly purified reconstituted cytochrome P-448 benzopyrene-3-monooxygenase activity in prolonged storage at 4°C. Applications for various immobilized enzymes and cells are assessed.     

Vitamin analysis with use of a yeast electrode

A vitamin B₁ (thiamin)-sensitive electrode has been devised by combining an oxygen electrode with a yeast-containing membrane. The assembly was used for assaying thiamin at concentrations down to 10^?11 gl^?1. The analytical procedure developed should allow the measurement of 10–20 samples per hour. The performance of the yeast electrode was improved when alginate membranes reinforced with a nylon network were used. An apparatus for preparing such membranes is described together with a magnetic membrane holder facilitating handling of membranes in combination with electrodes.     

Differentiation between the somatostatin inhibition and the post-somatostatin rebound observed on growth hormone secretion in vitro

A rebound in growth hormone secretion following somatostatin treatment has been shown in several systems where somatostatin suppresses secretion of the hormone. We have developed an in vitro system in which isolated and cultured pituitary cells were perfused after mild trypsinization. After washing, these cells retained their sensitivity and secreted growth hormone (GH) in response to physiological activators (norepinephrine, dopamine, serotonin) or inhibitors (somatostatin) as well as pharmacological activators (PGE2). The variation in GH secretion occurred within a minute after commencement of the infusion and was as rapidly reversible and repeatable minutes later. During somatostatin infusion the GH secretion was not totally suppressed (residual secretion (mean +/- S.D.) 34 +/- 7%). After the infusion a rapid rebound in GH secretion occurred, reaching levels in excess of the pretreatment value of 138 +/- 13%. This rebound effect occurred at doses higher than (10(-10)M) but not at lower doses, even when significant inhibition was observed. The inhibitory effect is of greater magnitude than the rebound effect (rebound = inhibition X 57 +/- 7% (mean +/- S.D.)). Furthermore, rebound was not enhanced by prolongation of somatostatin infusion. These latter results indicate that the rebound in secretion cannot be explained on the sole basis of storage of intracellular GH during somatostatin infusion and in fact suggest the involvement of a process of GH degradation and/or an inhibition of GH synthesis.     

Control of passive permeability of chinese hamster ovary cells by external and intracellular ATP

External ATP causes passive permeability change in several transformed cells, but not in untransformed cells. We studied the effect of external ATP on the passive permeability of CHO-K1 cells, a transformed clone of Chinese hamster ovary cells. Treatment of the cells with external ATP alone did not produce a permeability change, and this was observed only when a mitochondrial inhibitor, such as rotenone or oligomycin, was present together with ATP. These inhibitors reduced the concentration of intracellular ATP and a permeability change by external ATP was observed when intracellular ATP was decreased more than 70%. This requirement for permeability change of CHO-K1 cells was quite unique, since passive permeability change of other transformed cells so far tested was induced by ATP alone. Treatment of CHO-K1 cells with cyclic AMP analogues increased their sensitivity to external ATP about 2-fold. The roles of external and intracellular ATP in controlling passive permeability are discussed.     

Monensin blocks endocytosis of vesicular stomatitis virus

Monensin inhibits the infection of mouse cells by Vesicular Stomatitis Virus (VSV). At low drug concentrations (0.5 μM), endocytosis of VSV is inhibited whereas viral binding is unaffected. Monensin may be useful for analyzing the internalization of other viruses as well as soluble ligands.     

Actin acetylation in Drosophila tissue culture cells

In a permanent cell line derived from Drosophila embryos, cytoplasmic actin is produced as an unstable precursor, which is subsequently converted to a stable form. This conversion results in a reduction in isoelectric point, with no apparent change in molecular weight. The conversion involves an enzymatic acetylation, and results in an insensitivity to aminopeptidase digestion, suggesting N-terminal blockage. Both the acetylated and unacetylated actins can participate in the assembly of F-actin, but with different efficiencies.This work was supported by a grant from the NIH (GM 22866).     

Modification of the cytotoxicity of aflatoxin B1 in rat liver cells by steroids

The addition of steroids with aflatoxin B1 (AFB1) to rat liver cells in culture has been shown to increase the toxin's inhibitory action on growth and protein synthesis. In contrast the inhibition of RNA synthesis by AFB1 was unaffected. The steroid potentiates the direct action of AFB1 at initiation of translation.