N5-methyltetrahydrofolate: Homocysteine methyltransferase activity in extracts from normal,malignant and embryonic tissue culture cells

Malignant cells (J111, L1210, W-256) and human embryonic cells (FL) are unable to survive and grow when homocystine replaces methionine in tissue culture media containing excess vitamin B₁₂ and folic acid. Extracts of these same cells when grown in media containing methionine and more than adequate vitamin B₁₂ and folic acid have diminished N⁵-methyltetrahydrofolate: homocysteine methyltransferase activities in the absence of added cyanocobalamin when compared with extracts of normal cells (adult rat thymus and liver fibroblasts). Extracts of human monocytic leukemia (J111) and human amnion cells (FL) have normal enzymatic activity in the presence of added cyanocobalamin whereas the rodent malignant cells (W-256 and L1210) have abnormally low activity in the absence or presence of added vitamin B₁₂.     

Acetylcholinesterase in normal and malignant human cells

Acetylcholinesterase (AChE) activity was determined in normal and malignant human cell lines by histochemical methods. In normal human fibroblasts, no AChE activity could be demonstrated by any histochemical technique or substrate. Enzymic activity was observed in HT-1080 human fibrosarcoma cells, RD 2 human rhabdomyosarcoma cells, and SW 311 human colon carcinoma cells. Activity was localized around the nuclear envelope, in the cytoplasm and associated with the cortical region of most cells. The specificity of the reaction was shown through the use of specific cholinesterase inhibitors.     

Local uptake and synthesis of oestrone in normal and malignant postmenopausal breast tissues

Uptake and local formation of oestrone (E1) were studied in vivo by a double isotopic technique in normal and malignant breast tissues from 24 postmenopausal women with breast cancer. Active uptake of radio-labelled E1 beyond plasma was found both in normal and malignant tissue, the effect being significantly greater in non-malignant compared with cancer tissue. The presence of local E1 formation was also demonstrated in most samples. Both uptake and synthesis positively correlated with total amount of radioactive E1 found in the tissues. Uptake appeared to make a greater contribution to E1 levels within the breast than in situ synthesis, although there were marked variations between specimens from different patients and the relative proportion of synthesis to uptake was higher in tumour compared with non-malignant tissue. These results demonstrate quantitative differences in the different compartments by which postmenopausal breasts obtain oestrogen and highlight variations between individual breasts. This may be important in optimising oestrogen deprivation therapy for postmenopausal patients with hormone-dependent cancers.     

Laminin and fibronectin in normal and malignant neuroectodermal cells

Laminin and fibronectin, the major noncollagenous matrix glycoproteins, were studied in connection with normal brain cells and neuroectodermal cell lines. Laminin, a Mr 900,000 dalton matrix glycoprotein and an essential component of basement membranes, was found to be produced by cultured cells of several malignant cell lines of neuroectodermal origin. In cultured mouse C1300 neuroblastoma line cells laminin was localized, by immunoelectron microscopy, to the rough endoplasmic reticulum and, to sites of cell-to-cell and cell-to-substratum adhesion. Further experiments on the intracellular transport of this glycoprotein in C1300 cells confirmed that laminin is, at least partially, transported through the Golgi pathway. These results favor a role for laminin in attachment and cellular interactions of malignant neuronal cells. Laminin was also found in connection with neurons and glial cells from mammalian brain. In primary cultures from developing rat brain the vast majority of non-neuronal cells (80%) expressed immunoreactivity for the glial fibrillary acidic protein, a cytoskeletal protein specific for astrocytes. During the first week in culture all the glial fibrillary acidic protein-positive cells, with the exception of mature-looking star-shaped astrocytes, exhibited immunoreactivity for laminin. The intracellular laminin disappeared gradually after a few weeks in culture, but an extensive laminin matrix persisted and seemed to be localized on the upper surface of the non-neuronal cells. The neurofilament-positive neurons were negative for laminin. Pretreatment of the cultures with the ionophore monensin, caused accumulation of laminin-immunoreactivity within the Golgi region, which confirmed that laminin is, indeed, produced by cultured astrocytes and secreted through the Golgi complex. No fibronectin immunoreactivity was found in the majority of glial cells. However, under culture conditions where fibronectin was omitted from the culture medium there was, in the primary cultures, a minor population of glial fibrillary acidic protein-positive flat glial cells that exhibited intracytoplasmic immunofluorescence for fibronectin. In the presence of fibronectin in culture medium no fibronectin-positive glial cells could be detected. It thus appears that laminin, and to a minor extent fibronectin, are proteins that normal glial cells are capable of producing under specific conditions. Laminin and fibronectin were localized in adult rat brain in capillary and meningeal structures.(ABSTRACT TRUNCATED AT 400 WORDS)     

Quantitative cellular uptake, localization and cytotoxicity of curcumin in normal and tumor cells

Using absorption and fluorescence spectroscopic methods, quantitative cellular uptake of curcumin, an antioxidant and anti-tumor agent from Curcuma longa, was calculated in two types of normal cells: spleen lymphocytes, and NIH3T3 and two tumor cell lines: EL4 and MCF7. Both the uptake and fluorescence intensity of curcumin were significantly higher in tumor cells compared to the normal cells. A linear dependency on the uptake was observed with treatment concentration of curcumin. Using laser confocal microscopy, intracellular localization of curcumin was monitored and the results indicated that curcumin is located both in the cell membrane and the nucleus. Sub-cellular fractionation of curcumin-loaded MCF7 cells supported the differential distribution of curcumin in membrane, cytoplasm and nuclear compartments of cell with maximum localization in the membrane. Cytotoxicity studies in different cell lines indicated that the toxicity of curcumin increased with increasing uptake.     

IGF-I mediated survival pathways in normal and malignant cells

The type-I and -II insulin-like growth factors (IGF-I, II) are now established as survival- or proliferation-factors in many in vitro systems. Of note IGFs provide trophic support for multiple cell types or organ cultures explanted from various species, and delay the onset of programmed cell death (apoptosis) through the mitochondrial (intrinsic pathway) or by antagonizing activation of cytotoxic cytokine signaling (extrinsic pathway). In some instances, IGFs protect against other forms of death such as necrosis or autophagy. The effect of IGFs on cell survival appears to be context specific, being determined both by the cell origin (tissue specific) and the cellular stress that induces loss of cellular viability. In many human cancers, there is a strong association with dysregulated IGF signaling, and this association has been extensively reviewed recently. IGF-regulation is also disrupted in childhood cancers as a consequence of chromosomal translocations. IGFs are implicated also in acute renal failure, traumatic injury to brain tissue, and cardiac disease. This article focuses on the role of IGFs and their cellular signaling pathways that provide survival signals in stressed cells.     

Proliferation control in cloned normal and malignant human cells

A method for cloning of a variety of normal and malignant human cells in culture is described. Palladium is precipitated on agarose through masks prefabricated by a photolithographic technique. Practically any pattern can be produced where cells exclusively settle and multiply as ‘miniclones’ on the metal ‘islands’. This communication establishes

1. 1. that ‘miniclones’ settle and multiply with the same efficiency as cells of a mass population, thus constituting an unbiased cell sample

2. 2. that certain malignant lines cannot be studied with the unmodified technique suitable for normal glia-like cells because of excessive translocation between islands

3. 3. that this can be circumvented by the interposition of a thin agar overlay between the cells and the fluid medium. Proliferation of normal glia-like cells was inhibited on palladium islands in a cell density-dependent way. On sufficiently small islands even single cells could be prevented from dividing. Circles of about 3 000 μm² prevented multiplication of about 50% of single cells otherwise capable of division, suggesting that cell-to-cell contacts may not be necessary for proliferation inhibition of normal cells.

     

GABA uptake in embryonic palate mesenchymal cells of two mouse strains

To obtain further evidence that the inhibitory neurotransmitter GABA functions in palate development, the presence of an active GABA uptake mechanism was sought using primary cultures of embryonic palate mesenchymal cells. Uptake was compared from cells of two inbred mouse strains in which the SWV strain shows greater sensitivity than the AJ strain to effects of GABA on palate morphogenesis and of diazepam in producing cleft palate (1). Palate cells were capable of accumulating [³H]GABA by saturable uptake mechanisms characteristic of a high and a low affinity active transport as indicated by temperature, Na⁺ ion and carrier dependence as well asK _m andV _max values that were comparable to other biological systems. TheV _max of the high-affinity uptake system from cells of the SWV strain was 1.8 fold higher than that of the AJ. GABA uptake was also observed in fibroblasts from various sources including embryonic mouse limb cells, human skin fibroblasts and 3T3 cells When active GABA uptake was measured in skin fibroblasts from the mouse SWV and AJ strains, the rate of uptake from SWV cells under high affinity conditions was also 1.8 fold greater than in AJ cells. Thus active GABA uptake appears to be genetically regulated in non-neural cells which may contribute to differential resonses to GABA.     

Patterns of plasminogen activator production in cultured normal embryonic cells

Cultured normal low-passage embryo fibroblasts, from a number of species, and two untransformed clones of a Balb/3T3 line elaborate increasing amounts of plasminogen activator (PA) as they approach confluence; the low-passage cells then lose this PA activity after reaching confluence, while the 3T3 cells retain it indefinitely. Even at their peaks, however, the PA activities of the low-passage cells remain well below those of the corresponding virally or spontaneously transformed cells. The PA increases in normal cells are probably a result of PA production rather than of adsorption of secreted PA to the cell surface, or of changes in cell-associated protease inhibitors. The elaboration of PA by normal cells is dependent upon their metabolic activity, such that the level of serum supplementation and the growth phase of the culture directly influence the level of cell-associated PA observed. In addition, there may be a component of serum which exerts a negative control on PA production and which is not an acid-labile protease inhibitor.     

Non-transferrin-bound iron uptake in Belgrade and normal rat erythroid cells

Belgrade (b) rats have an autosomal recessive, microcytic, hypochromic anemia. Transferrin (Tf)-dependent iron uptake is defective because of a mutation in DMT1 (Nramp2), blocking endosomal iron efflux. This experiment of nature permits the present study to address whether the mutation also affects non-Tf-bound iron (NTBI) uptake and to use NTBI uptake compared to Tf-Fe utilization to increase understanding of the phenotype of the b mutation. The distribution of ⁵⁹Fe²⁺ into intact erythroid cells and cytosolic, stromal, heme, and nonheme fractions was different after NTBI uptake vs. Tf-Fe uptake, with the former exhibiting less iron into heme but more into stromal and nonheme fractions. Both reticulocytes and erythrocytes exhibit NTBI uptake. Only reticulocytes had heme incorporation after NTBI uptake. Properly normalized, incorporation into b/b heme was ∼20% of +/b, a decrease similar to that for Tf-Fe utilization. NTBI uptake into heme was inhibited by bafilomycin A1, concanamycin, NH₄Cl, or chloroquine, consistent with the endosomal location of the transporter; cellular uptake was uninhibited. NTBI uptake was unaffected after removal of Tf receptors by Pronase or depletion of endogenous Tf. Concentration dependence revealed that NTBI uptake into cells, cytosol, stroma, and the nonheme fraction had an apparent low affinity for iron; heme incorporation behaved like a high-affinity process, as did an expression assay for DMT1. DMT1 serves in both apparent high-affinity NTBI membrane transport and the exit of iron from the endosome during Tf delivery of iron in rat reticulocytes; the low-affinity membrane transporter, however, exhibits little dependence on DMT1. J. Cell. Physiol. 178:349–358, 1999. © 1999 Wiley-Liss, Inc.     

Quantitation and potential function of nucleolar phosphoprotein pp 105 in mouse tumor cells, embryonic cells and normal tissues

1. Nucleolar phosphoprotein pp 105 was determined in various mouse cell and tissue extracts using a highly sensitive ELISA. The results indicate that the highest relative amounts of pp 105 correlate with cells and tissues of high growth rate such as tumor cell lines, solid tumors and embryonic tissues. 2. The specific phosphorylation of pp 105 was compared in a 1 min endogenous phosphorylation assay with native cell and tissue extracts. 3. The mitogenic activity of highly purified pp 105 was demonstrated in cultures of resting mouse embryonic cells and mouse thymocytes.     

Multiple interconvertible forms of cAMP phosphodiesterase in normal, malignant and hybrid mammalian cells

cAMP-Phosphodiesterase has been studied in a mouse lymphoma cell line (P388F-36), a Chinese Hamster cell line (CH23), and the hybrid of these two (PCM3). This hybrid is unusual in that, at any one time, it exhibits both monolayer and suspension states of growth. Resolution of the crude cell extracts by sucrose density gradient centrifugation gave rise to three activity peaks (I, II, and III) when assayed at 100 μM cAMP, indicating the presence of three enzyme forms with molecular size increasing from peak I to III. Each peak on recentrifugation regenerated the three peaks suggesting that the molecular forms are interconvertible and may exist in equilibrium. The parental cells differ with regard to the relative sizes of the three peaks. Peak III, presumably corresponding to a low K_m enzyme, was predominant in the malignant cell line P388F-36 and represented about 80% of total activity. Both the monolayer hybrid PCM3(M) and the suspension hybrid PCM3(S) exhibited some parental characteristics but were different from each other. These results were used to confirm or explain previous investigations on these cell lines especially with regard to basal cAMP concentration and some kinetic parameters. Resolution of the cell extracts by isoelectric focusing and DEAE-cellulose column chromatography gave a variable number of peaks depending on the experimental conditions. An explanation is given in every case.     

ANG II increases 2-deoxyglucose uptake in mouse embryonic stem cells

It is now suggested that all components of the renin-angiotensin system are present in many tissues, including the embryo and may play a major role in embryo development and differentiation. However, little is known regarding whether ANG II regulates glucose transport in mouse embryonic stem (ES) cells. Thus, the effects of ANG II on [3H]-2-deoxyglucose (2-DG) uptake and its related signal pathways were examined in mouse ES cells. ANG II significantly increased cell proliferation and 2-DG uptake in concentration- and time-dependent manner (>18 h, >10(-8) M) and increased mRNA and protein level of GLUT1 by 31+/-7% and 22+/-5% compared to control, respectively. Actinomycin D and cycloheximide completely blocked the effect of ANG II on 2-DG uptake. ANG II-induced increase of 2-DG uptake was blocked by losartan, an ANG II type 1 (AT1) receptor blocker, but not by PD 123319, an ANG II type 2 (AT2) receptor blocker. In addition, ANG II-induced stimulation of 2-DG uptake was attenuated by phospholipase C (PLC) inhibitors, neomycin and U 73122 and ANG II increased inositol phosphates (IPs) formation by 37+/-8% of control. Protein kinase C (PKC) inhibitors, staurosporine, bisindolylmaleimide I, and H-7 also blocked ANG II-induced stimulation of 2-DG uptake. Indeed, ANG II activated a PKC translocation from the cytosolic to membrane fraction, suggesting a role of PKC. A 23187 (Ca2+ ionophore) increased 2-DG uptake and nifedifine (L-type Ca2+ channel blocker) blocked it. In conclusion, ANG II increased 2-DG uptake by PKC activation via AT1 receptor in mouse ES cells.