Expression of external-surface membrane proteins in differentiated and undifferentiated mouse neuroblastoma cells.

Murine neuroblastoma cultures were labeled externally with the cationic reagent N,N,N-[3H]-trimethylamino-beta-alanyl-N-hydroxy-succinimide ester ([3H]Me3N-beta Ala-NSuc) or with 125I/lactoperoxidase. The cells were labeled in the logarithmic and confluent growth phases as well as in a highly differentiated state following treatment with 2% dimethylsulfoxide. The labeled exterior membrane proteins were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Major changes in the exterior membrane proteins were observed during maturation and differentiation of the cells. Most of these changes were clonal-specific, while others were common to several clones. Two proteins of Mr 55,000 and 65,000 were labeled by both 125I/lactoperoxidase and Me3N-[3H]-beta Ala-NSuc. The level of labeling was dependent on the clonal lines used and the state of the cell maturation. A group of proteins displaying a molecular weight between 150,000 and 200,000 was found to be related to the transition of a culture from logarithmic to confluent growth phases. An additional protein, with an apparent molecular weight of 95,000, was common to differentiated cells of the two inducible clones used. In general the maturation of logarithmic phase cells into confluent cells resulted in a less complex electrophoretic distribution of the pattern of labeling. After dimethyl-sulfoxide treatment, further reduction in the complexity of the externally labeled proteins was observed.     

Calcium currents of differentiated mouse neuroblastoma cells

Differentiated mouse neuroblastoma cells (line C 1300, clone N-18-TG₂A₁) were investigated by intracellular dialysis. A slow component was found in the potential-dependent inward current of these cells. The results of investigation of changes in amplitude of this component during variation of the ionic composition of the external and internal solutions showed that this component is due to transport of calcium ions. A calcium current was observed in all cells tested. The region of its activation was between –70 and –65 mV; maximal values of this current were reached when the membrane potential was between –30 and –40 mV. The kinetic characteristics of this current were examined. In its kinetics and potential-dependence, this calcium current of the mouse neuroblastoma cell membrane is analogous to the fast component of the calcium current in normal neurons of rat spinal ganglia.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 16, No. 4, pp. 527–531, July–August, 1984.     

Expression of differentiated functions in dexamethasone-resistant hepatoma cells

The expression of liver-specific functions of different dexamethasone-resistant variants derived from a well-differentiated dexamethasone-sensitive Reuber H35 rat hepatoma cell line (Faza 967) was examined during long-term cultivation. The dexamethasone-sensitive Faza 967 cells are characterized by the activity of tyrosine aminotransferase (TAT) and gluconeogenic enzymes, secretion of serum albumin, and the presence of liver isozymes of alcohol dehydrogenase (L-ADH), aldolase (aldolase-B), and five isoenzymes of lactate dehydrogenase (LDH). The hormone-resistant cells undergo a very dramatic change in expression of most liver-specific functions (dedifferentiation) during long-term culture, in contrast to the sensitive cells in which only certain functions (TAT activity, inducibility, and synthesis of serum albumin) exhibit considerable changes. The hormone-dependent growth sensitivity and the expression of other differentiated functions is not controlled in coordinated way in Faza 967 cells. The time course of the expression of liver-specific functions shows that the cells are resistant before they became 'dedifferentiated', i.e., loss of these liver-specific functions is not a prerequisite of the establishment of the hormone-resistant state.     

Expression of differentiated functions in dexamethasone-resistant hepatoma cells

Abstract. The expression of liver-specific functions of different dexamethasone-resistant variants derived from a well-differentiated dexamethasone-sensitive Reuber H35 rat hepatoma cell line (Faza 967) was examined during long-term cultivation. The dexamethasone-sensitive Faza 967 cells are characterized by the activity of tyrosine aminotransferase (TAT) and gluconeogenic enzymes, secretion of serum albumin, and the presence of liver isozymes of alcohol dehydrogenase (L-ADH), aldolase (aldolase-B), and five isoenzymes of lactate dehydrogenase (LDH). The hormone-resistant cells undergo a very dramatic change in expression of most liver-specific functions (dedifferentiation) during long-term culture, in contrast to the sensitive cells in which only certain functions (TAT activity, inducibility, and synthesis of serum albumin) exhibit considerable changes. The hormone-dependent growth sensitivity and the expression of other differentiated functions is not controlled in coordinated way in Faza 967 cells. The time course of the expression of liver-specific functions shows that the cells are resistant before they became 'dedifferentiated', i.e., loss of these liver-specific functions is not a prerequisite of the establishment of the hormone-resistant state.     

Expression of c-kit protooncogene is stimulated by cAMP in differentiated F9 mouse teratocarcinoma cells.

Protooncogene c-kit, a transmembrane tyrosine kinase receptor, was recently shown to map to the dominant white spotting locus (W) of the mouse. W mutations affect melanogenesis, gametogenesis, and hematopoiesis during development and in adult life. In order to determine the regulation of the c-kit gene in cell differentiation, we investigated its expression during the differentiation of F9 cells. Undifferentiated F9 cells and F9 cells treated with retinoic acid (RA) alone or dbcAMP alone showed little expression of c-kit mRNA if any. The subsequent addition of dbcAMP to F9 cells treated with RA markedly increased the expression of c-kit mRNA. Furthermore, the effect of dbcAMP on c-kit expression is reversible. In differentiated cells treated with RA, c-kit gene expression is induced by agents such as forskolin or theophylline, which are known to elevate cellular cAMP level. These results indicate that the expression of the c-kit gene is regulated by the level of intracellular cAMP in differentiated F9 cells induced by RA.     

Estrogen synthesis in human colon cancer epithelial cells

Epidemiological and experimental data suggest an involvement of estrogen in the development and progression of colorectal cancer. In order to determine whether local synthesis of estrogen occurred in human colonic cancer cells, two colorectal cancer cell lines, HCT8 and HCT116, were evaluated for gene expression and enzyme activity of cytochrome P450 aromatase. In addition, the effect on aromatase expression of charcoal-stripped fetal calf serum, of quercetin and genistein and of tamoxifen and raloxifene was investigated in both cell lines. RT-PCR analysis revealed that colorectal adenocarcinoma cell lines contain aromatase as a major component. The conversion of [³H]-androstenedione to estrone and labeled water was dose-dependently inhibited by 4-hydroxyandrostenedione and obeyed Michaelis–Menten kinetic with apparent Km values of 20 nM and V_max values of approx. 200 and 500 fmol/mg protein/h for HCT8 and HCT116 cells, respectively. After 24 h incubation, genistein (1 μM) significantly increased aromatase activity in HCT8 cells, with no effect on HCT116 cells. In accord with previous observation in reproductive tissues, quercetin (1 μM) significantly inhibited the enzyme activity in both cell lines. Also tamoxifen (100 nM) acted as inhibitor, while raloxifene (10 nM) decreased the enzyme activity only in HCT116 cells. The aromatase gene expression modulation by these effective agents was consistent with their effects on enzyme activity. These findings demonstrate for the first time that colorectal adenocarcinoma cell lines express aromatase. Interestingly, the enzyme activity was inhibited by quercetin, one major dietary flavonoid, by tamoxifen, a hormonal therapeutic agent for breast cancer, and by raloxifene, used in the prevention of postmenopausal osteoporosis.     

Collagenous substrata regulate the nature and distribution of glycosaminoglycans produced by differentiated cultures of mouse mammary epithelial cells

We have investigated the influence of culture substrata upon glycosaminoglycans produced in primary cultures of mouse mammary epithelial cells isolated from the glands of late pregnant mice. Three substrata have been used for experiments: tissue culture plastic, collagen (type I) gels attached to culture dishes, and collagen (type I) gels that have been floated in the culture medium after cell attachment. These latter gels contract significantly. Cells cultured on all three substrata produce hyaluronic acid, heparan sulfate, chondroitin sulfates and dermatan sulfate but the relative quantities accumulated and their distribution among cellular and extracellular compartments differ according to the nature of the culture substratum. Notably most of the glycosaminoglycans accumulated by cells on plastic are secreted into the culture medium, while cells on floating gels incorporate almost all their glycosaminoglycans into an extracellular matrix fraction. Cells on attached collagen gels secrete approx. 30% of their glycosaminoglycans and assemble most of the remainder into an extracellular matrix. Hyaluronic acid is produced in significant quantities by cells on plastic and attached gels but in relatively reduced quantity by cells on floating gels. In contrast, iduronyl-rich dermatan sulfate is accumulated by cells on floating gels, where it is primarily associated with the extracellular matrix fraction, but is proportionally reduced in cells on plastic and attached gels. The results are discussed in terms of polarized assembly of a morphologically distinct basal lamina, a process that occurs primarily when cells are on floating gels. In addition, as these cultures secrete certain milk proteins only when cultured on floating gels, we discuss the possibility that cell synthesized glycosaminoglycans and proteoglycans may play a role in the maintenance of a differentiated phenotype.     

Expression of amiloride-sensitive epithelial sodium channels in mouse taste cells after chorda tympani nerve crush

Our previous electrophysiological study demonstrated that amiloride-sensitive (AS) and -insensitive (AI) components of NaCl responses recovered differentially after the mouse chorda tympani (CT) was crushed. AI responses reappeared earlier (at 3 weeks after the nerve crush) than did AS ones (at 4 weeks). This and other results suggested that two salt-responsive systems were differentially and independently reformed after nerve crush. To investigate the molecular mechanisms of formation of the salt responsive systems, we examined expression patterns of three subunits (alpha, beta and gamma) of the amiloride-sensitive epithelial Na(+) channel (ENaC) in mouse taste cells after CT nerve crush by using in situ hybridization (ISH) analysis. The results showed that all three ENaC subunits, as well as alpha-gustducin, a marker of differentiated taste cells, were expressed in a subset of taste bud cells from an early stage (1-2 weeks) after nerve crush, although these taste buds were smaller and fewer in number than for control mice. At 3 weeks, the mean number of each ENaC subunit and alpha-gustducin mRNA-positive cells per taste bud reached the control level. Also, the size of taste buds became similar to those of the control mice at this time. Our previous electrophysiological study demonstrated that at 2 weeks no significant response of the nerve to chemical stimuli was observed. Thus ENaC subunits appear to be expressed prior to the reappearance of AI and AS neural responses after CT nerve crush. These results support the view that differentiation of taste cells into AS or AI cells is initiated prior to synapse formation.     

Transfer into the mouse of mast cells differentiated in vitro

A population of mast cells can be derived in vitro by culturing normal spleen cells from C57BL/6 mice with the factor Interleukin 3. These mast cells share the morphological and histochemical features of mast cells at different stages of maturation. We have labelled these in vitro produced mast cells with 111In-Ox and injected them i. v. into normal syngeneic mice. The localisation of labelled cells has been determined 24 and 96 hours after the injection in the spleen, thymus and lymph-nodes.     

Microtubular system in cultured mouse epithelial cells

Previous observations had shown that colcemid does not affect locomotion of epithelial cells. Nevertheless, cultured mouse kidney cells forming epithelial sheets were found to contain a well-developed microtubular system sensitive to colcemid. The orientation of microtubules in the epithelial cells was not correlated with the stable or active state of the cell edges. It is suggested that microtubular system of the epithelial cells forming coherent sheets, in contrast to that of individually moving fibroblasts, is not essential for stabilization of the lateral cell edges.     

Direct evidence for transformation of differentiated iris epithelial cells into lens cells

Although it is generally assumed that the lens regenerated in the newt eye after complete lentectomy is formed by cells derived from the dorsal iris epithelium, experimental evidence so far obtained for this transformation does not rule out participation of cells from the dorsal iris stroma. When the normal dorsal iris epithelium of adult Notophthalmus (Triturus) viridescens was isolated and cultured in the presence of frog retinal complex, newt lens tissue was produced in 88% of cultures. These lens tissues were positive for immunofluorescence for lens-fiber-specific gamma crystallins as well as for total lens protein. On the basis of a study of stromal cells contaminating the samples of dorsal iris epithelium and a test for the lens-forming capacity in vitro of the dorsal iris stroma in the presence of frog retinal complex, it is concluded that lens formation observed in the above experiment is not dependent on the contaminating stromal cells. This implies that, in Wolffian lens regeneration, fully differentiated adult cells completely withdrawn from the cell cycle are transformed into another cell type. An additional culture experiment demonstrated that lens-forming capacity is not restricted to the dorsal half of the iris epithelium, but extends into its ventral half.     

小鼠胚胎干细胞分化为血管内皮细胞的永生化研究

本文探讨了小鼠胚胎干细胞（ES细胞）、诱导分化的血管内皮细胞永生化。在体外培养系统中,以维甲酸（RA）和转化生长因子－β1（TGF－β1）诱导小鼠胚胎干细胞（ES细胞）的拟胚体（EB）分化为“圆形细胞”和由这些“圆形细胞”组成的血管样结构。经光学和扫描电镜及免疫荧光等法分析检测,证明组成血管样结构的细胞具有专一性vWF荧光染色,表明是血管内皮样细胞。利用脂质体将人端粒酶催化亚基逆转录酶（hTERT)基因转染诱导分化中的“圆形细胞”。应用Dot-blot,RT－PCR,Western blot及免疫组织化学等方法分析、观察和证明了诱导分化的组成血管样结构的园形细胞和被hTERT基因转染的“圆形”细胞的形态和生物学特性。结果表明,携带hTERT基因的从ES细胞分化来的圆形细胞在体外可大量增殖,持续传代,95％具有血管内皮细胞的一些特有标志和管道化生长特征。因此,通过人端粒酶基因的转染途径可解决由ES细胞诱导分化而来的内皮细胞扩增和永生化问题,为构建组织工程化血管及其它人工血管的内皮化提供种子细胞来源打下基础。     

Activation of NADPH oxidase 1 in tumour colon epithelial cells

In the plasma membrane fraction from Caco-2 human colon carcinoma cells, active Nox1 (NADPH oxidase 1) endogenously co-localizes with its regulatory components p22(phox), NOXO1, NOXA1 and Rac1. NADPH-specific superoxide generating activity was reduced by 80% in the presence of either a flavoenzyme inhibitor DPI (diphenyleneiodonium) or NADP(+). The plasma membranes from PMA-stimulated cells showed an increased amount of Rac1 (19.6 pmol/mg), as compared with the membranes from unstimulated Caco-2 cells (15.1 pmol/mg), but other components did not change before and after the stimulation by PMA. Spectrophotometric analysis found approx. 36 pmol of FAD and 43 pmol of haem per mg of membrane and the turnover of superoxide generation in a cell-free system consisting of the membrane and FAD was 10 mol/s per mol of haem. When the constitutively active form of Rac, Rac1(Q61L) or GTP-bound Rac1 was added exogenously to the membrane, O(2)(-)-producing activity was enhanced up to 1.5-fold above the basal level, but GDP-loaded Rac1 did not affect superoxide-generating kinetics. A fusion protein [NOXA1N-Rac1(Q61L)] between truncated NOXA1(1-211) and Rac1-(Q61L) exhibited a 6-fold increase of the basal Nox1 activity, but NOXO1N(1-292) [C-terminal truncated NOXO1(1-292)] alone showed little effect on the activity. The activated forms of Rac1 and NOXA1 are essentially involved in Nox1 activation and their interactions might be responsible for regulating the O(2)(-)-producing activity in Caco-2 cells.     

Presynaptic dopaminergic properties of differentiated mouse embryonic stem cells

This study characterized the presynaptic dopaminergic properties of neuronally differentiated mouse embryonic stem (ES) cells. Approximately 30% of the ES cells expressed tyrosine hydroxylase (TH) immunoreactivity when co-cultured with PA6 cells. These cultures expressed high affinity, sodium-dependent dopamine uptake as well as depolarization-induced and calcium-dependent dopamine release of this transmitter. These and other important dopaminergic genes found expressed in these cultures by RT-PCR included Nurr1, vesicular monoamine transporter 2 (VMAT2), TH, dopamine transporter (DAT), and glial cell line-derived neurotrophic factor (GDNF) receptors c-Ret and GFRalpha1. These results demonstrate that differentiated ES cells have the presynaptic functions for maintaining dopaminergic homeostasis, which may be essential for their long-term use in restoring CNS levels of this transmitter.     

Localization of the GLUT1 glucose transporter to brefeldin A-sensitive vesicles of differentiated CIT3 mouse mammary epithelial cells

Glucose is a precursor of lactose, the major carbohydrate and osmotic constituent of human milk, which is synthesized in the Golgi. The GLUT1 glucose transporter is the only glucose transporter isoform expressed in the mammary gland. The hypothesis that lactogenic hormones induce GLUT1 and cause its localization to the Golgi of mammary epithelial cells was tested in CIT(3)mouse mammary epithelial cells. Treatment with prolactin and hydrocortisone caused a 15-fold induction of GLUT1 by Western blotting, but 2-deoxyglucose uptake decreased. Subcellular fractionation and density gradient centrifugation demonstrated enrichment of Golgi fractions with GLUT1. Lactogenic hormones enhanced GLUT1 glycosylation, but did not determine whether GLUT1 was targeted to plasma membrane or to Golgi. Confocal microscopy revealed that lactogenic hormones alter GLUT1 targeting from a plasma membrane pattern to a predominant perinuclear distribution with punctate scattering through the cytoplasm. GLUT1 is targeted to a compartment which is more sensitive to Brefeldin A than the compartments in which GM130 and beta-COP reside. Targeting of GLUT1 to endosomes was specifically excluded. We conclude that prolactin and hydrocortisone induce GLUT1, enhance GLUT1 glycosylation, and cause glycosylation-independent targeting of GLUT1 to Brefeldin A-sensitive vesicles which may represent a subcompartment of cis-Golgi. These results demonstrate a hormonally-regulated targeting mechanism for GLUT1 and are consistent with an important role for GLUT1 in the provision of substrate for lactose synthesis.     

Growth of mouse vaginal epithelial cells in vitro

Summary Pieces of adult mouse vagina (comprising epithelium and connective tissue), when explanted onto glass coverslips, gave rise to outgrowing sheets of pure epithelium whose cells had ultrastructural features in common with the cells of origin in vivo. Epithelial outgrowths from vaginas of estradiol-primed and nonprimed ovariectomized mice were studied. After the first 5 days in vitro, in the absence of estradiol, the labeling index and length of the cell cycle were similar in both types of cultures. The values were similar to those reported by others in vivo in response to estrogen. Thus, proliferative activity of cells from nonprimed mice was stimulated merely by in vitro conditions, while proliferation of cells from primed mice continued at the high level existing prior to explantation. The high rate of proliferation wasnot associated with keratinization of any cells. In the continued absence of estrogen, cells from both kinds of cultures showed a progressive decrease in proliferative activity between 5 and 14 days, also associated with inability of cells to keratinize. Addition of estradiol didnot reverse the mitotic drop or promote keratinization. Supplementation with hydrocortisone and insulin had no effect. The results suggest that (a) vaginal epithelial cells in vitro require factors in addition to estradiol in order to maintain a high level of proliferative activity or to differentiate fully by keratinization and (b) keratinization is not dependent on rate of cell proliferation. Supported by grants from the National Cancer Institute (1 PO 1 CA 11536) and the National Institute of Arthritis and Metabolic Diseases (1 P0 1 AM 15515).