Induction of apoptosis in proliferating lymphocytes by tricyclic antidepressants

We have previously found that tricyclic antidepressants (TCAs) induce apoptosis in quiescent human lymphocytes. The aim of the present study was to evaluate if TCAs induce apoptosis in proliferating human lymphocytes and in established blastoid lymphocytes also. The development of conA-induced lymphoblast populations was followed by measuring the CD25 membrane expression. Three TCA compounds were run with the following concentrations: imipramine (10, 20, 30, 40, 60 M), clomipramine (1, 10, 20, 30, 40 M) and citalopram (40, 60, 80, 100, 180 M). They all induced a dose-dependent apoptosis both in continuously transformed, as well as in established lymphoblasts. Preincubation of the TCA up to 48 h did not significantly increase induction of apoptosis. The three drugs tested were found to be potent inducers of apoptosis in proliferating lymphocytes. Furthermore, we found that the apoptotic populations in proliferating and in established blastoid lymphocytes were of f airly the same magnitude than in the corresponding population in TCA-incubated resting lymphocytes. In conclusion, we demonstrate that TCAs induce apoptosis in proliferating lymphocytes, as they do in quiescent lymphocytes. Furthermore, the exent of apoptosis was even more pronounced in TCA-incubated lymphoblasts compared to TCA-treated resting lymphocytes.     

Induction of apoptosis by novel synthesized acylamides of human lymphocytes

To investigate a pathway to apoptosis which may involve ceramides and to elucidate the minimum structure which leads to apoptosis, we synthesized several novel acylamides. Although the four synthesized compounds were different in structure from C2-ceramide, they caused Jurkat cells to undergo apoptosis. The most effective of them was N-myristoyl-D-alaninol (D-MA), as shown by DNA fragmentation (detected with propidium iodide) and a decrease in the mitochondrial transmembrane potential (DeltaPsi(m)) (detected with rhodamine 123). Nevertheless, peripheral blood leukocytes exhibited no change after D-MA exposure, like after C2-ceramide or anti-Fas antibody treatment. The DNA fragmentation and DeltaPsi(m) caused by D-MA were blocked by a caspase-3 specific inhibitor as in the case of anti-Fas antibody stimulation. Quantification of ceramides by metabolic labeling with [(14)C]palmitic acid and HPTLC showed no increases in the ceramide levels on stimulation with D-MA, C2-ceramide or anti-Fas antibodies. Furthermore, D-MA had an apoptosis-inducing effect on an anti-Fas-resistant subline of Jurkat cells. These data suggest that D-MA may cause apoptosis of Jurkat cells without distinct ceramide formation and that this apoptotic pathway is very comparable, i.e. not identical, to that induced by anti-Fas antibodies.     

Induction of apoptosis of splenic lymphocytes in mice by accelerated carbon ions

To assess the capacity of heavy ions to induce apoptosis in lymphocytes, mice have been irradiated with accelerated carbon ions (95 MeV/nucleon) at doses ranging from 0.1 to 4 Gy. Their spleens were removed 24 h later and gently dissociated to prepare a single cell suspension. Mononuclear cells were then maintained in culture at 37 degrees C, and the occurrence of apoptosis in these cells was analysed 24 h later. Lymphocytes were also irradiated in vitro, in the presence of Ac-DEVD-CHO, a potent caspase-3 and -7 inhibitor. Results from three experiments performed at the Grand Accelerateur National d'Ions Lourds (GANIL, Caen, France) are reported here. They indicate that carbon ions induce a marked, dose-dependent, reduction of the spleen weight and cellularity. However, in sharp contrast with spleen cells prepared from X-ray irradiated mice, only a slight increase of apoptosis is evidenced in cultured lymphocytes from mice irradiated with heavy ions. The significance of such results is discussed. So far, few data exist concerning the biological effects of heavy ions, in particular their capacity to induce apoptosis in lymphocytes; the present study provides useful clues for further investigations.     

Induction of apoptosis in granulosa cells by TNF alpha and its attenuation by glucocorticoids involve modulation of Bcl-2

Tumor necrosis factor alpha (TNF alpha) plays a role in mammalian ovarian follicular development, steroidogenesis, ovulation, luteolysis, and atresia, but the exact mechanism of TNF alpha action is not completely understood. Induction of apoptosis and suppression of steroidogenesis by TNF alpha in primary preovulatory rat and human granulosa cells, as well as, in human granulosa cells immortalized by mutated p53, were characterized in the present work. Dexamethasone (Dex) and hydrocortisone efficiently suppressed TNF alpha-induced apoptosis in granulosa cells. TNF alpha dramatically reduced intracellular levels of Bcl-2, while Dex abrogated this reduction. TNF alpha reduced considerably intracellular levels of StAR protein, a key regulating factor in steroidogenesis. This reduction can be explained only in part by elimination of cells through apoptosis, since loss of steroidogenic capacity was much higher and faster than the rate and extent of loss of cell viability induced by TNF alpha, suggesting independent mechanisms for TNF alpha-induction of apoptosis and TNF alpha-suppression of steroidogenesis.     

Power functions in physiology and pharmacology

The plasma clearance curves of certain substances which are removed from the circulation by the liver with high extraction ratios are best described by a power function of the form At^?a. This is most significant both in the calculation of metabolic turnover, and in the elucidation of the function of the liver.     

Ion-channels on parasite muscle: pharmacology and physiology

Ion-channels are essential components of excitable cells. This fact has been exploited in the development of anthelmintic agents; the majority of which act on nematode ion channels. The purpose of this review is to describe the site of action of some frequently used anthelmintic compounds: nAChRs and levamisole/pyrantel; Glu-Cls and avermectins/mylbemycins; GABA receptors and piperazine. Also described is some of the physiological and pharmacological data on other nematode muscle ion-channels which may prove attractive targets for future anthelmintic development: Ca²⁺ activated Cl⁻ channels; peptide gated chloride Cl⁻ channels; Ca²⁺ channels and potassium channels. Emphasis is placed on the pharmacological and physiological data from parasite tissue. Information on the genes involved in ion-channel formation and modulation are reviewed in detail elsewhere in this issue.     

Induction of hepatic microsomal calcium uptake by glucocorticoids

Hepatic microsomes take up calcium in the presence of ATP and oxalate. In either fed or fasted adrenalectomized rats injections of dexamethasone 18 hours and then again 1 hour prior to sacrifice increased uptake of calcium by microsomes. Injections of estradiol had no similar effect indicating that the stimulation might be specific to glucocorticoids. Injection of Actinomycin D, an inhibitor of protein synthesis, 1 hour prior to dexamethasone administration resulted in a complete block of the stimulation. It is therefore likely that the increased calcium uptake is due to the induction of the microsomal calcium activated ATP-ase. The onset of this effect occurred later than the induction of tyrosine-amino transferase (TAT). The present data, in conjunction with the previous demonstration of glucagon stimulation and insulin inhibition of this system, indicates that microsomes might serve as a modifier of intracellular calcium distribution.     

Induction of programmed cell death (apoptosis) in mature lymphocytes

The apoptosis of human peripheral blood lymphocytes was analyzed by the breakdown of DNA into oligonucleosome-sized fragments. The mature lymphocytes were rendered sensitive to apoptosis by either the omission of fetal bovine serum in the culture medium or the addition of polymyxin B. In the first case it was counteracted by phorbol myristate acetate. The possible involvement of protein kinase C in cell survival is pointed out.     

Two dopamine receptors: biochemistry, physiology and pharmacology

In 1979, two categories of dopamine (DA) receptors (designated as D-1 and D-2) were identified on the basis of the ability of a limited number of agonists and antagonists to discriminate between these two entities. In the past 5 years agonists and antagonists selective for each category of receptor have been identified. Using these selective drugs it has been possible to attribute the effects of DA upon physiological and biochemical processes to the stimulation of either a D-1 or a D-2 receptor. Thus, DA-induced enhancement of both hormone release from bovine parathyroid gland and firing of neurosecretory cells in the CNS of Lymnaea stagnalis has been attributed to stimulation of a D-1 receptor. Likewise, the DA-induced inhibition of the release of prolactin and alpha-MSH from the pituitary gland, as well as of acetylcholine, DA and beta-endorphin from brain, the DA-induced inhibition of chemo-sensory discharge in rabbit carotid body and the DA-induced hyperpolarization of neurosecretory cells in the CNS of Lymnaea stagnalis have been attributed to stimulation of a D-2 receptor. Independently two categories of DA receptors (designated as DA-1 and DA-2) were identified in the cardiovascular system. Stimulation of a DA-1 receptor increases the vascular cyclic AMP content and causes a relaxation of vascular smooth muscle in renal blood vessels, whereas stimulation of a DA-2 receptor inhibits the release of norepinephrine from certain postganglionic sympathetic neurons. Recent studies with the newly developed drugs discriminating between D-1 and D-2 receptors suggest however that the independently developed schemata for classification of dopamine receptors in either the central nervous and endocrine systems or the cardiovascular system are similar although maybe not completely identical.     

Induction of apoptosis by aryl-substituted diamines: role of aromatic group substituents and distance between nitrogens

A series of aromatic substituted diamines was synthesized and characterized for their cytotoxic profiles against human breast and prostate tumor cell lines. Following a structure function analysis of the effects of changes of the benzyl substituents and the distance between amino groups the most potent analogues were analyzed biologically and were shown to induce apoptosis. These compounds do not induce the enzyme SSAT or deplete intracellular polyamine levels, mechanisms demonstrated by other cytotoxic polyamine analogues.     

Rat genetics: attaching physiology and pharmacology to the genome

During the past five years, the Rat Genome Project has been rapidly gaining momentum, especially since the announcement in August 2000 of plans to sequence the rat genome. Combined with the wealth of physiological and pharmacological data for the rat, the genome sequence should facilitate the discovery of mammalian genes that underlie the physiological pathways that are involved in disease. Most importantly, this combined physiological and genomic information should also lead to the development of better pre-clinical models of human disease, which will aid in the development of new therapeutics.     

Oxidant-mediated mitochondrial injury in eosinophil apoptosis: enhancement by glucocorticoids and inhibition by granulocyte-macrophage colony-stimulating factor

The mainstay of asthma therapy, glucocorticosteroids (GCs) have among their therapeutic effects the inhibition of inflammatory cytokine production and induction of eosinophil apoptosis. In the absence of prosurvival cytokines (e.g., GM-CSF), eosinophils appear to be short-lived, undergoing apoptosis over 96 h in vitro. In a dose-dependent manner, GC further enhances apoptosis, while prosurvival cytokines inhibit apoptosis and antagonize the effect of GC. The mechanisms of eosinophil apoptosis, its enhancement by GC, and antagonism of GC by GM-CSF are not well-understood. As demonstrated in this study, baseline apoptosis of eosinophils resulted from oxidant-mediated mitochondrial injury that was significantly enhanced by GC. Mitochondrial injury was detected by early and progressive loss of mitochondrial membrane potential and the antioxidant protein, Mn superoxide dismutase (SOD). Also observed was the activation/translocation of the proapoptotic protein, Bax, to mitochondria. Underscoring the role of oxidants was the inhibition of mitochondrial changes and apoptosis with culture in hypoxia, or pretreatment with a flavoprotein inhibitor or a SOD mimic. GCs demonstrated early (40 min) and late (16 h) activation of proapoptotic c-Jun NH2-terminal kinase (JNK) and decreased the antiapoptotic protein X-linked inhibitor of apoptosis, a recently demonstrated inhibitor of JNK activation. Similarly, inhibition of JNK prevented GC-enhanced mitochondrial injury and apoptosis. Importantly, GM-CSF prevented GC-induced loss of X-linked inhibitor of apoptosis protein, late activation of JNK, and mitochondrial injury even in the face of unchanged oxidant production, loss of MnSOD, and early JNK activation. These data demonstrate that oxidant-induced mitochondrial injury is pivotal in eosinophil apoptosis, and is enhanced by GC-induced prolonged JNK activation that is in turn inhibited by GM-CSF.     

Induction of apoptosis by Drosophila Myc

Myc proteins are essential regulators of cellular growth and proliferation during normal development. Activating mutations in myc genes result in excessive growth and are frequently associated with human cancers. At the same time, forced expression of Myc sensitizes vertebrate cells towards different pro-apoptotic stimuli. Recently, the ability of overexpressed Myc to induce cell-autonomous apoptosis has been shown to be evolutionarily conserved in Drosophila Myc (dMyc). Here, we show that dMyc induced apoptosis is accompanied by the induction of Drosophila p53 mRNA, but that dp53 activity is not essential for dMyc's ability to induce apoptosis. Conversely, larvae carrying a hypomorphic dmyc mutation are more resistant to the apoptosis-promoting effects of X-irradiation. These data suggest that the control of apoptosis is a physiological function of Myc and that dMyc might play a role in the response to DNA damage.     

Induction of apoptosis by cancer chemotherapy

Studies performed over the past five years have demonstrated that there are two major cell-intrinsic pathways for inducing apoptosis, one that begins with ligation of cell surface death receptors and another that involves mitochondrial release of cytochrome c. Several reports have suggested that anticancer drugs kill susceptible cells by inducing expression of death receptor ligands, especially Fas ligand (FasL). Other reports have indicated that chemotherapeutic agents trigger apoptosis by inducing release of cytochrome c from mitochondria. In this review, we describe the two prototypic death pathways, indicate experimental approaches for distinguishing whether chemotherapeutic agents trigger one pathway or the other, summarize current understanding of the role of the two pathways in chemotherapy-induced apoptosis, and discuss the implications of these studies for mechanisms of resistance to chemotherapeutic agents.