Practical characteristics of adaptive design in phase 2 and 3 clinical trials.

WHAT IS KNOWN AND OBJECTIVE: Adaptive design methods are expected to be ethical, reflect real medical practice, increase the likelihood of research and development success and reduce the allocation of patients into ineffective treatment groups by the early termination of clinical trials. However, the comprehensive details regarding which types of clinical trials will include adaptive designs remain unclear. We examined the practical characteristics of adaptive design used in clinical trials. METHODS: We conducted a literature search of adaptive design clinical trials published from 2012 to 2015 using PubMed, EMBASE, and the Cochrane Central Register of Controlled Trials, with common search terms related to adaptive design. We systematically assessed the types and characteristics of adaptive designs and disease areas employed in the adaptive design trials. RESULTS AND DISCUSSION: Our survey identified 245 adaptive design clinical trials. The number of trials by the publication year increased from 2012 to 2013 and did not greatly change afterwards. The most frequently used adaptive design was group sequential design (n = 222, 90.6%), especially for neoplasm or cardiovascular disease trials. Among the other types of adaptive design, adaptive dose/treatment group selection (n = 21, 8.6%) and adaptive sample-size adjustment (n = 19, 7.8%) were frequently used. The adaptive randomization (n = 8, 3.3%) and adaptive seamless design (n = 6, 2.4%) were less frequent. Adaptive dose/treatment group selection and adaptive sample-size adjustment were frequently used (up to 23%) in "certain infectious and parasitic diseases," "diseases of nervous system," and "mental and behavioural disorders" in comparison with "neoplasms" (<6.6%). For "mental and behavioural disorders," adaptive randomization was used in two trials of eight trials in total (25%). Group sequential design and adaptive sample-size adjustment were used frequently in phase 3 trials or in trials where study phase was not specified, whereas the other types of adaptive designs were used more in phase 2 trials. Approximately 82% (202 of 245 trials) resulted in early termination at the interim analysis. Among the 202 trials, 132 (54% of 245 trials) had fewer randomized patients than initially planned. This result supports the motive to use adaptive design to make study durations shorter and include a smaller number of subjects. WHAT IS NEW AND CONCLUSION: We found that adaptive designs have been applied to clinical trials in various therapeutic areas and interventions. The applications were frequently reported in neoplasm or cardiovascular clinical trials. The adaptive dose/treatment group selection and sample-size adjustment are increasingly common, and these adaptations generally follow the Food and Drug Administration's (FDA's) recommendations.

The comparative anatomy of the folds, fossae, and adhesions around the duodenojejunal flexure in mammals.

BACKGROUND: Anatomical knowledge of the duodenojejunal flexure is necessary for abdominal surgeries, and also important for physiologic studies about the duodenum. But little is known about the anatomy of this region in mammals. Here, we examined comparative anatomy to understand the anatomical formation of the duodenojejunal flexure in mammals. MATERIALS AND METHODS: The areas around the duonenojejunal flexure were ob-served in mouse, rat, dog, pig, and human, and the anatomical structures around the duodenojejunal junction in the animals were compared with those in human. RESULTS: The superior and inferior duodenal folds, and the superior and inferior duodenal fossae were identified in all examined humans. In pig, the structures were not clearly identified because the duodenum strongly adhered to the retroperitoneum and to the mesocolon. In mouse, rat, and dog, only the plica duodenocolica, which is regarded as the animal counterpart of the superior duo-denal fold in human, was identified, and other folds or fossae were not observed, probably because the duodenum was not fixed to the parietal peritoneum in those animals. Transection of the plica duodenocolica could return the normally rotated intestine back to the state of non-rotation in rat. CONCLUSIONS: This study showed the anatomical similarities and dissimilarities of the duodenojejunal flexure among the mammals. Anatomical knowledge of the area is useful for duodenal and pancreatic surgeries, and for animal studies about the duodenum. (Folia Morphol 2018; 77, 2: 286-292).

HIV-1 Vpr induces ATM-dependent cellular signal with enhanced homologous recombination.

An ATM-dependent cellular signal, a DNA-damage response, has been shown to be involved during infection of human immunodeficiency virus type-1 (HIV-1), and a high incidence of malignant tumor development has been observed in HIV-1-positive patients. Vpr, an accessory gene product of HIV-1, delays the progression of the cell cycle at the G2/M phase, and ATR-Chk1-Wee-1, another DNA-damage signal, is a proposed cellular pathway responsible for the Vpr-induced cell cycle arrest. In this study, we present evidence that Vpr also activates ATM, and induces expression of gamma-H2AX and phosphorylation of Chk2. Strikingly, Vpr was found to stimulate the focus formation of Rad51 and BRCA1, which are involved in repair of DNA double-strand breaks (DSBs) by homologous recombination (HR), and biochemical analysis revealed that Vpr dissociates the interaction of p53 and Rad51 in the chromatin fraction, as observed under irradiation-induced DSBs. Vpr was consistently found to increase the rate of HR in the locus of I-SceI, a rare cutting-enzyme site that had been introduced into the genome. An increase of the HR rate enhanced by Vpr was attenuated by an ATM inhibitor, KU55933, suggesting that Vpr-induced DSBs activate ATM-dependent cellular signal that enhances the intracellular recombination potential. In context with a recent report that KU55933 attenuated the integration of HIV-1 into host genomes, we discuss the possible role of Vpr-induced DSBs in viral integration and also in HIV-1 associated malignancy.

Micronuclei formation with chromosome breaks and gene amplification caused by Vpr, an accessory gene of human immunodeficiency virus.

Vpr, an accessory gene of human immunodeficiency virus, induces cell cycle abnormality by accumulating cells at the G2-M phase. We reported recently that Vpr caused both micronuclei formation and aneuploidy. Here, we show that Vpr also induced chromosome breaks and gene amplification. Expression of Vpr induced more than 10-fold increase of colonies resistant to N-(phosphonacetyl)-L-aspartate, an inhibitor of pyrimidine de novo synthesis. Fluorescence in situ hybridization analysis detected that 4 of 10 N-(phosphonacetyl)-L-aspartate resistant clones studied had intrachromosomal amplification of carbamyl-phosphate synthetase/aspartate transcarbamoylase/dihydroorotase gene. Another single clone had dicentrics. Data suggested that the Vpr-induced chromosome breaks leading to gene amplification, followed by bridge-breakage-fusion cycle, were one of the possible mechanisms of Vpr-induced genomic instability.

Molecular cloning of dihydrolipoamide acetyltransferase of the rat pyruvate dehydrogenase complex: sequence comparison and evolutionary relationship to other dihydrolipoamide acyltransferases.

A complementary DNA (cDNA) clone of dihydrolipoamide acetyltransferase (E2) of the rat pyruvate dehydrogenase complex (PDC) was isolated from a lambda gt11 rat heart cDNA library. The amino acid sequence of a full mature protein of rat PDC-E2 was predicted by combination of the cDNA nucleotide sequence and the N-terminal amino acid sequence determined chemically. The amino acid sequence of rat PDC-E2 was well consistent with those of the E2 components of other alpha-ketoacid dehydrogenase complexes. These E2 components possess the sequence G-X-G-X-X-G, which is the consensus sequence for nucleotide binding sites of nucleotide binding proteins, in the E3 and/or E1 binding domains. The E2 components of the three alpha-ketoacid dehydrogenase complexes are suggested to be classified into three clusters separated during evolution.

Induction of apoptosis in human hematopoietic U937 cells by granulocyte-macrophage colony-stimulating factor: possible existence of caspase 3-like pathway.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) induced apoptosis in human hematopoietic U937 cells by itself and in a synergistic manner with tumor necrosis factor (TNF). GM-CSF-induced apoptosis was not inhibited by caspase inhibitors YVAD-CMK, DEVD-CHO and z-VAD-FMK, under the condition that these inhibitors potently suppressed TNF-induced apoptosis. Both GM-CSF and TNF induced caspase 3-like activity in this cell line though the time course was distinct between two cytokines, and combined stimulation of cells with GM-CSF plus TNF induced additive or synergistic activation of caspase 3-like activity. Amount of immunoreactive cleaved forms of caspase 3 recognized by specific antibody was completely dissociated with its enzymatic activity when the cells were stimulated with GM-CSF, but not with TNF. These results indicate that GM-CSF induces apoptosis of U937 cells via unknown pathway, which seems to be mediated by caspase 3-like activity, yet not caspase 3 itself, resistant to the caspase inhibitors, and synergistically interacts with conventional caspase 3 pathway of TNF. Possible involvement of caspases 1 and 8 (-like activity) but not caspase 7 in this pathway was also suggested.

Oxidative stress as a necessary factor in room temperature-induced apoptosis of HL-60 cells.

HL-60 cells undergo apoptosis when placed at room temperature (RT) [Shimura et al. (1997) FEBS Lett. 417, 379-384]. We report that superoxide anion radical, one of the reactive oxygen species (ROS), was produced after RT treatment. Affinity blot analysis with a biotinylated YVAD-CHO detected the generation of processed peptides with molecular masses of 15-25 kDa. Activation of such an ICE-like protease was completely abolished by N-acetylcysteine and exogenously expressed Bcl-2, known as antioxidants. We concluded that oxidative stress was a critical factor in the signal cascade of the apoptosis. Western blot analysis and experiments using tetrapeptide inhibitors suggested that caspases-1, -3, -4, -6, and -9 did not have an essential role in the apoptotic cascade. It is interesting that cyclosporin A (CsA) blocked RT-induced apoptosis with an inhibition of cytochrome c release from mitochondria. CsA, however, generated a significant amount of ROS with considerable reduction of mitochondrial membrane potential, implying that oxidative stress was one necessary factor for RT-induced apoptosis. It is also likely that mitochondrial membrane potential and the release of apoptotic factors from cytoplasm are differently regulated. Taken together with the reports that some Burkitt lymphoma cells showed apoptosis when exposed at low temperature followed by rewarming, and that hepatocytes or liver endothelial cells are susceptible to cold-induced apoptosis through the ROS function, we propose that studying the mechanism of RT-induced apoptosis of HL-60 cells may provide a therapeutic strategy for pathological conditions involving ROS, such as neurodegenerative diseases and ischemia.

Effects of Pathogen Density, Soil Moisture, and Soil pH on Biological Control of Clubroot in Chinese Cabbage by Heteroconium chaetospira.

The effects of soil moisture and pH, and pathogen resting spore density, on the effectiveness of the biological control of clubroot by the fungal endophyte Heteroconium chaetospira was evaluated in greenhouse and field experiments. Conditions favoring disease development included low pH (5.5) and high soil moisture content (80%), with significant reductions in the disease being observed at a higher pH (6.3 and 7.2) and lower soil moisture content (40 and 60%). In greenhouse tests, H. chaetospira effectively controlled clubroot (reducing the disease by 90 to 100%) at pathogen resting spore densities of 104 and 105 spores/g of soil at all soil pHs tested (5.5, 6.3, and 7.2). However, when the resting spore density was 106 spores/g of soil, plants were severely diseased, regardless of treatment, and H. chaetospira had no effect on disease. At a soil moisture content of 40%, disease occurrence was low, regardless of pathogen spore density, but disease was significantly lower in H. chaetospira-treated plants at pathogen spore density of 105 spores/g of soil. At 60% soil moisture content, H. chaetospira significantly could affect at pathogen spore densities of 104 and 105 but not 104/g of soil. At 80% soil moisture content, there was no effect of H. chaetospira at pathogen density. In situ, the soil moisture contents were constantly adjusted to relatively low to moderate (pF 2.2 to 2.4 and pF 2.0 to 2.2) and high (pF 1.6 to 1.8). Other environmental conditions, such as resting spore density and soil pH, were maintained at constant levels. Control plants (not treated with H. chaetospira) showed uniformly high disease levels and proportions of diseased plants across all three moisture treatments (disease index = 72 to 80, proportion of diseased plants 85 to 97%). In the field, H. chaetospira-treated plants at low soil moisture (pF 2.2 to 2.4, plot 1) had 68% disease reduction compared with untreated controls and 49% reduction at moderate moisture pF (pF 2.0 to 2.2, plot 2). There was no effect on disease by H. chaetospira at high soil moisture (pF 1.6 to 1.8, plot 3). Based on our results, H. chaetospira is an effective biocontrol agent against clubroot in Chinese cabbage at a low to moderate soil moisture range and a pathogen resting spore density of 105 (or lower resting spores per gram of soil in situ.

Different change in lipoprotein(a) levels from lipid levels of other lipoproteins with improved glycemic control in patients with NIDDM.

OBJECTIVE: To evaluate change both in lipoprotein(a) [Lp(a)] and lipid levels in other lipoproteins in non-insulin-dependent diabetes mellitus (NIDDM) after short-term improvement of glycemic control. RESEARCH DESIGN AND METHODS: We compared Lp(a) levels in 210 NIDDM patients with those in 46 control subjects and evaluated the relationship between glycemic control and Lp(a) levels in diabetic patients. In addition, changes in Lp(a) levels and lipid levels were assessed after the improvement of glycemic control in 54 poorly controlled NIDDM patients. RESULTS: In NIDDM, Lp(a) levels in all patients, 62 patients with HbA1c < 6.0%, and 75 patients with HbA1c between 6.0 and 8.0%, were significantly higher than those in control subjects (19.1 [1.7-106.6], 19.2 [6.0-106.6], and 20.3 [2.7-75.3] vs. 15.4 [2.0-61.7] mg/dl, median [range], P < 0.05). Lp(a) levels in 73 patients with HbA1c of > or = 8.0% (18.7 [1.7-58.8] mg/dl) were not significantly different from those in control subjects. After glycemic control, lipid levels in plasma and in other lipoproteins fell significantly, but Lp(a) did not change (from 18.3 [1.7-58.8] to 18.4 [6.6-95.3] mg/dl). Changes in lipid levels, including Lp(a), did not correlate with those in fasting plasma glucose or HbA1c. CONCLUSIONS: These results suggest that elevated Lp(a) levels do not reflect poor glycemic control and that Lp(a) levels are independent of lipid levels in other lipoproteins after improved glycemic control in NIDDM.

Improved gene transfer to neuroblastoma cells by a monoclonal antibody targeting RET, a receptor tyrosine kinase.

Receptor-mediated gene transfer is an effective strategy among nonviral vector systems. It is, however, crucial to develop various types of monoclonal antibodies satisfying both the binding specificity for cell targeting and the capacity of endocytosis required for gene transfer. In the present study, we generated a novel monoclonal antibody (NBL-1) to RET, a receptor tyrosine kinase expressed in both neuroblastoma cells and cells present in substantia nigra, a responsive locus of Parkinson's disease. NBL-1, when added to the culture medium of the neuroblastoma cells, was incorporated by endocytosis in a wortmannin-sensitive manner. Using a biotinylated NBL-1 complexed with plasmid DNAs based on electrostatic interaction through avidin-conjugated polylysines, exogenous luciferase genes were expressed in neuroblastoma cells at a more than 10-fold higher level. The expression level of the gene based on NBL-1 was comparable to that obtained by a geneporter system, an improved nonviral gene transduction method. Furthermore, the NBL-1-based gene transfer mediated the formation of more than 20-fold higher numbers of drug-resistant colonies. In contrast, RET-negative cells, which included HeLa, HT1080, Caco-2, and Colo205 cells, did not show any increased expression of an exogenous gene by NBL-1. These data suggest that the RET molecules enable selective gene transduction, and that NBL-1 may possibly be applied to gene therapy for neuroblastomas and Parkinson's disease.

Characterization of room temperature induced apoptosis in HL-60.

We found that exposure to room temperature (RT/21 degrees C) causes apoptosis in HL-60 cells. Here we characterized RT-induced apoptosis in HL-60. After exposure to RT, apoptosis starts within 6 h and more than 80% of the cells underwent apoptosis within 20 h. All cells, however, were committed to apoptosis after 16 h and no viable cells could be recovered. The caspase-1 inhibitor (YVAD-CHO) effectively blocked apoptosis, whereas the caspase-3 inhibitor (DEVD-CHO) did not. About 20% of newly obtained early passage HL-60 cells (passage 10) also underwent apoptosis by RT treatment. These data suggest that some population in HL-60 which responds to RT with apoptosis became dominant during passaging.

Functional sites of cytochrome c and other electron carrier proteins: semi-empirical molecular orbital program PM3 applied to the conformational analysis of Cys-X1-X2-Cys peptides.

The semi-empirical molecular orbital program, MOPAC version 6.0, PM3 was applied to estimate the distance between two sulfur atoms of two Cys side chains in optimized conformers of Cys-X1-X2-Cys sequences, as well as the total energy of that conformer relative to the most stable one. Some Cys-X1-X2-Cys tetrapeptides found in cytochrome c were optimized to conformers whose sulfur-sulfur distances were just suitable for binding to heme, whereas some tetrapeptides not found in cytochrome c were unable to be optimized to heme-binding conformers. Similarly, Cys-X1-X2-Cys tetrapeptides found in [4Fe-4S]ferredoxin were optimized to [4Fe-4S]-binding conformers, etc. The tetrapeptides found in the redox site of thioredoxin were optimized to conformers in which the two sulfur atoms were in van der Waals contact, so that a disulfide bond may be formed during the function. The conclusion has been drawn that the combination of X1 and X2 in a Cys-X1-X2-Cys sequence may be determining for that sequence to be a functional redox site in an electron carrier protein.

Characterization of the electrogenic Na+ -K+ pump in bipolar cells isolated from carp retina.

The electrogenic Na+ -K+ pump current (Ip) in carp bipolar cells was investigated under voltage-clamp conditions. The Ip was activated in a concentration-dependent manner by adding external K+ (Ko+) and was completely suppressed with 10(-4) M ouabain (EC50=1.23 mM; Hill coefficient=1.36). The Ip was suppressed in a concentration-dependent manner by ouabain (IC50=1.90 mM; Hill coefficient=0.93). The Ip did not show a distinct voltage dependency either with or without Na(o)+. A large outward shift of the holding current was observed by completely removing Na(o)+. In the presence of Na(o)+, a steady Ip was observed even in the absence of internal Na+ (Na(i)+). These results suggest that continuous Na+ influxes exist across the membrane. When external and internal Na+ was removed, a transient Ip was observed (half decay time (t1/2) was 5.0+/-0.6 s), thus indicating that the transient Ip was activated by the residual Na(i)+. In the absence of Na(o)+, the transient Ip was also observed with lower than 8 mM Na(i)+. The t1/2 depended on Na(i)+. However, a steady Ip was observed with 10 mM Na(i)+ or more. The functional properties of the Ip are discussed.

Characterization of the electrogenic Na+-K+ pump in horizontal cells isolated from the carp retina.

The electrogenic Na+-K+ pump current in horizontal cells acutely dissociated from the carp retina was investigated using a nystatin-perforated patch recording configuration under voltage-clamp conditions. In the presence of suitable blockers for known voltage-dependent Na+, K+ and Ca2+ conductances, the pump current was activated in a concentration-dependent manner by adding K+ ions to external solution. The EC50 value and Hill coefficient for the external K+ concentration were 0.66 mM and 1.39, respectively. The pump current did not show any significant voltage dependency at the physiological potential range between -90 and 20 mV either with or without external Na+ ions. In the presence of 120 mM external Na+ concentration, the addition of 3 mM K+ to the external solution induced a steady outward pump current even when the patch-pipette (internal) solution did not contain Na+. A large outward shift of the holding current was observed by removing external Na+. The result thus suggests that continuous Na+ influxes exist across the plasma membrane in the presence of external Na+. When Na+ was removed from both external and internal solutions, a transient outward pump current was observed by adding K+ to the external solution, thus indicating that the transient pump current was activated by the residual intracellular Na+ ions. The pump current was suppressed by ouabain in a concentration-dependent manner, and the ouabain-sensitive inhibition curve was fitted by two components. The IC50 values of high- and low-sensitive pump currents for ouabain were 20 nM and 10.4 microM, respectively, indicating the existence of at least two isoforms of the pump in the horizontal cells.

Na+,K+-ATPase activity in cultured bovine retinal pigment epithelium.

PURPOSE: To characterize electrogenic Na+,K+-ATPase activity in cultured bovine retinal pigment epithelium (RPE). METHODS: Cultured bovine RPE cells from passages 3 through 5 were dissociated enzymatically. Na+,K+-adenosine triphosphatase (ATPase)-activated currents (Ip) were measured by using a nystatin perforated-patch recording technique under voltage- clamp conditions. In the presence of suitable blockers for known voltage-dependent Na+, K+, and Ca2+ conductances, the Ip was activated in a concentration-dependent manner by adding K to the external solution. RESULTS: The median effective concentration (EC50) and Hill coefficient for external K+ concentration ([K+]o) were 1.06 mM and 2.55, respectively. The Ip showed no significant voltage dependency. A large outward shift of holding current was observed when [Na+]o, was removed. In the presence of [Na+]o, the addition of K+ to the external solution induced Ip, even when the internal solution did not contain Na+, suggesting the existence of a continuous Na+ influx across the plasma membrane in the presence of [Na+]o,. When Na+ was removed from the external and internal solutions, a transient Ip was observed, indicating that the transient Ip was activated by the intracellular residual Na+. The Ip was concentration-dependently suppressed by ouabain. The 50% inhibitory concentration (IC50) value and Hill coefficient for ouabain were 5.98 microM and 1.12, respectively. CONCLUSIONS: The present study is the first to reported the functional properties of electrogenic Na+,K+-ATPase activity in cultured bovine RPE.

Outcome of disseminated intravascular coagulation in relation to the score when treatment was begun. Mie DIC Study Group.

We examined 395 patients with disseminated intravascular coagulation (DIC) divided into two groups: non-leukemic and leukemic. In 58% of the patients as a whole, treatment of DIC resulted in complete or partial remission, while exacerbation and death occurred in 31%. The efficacy of DIC treatment in the non-leukemic group was less than that in the leukemic group, indicating that the outcome of DIC depended, in part, on the underlying disease. We examined hemostatic indicators in relation to DIC score: prothrombin time (PT) ratio, FDP, platelet count, and fibrinogen levels were found to be important indicators for the diagnosis of DIC, but not for Pre-DIC. Plasma levels of fibrin-D-dimer, thrombin-antithrombin complex (TAT), and plasmin-plasmin inhibitor complex (PPIC) were significantly increased in pre-DIC. The efficacy of treatment in relation to the DIC score when the treatment was begun showed that greater efficacy was achieved in pre-DIC than in DIC patients. The outcome was poorer with increasing DIC score, suggesting that early diagnosis and early treatment are important. On examining the relationship between outcome and hemostatic indicators, we found that the PT ratio and the levels of antithrombin, plasminogen, PPIC, the PPIC/TAT ratio, and thrombomodulin were related to outcome, suggesting that very high consumption of blood coagulation factors, liver dysfunction, hypofibrinolysis, or organ failure caused a poor outcome. Although the outcome in DIC patients may not depend substantially on plasma levels of TAT and fibrin-D-dimer, we can use these indicators to treat DIC patients at an early stage.

Decreased plasma tissue factor pathway inhibitor levels in patients with thrombotic thrombocytopenic purpura.

We measured plasma levels of tissue factor (TF) and tissue factor pathway inhibitor (TFPI) in patients with thrombotic thrombocytopenic purpura (TTP) and disseminated intravascular coagulation (DIC) to examine the relationship between TFPI and vascular endothelial cell injury. TF antigen was detected in the plasma of healthy volunteers, and the levels were significantly increased in the patients with DIC, but decreased slightly in those with TTP. Plasma TFPI levels were significantly decreased in patients with TTP compared with those in healthy volunteers. The concentration of plasma thrombomodulin (TM) antigen was significantly higher in those with TTP than in normal volunteers. One month after treatment, TTP patients showed a significant decrease in plasma TM levels, and a significant increase in plasma TFPI levels, but plasma levels of TF antigen were not significantly increased. As plasma TFPI/TF ratio was significantly increased after treatment, the hypercoagulable state was therefore improved after treatment. There was no significant difference in plasma TF and TFPI levels between those who achieved complete remission (CR) and those who died. However, plasma TM levels were significantly higher in those who died than in those who achieved CR. Plasma TFPI levels might reflect injury of vascular endothelial cells as do plasma TM levels, and decreased plasma TFPI/TF ratio and vascular endothelial cell injuries might play causative roles in TTP.

Room temperature-induced apoptosis of Jurkat cells sensitive to both caspase-1 and caspase-3 inhibitors.

We recently reported that HL-60 cells underwent apoptosis when exposed to room temperature (RT) (21 degrees C). RT-induced apoptosis of HL-60 cells is inhibited by the caspase-1 inhibitor (YVAD-CMK), but not by the caspase-3 inhibitor (DEVD-CHO). In this study, we studied RT-induced apoptosis in 15 human cell lines of hematopoietic lineage and found that the Jurkat cell line also responded to RT by a different apoptotic process. RT-induced apoptosis of Jurkat cells was attenuated by YVAD-CMK as well as DEVD-CHO. Increased caspase activity on DEVD-AMC, which was inhibited by both YVAD-CMK and DEVD-CHO added to the cell culture, was also detected. The involvement of caspase-3 itself, however, was not recognized by Western blot analysis. In contrast, the processing of caspase-3 was observed in the apoptotic HL-60 cells. These data implicate the presence of the redundant processes of apoptosis induced by RT treatment.

p53 proteins accumulated by heat stress associate with heat shock proteins HSP72/HSC73 in human glioblastoma cell lines.

We investigated the accumulation of p53 proteins after heat stress and their association with HSP72/HSC73 using four human glioblastoma cell lines. Human glioblastoma cell lines U-87MG and A-172 exhibited no mutation in the region between the 2nd and 11th exons of the p53 gene, whereas A-7 and T98G had mutations in exon 5 and exon 7 of the p53 gene, respectively. In U-87MG and A-172, the levels of wild-type p53 protein were slightly increased by heat stress. Levels of mutant p53 protein were apparently increased by heat stress in A-7, but not in T98G. Furthermore, wild-type p53 proteins in both U-87MG and A-172 co-immunoprecipitated with anti-HSP72/HSC73 antibody and HSP72 and HSC73 in them co-immunoprecipitated with anti-p53 antibody as did the mutant p53 proteins. These findings suggest that p53 proteins accumulated by heat stress are associated with HSP72 and HSC73.

Possible involvement of protein phosphatase type 2A in tumor necrosis factor alpha-induced tissue factor expression in endothelial cells.

The involvement of protein Ser/Thr phosphatase types 2A (PP2A) and 1 (PP1) in tumor necrosis factor alpha (TNF alpha)-induced tissue factor (TF) expression of human umbilical vein endothelial cells (HUVEC) was investigated. PP2A was more abundant than PP1 in the cytosol of HUVEC. CAL-A (0.5 nM) and OKA (2 nM), cell permeable inhibitors of PP1 and PP2A, augmented TNF alpha-induced TF expression, although TF expression induced by either TPA or thrombin was unchanged in the presence of GAL-A. Addition of CAL-A (0.5 nM) to TNF alpha-stimulated cultures led to an increase in the accumulation of TF transcripts. CAL-A (0.5 nM) also augmented the TNF alpha-induced phosphorylation of I kappa B-alpha, an inhibitor of NF-kappa B. Since PP2A is more sensitive to OKA than PP1, these results suggest that PP2A may be involved in regulating TNF alpha-induced TF expression in HUVEC through I kappa B-alpha dephosphorylation.