Role of carbohydrate moiety of bleomycin-A2 in caspase-3 activation and internucleosomal chromatin fragmentation in apoptosis of laryngeal carcinoma cells

Bleomycin (BLM), an antitumor antibiotic, is currently used during anticancer therapy. The therapeutic efficiency of BLM for the treatment of malignant tumors is related to its ability to cleave DNA. However, little is known about the biological activity of the glycannic moiety in BLM-induced cytotoxicity. In this study, cell death induced by BLM-A2 and deglycosylated BLM-A2 was studied in a laryngeal carcinoma cell line (HEp-2 cells). Our results indicate that HEp-2 cells showed morphological and biochemical changes associated with apoptosis in the presence of low concentrations of BLM-A2. In contrast, the same changes, except activation of caspase-3 and internucleosomal digestion of genomic DNA, were observed when cells were exposed to high concentrations of deglycosylated BLM-A2. These observations indicate that the glycannic moiety from the bleomycin molecule has important biological effects on the cytotoxicity of the drug.     

Outer membrane protein 38 of Acinetobacter baumannii localizes to the mitochondria and induces apoptosis of epithelial cells

Acinetobacter baumannii is an important opportunistic pathogen responsible for nosocomial infection. Despite considerable clinical and epidemiological data regarding the role of A. baumannii in nosocomial infection, the specific virulence factor or pathogenic mechanism of this organism has yet to be elucidated. This study investigated the molecular mechanism of apoptosis on the infection of human laryngeal epithelial HEp-2 cells with A. baumannii and examined the contribution of outer membrane protein 38 (Omp38) on the ability of A. baumannii to induce apoptosis of epithelial cells. A. baumannii induced apoptosis of HEp-2 cells through cell surface death receptors and mitochondrial disintegration. The Omp38-deficient mutant was not as able to induce apoptosis as the wild-type A. baumannii strain. Purified Omp38 entered the cells and was localized to the mitochondria, which led to a release of proapoptotic molecules such as cytochrome c and apoptosis-inducing factor (AIF). The activation of caspase-3, which is activated by caspase-9, degraded DNA approximately 180 bp in size, which resulted in the appearance of a characteristic DNA ladder. AIF degraded chromosomal DNA approximately 50 kb in size, which resulted in large-scale DNA fragmentation. These results demonstrate that Omp38 may act as a potential virulence factor to induce apoptosis of epithelial cells in the early stage of A. baumannii infection.     

Tissue transglutaminase induces the release of apoptosis inducing factor and results in apoptotic death of pancreatic cancer cells

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignant disease with poor long-term survival rates. Major reason for poor disease outcome is the profound intrinsic resistance of PDAC cells to currently available treatment regimens. We recently found that a great majority of PDAC tumors and tumor cell lines express high basal level of tissue transglutaminase (TG2), a multifunctional protein implicated in apoptosis, cell attachment, cell survival, and cell motility functions. Based on these observations, we hypothesized that activation of endogenous TG2 can induce spontaneous apoptosis in PDAC cells. The results obtained suggested that activation of endogenous TG2 by calcium ionophore A23187 induced rapid and spontaneous apoptosis in PDAC cells. TG2-induced apoptosis was associated with release of apoptosis-inducing factor (AIF). The release of AIF from mitochondria led to its translocation to the nucleus and subsequent apoptosis of PDAC cells in caspase-independent manner. In conclusion, our results provide first evidence that TG2 can induce apoptosis in PDAC cells in an AIF-dependent and caspase-independent manner.     

Wild-type herpes simplex virus 1 blocks programmed cell death and release of cytochrome c but not the translocation of mitochondrial apoptosis-inducing factor to the nuclei of human embryonic lung fibroblasts

Programmed cell death activated by herpes simplex virus 1 mutants can be caspase dependent or independent depending on the nature of the infected cell. The recently discovered mitochondrial apoptosis-inducing factor (AIF) on activation is translocated to the nucleus and induces programmed cell death that is caspase independent. To assess the role of AIF and also to assay apoptosis-related events in primary human embryonic lung (HEL) fibroblasts, cells were mock infected or infected with wild-type virus previously shown not to induce apoptosis in continuous lines of primate cells or with the d120 mutant lacking infected cell protein no. 4 (ICP4) and were shown to induce apoptosis in all cell lines tested. Cells exposed to dexamethasone or osmotic shock induced by sorbitol were the positive controls. The results were as follows: (i) AIF was translocated to the nucleus in all infected cell cultures and in cells treated with dexamethasone or sorbitol, but cells infected with the wild type-virus showed no evidence of undergoing programmed death. (ii) Cytochrome c was released from mitochondria of cells infected with the d120 mutant or exposed to dexamethasone or sorbitol but not from mitochondria in cells treated with sorbitol and infected with the wild-type virus. (iii) Poly(ADP-ribose) polymerase was cleaved in mock-infected cells exposed to sorbitol or dexamethasone and in cells infected with the d120 mutant but not in either untreated cells infected with wild-type virus or cells exposed to sorbitol and then infected with wild-type virus. In contrast to HEp-2 cells, neither d120 infection nor treatment with dexamethasone or sorbitol caused fragmentation of DNA in HEL fibroblasts. Electron microscopic examination showed chromatin condensation and vacuolization in a fraction of cells infected with d120 but not in wild-type virus-infected cells or cells treated with dexamethasone or sorbitol. We conclude that AIF is translocated to the nucleus in infected cells but apoptosis does not ensue in wild-type-infected cells. HEL fibroblasts infected with the d120 virus exhibit symptoms of classical apoptosis, such as cytochrome c release and cleavage of poly(ADP-ribose) polymerase observed also in cells undergoing caspase 3-dependent programmed cell death in which AIF is either not involved or not a contributory factor.     

The role of the gulose-mannose part of bleomycin in activation of iron-molecular oxygen complexes.

A comparison of the complexing properties of metal ions and O2 activation by bleomycin-A2 (BLM-A2) and deglyco-BLM-A2 is presented. Deglyco-BLM-A2 is obtained from the parent derivative by HF cleavage of the sugar moiety followed by h.p.l.c. purification. Complexing of Cu(II) and Fe(III) is studied by using c.d. and e.s.r. spectroscopy. Spin-trapping experiments in the presence of phenyl N-t-butylnitrone indicated lower production of free radicals by deglyco-BLM-A2. Finally, a proposal is made to explain this discrepancy, focusing on the probable role of the gulose-mannose moiety acting as a protecting pocket, comparable with the pocket and picket-fence porphyrins described for haemoproteins.     

ROS and protein oxidation in early stages of cytotoxic drug induced apoptosis

Cytotoxic drugs induce cell death through induction of apoptosis. This can be due to activation of a number of cell death pathways. While the downstream events in drug induced cell death are well understood, the early events are less clear. We therefore used a proteomic approach to investigate the early events in apoptosis induced by a variety of drugs in HL60 cells. Using 2D-gel electrophoresis, we were able to identify a number of protein changes that were conserved between different drug treatments. Identification of post-translational modifications (PTM) responsible for these proteome changes revealed an increase in protein oxidation in drug treated cells, as well as changes in protein phosphorylation. We demonstrate an accumulation of oxidised proteins within the ER, which lead to ER stress and calcium release and may result in the induction of apoptosis. This study demonstrates the importance of ROS mediated protein modifications in the induction of the early stages of apoptosis in response to chemotherapeutic drug treatment.     

Different patterns of apoptosis in response to cisplatin in B50 neuroblastoma rat cells

Cisplatin (cisPt) is a chemotherapeutic drug used for several human malignancies. CisPt cytotoxicity is primarily mediated by its ability to cause DNA damage and subsequent apoptotic cell death. DNA is the primary target of cisPt; however, recent data have shown that cisPt may have important direct interactions with mitochondria, which can induce apoptosis and may account for a significant part of the clinical activity associated with this drug. We have previously demonstrated that in the rat neuronal cell line B50, at 20 h-treatment with cisPt activates apoptosis through an intrinsic pathway involving an alteration of mitochondrial membrane permeability and the release of cytochrome c. The present study investigates different death pathways induced in the same cell line by a prolonged treatment with 40 μM cisPt for 48 h. To address this issue, we focused on caspases-8 and -12, and on the mitochondrial apoptosis inducing factor (AIF), which translocates to the nucleus and induces cell death via caspase-independent pathway. We found that cisPt activates different forms of cell death, i.e. the receptor-mediated apoptotic extrinsic pathway and a death process mediated by endoplasmic reticulum stress. Moreover, we demonstrated that AIF-mediated death occurs, being characterized by the translocation of AIF from mitochondria to the nucleus. On the whole, we provided evidence that prolonged cisPt treatment is able to activate both caspase-dependent and caspase-independent apoptotic pathways in B50 rat neuronal cells.     

Flavopiridol induces mitochondrial-mediated apoptosis in murine glioma GL261 cells via release of cytochrome c and apoptosis inducing factor

Glioblastoma (GBM) remains one of the most challenging solid cancers to treat due to its highly proliferative, angiogenic and invasive nature. The small molecule CDK inhibitor, flavopiridol, has demonstrated antitumor activity in human xenograft models and is currently in clinical trials showing efficacy in patients with advanced disease. We have developed an experimental animal model using the murine glioma GL261 cells as a novel in vivo system to screen potential therapeutic agents for GBM. Results of in vitro testing demonstrate that flavopiridol has several relevant clinical characteristics such as its ability to: 1. inhibit cell growth; 2. inhibit cell migration; 3. decrease expression of cyclin D1, CDK4 and p21; 4. induce apoptosis in cells with high levels of p27 expression; and 5. decrease the expression of the anti-apoptotic protein Bcl-2. The mechanism by which flavopiridol induces apoptosis is mitochondrial-mediated. We demonstrate by electron microscopy and immunohistochemistry that drug treatment induces mitochondrial damage that was accompanied by the release of cytochrome c into the cytosol together with the translocation of apoptosis inducing factor (AIF) into the nucleus. This finding in murine glioma cells differs from the mechanism of flavopiridolinduced cell death reported by us for human glioma cells (Alonso et al., Mol Cancer Ther 2003; 2:139) where drug treatment induced a caspase- and cytochrome c-independent pathway in the absence of detectable damage to mitochondria. In apoptotic human glioma cells only translocation of AIF into the nucleus occurred. Thus, the same drug kills different types of glioma cells by different mitochondrial-dependent pathways.     

ROS and protein oxidation in early stages of cytotoxic drug induced apoptosis

Cytotoxic drugs induce cell death through induction of apoptosis. This can be due to activation of a number of cell death pathways. While the downstream events in drug induced cell death are well understood, the early events are less clear. We therefore used a proteomic approach to investigate the early events in apoptosis induced by a variety of drugs in HL60 cells. Using 2D-gel electrophoresis, we were able to identify a number of protein changes that were conserved between different drug treatments. Identification of post-translational modifications (PTM) responsible for these proteome changes revealed an increase in protein oxidation in drug treated cells, as well as changes in protein phosphorylation. We demonstrate an accumulation of oxidised proteins within the ER, which lead to ER stress and calcium release and may result in the induction of apoptosis. This study demonstrates the importance of ROS mediated protein modifications in the induction of the early stages of apoptosis in response to chemotherapeutic drug treatment.     

Vibrio cholerae Porin OmpU Induces Caspase-independent Programmed Cell Death upon Translocation to the Host Cell Mitochondria

Porins, a major class of outer membrane proteins in Gram-negative bacteria, primarily act as transport channels. OmpU is one of the major porins of human pathogen, Vibrio cholerae. In the present study, we show that V. cholerae OmpU has the ability to induce target cell death. Although OmpU-mediated cell death shows some characteristics of apoptosis, such as flipping of phosphatidylserine in the membrane as well as cell size shrinkage and increased cell granularity, it does not show the caspase-3 activation and DNA laddering pattern typical of apoptotic cells. Increased release of lactate dehydrogenase in OmpU-treated cells indicates that the OmpU-mediated cell death also has characteristics of necrosis. Further, we show that the mechanism of OmpU-mediated cell death involves major mitochondrial changes in the target cells. We observe that OmpU treatment leads to the disruption of mitochondrial membrane potential, resulting in the release of cytochrome c and apoptosis-inducing factor (AIF). AIF translocates to the host cell nucleus, implying that it has a crucial role in OmpU-mediated cell death. Finally, we observe that OmpU translocates to the target cell mitochondria, where it directly initiates mitochondrial changes leading to mitochondrial membrane permeability transition and AIF release. Partial blocking of AIF release by cyclosporine A in OmpU-treated cells further suggests that OmpU may be inducing the opening of the mitochondrial permeability transition pore. All of these results lead us to the conclusion that OmpU induces cell death in target cells in a programmed manner in which mitochondria play a central role.     

Arsenic trioxide and paclitaxel induce apoptosis by different mechanisms

Arsenic trioxide (ATO) and paclitaxel (TAXOL) are effective in the treatment of various types of cancers. Both drugs induce G2/M arrest. We have previously shown that ATO is a potent inducer of apoptosis in myeloma cells expressing mutant p53 engaging both the intrinsic and extrinsic apoptotic pathways. Here we compared the effect of ATO and TAXOL on myeloma cells expressing mutant p53 and varying levels of Bcl-2. ATO rapidly induced Apo2/TRAIL, activation of caspase 8, cleavage of BID, depolarization of mitochondrial membrane (MM) and release of AIF from mitochondria in a Bcl-2 independent fashion. Apoptosis was associated with early formation of ring-like perinuclear condensed chromatin colocalized with AIF. In contrast, paclitaxel-induced apoptosis MM depolarization, cytochrome C release and activation of caspase 9 were all blocked by Bcl-2. Apoptosis was associated with a random chromatin condensation and nuclear fragmentation with no early involvement of AIF.     

Common regulation of apoptosis signaling induced by CD95 and the DNA-damaging stimuli etoposide and gamma-radiation downstream from caspase-8 activation.

The death receptor CD95 (APO-1/Fas), the anticancer drug etoposide, and gamma-radiation induce apoptosis in the human T cell line Jurkat. Variant clones selected for resistance to CD95-induced apoptosis proved cross-resistant to etoposide- and radiation-induced apoptosis, suggesting that the apoptosis pathways induced by these distinct stimuli have critical component(s) in common. The pathways do not converge at the level of CD95 ligation or caspase-8 signaling. Whereas caspase-8 function was required for CD95-mediated cytochrome c release, effector caspase activation, and apoptosis, these responses were unaffected in etoposide-treated and irradiated cells when caspase-8 was inhibited by FLIPL. Both effector caspase processing and cytochrome c release were inhibited in the resistant variant cells as well as in Bcl-2 transfectants, suggesting that, in Jurkat cells, the apoptosis signaling pathways activated by CD95, etoposide, and gamma-radiation are under common mitochondrial control. All three stimuli induced ceramide production in wild-type cells, but not in resistant variant cells. Exogenous ceramide bypassed apoptosis resistance in the variant cells, but not in Bcl-2-transfected cells, suggesting that apoptosis signaling induced by CD95, etoposide, and gamma-radiation is subject to common regulation at a level different from that targeted by Bcl-2.     

LPS-induced apoptosis is dependent upon mitochondrial dysfunction

Bacterial infection induces apoptotic cell death in human monoblastic U937 cells that have been pretreated with interferon gamma (U937IFN). Apoptosis occurs in a manner that is independent of bacterial virulence proteins. In the present study, we show that lipopolysaccharide (LPS), a membrane constituent of gram-negative bacteria, also induces apoptosis in U937IFN cells. LPS treatment led to the appearance of characteristic markers of apoptosis such as nuclear fragmentation and activation of caspases. While the caspase inhibitor Z-VAD-fmk prevented LPS-induced apoptosis as judged by its inhibition of nuclear fragmentation, it failed to inhibit cytochrome c release and loss of mitochondrial membrane potential. Transfection of peptides containing the BH4 (Bcl-2 homology 4) domain derived from the anti-apoptotic protein Bcl-XL blocked LPS-induced nuclear fragmentation and the limited digestion of PARP. These results suggest that LPS does not require caspase activation to induce mitochondrial dysfunction and that mitochondria play a crucial role in the regulation of LPS-mediated apoptosis in U937IFN cells.     

A comparative study of proteasomal inhibition and apoptosis induced in N27 mesencephalic cells by dopamine and MG132

Dopamine (DA) and its metabolites have been implicated in the pathogenesis of Parkinson's disease. DA can produce reactive-oxygen species and DA-derived quinones such as aminochrome can induce proteasomal inhibition. We therefore examined the ability of DA and MG132 to induce apoptosis and proteasomal inhibition in N27 rat dopaminergic cells. DA (0-500 micromol/L, 0-24 h) and MG132 (0-5 micromol/L, 0-24 h) treated N27 cells resulted in time- and concentration-dependent apoptosis. To better define DA and MG132-induced apoptosis, the activation of initiator caspases 2 and caspase 9 and the executioner caspase 3 was investigated. Activation of caspase 2, caspase 9, and caspase 3 occurred early and prior to cell death. In addition, N-acetylcysteine (NAC) blocked DA but not MG132-induced apoptosis and mitochondrial membrane potential loss. NAC can react with both reactive-oxygen and quinoid metabolites and its inhibitory activity suggests a role for reactive species in DA-induced apoptosis. Proteasomal inhibition was detected after DA treatment in N27 cells which occurred prior to cell death and was abrogated by NAC. Our results implicate DA-derived reactive species in proteasomal inhibition and caspase-dependent apoptosis in N27 cells. The ability of endogenous DA-derived metabolites to induce proteasomal inhibition and apoptosis may contribute to the selective loss of dopaminergic neurons in Parkinson's disease.     

CD437, a synthetic retinoid, induces apoptosis in human respiratory epithelial cells via caspase-independent mitochondrial and caspase-8-dependent pathways both up-regulated by JNK signaling pathway

The synthetic retinoid-related molecule CD437-induced apoptosis in human epithelial airway respiratory cells: the 16HBE bronchial cell line and normal nasal epithelial cells. CD437 caused apoptosis in S-phase cells and cell cycle arrest in S phase. Apoptosis was abolished by caspase-8 inhibitor z-IETD-fmk which preserved S-phase cells but was weakly inhibited by others selective caspase-inhibitors, indicating that caspase-8 activation was involved. z-VAD and z-IETD prevented the nuclear envelope fragmentation but did not block the chromatin condensation. The disruption of mitochondrial transmembrane potential was also induced by CD437 treatment. The translocation of Bax to mitochondria was demonstrated, as well as the release of cytochrome c into the cytosol and of apoptosis-inducing factor (AIF) translocated into the nucleus. z-VAD and z-IETD did not inhibit mitochondrial depolarization, Bax translocation or release of cytochrome c and AIF from mitochondria. These results suggest that CD437-induced apoptosis is executed by two converging pathways. AIF release is responsible for chromatin condensation, the first stage of apoptotic cell, via a mitochondrial pathway independent of caspase. But final stage of apoptosis requires the caspase-8-dependent nuclear envelope fragmentation. In addition, using SP600125, JNK inhibitor, we demonstrated that CD437 activates the JNK-MAP kinase signaling pathway upstream to mitochondrial and caspase-8 pathways. Conversely, JNK pathway inhibition, which suppresses S-phase apoptosis, did not prevent cell cycle arrest within S phase, confirming that these processes are triggered by distinct mechanisms.     

Fibroblast apoptosis induced by Porphyromonas gingivalis is stimulated by a gingipain and caspase-independent pathway that involves apoptosis-inducing factor

Porphyromonas gingivalis is an oral bacterium that causes pathology in a number of dental infections that are associated with increased fibroblast cell death. Studies presented here demonstrated that P. gingivalis stimulates cell death by apoptosis rather than necrosis. Unlike previous studies apoptosis was induced independent of proteolytic activity and was also independent of caspase activity because a pancaspase inhibitor, Z-VAD-fmk, had little effect. Moreover, P. gingivalis downregulated caspase-3 mRNA levels and caspase-3 activity. The consequence of this downregulation was a significant reduction in tumour necrosis factor-alpha-induced apoptosis, which is caspase-3-dependent. Immunofluorescence and immunoblot analysis revealed P. gingivalis-induced translocation of apoptosis-inducing factor (AIF) from the cytoplasm to the nucleus. siRNA studies were undertaken and demonstrated that P. gingivalis stimulated cell death was significantly reduced when AIF was silenced (P < 0.05). Treatment of human gingival fibroblasts with H-89, a protein kinase A inhibitor that blocks AIF activation also reduced P. gingivalis-induced apoptosis (P < 0.05). These results indicate that P. gingivalis causes fibroblast apoptosis through a pathway that involves protein kinase A and AIF, is not dependent upon bacterial proteolytic activity and is also independent of the classic apoptotic pathways involving caspase-3.     

Caspase-2 induces apoptosis by releasing proapoptotic proteins from mitochondria

Caspase-2 is one of the earliest identified caspases, but the mechanism of caspase-2-induced apoptosis remains unknown. We show here that caspase-2 engages the mitochondria-dependent apoptotic pathway by inducing the release of cytochrome c (Cyt c) and other mitochondrial apoptogenic factors into the cell cytoplasm. In support of these observations we found that Bcl-2 and Bcl-xL can block caspase-2- and CRADD (caspase and RIP adaptor with death domain)-induced cell death. Unlike caspase-8, which can process all known caspase zymogens directly, caspase-2 is completely inactive toward other caspase zymogens. However, like caspase-8, physiological levels of purified caspase-2 can cleave cytosolic Bid protein, which in turn can trigger the release of Cyt c from isolated mitochondria. Interestingly, caspase-2 can also induce directly the release of Cyt c, AIF (apoptosis-inducing factor), and Smac (second mitochondria-derived activator of caspases protein) from isolated mitochondria independent of Bid or other cytosolic factors. The caspase-2-released Cyt c is sufficient to activate the Apaf-caspase-9 apoptosome in vitro. In combination, our data suggest that caspase-2 is a direct effector of the mitochondrial apoptotic pathway.     

Mitochondrial dysfunction in CD47-mediated caspase-independent cell death: ROS production in the absence of cytochrome c and AIF release

Ligation of CD47 by its natural ligand thrombospondin (TSP), or cross-linking by CD47 antibodies, triggers caspase-independent cell death in normal and leukemic cells. This kind of cell death is characterised by the cytoplasmic events of apoptosis including externalisation of phosphatidylserines and mitochondria swelling. We report herein selective mitochondrial changes in CD47-dependent cell death of T cells. After T cell stimulation via CD47, a rapid mitochondrial transmembrane potential (deltapsi(m)) disruption is accompanied by the production of reactive oxygen species (ROS) and phosphatidylserine exposure. Surprisingly, mitochondrial dysfunction does not induce cytochrome c or AIF release. Moreover, the dying cells do not exhibit caspase-3 activation and display intact nuclei without any large-scale, or oligonucleosomal DNA fragmentation. We conclude that DeltaPsi(m) loss and ROS production are an early step in CD47-dependent killing and neither cytochrome c, nor AIF are implicated in this new cell death pathway.     

Spatiotemporal activation of caspase‐dependent and ‐independent pathways in staurosporine‐induced apoptosis of p53wt and p53mt human cervical carcinoma cells

Background information. Caspase‐dependent and ‐independent death mechanisms are involved in apoptosis in a variety of human carcinoma cells treated with antineoplastic compounds. Our laboratory has reported that p53 is a key contributor of mitochondrial apoptosis in cervical carcinoma cells after staurosporine exposure. However, higher mitochondrial membrane potential dissipation and greater DNA fragmentation were observed in p53^wt (wild‐type p53) HeLa cells compared with p53^mt (mutated p53) C‐33A cells. Here, we have studied events linked to the mitochondrial apoptotic pathway. Results. Staurosporine can induce death of HeLa cells via a cytochrome c/caspase‐9/caspase‐3 mitochondrial‐dependent apoptotic pathway and via a delayed caspase‐independent pathway. In contrast with p53^wt cells, p53^mt C‐33A cells exhibit firstly caspase‐8 activation leading to caspase‐3 activation and Bid cleavage followed by cytochrome c release. Attenuation of PARP‐1 [poly(ADP‐ribose) polymerase‐1] cleavage as well as oligonucleosomal DNA fragmentation in the presence of z‐VAD‐fmk points toward a major involvement of a caspase‐dependent pathway in staurosporine‐induced apoptosis in p53^wt HeLa cells, which is not the case in p53^mt C‐33A cells. Meanwhile, the use of 3‐aminobenzamide, a PARP‐1 inhibitor known to prevent AIF (apoptosis‐inducing factor) release, significantly decreases staurosporine‐induced death in these p53^mt carcinoma cells, suggesting a preferential implication of caspase‐independent apoptosis. On the other hand, we show that p53, whose activity is modulated by pifithrin‐α, isolated as a suppressor of p53‐mediated transactivation, or by PRIMA‐1 (p53 reactivation and induction of massive apoptosis), that reactivates mutant p53, causes cytochrome c release as well as mitochondrio—nuclear AIF translocation in staurosporine‐induced apoptosis of cervical carcinoma cells. Conclusions. The present paper highlights that staurosporine engages the intrinsic mitochondrial apoptotic pathway via caspase‐8 or caspase‐9 signalling cascades and via caspase‐independent cell death, as well as through p53 activity.     

Cadmium activates CaMK-II and initiates CaMK-II-dependent apoptosis in mesangial cells

Cadmium is a toxic metal that initiates both mitogenic responses and cell death. We show that Cd(2+) increases phosphorylation and activity of Ca(2+)/calmodulin-dependent protein kinase II (CaMK-II) in mesangial cells, in a concentration-dependent manner. Activation is biphasic with peaks at 1-5 min and 4-6 h. Cadmium also activates Erk, but this appears to be independent of CaMK-II. At 10-20 microM, Cd(2+) initiates apoptosis in 25-55% of mesangial cells by 6h. Inhibition of CaMK-II, but not of Erk, suppresses Cd(2+)-induced apoptosis. We conclude that activation of CaMK-II by Cd(2+) contributes to apoptotic cell death, independent of Erk activation.