Altered Regulation of the Glucose Transporter GLUT3 in PRDX1 Null Cells Caused Hypersensitivity to Arsenite.

Targeting tumour metabolism through glucose transporters is an attractive approach. However, the role these transporters play through interaction with other signalling proteins is not yet defined. The glucose transporter SLC2A3 (GLUT3) is a member of the solute carrier transporter proteins. GLUT3 has a high affinity for D-glucose and regulates glucose uptake in the neurons, as well as other tissues. Herein, we show that GLUT3 is involved in the uptake of arsenite, and its level is regulated by peroxiredoxin 1 (PRDX1). In the absence of PRDX1, GLUT3 mRNA and protein expression levels are low, but they are increased upon arsenite treatment, correlating with an increased uptake of glucose. The downregulation of GLUT3 by siRNA or deletion of the gene by CRISPR cas-9 confers resistance to arsenite. Additionally, the overexpression of GLUT3 sensitises the cells to arsenite. We further show that GLUT3 interacts with PRDX1, and it forms nuclear foci, which are redistributed upon arsenite exposure, as revealed by immunofluorescence analysis. We propose that GLUT3 plays a role in mediating the uptake of arsenite into cells, and its homeostatic and redox states are tightly regulated by PRDX1. As such, GLUT3 and PRDX1 are likely to be novel targets for arsenite-based cancer therapy.

The human cathelicidin, LL-37, induces granzyme-mediated apoptosis in regulatory T cells.

LL-37 is a human cationic host defense peptide (antimicrobial peptide) belonging to the cathelicidin family of peptides. In this study, LL-37 was shown to kill stimulated and nonstimulated CD4(+)CD25(+)FoxP3(+) T cells (regulatory T cells; Tregs) through apoptosis, while having no cytotoxic effect on CD4(+)CD25(-) T cells at the same LL-37 concentrations. Of interest, Tregs were much more sensitive to LL-37 than many other cells, dying at 10-fold lower concentrations than other cell types tested. LL-37 exposure resulted in DNA fragmentation, chromatin condensation, and apoptotic body formation, all indicative of an apoptotic form of cell death. The importance of granzyme family members in the apoptosis of Tregs after LL-37 treatment was analyzed by using C57Bl/6 lymphocytes obtained from mice that were homozygous for null mutations in the granzyme B gene, and both the granzyme A and B genes. Granzyme A and granzyme B were both shown to play a role in LL-37-induced apoptosis of Tregs. Further analysis showed that apoptosis occurred primarily through caspase-dependent apoptosis at high LL-37 concentrations. However, grA-dependent/caspase-independent cell death was also observed. This suggests that LL-37 induces apoptosis in Tregs through multiple different mechanisms, initiated by the LL-37-induced leakage of granzymes from cytolytic granules. Our results imply that LL-37 administered at the site of a tumor could influence the adaptive antitumor immune response by killing Tregs and thus inhibiting their suppressor activity.

Contrasting role of phospholipase C-gamma1 in the expression of immediate early genes induced by epidermal or platelet-derived growth factors.

While significant progress has been achieved in identifying the signal transduction elements that operate downstream of activated receptor tyrosine kinases, it remains unclear how different receptors utilize these signaling elements to achieve a common response. This study compares the capacity of epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) to elicit the induction of immediate early gene (IEG) mRNAs in the presence or absence of phospholipase C-gamma1 (PLC-gamma1). The results show that while PDGF induction of nearly all IEG mRNAs is abrogated in plcg1 null cells, EGF induction of the same genes is variable in the null cells and exhibits three distinct responses. Five IEG mRNAs (Nup475, Cyr61, TF, Gly, TS7) are completely inducible by EGF in the presence or absence of PLC-gamma1, while three others (JE, KC, FIC) exhibit a stringent requirement for the presence of PLC-gamma1. The third type of response is exhibited by c-fos and COX-2. While these mRNAs are completely induced by EGF in the absence of PLC-gamma1, the time course of their accumulation is significantly delayed. No IEG was identified as completely inducible by EGF and PDGF in the absence of PLC-gamma1. Electrophoretic mobility shift assays (EMSA) demonstrate that PLC-gamma1 is necessary for nuclear extracts from PDGF-treated cells, but not EGF-treated cells, to interact with probes for AP-1 or NF-kappaB.

Influence of low-dose oral contraception on peripheral blood lymphocyte subsets at particular phases of the hormonal cycle.

OBJECTIVE: To investigate the effects of low-dose oral hormonal contraception on the immune system during certain phases of the hormonal cycle. DESIGN: Prospective, nonrandomized, controlled study. SETTING: Academic research setting. PATIENT(S): Women with regular menstrual cycle using hormonal oral contraception (OC; Cileste, 250 microg of norgestimat and 35 microg of ethinylestradiol, or Marvelon, 150 microg of desogestrel and 30 microg of ethinylestradiol) and women not using hormonal or other forms of contraception. INTERVENTION(S): Peripheral blood lymphocyte subsets were determined by flow cytometry on the first day of menstruation (day 1), in the follicular phase (day 8), midcycle (day 15), and in the luteal phase (day 22). MAIN OUTCOME MEASURE(S): Levels of lymphocyte subpopulations. RESULT(S): Women using OC had significantly higher levels of CD3+ CD8+ cells throughout their pill cycle compared to controls. Furthermore, women taking Cileste had lower levels of natural killer (NK) cells during their cycle and also women taking Marvelon but only from days 8-15. Within the pill cycle of Cileste we observed an increase in CD20+ and CD20+ CD5+ cells from days 1-8. CONCLUSION(S): Cytotoxic lymphocytes, which are responsible for first-line immune defense, and B cells, which are involved in autoimmune disorders, are affected by OC.

The effect of 2-acetyl-4-tetrahydroxybutylimidazole on lymphocyte subsets in peripheral blood of the rat.

A constituent of Ammonia Caramel, 2-acetyl-4-tetrahydroxybutylimidazole (THI), is known to cause a reduction in the number of circulating lymphocytes when fed to rats. In the present study the effect of giving THI 1 mg/kg by gavage daily for 7 days on the numbers of lymphocytes in subsets has been monitored in peripheral blood. Both immunoglobulin light chain-bearing B-cells (MARK-1+) and CD5 marker-bearing T-cells (OX-19+) were reduced in number within 1 day of treatment. Within the pan-T-cell population, Class II MHC reactive helper T-lymphocytes (W3/25-) were more reduced than the Class I MHC reactive cytotoxic/suppressor T cells (OX-8+). The number of null cells (MARK-1-, OX-19-) was not affected; the majority of these cells appeared to be large granular lymphocytes.

Natural suppressor (NS) activity from murine neonatal spleen is responsive to IFN-gamma.

Natural suppressor (NS) cell activity is the ability of apparently unprimed "null" cells to nonspecifically suppress immune responses. Previously we have shown that NS cell activity from the spleens of mice undergoing chronic graft-vs-host disease (GVHD) is enhanced in vitro by activated T cell signals (e.g., Con A supernatant [CAS]). Here we asked if the naturally occurring suppressor activity found in the neonatal mouse spleen is caused by NS cells, and if so whether this NS activity is also responsive to T cell signals. Finally, we wanted to identify the material in the CAS to which the NS cells respond. Spleen cells from (BALB/c X B10.D2)F1 neonates contain potent, genetically unrestricted suppressor activity toward normal mitogen responses. The cells responsible for this suppression are nonadherent, Thy-, Ig- and are thus by definition NS cells. Neonatal spleen NS cells suppress the indicator Con A response of all mouse strains tested, but their behavior with regard to LPS responses is different. They significantly inhibit the indicator LPS response of allogeneic strains, but are less inhibitory of LPS-stimulated syngeneic (BALB/c X B10.D2)F1 and parental strains. However, the addition of CAS to these latter cultures enhances the NS inhibition of the LPS response to the level of suppression seen with a Con A response. Two lymphokines were able to replace the CAS. Recombinant interferon-gamma (rIFN-gamma) closely mimics the activity found with whole CAS, with low concentrations (1 U/well) being capable of enhancing the neonatal NS activity to near-maximal levels. Recombinant interleukin 2 (rIL 2) is also capable of stimulating the neonatal NS activity to near maximum. However, the rIL 2 must be added at much higher concentrations, taking greater than 50 U/well to get maximum activation of NS suppression. The addition of anti-IFN-gamma antiserum to these LPS suppression assays removes the ability of CAS to activate the neonatal NS cells. Anti-IFN-gamma antiserum also removes the ability of rIL 2 as well as rIFN-gamma to activate the NS cells. It thus appears that the rIL 2 is working by its ability to stimulate IFN-gamma production. Anti-IFN-gamma also removes the ability of the neonatal NS cells to suppress a Con A response. Therefore, it appears that neonatal splenic NS cells respond to, and are activated by, IFN-gamma to carry out their suppressive activity.(ABSTRACT TRUNCATED AT 400 WORDS)

The molecular basis for the differential sensitivity of B and T lymphocytes to growth inhibition by thymidine and 5-fluorouracil.

Cultured leukemic lymphocytes originating from patients with T, B and non-T, non-B (null) leukemia were tested for their sensitivity to thymidine and 5-fluorouracil. T cells were found to be 5-7 fold more sensitive to thymidine growth inhibition than B-cells. At 10(-3) M concentration of thymidine, T cells showed a progressive (up to 75%) decline in the populating trypan blue-excluding cells, after 72 h. At this concentration of thymidine B cells showed slight inhibition at 24 and 48 h, then at 72 h the surviving cell level returned almost to the level of unperturbed cells. Thymidine at 10(-5) M concentration, caused 40% cell growth inhibition of T cells, however, at this concentration it had little or no effect on B cells. 5-fluorouracil effects on B and T lymphocytes are opposite to that of thymidine. B cells were on an average 5-7 times more sensitive to 5-FU than T cells. 5-FU at 10(-6) M caused up to 45% inhibition of B-cell growth but at this concentration it had no effect on the growth of T cells. B-, T- and null-lymphocytes sensitivity to thymidine and 5-FU was correlated with the level of the catabolic enzyme thymidine phosphorylase. B cells had, on average, 5-fold more thymidine phosphorylase than T or null cells. Furthermore, the enzyme from the B-cell line (HR1K) chromatographed differently on DEAE-Sephadex than the normal peripheral blood lymphocytes enzyme. The normal enzyme from peripheral blood lymphocytes when adsorbed to DEAE-Sephadex was eluted at a salt concentration of 0.3 M KCI, Enzyme activities of HR1K did not adsorb to the DEAE-Sephadex column but were adsorbed to a phosphocellulose column. Enzyme from normal and leukemic lymphocytes showed similar molecular weights of 130,000 dalton as determined by gel filtration.

Response of non-T, non-B acute lymphocytic leukemia cells to phorbol ester.

Non-T, Non-B acute lymphocytic leukemia cells were cultured in vitro with or without the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), a potential modulator of differentiation. The eight cases studied were representative of non-T, non-B acute lymphocytic leukemia (ALL) cells and expressed amounts of la antigens varying from 0.9 X 10(5) to 7.1 X 10(5) molecules/cell; these levels were measured in a cellular radioimmunoassay with 21w4 monoclonal antibody directed at a monomorphic human la determinant. With all cases, TPA caused a significant increase in the level of la. Cultures with TPA expressed 4.3 times the amount of la found on fresh ALL cells, and a correlation was observed (r = 0.92) between the level of la following culture with TPA and that found on fresh ALL cells. A 25% increase in the modal volume of ALL cells was also caused by TPA. There was no detectable induction of surface or cytoplasmic immunoglobulin and no change in the expression of the common ALL antigen. Inhibition of [3H]thymidine incorporation and stimulation of 14C-labeled amino acid incorporation were observed in the presence of TPA, suggesting that the increase in la level occurs concurrently with an increase in protein synthesis induced by phorbol ester. Following culture with TPA, a substantial increase in the ability of the ALL cells to stimulate in a mixed-lymphocyte reaction was obtained. These results suggest that ALL cells, like other cell types, are susceptible to the effects of TPA and respond by changes in cell volume, surface antigen expression, and mixed-lymphocyte reaction stimulating capacity.

Effects of antimalarial drugs on human natural killer cell activity.

Separation of null cell fraction from the other cellular components of human peripheral blood obtained from normal healthy individuals was effected through the Ficoll-Hypaque density gradient centrifugation, carbonyl iron phagocytosis-magnet application, E-rosette forming and binding to 19S-EAC respectively. The null cells were used as effector cells in the cytotoxic assay. The spontaneous cell-mediated cytotoxicity assay was employed and the highly NK-sensitive K562 labelled with Na251 CrO4 were used as targets. The null cell fraction was divided into several portions to allow for normal control, diluent control and tests. The test portions were those exposed to the various antimalarial drugs employed. It was observed that the T cell, B cells and null cell fractions accounted for 72%, 18% and 10% of the total lymphocyte population respectively. The mean cytotoxicity generated by the natural killer subset was 63%. The antimalarial drugs/drug combination used were chloroquine, quinine, pyrimethamine and sulfadoxine/pyrimethamine combination. Concentrations used were their respective minimal inhibitory concentration (MIC) and corresponding 5 X MIC. The inhibitory effects on natural killer cell activity of these drugs were observed. The possible reasons for these observations are discussed.

Production of a monoclonal antibody-bleomycin conjugate utilizing dextran T-40 and the antigen-targeting cytotoxicity of the conjugate.

Bleomycin (BLM), a potent anticancer glycopeptide antibiotic, was linked covalently to murine monoclonal anti-HLA IgG1 antibody (H-1) with the use of dextran T-40. As determined spectrophotometrically, the conjugate was composed of 57.5 moles BLM per mole antibody. Of the substituted BLM, 18.4% (10.6 moles BLM per mole antibody) exhibited antimicrobial activity. The BLM-(H-1) conjugate showed stronger cytotoxicity than BLM alone against HLA-bearing cells in cultivation after a 30-min exposure to the drugs. In the same experiment, the conjugate was less toxic than BLM against cells lacking HLA.

An apparent lack of HLA restriction in the stimulation of granulocyte-macrophage colony formation from normal human null cells by helper T lymphocytes.

Haemopoietic progenitor cells capable of producing granulocyte-macrophage (GM) colonies have been demonstrated in the 'null' lymphocyte population of normal peripheral blood. The helper and suppressor roles of different T cell subpopulations have been implicated in the regulation of granulopoiesis in disease as well as in normal individuals. However, it is not certain whether such interactions between T cells and progenitor cells are HLA-restricted. We undertook further investigation of the effect of T cell subpopulations (TG and TnonG) on GM colony formation in vitro. In particular, we studied the possibility of HLA restriction in this process. Our results demonstrate that the enhancement of GM colony growth by T lymphocytes is not restricted by HLA compatibility between T cells and null cells, that such stimulation is radio-sensitive and that it is provided by the TnonG cell subpopulation.

Immunoregulation by blasts from null cell and T-cell leukemias: help and suppression of T-cell proliferative responses to mitogens.

The immunoregulatory activity of bone marrow leukemic blasts from five patients with null-cell acute lymphoblastic leukemia (ALL) and two children with T-cell ALL was evaluated. Blasts were studied in co-culture for their effects on the proliferative responses of normal allogeneic peripheral blood lymphocytes to phytohemagglutinin. Mitomycin C-treated null cell ALL blasts from two of five children suppressed the proliferative responses of normal responder cells by 80% and 58% whereas those from two other children enhanced the responses by 118% and 31%. T-cell leukemic blasts from one patient with T-cell ALL exhibited helper cell activity (26%) and T-leukemic blasts from the other demonstrated suppressor cell activity (53%). The helper cell activity of leukemic blasts was associated with stimulating capacity of null blasts in one-way mixed lymphocyte reactions. In six of seven cases, incubation of blast cells with concanavalin A (20 microgram/ml) for 48 hours prior to co-culture with responder cells did not result in the generation of significant suppressor cell activity. Our study suggests that leukemic blasts from certain patients with 'null' and T-cell ALL may possess spontaneous helper or suppressor cell immunoregulatory activity. This functional heterogeneity of leukemic blasts may help in subclassifying the ALL.