Intact B-cell activity is essential for complete expression of experimental allergic encephalomyelitis in Lewis rats

The mechanisms of the inhibitory effect of the serum thymic factor (FTS) on allograft immunity have been studied on both cellular and humoral immune responses of skin allografted mice. FTS-induced suppression of allogeneic skin graft rejection was correlated with a transient diminution of in vivo alloreactive CTL production in the spleen, whereas the generation of allo-anti-H-2 antibodies was not affected. The involvement of suppressor cells in the effect of FTS was supported by the observation that irradiated spleen cells from FTS-treated recipients (bearing a 9-day allograft) suppressed the in vitro CTL generation.     

In vitro modulation of mouse natural killer (NK) cell activity by the serum thymic factor (FTS)

Previous studies indicated that the serum thymic factor (FTS) could modulate in vivo the level of splenic natural killer (NK) cell activity in mice. The present report shows that such an effect is also observed after a short term in vitro incubation of the effector cells with FTS. The regulatory effects of FTS result in an increase or a decrease of the splenic NK cell cytotoxicity depending upon the age and the mouse strain. Furthermore, FTS is able to enhance the NK cell activity of thymus and bone marrow cells which are known to be weakly reactive in NK cytotoxicity. Depletion experiments demonstrated that the FTS-induced increase of NK cell activity was not mediated by Thy 1+ cells nor macrophages, thus suggesting a direct action of FTS on the effector cells. Comparative studies using other thymic hormones revealed similar patterns of reactivity. These results favor the hypothesis of a close relationship between the thymus and NK cells.     

Regulation of aldosterone synthesis

The effects of angiotensin II and ACTH on cyclic AMP and aldosterone synthesis were studied in cells isolated from the bovine adrenal cortex. Angiotensin is a more potent stimulus of aldosterone synthesis than ACTH and the action of ACTH on aldosterone synthesis in cells from the glomerulosa is augmented by the presence of cells from the fasciculata. Angiotensin stimulates aldosterone synthesis in the absence of detectable changes in cyclic AMP, but the cells do respond to dibutyryl cyclic AMP leaving open the possibility that a cyclic nucleotide may play a role in the steroidogenic action of this hormone in the outer zone of the bovine adrenal cortex.     

FTS and 2-DG induce pancreatic cancer cell death and tumor shrinkage in mice

The Ras inhibitor S-trans-trans farnesylthiosalicylic acid (FTS) inhibits active Ras, which controls cell proliferation, differentiation, survival, and metabolism. FTS also inhibits HIF1α expression in cancer cells, leading to an energy crisis. The synthetic glucose analog 2-deoxy-D-glucose (2-DG), which inhibits glycolysis, is selectively directed to tumor cells that exhibit increased glucose consumption. The 2-DG enters tumor cells, where it competes with glucose for glycolytic enzymes. In cancer models, as well as in human phase 1 trials, 2-DG inhibits tumor growth without toxicity. We postulated that under normoxic conditions, tumor cells treated with FTS would be more sensitive than normal cells to 2-DG. We show here that combined treatment with FTS and 2-DG inhibited cancer cell proliferation additively, yet induced apoptotic cell death synergistically both in vitro and in vivo. The induced apoptosis was inferred from QVD-OPH inhibition, an increase in cleaved caspase 3, and loss of survivin. FTS and 2-DG when combined, but not separately, also induced an increase in fibrosis of the tumor tissue, chronic inflammation, and tumor shrinkage. Overall, these results suggest a possible new treatment of pancreatic tumors by the combined administration of FTS and 2-DG, which together induce pancreatic tumor cell death and tumor shrinkage under non-toxic conditions.     

An FTS/Hook/p107(FHIP) complex interacts with and promotes endosomal clustering by the homotypic vacuolar protein sorting complex

Fused Toes (FTS) is a member of a small group of inactive variant E2 ubiquitin-conjugating enzyme domain-containing proteins of unknown function. Through proteomic analysis of FTS complexes purified from human embryonic kidney 293T cells, we identified a new multiprotein complex, the FHF complex, containing FTS, members of the microtubule-binding Hook family of coiled-coil proteins (Hook1, Hook2, and Hook3), and a previously uncharacterized 107-kDa protein, FTS and Hook Interacting Protein (FHIP). FTS associated with a conserved C-terminal motif in Hook proteins in the yeast two-hybrid system and in tissue culture cells, and Hook proteins were found to form homo- and heterodimers. The approximately 500-kDa FHF complex contained all three Hook proteins, and small interfering RNA depletion experiments suggest that Hook proteins can interact interchangeably within this complex. Hook proteins as well as FTS interact with members of both the class B and class C components of the homotypic vesicular protein sorting (HOPS) complex. Depletion of FTS by RNA interference affects both the trafficking of epidermal growth factor from early-to-late endosome/lysosomes and the efficiency by which overexpression of the HOPS component Vps18 promotes clustering of lysosomal-associated membrane protein 1-positive endosome/lysosomes. These data suggest that the FTS/Hook/FHIP complex functions to promote vesicle trafficking and/or fusion via the HOPS complex.     

Structural study of circulating thymic factor: a peptide isolated from pig serum. I. Isolation and purification.

A circulating thymic factor (FTS) has been characterized by a bioassay based on its ability to render theta-negative rosette-forming cells theta-positive and azathioprine-sensitive. FTS was sequentially purified and finally isolated from 1000 liters of pig serum by ultrafiltration, gel filtration, and ion exchange chromatography. Its amino acid composition and apparent molecular weight estimated by Sephadex G-25 chromatography, indicate that FTS is a nonapeptide of composition lysine, aspartic acid (or asparagine), serine 2, glutamic acid (or glutamine) 2, glycine 2, and alanine.     

Stimulation of neutrophils by prenylcysteine analogs: Ca(2+) release and influx.

Farnesylthiosalicylic acid (FTS), a synthetic analog of the terminal prenylcysteine present in signaling proteins induces generation of superoxide ions, phospholipase C-driven hydrolysis of inositol lipids and calcium elevation in human neutrophils and DMSO-differentiated HL60 cells. These effects were ascribed to an interaction of the analog with elements responsible for recognition of specific prenylated proteins. The present study demonstrated that in addition to the release of intracellular calcium stores, FTS enhanced entry of Ca(2+) and Mn(2+) from the medium. The biphasic dependence of the influx on the concentration of FTS, as well as its insensitivity to inhibition by PMA and La(3+) suggest that the influx pathway activated by FTS is distinct from the previously described store-operated calcium channels of neutrophils. Consistent with the participation of a cellular membrane component in the interaction, FTS enhanced (45)Ca uptake in neutrophils and neutrophil cell membranes, but not in multilamellar vesicles. To establish specificity of the farnesyl moiety of FTS (C(15)), effects of three other analogs, geranylthiosalicylate, GTS (C(10)), geranylgeranylthiosalicylate, GGTS (C(20)), as well as the carboxymethyl ester FTS-Me on calcium homeostasis and superoxide production were investigated. GGTS dose-dependently elevated [Ca(2+)](i), induced quenching of the 360 nm Fura-2-calcium fluorescence by Mn(2+) and stimulated superoxide release, while GTS and FTS-Me were inactive. These results defined specific structural requirements for the functional interaction of prenylcysteine analogs with myeloid cells.     

Lymphocyte function in experimental African trypanosomiasis. VIII. Loss of suppressor T cell function in lymph nodes

The immunosuppression that occurs in mice experimentally infected with African trypanosomiasis has been examined further. In the present study we have examined lymph node cells from Trypanosoma rhodesiense-infected C57Bl/6J mice for the ability to produce mitogen induced antigen-nonspecific suppressor T cells (Ts). Inguinal, mesenteric, and brachial lymph node cells were harvested from uninfected control mice and from mice at different periods of infection. These cells were cultured with or without concanavalin A (Con A) for 48 hr to induce Ts activity. After stimulation, the control and infected lymph node cells were passed over Sephadex G-10 columns to remove suppressor macrophages that arise during the infection from Con A-induced Ts. The column passed cells were then added to normal mouse responder spleen cells in a primary in vitro antibody response culture system with sheep erythrocytes (SRBC) as antigen. The resultant plaque-forming cell responses to SRBC indicated that Ts function was not induced in infected lymph node cell populations. However, early in the infection, a stimulatory signal was provided by both the untreated and Con A-treated infected lymph node cells, which was lost in the terminal stage. Determinations of T cell subpopulations revealed that the infected Lyt 2.2-bearing subpopulation was not significantly altered from normal controls. We conclude that T. rhodesense infected mice fail to mount normal lymph node cell antigen nonspecific Ts responses and that this loss of activity may be due to an intrinsic dysfunction in the suppressor T cell population.     

Targeting of K-Ras 4B by S-trans,trans-farnesyl thiosalicylic acid

Ras proteins regulate cell growth, differentiation and apoptosis. Their activities depend on their anchorage to the inner surface of the plasma membrane, which is promoted by their common carboxy-terminal S-farnesylcysteine and either a stretch of lysine residues (K-Ras 4B) or S-palmitoyl moieties (H-Ras, N-Ras and K-Ras 4A). We previously demonstrated dislodgment of H-Ras from EJ cell membranes by S-trans,trans-farnesylthiosalicylic acid (FTS), and proposed that FTS disrupts the interactions between the S-prenyl moiety of Ras and the membrane anchorage domains. In support of this hypothesis, we now show that FTS, which is not a farnesyltransferase inhibitor, inhibits growth of NIH3T3 cells transformed by the non-palmitoylated K-Ras 4B(12V) or by its farnesylated, but unmethylated, K-Ras 4B(12) CVYM mutant. The growth-inhibitory effects of FTS followed the dislodgment and accelerated degradation of K-Ras 4B(12V), leading in turn to a decrease in its amount in the cells and inhibition of MAPK activity. FTS did not affect the rate of degradation of the K-Ras 4B, SVIM mutant which is not modified post-translationally, suggesting that only farnesylated Ras isoforms are substrates for facilitated degradation. The putative Ras-recognition sites (within domains in the cell membrane) appear to tolerate both C(15) and C(20) S-prenyl moeities, since geranylgeranyl thiosalicylic acid mimicked the growth-inhibitory effects of FTS in K-Ras 4B(12V)-transformed cells and FTS inhibited the growth of cells transformed by the geranylgeranylated K-Ras 4B(12V) CVIL isoform. The results suggest that FTS acts as a domain-targeted compound that disrupts Ras-membrane interactions. The fact that FTS can target K-Ras 4B(12V), which is insensitive to inhibition by farnesyltransfarase inhibitors, suggests that FTS may target Ras (and other prenylated proteins important for transformed cell growth) in an efficient manner that speaks well for its potential as an anticancer therapeutic agent.     

Endocrine control of thymic serum factor production in young-adult and old mice

The influence of different endocrinological manipulations on the blood concentration of serum thymic factor (FTS) was studied in young-adult and old mice. Among the experimentally induced endocrinopathies in youth, hypothyroidism and diabetes caused strong reductions of FTS levels, which were restored to normal by the appropriate hormonal substitutive therapy. Removal of adrenals or gonads has no significant effect on FTS level. Old mice, which show undetectable levels of FTS and low levels of thyroxine, can regain the capacity to produce FTS, provided they are treated with thyroxine. The variations of FTS blood levels in the course of endocrinological manipulations were due to a direct or indirect effect exerted on the recipient thymus. Hormonal treatment of thymectomized mice did not induce any FTS-like activity in their sera, nor did hormones interfere in vitro with the bioassay used to test for FTS. These data suggest that the neuroendocrine balance modulates the synthesis and/or the release of FTS from the thymus during the whole life of the organism and that the decline of FTS production with advancing age is largely dependent on age-associated endocrinological imbalances.     

Radioprotective effects of serum thymic factor in mice.

Serum thymic factor (FTS) reduced mortality of mice after total-body irradiation with 7.56 Gy X rays. The radioprotective effect was achieved by daily repeated subcutaneous injections of 3-100 micrograms FTS, while doses higher than 300 micrograms/day/mouse were neither radioprotective nor toxic. Similarly, degeneration of the spleen was moderated by 3-100 micrograms FTS but not by 500 micrograms FTS in sublethally (3.78 Gy) irradiated mice. Histological examination showed that hematopoiesis was enhanced in the spleen by daily injections of 10 micrograms FTS. Spleen cells from the FTS-treated mice incorporated more [3H]thymidine in culture with or without concanavalin A. The treatment with FTS increased the production of colony-stimulating factor in the spleen as well as in peritoneal macrophage-like cells, and caused a significant increase in the number of granulocyte-macrophage colony-forming cells both in the spleen and in the femoral bone marrow. Furthermore, FTS prevented a decrease in circulating neutrophils in the sublethally irradiated mice. Prominent overshoot recovery of myelopoiesis, which occurred occasionally in sublethally irradiated mice, did not occur in the FTS-treated mice. The decrease in blood erythrocytes was also significantly reduced. These observations imply that this thymic hormone has potential as a radioprotector.     

The effect of cyclic nucleotides on purine biosynthesis and the induction of PRPP synthetase during lymphocyte activation

The activation of lymphocytes has been used to study the regulation of mammalian gene expression. Concanavalin A (Con A) added to mouse spleen lymphocytes in serum-free medium leads to an increase in the rate of DNA synthesis as great as 1000 fold, commencing 20 hr after its addition. Prior to 20 hr, the rate of purine synthesis increases 10–100 fold as measured by accumulation of the purine intermediate, formyl glycineamide ribonucleotide (FGAR). Addition of dibutyryl cyclic GMP to the lymphocyte suspensions results in a 10 fold increase in the rate of DNA synthesis in the absence of Con A and enhances both purine synthesis and DNA synthesis in its presence. The activity of phosphoribosyl pyrophosphate synthetase (PRPP synthetase), an enzyme central to purine and pyrimidine biosynthesis, is increased 2–10 fold during the activation. The increase begins to appear 8 hr after Con A addition and requires concomitant protein synthesis. The induced PRPP synthetase activity is stimulated by the presence of cyclic GMP in the enzyme assay. Addition of dibutyryl cyclic AMP to Con A-stimulated lymphocytes inhibits FGAR production, the stimulation of DNA synthesis, and the appearance of cyclic GMP-sensitive PRPP synthetase. These studies suggest that cyclic nucleotides play a significant role in the molecular mechanism of lymphocyte activation, the regulation of purine biosynthesis, and of eucaryotic genetic expression.     

Fused Toes Homolog modulates radiation cytotoxicity in uterine cervical cancer cells

Radiotherapy is the major treatment modality for uterine cervical cancer, but in some cases, the disease is radioresistant. Defining the molecular events that contribute to radioresistance and progression of cancer are of critical importance. Here we evaluated the role of Fused Toes Homolog (FTS) in radiation resistance of cervical carcinoma. Immunostaning of cervical cancer cells and tissues revealed that FTS localization and expression was changed after radiation. Targeted stable knockdown of FTS in HeLa cells led to the growth inhibition after radiation. Radiation induced AKT mediated cytoprotective effect was countered by FTS knockdown which leads to PARP cleavage and caspase-3 activation leading to cell death. FTS knockdown promotes radiation induced cell cycle arrest at G0/G1 and apoptosis of HeLa cells with concurrent alterations in the display of cell cycle regulatory proteins. This study revealed FTS is involved in radioresistance of cervical cancer. Targeted inhibition of FTS led to the shutdown of key elemental characteristics of cervical cancer and could lead to an effective therapeutic strategy.     

Effects of cells of epithelial rests of Malassez and endothelial cells on synthesis of glycosaminoglycans by periodontal ligament fibroblasts in vitro

Cultures of fibroblast-like cells (PLF) and epithelial rest cells (PLE) prepared from explants of porcine periodontal ligament synthesized and secreted four glycosaminoglycans (GAG) in differing proportions. The PLF produced predominantly chondroitin sulfate (greater than 60%) with smaller amounts of hyaluronic acid (HA) (17%), dermatan sulfate (13%), and heparan sulfate (7%), whereas PLE produced predominantly HA (greater than 80%). In coculture and under conditions of reciprocal transfer of conditioned media neither cell type affected the other's GAG synthesis. Endothelial cells (EC), however, or their conditioned growth media, were able to stimulate increased GAG synthesis, especially HA, in PLF. A similar result was obtained with smooth muscles cells (SMC) cultured in EC growth media but here again PLE were unable to stimulate GAG synthesis by SMC. These findings suggest that the spectrum of GAG found in whole ligament results both from independent production by, and from interaction between, the different cell types within the ligament. The results also provide support for a general hypothesis that loose connective tissues, which are rich in HA, are formed and maintained under the influence of epithelial, including endothelial, cells.     

A Thymic Hormone Protects Mice from Enteropathy during Acute Graft-versus-Host Disease

We have previously reported that a nonapeptide thymic hormone, facteur thymique serique (FTS), is involved in the differentiation and activation of intestinal intraepithelial lymphocytes (i-IEL) in mice. In this study, we examined the effect of FTS treatment on enteropathy in a murine model for acute graft-vs.-host disease (GVHD) induced by injection of parental C57BL/6 splenocytes into unirradiated (C57BL/6XDBA/2) F₁ hybrids. FTS treatment significantly protected mice from developing acute GVHD as assessed by mortality rate, splenomegaly and enteropathy. The infiltration of donor-derived TCRαβ i-IEL bearing CD8αβ was significantly inhibited in the small intestine of FTS-treated mice, and the frequencies of apoptosis of crypt cells in the intestinal mucosa were decreased in these mice during acute GVHD. These results suggest that FTS treatment contributes to protection against enteropathy of acute GVHD. Thus, FTS may provide a useful approach to control acute GVHD after blood transfusion or bone marrow transplantation.     

The ionic control of 1,25-dihydroxyvitamin D-3 synthesis in isolated chick renal mitochondria: The role of potassium

In previous studies it was found that change in the concentrations of Ca²⁺, H⁺, and HPO₄²⁻ in the incubation medium altered the rates of synthesis of 1,25-dihydroxyvitamin D-3 (1,25(OH)₂D-3) by isolated renal mitochondria obtained from D-deficient chicks. The present studies demonstrate that raising the medium concentration of K⁺ from 1 to 50 mM leads to a 6-fold increase in rate of 1,25(OH)₂D-3 synthesis by isolated chick mitochondria; that the magnitude of this K⁺-dependent stimulation is enhanced by optimal concentrations of calcium (pCa = 5) and phosphate (pP_i = 3) (3 mM) but not by pH (from 6.8 to 7.4); that the effect is not produced by similar changes in media Na⁺ concentration; and that the stimulatory effect of K⁺ is not blocked by ruthenium red, an inhibitor of calcium transport and of the calcium-dependent stimulation of mitochondrial 1,25(OH)₂D-3 synthesis. It was also found taht valinomycin, a K⁺-specific ionophore, enhanced the sensitivity of the mitochondrial 1α-hydroxylase activity to K⁺. In the presence of valinomycin, an increase of pK⁺ to 3 was sufficient to cause a significant stimulation of 1,25(OH)₂D-3 synthesis. It was concluded that changes in the ion content of the mitochondrial matrix space regulate the activity of the 1α-hydroxylase.     

Inhibition of DNA synthesis in senescent-proliferating human cybrids is mediated by endogenous proteins

Cytoplasts were prepared from senescent human diploid fibroblasts. Brief treatments of the senescent cells with cycloheximide or puromycin prior to or after enucleation eliminated the ability of senescent cytoplasts to block initiation of DNA synthesis in senescent-young cybrids. Senescent cells treated with cycloheximide, enucleated and allowed to recover in complete medium without cycloheximide, regained the ability to block initiation of DNA synthesis in senescent-young cybrids. These results support the hypothesis that senescent cells synthesize an inhibitor of DNA synthesis which is either a protein(s) or its activity is mediated by a protein(s) found in the cytoplasm of the senescent cell.