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.     

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.     

Molecular characterization of an anthocyanin-related glutathione S-transferase gene in cyclamen

Anthocyanins are a subclass of flavonoids and are a major contributor to flower colors ranging from red to blue and purple. Previous studies in model and ornamental plants indicate a member of the glutathione S-transferase (GST) gene family is involved in vacuolar accumulation of anthocyanins. In order to identify the anthocyanin-related GST in cyclamen, degenerate PCR was performed using total RNA from immature young petals. Four candidates of GSTs (CkmGST1 to CkmGST4) were isolated. Phylogenetic analysis indicated that CkmGST3 was closely related to PhAN9, an anthocyanin-related GST of petunia, and this clade was clustered with other known anthocyanin-related GSTs. Expression analysis at different developmental stages of petals revealed that CkmGST3 was strongly expressed in paler pigmented petals than in fully pigmented petals, in contrast to the constitutive expression of the other three candidates during petal development. This expression pattern of CkmGST3 was correlated with those of other anthocyanin biosynthetic genes such as CkmF3'5'H and CkmDFR2. Molecular complementation of Arabidopsis tt19, a knockout mutant of an anthocyanin-related GST gene, demonstrated that CkmGST3 could complement the anthocyanin-less phenotype of tt19. Transgenic plants that expressed the other three CkmGSTs did not show anthocyanin accumulation. These results indicate CkmGST3 functions in anthocyanin accumulation in cyclamen.     

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.     

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.     

Generation of reactive oxygen species is an early event in dolichyl phosphate-induced apoptosis

The mechanism of induction of apoptosis by dolichyl phosphate (Dol-P) was investigated in U937 cells. Studies using isolated mitochondria revealed that the respiratory complex II activity was almost completely inhibited by 20 microg/ml of Dol-P but not by the same concentration of dolichol. Activities of complex I and III were also inhibited by Dol-P, but nearly 50% of activity still remained at 20 microg/ml. Dol-P induced release of cytochrome-c from the isolated mitochondria. Fluorometric microtiter plate assay revealed that generation of reactive oxygen species (ROS) increased in a time-dependent manner. Flow cytometric analysis also indicated that Dol-P caused loss of mitochondrial membrane potential (Deltapsi(m)) and increased ROS generation. The addition of the antioxidant pyrrolidine dithiocarbamate (PDTC) significantly inhibited Dol-P-induced ROS generation and activation of caspase-3. A specific inhibitor of respiratory complex II, thenoyltrifluoroacetone (TTFA), increased ROS generation, potentially mimicking the consequence of inhibition of electron flow at complex II by Dol-P in U937 cells. Electron microscopy revealed that mitochondria became swollen and spherical in shape by the treatment with Dol-P. Neither the tyrosine kinase inhibitor k252a nor mitogen activated protein kinase/extracellular signal-regulated kinase kinase (MEK) inhibitors PD98059 and U0126 inhibited the Dol-P-induced apoptosis. Together, these results suggest that the direct disruption of mitochondrial respiratory complexes and the consequent ROS generation play a critical role in the initiation of Dol-P-induced apoptosis.     

The Drosophila glutathione S-transferase 1-1 is encoded by an intronless gene at 87B

The Drosophila glutathione S-transferase 1-1 is a dimer of a 209 amino acid subunit, designated DmGST1. DmGST1 is encoded by a member of a multigene family. Sequence analysis of a genomic clone for GST1 revealed that it is encoded by an intronless gene. We designate this gene and its other family members the GST D genes in the glutathione S-transferase gene superfamily. The Drosophila GST D genes are mapped by in situ hybridization to chromosome 3R at 87B of the polytene chromosome, which is flanked by the two clusters of hsp70 genes at 87A7 and 87C1. Cytogenetic data in the literature indicated that a puff occurred in this region under heat shock. We report that the glutathione S-transferase activity in Kco cells as determined by conjugation with 1-chloro-2,4-dinitrobenzene is elevated slightly to two-fold under heat shock. The implication of this finding is discussed.     

The gene for the microsomal glutathione S-transferase is on human chromosome 12

The microsomal glutathione S-transferase (GST) is a unique membrane-bound GST structurally distinct from the cytosolic GSTs. A cDNA encoding this 154 amino acid protein has recently been isolated and characterized. Using the cDNA as the hybridization probe, we now report the assignment of the human microsomal GST gene to chromosome 12 through the use of a panel of mouse-human somatic cell hybrid lines. This locus has recently been designated as GST 12. In addition, genomic Southern blotting data suggest that the human microsomal GST is encoded by a single- or very-low-copy gene. Therefore, the human GST gene superfamily resides on at least four separate chromosomes: 1 (GST 1), 6 (GST 2), 11 (GST 3), and 12 (GST 12).     

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.     

Perturbation of membrane dynamics in nerve cells as an early event during bilirubin-induced apoptosis

Increased levels of unconjugated bilirubin, the end product of heme catabolism, impair crucial aspects of nerve cell function. In previous studies, we demonstrated that bilirubin toxicity may be due to cell death by apoptosis. To characterize the sequence of events leading to neurotoxicity, we exposed developing rat brain astrocytes and neurons to unconjugated bilirubin and investigated whether changes in membrane dynamic properties can mediate apoptosis. Bilirubin induced a rapid, dose-dependent increase in apoptosis, which was nevertheless preceded by impaired mitochondrial metabolism. Using spin labels and electron paramagnetic resonance spectroscopy analysis of whole cell and isolated mitochondrial membranes exposed to bilirubin, we detected major membrane perturbation. By physically interacting with cell membranes, bilirubin induced an almost immediate increase in lipid polarity sensed at a superficial level. The enhanced membrane permeability coincided with an increase in lipid fluidity and protein mobility and was associated with significant oxidative injury to membrane lipids. In conclusion, apoptosis of nerve cells induced by bilirubin is mediated by its primary effect at physically perturbing the cell membrane. Bilirubin directly interacts with membranes influencing lipid polarity and fluidity, protein order, and redox status. These data suggest that nerve cell membranes are primary targets of bilirubin toxicity.     

Isolation and characterization of an auxin-inducible glutathione S-transferase gene of Arabidopsis thaliana

Genes homologous to the auxin-inducible Nt103 glutathione S-transferase (GST) gene of tobacco, were isolated from a genomic library of Arabidopsis thaliana. We isolated a clone containing an auxin-inducible gene, At103-1a, and part of a constitutively expressed gene, At103-1b. The coding regions of the Arabidopsis genes were highly homologous to each other and to the coding region of the tobacco gene but distinct from the GST genes that have been isolated from arabidopsis thusfar. Overexpression of a cDNA clone in Escherichia coli revealed that the AT103-1A protein had GST activity.     

cFLIP downregulation is an early event required for endoplasmic reticulum stress-induced apoptosis in tumor cells

Protein misfolding or unfolding and the resulting endoplasmic reticulum (ER) stress frequently occur in highly proliferative tumors. How tumor cells escape cell death by apoptosis after chronic ER stress remains poorly understood. We have investigated in both two-dimensional (2D) cultures and multicellular tumor spheroids (MCTSs) the role of caspase-8 inhibitor cFLIP as a regulator of the balance between apoptosis and survival in colon cancer cells undergoing ER stress. We report that downregulation of cFLIP proteins levels is an early event upon treatment of 2D cultures of colon cancer cells with ER stress inducers, preceding TNF-related apoptosis-inducing ligand receptor 2 (TRAIL-R2) upregulation, caspase-8 activation, and apoptosis. Maintaining high cFLIP levels during ER stress by ectopic expression of cFLIP markedly inhibits ER stress-induced caspase-8 activation and apoptosis. Conversely, cFLIP knockdown by RNA interference significantly accelerates caspase-8 activation and apoptosis upon ER stress. Despite activation of the proapoptotic PERK branch of the unfolded protein response (UPR) and upregulation of TRAIL-R2, MCTSs are markedly more resistant to ER stress than 2D cultures of tumor cells. Resistance of MCTSs to ER stress-induced apoptosis correlates with sustained cFLIP_L expression. Interestingly, resistance to ER stress-induced apoptosis is abolished in MCTSs generated from cFLIP_L knockdown tumor cells. Overall, our results suggest that controlling cFLIP levels in tumors is an adaptive strategy to prevent tumor cell’s demise in the unfavorable conditions of the tumor microenvironment.Subject terms: Cancer microenvironment, Apoptosis     

The human liver glutathione S-transferase gene superfamily: expression and chromosome mapping of an Hb subunit cDNA.

We have isolated from a lambda gt10 cDNA library a clone lambda GTH4 which encodes a human liver glutathione S-transferase Hb subunit, designated as subunit 4. Expression of this cDNA in E. coli and subsequent purification and immunoblotting analysis provided a definitive assignment of a structure and function relationship. RNA blot hybridization with human liver poly(A) RNA revealed a single band of approximately 1200 nucleotides, comparable in size to the rat brain Yb3 mRNA. Divergence analysis of amino acid replacement sites in subunit 4 relative to the four rat Yb subunits revealed that it is most closely related to the brain-specific Yb3 subunit. This conclusion is further substantiated by the nucleotide sequence homology between lambda GTH4 and the Yb3 cDNA in their 3' untranslated region. In situ chromosome mapping has located this glutathione S-transferase gene in the region of p31 on chromosome 1. Results from many laboratories, including ours, indicate that the human glutathione S-transferases are encoded by a gene superfamily which is located on at least two different chromosomes.