Interference with tolerance induction of primed B cells by the Fc fragment of immunoglobulin

The capacity to interfere with tolerance induction in primed B cells was examined. Previous work had shown that TNP-specific splenic B cells from mice primed and boosted with TNP-KLH are highly susceptible to in vitro tolerization upon a brief exposure to TNP on a carrier unrelated to KLH. In the present work it was found that tolerance induction in these primed B cells could be partially disrupted by addition of the Fc fragment of immunoglobulin, a B-cell mitogen, and adjuvant, during exposure of the B cells to tolerogen. Addition of Fc fragments prepared by papain digestion of human IgG interfered with tolerization routinely in approximately 30-60% of the spleen cells susceptible to tolerogen. Addition of whole IgG or Fab fragments had no effect on tolerance induction. As little as 5 micrograms/ml of the Fc fragment preparation significantly interfered with tolerization and 32-64 micrograms/ml was optimal. Disruption of tolerization was most effective when the Fc fragment was added to the spleen cells either 4 hr prior to tolerogen or simultaneously with tolerogen; addition of the Fc fragment 4 hr after exposure to tolerogen was significantly less effective. Disruption of tolerization by the Fc fragment was not through polyclonal activation of B cells, as antigen was required for generation of significant numbers of PFC to TNP. Also, disruption was not through expansion of low avidity clones of B cells insusceptible to tolerogen, as the avidity of the antibody produced with and without Fc fragments present was approximately the same. These results show that the Fc fragment of IgG can partially interfere with tolerization of primed B cells. The manner in which Fc fragments may function to prevent tolerization through its lymphoid cell stimulatory capacities is discussed.     

Changes in endogenous cell surface galactosyltransferase activity during in vitro limb bud chondrogenesis

When the subridge mesoderm of the embryonic chick limb bud is cultured in the absence of the apical ectodermal ridge and adjacent ectoderm, the cells rapidly progress through the various stages of chondrogenesis. During the first day of culture, the cells initiate condensation, and during subsequent days, deposit a cartilage matrix. In the present study, we show that early in the first day there is a progressive 2-fold increase in cell surface galactosyltransferase activity towards endogenous acceptors. Later in the first day, although the cells are still in condensation, endogenous galactosyltransferase activity has decreased, suggesting in situ galactosylation of surface acceptors. During subsequent development, when cartilage matrix is being deposited, surface galactosyltransferase activity remains low. All controls have been performed to insure cell surface localization of enzyme activity. Two other surface glycosyltransferases show very low levels of activity, which do not change significantly during culture. We suggest that during cellular condensation, an interaction between surface galactosyltransferases and acceptors on adjacent cells occurs, and this interaction may be causally related to subsequent chondrogenic differentiation.     

An inhibitory effect of tumour promoters on human epithelial cell growth can be dissociated from an effect on junctional communication

Studies with rodent cells have indicated that the abilities of various tumour promoters to inhibit metabolic cooperation correlate with their potencies as mitogens. Here we have examined the effects of the most potent phorbol ester tumour promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), on metabolic cooperation and growth of human epidermal cells transformed by SV40 (SVK14 cells). In this system, TPA inhibits Junctional communication and at the same concentration also inhibits growth in a reversible fashion. These effects appear to be mediated by binding of phorbol ester to a single class of high affinity binding site with a Kd similar to that reported for rodent cells (K_d = 20.9 nM at 4 °C). Further studies on the effects of phorbol esters on other human epithelial cell lines reveal that the inhibitory effects of TPA on growth and metabolic cooperation may be completely dissociated. Alternative mechanisms by which TPA may exert its growth-inhibitory effects are discussed.     

The effects of cyclosporins on the cell cycle of T-lymphoid cell lines

Cyclosporin A (CsA) is a cyclic endecapeptide of fungal origin displaying strong immunosuppressive properties. CsA and another active member of the cyclosporin (Cs) family, but not an inactive one, can interfere with the proliferation of some, but not all, T-lymphoid cell lines. Cells from Cs-sensitive lines accumulate in the G1 phase of the cell cycle. No effect is detected on the cycle of Cs-resistant lines. Both Cs-sensitive and Cs-resistant lines are arrested by another G1 blocker (actinomycin D) and DNA synthesis inhibitors (cytosine arabinoside, hydroxyurea), become multinucleated/polyploid when exposed to cytochalasin B (CB), are arrested in mitosis by colchicine and accumulate in G2 phase in the presence of Taxol. The effect of Cs is best evidenced when the drug is applied to cells which were already delayed in G1 by saturation density cultivation or serum deprivation. By the combined use of Cs and of other drugs working at a later phase of the cycle, results were obtained which suggest that the effect of Cs is either to delay very much the cells throughout the G1 phase or to arrest them at that G1 phase or at the following one. A correlation of the G1-blocking property of Cs with their immunosuppressive properties may be possible but is still speculative.     

The effect of diabetes on hepatocyte plasma membrane fluidity and concanavalin A-induced agglutination

The techniques of fluorescence polarization and lectin-induced agglutination have been utilized to investigate the effects of the diabetic state on some of the dynamic properties of cell membranes. Hepatocyte plasma membranes from streptozotocin-induced diabetic rats exhibited a significant decrease in cholesterol and sialic acid with no alteration in phospholipid content. This membrane system also exhibited a decrease in fluorescence polarization, using the fluorescent probe, 1,6-di-phenyl-1, 3,5-hexatriene, suggesting an increase in membrane fluidity over the value observed in normal hepatocytes. When normal hepatocytes were incubated in the presence of the lectin, concanavalin A (ConA), no significant agglutination was observed. In contrast, hepatocytes from diabetic rats which exhibited a slightly decreased lectin-binding capacity underwent extensive agglutination. In addition, normal hepatocytes which were pretreated with 0.1 mM tetracaine also underwent extensive agglutination with no measurable increase in lectin-binding capacity. These results suggest that altered membrane lipid fluidity and/or cytoskeletal organization may have a profound effect on cell surface dynamics and could result in the uncoupling of the insulin receptor complex from the membrane-associated effector system(s), a defect which may play a role in the problem of insulin resistance observed in some forms of diabetes.     

The distribution of tau and HMW microtubule-associated proteins in different cell types

To investigate the distribution of the tau and HMW microtubule-associated proteins (MAPS) and their relationship to microtubules in vivo, we have examined a wide variety of avian and mammalian cell types by immunofluorescence with antisera to these two proteins. Anti-HMW serum stains cytoplasmic microtubules in all mammalian cell types so far examined. However, anti-tau serum did not stain cytoplasmic microtubules in rat glial cells or in pig kidney cells. In mammalian neurons, fibroblasts and neuroblastoma cells, the staining of microtubules with both sera was similar. Anti-HMW serum did not stain primary cilia or cilia on isolated tracheal epithelial cells, whereas anti-tau serum did stain these ciliary microtubules. We believe these results indicate that some types of microtubules may be associated with only the tau or the HMW protein, whereas others may be associated with both tau and HMW protein. With respect to avian cells, anti-HMW serum did not stain microtubules in any of the three cell types examined, whereas the anti-tau serum stained them in two cell types. Furthermore, double diffusion tests indicated that anti-pig tau serum will precipitate both pig brain tau and tau protein isolated from chick brain, whereas anti-HMW serum will precipitate only pig brain and not chick brain HMW protein. We believe tau protein is antigenically similar in both avian and mammalian cells, whereas the HMW protein from these two sources is antigenically distinct.     

Doubling of cell mass is not necessary in order to achieve cell division in cultured human cells

When exponentially growing NHIK 3025 cells were shifted from medium containing 30% serum to medium containing 0.03% serum the rate of net protein accumulation was reduced due to both a reduction in the rate of protein synthesis and an increase in the rate of protein degradation. This change in growth conditions increased the protein doubling time from 18 to 140 h. The cell cycle duration of cells synchronized by mitotic selection was, however, only increased from 17 to 26 h by this treatment. Therefore, when the cells divide by the end of the first cell cycle following synchronization, the cells shifted to 0.03% serum contained far less protein than those growing continuously in 30% serum. Hence, the attainment of a critical cell mass is probably not controlling cell division for cells growing in a balanced state.     

The effect of serum on the calcium content of BALB/c 3T3 mouse cells

The 5'-termini of purified rat liver nucleolar and cytoplasmic 28S ribosomal RNA (rRNA) are precisely located within the homologous rDNA sequence by S1 nuclease protection mapping using an appropriate rDNA restriction fragment. The 5'-termini of nucleolar 28S rRNA are heterogeneous in length. The bulk of the nucleolar 28S rRNA map within two CTC motifs in rDNA located in the internal transcribed spacer 2 at the 50-60 and 5-15 bp upstream from the site of the homogeneous 5'-terminus of the cytoplasmic 28S rRNA. These results provide direct proof that nucleolar 28S rRNA molecules contain excess sequences at their 5'-termini and require further processing to generate the mature cytoplasmic 28S rRNA.     

Effects of 3-aminobenzamide on DNA synthesis and cell cycle progression in Chinese hamster ovary cells

3-Aminobenzamide (3AB), an inhibitor of poly(ADP-ribose) polymerase, is a potent inducer of sister chromatid exchanges (SCEs). Because of the possible relation between SCEs and DNA synthesis, the effects of 3AB on DNA synthesis and cell cycle progression in Chinese hamster ovary (CHO) cells were examined. Unlike all other SCE-inducing agents whose effects on DNA synthesis have been studied, short term exposures (30–120 min) of 3AB did not inhibit the overall rate of DNA synthesis and this result was independent of the amount of bromodeoxyuridine (BrdU) in the DNA. Longer exposure times (>24 h) did result in an extended S phase, but this was not due to an effect on the rate of DNA chain elongation. 3AB also delayed the entry of cells into S phase. The overall cell cycle delay was dose dependent, approaching 9 h after a 54 h exposure to 10 mM 3AB. Earlier reports that 3AB is neither mutagenic nor cytotoxic were confirmed. Thus 3AB acts to increase SCE frequency by a mechanism distinct from that which causes cytotoxicity and mutagenicity, and does not involve any inhibition in the rate of DNA chain growth.     

Comparative study of the cellular replication kinetics of rat mammary epithelial cells in vivo and in vitro

Ts-131b, one of the temperature-sensitive (ts) mutants isolated from mouse FM3A cells, was found to be defective in DNA replication at a non-permissive temperature. After the cells were transferred to 39.5 °C, the cell number increased by only 10% and the rate of incorporation of precursors into cellular DNA decreased rapidly. Cell cycle analysis by a flow cytometric method with the cells incubated at 39.5 °C revealed that progression of the cells through the S phase was inhibited and most of the cells were arrested in the S phase. To study the defect in DNA replication of this ts-mutant at 39.5 °C, DNA-fiber autoradiography was performed to measure the rate of DNA-chain elongation. The results showed that the rate of DNA-chain elongation was decreased at 6 h after the temperature shift. However, since the decrease in the rate of DNA-chain elongation was not sufficient to account for the decrease in the rate of incorporation of the precursors, it was suggested that there was also a decrease in the rate of initiation of DNA replication at some of the replicon origins.     

The culture of chick embryo chondrocytes and the control of their differentiated functions in vitro. I. Characterization of the chondrocyte-specific phenotypes

We have maintained chick embryo chondrocytes in culture for more than 2 months, passaging the floating cells in the absence of ascorbic acid. Throughout the culture period some of the cells attached to the dish, assuming an epithelial-like morphology and subsequently giving rise to new floating cells. The interconversion of the two cell populations was highest in primaries and decreased with the aging of the culture. Cartilage cells synthesized pro-alpha 1 (II) collagen and sulphated proteoglycans in vitro; compared with floaters, the epithelial-like cells secreted relatively large amounts of fibronectin. When ascorbic acid was added to the medium, all cells attached, maintaining their rounded shape; in this condition the pro-alpha, (II) collagen was matured and collagen fibres were detectable outside the cells. Other specific proteins synthesized by the chondrocytes in culture were also identified. One of these, a 64 K collagenase-sensitive protein, was not related to the type II collagen and may represent a new collagen type.     

Effect of n-butyrate on cell cycle progression and in situ chromatin structure of L1210 cells

In the presence of 1–5 mM n-butyrate, murine leukemic L1210 cells cease proliferation and become arrested in the G1A compartment of the G1 phase. Cells in this compartment, in comparison with the remaining cells of the G1 phase (G1B), are characterized by low RNA content and more condensed chromatin. During unperturbed growth the cell residence times in G1A are of indeterminate duration (exponentially distributed); the half-time of L1210 cell residence in G1A is about 1.4 h. The effect of n-butyrate in arresting cells in G1A was concentration-dependent. However, the sensitivity of L1210 cells to this drug was markedly enhanced when cells were treated for longer than one generation (12 h). Cells arrested in G1A remained viable and when n-butyrate was removed, after a lag period, they resumed progression through the cycle.The effect of n-butyrate on cell progression through various parts of the cycle was studied in a stathmokinetic experiment. The rate of cell entrance into mitosis was decreased by 30, 60 and 110%, in the presence of 1, 2.5 and 5 mM n-butyrate respectively, thus indicating a slowdown in cell progression through G2 and S. The duration of G2 was prolonged by 20, 70 and 140% at 1, 2.5 and 5 mM n-butyrate respectively. The half-time of cell residence in G1A was increased by as much as 1.5-, 6.3- and 15.6-fold by 1, 2.5 and 5 mM n-butyrate. Progression through late G1 (G1B) was not affected at 1 mM, and could not be estimated at higher drug concentrations. The effects on cell cycle progression were evident 1 h after addition of n-butyrate.DNA in situ in nuclei of n-butyrate-treated cells had lowered (by 2–8 °C) stability to thermal denaturation and increased (by 15%) accessibility to DNase I. The decrease in DNA stability to heat was more pronounced when permealized cells were heated in the presence of 1 mM MgCl₂ rather than EDTA. DNA in situ in the nuclei of n-butyrate-treated cells also showed decreased sensitivity to acid-induced denaturation. Changes in chromatin were seen in all cells, regardless of cell cycle phase, within the first hours after addition of n-butyrate. Mitotic cells, however, reacted to n-butyrate more rapidly than interphase cells. The observed changes in L1210 cells are most likely a consequence of histone modifications (acetylation of inner histones, dephosphorylation of histone H1) induced by n-butyrate.     

Retinoic acid-induced modifications in the growth and cell surface components of a human carcinoma (HeLa) cell line

The addition of retinoic acid to cultures of HeLa-S3 cells caused a reduction in cell proliferation rate which became apparent after 72 h and was linearly dependent on retinoic acid concentration in the range 10⁻⁹–10⁻⁵ M. After 72 h of exposure to retinoic acid, the cells assumed a flattened appearance and no longer formed multilayers. These changes were reversed within 48 h after removal of retinoic acid from the medium. Structural analogs of retinoic acid with a free ---COOH group at C-15 were usually more potent in growth inhibition than compounds with an alcohol, aldehyde, ether or ester group. A cellular retinoic acid-binding protein was detected in cell homogenates, and the binding of [³H]retinoic acid to the binding protein was inhibited by most, but not all, analogs possessing a free terminal ---COOH group. For example, the 4-oxo analog of retinoic acid, while capable of inhibiting cellular proliferation, failed to bind to the retinoic acid-binding protein. Analysis of cell surface and cellular glycoproteins by lactoperoxidase-catalysed ¹²⁵I iodination and by metabolic labeling with [³H]glucosamine revealed that a 190000 D glycoprotein which was labeled by both methods and a 230000 D glycoprotein which was labeled only with [³H]glucosamine were labeled more intensely in retinoic acid-treated cells compared with untreated cells. The electrophoretic mobility of the 230000 D glycoprotein could be modified by treatment of intact cells with either neuraminidase or proteolytic enzymes, suggesting that this glycoprotein is also exposed on the cell surface. The cell surface alterations were detected much earlier than the onset of growth inhibition and appeared as early as 24 h after exposure to retinoic acid. The possible relationship between retinoic acid-induced changes in cell membrane structure, cell morphology, and cell proliferation is discussed.     

Expression and extinction of glial properties in glioma X neuroblastoma cell hybrids

Rat glioma cells (clone C₆TK⁻) were hybridized with mouse neuroblastoma cells (clone NA), and 18 primary and secondary hybrid clones containing one chromosome set from each parent were isolated. The hybrids were assayed for the glial marker enzymes 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNP) and glycerol-3-phosphate dehydrogenase (GPDH). In many of the hybrid clones, the levels of CNP and GPDH were reduced to 5–20% of the activity of C₆TK⁻, as has been observed in other classes of glial X non-glial cell hybrids. In some hybrid clones, however, GPDH and CNP were expressed at high activity. Rat (glial) GPDH activity was not reduced in these clones, but mouse GPDH activity remained low, and was not “de-repressed” or “activated”. This suggests that the controls governing differentiation in neuroblastoma cells and extinction in hybrids may differ in some important details. There was a strong positive correlation between the specific activities of CNP and GPDH in the hybrid clones, suggesting that a mechanism regulates the activity of these two glial enzymes coordinately.     

The tumor promoter, 12-O-tetradecanoylphorbol-13-acetate accelerates keratinocyte differentiation and stimulates growth of an unidentified cell type in cultured human epidermis

Studies were conducted to determine the effects of the mouse skin tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) on cultured human epidermal cells for comparison with known effects on mouse keratinocytes. In contrast to its effect on mouse cells, TPA did not stimulate human epidermal cell DNA synthesis. TPA stimulated differentiation in human keratinocytes resulting in sloughing of many cells by the 3rd day after exposure. Quantitative assays revealed that 50% of the TPA-exposed population was composed of cornified cells as opposed to 8% in untreated controls. A morphologically distinct cell type (TT cell) emerged after TPA treatment which was triangular in shape, did not stratify, appeared to proliferate rapidly and at most TPA concentrations became the predominant cell type within 1–2 weeks. Cultures composed predominantly of TT cells formed few cornified envelopes, grew well in the absence of TPA and formed colonies at low cell input. In contrast to its effect on keratinocytes, TPA enhanced TT colony formation 3–4-fold and decreased the doubling time of TT cells. Studies were performed to determine the origin of TT cells. Immunofluorescent staining indicated that TT cells lacked the keratinocyte antigens keratin, pemphigus and pemphigoid. Tonofilaments and desmosomes were not seen by electron microscopy. The lack of both melanosomes and standard histochemical DOPA oxidase staining indicated that TT cells were probably not of melanocyte origin. Tests used to identify Langerhans cells were negative. Whereas TT cells, as well as dermal fibroblasts, yielded positive immunofluorescence with antibodies to vimentin, TT cells gave a weak histochemical leucine aminopeptidase reaction, while the reaction of fibroblasts exposed to TPA was strong. Treatment of human dermal fibroblasts with TPA did not yield TT cells. The endothelial cell antigen factor VIII-associated protein was absent by immunofluorescence. These results suggest that the primary effect of TPA on cultured human epidermis is to accelerate terminal differentiation in the keratinocyte population and to stimulate growth of an as yet unidentified cell type.     

Adult human retinal cells in culture. Identification of cell types and expression of differentiated properties

A method for culturing adult mammalian retinal neurons in serum-free N2 medium supplemented with nerve growth factor (NGF) is described. Identification of neurons in cultures of dispersed human retina was based upon morphology, immunocytochemical localization of bound tetanus toxin, and autoradiographic localization of 3H-neurotransmitter candidates (gamma-aminobutyric acid, glycine, dopamine) accumulated by high-affinity uptake mechanisms. Neurons would not attach to glass or plastic substrates, consequently the present studies were performed using neurons plated upon a feeder layer. Serum was required for the initial phase of attachment. The feeder layer was derived from retinal cells that had been plated on glass or plastic in the presence of serum and had later been passaged. Since these cells exhibited glial fibrillary acidic protein (GFAP) immunoreactivity, they were tentatively identified as being glial in origin. Under these conditions, neuron- and glia-specific properties were retained up to 28 days. The presence of interstitial retinol-binding protein (IRBP) in medium of cultures of neuronal cells on feeder layers was demonstrated by an immunoblot technique using rabbit antibovine IRBP antibodies. No IRBP was detected in medium in which the feeder layers alone had been cultured. IRBP biosynthesis was demonstrated by incubation of the cultures with [35S]methionine. Immunoprecipitable [35S]IRBP was detected only in medium from cultures containing neurons; cells of the feeder layer did not synthesize and secrete this glycoprotein. These findings are consistent with the hypothesis that IRBP, a 135K constituent of the interphotoreceptor matrix, is synthesized in vivo by a neuronal cell, specifically, the photoreceptors.     

A gene function required for cell cycle progression during the G1 portion of the cell cycle and for maintenance of macronuclear DNA synthesis in Paramecium tetraurelia

The ccl mutation in Paramecium tetraurelia reversibly and rapidly blocks cell cycle progression and DNA synthesis at the restrictive temperature. Progression through the cell cycle is blocked during both the G1 and S portions of the cell cycle, while at the restrictive temperature there is neither residual cell cycle progression nor induction of excess delay of subsequent cell cycle events. DNA synthesis activity is reduced to 50% of the normal level in about 5 min and is completely blocked at 30 min after a shift to restrictive temperature. On return to permissive conditions, DNA synthesis is reactivated with similar kinetics.     

Regulation of low-density lipoprotein receptors in the human hepatoma cell line Hep G2

The human hepatoma cell line Hep G2 can be maintained in continuous culture and secretes numerous plasma proteins and lipoproteins into the medium. To better characterize cholesterol homeostasis in these cells we have examined the binding, internalization and degradation of [125I]LDL by cultured Hep G2 cells. Hep G2 cells express high-affinity low-density lipoprotein (LDL) receptors which facilitate the binding, internalization and degradation of [125I]LDL; these receptors can be induced by growth in LDL-depleted medium and repressed by further incubation in medium supplemented with LDL. The degradation of [125I]LDL by derepressed Hep G2 cells was inhibited by greater than 90% by monensin. Incubation of Hep G2 cells in the presence of increasing concentrations of LDL also inhibited cholesterol biosynthesis. Our results indicate that Hep G2 cells possess high affinity LDL receptors which are subject to metabolic regulation and suggest that this cell line affords a valuable model to further examine cholesterol and lipoprotein metabolism in human liver cells.     

Induction of differentiation in a human promyelocytic leukemic cell line (HL-60). Production of granule proteins

Serum fibronectin inhibits the adhesion of neutrophil granulocytes (PMNs) to clean glass, HSA-coated glass, and gelatin-coated glass. It does not affect adhesion to collagen-coated glass which itself provides a substratum of low adhesiveness for PMNs. Cell-cell adhesion is not affected. During the acute inflammatory response in vivo, PMNs must migrate through the fibronectin and collagen containing extracellular matrix: reducing cell-substratum adhesion in these circumstances might facilitate locomotion towards inflammatory foci.