Morphological and functional polarity of an epithelial thyroid cell line

The thyroid epithelial cell line FRT in monolayer culture appeared to be strongly polarized by morphological criteria. Cells were connected by tight junctions, exposed microvilli toward the culture medium and formed domes at confluency. FRT cells were infected with vesicular stomatitis virus (VSV) and Sindbis virus and the budding polarity was examined 8 and 16 h after infection, respectively. VSV budding occurred preferentially from the basolateral domain of plasma membrane, while Sindbis virus budding was mostly apical. The distribution of VSV and Sindbis virus glycoproteins, as determined by the immuno-gold technique, correlated well with the budding polarity. Polarized budding was not observed in isolated cells in suspension.     

Viruses budding from either the apical or the basolateral plasma membrane domain of MDCK cells have unique phospholipid compositions.

Influenza virus and vesicular stomatitis virus (VSV) obtain their lipid envelope by budding through the plasma membrane of infected cells. When monolayers of Madin-Darby canine kidney (MDCK) cells, a polarized epithelial cell line, are infected with fowl plague virus (FPV), an avian influenza virus, or with VSV, new FPV buds through the apical plasma membrane whereas VSV progeny is formed by budding through the basolateral plasma membrane. FPV and VSV were isolated from MDCK host cells prelabeled with [32P]orthophosphate and their phospholipid compositions were compared. Infection was carried out at 31 degrees C to delay cytopathic effects of the virus infection, which lead to depolarization of the cell surface. 32P-labeled FPV was isolated from the culture medium, whereas 32P-labeled VSV was released from below the cell monolayer by scraping the cells from the culture dish 8 h after infection. At this time little VSV was found in the culture medium, indicating that the cells were still polarized. The phospholipid composition of the two viruses was distinctly different. FPV was enriched in phosphatidylethanolamine and phosphatidylserine and VSV in phosphatidylcholine, sphingomyelin, and phosphatidylinositol. When MDCK cells were trypsinized after infection and replated, non-infected control cells attached to reform a confluent monolayer within 4 h, whereas infected cells remained in suspension. FPV and VSV could be isolated from the cells in suspension and under these conditions the phospholipid composition of the two viruses was very similar. We conclude that the two viruses obtain their lipids from the plasma membrane in the same way and that the different phospholipid compositions of the viruses from polarized cells reflect differences in the phospholipid composition of the two plasma membrane domains.     

促甲状腺激素对甲状腺细胞胞内游离钙和钙调蛋白的影响

本文研究TSH和forskolin对原代培养的猪甲状腺细胞[Ca~(2+)]_i和钙调蛋白的影响。结果表明,TSH可引起甲状腺细胞[Ca~(2+)]_1急性升高。此反应是剂量依赖关系,而与细胞外钙的存在与否无关。其反应性在细胞单层高于细胞是液,近汇合细胞单层高于汇合细胞单层。TSH作用3天,可使甲状腺细胞的钙调蛋白含量增高,此作用与TSH对甲状腺细胞数的影响无关。Forskolin对甲状腺细胞的[Ca~(2+)]_i和钙调蛋白均无明显的影响。     

Inhibition of vesicular stomatitis virus replication in dexamethasone-treated L929 cells

We previously demonstrated that dexamethasone treatment of L929 cells inhibited plaque formation by vesicular stomatitis virus (VSV), encephalomyocarditis virus, or vaccinia virus. We now have characterized the antiviral effects of glucocorticoids in L929 cells. Dexamethasone did not directly inactivate VSV nor did steroid treatment of L929 cells affect virion adsorption or penetration. The VSV yield in L929 cells treated with dexamethasone for a period of only 4 or 8 hr was decreased by 50% when cells were infected the day following steroid treatment. Treating L929 cells with dexamethasone for a longer period resulted in greater inhibitions of virus synthesis. Interferon activity (less than 5 units/ml) was not detected in L929 cell culture fluids and cell sonicates from steroid-treated cells and the addition of antiserum to murine alpha/beta-interferon had no effect on the ability of dexamethasone to inhibit VSV replication. Dexamethasone treatment of L929 cells did not induce the production of double-stranded RNA-dependent protein kinase but did result in a slight elevation of 2-5A oligoadenylate synthetase activity, two enzymatic activities associated with the antiviral state induced by interferon. However, the elevated 2-5A synthetase activity was not associated with an inhibition of VSV RNA accumulation in dexamethasone-treated L929 cells. By contrast, the synthesis of all five VSV proteins was reduced by 50-75% in dexamethasone-treated L929 cells as early as 4 hr after infection. Thus, the dexamethasone-mediated inhibition of VSV replication in L929 cells is associated with decreased production of VSV structural proteins.     

Monensin-resistant mouse Balb/3T3 cell mutant with aberrant penetration of vesicular stomatitis virus

A mutant (MO-5) resistant to monensin (an ionophoric antibiotic) derived from the mouse Balb/3T3 cell line, was a poor host for vesicular stomatitis virus (VSV) or semliki forest virus (SFV) multiplication. The yield of VSV particles in MO-5 is one 100-fold reduced as is VSV-dependent RNA synthesis. In contrast to a pH-remedial mutant, the abortive production of infectious VSV particles in MO-5 cells was not restored by low pH treatment. The pH values in the endosome and the lysosome of MO-5 cells were 5.2 and 5.4, respectively, values that were comparable to the pH value in Balb/3T3 cells. Assays with [3H]uridine-labeled VSV indicated similar binding of VSV in MO-5: percoll gradient centrifugation analysis of [35S]methionine-labeled VSV-infected Balb/3T3 showed accumulation of VSV in the lysosome fraction 20 min after VSV infection, whereas VSV can be found mainly in endosome/Golgi fraction of MO-5 cells after 40 to 60 min on the percoll gradients. Degradation of [35S]methionine-labeled VSV was observed at a significant rate in Balb/3T3 cells, but not in MO-5 cells. The monensin-resistant somatic cell may thus provide a genetic route to study the mechanism of endocytosis or transport of enveloped viruses.     

Interactions of vesicular stomatitis virus with murine cell surface antigens.

The process of maturation of vesicular stomatitis virus (VSV) results in the loss of 70% of the H-2k antigenic activity from L-cell plasma membranes. This phenomenon is also demonstrated during VSV infection of cells of the H-2d haplotype. Using the method of inhibition of immune cytolysis, VSV-infected L5178Y tissue culture cells and VSV-infected METH A fibrosarcoma cells grown in vivo show a loss of H-2d activity of 73 and 76%, respectively. Using monospecific antisera, it is seen that VSV infection results in a significant loss of antigenic activity of the gene products of both the H-2D and H-2K regions in cells of the H-2d and H-2k haplotypes. In hybrid cells expressing H-2k as well as H-2b, VSV infection results in the decrease of both H-2 antigenic activities to the same extent. VSV purified from L cells shows considerable H-2k activity, but the reaction of this virus with anti-H-2k serum does not prevent a normal subsequent infection with this virus. VSV may associate with H-2 antigen in the culture medium, but the results of mixing VSV with uninfected H-2-containing homogenates suggest that this association occurs only when the host cell and the cell homogenate share the same H-2 haplotype. Velocity sedimentation of VSV, which would remove contaminating cellular membrane fragments, does not separate H-2 activity from VSV. H-2 activity is also stably associated with VSV throughout sequential sucrose gradient centrifugation steps. It is possible that H-2 antigen is a structural component of VSV grown in murine cells.     

Hexamethylene bisacetamide (HMBA) increases thyroglobulin levels in porcine thyroid cells without increasing cyclic-AMP.

The polar planar compound hexamethylene bisacetamide (HMBA) is an inducer of terminal differentiation which has been extensively studied in the murine erythroleukemia cells (MELC). We have tested this compound in normal porcine thyroid cells in primary culture where it either activates or inhibits the major tissue specific functions of these cells: it induces the reorganization of cells into follicles, prevents the loss of thyrotropin sensitivity in monolayer cells, activates cell growth but inhibits their iodide metabolism. In this paper, we demonstrate that HMBA acts on the total thyroglobulin levels measured in cell layers plus media. This specific marker of thyroid tissue is increased by HMBA both in kinetics and in concentration-response experiments. HMBA per se does not increase the total cyclic AMP measured either during the first hours after stimulation or in the following days when compared to controls. As expected, cyclic AMP in the same experiment increased rapidly within minutes after the cells were challenged by TSH (positive control). Altogether, the results show that the drug HMBA mimics thyrotropin effects on thyroglobulin levels measured in porcine thyroid cells in culture. This modulation cannot be explained by an increase in cyclic AMP, indicating that despite similarities between TSH and HMBA effects, the mechanism of the mode of action of these two molecules is very different.     

Synthesis and infectivity of vesicular stomatitis virus containing nonglycosylated G protein.

The replication of vesicular stomatitis virus (VSV) is inhibited by tunicamycin (TM), an antibiotic that blocks the formation of N-acetylglucosaminelipid intermediates. We had shown previously that the viral glycoprotein (G) synthesized in cells treated with TM is not glycosylated and is not found on the outer surface of the cell plasma membrane. In this report, we shown that cells exposed to TM produce a low yield of infectious particles. The yield is increased when the temperature during infection is lowered from 37 to 30 degrees C. At 30 degrees C in the presence of TM, both wild-type VSV and the temperature-sensitive mutant ts045 produce particles that do not bind to concanavalin A Sepharose and contain only the nonglycosylated form of G. These particles have a specific infectivity (pfu/cpm) comparable to that of VSV containing glycosylated G.     

Effect of monensin on the synthesis, maturation and secretion of vesicular stomatitis virus proteins in a monensin-resistant Chinese hamster ovary cell line

We compared the effects of the cationic ionophore, monensin, on the synthesis, maturation and release of vesicular stomatitis virus (VSV) in cultures of Chinese hamster ovary (CHO) cells and the monensin-resistant clone, MonR-31. Our results depended on the dose and time of the addition of monensin to the infected cells, from 1 h prior to VSV infection to 1 h after infection. VSV production was more resistant in MonR-31 than in CHO cells when the ionophore was added 1 h prior to VSV infection. Monensin added 1 h after VSV infection showed the opposite phenomenon; release of virus particles into the medium was 10- to 10(5)-fold less in MonR-31 cells than in CHO cells, and the intracellular virus number in the resistant cells was one-third to one-fourth of that in the parental CHO cells. Syntheses of all virus-associated G, N and M proteins were inhibited in both cell lines by monensin, but especially so in the MonR-31 cells. There were no marked qualitative changes in the biochemical properties of viral glycoprotein G in virus-infected CHO and MonR-31 cells treated with monensin after virus infection. An endoglycosidase H-resistant G with a molecular weight smaller than that of normal G and attachments of palmitate or fucose on the truncated G protein appeared. Alteration of the secretion of as well as the synthesis of the enveloped virus is discussed in relation to the monensin susceptibility of the resistant MonR-31 clone.     

Differential regulation of thyroid cell-cell and cell-substrate adhesion by thyrotropin

Preservation of cell aggregation is necessary for thyroid follicular differentiation in vitro and requires stimulation by thyrotropin (TSH). We have tested the hypothesis that TSH preferentially increases thyroid cell-cell adhesion relative to cell-substrate adhesion. Cell-cell adhesion was measured in short-term suspension cultures by the decrease in the fraction of single cells remaining in culture (free cell ratio, FCR). When incubated in medium alone freshly isolated cells showed a progressive fall in FCR but this was accelerated by TSH and the cyclic AMP analog, 8-(4-chlorophenylthio)cyclic AMP. Aggregation was dependent upon extracellular Ca2+ and also promoted by a cell-free membrane extract. In contrast, attachment of cells to plastic dishes treated for tissue culture was not affected by TSH. We conclude that thyroid cells possess a TSH-sensitive cell adhesion system. The preferential increase in cell-cell adhesion may be one mechanism by which TSH stimulates the formation and preservation of follicles in vitro.     

Sensitivity of Ribonucleic Acid and Deoxyribonucleic Acid Viruses to Different Species of Interferon in Cell Cultures

Although two deoxyribonucleic acid (DNA) viruses, pseudorabies (PsRV) and vaccinia, are as susceptible as a ribonucleic acid (RNA) virus, vesicular stomatitis (VSV), to interferon when tested in chicken or mouse cells, they are refractory to inhibition in interferon-treated primary rabbit kidney cells and in a continuous line (RK-13) of rabbit kidney cells. Superinfection with VSV of RK-13 cells first infected with PsRV completely blocks the replication of PsRV with no effect on VSV yield. When the same experiment is carried out in RK-13 cells pretreated with 1,000 units of interferon, VSV replication is inhibited, which permits PsRV to replicate normally. These findings demonstrate that in the same cell one virus (PsRV) can be refractory to interferon and a second virus (VSV) can be susceptible. These experiments show that rabbit kidney cell cultures are deficient in the synthesis of resistance factors active against the DNA viruses tested and raise the possibility that separate resistance factors may exist for RNA and DNA viruses. In the case of sequential infection of interferon-treated RK-13 cells with vaccinia and VSV, it was found that not only was vaccinia replication refractory to inhibition by interferon, but also that prior infection with vaccinia was able to partially reverse the effect of the inhibitor on the replication of the VSV used for superinfection. On the basis of these and other data it is postulated that a vaccinia virion component or a replication product of vaccinia virus, or both, enables VSV to escape the inhibiting action of interferoninduced resistance factors.     

Vesicular stomatitis virus growth in Drosophila melanogaster cells: G protein deficiency.

In cultured Drosophila melanogaster cells, vesicular stomatitis virus (VSV) established a persistent, noncytopathic infection. No inhibition of host protein synthesis occurred even though all cells were initially infected. No defective interfering particles were detected, which would explain the establishment of the carrier state. In studies of the time course of viral protein synthesis in Drosophila cells, N, NS, and M viral polypeptides were readily detected within 1 h of infection. The yield of G protein and one of its precursors; G1, was very low at any time of the virus cycle; the released viruses always contained four to five times less G than those produced by chicken embryo cells, whatever the VSV strain or serotype used for infection and whatever the Drosophila cell line used as host. Actinomycin D added to the cells before infection enhanced VSV growth up to eight times. G and G1 synthesis increased much more than that of the other viral proteins when the cells were pretreated with the drug; nevertheless, the released viruses exhibited the same deficiency in G protein as the VSV released from untreated cells. Host cell control on both G-protein maturation process and synthesis at traduction level is discussed in relation to G biological properties.     

Role of heterologous and homologous glycoproteins in phenotypic mixing between Sendai virus and vesicular stomatitis virus.

Phenotypic mixing between Sendai virus and vesicular stomatitis virus (VSV) or the mutant VSV ts045 was studied. Conditions were optimized for double infection, as shown by immunofluorescence microscopy. Virions from double-infected cells were separated by sequential velocity and isopycnic gradient centrifugations. Two types of particles with mixed protein compositions were found. One type was VSV particles with Sendai virus spikes, i.e., phenotypically mixed particles. A second type was Sendai virus-VSV associations, which in plaque assays also behaved as phenotypically mixed particles. The ratio of VSV G protein to Sendai virus glycoproteins on the cell surface was varied, using the VSV mutant ts045 in double infections. Thus, different amounts of the VSV G protein were allowed to reach the cell surface at 32, 38, and 39 degrees C in Sendai virus-infected cells. However, a fixed number of Sendai virus spikes was always found in the ts045 virions. This represented 12 to 16% of the number of G proteins present in normal VSV. Furthermore, the yield of ts045 virions was radically reduced during double infection when the temperature was raised to block G-protein transport to the cell surface, suggesting that the Sendai virus glycoproteins were not able to compensate for G protein in budding. These results emphasize the role of the G protein in VSV assembly.     

Vesicular stomatitis virus oncolysis of T lymphocytes requires cell cycle entry and translation initiation

Vesicular stomatitis virus (VSV) is a candidate oncolytic virus that replicates and induces cell death in cancer cells while sparing normal cells. Although defects in the interferon antiviral response facilitate VSV oncolysis, other host factors, including translational and growth regulatory mechanisms, also appear to influence oncolytic virus activity. We previously demonstrated that VSV infection induces apoptosis in proliferating CD4(+) T lymphocytes from adult T-cell leukemia samples but not in resting T lymphocytes or primary chronic lymphocytic leukemia cells that remain arrested in G(0). Activation of primary CD4(+) T lymphocytes with anti-CD3/CD28 is sufficient to induce VSV replication and cell death in a manner dependent on activation of the MEK1/2, c-Jun NH(2)-terminal kinase, or phosphatidylinositol 3-kinase pathway but not p38. VSV replication is specifically impaired by the cell cycle inhibitor olomoucine or rapamycin, which induces early G(1) arrest, but not by aphidicolin or Taxol, which blocks at the G(1)1S or G(2)1M phase, respectively; this result suggests a requirement for cell cycle entry for efficient VSV replication. The relationship between increased protein translation following G(0)/G(1) transition and VSV permissiveness is highlighted by the absence of mTOR and/or eIF4E phosphorylation whenever VSV replication is impaired. Furthermore, VSV protein production in activated T cells is diminished by small interfering RNA-mediated eIF4E knockdown. These results demonstrate that VSV replication in primary T lymphocytes relies on cell cycle transition from the G(0) phase to the G(1) phase, which is characterized by a sharp increase in ribogenesis and protein synthesis.     

Effects of thyroid-stimulating hormone and phorbol ester on glycosaminoglycan synthesis in porcine thyroid epithelial cells in primary culture

The effects of thyroid-stimulating hormone (TSH) and a tumor promoter: 12-0-tetradecanoyl-phorbol-13-acetate on glycosaminoglycan (GAG) synthesis were studied in porcine thyroid epithelial cells in primary culture. TSH is known to involve cyclic AMP mechanism and phorbol ester to act by protein kinase C pathway. Chronic treatment of cells with TSH increased the synthesis of heparan sulphate associated with the cell layer and hyaluronic acid in the culture medium. Phorbol ester increased the radioactivity of total GAGs in the culture medium but had no effect on GAGs associated with the cell layer. It inhibited the positive effect of TSH on heparan sulphate synthesis. These results suggest that in thyroid epithelial cells the synthesis of the GAGs associated with the cell layer and those secreted into the culture medium are regulated by different intracellular mechanisms.     

N protein is the predominant antigen recognized by vesicular stomatitis virus-specific cytotoxic T cells.

The specificity of anti-vesicular stomatitis virus (VSV)-specific cytotoxic T cells was explored with cell lines expressing VSV genes introduced by electroporation. Low levels of nucleocapsid (N) protein were detected on the surface of VSV-infected cells, but N protein could not be detected on the plasma membrane of transfected EL4 cells. Intracellular N protein was detectable by enzyme-linked immunosorbent assay or immunoprecipitation in some of the transfected cell lines but not in others, unless the transfected genes were induced by sodium butyrate. However, all of the stably transfected EL4 cell lines expressing the VSV-Indiana N protein were efficiently lysed by serotype-specific and cross-reactive anti-VSV cytotoxic T cells (CTLs). Primary cross-reactive anti-VSV CTLs appeared to be specific solely for N protein, based on cold-target competition assays using infected and transfected target cells. Cell lines expressing 100- to 1,000-fold less N protein than did VSV-infected cells were efficiently lysed by both primary and secondary anti-VSV CTLs. Cell lines expressing 100-fold less G protein than did VSV-infected cells were not lysed by either population of effectors. Significantly, cold-target competition studies with secondary CTLs demonstrated that N protein-expressing cell lines were more efficient competitors than were VSV-infected cells even though the latter expressed 100- to 1,000-fold more N protein. This was not an artifact of viral infection since infection of the transfected cell lines did not affect their ability to compete. The possibility that cell lines constitutively expressing internal virus proteins present antigen more effectively than infected cells do is discussed.     

L'hexaméthylène bisacétamide (hmba) supprime la perte de sensibilité des cellules thyroïdiennes monocouches de porc à la thyrotropine: Hexamethylenebisacetamide (HMBA) prevents the loss of TSH sensitivity in porcine thyroid monolayer cells

The polar coumpound hexamethylenebisacetamide (HMBA) is a differentiating agent in the murine erythroleukemia cell system (MELC). It induces, like dimethylsulfoxide, the commitment to terminal differentiation leading to a recovery in the expression of several genes like the globin gene. This molecule which also induces differentiation in other cellular types is a growth agent for human, ovine and porcine thyroid cells. Forty-eight hours after the onset of culture, porcine thyroid monolayer cells do not respond to thyrotropin (TSH). We demonstrate that a pretreatment from the onset of culture with HMBA of porcine thyroid cells prevents the loss of TSH-sensitivity. The TSH-sensitivity is concentration-dependent in HMBA and leads to the reorganization of cells into follicles, even in the presence of HMBA However, the withdrawal of HMBA when TSH is added is absolutely required to obtain a total recovery in iodide trapping and organification. If HMBA is present during TSH-stimulation, it inhibits iodide trapping partially but iodide organification completely. Cells remain sensitive to TSH for at least 12 days if HMBA treated, and their sensitivity is totally restored after 3, 6 or 9 days of TSH-stimulation. HMBA, which is, like TSH, a growth agent for the thyroid cell and an agent that maintains some of the specialized functions, could be a putative candidate to obtain normal human thyroid cell lines.     

Thyrotropin stimulation of cultured thyroid cells increases steady state levels of the messenger ribonucleic acid for thyroid peroxidase

We have examined the effect of TSH on thyroid peroxidase (TPO) mRNA levels in dog thyroid cell primary cultures. Freshly dispersed dog thyroid cells were cultured for up to 5 days in the absence or presence of 5 mU/ml bovine TSH. At the outset of culture, and at daily intervals thereafter, total cytoplasmic RNA was extracted and applied to Nytran paper using a slot-blot apparatus. A nick-translated cDNA fragment of the porcine TPO gene was used to probe these filters. Autoradiographs were quantified by densitometry. Nonspecific binding was negligible as determined using a pUC18 probe. During the first 2 days of culture, TPO mRNA levels declined irrespective of whether or not TSH was present in the medium. TSH did not affect this decline. Between 3 and 5 days of culture, TPO mRNA levels in control (no TSH) cells increased to 3 times the initial level (expressed relative to cellular DNA). However, during the same period TSH stimulated TPO mRNA levels 8-fold above the initial level. To confirm that the signal with the cDNA probe was actually that of dog TPO mRNA, cellular RNA (day 4 of culture) was subjected to Northern blot analysis using the same cDNA probe. Specific bands of 2.9 kilobases were detected corresponding to the known size of TPO mRNA in pig thyroid tissue. The signal of this 2.9 kilobase species was enhanced by TSH. In conclusion, the data indicate that chronic TSH stimulation raises steady state levels of TPO mRNA and provide an explanation, at least in part, for the mechanism by which TSH enhances TPO bioactivity in thyroid tissue.     

An alpha-subunit-secreting cell line derived from a mouse thyrotrope tumor.

The anterior pituitary contains multiple distinct endocrine cell types that secrete individual hormones. To derive a pure cell culture population in which to study the regulation of the alpha-subunit of TSH free of other hormones and cell types, we have developed a clonal continuous cell line from the transplantable thyrotrope tumor MGH101A. This cell line expresses alpha-subunit mRNA, secretes alpha-subunit protein, and has maintained a stable phenotype for over 3 yr in culture. However, as is the case for the transplantable tumor from which they are derived, these cells do not express the beta-subunit of TSH or respond to TRH or thyroid hormone. We have used this cell line to investigate regulation of the alpha-subunit mRNA by the second messengers, cAMP and phorbol esters, and by glucocorticoids. Phorbol esters increase alpha-subunit mRNA levels significantly (3.5-fold), as does cAMP (1.8-fold). In contrast, glucocorticoids decrease mRNA levels from cAMP-induced or basal levels (2-fold). These cells should prove valuable for study of alpha-subunit gene expression in an isolated renewable clonal cell culture system.