Effect of Bcl-2 overexpression on apoptosis, cell growth and telomerase activity.
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Apoptosis↗
Biomedical subjects
S C Chow
Publications and source records attributed to S C Chow.
Simultaneous cross-linking of CD6 and CD28 induces cell proliferation in resting T cells.
In the present study, we showed that simultaneous ligation of the monoclonal antibodies (mAb) against CD6 and CD28 induces T-cell proliferation in purified resting T lymphocytes in the absence of T-cell receptor (TCR) occupancy. No cell proliferation was observed when the mAb were cross-linked alone or used simultaneously in the soluble form. T-cell proliferation mediated through CD6/CD28 is accompanied by the up-regulation of interleukin-2 (IL-2) mRNA and expression of IL-2 receptors on the cell surface. In the presence of IL-2-neutralizing mAb the proliferative response of the T cell induced through CD6/CD28 was inhibited dose dependently. Cross-linking mAb to CD6 and CD28 alone or together did not down-regulate the CD3/TCR complex. T-cell proliferation mediated through CD6/CD28 was only partially blocked by the immunosuppressive drug, cyclosporin A (CsA), whereas anti-CD28-induced T-cell proliferation in the presence of the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), was unaffected. In sharp contrast T-cell proliferation mediated by anti-CD6 in the presence of TPA was efficiently blocked by CsA. In addition, two protein kinase C (PKC) inhibitors, GF 109203X and H-7 dose-dependently inhibited T-cell proliferation mediated through CD6/CD28, suggesting that PKC activation may be involved. Furthermore, there was a marked differential dose-dependent inhibitory effect of the PKC inhibitors on T-cell proliferation mediated by the co-ligation of anti-CD6 or anti-CD28 in the presence of anti-CD3, with the former being more sensitive to PKC inhibition. Taken collectively, our results suggest that T-cell activation can occur through an antigen-independent pathway by cross-linking the accessory molecules, CD6 and CD28, and that these two cell surface antigens may have distinct signalling pathways.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
Thioredoxin is transcriptionally induced upon activation of heat shock factor 2.
Heat shock gene expression is differentially regulated in cells exposed to stress stimuli and in cells undergoing processes of differentiation and development. Regulation of the classical heat shock response is mediated by heat shock factor 1 (HSF1), whereas heat shock factor 2 (HSF2) is activated in certain differentiating cells, for example during hemin-mediated differentiation of human K562 erythroleukemia cells. Hence, the signaling pathways leading to induction of heat shock gene expression upon different stimuli are likely to be distinct. We have used RNA arbitrarily primed polymerase chain reaction to identify genes that are differentially regulated upon activation of HSF1 and HSF2. In this study, we report that thioredoxin (TRX) expression is induced in K562 cells in response to hemin in an HSF2-dependent manner. Increased TRX expression was primarily detected on the transcriptional level, subsequently leading to elevated TRX mRNA and protein levels. Hemin treatment caused no reduction in cellular glutathione concentrations, indicating that the increased TRX expression was not due to oxidative stress. Studies using cell lines where overexpression of the HSF2-beta isoform represses HSF2 activation implied that active HSF2 is required for transcriptional induction of TRX. Unlike HSF2, activation of HSF1 did not induce TRX expression. Taken together, our results suggest that HSF1 and HSF2 may regulate distinct target genes, and activation of HSF2 could be involved in the regulation of TRX expression during hemin-mediated differentiation of K562 cells.
Cell Differentiation↗
Induction of apoptosis and potentiation of TNF- and Fas-mediated apoptosis in U937 cells by the xanthogenate compound D609.
Apoptosis induced by ligation of either the tumor necrosis factor (TNF) p55 receptor or the Fas/APO-1/CD95 receptor has been suggested to require ceramide as a signaling molecule. Ceramide is formed as a result of sphingomyelinase (SMase) activation in the sphingomyelin cycle, and ligation of TNF and Fas receptors has been reported to stimulate SMase activity. We have studied the effects of D609, a xanthogenate compound with antitumoral properties, on TNF- or Fas-induced apoptosis of monocytic U937 cells. First, the effects of D609 on SMase activity were assessed using in vitro assays for neutral and acidic SMase, and the results suggested that D609 caused a modest stimulation of the activity of both SMases in U937 cells. Exposure of U937 cells for 6 h to TNF or anti-Fas mAb induced apoptosis in 40-45% of the cells, as measured by fluorescent staining of nuclear chromatin. Cotreatment with D609 potentiated TNF- as well as Fas-mediated apoptosis up to 70 and 90%, respectively. Furthermore, in incubations with D609 alone, 60% of the cells became apoptotic within 16 h. Since D609 has been reported to inhibit protein kinase C (PKC) activity, the effect of phorbol 12-myristate 13-acetate (PMA) on D609-induced apoptosis was investigated. PMA was found to inhibit D609-induced apoptosis in U937 cells as well as cell death induced by TNF and anti-Fas mAb. Thus, PKC inactivation may play an important role in the regulation of apoptosis in U937 cells. In summary, the present results show that D609 stimulates SMase activity, potentiates TNF- and Fas-induced apoptosis, and induces apoptosis on its own in U937 cells.
Antibodies, Monoclonal↗
Susceptibility of different subsets of immature thymocytes to apoptosis.
In the present study the susceptibility of different subsets of immature rat thymocytes to undergo apoptosis was examined. Unfractionated rat thymocytes were negatively enriched into immature double positive (CD4+ CD8+), immature single positive (CD4- CD8+ CD3-) and triple negative (CD4- CD8- CD3-) thymocytes. These enriched subsets of immature thymocytes were then exposed to various apoptotic stimuli such as dexamethasone, etoposide and thapsigargin which readily induced apoptosis in unfractionated rat thymocytes. We found that the double positive thymocytes and their precursor cells, i.e. the single positive immature thymocytes, were equally sensitive to apoptosis after treatment with the apoptotic stimuli. In sharp contrast, the early migrants or precursor-containing thymocytes which are triple negative have a lower spontaneous apoptosis rate and were relatively resistant to all the apoptotic stimuli. These findings showed a breakpoint in thymocyte sensitivity to apoptosis which occurs after the onset of CD8 expression, suggesting that susceptibility of thymocytes to apoptosis is developmentally regulated.
Animals↗
Statistical methods for two-sequence three-period cross-over designs with incomplete data.
In clinical trials, and in bioavailability and bioequivalence studies, one often encounters replicate cross-over designs such as a two-sequence three-period cross-over design to assess treatment and carry-over effects of two formulations of a drug product. Because of the potential dropout (or for some administrative reason), however, the observed data set from a replicate cross-over design is incomplete or unbalanced so that standard statistical methods for a cross-over design may not apply directly. For inference on the treatment and carry-over effects, we propose a method based on differences of the observations that eliminates the random subject effects and thus does not require any distributional condition on the random subject effects. When no datum is missing, this method provides the same results as the ordinary least squares method. When there are missing data, the proposed method still provides exact confidence intervals for the treatment and carry-over effects, as long as the dropout is independent of the measurement errors. We provide an example for illustration.
Clinical Trials as Topic↗
Purines and their roles in apoptosis.
Purines are ubiquitous endogenous metabolites, and their roles as signalling molecules, especially in the case of adenosine and ATP, are well documented. The release of purines is increased when cells are highly activated, stressed or damaged, and this is known to have profound effects on various organ systems. Recently, purines like adenosine and ATP have been shown to be cytotoxic. Current evidence suggests that adenosine induces cell death by apoptosis, whereas ATP appears to cause both necrosis and apoptosis. Apoptosis is an important physiological process during normal tissue turnover and in the maturation of the immune system, embryogenesis, metamorphosis, endocrine-dependent tissue atrophy, etc. Recently, many of the key components of the apoptotic cell death cascade have become unravelled. In particular, proteases belonging to the interleukin-1 beta-converting (ICE) enzyme family, also known as caspases, have been shown to act as an intracellular convergence point that orchestrates the morphological and biochemical features of apoptosis. However, little is known about the signalling or the biochemical mechanisms of purine-mediated cell death. Adenosine appears to act through P1 purinoceptors, although the subtype involved remains controversial, whereas ATP may involve both P2X1 and P2X7 purinoceptors. More recent evidence suggests that the intracellular levels of purines, in addition to the cell surface receptor-mediated responses, may also play a critical role by modulating other apoptotic cell death signals. Here, we review our current understanding about purines in mediating cell death and raise a number of questions as to the possible mechanisms involved.
Adenosine↗
A note on sample size determination for bioequivalence studies with high-order crossover designs.
Similar to Liu and Chow, approximate formulas for sample size determination are derived based on Schuirmann's two one-sided tests procedure for bioequivalence studies for the additive and the multiplicative models under various higher order crossover designs for comparing two formulations of a drug product. The higher order crossover designs under study include Balaam's design, the two-sequence dual design, and two four-period designs (with two and four sequences), which are commonly used for assessment of bioequivalence between formulations. The derived formulas are simple enough to be carried out with a pocket calculator. The number of subjects required for each of the four higher order designs are tabulated for selected powers and various parameter values.
Clinical Trials as Topic↗
The role of proteolysis in T cell apoptosis triggered by chelation of intracellular Zn2+.
Our previous work showed that chelation of intracellular Zn2+ with N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) induces apoptosis in rat thymocytes. The molecular mechanism involved in TPEN-triggered apoptosis remains unknown, except that it is a Ca2+-independent process. In the present study, we show that TPEN is unable to induce DNA fragmentation when added to isolated thymocyte nuclei, indicating that activation of a cytoplasmic component is essential for TPEN-induced apoptosis. Since cytosolic proteases related to interleukin-1beta-converting enzyme (ICE) are implicated as key activators of apoptosis in many different systems, we investigated the possible involvement of such proteases in TPEN-induced apoptosis. We found that treatment of thymocytes with TPEN caused an early degradation of nuclear poly(ADP-ribose) polymerase (PARP) and lamin prior to DNA cleavage. This could be inhibited by Z-Val-Ala-Asp-chloromethylketone (VADcmk), an inhibitor of ICE-like proteases, but not by an inhibitor of Ca2+-regulated serine protease. Jurkat T cells also underwent extensive DNA fragmentation when incubated with TPEN. A cytosolic fraction, prepared from TPEN-treated Jurkat cells, produced extensive DNA fragmentation when applied to isolated thymocyte nuclei, whereas the cytoplasmic extract from untreated cells was ineffective either alone or together with TPEN. The apoptosis-inducing activity in cytosolic fraction from TPEN-treated Jurkat cells was blocked by incubating cells in the presence of VADcmk or another inhibitor of ICE-like proteases, Ac - Asp - Glu - Val - Asp-aldehyde (DEVD-CHO), which has been found to competitively inhibit CPP32/apopain. An increase in enzyme activity that cleaves Ac-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (DEVD-AMC), a fluorogenic substrate of CPP32/apopain and Mch3alpha, was detected in TPEN-treated thymocytes and Jurkat cells. In addition, the proteolytic cleavage of CPP32 resulting in the formation of two active fragments (p17 and p12) was observed in cytosolic extracts from TPEN-treated Jurkat cells, but not in extracts which were prepared from cells treated with TPEN in the presence of VADcmk or DEVD-CHO. Our results suggest that activation of cytosolic ICE-like proteases is an essential step in TPEN-induced apoptosis, and that CPP32/apopain is critically involved in this process.
Journal Article↗
Some thoughts on individual bioequivalence.
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Humans↗
A two one-sided tests procedure for assessment of individual bioequivalence.
In this paper we propose a two one-sided tests procedure for assessment of individual bioequivalence based on the concept of individual equivalence ratios proposed by Anderson and Hauck. The proposed procedure is derived under the normality assumption for the logarithmic transformation of pharmacokinetic responses obtained from a standard two-sequence, two-period crossover design. We show that the hypotheses for individual bioequivalence are equivalent to the hypotheses for testing whether the upper (or lower) pth quantile of the distribution of the differences between the test and reference formulations from the same subject is not greater (or not smaller) than some prespecified equivalence limits. Under this setting, we examine the relationship between average and individual bioequivalence. There exists the uniformly most powerful invariant test for each of the two one-sided hypotheses. In addition, the proposed two one-sided tests procedure is a test of size alpha (i.e., < or = alpha). We demonstrate that the determination of critical values, the enumeration of power, and the estimation of sample sizes requires noncentral t-distributions but does not necessarily require the estimation of unknown population mean and variance for noncentrality parameters. We discuss possible extensions to other crossover and replicated crossover designs. A numerical example illustrates the proposed procedure.
Biotransformation↗
Meta-analysis for bioequivalence review.
The problem of drug interchangeability among a brand-name drug and its generic copies is considered. Under current Food and Drug Administration (FDA) regulation, a patient may switch from the brand-name drug to a generic drug if the generic drug is shown to be bioequivalent to the brand-name drug based on bioequivalence testing. After the patent of a brand-name drug is expired, usually there will be a number of generic copies available on the market. The FDA does not indicate that a patient may switch from a generic to another even though both of the generic drugs are bioequivalent to the brand-name drug. As a result, drug interchangeability among the brand-name and its generic copies is a safety concern. In this paper, we propose to perform a meta-analysis for an overview of bioequivalence. The proposed meta-analysis provides an assessment of bioequivalence among generic copies of a brand-name that can be used as a tool to monitor the performance of the approved generic copies of the brand-name drug. In addition, it provides more accurate estimates of inter- and intrasubject variabilities of the drug product.
Humans↗
An adjusted two one-sided t-test for the assessment of bioequivalence with multiple doses.
In medical practice, it has been realized that noncompliance may have an impact on the therapeutic effect of a drug therapy, regardless of race, gender, and education of patients. Therefore, it is of interest to study the impact of noncompliance on drug absorption through in vivo testing. Efron and Feldman examined dose-response relationship when noncompliance is an issue. In this paper, we study bias and variation induced by noncompliance for pharmacokinetic parameters such as the area under the curve (AUC). We use bioequivalence testing as an example to demonstrate that a false conclusion could be drawn if one ignores the effect of noncompliance. We propose a new test for the assessment of bioequivalence in multiple doses. The proposed test appears to have a substantial improvement over the usual two one-sided tests based on a simulation study.
Area Under Curve↗
Statistical comparison between dissolution profiles of drug products.
The problem for assessment of similarity between dissolution profiles of two drug products is considered. The commonly used classical methods including model-dependent and model-independent approaches are reviewed. Most methods encountered the difficulties of no well-defined equivalence limits and the correlation between consecutive time points. Several methods have been proposed to account for these difficulties, e.g., the method of repeated measures (1) and the multivariate analysis approach (2). These methods, however, are sensitive to the selection of time points and complex covariance error structure. In this paper, we propose equivalence limits for similarity based on the amount of dissolved active ingredient as specified in the United States Pharmacopeia and National Formulary (USP/NF). Accordingly, we proposed a time series approach, which accounts for correlation between dissolution results at different time points. The proposed model is shown to be useful in assessment of similarity between dissolution profiles of two drug products. An example concerning dissolution testing of two lots of a drug product is used to illustrate the proposed equivalence limits and statistical methodology.
Analysis of Variance↗
Chromatin condensation during apoptosis requires ATP.
The processes leading to morphological changes of the chromatin in cells that undergo apoptosis are presently unclear. We have recently shown that chromatin fragmentation and the nuclear morphological changes typically seen in apoptosis were reproduced in an in vitro system comprised of isolated rat thymocyte nuclei incubated in the presence of a lysate from Fas/APO-1-stimulated JURKAT cells [Chow, Weis, Kass, Holmström, Eriksson and Orrenius (1995) FEBS Lett. 364, 134-138]. Using this in vitro system, we now report that the presence of ATP is necessary for chromatin condensation, its movement to the nuclear periphery and apoptotic body formation. In clear contrast, chromatin cleavage into high-molecular-mass and oligonucleosomal-length DNA fragments induced by lysates derived from Fas/APO-1-activated JURKAT cells did not require the presence of ATP. The induction of these morphological changes by ATP could not be substituted by the analogues, adenosine 5'-[beta, gamma-methylene]triphosphate and adenosine 5'-[alpha, beta-methylene]-triphosphate, AMP, cAMP and UTP. However, adenosine 5'-[gamma-thio]triphosphate, and to a lesser degree GTP and ADP, could partially replace ATP in inducing nuclear apoptotic morphological changes. It is concluded that ATP is essential for the morphological changes occurring in nuclei during apoptosis, but not for DNA fragmentation.
Adenosine Triphosphate↗
Adenosine stimulates calcium influx in isolated rat hepatocytes.
The mechanism of stimulation of Ca2+ entry into hepatocytes by adenosine was investigated. When Fura-2-loaded hepatocytes were suspended in a nominally Ca(2+)-free buffer, adenosine produced only a small transient increase in the cytosolic free Ca2+ concentration ([Ca2+)i). However, on restoration of an extracellular Ca2+ concentration of 1.3 mM, a rapid increase in [Ca2+]i occurred, which indicates activation of a Ca(2+)-influx pathway. Adenosine augmented the rate of Ca2+ influx triggered by maximally effective concentrations of thapsigargin or cAMP, but was without effect on the rate of Ca2+ entry that resulted from phospholipase-C-linked-receptor activation by maximally effective concentrations of vasopressin or ATP. However, in contrast to vasopression and ATP, adenosine did not stimulate Mn2+ entry. The rate of Mn2+ influx after stimulation of the hepatocytes with vasopressin was not increased by adenosine treatment. The stimulation of hepatocytes with adenosine did not result in significant accumulation of inositol phosphates or cAMP. Furthermore, the rate of adenosine-induced Ca2+ entry in hepatocytes was only slightly reduced in the presence of the P1 purinoceptor antagonist 8-phenyltheophylline. In contrast, the receptor-mediated-Ca(2+)-entry antagonist SK&F 96365 nearly completely blocked the Ca(2+)-entry response without any effect on internal-Ca(2+)-pool mobilisation by adenosine. It is concluded that adenosine activates the internal-pool-regulated pathway of Ca2+ entry and an additional pathway that appears comparable to the previously reported receptor-dependent pathway, except that Mn2+ entry is not stimulated.
Adenosine↗
Benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD.FMK) inhibits apoptosis by blocking the processing of CPP32.
Interleukin-1 beta converting enzyme (ICE)-like proteases, which are synthesized as inactive precursors, play a key role in the induction of apoptosis. We now demonstrate that benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD.FMK), an ICE-like protease inhibitor, inhibits apoptosis by preventing the processing of CPP32 to its active form. These results suggest that novel inhibitors of apoptosis can be developed which prevent processing of proforms of ICE-like proteases.
Amino Acid Chloromethyl Ketones↗
The impact of patient compliance on drug concentration profile in multiple doses.
Physicians commonly prescribe drugs in a multiple dosage regimen for prolonged therapeutic activity. To study the effect of multiple dosing on drug concentration in blood, researchers often use deterministic models with the assumption that drugs are administered at a fixed dosage, with equal or unequal (fixed) dosing intervals. In practice, many patients do not comply with such a rigid schedule. Hence, two possible scenarios might occur: patients might not take the prescribed dosing amount, resulting in erratic dosing sizes; they might not adhere to the dosing schedule, resulting in erratic dosing times. We propose separate statistical models for these two scenarios and study their impact on blood serum/plasma concentration. With non-compliance, some basic concepts such as steady state need new definition. We provide a rigorous formulation for the principle of superposition which enables us to generalize the concept of steady state. Applying the proposed models, we demonstrate that non-compliance causes the drug concentration time curve to exhibit an increase in fluctuation. The increase in fluctuation due to non-compliance cannot be explained with use of the classical deterministic multiple dose model.
Bias↗