DNA damage is an early event in doxorubicin-induced cardiac myocyte death

Anthracyclines are antitumor agents the main clinical limitation of which is cardiac toxicity. The mechanism of this cardiotoxicity is thought to be related to generation of oxidative stress, causing lethal injury to cardiac myocytes. Although protein and lipid oxidation have been documented in anthracycline-treated cardiac myocytes, DNA damage has not been directly demonstrated. This study was undertaken to determine whether anthracyclines induce cardiac myocyte DNA damage and whether this damage is linked to a signaling pathway culminating in cell death. H9c2 cardiac myocytes were treated with the anthracycline doxorubicin at clinically relevant concentrations, and DNA damage was assessed using the alkaline comet assay. Doxorubicin induced DNA damage, as shown by a significant increase in the mean tail moment above control, an effect ameliorated by inclusion of a free radical scavenger. Repair of DNA damage was incomplete after doxorubicin treatment in contrast to the complete repair observed in H2O2-treated myocytes after removal of the agent. Immunoblot analysis revealed that p53 activation occurred subsequent in time to DNA damage. By a fluorescent assay, doxorubicin induced loss of mitochondrial membrane potential after p53 activation. Chemical inhibition of p53 prevented doxorubicin-induced cell death and loss of mitochondrial membrane potential without preventing DNA damage, indicating that DNA damage was proximal in the events leading from doxorubicin treatment to cardiac myocyte death. Specific doxorubicin-induced DNA lesions included oxidized pyrimidines and 8-hydroxyguanine. DNA damage therefore appears to play an important early role in anthracycline-induced lethal cardiac myocyte injury through a pathway involving p53 and the mitochondria.     

Massive telomere loss is an early event of DNA damage-induced apoptosis

Chromosomal stability and cell viability require a proficient telomeric end-capping function. In particular, telomere dysfunction because of either critical telomere shortening or because of mutation of telomere-binding proteins results in increased apoptosis and/or cell arrest. Here, we show that, in turn, DNA damage-induced apoptosis results in a dramatic telomere loss. In particular, using flow cytometry for simultaneous detection of telomere length and apoptosis, we show that cells undergoing apoptosis upon DNA damage also exhibit a rapid and dramatic loss of telomeric sequences. This telomere loss occurs at early stages of apoptosis, because it does not require caspase-3 activation, and it is induced by loss of the mitochondrial membrane potential (Deltapsi(m)) and production of reactive oxygen species. These observations suggest a direct effect of mitochondrial dysfunction on telomeres.     

Polarization of the endomembrane system is an early event in fucoid zygote development

Background  

Fucoid zygotes are excellent experimental organisms for investigating mechanisms that establish cell polarity and determine the site of tip growth. A common feature of polarity establishment is targeting endocytosis and exocytosis (secretion) to localized cortical domains. We have investigated the spatiotemporal development of endomembrane asymmetry in photopolarizing zygotes, and examined the underlying cellular physiology.     

Plasma membrane depolarization without repolarization is an early molecular event in anti-Fas-induced apoptosis

The movement of intracellular monovalent cations has previously been shown to play a critical role in events leading to the characteristics associated with apoptosis. A loss of intracellular potassium and sodium occurs during apoptotic cell shrinkage establishing an intracellular environment favorable for nuclease activity and caspase activation. We have now investigated the potential movement of monovalent ions in Jurkat cells that occur prior to cell shrinkage following the induction of apoptosis. A rapid increase in intracellular sodium occurs early after apoptotic stimuli suggesting that the normal negative plasma membrane potential may change during cell death. We report here that diverse apoptotic stimuli caused a rapid cellular depolarization of Jurkat T-cells that occurs prior to and after cell shrinkage. In addition to the early increase in intracellular Na(+), (86)Rb(+) studies reveal a rapid inhibition of K(+) uptake in response to anti-Fas. These effects on Na(+) and K(+) ions were accounted for by the inactivation of the Na(+)/K(+)-ATPase protein and its activity. Furthermore, ouabain, a cardiac glycoside inhibitor of the Na(+)/K(+)-ATPase, potentiated anti-Fas-induced apoptosis. Finally, activation of an anti-apoptotic signal, i.e. protein kinase C, prevented both cellular depolarization in response to anti-Fas and all downstream characteristics associated with apoptosis. Thus cellular depolarization is an important early event in anti-Fas-induced apoptosis, and the inability of cells to repolarize via inhibition of the Na(+)/K(+)-ATPase is a likely regulatory component of the death process.     

Focal adhesion disassembly is an essential early event in hepatic stellate cell chemotaxis

Chemotaxis (i.e., directed migration) of hepatic stellate cells to areas of inflammation is a requisite event in the liver's response to injury. Previous studies of signaling pathways that regulate stellate cell migration suggest a key role for focal adhesions, but the exact function of these protein complexes in motility remains unclear. Focal adhesions attach a cell to its substrate and therefore must be regulated in a highly coordinated manner during migration. To test the hypothesis that focal adhesion turnover is an essential early event for chemotaxis in stellate cells, we employed a live-cell imaging technique in which chemotaxis was induced by locally stimulating the tips of rat stellate cell protrusions with platelet-derived growth factor-BB (PDGF). Focal adhesions were visualized with an antibody directed against vinculin, a structural component of the focal adhesion complex. PDGF triggered rapid disassembly of adhesions within 6.25 min, subsequent reassembly by 12.5 min, and continued adhesion assembly in concert with the spreading protrusion until the completion of chemotaxis. Blockade of adhesion disassembly by growing cells on fibronectin or treatment with nocodazole prevented a chemotactic response to PDGF. Augmentation of adhesion disassembly with ML-7 enhanced the chemotactic response to PDGF. These data suggest that focal adhesion disassembly is an essential early event in stellate cell chemotaxis in response to PDGF.     

Phosphorylation of nuclear protein is an early event in TGF beta 1 action.

Transforming growth factor beta (TGF beta) is a family of polypeptides that modulate growth and differentiation. TGF beta exerts its effects on target cells through interaction with specific cell surface receptors, but the signal transduction pathways are as yet largely unresolved. In this study we report that the growth inhibitory action of TGF beta on mink lung CCl 64 cells is associated with a rapid and transient phosphorylation of a number of nuclear proteins. In parallel, a transient expression of the immediate early gene jun B is observed. The expression of jun B can be inhibited by the protein kinase inhibitor H7 and can be augmented by the phosphatase inhibitor okadaic acid. Thus, protein phosphorylation can be a possible mechanism through which TGF beta 1 initiates early genomic responses.     

Reduction in mitochondrial membrane potential is an early event in Fas-independent CTL-mediated apoptosis.

Cytotoxic T lymphocytes (CTL) can destroy target cells via the Fas-mediated pathway or the granule-mediated pathway. We used Fas-negative target cells to examine for target-cell reduction in mitochondrial membrane potential (DeltaPsi(m)) induced by intact CTL via the granule-mediated pathway. We find that reduction in DeltaPsi(m) is an early step in Fas-independent CTL killing of target cells that precedes phosphatidyl serine translocation, cytosolic protein release, or loss of plasma membrane integrity. Target-cell reduction in DeltaPsi(m) and cytoplasmic protein release in Fas-independent CTL killing were inhibited by N-carbobenzoxy-Ala-Pro-Phe chloromethyl ketone, but not by caspase inhibitors N-carbobenzoxy-Val-Ala-Asp fluoromethyl ketone (z-VAD-fmk) or N-carbobenzoxy-Asp-Glu-Val-Asp fluoromethyl ketone (z-DEVD-fmk). This contrasts with Fas-mediated apoptosis, in which the reduction in DeltaPsi(m) can be inhibited by z-VAD-fmk or z-DEVD-fmk. Assessing the changes in target-cell DeltaPsi(m) can provide for a sensitive and rapid means with which to monitor CTL activity.     

HVEM signaling in monocytes is mediated by intracellular calcium mobilization

Herpes virus entry mediator (HVEM) is a member of the TNF receptor (TNFR) superfamily and is expressed on many immune cells, including T and B cells, NK cells, monocytes, and neutrophils. Interaction of HVEM with its ligand, LIGHT, costimulates T cells and increases the bactericidal activity of monocytes and neutrophils. The interaction recruits cytoplasmic TNFR-associated factor adaptor proteins to the intracellular domain of HVEM. This leads to NFkappaB activation as a result of IkappaBalpha degradation and/or JNK/AP-1 activation, and ultimately results in the expression of genes required for cell survival, cytokine production, or cell proliferation. In this study, we show that treatment of human monocytes with recombinant human LIGHT (rhLIGHT) induces rapid elevation of intracellular calcium concentration ([Ca(2+)](i)) in a HVEM-specific manner in parallel with TNF-alpha production, and enhances the bactericidal activities of monocytes. Immunoprecipitation and Western blotting analyses revealed phosphorylation of phospholipase Cgamma1 (PLCgamma1) but not PLCgamma2. rhLIGHT-induced Ca(2+)response was completely abolished by silencing PLCgamma1, or preincubating monocytes with PLC inhibitors, antagonists of the inositol-1,4,5-triphosphate receptor, or [Ca(2+)](i) chelators. Furthermore, these PLC/Ca(2+) inhibitors also blocked rhLIGHT-mediated IkappaBalpha degradation, generation of reactive oxygen species, TNF-alpha production and the bactericidal activities of monocytes. Our results indicate that Ca(2+)is a downstream mediator of the LIGHT/HVEM interaction in monocytes.     

Inositol 1,4,5-trisphosphate-induced calcium mobilization is localized in Xenopus oocytes

Injection of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) into the animal pole of Xenopus oocytes induced membrane depolarization due to the internal mobilization of calcium, which activates a chloride conductance. Repetitive injections of Ins(1,4,5)P3 results in desensitization probably as a result of depletion of the internal store of calcium. Desensitization was restricted to the region surrounding the site of injection. Injection of Ins(1,4,5)P3 at one position induced desensitization, which failed to spread to a neighbouring region (ca. 200 microns away). Even when sufficient Ins(1,4,5)P3 was injected to induce calcium oscillations, there was still no evidence for the effects of Ins(1,4,5)P3 spreading to neighbouring regions. The fact that periodic calcium transients could also be established by the repetitive injection of small amounts of Ins(1,4,5)P3 suggests that calcium oscillations may also be localized. It is concluded that the Ins(1,4,5)P3-sensitive store of calcium comprises separate local compartments that can be activated independently of each other.     

Increased phosphatidylinositol metabolism is an important but not an obligatory early event in B lymphocyte activation

The phosphatidylinositol (PI) response has been implicated in membrane signaling and cell activation. The role of phospholipid metabolism among the early events in B cell activation has not been clear. We have treated murine B cells with anti-Ig antibody and lipopolysaccharide (LPS) and have found that, although anti-IgM induces the PI response, LPS does not. The increase in metabolic labeling of PI is specific to PI, and not the phosphatidylinositols. Anti-IgM unresponsive B cells from CBA/N mice, which may correspond to a specific functional subpopulation of normal B cells, do not increase PI metabolism in response to anti-IgM, nor do they undergo blastogenesis or DNA synthesis. Moreover, when these deficient B cells are given a stimulus sufficient to drive them into S (LPS + anti-IgM), there is still no corresponding activation of PI metabolism. These results are consistent with a two-signal model of xid B cell activation by anti-IgM. One very early signal primes the cells but does not induce the PI response. A second early signal is supplied by LPS. This signal sustains cells in the activated state, allowing them to receive yet other signals to proceed through G1 and progress further along the cell cycle. A similar sequence of events may occur in the normal B cell, with the first signal provided by priming with anti-IgM, and the second signal, the PI response, supported by a sufficiently high dose of anti-IgM to induce PI turnover and maintain the cell in G1.     

Retinoblastoma protein dephosphorylation is an early event of cellular response to prooxidant conditions

The modification of intracellular redox conditions with diethylmaleate (DEM), a glutathione-depleting agent, induces a p53-independent growth arrest mediated by the accumulation of p21(waf1) mRNA and protein. The same treatment also induces the retinoblastoma protein (pRb) dephosphorylation. This dephosphorylation (i) is very fast, being observed already 5 min after the exposure of the cells to DEM, (ii) is dependent on the prooxidant effects of DEM, being prevented by the treatment with N-acetylcysteine and (iii) is completely reversible, since the rephosphorylation of pRb is promptly obtained upon the removal of the glutathione-depleting agent from the culture medium. The dephosphorylation of pRb is independent of the accumulation of p21(waf1) induced by DEM; in fact, p21(waf1) levels start to increase much later after DEM treatment and accordingly cyclin-dependent kinase activities are not yet induced when pRb is already dephosphorylated following DEM treatment. Finally, pRb dephosphorylation is catalyzed by phosphatases activated by DEM treatment.     

Collapsin response mediator protein-2 hyperphosphorylation is an early event in Alzheimer's disease progression

Collapsin response mediator protein 2 (CRMP2) is an abundant brain-enriched protein that can regulate microtubule assembly in neurons. This function of CRMP2 is regulated by phosphorylation by glycogen synthase kinase 3 (GSK3) and cyclin-dependent kinase 5 (Cdk5). Here, using novel phosphospecific antibodies, we demonstrate that phosphorylation of CRMP2 at Ser522 (Cdk5-mediated) is increased in Alzheimer's disease (AD) brain, while CRMP2 expression and phosphorylation of the closely related isoform CRMP4 are not altered. In addition, CRMP2 phosphorylation at the Cdk5 and GSK3 sites is increased in cortex and hippocampus of the triple transgenic mouse [presenilin-1 (PS1)(M146V)KI; Thy1.2-amyloid precursor protein (APP)(swe); Thy1.2tau(P301L)] that develops AD-like plaques and tangles, as well as the double (PS1(M146V)KI; Thy1.2-APP(swe)) transgenic mouse. The hyperphosphorylation is similar in magnitude to that in human AD and is evident by 2 months of age, ahead of plaque or tangle formation. Meanwhile, there is no change in CRMP2 phosphorylation in two other transgenic mouse lines that display elevated amyloid beta peptide levels (Tg2576 and APP/amyloid beta-binding alcohol dehydrogenase). Similarly, CRMP2 phosphorylation is normal in hippocampus and cortex of Tau(P301L) mice that develop tangles but not plaques. These observations implicate hyperphosphorylation of CRMP2 as an early event in the development of AD and suggest that it can be induced by a severe APP over-expression and/or processing defect.     

Elevation in phosphatidylethanolamine is an early but not essential event for cardiac cell differentiation

The biosynthesis of phosphatidylethanolamine was examined during differentiation of P19 teratocarcinoma cells into cardiac myocytes. P19 cells were induced to undergo differentiation into cardiac myocytes by the addition of dimethyl sulfoxide to the medium. Immunofluorescence labeling confirmed the expression of striated myosin 10 days postinduction of differentiation. The content of phosphatidylethanolamine increased significantly within the first 2 days of differentiation. [1,3-(3)H]Glycerol incorporation into phosphatidylethanolamine was increased 7.2-fold during differentiation, indicating an elevation in de novo synthesis from 1, 2-diacyl-sn-glycerol. The mechanism for the increase in phosphatidylethanolamine levels during cardiac cell differentiation was a 2.8-fold increase in the activity of ethanolaminephosphotransferase, the 1,2-diacyl-sn-glycerol utilizing reaction of the cytidine 5'-diphosphate-ethanolamine pathway of phosphatidylethanolamine biosynthesis. Incubation of P19 cells with the phosphatidylethanolamine biosynthesis inhibitor 8-(4-chlorophenylthio)-cAMP inhibited the differentiation-induced elevation in phosphatidylethanolamine levels but did not affect the expression of striated myosin. The results suggest that elevation in phosphatidylethanolamine is an early event of P19 cell differentiation into cardiac myocytes, but is not essential for differentiation to proceed.     

Tyrosine phosphorylation is an early and specific event involved in primary keratinocyte differentiation.

Very little is known about early molecular events triggering epithelial cell differentiation. We have examined the possible role of tyrosine phosphorylation in this process, as observed in cultures of primary mouse keratinocytes after exposure to calcium or 12-O-tetradecanoylphorbol-13-acetate (TPA). Immunoblotting with phosphotyrosine-specific antibodies as well as direct phosphoamino acid analysis revealed that induction of tyrosine phosphorylation occurs as a very early and specific event in keratinocyte differentiation. Very little or no induction of tyrosine phosphorylation was observed in a keratinocyte cell line resistant to the differentiating effects of calcium. Treatment of cells with tyrosine kinase inhibitors prevented induction of tyrosine phosphorylation by calcium and TPA and interfered with the differentiative effects of these agents. These results suggest that specific activation of tyrosine kinase(s) may play an important regulatory role in keratinocyte differentiation.     

Decreased intracellular Na+ concentration is an early event in murine erythroleukemic cell differentiation

A decrease in Na+/K+-pump activity is an early event of Friend murine erythroleukemic (MEL) cell differentiation along the erythroid pathway. This decreased Na+/K+-pump activity has been proposed to be an essential step in differentiation which would cause a rise in intracellular Na+ concentration and then, by means of Na+/Ca2+ exchange, an increase in intracellular Ca2+. An increase in intracellular Ca2+ has been proposed to be essential for induction of differentiation. A critical prediction of this Na+-Ca2+ hypothesis is the rise in intracellular Na+. To test this prediction we have measured intracellular Na+ using a novel triple isotope method involving 3H2O, [14C]sucrose, and 22Na to measure total water, extracellular fluid, and Na+, respectively. 22Na equilibration occurred in less than 10 min. In uninduced cells, intracellular Na+ was 15.2 +/- 2.2 mM (S.D., n = 22); after induction for 14-16 h with dimethyl sulfoxide, intracellular Na+ decreased significantly (p less than 0.0001) to 8.4 +/- 1.4 mM (n = 21). The time course of the decline in intracellular Na+ paralleled that of the decrease in the Na+/K+-pump activity. These results are in direct contradiction to the Na+-Ca2+ hypothesis and suggest that observed changes in Na+/K+-pump activity can be explained solely on the basis of changes in intracellular Na+. The drop in intracellular Na+ is due to a decrease in Na+ influx. We suggest, however, that the decrease in the Na+ influx is not itself an essential event of differentiation, but may be induced by a change in the flux of another ion coupled to Na+.     

Disruption of mitochondria is an early event during dolichyl monophosphate-induced apoptosis in U937 cells

Dolichyl monophosphate (Dol-P) is involved in the attachment of carbohydrate chains to proteins in the formation of N-linked glycoprotein. We found that this compound induces apoptosis in human leukemia U937 cells. During this apoptotic execution, the increase of plasma membrane fluidity (5-20 min), reduction in mitochondrial transmembrane potential (delta psi m) and translocation of apoptosis-inducing factor (1-3 hr), caspase-3-like protease activation (2-4 hr), chromatin condensation and DNA ladder formation (3-4 hr) were observed successively. In this study, we examined mitochondrial morphological changes by electron microscopy and delta psi m by JC-1 from immediately after treatment of Dol-P. After 5 min of treatment, we observed clearly that mitochondrial cristae began to be disrupted ultrastructurally and almost all the cristae were disintegrated after 1 hr of treatment. The delta psi m of Dol-P treated cells was reduced to 34% as compared with that of control cells immediately after treatment and was quartered within 1 hr. The reduction in delta psi m was not inhibited by cyclosporin A, N-acetyl-L-cysteine and vitamin E. These results indicate that mitochondrial disruption is one of the first triggering events of Dol-P-induced apoptosis.