Ian J. Forbes

Video image analysis of labile zinc in viable pancreatic islet cells using a specific fluorescent probe for zinc.

We used an intracellular zinc-specific fluorophore, Zinquin, in conjunction with fluorescence video image analysis, to reveal labile zinc in pancreatic islet cells, which concentrate this metal for use in synthesis, storage, and secretion of insulin. Zinquin vividly demonstrated zinc in the islet cell secretory granules, which formed a brightly labeled crescent in the cytoplasm between one side of the nucleus and the plasma membrane. Lower but still appreciable amounts of zinc were detected in the remaining cytoplasm, but there was little labeling in the nucleus. Fluorescence intensity varied among islet cells, suggesting differences in zinc content. Their average fluorescence intensity greatly surpassed that of the surrounding pancreatic acinar cells in frozen sections of pancreas and in all other types of cell studied, including lymphocytes, neutrophils, fibroblasts, and erythrocytes. Less labile zinc was detected in cells of the mouse insulinoma cell line NIT-1, regardless of whether they were maintained in long-term culture in the presence or absence of exogenous extracellular zinc. Exposure of islet or insulinoma cells to a high concentration of glucose or other secretagogue decreased the content of labile zinc. Zinquin should be a useful probe for revealing changes in zinc homeostasis in islet B-cells that may be important in their dysfunction and death during diabetes.

DEPRESSION OF IMMUNOLOGICAL FUNCTION IN PATIENTS TREATED WITH PHENYTOIN SODIUM (SODIUM DIPHENYLHYDANTOIN)

63 patients on long-term oral therapy with phenytoin sodium (sodium diphenylhydantoin) were screened for abnormalities of immunological function. They were compared with 92 controls and 28 patients with lymphoma. Depression of cellular or humoral immunity, or both, was found in a significant number of phenytoin-treated and lymphoma subjects. Phenytoin therapy was associated with low immunoglobulin A (21%), failure of antibody response to Salmonella typhi antigen (9%), absence of delayed hypersensitivity (D.H.S.) to three common skin-test antigens (22%), and depression of in-vitro lymphocyte transformation by phytohaemagglutinin (27%). Lymphoma patients manifested low IgM (22%), and inability to make antibody to S. typhi (11%) and to tetanus toxoid (21%). D.H.S. was absent in 36%; lymphocyte transformation was depressed in 17%. Abnormal lymphocyte transformation did not correlate with depression of cellular or humoral immunity in either group.

Flux of intracellular labile zinc during apoptosis (gene-directed cell death) revealed by a specific chemical probe, Zinquin

BACKGROUND The transition metal Zn(II) is thought to regulate cell and tissue growth by enhancing mitosis (cell proliferation) and suppressing the counterbalancing process of apoptosis (gene-directed cell death). To investigate the role of Zn(II) further, we have used a UV-excitable Zn(II)-specific fluorophore, Zinquin. The ester group of Zinquin is hydrolyzed by living cells, ensuring its intracellular retention; this allows the visualization and measurement of free or loosely-bound (labile) intracellular Zn(II) by fluorescence video image analysis or fluorimetric spectroscopy. RESULTS Here we show that in cells undergoing early events of apoptosis, induced spontaneously or by diverse agents, there is a substantial increase in their Zinquin-detectable Zn(II). This increase occurred in the absence of exogenous Zn(II) and before changes in membrane permeability, consistent with a release of Zn(II) from intracellular stores or metalloproteins rather than enhanced uptake from the medium. We propose that there is a major redistribution of Zn(II) during the induction of apoptosis, which may influence or precipitate some of the later biochemical and morphological changes. CONCLUSIONS The phenomenon of Zn(II) mobilization, revealed by Zinquin, presents a new element in the process of apoptosis for investigation and may permit rapid and sensitive identification of apoptotic cells, particularly in those tissues where their frequency is low.

Ca2+Mg2+-dependent nuclease: Tissue distribution, relationship to inter-nucleosomal DNA fragmentation and inhibition by Zn2+

Ca2+/Mg(2+)-dependent endonuclease has been implicated in the extensive internucleosomal DNA fragmentation that accompanies apoptosis (gene-directed cell death). We present further evidence that this enzyme is involved in apoptosis. Ca2+/Mg2+ nuclease activity was increased about 6-fold during colchicine-induced apoptosis in human chronic lymphocytic leukaemia cells. The increase in activity coincided with onset of DNA fragmentation. Spleen, liver, kidney and thymus expressed high levels of this enzyme while lung, brain, heart and testis contained little activity. Cells from tissues with high Ca2+/Mg2+ nuclease activity underwent rapid DNA fragmentation in response to a Ca2+ flux. Physiological concentrations of Zn2+ known to inhibit both apoptosis and DNA fragmentation also inhibited Ca2+/Mg2+ nuclease activity.

Induction of apoptosis in chronic lymphocytic leukemia cells and its prevention by phorbol ester

Chronic lymphocytic leukemia lymphocytes were used to study mechanisms involved in apoptosis (programmed cell death). Apoptosis, which was determined by morphological changes including cell death and by internucleosomal DNA fragmentation, occurred during culture for 1 to 2 days in a portion of the cells from three of the four patients tested. Most of the cells underwent apoptosis and DNA fragmentation was greatly enhanced when cells were cultured in the presence of the microtubule inhibitor colchicine, the topoisomerase II inhibitor etoposide, or the glucocorticoid methylprednisolone. Tumor-promoting phorbol esters inhibited spontaneous DNA fragmentation and cell death including that induced by colchicine, etoposide, and methylprednisolone, indicating that they act on an event common to apoptosis caused by diverse stimuli. Phorbol esters probably act through protein phosphorylation, since they were effective at concentrations which modulated protein kinase C (PKC) and their action was prevented by H-7, which binds to and inactivates the catalytic site of PKC. In the absence of phorbol ester, H-7 itself induced some apoptosis. These findings implicate PKC in the suppression of apoptosis, but its precise role requires systematic investigation.

OXYGEN DEPENDENCY OF PHOTOCYTOTOXICITY WITH HAEMATOPORPHYRIN DERIVATIVE

Abstract— Exposure of Raji cells to haematoporphyrin derivative (HPD) and red light caused marked cytotoxicity. This was completely inhibited under anaerobic conditions. By using sodium dithionite in aqueous solutions, precise and graded oxygen concentrations could be achieved. Cytotoxicity was directly proportional to the oxygen concentration of the medium until a maximum was reached at a pO2 of 90 mm Hg. Sodium dithionite did not affect the viability of test cells and did not alter the chromatographic profile of HPD. Dithionite did not interfere with the uptake of HPD by cells. Dependency of phototoxicity upon aerobic conditions suggests that the cytotoxic agent is derived from oxygen and is consistent with the hypothesis that singlet oxygen and/or oxygen‐derived free radicals play an important role in photochemotherapy with HPD.

Synergy between zinc and phorbol ester in translocation of protein kinase C to cytoskeleton.

Protein kinase C was measured in the cytoskeletal fraction of lymphocytes, platelets and HL60 cells, by specific binding of [3H]phorbol dibutyrate and by immunoblotting with antibody to a consensus sequence in the regulatory domain of α‐, β‐ and γ‐isozymes of protein kinase C. Treatment of cells for 40 min with a combination of zinc (2–50 μM), zinc ionophore pyrithione and unlabelled phorbol dibutyrate (200 nM) caused up to a ten‐fold increase in cytoskeletal protein kinase C and a corresponding decrease in other cellular compartments. Omission of any of the reagents resulted in much less or no translocation. These effects were inhibited by 1,10‐phenanthroline, which chelates zinc, and were not seen with calcium. Increase in cytoskeletal protein kinase C persisted for several hours and appeared to involve attachment of the enzyme to actin microfilaments. We propose that zinc, like calcium, regulates the distribution of PKC in cells. However, unlike calcium which controls the binding of PKC to the lipid component on cell membranes, zinc controls the distribution of PKC to membrane cytoskeleton, possibly actin.

CHANGES IN LYMPHOCYTE FUNCTION DURING PREGNANCY

A gradual increase in spontaneous lymphocyte DNA synthesis was demonstrated in each trimester of pregnancy. Autoradiographic studies indicated that lymphocytes were primarily responsible for this activity. PHA‐induced lymphocyte transformation in both fetal calf serum and autologous serum was significantly reduced in the second and third trimesters of pregnancy. Spontaneous lymphocyte DNA synthesis was significantly reduced in patients with mild pre‐eclampsia. However, no significant differences were seen in patients with severe pre‐eclampsia in the third trimester of pregnancy compared with the normal control subjects. No evidence was adduced to implicate inhibitory humoral factors affecting the peripheral blood lymphocytes in pregnant patients in experiments in which washed lymphocytes were cultured in medium containing heterologous serum. In vitro experiments demonstrated that cortisol, progesterone and HPL caused a significant reduction in lymphocyte DNA synthesis, and HGH and HCG had a variable effect. However, only cortisol was regularly inhibitory at physiological concentrations. The progesterone effect was dose‐related, producing 90 per cent inhibition of activity at a concentration of 10 μg/ml. No synergism could be shown between HPL and progesterone on lymphocyte transformation. The increase in activity of circulating immunoreactive cells during pregnancy and its depression with the onset of pre‐eclampsia is discussed.

Interaction between protein kinase C and regulatory ligand is enhanced by a chelatable pool of cellular zinc

At micromolar concentrations, zinc (Zn) and cadmium, but not other metals, greatly augmented binding of [3H]phorbol dibutyrate ([3H]PDBu) to protein kinase C (PKC) in cell homogenates and intact cells (in the presence of ionophore). Increased binding persisted for several hours. The heavy-metal chelating agent 1,10-phenanthroline completely reversed the increased [3H]PDBu binding in cells pretreated with 65Zn and ionophore and this was associated with a decline of about 20% in cell-associated 65Zn, suggesting that a relatively small pool of intracellular Zn acts on PKC. This may be a membrane-associated pool, since 65Zn readily bound to isolated erythrocyte inside-out membranes. Phenanthroline also partially inhibited binding of [3H]PDBu to PKC in untreated cells and extracts in a Zn-reversible manner. Therefore, cellular Zn appears to regulate the interaction of ligand with PKC. PKC bound to a Zn affinity column and was eluted by metal-chelator, confirming that Zn interacts directly with PKC.

COMPARISON OF THE EFFICACY OF PULSED AND CONTINUOUS‐WAVE RED LASER LIGHT IN INDUCTION OF PHOTOCYTOTOXICITY BY HAEMATOPORPHYRIN DERIVATIVE

Abstract— The efficiency of pulsed and continuous wave laser light to induce photodynamic activity in haematoporphyrin derivative (Hpd) was compared in two systems, a tissue culture assay and a transplantable mouse tumour. No difference was found.