Live Monitoring of Microenvironmental pH Based on Extracellular Acidosis around Cancer Cells with Cell-Coupled Gate Ion-Sensitive Field-Effect Transistor.

We demonstrated the live monitoring of cellular respiration using an ion-sensitive field-effect transistor (ISFET), focusing on different types of living cells, namely cancer and normal cells. In particular, we realized the label-free, real-time, and noninvasive monitoring of microenvironmental pH behavior based on extracellular acidosis around cancer cells in the long term and in situ. The change in interfacial pH (ΔpHint), which was analyzed based on the change in interfacial potential (Δ Vout) at the cell/gate nanogap interface gradually decreased for every cell-based ISFET. Moreover, the ΔpHint for cancer cells shifted by a factor of 5 to 6, which was larger than that for normal cells. This is because cancer cells cause dysbolism and are activated, thereby suppressing oxidative phosphorylation in mitochondria so as not to induce their apoptosis. Therefore, cancer cellular respiration proceeds via the glycolysis pathway, through which lactic acid is eventually released. Additionally, the pH sensitivity of the ISFET device was maintained even when the device was immersed into a cell culture medium for 24 h and 1 w; thus, the effect of nonspecific adsorption of proteins contained in the medium on the pH sensitivity of the ISFET device was negligible in the live monitoring of cellular respiration.

Dysregulation of a potassium channel, THIK-1, targeted by caspase-8 accelerates cell shrinkage.

Activation of caspases is crucial for the execution of apoptosis. Although the caspase cascade associated with activation of the initiator caspase-8 (CASP8) has been investigated in molecular and biochemical detail, the physiological role of CASP8 is not fully understood. Here, we identified a two-pore domain potassium channel, tandem-pore domain halothane-inhibited K+ channel 1 (THIK-1), as a novel CASP8 substrate. The intracellular region of THIK-1 was cleaved by CASP8 in apoptotic cells. Overexpression of THIK-1, but not its mutant lacking the CASP8-target sequence in the intracellular portion, accelerated cell shrinkage in response to apoptotic stimuli. In contrast, knockdown of endogenous THIK-1 by RNA interference resulted in delayed shrinkage and potassium efflux. Furthermore, a truncated THIK-1 mutant lacking the intracellular region, which mimics the form cleaved by CASP8, led to a decrease of cell volume of cultured cells without apoptotic stimulation and excessively promoted irregular development of Xenopus embryos. Taken together, these results indicate that THIK-1 is involved in the acceleration of cell shrinkage. Thus, we have demonstrated a novel physiological role of CASP8: creating a cascade that advances the cell to the next stage in the apoptotic process.

Single embryo-coupled gate field effect transistor for elective single embryo transfer.

In this study, we have proposed and demonstrated experimentally a novel monitoring device of single mouse embryo activity after in vitro fertilization (IVF) using a semiconductor-based field effect transistor (FET). The FET biosensor realized to detect it noninvasively, quantitatively, and continuously by change of hydrogen ions with positive charges, which were induced by dissolved carbon dioxide due to cellular respiration activity during cleavage. The electrical signal of FET biosensor should become an effective indication to evaluate objectively single embryo activity as its morphology is observed subjectively after IVF. The platform based on the FET biosensor will contribute to promote elective single embryo transfer (eSET) in human assisted reproductive technology (ART).

Overexpressed NF-kappaB-inducing kinase contributes to the tumorigenesis of adult T-cell leukemia and Hodgkin Reed-Sternberg cells.

The nuclear factor-kappaB (NF-kappaB) transcription factors play important roles in cancer development by preventing apoptosis and facilitating the tumor cell growth. However, the precise mechanisms by which NF-kappaB is constitutively activated in specific cancer cells remain largely unknown. In our current study, we now report that NF-kappaB-inducing kinase (NIK) is overexpressed at the pretranslational level in adult T-cell leukemia (ATL) and Hodgkin Reed-Sternberg cells (H-RS) that do not express viral regulatory proteins. The overexpression of NIK causes cell transformation in rat fibroblasts, which is abolished by a super-repressor form of IkappaBalpha. Notably, depletion of NIK in ATL cells by RNA interference reduces the DNA-binding activity of NF-kappaB and NF-kappaB-dependent transcriptional activity, and efficiently suppresses tumor growth in NOD/SCID/gammac(null) mice. These results indicate that the deregulated expression of NIK plays a critical role in constitutive NF-kappaB activation in ATL and H-RS cells, and suggest also that NIK is an attractive molecular target for cancer therapy.

Role for protein geranylgeranylation in adult T-cell leukemia cell survival.

Adult T-cell leukemia (ATL) is a fatal lymphoproliferative disease that develops in human T-cell leukemia virus type I (HTLV-I)-infected individuals. Despite the accumulating knowledge of the molecular biology of HTLV-I-infected cells, effective therapeutic strategies remain to be established. Recent reports showed that the hydroxyl-3-methylglutaryl (HMG)-CoA reductase inhibitor statins have anti-proliferative and apoptotic effects on certain tumor cells through inhibition of protein prenylation. Here, we report that statins hinder the survival of ATL cells and induce apoptotic cell death. Inhibition of protein geranylgeranylation is responsible for these effects, since simultaneous treatment with isoprenoid precursors, geranylgeranyl pyrophosphate or farnesyl pyrophosphate, but not a cholesterol precursor squalene, restored the viability of ATL cells. Simvastatin inhibited geranylgeranylation of small GTPases Rab5B and Rac1 in ATL cells, and a geranylgeranyl transferase inhibitor GGTI-298 reduced ATL cell viability more efficiently than a farnesyl transferase inhibitor FTI-277. These results not only unveil an important role for protein geranylgeranylation in ATL cell survival, but also implicate therapeutic potentials of statins in the treatment of ATL.

In situ measurement of autophagy under nutrient starvation based on interfacial pH sensing.

In this study, we report a novel method for the in situ measurement of autophagy under nutrient starvation using a principle of semiconductor technology. A semiconductor-based field-effect transistor (FET) biosensor enables the direct detection of ionic or molecular charges under biological conditions. In particular, cellular respiration accompanied by the generation of carbon dioxide can be continuously and directly monitored as a change in pH at a cell/sensor interface. When autophagy was induced in HeLa cells on a FET biosensor under nutrient starvation, the surface potential increased more significantly for about 15 h than that for nonstarved cells. This positive shift indicates an increase in the number of hydrogen ions produced from the respiration of starved cells because the sensing surface was previously designed to be sensitive to pH variation. Therefore, we have found that cellular respiration is more activated by autophagy under nutrient starvation because the amino acids that decomposed from proteins in autophagic cells would have been rapidly spent in cellular respiration.

Macrovascular changes in mice overexpressing human semicarbazide-sensitive amine oxidase in smooth muscle cells.

BACKGROUND: The catalytic activity of semicarbazide-sensitive amine oxidase (SSAO) is increased in diabetes, as well as in other disorders of cardiovascular origin. Our hypothesis is that SSAO is involved in the synthesis or maturation of elastin in vascular tissue. An increased SSAO activity can thereby be involved in the development of vascular damage. METHODS: Elastin quantification was performed in aorta of transgenic mice overexpressing the human form of SSAO, using electron microscopy. Furthermore, lung capacity was measured using a spirometry-mimicking method, developed for mice. The effect of vasoactive substances was estimated by measuring mean arterial pressure and pulse pressure under anesthesia. RESULTS: No differences in elastin quantity or lung capacity could be observed between transgenic or nontransgenic littermates. Pulse pressure was higher in transgenic mice, and electron microscopy of aorta showed elastin fibers parallel with the aorta wall (ie, straight fibers instead of folded compared with control mice). No difference in the response to adrenaline or sodium chloride was observed between the transgenic and control mice. The control mice had a clear decrease in blood pressure (BP) with a longer duration as a response to injection of a nitric oxide (NO) donor, sodium nitroprusside, compared with transgenic mice where only a minor response was observed. The SSAO activity in serum of control mice was elevated in response to injection of the NO donor, but not in response to a ganglion blocker. CONCLUSIONS: An elevated pulse pressure, together with an abnormal elastin structure in the aorta, suggests a rigidity of large arteries as a result of an elevated SSAO activity as well as a physiologic role for SSAO in elastin maturation.

Absence of tissue-bound semicarbazide-sensitive amine oxidase activity in carp tissues.

We have previously reported that carp (Cyprinus carpio) tissue mitochondria contain a novel form of monoamine oxidase (MAO), which belongs neither to MAO-A nor to MAO-B of the mammalian enzyme. This conclusion results from the findings that the carp MAO was equally sensitive to a selective MAO-A inhibitor clorgyline and to the MAO-B selective inhibitor l-deprenyl, when tyramine, a substrate for both forms, serotonin or beta-phenylethylamine, a substrate for either A or B-form of mammalian MAO, was used. In the present study, we tried to detect another amine oxidase, termed tissue-bound semicarbazide-sensitive amine oxidase (SSAO), activity in carp tissues. As definition of SSAO was used, such as insensitivity to inhibition of the kynuramine oxidizing activity by an MAO inhibitor pargyline and high sensitivity to the SSAO inhibitor semicarbazide. The results indicated that the oxidizing activity was selectively and almost completely inhibited by 0.1 mM pargyline alone or a combination of 0.1 mM pargyline plus 0.1 mM semicarbazide, but not by 0.1 mM semicarbazide alone. We also tried to detect any SSAO activity by changing experimental conditions, such as lower incubation temperature, higher enzyme protein concentration, a lower substrate concentration and different pH's in the reaction, as the enzyme source. However, still no SSAO activity could be detected in the tissues. These results conclusively indicate that carp tissues so far examined do not contain SSAO activity.

Selective inhibitors of membrane-bound semicarbazide-sensitive amine oxidase (SSAO) activity in mammalian tissues.

Semicarbazide-sensitive amine oxidase (SSAO, EC 1.4.3.6) is a group of enzymes highly sensitive to inhibition by semicarbazide. This high sensitivity distinguishes these enzymes from monoamine oxidase (MAO). Various mammalian tissues contain membrane-bound SSAO which metabolizes only the primary monoamines. Vascular and non-vascular smooth muscle cells have particularly high SSAO activity, but recently the enzyme activity has also been found in non-vascular smooth muscle cells. The substrate specificity of SSAO shows considerable species-related variations. A variety of compounds inhibiting MAO activity has also been identified as SSAO inhibitors. Among inhibitors, there is no specific SSAO inhibitor so far tested. Many studies reinforce the conclusion that inhibitory properties of some compounds against MAO activities has been markedly differed from their properties as SSAO inhibitors. 2-bromoethylamine has been recently developed with a potent, selective and suicide SSAO inhibitor without any inhibitory effect on MAO activity Using this inhibitor, it is possible to study the role of the enzyme in mammalian tissues. As physiological role the increased concentrations of SSAO, especially in blood plasma, have been found in diabetic patients and experimental animals. This enzyme was found to be associated with translocation of the glucose transporter GLUT 4 into the adipose cell surface and involved in the signaling of glucose uptake. Recent studies showed that vascular SSAO metabolizes endogenous primary amines, allylamine, methylamine and aminoacetone, to the corresponding cytotoxic aldehydes. These aldehydes have been linked to the ability of diabetic complications such as neuropathy, retinopathy and nephropathy. Overproduction of such toxic aldehydes produced by increased SSAO activity was proposed to be potentially hazardous in diabetic complications. Thus, reduction or inhibition of SSAO may be beneficial in these pathological conditions. Clearly species-related differences in properties of SSAO must be taken into account in this respect, particularly when assessing if SSAO inhibition may have great application in human.

Molecular characteristics of a single and novel form of carp (Cyprinus carpio) monoamine oxidase.

Two mammalian monoamine oxidases (MAO), MAO-A and MAO-B, are similar in primary structures but have unique substrate/inhibitor selectivities. Carp (Cyprinus carpio) contains a MAO enzyme (C-MAO) with properties different from MAO-A and MAO-B. To determine the molecular characteristics of C-MAO and its phylogenetic relationship with other fish and mammalian MAOs, the primary structure of C-MAO was estimated. The putative C-MAO cDNA encodes 526 amino acids with 59.001 Da, and the deduced amino acid sequence showed as much as 68.9% homology with some mammalian MAO-A proteins, 69.8% homology with some mammalian MAO-B proteins, and as much as 92.4% homology with some fish MAOs. Comparison of two regions in the polypeptide sequence of C-MAO determining possible substrate/inhibitor preferences of MAO-A and MAO-B showed both 79.5% homologies.

Nucleotide sequences of putative cDNAs for guinea-pig monoamine oxidase.

To study the molecular structure of guinea pig monoamine oxidase (MAO) and its phylogenetic relationship with other mammalian MAOs, we determined nucleotide sequences of putative MAO cDNAs isolated from guinea pig tissues. Both the 5'- and 3'-ends of the cDNAs were amplified using the RACE (rapid amplification of cDNA ends) method. The sequence (1924 bp) of a putative guinea-pig MAO-B cDNA covers a complete coding region that corresponds to 521 amino acids. We also analyzed a partial sequence of a putative guinea-pig MAO-A cDNA, which corresponds to 506 amino acids, but have left the region of 66 bp at the 3'-end undetermined. The nucleotide and deduced amino-acid sequences of the putative guinea-pig MAO cDNAs showed the highest homology with that of human MAO cDNAs among the known mammalian MAO sequences. These results suggest that guinea-pig MAOs are structurally similar to human MAOs. Our molecular phylogenetic data support the idea that guinea pigs and rodents diverged before the separation between rodents and other lineage leading to Primates and Artiodactyla.