Caspase-3/CPP32-like activity is not sufficient to mediate apoptosis in an IL-2 dependent T cell line

CTLL cells undergo apoptosis when cultured in the absence of IL-2. The IL-1-converting-enzyme (ICE)/ caspase family has been implicated as an integral component of some forms of apoptosis. Numerous members of the caspase family have been identified, and it appears as if caspase-3/CPP32 plays a critical role. Previously we demonstrated that ICE/caspase-1 expression increases in CTLL cells during apoptosis; however, inhibition of ICE activity did not abrogate apoptotic death. The purpose of this report is to determine if other members of the caspase family are involved in T cell apoptosis induced by growth factor starvation. We show that cytosolic CPP32-like activity, as measured by the cleavage of DEVD-pNA and poly(ADP-ribose) polymerase (PARP), increases during apoptosis following growth factor deprivation. Cytosolic CPP32-like activity is inhibited in cells treated with the broad spectrum ICE family inhibitor boc-aspartyl(OMe)-fluoromethylketone (D-FMK) and by VAD-FMK and DEVD-FMK which have greater specificity for CPP32-like ICE homologs; however, only the broad spectrum ICE inhibitor D-FMK inhibited apoptosis. Our results suggest that apoptosis induced by growth factor deprivation involves the caspase family, but increased CPP32-like activity is not sufficient to mediate apoptosis induced by IL-2 starvation.     

人肺癌细胞CPP32基因的克隆及表达

蛋白酶尤其是ＩＣＥ家族的蛋白酶是细胞死亡机制的核心成分．ＩＣＥ蛋白酶家族中,ＣＰＰ３２（又称Ｙａｍａ,ａｐｏｐａｉｎ）在不同形式的凋亡途径中起核心作用．为深入研究ＣＰＰ３２的结构与功能,克隆了ＣＰＰ３２基因,并在大肠杆菌中进行了表达．采用ＲＴ－ＰＣＲ技术从人肺癌细胞株中获得了ＣＰＰ３２蛋白酶基因．ＤＮＡ序列分析表明,该基因由已报道的编码ＣＰＰ３２αｐ２０亚单位和ＣＰＰ３２βｐ１０亚单位的核苷酸组成,提示ＩＣＥ家族蛋白酶寡聚化可能受ＤＮＡ水平调控．将获得的ＣＰＰ３２基因分别重组到ｐＢＶ３２１和ｐＥＸ３１Ｂ载体上,并分别转化到大肠杆菌中,均获得了ＣＰＰ３２基因的较高表达,表达产物主要以包涵体形式存在．     

CPP32 cDNA的克隆及其表达和活性检测

CPP32在细胞编程死亡调控中起重要的作用.利用CPP32专一性引物,用RT-PCR方法从人鼻咽癌CNE细胞中扩增出约830 bp的片段,经序列分析证明与已发表的CPP32序列完全一致.将扩增出的编码人全长CPP32的cDNA片段克隆入pGEX-2T中,转化大肠杆菌DH5α.转化菌经诱导表达出较高含量的GST-CPP32融合蛋白.进一步研究显示,细菌中表达的CPP32蛋白能自我切割,而且能裂解体外翻译的PARP,从而证明其具有生物活性.     

The role of thiol reduction in hydroquinone-induced apoptosis in HEK293 cells

Hydroquinone (HQ) is a chemical used as a reducing agent, antioxidant, polymerization inhibitor, and chemical intermediate. It has a minor use as a bleaching agent in dermatologic preparations. HQ also occurs as a main metabolite of benzene. In the present study, HQ-induced apoptosis was evaluated by cell morphology changes, determination of phosphatidylserine (PS) externalization and analysis of sub-G1 cells. The effect of HQ on intracellular thiol concentration, including glutathione and protein thiol, and the effect of N-acetylcysteine (NAC) and buthionine sulfoximine (BSO) pretreatment on HQ-induced apoptosis were investigated. The results showed that HQ was able to induce typical apoptosis in HEK293 cells (human embryonic kidney cells) in a dose-dependent manner. Intracellular thiol, including glutathione and protein thiol, was decreased following treatment with HQ. NAC, a precursor of intracellular GSH synthesis, significantly inhibited HQ-induced apoptosis. However, BSO, a specific inhibitor of intracellular GSH synthesis, enhanced HQ-induced apoptosis significantly. Taken together, the present study demonstrates that HQ is able to induce apoptosis in HEK293 cells, most probably through depletion of intracellular thiol. The results also suggest that, at least in HEK293 cells, the control of intracellular redox homeostasis has a central role in the regulation of cell death induced by HQ.     

Characterization of CPP32-Like Protease Activity Following Apoptotic Challenge in SH-SY5Y Neuroblastoma Cells

Abstract: We characterized the activation of interleukin-1β-converting enzyme (ICE)-like proteases (caspases) in human neuroblastoma cells (SH-SY5Y) following challenge with staurosporine, an established agent known to induce apoptosis. Time course analyses of lactate dehydrogenase release detected a significant increase in cell death as early as 6 h that continued at least until 24 h following staurosporine treatment. Western blot analyses using anti-poly(ADP-ribose) polymerase (anti-PARP) and anti-CPP32 antibodies revealed proteolytic processing of CPP32 (an ICE homologue) as well as fragmentation of PARP as early as 3 h following staurosporine challenge. Furthermore, the hydrolysis of the CPP32 substrate acetyl-DEVD-7-amido-4-methylcoumarin was detected as early as 3 h and became maximal at 6 h after staurosporine challenge, suggesting a delayed and sustained period of CPP32-like activation. In addition, we used the first immunohistochemical examination of CPP32 and PARP in cells following an apoptotic challenge. The localization of CPP32 in untreated SH-SY5Y cells was exclusively restricted to the cytoplasm. Following staurosporine challenge there was a condensing of CPP32 immunofluorescence from the cytoplasm to a region adjacent to the plasma membrane. In contrast, PARP immunofluorescence was evenly distributed in the nucleus in untreated SH-SY5Y cells and on staurosporine challenge was found to be associated with condensed chromatin. It is important that a pan ICE inhibitor [carbobenzoxy-Asp-CH₂OC(O)-2,6-dichlorobenzene] was able to attenuate lactate dehydrogenase release and PARP and CPP32 cleavage and altered immunohistochemical staining patterns for PARP and CPP32 following staurosporine challenge.     

Improvement of a mammalian cell culture process by adaptive,model-based dialysis fed-batch cultivation and suppression of apoptosis

Both conventional and genetic engineering techniques can significantly improve the performance of animal cell cultures for the large-scale production of pharmaceutical products. In this paper, the effect of such techniques on cell yield and antibody production of two NS0 cell lines is presented. On the one hand, the effect of fed-batch cultivation using dialysis is compared to cultivation without dialysis. Maximum cell density could be increased by a factor of ~5–7 by dialysis fed-batch cultivation. On the other hand, suppression of apoptosis in the NS0 cell line 6A1 bcl-2 resulted in a prolonged growth phase and a higher viability and maximum cell density in fed-batch cultivation in contrast to the control cell line 6A1 (100)3. These factors resulted in more product formation (by a factor ~2). Finally, the adaptive model-based OLFO controller, developed as a general tool for cell culture fed-batch processes, was able to control the fed-batch and dialysis fed-batch cultivations of both cell lines.Abbreviations A membrane area (dm²) - c _Glc,F glucose concentration in nutrient feed (mmol L^–1) - c _Glc,FD glucose concentration in dialysis feed (mmol L^–1) - c _Glc,i glucose concentration in inner reactor chamber (mmol L^–1) - c _Glc,o glucose concentration in outer reactor chamber (dialysis chamber) (mmol L^–1) - c _Lac,FD lactate concentration in dialysis feed (mmol L^–1) - c _Lac,i lactate concentration in inner reactor chamber (mmol L^–1) - c _Lac,o lactate concentration in outer reactor chamber (dialysis chamber) (mmol L^–1) - c _LS,FD limiting substrate concentration in dialysis feed (mmol L^–1) - c _LS,i limiting substrate concentration in inner reactor chamber (mmol L^–1) - c _LS,o limiting substrate concentration in outer reactor chamber (dialysis chamber) (mmol L^–1) - c _Mab monoclonal antibody concentration (mg L^–1) - F _D feed rate of dialysis feed (L h^–1) - F _Glc feed rate of nutrient concentrate feed (L h^–1) - K _d maximum death constant (h^–1) - k _d,LS death rate constant for limiting substrate (mmol L^–1) - k _Glc monod kinetic constant for glucose uptake (mmol L^–1) - k _Lac monod kinetic constant for lactate uptake (mmol L^–1) - k _LS monod kinetic constant for limiting substrate uptake (mmol L^–1) - K _Lys cell lysis constant (h^–1) - K _S,Glc monod kinetic constant for glucose (mmol L^–1) - K _S,LS monod kinetic constant for limiting substrate (mmol L^–1) - µ cell-specific growth rate (h^–1) - µ _d cell-specific death rate (h^–1) - µ _d,min minimum cell-specific death rate (h^–1) - µ _max maximum cell-specific growth rate (h^–1) - P _Glc membrane permeation coefficient for glucose (dm h^–1) - P _Lac membrane permeation coefficient for lactate (dm h^–1) - P _LS membrane permeation coefficient for limiting substrate (dm h^–1) - q _Glc cell-specific glucose uptake rate (mmol cell^–1 h^–1) - q _Glc,max maximum cell-specific glucose uptake rate (mmol cell^–1 h^–1) - q _Lac cell-specific lactate uptake/production rate (mmol cell^–1 h^–1) - q _Lac,max maximum cell-specific lactate uptake rate (mmol cell^–1 h^–1) - q _LS cell-specific limiting substrate uptake rate (mmol cell^–1 h^–1) - q _LS,max maximum cell-specific limiting substrate uptake rate (mmol cell ^–1 h^–1) - q _Mab cell-specific antibody production rate (mg cell^–1 h^–1) - q _MAb,max maximum cell-specific antibody production rate (mg cell^–1 h^–1) - t time (h) - V _i volume of inner reactor chamber (culture chamber) (L) - V _o volume of outer reactor chamber (dialysis chamber) (L) - X _t total cell concentration (cells L^–1) - X viable cell concentration (cells L^–1) - Y _Lac/Glc kinetic production constant (stoichiometric ratio of lactate production and glucose uptake) (–)     

Role of apoptosis and apoptosis-related proteins in the cisplatin-resistant phenotype of human tumor cell lines

Since apoptosis is the primary mode of cell death induced by cisplatin, the role of apoptosis and apoptosis-related gene products in cisplatin resistance was investigated in four human cisplatin-resistant cell lines of different tumour type. A common feature of the resistant sublines was a reduced susceptibility to drug-induced apoptosis compared to parental sensitive lines. Loss of wild-type p53 function was not a general event associated with the development of drug resistance. An increased bcl-2 expression was found in resistant cells characterized by mutant p53 (A431/Pt and IGROV-1/Pt), whereas in osteosarcoma (U2-OS/Pt) and in ovarian carcinoma (A2780/CP) cells with wild-type p53, bcl-2 levels were markedly reduced. U2-OS/Pt cells had a 16-fold increase in the level of Bcl-xL protein. Stable transfection of U2-OS cells with bcl-xL cDNA conferred a low level of drug resistance to cisplatin, suggesting that overexpression of this gene contributes to the ci splatin-resistant phenotype of this osteosarcoma cell system. In conclusion, these observations suggest a variable contribution of apoptosis-related genes to cisplatin resistance depending on the biological background of the cell system and presumably reflecting different pathways of apoptosis. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cytochrome c activation of CPP32-like proteolysis plays a critical role in a Xenopus cell-free apoptosis system.

In a cell-free system based on Xenopus egg extracts, Bcl-2 blocks apoptotic activity by preventing cytochrome c release from mitochondria. We now describe in detail the crucial role of cytochrome c in this system. The mitochondrial fraction, when incubated with cytosol, releases cytochrome c. Cytochrome c in turn induces the activation of protease(s) resembling caspase-3 (CPP32), leading to downstream apoptotic events, including the cleavage of fodrin and lamin B1. CPP32-like protease activity plays an essential role in this system, as the caspase inhibitor, Ac-DEVD-CHO, strongly inhibited fodrin and lamin B1 cleavage, as well as nuclear morphology changes. Cytochrome c preparations from various vertebrate species, but not from Saccharomyces cerevisiae, were able to initiate all signs of apoptosis. Cytochrome c by itself was unable to process the precursor form of CPP32; the presence of cytosol was required. The electron transport activity of cytochrome c is not required for its pro-apoptotic function, as Cu- and Zn-substituted cytochrome c had strong pro-apoptotic activity, despite being redox-inactive. However, certain structural features of the molecule were required for this activity. Thus, in the Xenopus cell-free system, cytosol-dependent mitochondrial release of cytochrome c induces apoptosis by activating CPP32-like caspases, via unknown cytosolic factors.     

Mesangial cell apoptosis induced by a fibronectin fragment

We previously showed that in passive Heymann nephritis (PHN) rats, a large quantity of fibronectin (FN) fragments containing the central cell-binding (CCB) domain and adjacent domains are generated in the kidney and excreted into urine (Nishizawa et al., Biol Pharm Bull 1998; 21: 429–433). To ascertain whether the FN fragments could affect the progression of PHN, we investigated the effect of a 150 K FN fragment containing the CCB and carboxyl-terminal heparin-binding (Hep 2) domains on cultured rat mesangial cells. When rat mesangial cells cultured on FN-coated plates were exposed to the 150 K FN fragment, some mesangial cells detached from the FN substrate and then underwent apoptosis as judged by nuclear and DNA fragmentations. The 150 K FN fragment competitively inhibited the mesangial cell adhesion to the FN substrate in a dose-dependent manner. Furthermore, gelatinzymography of the conditioned medium of mesangial cells showed that the 150 K FN fragment induced and/or poteintiated the extracellular matrix (ECM)-degrading proteinases including matrix metalloproteinases (MMPs) of mesangial cells. These results indicate that the 150 K FN fragment may elicit mesangial cell apoptosis by disrupting the mesangial cell adhesion through two distinct ways: the inhibition of mesangial cell adhesion and the ECM-degradation by the 150 K FN fragment-induced MMPs. Thus, FN fragments containing the CCB and adjacent domains generated in the kidneys of PHN rats may be involved in the evolution of the renal injury.     

Protein expression profiles in Saccharomyces cerevisiae during apoptosis induced by H2O2

We identified the proteins involved during apoptosis induced by H2O2 in Saccharomyces cerevisiae, and analyzed the global protein pattern by 2-DE. We analyzed classical parameters of apoptosis such as chromatin condensation, DNA fragmentation, and morphology changes of cells. Exposure of yeast cells to nonphysiological doses of peroxides decreases the expression (or increases degradation) of enzymes involved in protection against oxidative stress. This leads the yeast cells to a reduction of their antioxidant defense and makes the cells more prone to apoptosis. In our data the down expression of peroxiredoxin II and GST I, could induce a perturbation of mitochondrial function with an alteration of permeability of the membrane leading to the mitochondria-mediated apoptosis. Moreover, we identified a new spot of a classical glycolytic enzyme: the glyceraldehyde 3-phosphate dehydrogenase during apoptosis. It is known that GAPDH is an extremely abundant glycolytic enzyme with multiple functions and that its overexpression is evident during apoptosis induced by a variety of stimuli. Our results confirm that it is a major intracellular messenger mediating apoptotic death and that this new spot of GAPDH could be an intracellular sensor of oxidative stress during apoptosis induced by H2O2 in S. cerevisiae.     

Nonylphenol enhances apoptosis induced by serum deprivation in PC12 cells

Although nonylphenol is well known as an endocrine disrupting chemical, there is little information concerning biological effect of nonylphenol. In this study, we investigated effect of nonylphenol on apoptosis induced by serum deprivation in PC12 cells using TUNEL and DNA fragmentation assays. In addition, changes in contents of proapoptotic factors, Bad and Bax, and antiapoptotic factor, Bcl-2, and enzyme activity of caspase-3 were studied. Below 100 ng/ml of nonylphenol increased TUNEL signals, DNA fragmentation and content of proapoptotic factor, Bad as compared to those by serum deprivation without nonylphenol. Furthermore, addition of nonylphenol enhanced caspase-3 activity and Z-VAD, caspase-3 inhibitor, diminished such effect. These results indicated that below 100 ng/ml of nonylphenol enhanced apoptosis induced by serum deprivation via caspase-3 activation in PC12 cell.     

Proteolytic cleavage and activation of PAK2 during UV irradiation-induced apoptosis in A431 cells

Exposure of mammalian cells to ultraviolet (UV) light elicits a cellular response and can also lead to apoptotic cell death. In this report, we show that a 36-kDa myelin basic protein (MBP) kinase detected by an in-gel kinase assay can be dramatically activated during the early stages of UV irradiation-triggered apoptosis of A431 cells. Immunoblot analysis revealed that this 36-kDa MBP kinase could be recognized by an antibody against the C-terminal regions of a family of p21^Cdc42/Rac-activated kinases (PAKs). By using this antibody and a PAK2-specific antibody against the N-terminal region of PAK2 as studying tools, we further demonstrated that UV irradiation caused cleavage of PAK2 to generate a 36-kDa C-terminal catalytic fragment and a 30-kDa N-terminal fragment in A431 cells. The appearance of the 36-kDa C-terminal catalytic fragment of PAK2 matched exactly with the activation of the 36-kDa MBP kinase in A431 cells upon UV irradiation. In addition, UV irradiation also led to activation of CPP32/caspase-3, but not ICH-1L/caspase-2 and ICE/caspase-1, in A431 cells and the kinetics of activation of CPP32/caspase-3 appeared to correlate well with that of DNA fragmentation and of cleavage/activation of PAK2, respectively. Moreover, blockage of activation of CPP32/caspase-3 by pretreating the cells with two specific tetrapeptidic inhibitors for caspases (Ac-DEVD-cho and Ac-YVAD-cmk) could significantly attenuate the extent of cleavage/activation of PAK2 induced by UV irradiation. Collectively, the results demonstrate that cleavage and activation of PAK2 can be induced during the early stages of UV irradiation-triggered apoptosis and indicate the involvement of CPP32/caspase-3 in this process. J. Cell. Biochem. 70:442-454, 1998. © 1998 Wiley-Liss, Inc.     

Characterization of a cDNA from Arabidopsis thaliana encoding a potential thiol protease whose expression is induced independently by wilting and abscisic acid

The sequence and expression characteristics are described of a wilt-inducible gene in Arabidopsis thaliana. A 1494 encodes a potential thiol protease whose mRNA accumulates rapidly in shoot tissue upon the loss of turgor. A1494 mRNA levels peaked after ca. 4 h and declined thereafter. Dehydration also induced rapid biosynthesis of the phytohormone abscisic acid (ABA), which continued for at least 9 h. Exogenous ABA induced the accumulation of A1494 mRNA, with kinetics similar to those after wilting. Rehydration of wilted shoots led to a rapid decline in the content of both ABA and A1494 mRNA. Wilting and ABA independently induced A1494 expression as evidenced by the effects of ABA and wilting on the ABA-deficient aba-1 and ABA-insensitive abi-1 and abi-3 genotypes. A1494 mRNA was not detectable in aba-1 shoots but accumulated rapidly after either wilting or ABA treatment, whereas the shoot ABA content was increased only by ABA treatment. ABA had no effect on A1494 mRNA levels in the abi-1 and abi-3 mutants but wilting did result in enhanced A1494 expression. Heat shock had only a minor effect on A1494 mRNA levels, whereas exposure to low temperature resulted in substantial accumulation of A1494 mRNA in wild-type shoots. However, this latter response, unlike that to drought, was mediated exclusively via ABA synthesis as demonstrated by the lack of A1494 mRNA accumulation in cold-treated aba-1 shoots.     

Enhancement of lysosomal proton permeability induced by photooxidation of membrane thiol groups

Effects of photooxidation of membrane thiol groups on lysosomal proton permeability were studied by measuring intralysosomal pH with fluorescein isothiocyanate-dextran and monitoring proton leakage with p-nitrophenol. Methylene blue-mediated photooxidation of lysosomes decreased their membrane thiol groups and produced cross-linking of the membrane proteins, which was established by the measurement of residual membrane thiol groups with 5,5'-dithio-bis(2-nitrobenzoic acid) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively. The cross-linking of proteins could be abolished by subsequent treatment of the photodamaged lysosomes with dithiothreitol, indicating that the proteins were linked via disulfide bonds. In addition, the photodamage of lysosomes raised the intralysosomal pH and caused leakage of the lysosomal protons, which could also be reversed by subsequent dithiothreitol treatment. This indicates that lysosomal proton permeability can be increased by photooxidation of the membrane thiol groups and recovered to the normal level by reduction of the groups.     

A DEVD-Inhibited Caspase Other than CPP32 Is Involved in the Commitment of Cerebellar Granule Neurons to Apoptosis Induced by K+ Deprivation

Abstract: Cultured cerebellar granule neurons undergo apoptosis when switched from a medium containing depolarizing levels of K⁺ (25 mM KCI) to medium containing lower levels of K⁺ (5 mM KCI). We used this paradigm to investigate the role of caspases in the death process. Two broad-spectrum caspase inhibitors, tert-butoxycarbonyl-Asp·(O-methyl)·fluoromethyl ketone and benzyloxycarbonyl-Val-Ala-Asp·fluoromethyl ketone, significantly reduced cell death (90 and 60%, respectively) at relatively low concentrations (10–25 µM), suggesting that caspase activation is involved in the apoptotic process. DNA fragmentation, a hallmark of apoptosis, was also reduced by these caspase inhibitors, suggesting that caspase activation occurred upstream of DNA cleavage in the sequence of events leading to cell death. As a step toward identifying the caspase(s) involved, the effects of N-acetyl Tyr-Val-Ala-Asp·chloromethyl ketone (YVAD·cmk), an interleukin-1β converting enzyme-preferring inhibitor, and N-acetyl Asp-Glu-Val-Asp·fluoromethyl ketone (DEVD·fmk), a CPP32-preferring inhibitor, were also evaluated. YVAD·cmk provided only modest (<20%) protection and only at the highest concentration (100 µM) tested, suggesting that interleukin-1β converting enzyme and/or closely related caspases were not involved. In comparison, DEVD·fmk inhibited cell death by up to 50%. Western blot analyses, however, failed to detect an increase in processing/activation of CPP32 or in the proteolysis of a CPP32 substrate, poly(ADP-ribose) polymerase, during the induction of apoptosis in granule neurons. Similarly, the levels of Nedd2, a caspase that is highly expressed in the brain and that is partially inhibited by DEVD·fmk, also remained unaffected in apoptotic neurons undergoing apoptosis. These results suggest that a DEVD-sensitive caspase other than CPP32 or Nedd2 mediates the induction of apoptosis in K⁺-deprived granule neurons.     

Induction of apoptosis and c-myc in L1210 lymphocytic leukemia cells by adenosine

High concentrations of adenosine (Ado), when added to L1210 lymphocytic leukemia cells, resulted in apoptosis or programmed cell death. The apoptotic process was accompanied by distinct morphological changes including chromatin condensation and blebbing of plasma membranes. Extensive DNA fragmentation was correlated with Ado concentrations. Furthermore, apoptosis in these cells was preceded by an early but transient expression of c-myc proto-oncogene, and was not influenced by homocysteine thiolactone added to the cells. Since severe combined immunodeficiency (SCID) is associated with a deficiency of adenosine deaminase, leading to defects in both cellular and humoral immunity, Ado-induced apoptosis may thus be a contributing factor in the pathology of SCID.     

Caspase-3 immunohistochemical expression is a marker of apoptosis,increased grade and early recurrence in intracranial meningiomas

Caspase-3 is the ultimate executioner caspase that is essential for the nuclear changes associated with apoptosis. We investigated caspase-3 immunohistochemical expression in 58 primary intracranial meningiomas, using one monoclonal antibody detecting both precursor and cleaved caspase-3 (CPP32) and a second recognizing only the cleaved activated form (ASP175). Caspase-3 expression was analyzed in relation to baseline apoptosis—as illustrated by the expression of anti-single stranded DNA (ss-DNA), the antiapoptotic protein bcl-2, proliferation indices (Ki-67, PCNA, topoisomerase IIa, mitosin C), hormonal status (estrogen, progesterone, androgen receptors), standard clinicopathological parameters and patients’ disease-free survival. Caspase-3 immunostaining was observed in 62% of cases for CPP32 and in 24% for ASP175. In both instances, the labeling index (LI) was significantly correlated with ss-DNA LI (p=0.038 and p=0.018). CPP32 but not ASP175 LI positively correlated with the mitotic index (p=0.001) and PCNA LI (p=0.004). Both CPP32 and ASP175 LIs were increased in nonbenign meningiomas (p<0.0001 and p=0.0035 respectively). In univariate and multivariate survival analyses, caspase-3 predicted meningioma recurrence, independently affecting disease-free survival (p=0.011 and p=0.047 respectively for CPP32; p<0.0001 and p=0.012 respectively for ASP175). Caspase-3 may prove to be a useful predictor of early recurrence in a group of neoplasms characterized by the frequent discordance between histology and clinical behavior.     

Existence of two kinds of thiol protease inhibitors in rat serum

In rat serum two kinds of thiol protease inhibitors were found. One had molecular weight about 103,000 and the other about 16,000. Though both inhibitors inhibited all thiol proteases examined, the high molecular weight inhibitor showed stronger inhibition on papain than cathepsin H. But the low molecular weight inhibitor equally inhibited papain and cathepsin H. The isoelectric point of low molecular weight inhibitor was pH 9.16 and that of high molecular one was pH 4.76.     

Role of vitamins in determining apoptosis and extent of suppression by bcl-2 during hybridoma cell culture

The identification of cell culture media components that may instigate apoptosis in cell lines used for the production of commercial antibodies and recombinant proteins, is crucial to aid the development of improved media for reduced cell death and to understand the role of nutrient components in cell survival and maintenance. Here we determine the impact of depriving all or individual B-group media vitamins either, D-CaPantothenate (DCaP), choline chloride (CC), riboflavin (Rb), i-inositol, nicotinamide (NAM), pyridoxal hydrochloride (PyrHCl), folic acid (FA), or thiamine hydrochloride (ThHCl) on hybridoma cell growth and viability using fluorescence microscopy techniques. Cultivation in media deprived of all these vitamins prevented cell proliferation from reaching maximum capacity while increasing cell death rate, predominantly via apoptosis. Deletion of either DCaP, CC, or Rb showed that these components were most likely responsible for the development of apoptosis. Exclusion of either i-inositol, NAM or PyrHCl failed to inhibit cell growth and viability, while marginal improvements in viability were noted by ThHCl deprivation and more so by FA exclusion. Over-expression of the anti-apoptotic gene bcl-2 suppressed cell death initiated by all or single vitamin (either DCaP, CC or Rb) deprivation. The involvement of bcl-2 activity, established a close association between small vitamin molecules particularly DCaP, CC or Rb and the biochemical activation of apoptosis.