Cell electrophoretic mobility and cationic flux

The net negativity of the surfaces of Ehrlich ascites cells was reduced by treating them with either neuraminidase or ribonuclease. Neither enzyme treatment affected the Na⁺ or K⁺ content of the cells, before or after cooling at 4°C. Experiments with K⁴² revealed a reduction (9.5 to 17%) in unidirectional K⁺-fluxes following incubation with neuraminidase, but no change after ribonuclease-treatment. Our data suggest that surface anionic sites associated with RNA and sialic acid moieties are not of major quantitative importance in regulating either intracellular Na⁺ and K⁺ concentrations, or unidirectional transmembrane K⁺-flux. Our results do not enable us to determine whether ion-binding to anionic sites at the electro-kinetic surface is not an essential prerequisite to transmembrane movement, or whether it is essential, but occurs through the 40% of cell surface net negativity which is unaffected by ribonuclease- and neuraminidase-treatment.     

Molecular and functional analysis of glutamine uptake in human hepatoma and liver-derived cells

Human hepatoma cells take up glutamine at rates severalfold faster than the system N-mediated transport rates observed in normal human hepatocytes. Amino acid inhibition, kinetic, Northern blotting, RT-PCR, and restriction enzyme analyses collectively identified the transporter responsible in six human hepatoma cell lines as amino acid transporter B(0) (ATB(0)), the human ortholog of rodent ASCT2. The majority of glutamine uptake in liver fibroblasts and an immortalized human liver epithelial cell line (THLE-5B) was also mediated by ATB(0). The 2.9-kb ATB(0) mRNA was equally expressed in all cell lines, whereas expression of the system A transporters ATA2 and ATA3 was variable. In contrast, the system N isoforms (SN1 and SN2) were expressed only in well-differentiated hepatomas. ATB(0) mRNA was also expressed in cirrhotic liver and adult and pediatric liver cancer biopsies but was not detectable in isolated human hepatocytes or fetal liver. Although the growth of all hepatomas was glutamine dependent, competitive inhibition of ATB(0)-mediated glutamine uptake blocked proliferation only in poorly differentiated cells lacking SN1 or SN2 expression and exhibiting low glutamine synthetase mRNA levels.     

The hypoxanthine transporter of Novikoff rat hepatoma cells exhibits directional symmetry and equal mobility when empty or substrate-loaded

The kinetics of hypoxanthine transport were measured in hypoxanthine phosphoribosyltransferase-deficient Novikoff cells by rapid kinetic techniques applying both zero-trans and equilibrium exchange protocols. The data indicate operation of a simple carrier with directional symmetry and equal mobility when substrate loaded and empty. Zero-trans influx and efflux were about equivalent and so were zero-trans influx and equilibrium exchange flux. The apparent Michaelis-Menten constant and maximum velocity were about 500 μM and 100 pmol/s per μl cell H₂O, respectively. The time courses of accumulation of radioactively labeled hypoxanthine at a concentration above the Michaelis-Menten constant differed noticeably in zero-trans and equilibrium exchange mode, but computer simulations showed that the difference is predicted by the symmetrical carrier model and does not reflect trans-stimulation.     

Molecular mobility of soluble collagen

Conformation structure of soluble collagen in anhydrous form, films and gels was studied by broad line NMR. An analysis of spectra points to partial ordering of polymer chains in the films and possible formation of secondary structure of collagen molecules by alpha-helix type. Distinction of gel spectra from those of films is explained by unordered rotation movements of the chain fragments at the expense of "superspiralization" of collagen molecules.     

Active taurocholic acid flux through hepatoma cells increases the cellular pool of unesterified cholesterol derived from lipoproteins

The effect of bile acid flux on the fate of lipoprotein-derived cholesterol was studied in bile acid-transporting McNtcp.18 hepatoma cells. The intracellular unesterified cholesterol (UC) concentration rose when McNtcp.18 cells grown in the presence of either high density lipoproteins (HDL) or low density lipoproteins (LDL) were incubated with taurocholic acid (TCA). This effect was more pronounced when the exogenous source of cholesterol was HDL. The presence of TCA in the culture medium of McNtcp.18 cells had no discernible effect on the uptake of cholesteryl esters (CE) from either lipoprotein. TCA treatment of cells preincubated with either lipoprotein did not affect cholesterol synthesis but antagonized the stimulation of cholesterol esterification in cells that were incubated with LDL. The CE concentration in cells treated with TCA was decreased, relative to cells not incubated with TCA, suggesting that cellular CE stores were also hydrolyzed. The TCA treatment reduced the amount of total cholesterol released into the medium by the lipoprotein-treated cells, which was coincident with the reduction in the amount of apolipoprotein B in the culture medium. However, the proportion of UC released into the medium by the lipoprotein-treated cells was increased in cells capable of active bile acid transport. The results indicate that active bile acid flux through hepatoma cells increases the cellular pool of UC derived from lipoproteins. The UC released by the cells into the culture medium under this condition may represent cholesterol destined for direct biliary secretion.     

固脾消积饮诱导人肝癌HepG2细胞凋亡的分子机制*

目的: 探讨固脾消积饮对人肝癌细胞HepG2凋亡的分子机制。方法: 将HepG2细胞分为4组:对照组(Control)、空白血清组(Blank)、固脾消积饮血清组(GPXJY)和顺铂组(Positive),每组设置8个复孔。固脾消积饮含药血清和顺铂干预24 h后,检测细胞活性、活细胞数量、细胞凋亡状态、细胞周期以及线粒体膜电位状况,检测细胞的脂质过氧化(MDA)水平、糖酵解速率和凋亡Bax、Bcl-2、Caspase-3蛋白的表达,检测细胞上清液三酰甘油(TG)、胆固醇(TC)、丙酮酸和葡萄糖的含量。结果: 与Control组比较,GPXJY组(细胞的)抑制率增加、细胞数量减少、凋亡阳性细胞数增多(P＜0.01),G1期细胞数目显著增加(P＜0.05),细胞线粒体膜电位降低(P＜0.01),糖酵解功能显著抑制,细胞中MDA水平升高,细胞Bax、Caspase-3的表达升高、Bcl-2的表达下降(P＜0.05,P＜0.01),细胞上清液中TC、TG、葡萄糖含量显著减少、丙酮酸含量显著增加(P＜0.05,P＜0.01)。结论: 固脾消积饮可诱导HepG2细胞发生凋亡,可能对能量代谢发挥作用。     

Zajdela hepatoma cells cultured in vitro

The goal of this work was to study cellular mechanisms of tumor progression and metastasizing. As a result of explantation of cells of rat Zajdela ascitic hepatoma, we obtained two transplantable cell cultures—monolayer (ZH-ad) and suspension (ZH-fl)—that differ in levels of cell differentiation and tumorigenicity. By using tumor-specific immune serum, we revealed tumor-associated antigens, synthesis of which is reduced or inhibited in ZH-ad cells, in outer membranes of the ZH-fl cells. Intraperitoneal injection into rat of 0.5–12 × 10⁶ ZH-fl cells leads to development of an ascitic tumor and death of 100% of animals, whereas, in the case of administration of ZH-ad cells, to achieve a tumorigenic effect, the minimal dose needs to be elevated to 20 × 10⁶ cells. Clonogenic analysis of the ZH-fl cells revealed three types of the formed clones—nonadhesive sphere colonies and two types of monolayer clones differing in proliferative potential, shape of colonies, and cell composition. Upon reaching a critical size, the spheres disintegrated, with separation of single cells and islands of different sizes, some of them being attached with monolayer formation. Three clonal cell lines were obtained: 1C as a result of expansion of a spherical clone and 4G and 10E from monolayer clones. We established that there is tumorigenicity of the 1C cell line, which, at a dose of 107 cells, led to the development of ascites and to the death of 50% of animals. The presented results indicate the existence in the ZH-fl cell population of tumor-initiating cells generating spherical clones—floating multicellular islets that, in culturing in the complete growth medium, are partly differentiated and are attached with monolayer formation.     

Reversibly permeable hepatoma cells in culture

A brief treatment of H35 hepatoma cells with lysolecithin resulted in a cell population which is permeable to low-molecular weight charged molecules that cannot normally cross the plasma membrane. These include deoxynucleotide and nucleotide triphosphates, folyl and methotrexate polyglutamates, and trypan blue. As a result dTTP can be incorporated into the DNA of the permeable cells, providing the required nucleotides and deoxynucleotides are added to the medium. This result, combined with only a slight observed loss (20–25%) in total cell protein, lactate dehydrogenase (EC 1.1.1.27) activity and tyrosine aminotransferase (EC 2.6.1.5) activity, demonstrated that permeation of the cells does not extensively disrupt membrane integrity. Further support for this view comes from the fact that the permeable cells could seal when placed in enriched medium. The process of sealing was inhibited by cycloheximide and tunicamycin. The sealed cells, whose surfaces appeared identical to those of untreated cells by scanning electron microscopy, were fully capable of cell division when exposed to serum. Values for several other parameters, including dexamethasone-dependent tyrosine aminotransferase induction, thymidine incorporation into DNA, leucine incorporation into protein and folate coenzyme transport, supported the conclusion that sealed cells and untreated H35 cells have identical properties. Based on the characteristics of the permeable and sealed H35 cells, a discussion of the experimental potential of these preparations for studying macromolecular synthesis, investigating enzymes in situ and depleting cells of folate coenzymes is presented.     

Molecular cloning of an amino acid-regulated mRNA (amino acid starvation-induced) in rat hepatoma cells

Using the combination of a subtracted library and differential hybridization, a 409-base pair cDNA was identified that corresponds to a mRNA that is induced 2-3-fold when rat Fao hepatoma cells are subjected to amino acid starvation for 12 h. While this mRNA species was induced during starvation, others such as beta-actin, Cu-Zn superoxide dismutase, glyceraldehyde-3-P, and histone H4 were decreased in abundance to 25-50% of their original levels. The induction of the amino acid starvation-induced (ASI) mRNA was repressed when starved cells were returned to a medium supplemented with amino acids. Tissue distribution analysis showed the ASI mRNA, approximately 650 base pairs in length, to be present in every rat tissue tested. The cDNA clone has been sequenced and appears to correspond to the 3'-most end of the mRNA. The cDNA sequence includes the poly(A) tail, two potential polyadenylation signal sequences, and an open reading frame that we presume to be a portion of the coding sequence. The ASI cDNA will be used to investigate the molecular mechanisms for amino acid-dependent regulation of protein expression by mammalian cells.     

Nucleo-cytoplasmic flux and intracellular mobility in single hepatocytes measured by fluorescence microphotolysis.

Fluorescence microphotolysis was used to measure nucleocytoplasmic flux in single rat hepatocytes for a series of dextrans ranging in molecular mass from 3 to 150 kd. The cytoplasmic translational diffusion coefficient DC and the nucleoplasmic diffusion coefficient DN of a 62-kd dextran were also determined. DC was approximately 2 X 10(-8) and DN approximately 3 X 10(-8) cm2/s, i.e., 1/20-1/15 of the value in free solution. The mobile fraction amounted to 0.7-0.8 in measurements of both intracellular diffusion and nucleo-cytoplasmic flux. The flux of dextrans from cytoplasm to nucleus depended inversely on molecular mass with an exclusion limit between 17 and 41 kd suggesting that the nuclear envelope has functions of a molecular sieve. Employing the Pappenheimer-Renkin equations, a functional pore radius of 50-56 A was derived. By comparison with recent measurements on isolated liver cell nuclei, large quantitative differences between the intracellularly located and the isolated nucleus were revealed.     

Molecular determinants of glucocorticoid receptor mobility in living cells: the importance of ligand affinity

The actions of glucocorticoids are mediated by the glucocorticoid receptor (GR), which is activated upon ligand binding, and can alter the expression of target genes either by transrepression or transactivation. We have applied FRAP (fluorescence recovery after photobleaching) to quantitatively assess the mobility of the yellow fluorescent protein (YFP)-tagged human GR alpha-isoform (hGRalpha) in the nucleus of transiently transfected COS-1 cells and to elucidate determinants of its mobility. Addition of the high-affinity agonist dexamethasone markedly decreases the mobility of the receptor in a concentration-dependent manner, whereas low-affinity ligands like corticosterone decrease the mobility to a much lesser extent. Analysis of other hGRalpha ligands differing in affinity suggests that it is the affinity of the ligand that is a major determinant of the decrease in mobility. Similar results were observed for two hGRalpha antagonists, the low-affinity antagonist ZK98299 and the high-affinity antagonist RU486. The effect of ligand affinity on mobility was confirmed with the hGRalpha mutant Q642V, which has an altered affinity for triamcinolone acetonide, dexamethasone, and corticosterone. Analysis of hGRalpha deletion mutants indicates that both the DNA-binding domain and the ligand-binding domain of the receptor are required for a maximal ligand-induced decrease in receptor mobility. Interestingly, the mobility of transfected hGRalpha differs among cell types. Finally, the proteasome inhibitor MG132 immobilizes a subpopulation of unliganded receptors, via a mechanism requiring the DNA-binding domain and the N-terminal part of the ligand-binding domain. Ligand binding makes the GR resistant to the immobilizing effect of MG132, and this effect depends on the affinity of the ligand. Our data suggest that ligand binding induces a conformational change of the receptor which is dependent on the affinity of the ligand. This altered conformation decreases the mobility of the receptor, probably by targeting the receptor to relatively immobile nuclear domains with which it transiently associates. In addition, this conformational change blocks immobilization of the receptor by MG132.     

Vitamin K uptake in hepatocytes and hepatoma cells

Hepatocellular carcinoma (HCC) or hepatoma cells have impaired ability to perform vitamin K-dependent carboxylation reactions. Vitamin K can also inhibit growth of HCC cells in vitro. Both carboxylation and growth inhibition are vitamin K dose dependent. We used rat hepatocytes, a vitamin K-growth sensitive (MH7777) and a vitamin K-growth resistant (H4IIE) rat hepatoma cell line to examine vitamin K uptake and vitamin K-mediated microsomal carboxylation. We found that vitamin K is taken up by normal rat hepatocytes against a saturable concentration gradient. The relative rates of uptake by rat hepatocytes and the two rat cell lines MH7777 and H4IIE correlated with their sensitivity to vitamin K-mediated cell growth inhibition. Pooled hepatocytes from liver nodules from rats treated with the hepatocarcinogen diethylnitrosamine (DEN) also had a reduced rate of vitamin K uptake. However, using a cell-free system, microsomes from both normal rat hepatocytes and the two rat hepatoma cell lines had a similar ability to support carboxylation mediated by exogenously added vitamin K. The results support the hypothesis that different sensitivity of hepatoma cells to vitamin K may be due to differences in vitamin K uptake and may be unrelated to the actions of vitamin K on carboxylation.