Reduced growth of Ehrlich ascites tumor cells in creatine depleted mice fed β-guanidinopropionic acid

The effect of implantation of Ehrlich ascites tumor (EAT) cells of creatine distribution was investigated. It was also studied how depletion of creatine by feeding creatine-analogue β-guanidinopropionic acid (β-GPA) affects the growth of EAT cells in mice. Enhanced mobilization of creatine from host tissues to EAT cells against a greater concentration gradient was observed. The creatine (but not creatinine) level in blood plasma was lowered to 22% of the normal value by β-GPA feeding alone and assimilation of ¹⁴C-creatine into EAT cells was inhibited. The growth of EAT cells was significantly reduced and the duration of survival of mice after implantation of EAT cells was extended when the creatine concentration was decreased. A decrease in daily food consumption and the degree of muscle atrophy after implantation of EAT cells was less in β-GPA than control groups. In the creatine-depleted mice, the rate of increase in total EAT cell number and the volume of abdominal ascites were approximately half of the control values, and more dead EAT cells were observed. These results suggest that supplementation of β-GPA inhibits creatine transfer to EAT cells and reduces the growth of cancer cells.     

Growth of a human yolk sac tumor cell line with yolk sac-derived functions in selenium-supplemented chemically defined synthetic medium

Summary A human yolk sac tumor cell line, TG1, which was established from a testicular yolk sac tumor, was found to replicate continuously in a chemically defined medium supplemented with Na₂SeO₃ (ISRPMI). TG1 produced several plasma proteins and growth factors: albumin, alpha-fetoprotein (AFP), ferritin, carcinoembryonic antigen, beta-2-microglobulin, polyamine, neuron specific enolase, tissue polypeptide antigen, transferrin (Tf), epidermal growth factor, and platelet derived growth factor. By analysis of lectin (LcHA)-affinity electrophoresis, to examine the microheterogeneity of carbohydrate chains of synthetic glycoproteins, TG1 cells cultured with ISRPMI produced only LcHA reactive Tf and AFP based on core fucose attached to asparagine-linkedN-acetylglucosamine residues instead of LcHA-nonreactive Tf and AFP produced by TG1 cells cultured with fetal bovine serum (FBS)-containing medium.α1-6 Fucosyltransferase activity was significantly greater in the TG1 cells cultured with ISRPMI (39.9±1.5 pmol · h⁻¹ · mg⁻¹ protein) than cultured with FBS-containing media (18.2±1.2 pmol · h⁻¹ · mg⁻¹ protein). These results have indicated that the selective increase ofα1-6 fucosyltransferase occurred when the cells were cultured with the FBS-free synthetic media.     

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.     

Microinjection of arginase into enzyme-deficient cells with the isolated glycoproteins of sendai virus as fusogen

Bumetanide is a potent diuretic drug which has some structural features in common with furosemide. The steady-state exchange of K⁺ and Cl^? was investigated in Ehrlich ascites tumor cells treated with bumetanide. This agent did not alter the cellular content of K⁺ or Cl^? but the self-exchange of both ions was depressed. K⁺ self-exchange was inhibited by 55% at bumetanide concentrations as low as 10^?6 M. Cl^? self-exchange was less sensitive to this drug but at low concentrations (between 10^?6 and 10^?3 M) bumetanide was a more effective inhibitor of Cl^? transfer than furosemide. The steady-state K⁺ flux of cells equilibrated in NO₃^? media was compared with the K⁺ flux in cells treated with 10^?4 or 10^?3 M bumetanide; the Cl^? -sensitive K⁺ exchange was equivalent to the bumetanide-sensitive K⁺ exchange. Since the results suggested that a bumetanide-sensitive (Cl^?, K⁺) cotransport could be operative in steady-state cells, the stoichiometry of the bumetanide-sensitive fluxes was determined by measuring Cl^? and K⁺ fluxes simultaneously in the same cell suspension. At $\text{5 · 10}{}^{\mathrm{?4}}$ and 10^?3 M bumetanide concentrations, the ratio of these fluxes was $\text{0.98 ? 0.07 (}\text{S.E.}\text{)}\text{and}\text{1.04 ? 0.06}$, respectively, consistent with the postulated cotransport mechanism. At 10^?4 and 10^?5 M, however, the ratio of the bumetanide-sensitive Cl^?/K⁺ flux was significantly less than 1.0. Since the magnitude of the bumetanide-sensitive K⁺ flux at 10^?4 M was close to that of the Cl^?-sensitive flux, a ratio of less than 1.0 at this drug level indicates that Cl^? sensitivity and drug sensitivity may not reflect inhibition of the same process under all circumstances.     

Ontogeny of murine T lymphocytes: I. Maturation of thymocytes induced in vitro by tumor necrosis factor-positive serum (TNF+)

One question which is unresolved in developmental immunology is whether cortical thymocytes are the precursor cells which give rise to medullary thymocytes and peripheral T cells. Cortical thymocytes display a characteristic surface antigen phenotype (high TL and Thy-1, low H-2, no Qa-2, no Qa-3), are agglutinated by peanut agglutinin (PNA), and are unresponsive to concanavalin A (Con-A). The functionally more mature medullary thymocytes express a surface phenotype more closely resembling peripheral T cells (no TL, low Thy-1, high H-2, and some Qa-2), are not agglutinated by PNA, and are responsive to Con-A. An in vitro induction system has been devised in which mouse thymocytes undergo quantitative changes in surface antigens in less than 24 hr and increase their mitogen response to Con-A. The phenotypic changes are characterized by a decrease of TL and Thy-1 and an increase in H-2, Qa-2, and Qa-3. Studies in which thymocytes were fractionated on BSA gradients and by PNA agglutination demonstrate that the inducible cells have the properties of cortical thymocytes. Our data show that a subpopulation of cortical thymocytes can acquire phenotypic characteristics similar to medullary thymocytes and peripheral T cells.     

Temperature-sensitive loss of tumor characteristics in a tobacco crown gall strain

Summary Three independently isolated tobacco crown gall strains incited byAgrobacterium tumefaciens C58 required phytohormone (auxin and cytokinin) supplements in the basal medium to grow, at 37°C. Six other tobacco crown gall strains incited, respectively, byA. tumefaciens IIBV7, B6, CGIC, A6NC, 27 and AT4 expressed, at 37°C, the tumor characteristic of ability to grow in vitro on medium lacking phytohormones. Nopaline was not detectable in C58 tumors cultured at 37°C, but octopine was produced by B6 tumor tissues incibated at the elevated temperature. C58 tumor strains kept at 37°C for 1 week or more lost the ability to express tumor characteristics at 27°C such as tissue morphology, growth on basal medium lacking phytohormones and nopaline production. Heat-treated C58 tissues also differed from the original tumor strain in regeneration ability and phytohormone requirements of explants; i.e. explants from regenerated, heart-treated C58 tumors required both auxin and cytokinin for growth in vitro.