Decline patterns and risk assessment of 10 multi-class pesticides in young sprout amaranth (Amaranthus mangostanus) under greenhouse growing conditions.

The present study was designed to investigate the residual decline pattern and the risk assessment of 10 different class pesticides, namely azoxystrobin, boscalid, diazinon, diethofencarb, difenoconazole, etofenprox, flubendiamide, paclobutrazol, and pyraclostrobin in young vegetative amaranth (Amaranthus mangostanus) sprayed once or twice under greenhouse growing conditions. Field-incurred samples, collected at 3, 7, or 10 days after application of both treatments, were extracted and purified with the quick, easy, cheap, effective, rugged, and safe "QuEChERS" citrate-buffered method and analyzed with liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS/MS) in positive ion mode. The linearity was satisfactory with determination coefficients (R 2) falling between 0.9817 and 0.9999 and limits of detection (LOD) and quantification (LOQ) values of 0.0007 and 0.002 mg/kg, respectively. The mean recovery rate at four spiking levels (equivalent to 5, 10, 50, and 100 × LOQ) ranged from 78.1 to 131.6% with a relative standard deviation (RSD) of < 11%. Substantial differences in the initial deposit between the tested analytes were observed and clearly indicated that the structure, as well as the initial concentration of applied products, greatly affected the residue deposit. From the obtained residual data, the provisional marginal maximum residue limits (MRLs) and the pre-harvest intervals (PHI) were proposed. Risk assessment was evaluated by comparing the theoretical maximum daily intake (TMDI) with the acceptable daily intake (ADI). Herein, the TMDI was lower than the ADI (TMDI/ADI ratio ≤ 80% set by the Korean Ministry of Food and Drug Safety) except for difenoconazole (80.92%, marginally higher), indicating that the vegetative amaranth is not hazardous and can be consumed safely by Korean consumers.

Hearing outcomes after stereotactic radiosurgery for unilateral intracanalicular vestibular schwannomas: implication of transient volume expansion.

PURPOSE: We evaluated the prognostic factors for hearing outcomes after stereotactic radiosurgery (SRS) for unilateral sporadic intracanalicular vestibular schwannomas (IC-VSs) as a clinical homogeneous group of VSs. METHODS AND MATERIALS: Sixty consecutive patients with unilateral sporadic IC-VSs, defined as tumors in the internal acoustic canal, and serviceable hearing (Gardner-Roberson grade 1 or 2) were treated with SRS as an initial treatment. The mean tumor volume was 0.34±0.03 cm3 (range, 0.03-1.00 cm3), and the mean marginal dose was 12.2±0.1 Gy (range, 11.5-13.0 Gy). The median follow-up duration was 62 months (range, 36-141 months). RESULTS: The actuarial rates of serviceable hearing preservation were 70%, 63%, and 55% at 1, 2, and 5 years after SRS, respectively. In multivariate analysis, transient volume expansion of ≥20% from initial tumor size was a statistically significant risk factor for loss of serviceable hearing and hearing deterioration (increase of pure tone average≥20 dB) (odds ratio=7.638; 95% confidence interval, 2.317-25.181; P=.001 and odds ratio=3.507; 95% confidence interval, 1.228-10.018; P=.019, respectively). The cochlear radiation dose did not reach statistical significance. CONCLUSIONS: Transient volume expansion after SRS for VSs seems to be correlated with hearing deterioration when defined properly in a clinically homogeneous group of patients.

Hearing preservation in patients with unilateral vestibular schwannoma who undergo stereotactic radiosurgery: Reinterpretation of the auditory brainstem response.

BACKGROUND: The objective of this study was to identify the prognostic factors for hearing preservation that would allow the more accurate stratification of patients who undergo stereotactic radiosurgery (SRS) for unilateral, sporadic vestibular schwannoma (VS). METHODS: In total, 119 patients with VS who had serviceable hearing underwent SRS as primary treatment. The mean (±standard deviation) patient age was 48 ± 11 years, and the mean (±standard deviation) follow-up duration was 55.2 ± 35.7 months. The median marginal radiotherapy dose was 12.0 grays (Gy), and the mean (±standard deviation) tumor volume was 1.95 ± 2.24 cm(3) . The mean (±standard deviation) pure tone average (PTA) score was 26 ± 12 decibels (dB) (range, 4-50 dB), and the mean (±standard deviation) maximum speech discrimination score was 91 ± 12% (range, 52-100%). The mean (±standard deviation) baseline values for the interlatency (IL) of waves I and III (IL I-III) and the IL of waves I through V (IL I-V) on auditory brainstem response were 2.58 ± 0.60 milliseconds (mS) (range, 1.92-4.30 mS) and 4.80 ± 0.61 mS (range, 3.80-6.40 mS), respectively. RESULTS: In multivariate analysis, the PTA score and IL I-V were significant and independent prognostic factors (hazard ratio, 1.072; 95% confidence interval, 1.046-1.098; P < .001; and hazard ratio, 1.534; 95% confidence interval, 1.008-2.336; P = .046, respectively). By using the PTA score and IL I-V, the patients were classified into 4 groups. The ratios of patients with serviceable hearing after SRS were 89.6%, 64.0%, 25.8%, and 6.7%, respectively, in Groups A through D (P < .001). CONCLUSIONS: The current results indicated that the classification system based on using the PTA score and the IL I-V of the auditory brainstem response may be useful and specific for predicting the rate of hearing preservation in each individual.

The risk factors of symptomatic communicating hydrocephalus after stereotactic radiosurgery for unilateral vestibular schwannoma: the implication of brain atrophy.

PURPOSE: To identify the effect of brain atrophy on the development of symptomatic communicating hydrocephalus (SCHCP) after stereotactic radiosurgery (SRS) for sporadic unilateral vestibular schwannomas (VS). METHODS AND MATERIALS: A total of 444 patients with VS were treated with SRS as a primary treatment. One hundred eighty-one patients (40.8%) were male, and the mean age of the patients was 53±13 years (range, 11-81 years). The mean follow-up duration was 56.8±35.8 months (range, 12-160 months). The mean tumor volume was 2.78±3.33 cm3 (range, 0.03-23.30 cm3). The cross-sectional area of the lateral ventricles (CALV), defined as the combined area of the lateral ventricles at the level of the mammillary body, was measured on coronal T1-weighted magnetic resonance images as an indicator of brain atrophy. RESULTS: At distant follow-up, a total of 25 (5.6%) patients had SCHCP. The median time to symptom development was 7 months (range, 1-48 months). The mean CALV was 334.0±194.0 mm2 (range, 44.70-1170 mm2). The intraclass correlation coefficient was 0.988 (95% confidence interval [CI], 0.976-0.994; p<0.001). In multivariate analysis, the CALV had a significant relationship with the development of SCHCP (p<0.001; odds ration [OR]=1.005; 95% CI, 1.002-1.007). Tumor volume and female sex also had a significant association (p<0.001; OR=1.246; 95% CI, 1.103-1.409; p<0.009; OR=7.256; 95% CI, 1.656-31.797, respectively). However, age failed to show any relationship with the development of SCHCP (p=0.364). CONCLUSION: Brain atrophy may be related to de novo SCHCP after SRS, especially in female patients with a large VS. Follow-up surveillance should be individualized, considering the risk factors involved for each patient, for prompt diagnosis of SCHCP.

Radiosurgery for para-IAC meningiomas: the effect of radiation dose to the cochlea on hearing outcome.

PURPOSE: This study was performed to assess the radiosurgical results of meningiomas extending into the internal acoustic canal (para-IAC meningiomas), with a particular focus on the effect of radiation dose to the cochlea on hearing outcome. METHODS AND MATERIALS: A total of 50 patients who underwent radiosurgery for para-IAC meningiomas between 1998 and 2009, which were followed for 2 years, were enrolled. The mean age was 55.8 years (range, 15-75). The mean tumor volume was 6.1 cm(3) (range, 1.0-19.0), the mean tumor length in the IAC was 6.9 mm (range, 1.3-13.3), and the mean prescribed marginal dose was 13.1 Gy (range, 10-15) at an isodose line of 50%. The mean follow-up duration was 46 months (range, 24-122). RESULTS: Eight (16.0%) patients had nonserviceable hearing at the time of surgery. At the last follow-up, the tumor control rate was 94%; unchanged in 17 patients, decreased in 30 patients, and increased in 3 patients. Among 42 patients with serviceable hearing at the time of radiosurgery, it was preserved in 41 (97.6%) patients at the last follow-up. The maximal and mean radiation doses to the cochleae of these 41 patients were 5.8 Gy ± 0.3 (range, 3.1-11.5) and 4.3 Gy ± 0.2 (range, 2.2-7.5), respectively. The maximal dose to the cochlea of the patient who lost hearing after radiosurgery was 4.7 Gy. CONCLUSIONS: The radiation dose to the cochlea may have the minimal toxic effect on the hearing outcome in patients who undergo radiosurgery for para-IAC meningiomas.

Prostaglandin A2 activates intrinsic apoptotic pathway by direct interaction with mitochondria in HL-60 cells.

HL-60 cells treated by prostaglandin (PG) A(2) showed characteristics of apoptosis such as accumulation of hypodiploid and annexin V positive cells, condensed and fragmented nuclei, cytochrome c (Cyt C) release from mitochondria and activation of caspase-1, -2, -3, -7 and -9. PGA(2)-induced cell death was rescued by inhibitors of caspase-9 and -3, but PGA(2)-induced Cyt C release was not prevented by caspase inhibitors. During Cyt C release by PGA(2), mitochondrial transmembrane potential was maintained and mitochondrial permeability transition pore was not formed. In addition, anti-apoptotic BCL-2 family proteins like BCL-2 and BCL-XL, and ROS scavengers including ascorbic acid and 2,2,6,6-tetramethyl-1-piperidinyloxy were not able to inhibit Cyt C release as well as apoptosis by PGA(2). Finally, it was shown that PGA(2)-induced Cyt C release in vitro from purified mitochondria in the absence of cytosolic components. Furthermore, thiol-containing compounds such as N-acetylcysteine, l-cysteine and monothioglycerol prevented Cyt C release, and hence induction of apoptosis. Taken together, these results suggest that PGA(2) activates intrinsic apoptotic pathway by directly stimulating mitochondrial outer membrane permeabilization to release Cyt C, in which thiol-reactivity of PGA(2) plays a pivotal role.

Induction of the Intrinsic Apoptotic Pathway by 3-Deazaadenosine Is Mediated by BAX Activation in HL-60 Cells.

3-Deazaadenosine (DZA), a potent inhibitor of S-adenosylhomocysteine hydrolase, was previously proposed to induce intrinsic apoptosis in human leukemic cells. In the present study, we analyzed the mechanism underlying the DZA-induced intrinsic apoptotic pathway. DZA activated typical caspase-dependent apoptosis in HL-60 cells, as demonstrated by an accumulation of hypo-diploidic cells, the processing of multiple procaspases and an inhibitory effect of z-VAD-Fmk on this cell death. During DZA-induced apoptosis, cytochrome c (cyt c) was released into the cytosol. This was neither prevented by z-VAD-Fmk and nor was it associated with the dissipation of mitochondrial membrane potential (ΔΨ(m)). Prior to the release of cyt c, BAX was translocated from the cytosol to mitochondria and underwent oligomerization. Finally, the overexpression of BCL-XL protected HL-60 cells from apoptosis by blocking both the cyt c release and BAX oligomerization. Collectively, these findings suggest that DZA may activate intrinsic apoptosis by stimulating BAX activation and thereby the release of cyt c.

Induction of unfolded protein response during neuronal induction of rat bone marrow stromal cells and mouse embryonic stem cells.

When we treated rat bone marrow stromal cells (rBMSCs) with neuronal differentiation induction media, typical unfolded protein response (UPR) was observed. BIP/GRP78 protein expression was time-dependently increased, and three branches of UPR were all activated. ATF6 increased the transcription of XBP1 which was successfully spliced by IRE1. PERK was phosphorylated and it was followed by eIF2alpha phosphorylation. Transcription of two downstream targets of eIF2alpha, ATF4 and CHOP/GADD153, were transiently up-regulated with the peak level at 24 h. Immunocytochemical study showed clear coexpression of BIP and ATF4 with NeuN and Map2, respectively. UPR was also observed during the neuronal differentiation of mouse embryonic stem (mES) cells. Finally, chemical endoplasmic reticulum (ER) stress inducers, thapsigargin, tunicamycin, and brefeldin A, dose-dependently increased both mRNA and protein expressions of NF-L, and, its expression was specific to BIP-positive rBMSCs. Our results showing the induction of UPR during neuronal differentiations of rBMSCs and mES cells as well as NF-L expression by ER stress inducers strongly suggest the potential role of UPR in neuronal differentiation.

Analysis of volatile compounds in fresh healthy and diseased peppers (Capsicum annuum L.) using solvent free solid injection coupled with gas chromatography-flame ionization detector and confirmation with mass spectrometry.

The characteristic volatile flavor compounds in healthy peppers (Capsicum annuum L.) were evaluated using a solvent-free solid injector coupled with a-gas chromatography-flame ionization detector (SFSI-GC-FID) and the results of evaluation were confirmed using GC-mass spectrometry (GC-MS). These compounds were compared with those obtained from peppers that were naturally infected or artificially inoculated with Colletotrichum spp. Parameters influencing the vaporization efficiency, including the injector temperature, pre-heating time and holding time, were optimized to improve the analytical efficiency. A total of 96 compounds (excluding eight capillary compounds), 17 of which were identified in healthy peppers, 49 of which were found in naturally infected peppers, and 61 of which were identified in artificially inoculated peppers, were separated and identified under the optimal conditions of an injector temperature of 250 degrees C and 7-min preheating and holding times. Acetic acid and 2-furanmethanol were the major compounds detected in the volatiles of the healthy and diseased peppers. The major compound detected in both the healthy and naturally infected peppers was 3-hydroxypyridine, while hexadecanoic acid was the primary compound identified in the artificially inoculated peppers. Indole derivatives (1H-indole, 4-methylindole and 1-ethylindole) were suggested to be the key factors contributing to the pepper infection caused by Colletotrichum spp. We conclude that SFSI in combination with GC is a suitable approach for distinguishing between healthy and diseased peppers by the investigation of their volatile compounds. It does not require the use of solvents and complicated equipment.

Protective effects of vacuolar H+ -ATPase c on hydrogen peroxide-induced cell death in C6 glioma cells.

We have isolated a gene, the c subunit (ATP6L) of vacuolar H(+)-ATPase, involved in oxidative stress response. In this study, we examined the role of ATP6L and its molecular mechanisms in glial cell death induced by H(2)O(2). Expression of the ATP6L gene was increased by H(2)O(2) treatment in C6 glial cells. ATP6L siRNA-transfected C6 cells treated with H(2)O(2) showed a significant decrease in viability. ATP6L siRNA-transfected cells that were pretreated with MEK1/2 inhibitor completely recovered cell viability. Pretreatment of the transfected cells with zVAD-fmk, a pan-specific caspase inhibitor, did not result in the recovery of cell viability, as determined by a H(2)O(2)-induced cytotoxicity assay. The ultrastructural morphology of the transfected cells as seen by the use of transmission electron microscopy showed numerous cytoplasmic autophagic vacuoles with double membrane. These results suggest that ATP6L has a protective role against H(2)O(2)-induced cytotoxicity via an inhibition of the Erk1/2 signaling pathway, leading to inhibition of autophagic cell death.

Overexpression of human 27 kDa heat shock protein in laryngeal cancer cells confers chemoresistance associated with cell growth delay.

PURPOSE: Among the family of heat shock proteins (HSPs), HSP70 and HSP27 have been implicated in tumorigenesis and chemoresistance, probably via the prevention of apoptosis. HSP27 levels are frequently increased in large populations of tumors of the head and neck, but the mechanism of its chemoresistance is not yet fully understood. In the present study, the role of HSP27 in the resistance to cytotoxic stress was studied in Hep-2 human laryngeal cancer cells. METHOD: We established a Hep-2 cell line overexpressing HSP27 and examined whether the expression of HSP27 provides resistance to heat shock and several cytotoxic agents using a MTT colorimetic assay. Cell cycle progression was assessed by flow cytometry and fluorescence staining was performed for F-actin filaments. RESULTS: HSP27 overexpression induced cellular resistance to heat shock at 45 degrees C for 1 h as well as against several cytotoxic agents, including cisplatin, staurosporin and H(2)O(2). However, no difference in sensitivity to irradiation or serum starvation was found. Moreover, HSP27 overexpressing Hep-2 cells showed a delayed cell growth, compared to control cells. To determine if the decreased cell proliferation in HSP27 overexpressing cells contributed to chemoresistance, control Hep-2 cells were synchronized at the late G1 phase by treatment with mimosine. The synchronized Hep-2 cells were resistant to cisplatin and H(2)O(2), but not to irradiation or serum starvation, correlating the protection effect shown in HSP27 overexpressing cells. These results suggest that the overexpression of HSP27 in Hep-2 cells confers chemoresistance which is associated with the delay in cell growth. We also propose that the stabilization of F-actin observed in Hep-2/hsp27 cells is partly related to the delay in cell cycle progression, by showing that the induction of actin polymerization in Hep-2/neo cells results in the retardation of cell growth as well as a cytoprotective effect as observed in Hep-2/hsp27.

Protein kinase A-dependent phosphorylation of B/K protein.

We have previously isolated a novel protein "B/K" that contains two C2-like domains. Here, we report the isolation and mRNA distribution of a human B/K isoform, and protein kinase A (PKA)-dependent phosphorylation of the B/K protein. The 1.5 kb human B/K cDNA clone exhibits 89% and 97% identities with rat B/K in the sequences of nucleotide and amino acid, respectively. Human B/K isoform encodes a 474 amino acid protein and shows structural features similar to the rat counterpart including two C2 domains, three consensus sequences for PKA, absence of a transmembrane region, and conservation of the N-terminal cysteine cluster. On Northern and dot blot analyses, a 3.0 kb B/K transcript was abundantly present in human brain, kidney, and prostate. Among the brain regions, strong signals were observed in the frontal and temporal lobes, the hippocampus, the hypothalamus, the amygdala, the substantia nigra, and the pituitary. Recombinant B/K proteins containing three consensus sites for PKA was very efficiently phosphorylated in vitro by PKA catalytic subunit. B/K protein which was overexpressed in LLC-PK1 cells was also strongly phosphorylated in vivo by vasopressin analog DDAVP, and PKA-specific inhibitor H 89 as well as type 2 vasopressin receptor antagonist specifically suppressed DDAVP-induced B/K phosphorylation. These results suggest that B/K proteins play a role as potential substrates for PKA in the area where they are expressed.

Prostaglandin A2 induces caspase-independent apoptosis in hepatocellular carcinoma cells.

BACKGROUND/AIMS: Prostaglandin (PG) A2 has been reported to inhibit the growth of hepatocellular carcinoma cells via activation of apoptosis, although the molecular mechanisms involved have not been clarified, yet. To investigate the mechanism of the PGA2-induced apoptosis, we analyzed the activation of caspases during the apoptosis of hepatoma cell lines. METHODS: Induction of apoptosis by PGA2 in hepatoma cell lines, Hep 3B and Hep G2, was assessed by DAPI staining of nuclei and agarose gel electrophoresis of genomic DNA. The involvement of caspases was analyzed by immunoblot analysis of poly ADP-ribose polymerase (PARP) and by checking the effect of caspase inhibitors on PGA2-induced apoptosis. RESULTS: PGA2 inhibited the growth of Hep 3B and Hep G2 cells, accompanying nuclear condensation and fragmentation, and genomic DNA laddering, which are the hallmarks of apoptosis. The PARP was not cleaved during the apoptosis of Hep 3B and Hep G2 cells and caspase inhibitors such as z-VAD-Fmk and z-DEVD-Fmk exerted no effect on the PGA2-induced apoptosis. CONCLUSIONS: These results suggest that PGA2 induces apoptosis in Hep 3B and Hep G2 cells via caspase-independent pathway.

Involvement of Sox-4 in the cytochrome c-dependent AIF-independent apoptotic pathway in HeLa cells induced by Delta12-prostaglandin J2.

Delta(12)-Prostaglandin (PG) J(2) is known to elicit an anti-neoplastic effects via apoptosis induction. Previous study showed Delta(12)-PGJ(2)-induced apoptosis utilized caspase cascade through cytochrome c-dependent pathways in HeLa cells. In this study, the cellular mechanism of Delta(12)-PGJ(2)- induced apoptosis in HeLa cells, specifically, the role of two mitochondrial factors; bcl-2 and apoptosis-inducing factor (AIF) was investigated. Bcl-2 attenuated Delta(12)-PGJ(2)-induced caspase activation, loss of mitochondrial transmembrane potential (Deltapsi(m)), nuclear fragmentation, DNA laddering, and growth curve inhibition for approximately 24 h, but not for longer time. AIF was not released from mitochondria, even if the Deltapsi(m) was dissipated. One of the earliest events observed in Delta(12)-PGJ(2)-induced apoptotic events was dissipation of Deltapsi(m), the process known to be inhibited by bcl-2. Pre-treatment of z-VAD- fmk, the pan-caspase inhibitor, resulted in the attenuation of ym depolarization in Delta(12)-PGJ(2)-induced apoptosis. Up-regulation of Sox-4 protein by Delta(12)-PGJ(2) was observed in HeLa and bcl-2 overexpressing HeLa B4 cell lines. Bcl-2 overexpression did not attenuate the expression of Sox-4 and its expression coincided with other apoptotic events. These results suggest that Delta(12)-PGJ(2) induced Sox-4 expression may activate another upstream caspases excluding the caspase 9-caspase 3 cascade of mitochondrial pathway. These and previous findings together suggest that Delta(12)-PGJ(2)-induced apoptosis in HeLa cells is caspase-dependent, AIF-independent events which may be affected by Sox-4 protein expression up-regulated by Delta(12)-PGJ(2).

Functional identification of the pro-apoptotic effector domain in human Sox4.

Recent studies provide evidence that Sox4 is involved in regulating apoptosis as well as tumorigenesis of various human cancers; however, its role in the apoptotic machinery is not fully understood. Here we describe that the central domain containing glycine-rich region in Sox4, named CD, is a pivotal pro-apoptotic domain to induce apoptotic cell death. Deletion of the DNA-binding domain or trans-activation domain in Sox4 did not significantly affect pro-apoptotic activity, whereas transient transfection of the high mobility group box or the serine-rich region abrogated the apoptotic activity. Moreover, overexpression of the CD construct (aa 166-342) revealed the apoptotic activity comparable to that of wild-type Sox4, approximately 60% of cell death. Our data suggest that the apoptotic activity of Sox4 can be dissociated from its transcriptional trans-activation and is mediated through its CD.

Expression of B/K protein in the hippocampus of kainate-induced rat seizure model.

B/K protein is a newly identified member of double C2-like domain protein family. We examined the expression of B/K protein in the hippocampus of kainate-induced rat seizure model. Intraperitoneal injection of kainate increased the immunoreactivity to B/K protein in the CA1 to CA3 of the hippocampus. B/K protein expression began to increase at 6 h, reached the maximum at 12 h, and then returned nearly to the normal level at 72 h after the injection of kainate (12 mg/kg), and it was also dependent on the dose of kainate between 4 and 16 mg/kg. In electron microscopic and subcellular fractionation studies, B/K protein was localized in the endoplasmic reticulum (ER) of the hippocampus. Kainate also induced the expression of BiP, a typical ER stress marker protein, in the hippocampus and the cortex, and it was coexpressed with B/K protein. Moreover, thapsigargin-induced ER stress caused upregulation of B/K protein expression in PC12 cells. In conclusion, our data showing the induction of both B/K protein expression and ER stress response in the hippocampus of kainate seizure model, and ER-specific expression and ER stress-induced expression of B/K strongly suggest the possible role of B/K protein in epileptogenesis or epilepsy-induced neuronal damage.

Cytochrome C-dependent Fas-independent apoptotic pathway in HeLa cells induced by delta12-prostaglandin J2.

Cyclopentenone prostaglandins (PGs) have antiproliferative activity on various tumor cell growth in vitro. Particularly, 9-deoxy-delta(9,12)-13,14-dihydro PGD(2) (delta(12)-PGJ(2)) was reported for its antineoplastic and apoptotic effects on various cancer cells, but its mechanism inducing apoptosis is still not clear. In this study, we have characterized apoptosis induced by delta(12)-PGJ(2) in HeLa cells. Treatment of delta(12)-PGJ(2) induced apoptosis as indicated by DNA fragmentation, chromatin condensation, and formation of apoptotic body. We also observed release of cytochrome c from mitochondria and activation of caspase cascade including caspase-3, -8, and -9. And the pan-caspase inhibitor z-Val-Ala-Asp (OMe) fluoromethyl-ketone (z-VAD-fmk) and Q-Val-Asp (OMe)-CH(2)-OPH (Q-VD (OMe)-OPH) prevented cell death induced by delta(12)-PGJ(2) showing participation of caspases in this process. However, protein expression level of Bcl-2 family was not altered by delta(12)-PGJ(2), seems to have no effect on HeLa cell apoptosis. And ZB4, an antagonistic Fas-antibody, exerted no effect on the activation of caspase 8 indicating that Fas receptor-ligand interaction was not involved in this pathway. Treatment of delta(12)-PGJ(2) also leads to suppression of nuclear factor kappaB (NF-kappaB) as indicated by nuclear translocation of p65/RelA and c-Rel and its DNA binding ability analyzed by EMSA. Taken together, our results suggest that delta(12)-PGJ(2)-induced apoptosis in HeLa cell utilized caspase cascade without Fas receptor-ligand interaction and accompanied with NF-kappaB inactivation.

Identification and differential expression of novel human cervical cancer oncogene HCCR-2 in human cancers and its involvement in p53 stabilization.

Basic studies of oncogenesis have demonstrated that either the elevated production of particular oncogene proteins or the occurrence of qualitative abnormalities in oncogenes can contribute to neoplastic cellular transformation. The purpose of this study was to identify unique oncogenes that are differentially expressed in human cancers and characterize their functions in tumorigenesis. To discover new putative oncogenes, the differential display RT-PCR method was applied using normal cervical tissues, cervical cancer cell lines, cervical cancer tissues, and metastatic tissues. We identified a new human cervical cancer oncogene HCCR-2 that was overexpressed in various human tumors including leukemia, lymphoma, and carcinomas of the breast, kidney, ovary, stomach, colon, and uterine cervix. Ectopic expression of HCCR-2 resulted in direct tumorigenic conversions of NIH/3T3 and Rat1 fibroblasts. Nude mice injected with NIH/3T3 cells stably transfected with HCCR-2 formed tumors in 4 weeks. The resultant tumors display characteristics of an epithelial carcinoma. In HCCR-2 transfected NCI-H460 cells and RKO cells, stabilization of the p53 tumor suppressor occurred without genetic mutation and correlated with functional impairment, as indicated by the defective induction of p53-induced p21(WAF1), MDM2, and bax. These results indicate that HCCR-2 probably represents a new oncogene that is related to tumorigenesis, functioning as a negative regulator of the p53 tumor suppressor.

Alterations in mRNA expression of ribosomal protein S9 in hydrogen peroxide-treated neurotumor cells and in rat hippocampus after transient ischemia.

This study was designed to isolate new genes related to apoptosis in rat pheochromocytoma (PC12) cells treated with hydrogen peroxide (H2O2), and to characterize the roles of the genes using both in vitro and in vivo models of oxidative injury. cDNA libraries were prepared from H2O2-treated and -untreated PC12 cells, and a ribosomal protein S9 (RPS9) clone was isolated by a differential screening method. Increase of RPS9 expression in both H2O2-treated PC12 and neuroblastoma (Neuro-2A) cells was shown by Northern blot analysis. Viability of the antisense-transfected Neuro-2A (RPS9-AS) cells following H2O2 treatment was significantly reduced in a dose-dependent manner. In an in vivo model of transient forebrain ischemia, an increase in RPS9 expression was prominent by 1 day postischemia in the granule cell layer neurons of the dentate gyrus. Both activation of caspase-3 and significant recovery of viability following pretreatment with cycloheximide were shown in RPS9-AS cells treated with H2O2. These data suggest that RPS9 plays a protective role in oxidative injury of neuronal cells.

Induction of apoptosis dependent on caspase activities and growth arrest in HL-60 cells by PGA2.

Prostaglandin (PG) A2 has been reported to inhibit the growth or induce apoptosis of various tumor cells. In the present study, PGA2 inhibited the growth of HL-60 cells and concomitantly-induced nuclear condensation and DNA fragmentation, characteristics of apoptosis. Down-regulation of c-myc mRNA, and activation of caspase-3 were observed in the PGA2 -treated cells. PGA2-induced DNA fragmentation was completely abolished in the presence of zVAD-Fmk or zDEVD-Fmk. But, relative cell survival was not improved up to that of untreated cells by pretreatment of caspase inhibitors, and c-myc down-regulation was not recovered by caspase inhibitors, either. Moreover, cytochrome c release and activation of caspase-9 was also observed in apoptotic cells and a specific inhibitor of caspase-9 (zLEHD-Fmk) prevented both DNA fragmentation and activation of caspase-3, but not relative cell survival, implying the upstream mitochondrial event of caspase-3 activation. In addition, antagonistic Fas antibody (ZB4) exerted no effect on the apoptosis. Taken together, these results suggest that PGA2 may induce the apoptosis as well as growth inhibition in HL-60 cells, and cytochrome c release and caspase activation seem to play a critical role in this apoptosis which might be independent or downstream of growth inhibition associated with c-myc down-regulation.