A novel turbidimetric immunoassay for fecal calprotectin optimized for routine chemistry analyzers.

BACKGROUND: Fecal calprotectin assays are widely used to exclude inflammatory bowel disease (IBD) in patients with suspected IBD. A problem with the fecal calprotectin assays is the rather long test-turnaround times. A particle enhanced turbidimetric immunoassays (PETIA) for fecal calprotectin would reduce test-turnaround times and would permit more laboratories to perform the measurements. The aim of this study was to evaluate a new feces calprotectin PETIA. METHOD: Using routine fecal samples the feces calprotectin PETIA was validated on two chemistry analyzers, Mindray BS-380 and Cobas 501. RESULTS: The assay is linear in the range 11-2000 µg/g, with a limit of quantitation of approximately 10 µg/g. No antigen excess hook effect was observed up to 10 000-15 000 µg/g depending on the instrument used. The turbidimetric method showed a good agreement with the Bühlmann ELISA. The total coefficient of variation was 3%-8% in the 50-100 µg/g range. CONCLUSION: The fecal calprotectin PETIA, fCal Turbo, is well suited for rapid analysis of fecal calprotectin on Mindray BS-380 or Cobas 501 clinical chemistry analyzers. The test results are commutable with Bühlmann fecal MRP8/14 ELISA.

The Roche Immunoturbidimetric Albumin Method on Cobas c 501 Gives Higher Values Than the Abbott and Roche BCP Methods When Analyzing Patient Plasma Samples.

BACKGROUND: Serum/plasma albumin is an important and widely used laboratory marker and it is important that we measure albumin correctly without bias. We had indications that the immunoturbidimetric method on Cobas c 501 and the bromocresol purple (BCP) method on Architect 16000 differed, so we decided to study these methods more closely. METHOD: A total of 1,951 patient requests with albumin measured with both the Architect BCP and Cobas immunoturbidimetric methods were extracted from the laboratory system. A comparison with fresh plasma samples was also performed that included immunoturbidimetric and BCP methods on Cobas c 501 and analysis of the international protein calibrator ERM-DA470k/IFCC. RESULTS: The median difference between the Abbott BCP and Roche immunoturbidimetric methods was 3.3 g/l and the Roche method overestimated ERM-DA470k/IFCC by 2.2 g/l. The Roche immunoturbidimetric method gave higher values than the Roche BCP method: y = 1.111x - 0.739, R² = 0.971. CONCLUSION: The Roche immunoturbidimetric albumin method gives clearly higher values than the Abbott and Roche BCP methods when analyzing fresh patient samples. The differences between the two methods were similar at normal and low albumin levels.

Chemical biology suggests a role for calcium signaling in mediating sustained JNK activation during apoptosis.

Calcium (Ca(2+)) is used as a signaling molecule to regulate many cellular processes. Calcium signaling generally involves transient elevations of the concentration of free Ca(2+) in the cytosol. More pronounced and sustained elevations of intracellular Ca(2+) concentrations are observed during apoptosis (programmed cell death). These Ca(2+) elevations have been shown to lead to the activation of proteases (calpains) and to changes in protein phosphorylation. Recent evidence, using chemical biology, has raised the possibility that calcium signaling is involved in sustained JNK activation during late phases of apoptosis. For at least some stimuli, calcium release leads to activation of calmodulin kinase II (CaMKII), apoptosis signaling kinase 1 (ASK1) and JNK. Calcium signaling may help to orchestrate the apoptotic response during the execution phase.

Induction of the lysosomal apoptosis pathway by inhibitors of the ubiquitin-proteasome system.

The lysosomal apoptosis pathway is a potentially interesting therapeutic target. Since apoptosis involving the lysosomal pathway has been described to involve cathepsins, we screened a drug library for agents that induce cathepsin-dependent apoptosis. Using pharmacological inhibitors and siRNA, we identified 2 structurally related agents (NSC687852 and NSC638646) that induced cathepsin D-dependent caspase-cleavage activity in human breast cancer cells. Both agents were found to induce the mitochondrial apoptosis pathway. NSC687852 and NSC638646 were found to inhibit the activity of ubiquitin isopeptidases and to induce the accumulation of high-molecular-mass ubiquitins in cells. We show that 3 other inhibitors of the proteasome degradation pathway induce lysosomal membrane permeabilization (LMP) and that cathepsin-D siRNA inhibits apoptosis induced by these agents. We conclude that a screen for cathepsin-dependent apoptosis-inducing agents resulted in the identification of ubiquitin isopeptidase inhibitors and that proteasome inhibitors with different mechanisms of action induce LMP and cathepsin D-dependent apoptosis.

Charting calcium-regulated apoptosis pathways using chemical biology: role of calmodulin kinase II.

BACKGROUND: Intracellular free calcium ([Ca2+]i) is a key element in apoptotic signaling and a number of calcium-dependent apoptosis pathways have been described. We here used a chemical biology strategy to elucidate the relative importance of such different pathways. RESULTS: A set of 40 agents ("bioprobes") that induce apoptosis was first identified by screening of a chemical library. Using p53, AP-1, NFAT and NF-kappaB reporter cell lines, these bioprobes were verified to induce different patterns of signaling. Experiments using the calcium chelator BAPTA-AM showed that Ca2+ was involved in induction of apoptosis by the majority of the bioprobes and that Ca2+ was in general required several hours into the apoptosis process. Further studies showed that the calmodulin pathway was an important mediator of the apoptotic response. Inhibition of calmodulin kinase II (CaMKII) resulted in more effective inhibition of apoptosis compared to inhibition of calpain, calcineurin/PP2B or DAP kinase. We used one of the bioprobes, the plant alkaloid helenalin, to study the role of CaMKII in apoptosis. Helenalin induced CaMKII, ASK1 and Jun-N-terminal kinase (JNK) activity, and inhibition of these kinases inhibited apoptosis. CONCLUSION: Our study shows that calcium signaling is generally not an early event during the apoptosis process and suggests that a CaMKII/ASK1 signaling mechanism is important for sustained JNK activation and apoptosis by some types of stimuli.

Mechanisms of action of DNA-damaging anticancer drugs in treatment of carcinomas: is acute apoptosis an "off-target" effect?

DNA damage induces apoptosis of cells of hematological origin. Apoptosis is also widely believed to be the major antiproliferative mechanism of DNA damaging anticancer drugs in other cell types, and a large number of laboratories have studied drug-induced acute apoptosis (within 24 hours) of carcinoma cells. It is, however, often overlooked that induction of apoptosis of carcinoma cells generally requires drug concentrations that are at least one order of magnitude higher than those required for loss of clonogenicity. This is true for different DNA damaging drugs such as cisplatin, doxorubicin and camptothecin. We here discuss apoptosis induction by DNA damaging agents using cisplatin as an example. Recent studies have shown that cisplatin induces caspase activation in enucleated cells (cytoplasts lacking a cell nucleus). Cisplatin-induced apoptosis in both cells and cytoplasts is associated with rapid induction of cellular reactive oxygen species and increases in [Ca(2+)](i). Cisplatin has also been reported to induce clustering of Fas/CD95 in the plasma membrane. Available data suggest that the primary responses to cisplatin-induced DNA damage are induction of long-term growth arrest ("premature cell senescence") and mitotic catastrophe, whereas acute apoptosis may be due to "off-target effects" not necessarily involving DNA damage.

Cytokeratin-18 is a useful serum biomarker for early determination of response of breast carcinomas to chemotherapy.

PURPOSE: With a widening arsenal of cancer therapies available, it is important to develop therapy-specific predictive markers and methods to rapidly assess treatment efficacy. We here evaluated the use of cytokeratin-18 (CK18) as a serum biomarker for monitoring chemotherapy-induced cell death in breast cancer. EXPERIMENTAL DESIGN: Different molecular forms of CK18 (caspase cleaved and total) were assessed by specific ELISA assays. Drug-induced release of CK18 was examined from breast carcinoma cells and tissue. CK18 protein composition was examined in serum. CK18 levels were determined in serum from 61 breast cancer patients during docetaxel or cyclophosphamide/epirubicin/5-fluorouracil (CEF) therapy. RESULTS: Caspase-cleaved CK18 molecules were released from monolayer cultures and tumor organ cultures to the extracellular compartment. CK18 was present in complexes with other cytokeratins in serum. Such CK18 protein complexes are remarkably stable, leading to favorable performance of CK18 biomarker assays for clinical investigations. Docetaxel induced increased levels of caspase-cleaved CK18 in serum from breast cancer patients, indicating apoptosis. CEF therapy led to increases predominantly in uncleaved CK18, indicating induction of necrotic cell death in many tumors. The increase in total CK18 at 24 h of the first treatment cycle correlated to the clinical response to CEF therapy (P < 0.0001). CONCLUSIONS: Induction of necrotic cell death may explain the clinical efficacy of anthracycline-based therapy for breast carcinomas with defective apoptosis pathways. We suggest that CK18 biomarkers are useful for early prediction of the response to CEF therapy in breast cancer and may be useful biomarkers for clinical trials.

Acute apoptosis by cisplatin requires induction of reactive oxygen species but is not associated with damage to nuclear DNA.

Cisplatin is a broad-spectrum anticancer drug that is also widely used in experimental studies on DNA damage-induced apoptosis. Induction of apoptosis within 24-48 hr requires cisplatin concentrations that are at least one order of magnitude higher than the IC(50). Here, we show that such high, apoptosis-inducing cisplatin concentrations induce cellular superoxide formation and that apoptosis is inhibited by superoxide scavengers. The same concentration limit and the requirement for superoxide are also true for induction of caspase activation in enucleated cells (cytoplasts), showing that cisplatin-induced apoptosis occurs independently of nuclear DNA damage. In contrast, lower cisplatin concentrations, which do not induce acute apoptosis, are sufficient for induction of DNA damage signaling. We propose that the antiproliferative effects of cisplatin at IC(50) doses involve premature senescence and secondary, nonstress-induced apoptosis. The higher doses currently used in in vitro studies lead to acute, stress-induced apoptosis that involves induction of superoxide but is largely DNA damage-independent.

Preferential cell death of CD8+ effector memory (CCR7-CD45RA-) T cells by hydrogen peroxide-induced oxidative stress.

T cells are used in many cell-based cancer treatments. However, oxidative stress that is induced during various chronic inflammatory conditions, such as cancer, can impair the immune system and have detrimental effects on T cell function. In this study, we have investigated the sensitivity of different human T cell subsets to H(2)O(2)-induced oxidative stress. We showed that central memory (CD45RA(-)CCR7(+)) and effector memory (CD45RA(-)CCR7(-)) T cells are more sensitive to H(2)O(2) as compared with naive (CD45RA(+)CCR7(+)) T cells. Furthermore, the study showed that CD8(+) effector memory T cells are more sensitive to low levels of H(2)O(2) (5 microM) compared with other types of T cells investigated. H(2)O(2)-exposed CD45RO(+) T cells showed mitochondrial depolarization prior to caspase 3 activity. Moreover, the pan-caspase inhibitor z-Val-Ala-Asp(OMe)-fluoromethylketone rescued cells from death. These experiments suggest that H(2)O(2)-induced cell death of CD45RO(+) T cells acts via the mitochondrial pathway and that caspase involvement is needed. This study suggests that oxidative stress in cancer patients can be disadvantageous for T cell-based adoptive cell transfer therapies, since effector memory T cells are the primary phenotype of the cells administered.

Phosphorylation of BAD at Ser-128 during mitosis and paclitaxel-induced apoptosis.

Phosphorylation of BCL-2 family member BAD at different residues triggers different physiological effects, either inhibiting or promoting apoptosis. The recently identified phosphorylation site at Ser-128 enhances the apoptotic activity of BAD. We here show that BAD becomes phosphorylated at Ser-128 in the mitotic phase of the cell cycle in NIH3T3 cells. We also show that BAD-S128 is phosphorylated in taxol-treated mouse fibroblasts and MDA-MB-231 human breast cancer cells. However, expression of a phosphorylation-defective dominant negative BAD mutant did not block taxol-induced apoptosis. These data support the view that the phosphorylation of BAD Serine 128 exerts cell-specific effects on apoptosis. Whereas the BAD Serine 128 phosphorylation induces apoptosis in neuronal cells, it does not appear to promote apoptosis in proliferating non-neural cells during mitosis or upon exposure to the antineoplastic agent taxol.

Determining tumor apoptosis and necrosis in patient serum using cytokeratin 18 as a biomarker.

Intracellular macromolecules are released from dying tumor cells and may subsequently be detected in patient blood. In this review, we will discuss the use of cytokeratin-18 as a serum biomarker for monitoring therapy-induced cell death. Cytokeratins are abundant intracellular proteins expressed by most types of carcinoma, but not by treatment-sensitive cells from bone marrow and other tissues. Release of cytokeratins into blood is therefore expected to show some specificity for tumor cell death. Cytokeratin-18 (CK18) is cleaved by caspases specifically during apoptosis, and the molecular form of this protein (caspase-cleaved vs. non-cleaved) released from dying tumor cells is therefore diagnostic as to the type of cell death (apoptosis vs. necrosis). Analyses of different CK18 forms in patient sera have suggested that tumor apoptosis may not necessarily be the dominating death mode in many tumors in vivo. Measurements of increased levels of CK18 in serum during therapy of prostate and breast cancer patients have been encouraging with regard to the possible future use of CK18 as a biomarker for monitoring therapy efficiency.