A TP is the primary utilizable source of high-energy phosphate bonds within the cell and acts as an allosteric effector of numerous cell processes. Most intracellular ATP is derived from cytosolic glycolysis and mitochondrial oxidative phosphorylation. The latter process couples the oxidation of reduced cofactors via the respiratory chain to ATP synthesis by mitochondrial ATP synthase. The supply of reduced cofactors (NADH, FADH 2 ) is ensured by mitochondrial oxidation of substrates derived from glucose, fatty acids, and amino acids via different metabolic pathways. Therefore, oxidative phosphorylation is a complex process regulated at different levels by the interactions of mitochondrial and cytosolic metabolism (1). In this crosstalk, mitochondrial Ca 2ϩ homeostasis, a process that has attracted a large interest in the past few years (2-11), appears to play a major role. Indeed, three dehydrogenases of the Krebs cycle (pyruvate, isocitrate, and ␣-ketoglutarate dehydrogenase) are modulated by [Ca 2ϩ ] in the micromolar range (12, 13). Recently, it has been shown that, despite the low affinity of the mitochondrial Ca 2ϩ uptake systems, large increases in matrix [Ca 2ϩ ] parallel the cytosolic Ca 2ϩ signals, thanks to the close contact between mitochondria and the intracellular Ca 2ϩ stores (14). The final outcome of this Ca 2ϩ transfer is expected to be the enhancement of mitochondrial ATP production to balance the increased ATP demand of a stimulated cell (15)(16)(17)(18).In this paper, we directly addressed these issues by investigating, in intact living cells, the effect of cytosolic and mitochondrial Ca 2ϩ signaling on intramitochondrial ATP concentration. For this purpose, we have utilized a targeted recombinant Ca 2ϩ probe (mitochondrial aequorin, mtAEQ) (2) and constructed a tool, a specifically targeted chimera of the ATP-sensitive photoprotein luciferase, with the aim of dynamically monitoring the ATP concentration in the mitochondrial matrix ([ATP] m ). The basis for this approach to the measurement of this key cellular parameter was the observation that the affinity of luciferase for ATP, which in vitro is in the micromolar range, is drastically lower in vivo, presumably because of the presence of competing proteins and anions (19). Indeed, in the cellular environment and in the presence of luciferin, luciferase light emission is a linear function of [ATP] in a concentration range between 10 Ϫ3 and 10 Ϫ2 M, i.e., in the physiological range (20,21). We thus constructed a chimeric cDNA, which allows the selective targeting of luciferase to the mitochondrial matrix. With this tool, we could not only directly demonstrate that agonist-dependent changes in mitochondrial Ca 2ϩ concentration correlate with an enhancement in mitochondrial ATP concentration, provided that oxidizable substrates are available, but also identify a phenomenon of long-term memory, allowing a prolonged metabolic ''priming'' that lasts longer than the mitochondrial [Ca 2ϩ ] increase. These observations may clarify how mitoch...

Targeted recombinant aequorins represent to date the most specific means of monitoring [Ca2+] in subcellular organelles (Rizzuto, R., Simpson, A. W. M., Brini, M., and Pozzan, T. (1992) Nature 358, 325-328; Brini, M., Murgia, M., Pasti, L., Picard, D., Pozzan, T., and Rizzuto, R. (1993) EMBO J. 12, 4813-4819; Kendall, J. M., Dormer, R. L., and Campbell, A. K. (1992) Biochem. Biophys. Res. Commun. 189, 1008-1016). Up until now, however, only limited attention has been paid to the use of recombinant photoproteins for measuring, in mammalian cells, the [Ca2+] in the cytoplasm, a compartment for which effective Ca2+ probes are already available. Here we describe this approach in detail, highlighting the advantages, under various experimental conditions, of using recombinant cytosolic aequorin (cytAEQ) instead of classical fluorescent indicators. We demonstrate that cytAEQ is expressed recombinantly at high levels in transiently transfected cell lines and primary cultures as well as in stably transfected clones, and we describe a simple algorithm for converting aequorin luminescence data into [Ca2+] values. We show that although fluorescent indicators at the usual intracellular concentrations (50-100 microM) are associated with a significant buffering of the [Ca2+]c transients, this problem is negligible with recombinantly expressed aequorin. The large dynamic range of the photoprotein also allows an accurate estimate of the large [Ca2+]c increases that are observed in some cell types such as neurons. Finally, cytAEQ appears to be an invaluable tool for measuring [Ca2+]c in cotransfection experiments. In particular, we show that when cotransfected with an alpha 1-adrenergic receptor (coupled to inositol 1,4,5-trisphosphate generation), cytAEQ faithfully monitors the subpopulation of cells expressing the receptor, whereas the signal of fura-2, at the population level, is dominated largely by that of the untransfected cells.

Abstract. Ca 2+ is a key regulator not only of multiple cytosolic enzymes, but also of a variety of metabolic pathways occurring within the lumen of intracellular organelles. Until recently, no technique to selectively monitor the Ca 2+ concentration within defined cellular compartments was available. We have recently proposed the use of molecularly engineered Ca2÷-sensitive photoproteins to obtain such a result and demonstrated the application of this methodology to the study of mitochondrial and nuclear Ca 2+ dynamics. We here describe in more detail the use of chimeric recombinant aequorin targeted to the mitochondria. The technique can be applied with equivalent results to different cell models, transiently or permanently transfected. In all the cell types we analyzed, mitochondrial Ca :+ concentration ([Ca2+]m) increases rapidly and transiently upon stimulation with agonists coupled to InsP3 generation. We confirm that the high speed of mitochondrial Ca 2+ accumulation with this type of stimuli depends on the generation of local gradients of Ca 2÷ in the cytosol, close to the channels sensitive to InsP3. In fact, only activation of these channels, but not the simple release from internal stores, as that elicited by blocking the intracellular Ca 2÷ ATPases, results in a fast mitochondrial Ca ~+ accumulation. We also provide evidence in favor of a microheterogeneity among mitochondria of the same cells, about 30% of them apparently sensing the microdomains of high cytosolic Ca 2+ concentration ([Ca2+]c). The changes in [Ca2+]m appear sufficiently large to induce a rapid activation of mitochondrial dehydrogenases, which can be followed by monitoring the level of NAD(P)H fluorescence. A general scheme can thus be envisaged by which the triggering of a plasma membrane receptor coupled to InsP3 generation raises the Ca 2+ concentration both in the cytoplasm (thereby triggering energy-consuming processes, such as cell proliferation, motility, secretion, etc.) and in the mitochondria, where it activates the metabolic activity according to the increased cell needs.T HE development of specific probes for measuring the Ca2. + concentration in the cytoplasm of living cells (lslen et al., 1982; Grynkiewicz et al., 1985) has led to major progress in the understanding of the mechanisms controlling cellular Ca 2+ homeostasis and of the role played by this cation in the chain of events coupling membrane receptor stimulation to cell activation (Berridge and Irvine, 1989;Pietrobon et al., 1990;Meldolesi et al., 1990). We now know that, in a variety of cell types, stimuli as diverse as hormones and growth factors, cell-cell interaction and synaptic transmission induce rises of the cytosolic free calcium concentration ([Ca2+]o). The Ca e+ ions then bind to a number of effector proteins, which, in turn, modulate a variety of cellular processes, such as motility, secretion, enzyme activation, etc. Rises in [Ca2+]c depend either on influx from the extracellular medium and/or release from intracellular stores. In both cases, Ca 2+...

Nutrient-stimulated insulin secretion is dependent upon the generation of metabolic coupling factors in the mitochondria of the pancreatic B cell.

Abstract, A molecularly tagged form of calreticulin (CR), a low affinity-high capacity Ca 2+ binding protein that resides in the ER lumen, was transiently transfected into HeLa cells to specifically modify the Ca 2÷ buffering capacity of the intracellular Ca 2÷ stores. Fluorescence and confocal microscope immunocytochemistry revealed the tagged protein to be expressed by over 40% of the cells and to overlap in its distribution the endogenous CR yielding a delicate cytoplasmic network, i.e., the typical pattern of ER. In contrast, no signal was observed associated with the plasmalemma (marked by ConA) and within the nucleus. One-and two-dimensional Western blots revealed the transfected to exceed the endogenous CR of ~3.5-fold and to maintain its Ca 2+ binding ability, whereas the expression of other ER proteins was unchanged. Ca 2÷ homeostasis in the transfected cells was investigated by three parallel approaches: (a) 45Ca equilibrium loading of cell populations; (b) [Ca2+]c measurement with fura-2 followed by quantitative immunocytochemistry of single cells and iii) [Ca2+]c measurement of cell population upon cotransfection with the Ca2+-sensitive photoprotein, aequorin. The three approaches revealed different aspects of Ca 2+ homeostasis, yielding results which were largely complementary. In particular, the following conclusions were established: (a) both endogenous and transfected CR participate in Ca 2÷ buffering within the IP3-sensitive, rapidly exchanging, Ca 2÷ stores; the other pools of the cells were in contrast unaffected by CR transfection; (b) the Ca 2+ capacity of the stores is not the main limiting factor of individual IP3-mediated Ca 2+ release responses triggered by receptor agonists; (c) in control cells, the contribution of CR to Ca 2+ buffering within the IP3-sensitive stores accounts for ~45 % of the total, the rest being probably contributed by the other lumenal (and also membrane) Ca 2÷ binding proteins; (d) the free [Ca z÷] within the lumen of the IP3-sensitive stores, revealed by the degree of Ca 2÷ binding to the transfected CR protein, amounts to values in (or approaching) the millimolar range; and (e) Ca 2+ influx across the plasmalemma activated by depletion of the stores is directly dependent on the lumenal [Ca2+]. T HE intracellular rapidly exchanging stores of Ca 2+have attracted increasing interest during the last several years (for reviews see Carafoli, 1987;Berridge, 1993;Pozzan et al., 1994). These structures are responsible for a fundamental step in many types of cell activation, the release of Ca 2+ to the cytosol taking place not at the surface but at multiple sites within the cytoplasm. Among cytoplasmic organelles, the ER is commonly identified as the cytological counterpart of the stores. Results in various cell types have however indicated that not the entire ER, but discrete areas (e.g., the sarcoplasmic reticuAddress correspondence to Prof. T. Pozzan, Dept. of Biomedical Sciences, University of Padova, Via Trieste, 75, 35121 Padova, Italy. Ph.: 39-49-8286568. Fax: 39-4...

Purpose: To explore intratumor heterogeneity in gene expression profiles from patients with cervical cancer. Experimental Design: A total of 33 biopsies were obtained from 11patients, sampling between two and five different areas for each tumor.The extracted RNA was hybridized onto theAffymetrix U133 Plus 2.0 oligonucleotide chip. The variance of expression within a patient (W), between patients (B) and the total variance (T = W + B) were calculated for each ProbeSet, and the ratio W/T was used as a measure of intratumor heterogeneity. Gene Ontology functional analysis was done to assess the function of genes that had high W/T (top 10%) and low W/T (bottom 10%) values.Results: In total, 448 ProbeSets (2.2% of the total) had W/T < 0.10, indicating low intratumor heterogeneity, and 537 ProbeSets (2.7% of the total) had W/T > 0.90, indicating high intratumor heterogeneity. In total 14,473 ProbeSets (72.4%) had higher intertumor than intratumor heterogeneity (W/T < 0.5). Genes with low intratumor heterogeneity were characterized by a statistically significant enrichment of immune-related functions (P < 0.0001). Genes with high intratumor heterogeneity were characterized by a significant tendency towards nuclear localization and nucleic acid binding (both P < 0.0001). For genes with W/T > 0.5, more than six biopsies would be required to minimize the intratumoral heterogeneity to <0.15; if W/T is 0.3 to 0.4, four biopsies are required; and for low W/T of 0.16 to 0.3, only two to three biopsies would be needed. Conclusion: Although the intratumor heterogeneity was low for the majority of the tested ProbeSets, for many genes, multiple biopsies are required to obtain a reliable estimate of gene expression.

Polo‐like kinase 1 (Plk1) is a critical regulator of many stages of mitosis; increasing evidence indicates that Plk1 overexpression correlates with poor clinical outcome, yet its mechanism of regulation remains unknown. Hence, a detailed evaluation was undertaken of Plk1 expression in human nasopharyngeal cancer (NPC), the cellular effects of targeting Plk1 using siRNA in combination with ionizing radiation (RT) and potential upstream microRNAs (miRs) that might regulate Plk1 expression. Using immunohistochemistry, Plk1 was observed to be overexpressed in 28 of 40 (70%) primary NPC biopsies, which in turn was associated with a higher likelihood of recurrence (p = 0.018). SiPlk1 significantly inhibited Plk1 mRNA and protein expression, and decreased Cdc25c levels in NPC cell lines. This depletion resulted in cytotoxicity of C666‐1 cells, enhanced by the addition of RT, mediated by G2/M arrest, increased DNA double‐strand breaks, apoptosis, and caspase activation. Immunofluorescence demonstrated that the G2/M arrest was associated with aberrant spindle formation, leading to mitotic arrest. In vivo, transfection of C666‐1 cells and systemic delivery of siPlk1 decreased tumour growth. MicroRNA‐100 (miR‐100) was predicted to target Plk1 mRNA, which was indeed underexpressed in C666‐1 cells, inversely correlating with Plk1 expression. Using luciferase constructs containing the 3′‐UTR of Plk1 sequence, we document that miR‐100 can directly target Plk1. Hence, our data demonstrate for the first time that underexpressed miR‐100 leads to Plk1 overexpression, which in turn contributes to NPC progression. Targeting Plk1 will cause mitotic catastrophe, with significant cytotoxicity both in vitro and in vivo, underscoring the important therapeutic opportunity of Plk1 in NPC.

A potential therapeutic agent for human head and neck cancer (HNC), cetrimonium bromide (CTAB), was identified through a cell-based phenotype-driven high-throughput screen (HTS) of 2000 biologically active or clinically used compounds, followed by in vitro and in vivo characterization of its antitumor efficacy. The preliminary and secondary screens were performed on FaDu (hypopharyngeal squamous cancer) and GM05757 (primary normal fibroblasts), respectively. Potential hit compounds were further evaluated for their anticancer specificity and efficacy in combination with standard therapeutics on a panel of normal and cancer cell lines. Mechanism of action, in vivo antitumor efficacy, and potential lead compound optimizations were also investigated. In vitro, CTAB interacted additively with ␥ radiation and cisplatin, two standard HNC therapeutic agents.CTAB exhibited anticancer cytotoxicity against several HNC cell lines, with minimal effects on normal fibroblasts; a selectivity that exploits cancer-specific metabolic aberrations. The central mode of cytotoxicity was mitochondria-mediated apoptosis via inhibition of H ϩ -ATP synthase activity and mitochondrial membrane potential depolarization, which in turn was associated with reduced intracellular ATP levels, caspase activation, elevated sub-G 1 cell population, and chromatin condensation. In vivo, CTAB ablated tumor-forming capacity of FaDu cells and delayed growth of established tumors. Thus, using an HTS approach, CTAB was identified as a potential apoptogenic quaternary ammonium compound possessing in vitro and in vivo efficacy against HNC models.