Hyperphosphorylation of histone H2A.X and dephosphorylation of histone H1 subtypes in the course of apoptosis.
(17/89)Chromatin condensation paralleled by DNA fragmentation is one of the most important nuclear events occurring during apoptosis. Histone modifications, and in particular phosphorylation, have been suggested to affect chromatin function and structure during both cell cycle and cell death. We report here that phosphate incorporation into all H1 subtypes decreased rapidly after induction of apoptosis, evidently causing a strong reduction in phosphorylated forms of main H1 histone subtypes. H1 dephosphorylation is accompanied by chromatin condensation preceding the onset of typical chromatin oligonucleosomal fragmentation, whereas H2A.X hyperphosphorylation is strongly correlated to apoptotic chromatin fragmentation. Using various kinase inhibitors we were able to exclude some of the possible kinases which can be involved directly or indirectly in phosphorylation of histone H2A.X. Neither DNA-dependent protein kinase, protein kinase A, protein kinase G, nor the kinases driven by the mitogen-activated protein kinase (MAP) pathway appear to be responsible for H2A.X phosphorylation. The protein kinase C activator phorbol 12-myristate 13-acetate (PMA), however, markedly reduced the induction of apoptosis in TNFalpha-treated cells with a simultaneous change in the phosphorylation pattern of histone H2A.X. Hyperphosphorylation of H2A.X in apoptotic cells depends indirectly on activation of caspases and nuclear scaffold proteases as shown in zVAD-(OMe)-fmk- or zAPF-cmk-treated cells, whereas the dephosphorylation of H1 subtypes seems to be influenced solely by caspase inhibitors. Together, these results illustrate that H1 dephosphorylation and H2A.X hyperphosphorylation are necessary steps on the apoptotic pathway. (+info)
Induction of mitochondrial alternative oxidase in response to a cell signal pathway down-regulating the cytochrome pathway prevents programmed cell death.
(18/89)Treatment of tobacco (Nicotiana tabacum L. cv Petit Havana SR1) cells with cysteine (Cys) triggers a signal pathway culminating in a large loss of mitochondrial cytochrome (cyt) pathway capacity. This down-regulation of the cyt path likely requires events outside the mitochondrion and is effectively blocked by cantharidin or endothall, indicating that protein dephosphorylation is one critical process involved. Generation of reactive oxygen species, cytosolic protein synthesis, and Ca(2+) flux from organelles also appear to be involved. Accompanying the loss of cyt path is a large induction of alternative oxidase (AOX) protein and capacity. Induction of AOX allows the cells to maintain high rates of respiration, indicating that the lesion triggered by Cys is in the cyt path downstream of ubiquinone. Consistent with this, transgenic (AS8) cells unable to induce AOX (due to the presence of an antisense transgene) lose all respiratory capacity upon Cys treatment. This initiates in AS8 a programmed cell death pathway, as evidenced by the accumulation of oligonucleosomal fragments of DNA as the culture dies. Alternatively, wild-type cells remain viable and eventually recover their cyt path. Induction of AOX in response to a chemical inhibition of the cyt path (by antimycin A) is also dependent upon protein dephosphorylation and the generation of reactive oxygen species. Common events required for both down-regulation of the cyt path and induction of AOX may represent a mechanism to coordinate the biogenesis of these two electron transport paths. Such coordinate regulation may be necessary, not only to satisfy metabolic demands, but also to modulate the initiation of a programmed cell death pathway responsive to mitochondrial respiratory status. (+info)
Transgenic plant cells lacking mitochondrial alternative oxidase have increased susceptibility to mitochondria-dependent and -independent pathways of programmed cell death.
(19/89)The plant mitochondrial electron transport chain is branched such that electrons at ubiquinol can be diverted to oxygen via the alternative oxidase (AOX). This pathway does not contribute to ATP synthesis but can dampen the mitochondrial generation of reactive oxygen species. Here, we establish that transgenic tobacco (Nicotiana tabacum L. cv Petit Havana SR1) cells lacking AOX (AS8 cells) show increased susceptibility to three different death-inducing compounds (H(2)O(2), salicylic acid [SA], and the protein phosphatase inhibitor cantharidin) in comparison with wild-type cells. The timing and extent of AS8 cell death are very similar among the three treatments and, in each case, are accompanied by the accumulation of oligonucleosomal fragments of DNA, indicative of programmed cell death. Death induced by H(2)O(2) or SA occurs by a mitochondria-dependent pathway characterized by cytochrome c release from the mitochondrion. Conversely, death induced by cantharidin occurs by a pathway without any obvious mitochondrial involvement. The ability of AOX to attenuate these death pathways may relate to its ability to maintain mitochondrial function after insult with a death-inducing compound or may relate to its ability to prevent chronic oxidative stress within the mitochondrion. In support of the latter, long-term treatment of AS8 cells with an antioxidant compound increased the resistance of AS8 cells to SA- or cantharidin-induced death. The results indicate that plants maintain both mitochondria-dependent and -independent pathways of programmed cell death and that AOX may act as an important mitochondrial "survival protein" against such death. (+info)
Role of protein phosphatases in hypoxic preconditioning.
(20/89)To find a protein kinase C (PKC)-independent preconditioning mechanism, hypoxic preconditioning (HP; i.e., 10-min anoxia and 10-min reoxygenation) was applied to isolated rat hearts before 60-min global ischemia. HP led to improved recovery of developed pressure and reduced end-diastolic pressure in the left ventricle during reperfusion. Protection was unaffected by the PKC inhibitor bisindolylmaleimide (BIM; 1 micromol/l). It was abolished by the inhibitor of protein phosphatases 1 and 2A cantharidin (20 or 5 micromol/l) and partially enhanced by the inhibitor of protein phosphatase 2A okadaic acid (5 nmol/l). In adult rat cardiomyocytes treated with BIM and exposed to 60-min simulated ischemia (anoxia, extracellular pH 6.4), HP led to attenuation of anoxic Na(+)/Ca(2+) overload and of hypercontracture, which developed on reoxygenation. This protection was prevented by treatment with cantharidin but not with okadaic acid. In conclusion, HP exerts PKC-independent protection on ischemic-reperfused rat hearts and cardiomyocytes. Protein phosphatase 1 seems a mediator of this protective mechanism. (+info)
Ca2+-ions and pattern control in Hydra.
(21/89)The fresh water polyp Hydraforms buds which develop a foot at their base and separate from the parent. In the strain H. vulgaris (Zurich), various compounds including phorbolesters, diacylglycerols, cantharidin and Li+-ions were found to prevent foot formation at the bud's base. Therewith, the bud transforms into a branch which persists at the parent. Other strains were found to be unaffected by such treatments. Here we show that a reduced Ca2+-ion concentration of the culture medium causes branch formation in the H. vulgaris (Zurich) strain but not in the other strains tested. However, all strains tested transformed their buds into branches when the medium was enriched with Ba2+ and Sr2+ ions. We suggest that the various treatments either reduce the internal concentration of Ca2+-ions by stimulating Ca2+-ion export or compete with Ca2+-ions at theirtarget. H. vulgaris (Zurich) isthe most sensitive strain tested and appears to have the most efficient Ca2+-pumps. This appears to be necessary forthese animals derived from a lake which is extremely rich in Ca2+ ions. (+info)
Antiadrenergic effects of adenosine A(1) receptor-mediated protein phosphatase 2a activation in the heart.
(22/89)The ability of adenosine A(1) receptors to activate type 2a protein phosphatase (PP2a) and account for antiadrenergic effects was investigated in rat myocardial preparations. We observed that the adenosine A(1) receptor agonist N(6)-cyclopentyladenosine (CPA) significantly reduces the isoproterenol-induced increase in left ventricular developed pressure of isolated heats, and this effect is blocked by pretreatment of hearts with the PP2a inhibitor cantharidin. CPA alone or given in conjunction with isoproterenol stimulation decreases phosphorylation of phospholamban and troponin I in ventricular myocytes. These dephosphorylations are blocked by an adenosine A(1) receptor antagonist and by PP2a inhibition with okadaic acid. Adenosine A(1) receptor activation was also shown to increase carboxymethylation of the PP2a catalytic subunit (PP2a-C) and cause translocation of PP2a-C to the particulate fraction in ventricular myocytes. These results support the hypothesis that adenosine A(1) receptor activation leads to methylation of PP2a-C and subsequent translocation of the PP2a holoenzyme. Increases in localized PP2a activity lead to dephosphorylation of key cardiac proteins responsible for the positive inotropic effects of beta-adrenergic stimulation. (+info)
Pharmacokinetic profile of meropenem, administered at 500 milligrams every 8 hours, in plasma and cantharidin-induced skin blister fluid.
(23/89)The pharmacokinetic disposition of meropenem, administered at 500 mg every 8 h, in plasma and cantharidin-induced blister fluid is described. Peak meropenem concentrations in blister fluid lagged behind peak meropenem concentrations in plasma, while a lower elimination rate from blister fluid was also noted. The mean penetration of meropenem into blister fluid was 67%. The pharmacokinetic profile of meropenem in blister fluid supports the utility of this dose in the management of skin and soft tissue infections. (+info)
Chemosensitivity of salivary gland and oral cancer cell lines.
(24/89)Chemosensitivity of salivary gland and oral cancer cell lines to 14 antitumor drugs was tested using a tetrazolium-based colorimetric assay and the relative antitumor activity (RAA) of the drugs was compared. Adriamycin (ADM), methotrexate and fluorouracil (5-FU) showed the most potent RAA against the cell lines while cantharidin did not. The rank orders of other 10 drugs against each cell line differed from each other. (+info)