Characteristics of hematin uptake in malignant, embryonic and normal cells

The characteristics of hematin uptake were examined in three malignant cell lines [L1210 leukemia, 745 murine erythroleukemia (MEL) and Walker carcinoma (W256)], a cell line derived from normal rat liver (BRL-3A) and a normal embryonic cell, chick embryo fibroblasts (CEF). Uptake in the normal liver cell line was slight and occurred at a slow rate in contrast to the rapid uptake, which was more rapid and of greater magnitude in the three tumor cell lines, Saturation of the heme uptake mechanism was observed in MEL cells at an extra-cellular hematin concentration of 160 micro M and in L1210 cells at 300 micro M. At saturation L1210 cells achieved a cellular heme concentration nine times as high as MEL cells. Hematin uptake in MEL cells was markedly augmented by pretreatment with DMSO, procaine, detergent or proteolytic enzymes or by increases in the pH of the medium from 8 to 9.5. In contrast to MEL cells where SA inhibits growth by lowering cellular heme, the inhibition of growth of L1210 cells by SA appears to operate by a mechanism independent of heme. In gradual increase in hematin uptake capacity in MEL cells over a period of days. Afer exposure of MEL cells to a high concentration of hematin in the medium, the egress of heme was followed under various conditions. Of the various agents studied, only cyanide produced a loss of heme from MEL cells.     

Relation between spontaneous activity and cholinesterase activity of dissociated embryonic and young rat-heart and embryonic chick-heart cells studied in tissue culture

Summary The number of spontaneously beating single cells obtained both from rat and chick hearts disaggregated by trypsin, is age-dependent: it is higher in younger than in older hearts. Cholinesterase activity and glycogen content are high in the spontaneously active cells. Being dependent on animal age, many Cholinesterase and PAS-positive cells are present in cultures prepared from early hearts. Succinicacid dehydrogenase-activity is equally feeble in spontaneously active and inactive heart cells. Only in older, already differentiated cells, devoid of spontaneous activity, is it intense.     

Nτ- and Nπ-histidinoalanine: Naturally occurring cross-linking amino acids in calcium-binding phosphoproteins

Two isomeric amino acid cross-links, N^τ-histidinoalanine and N^π-histidinoalanine have been isolated from calcium-binding phosphoprotein particles derived from the extrapallial fluid of the estuarine clam $\text{Rangia}\text{,}\text{cuneata}$. The cross-links were identified and compared by ¹³C and ¹H NMR spectroscopy and mass spectroscopy. In the phosphoprotein particles, 6% of the amino acid residues are involved in cross-linkages. This is the first demonstration of the occurrence of the isomer N^π-histidinoalanine.     

Prolonged culture of cells from normal human thyroid tissue and toxic goiters

Normal human thyroid cells and cells from patients with Grave's disease were cultured for 5 months (11 passages) in vitro. Both normal and diseased thyreocytes, similar in morphology, proliferated actively and responded to thyrotropin stimulation by cytoplasmic arborization of a part of the population. Slight inhibition of mitotic activity was present under the influence of thyrotropin.     

Lysosomal hydrolase activity in chorionic villi and embryonic cells in culture

Summary Fourteen lysosomal enzymes were compared in 20 cultured cell lines from chorionic biopsy and corresponding embryonic tissue after voluntary abortions. Enzymatic expression appears to be similar in cultured cells from both sources with some slightly higher levels for chorionic villi. We stress the importance of culturing chorionic villi especially in the case of enzymes (-L-iduronidase) or diseases (I cell disease) whose expression is unusual in fresh trophoblast tissue.     

Induction of betaine: Homocysteine methyltransferase in some murine cells cultured in vitro

Betaine when present in the culture medium could induce the activity of betaine: homocysteine methyltransferase (EC 2.1.1.5) in mouse L-cells, and leukemic L1210 cells, as well as in mouse embryo fibroblasts grown in vitro. We found this process to be time- and concentration-dependent. A persisting contact of the cells with betaine was indispensible for expressing and maintaining the enzyme activity. The treatment of cells with cycloheximide or actinomycin D abolished the process of induction. Methionine as well as homocysteine, when present either in the culture medium or in the reaction mixture, strongly depressed the activity of this enzyme. The L-cells with the induced betaine: homocysteine methyltransferase survived but did not multiply in the methionine-deficient medium, therefore, they did not become prototrophs with respect to methionine.     

Communication between normal and enzyme deficient cells in tissue culture

Correction of certain mutant phenotypes by intimate contact with normal cells, i.e. ‘metabolic cooperation’, is an easily studied form of cell communication. Metabolic cooperation between normal cells and mutant cells deficient in hypoxanthine-guanine or adenine phosphoribosyl transferase (HGPRTase and APRTase respectively) appears to be the result of transfer of the enzyme product, nucleotide or nucleotide derivative, from normal to mutant cells. This process shows selectivity in that mutant derivatives of mouse L cells are unable to function as recipients of HGPRTase or APRTase products, while hamster and human fibroblasts with these enzyme deficiencies, exhibit correction of the mutant phenotype, when in contact with normal donor cells. There is also selectivity with respect to substances transferred, since other mutant phenotypes, i.e. G-6 PD deficiency, are not corrected by contact with normal cells. Species specificities do not appear to influence metabolic cooperation, therefore heterospecific cell mixtures provide an opportunity to cytologically distinguish cells and study individual cell interactions.     

Neurotrophic and hepatotrophic stimulation of proliferation of embryonic chick muscle cells in vitro: Assay and partial characterization of mitogenic activity in chick embryonic organ and tissue extracts

Whole embryo extract is routinely employed as a growth-promoting supplement in chick embryonic muscle cell cultures. In assessing the effect of the extract on muscle cell cultures, extracts of various embryonic tissues and organs were substituted for whole embryo extract and the effects on proliferation of dissociated 12-day chick embryonic leg muscle cells were observed. The effects were measured according to [³H]thymidine incorporation into deoxyribonucleic acid (DNA) and were confirmed with total cell counts. Brain and liver extracts were found to be especially effective in stimulating muscle cell proliferation. The extracts were found to be heat and trypsin labile. Further analysis of activity in the extracts by dialysis and Sephadex G-25 fractionation revealed the presence of at least two classes of activity—one of high molecular weight (>5000) and one of low molecular weight (<5000)—which must be present together to yield the full activity of crude extracts from embryonic liver and brain. The results are discussed against the background of our interest in the neurotrophic phenomenon.     

Routine culturing of normal,dysplastic and malignant human mammary epithelial cells from small tissue samples

Summary We compared the growth and morphology of normal, dysplastic and malignant human mammary epithelial cells (HMEC) in medium containing 5% human serum, a serum-free medium (32) and serum-free medium with a low Ca⁺⁺ concentration. Tissues were dissociated and epithelial organoids or single cells were seeded onto collagen-coated dishes. The cells grew in serum-containing medium, but growth of fibroblasts was also stimulated. The serum-free medium consistently selected for and stimulated the growth of epithelial cells. There was little advantage in reducing the Ca⁺⁺ concentration to further increase cell yield. This serum-free primary culture system allows us to routinely prouce sufficient numbers of HMEC from small tissue samples for molecular biological investigations. Furthermore, the maintenance of cells in a defined medium can provide a system for evaluating the direct effects of factors on gene expression. This work was supported by a grant from the National Cancer Institute of Canada and funds contributed by Mr. B. T. Wharton in memory of his wife, Nadia.     

Trypsin modifies the activity of adenylate cyclase from normal, malignant and hybrid mammalian cells

Adenylate cyclase (ATP pyrophosphate-lyase (cylizing), EC 4.6.1.1) activity, measured in homogenates of normal, malignant and hybrid mammalian cell lines, is enhanced and subsequently inhibited by increasing concentrations of trypsin (EC 3.4.21.4). Treatment of intact cells with trypsin appears to cause latent activation of adenylate cyclase (i.e. activation which is only expressed after homogenization of the cells). Conversely, adenylate cyclase activity of a normal Chinese hamster fibroblast cell line is inhibited in intact cells by trypsin through the degradation of some site on the outer surface of the plasma membrane. The prostaglandin E1 receptor is not affected by trypsinization of cells.     

Three-dimensional culture models of normal and malignant breast epithelial cells

Extracellular matrix is a key regulator of normal homeostasis and tissue phenotype. Important signals are lost when cells are cultured ex vivo on two-dimensional plastic substrata. Many of these crucial microenvironmental cues may be restored using three-dimensional (3D) cultures of laminin-rich extracellular matrix (lrECM). These 3D culture assays allow phenotypic discrimination between nonmalignant and malignant mammary cells, as the former grown in a 3D context form polarized, growth-arrested acinus-like colonies whereas the latter form disorganized, proliferative and nonpolar colonies. Signaling pathways that function in parallel in cells cultured on plastic become reciprocally integrated when the cells are exposed to basement membrane-like gels. Appropriate 3D culture thus provides a more physiologically relevant approach to the analysis of gene function and cell phenotype ex vivo. We describe here a robust and generalized method for the culturing of various human breast cell lines in three dimensions and describe the preparation of cellular extracts from these cultures for molecular analyses. The procedure below describes the 3D 'embedded' assay, in which cells are cultured embedded in an lrECM gel (Fig. 1). By lrECM, we refer to the solubilized extract derived from the Engelbreth-Holm-Swarm mouse sarcoma cells. For a discussion of user options regarding 3D matrices, see Box 1. Alternatively, the 3D 'on-top' assay, in which cells are cultured on top of a thin lrECM gel overlaid with a dilute solution of lrECM, may be used as described in Box 2 (Fig. 1 and Fig. 2).     

In vitro culture of embryonic cells from the shrimp, Penaeus chinensis

Studies were carried out in our laboratory to develop cell cultures from embryonic tissue of Penaeus chinensis. Good yields of dissociated, uncontaminated, viable cell suspensions were obtained by physical disruption of harvested embryonic tissues. Primary cultures in the form of proliferating foci of cells were readily initiated using MPS medium (osmolality: 2.4%) with 20% heat-inactivated fetal bovine serum. Insulin-like growth factor-II (IGF-II) and basic fibroblast growth factor (bFGF) at different concentrations were also added into the culture medium. Passage of primary cultures resulted in rapid proliferation and good adherence of lymphocytes in the presence of IGF-II at 80 ng/ml and bFGF at 20 ng/ml. Cells maintained in subculture for up to 10 passages still had good cellular morphology and division rates.