Glutamate receptor overactivation contributes to neuron death after stroke, trauma, and epileptic seizures. Exposure of cultured rat hippocampal neurons to the selective glutamate receptor agonist N-methyl-D-aspartate (300 M, 5 min) or to the apoptosis-inducing protein kinase inhibitor staurosporine (300 nM) induced a delayed neuron death. In both cases, neuron death was preceded by the mitochondrial release of the pro-apoptotic factor cytochrome c. Unlike staurosporine, the Nmethyl-D-aspartate-induced release of cytochrome c did not lead to significant activation of caspase-3, the main caspase involved in the execution of neuronal apoptosis. In contrast, activation of the Ca 2؉ -activated neutral protease calpain I was readily detectable after the exposure to N-methyl-D-aspartate. In a neuronal cell-free apoptosis system, calpain I prevented the ability of cytochrome c to activate the caspase cascade by inhibiting the processing of procaspase-3 and -9 into their active subunits. In the hippocampal neuron cultures, the inhibition of calpain activity restored caspase-3-like protease activity after an exposure to N-methyl-D-aspartate. Our data demonstrate the existence of signal transduction pathways that prevent the entry of cells into a caspase-dependent cell death program after the mitochondrial release of cytochrome c.

Polymerase chain reaction and rapid amplification of cDNA ends were used to isolate cDNAs encoding a 5-hydroxytryptamine 3 (5-HT 3 ) receptor subunit and its splice variants from guinea pig intestine. The amino acid sequence predicted from this cDNA is 81% homologous to the murine 5-HT 3 receptor subunits cloned from NCB20 and N1E-115 cells. The splice variants code for two proteins differing by a deletion of six amino acids located in the large intracellular loop between transmembrane domains M3 and M4. For characterization, the cloned 5-HT 3 cDNA was expressed in HEK 293 cells, and the electrophysiological and pharmacological properties of the recombinant ion/channel/receptor complex were investigated by patch clamping. Our data reveal that the cloned cDNAs code for guinea pig 5-HT 3 receptors, which functionally assemble as homo-oligomers. The kinetic behavior of the ion channel and its sensitivity to several agonists and antagonists were markedly different from those of the cloned 5-HT 3 receptors from mouse and human under similar experimental conditions. The agonists used were 5-hydroxytryptamine, 2-methyl-5-hydroxytryptamine, 1-phenylbiguanide (PBG), m-chlorophenylbiguanide, and the antagonists tropisetron and metoclopramide. In addition, 5-HT, PBG, and tropisetron were investigated through radioligand binding to isolated membranes. Compared with the human and murine 5-HT 3 receptors, the guinea pig receptor showed prolonged desensitization kinetics. In addition, the guinea pig 5-HT 3 receptor did not respond to the selective 5-HT 3 receptor agonist PBG. Construction of chimeric receptors between guinea pig and human 5-HT 3 receptor sequences localized the differences in desensitization kinetics to the carboxyl-terminal domain and the ligand binding site to the aminoterminal domain of the receptor protein. Molecular determinants of the PBG binding site of the human 5-HT 3 receptor were localized to a 28-amino-acid spanning region adjacent to the M1 region.5-HT 3 Rs belong to the superfamily of ligand-gated ion channels that mediate fast synaptic transmission in the peripheral and central nervous systems (Peters et al., 1992;Yakel, 1992). These channels are composed of five identical or homologous subunits and their functional diversity generally is attributed to the presence of several different subunits that can coassemble to yield receptors with specific pharmacological and physiological properties (Betz, 1990). No such diversity has emerged for 5-HT 3 Rs. A single 5-HT 3 inotropic receptor subunit (5-HT 3 R-A) was cloned 6 years ago from the NCB20 neuroblastoma cell line (Maricq et al., 1991), but despite evidence for both pharmacological and biophysical variations between tissues and species, no further 5-HT 3 R subunits, like different ␣ or ␤ subunits, have been identified. 5-HT 3 R-A cDNA and a splice variant have been cloned from additional neuroblastoma cell lines and from mouse, rat, and human tissues. These subunits form functional homo-oligomeric 5-HT 3 Rs when expressed in oocytes or H...

During apoptotic and excitotoxic neuron death, challenged mitochondria release the pro-apoptotic factor cytochrome c. In the cytosol, cytochrome c is capable of binding to the apoptotic protease-activating factor-1 (APAF-1). This complex activates procaspase-9 in the presence of dATP, resulting in caspase-mediated execution of apoptotic neuron death. Many forms of Ca 21 -mediated neuron death, however, do not lead to prominent activation of the caspase cascade despite signi®cant release of cytochrome c from mitochondria. We demonstrate that elevation of cytosolic Ca 21 induced prominent degradation of APAF-1 in human SH-SY5Y neuroblastoma cells and in a neuronal cell-free apoptosis system. Loss of APAF-1 correlated with a reduced ability of cytochrome c to activate caspase-3-like proteases. Ca 21 induced the activation of calpains, monitored by the cleavage of full-length a-spectrin into a calpain-speci®c 150-kDa breakdown product. However, pharmacological inhibition of calpain activity indicated that APAF-1 degradation also occurred via calpainindependent pathways. Our data suggest that Ca 21 inhibits caspase activation during Ca 21 -mediated neuron death by triggering the degradation of the cytochrome c-binding protein APAF-1.

Circulating tumour cells (CTC) can be traced in patients with different types of cancer. The aim of this study was to detect CTC in patients with advanced colorectal cancer and whether CTC are still detectable after systemic chemotherapy. Blood from 34 patients with advanced colorectal cancer was analysed for the presence of CTC before chemotherapy was given and after 3 months. Eleven patients demonstrated a tumour remission after chemotherapy. In 6 cases CTC were detectable before but not after initiation of chemotherapy. Ten patients demonstrated a progression. In 5 cases CTC were detected before and after chemotherapy. Our data suggest that the detection of CTC will help to identify patients responding to chemotherapy or with a risk of a therapy failure.

Detection of disseminated tumor cells in the blood circulation is important in assessing tumor progression. The objective of this examination was to develop a highly specific and sensitive quantitative realtime reverse transcriptase-polymerase chain reaction (qRT-PCR) assay for the detection of relevant tumor-associated transcripts in patients' blood. The qRT-PCR assays detect the human epidermal growth factor receptor 2 (HER2) and CK20 transcripts of two tumor cells spiked into 5 mL of blood after an immunomagnetic tumor cell enrichment. Furthermore, the HER2 assay is only specific when enrichment is included. This procedure is a useful alternative to fluorescence in situ hybridization and immunocytochemistry for gene alteration analysis in human tumors. The analysis of the studied molecular markers of tumor cells in blood may be useful in the detection of disseminated tumor cells as well as for monitoring treatment response, early detection of relapse, and for stratification of patients with carcinoma.