Hans Jürgen Steinfelder

Activation of the Dual-Leucine-Zipper-Bearing Kinase and Induction of β-Cell Apoptosis by the Immunosuppressive Drug Cyclosporin A

Post-transplant diabetes is an untoward effect often observed under immunosuppressive therapy with cyclosporin A. Besides the development of peripheral insulin resistance and a decrease in insulin gene transcription, a β-cell toxic effect has been described. However, its molecular mechanism remains unknown. In the present study, the effect of cyclosporin A and the dual leucine-zipper-bearing kinase (DLK) on β-cell survival was investigated. Cyclosporin A decreased the viability of the insulin-producing pancreatic islet cell line HIT in a time- and concentration-dependent manner. Upon exposure to the immunosuppressant fragmentation of DNA, the activation of the effector caspase-3 and a decrease of full-length caspase-3 and BclXL were observed in HIT cells and in primary mature murine islets, respectively. Cyclosporin A and tacrolimus, both potent inhibitors of the calcium/calmodulin-dependent phosphatase calcineurin, stimulated the enzymatic activity of cellular DLK in an in vitro kinase assay. Immunocytochemistry revealed that the overexpression of DLK but not its kinase-dead mutant induced apoptosis and enhanced cyclosporin A-induced apoptosis to a higher extent than the drug alone. Moreover, in the presence of DLK, the effective concentration for cyclosporin A-caused apoptosis was similar to its known IC50 value for the inhibition of calcineurin activity in β cells. These data suggest that cyclosporin A through inhibition of calcineurin activates DLK, thereby leading to β-cell apoptosis. This action may thus be a novel mechanism through which cyclosporin A precipitates post-transplant diabetes.

Apoptosis induction by inhibitors of Ser/Thr phosphatases 1 and 2A is associated with transglutaminase activation in two different human epithelial tumour lines

Two epithelial tumour lines, HeLa and KB, were treated with okadaic acid and calyculin A, specific inhibitors of Ser/Thr phosphatases (PP), esp. PP1 and PP2A. Morphological criteria, analysis of DNA fragmentation and studies of membrane integrity revealed that both agents concentration‐ and time‐dependently induced apoptosis at nanomolar concentrations which in these cells was associated with the stimulation of a transglutaminase activity. Since a non‐functional derivative of okadaic acid did not affect cell viability apoptosis was apparently related to the inhibition of PP1 and PP2A. Membrane damage marker activity was delayed by at least 24 h when compared to nuclear alterations.

A fast and sensitive technique to study the kinetics and the concentration dependencies of DNA fragmentation during drug-induced apoptosis.

Apoptotic cell death with its characteristic coordinated cellular breakdown can be triggered by cytotoxic drugs. One prominent feature that differentiates apoptotic from necrotic cell death is the caspase-mediated activation of an endonuclease that internucleosomally cleaves DNA resulting in the so-called apoptotic DNA ladder. Here we report a new rapid, sensitive and inexpensive column separation technique to study drug-induced DNA fragmentation from 10(6) or less cells. This technique, which is based on a modified plasmid spin column kit, avoids the use of hazardous chemicals. With this procedure and subsequent densitometric analysis it was possible to study the concentration dependencies and the kinetics of drug-induced DNA fragmentation. The applicability of this technique is shown for okadaic acid- and cantharidic-acid-treated pituitary GH(3) cells as well as highly okadaic-acid-resistant sublines. These studies allowed us to compare as well as to differentiate the effects and potencies of these structurally different but functionally quite similar inhibitors of ser/thr phosphatases 1 and 2A.

Inhibitors of ser/thr phosphatases 1 and 2A induce apoptosis in pituitary GH3 cells

Two structurally different inhibitors of ser/thr phosphatases 1 and 2A, okadaic acid and calyculin A, time- and concentration-dependently stimulated and inhibited cell-specific function (hormone gene expression) in pituitary GH3 cells. The negative effect was associated with the appearance of apoptotic cell death. Nanomolar concentrations of both agents produced the characteristic morphological alterations and a DNA fragmentation ladder. Calyculin A treatment resulted in comparable changes with 10fold lower concentrations than okadaic acid. Observations with derivatives of okadaic acid with no or lower phosphatase inhibitory potency supported the conclusion that apoptosis induction is related to inhibition of ser/thr phosphatases, presumably types 1 and 2A. Membrane damage as measured by lactate dehydrogenase liberation into medium was significantly lower in apoptotic vs. necrotic cells. DNA fragmentation could be reduced by the addition of zinc but not by removal of extracellular calcium with EGTA. Apoptotic changes were reduced by the concomitant activation of protein kinase A by a membrane permeable cAMP analogue. Incubation of cells for 4 months in successively increased concentrations of okadaic acid resulted in a population that proliferated at the initially lethal concentration of 30 nM.

Michael Adducts of Ascorbic Acid as Inhibitors of Protein Phosphatase 2A and Inducers of Apoptosis

Michael adducts of ascorbic acid with alpha,beta-unsaturated carbonyl compounds have been shown to be potent inhibitors of protein phosphatase 1 (PP1) without affecting cell viability at the respective concentrations. Here we were able to show that higher concentrations can partially inhibit PP2A activity and concomitantly induce apoptotic cell death. A nitrostyrene adduct of ascorbic acid proved to be a more potent and effective inhibitor of PP2A as well as a stronger inducer of apoptosis. These adducts only slightly lost their cytotoxic potential in multidrug resistant cells that were 10-fold less sensitive to apoptosis induction by okadaic acid and vinblastine.

Characterization of two pituitary GH3 cell sublines partially resistant to apoptosis induction by okadaic acid.

Pituitary GH3 cells die by apoptosis when treated with okadaic acid, a specific inhibitor of ser/thr phosphatases. Incubations starting at concentrations of 5 and 12.5 nM followed by stepwise rises resulted in two populations (the S1 and S2 sublines) that proliferated at initially lethal 30 nM. Cells were partially resistant to higher concentrations of okadaic acid and its derivative methyl okadaate. Toxicity of the structurally distinct inhibitors cantharidic acid and calyculin A was differently affected in the two resistant lines. The enhanced expression of the P-glycoprotein was one mechanism of resistance in S1 and S2. Resistance was reversed completely (S1) or partially (S2) by the addition of verapamil. In addition, phosphatase activity, presumably PP2A, was increased in S2. Therefore, pharmacokinetic and pharmacodynamic mechanisms can protect pituitary GH3 cells from apoptotic cell death by okadaic acid.

Inhibition of insulin-stimulated glucose transport in rat adipocytes by nucleoside transport inhibitors

In isolated rat adipocytes, basal as well as insulin‐stimulated 3‐O‐methylglucose transport was inhibited nearly completely (maximal inhibition: 95%) by the nucleoside transport inhibitors dipyridamole (IC50 = 5 μM), nitrobenzylthioguanosine (20 μM), nitrobenzylthioinosine (35 μM) and papaverine (130 μM). Transport kinetics in the presence of 10 μM dipyridamole revealed a significant increase in the transport K m value of 3‐O‐methylglucose (3.45±0.6 vs 2.36±0.29 mM in the controls) as well as a decrease in the Vmax value (4.84±0.95 vs 9.03±1.19 pmol/s per μl lipid in the controls). Half‐maximally inhibiting concentrations of dipyridamole were one order of magnitude higher than those inhibiting nucleoside (thymidine) uptake (0.48 μM). The inhibitory effect of dipyridamole (5 μM) reached its maximum within 30 s. The agent failed to affect insulins half‐maximally stimulating concentration (0.075 nM) indicating that it did not interfere with the mechanism by which insulin stimulates glucose transport. Further, dipyridamole fully suppressed the glucose‐inhibitable cytochalasin B binding (IC50 = 1.65±0.05 μM). The data indicate that nucleoside transport inhibitors reduce glucose transport by a direct interaction with the transporter or a closely related protein. It is suggested that glucose and nucleoside transporters share structural, and possibly functional, features.

Hamster pancreatic beta cell lines with altered sensitivity towards apoptotic signalling by phosphatase inhibitors.

Specific inhibitors of serine/threonine phosphatases like okadaic acid can induce apoptotic cell death in the pancreatic beta cell line HIT. Cultivation in stepwise increased concentrations of okadaic acid enabled the isolation of HIT100R cells which proliferate at 100 nM okadaic acid (8–10 times the initially lethal concentration). These two cell lines were used to characterize the events triggered by okadaic acid that led to apoptosis. Biochemical markers, e.g. cytochrome c release from mitochondria and increase of caspase‐3‐like activity, revealed that induction of apoptosis by 100 nM okadaic acid in parental HIT cells started with the release of cytochrome c. In HIT100R cells 500 nM okadaic acid were necessary to induce alterations comparable to those observed with 100 nM okadaic acid in non‐resistant HIT cells. In contrast to okadaic acid, the potency of the structurally different phosphatase inhibitor cantharidic acid to induce cytochrome c release, increase of caspase‐3‐like activity and DNA fragmentation was comparable in HIT and HIT100R cells. Thus, no cross‐resistance between these phosphatase inhibitors seemed to exist. Phosphatase activity in extracts from HIT and HIT100R cells did not differ in its total amount or in its sensitivity for okadaic acid. Since higher concentrations of okadaic acid were needed to induce apoptosis in HIT100R cells, a compromised intracellular accumulation of the toxin appeared likely. Functional and structural analysis revealed that this was achieved by the development of the multidrug resistance phenotype in HIT100R cells. The underlying mechanism appeared to be the enhanced expression of the pgp1 but not the pgp2 gene.

Doxorubicin-resistant LoVo adenocarcinoma cells display resistance to apoptosis induction by some but not all inhibitors of ser/thr phosphatases 1 and 2A.

LoVo adenocarcinoma cells are fairly sensitive to cytostatic drugs, e.g. doxorubicin, but can develop drug resistance by expression of a P-glycoprotein-mediated MDR1 phenotype. LoVo cells respond with apoptosis to nanomolar concentrations of okadaic acid and micromolar concentrations of cantharidic acid. Interestingly, LoVoDx cells which had become about 10-fold less sensitive to doxorubicin by incubation in increasing concentrations of this cytostatic drug were also less sensitive to the toxicity of okadaic acid. Resistance to both agents was lost or significantly reduced by incubation in drug-free medium for about 4 months. On the other hand, LoVoDx cells did not lose responsiveness to the structurally different phosphatase inhibitor cantharidic acid but were about twofold more sensitive to the cytotoxic effect of this agent. Thus, MDR expression protects LoVo cells from the toxicity of phosphatase inhibitors that presumably are substrates of the P-glycoprotein, e.g. okadaic acid and its derivatives but not cantharidic acid, despite the fact that both agents are potent inducers of apoptotic cell death via ser/thr phosphatase inhibition.

Prostaglandin E2 differentiates between two forms of glucose transport inhibition by lipolytic agents

SummaryThe inhibition of insulin-stimulated glucose transport by lipolytic agents was studied in isolated rat adipose cells. Two different mechanisms for the inhibition of glucose transport by lipolytic hormones and agents were distinguished by use of the antilipolytic agent prostaglandin E2 (PGE2). The inhibition of glucose transport induced by lipolytic hormones such as glucagon, catecholamines or ACTH in the presence of adenosine deaminase was antagonized by PGE2. In contrast, inhibition of hexose transport by alkylxanthines was only partially (20–30%) attenuated by PGE2, although the eicosanoid had antagonized cyclic AMP accumulation and stimulation of lipolysis in response to all tested lipolytic agents. The inhibition of glucose transport by IBMX was immediate, whereas the lipolytic hormones (isoprenaline and ACTH) exhibited a latency of 2–3 min. In addition, the inhibition induced by the lypolytic hormones disappeared after cooling of the cells to 22°C, at which temperature IBMX was still inhibitory. Thus, the PGE2-sensitive component of the effect of lipolytic agents on glucose transport appears to be mediated by adenylate cyclase or its subunits Ns/Ni. The PGE2-insensitive effect of alkylxanthines probably reflects a direct interaction of the agents with a regulatory site at the transporter or a related protein.