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.

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.

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.