Modulation of anti-cancer drug sensitivity through the regulation of mitochondrial activity by adenylate kinase 4.

BACKGROUND: Adenylate kinase is a key enzyme in the high-energy phosphoryl transfer reaction in living cells. An isoform of this enzyme, adenylate kinase 4 (AK4), is localized in the mitochondrial matrix and is believed to be involved in stress, drug resistance, malignant transformation in cancer, and ATP regulation. However, the molecular basis for the AK4 functions remained to be determined. METHODS: HeLa cells were transiently transfected with an AK4 small interfering RNA (siRNA), an AK4 short hairpin RNA (shRNA) plasmid, a control shRNA plasmid, an AK4 expression vector, and a control expression vector to examine the effect of the AK4 expression on cell proliferation, sensitivity to anti-cancer drug, metabolome, gene expression, and mitochondrial activity. RESULTS: AK4 knockdown cells treated with short hairpin RNA increased ATP production and showed greater sensitivity to hypoxia and anti-cancer drug, cis-diamminedichloro-platinum (II) (CDDP). Subcutaneous grafting AK4 knockdown cells into nude mice revealed that the grafted cells exhibited both slower proliferation and reduced the tumor sizes in response to CDDP. AK4 knockdown cell showed a increased oxygen consumption rate with FCCP treatment, while AK4 overexpression lowered it. Metabolome analysis showed the increased levels of the tricarboxylic acid cycle intermediates, fumarate and malate in AK4 knockdown cells, while AK4 overexpression lowered them. Electron microscopy detected the increased mitochondrial numbers in AK4 knockdown cells. Microarray analysis detected the increased gene expression of two key enzymes in TCA cycle, succinate dehydrogenase A (SDHA) and oxoglutarate dehydrogenease L (OGDHL), which are components of SDH complex and OGDH complex, supporting the metabolomic results. CONCLUSIONS: We found that AK4 was involved in hypoxia tolerance, resistance to anti-tumor drug, and the regulation of mitochondrial activity. These findings provide a new potential target for efficient anticancer therapies by controlling AK4 expression.

Effects of varying virus-spiking conditions on a virus-removal filter Planova™ 20N in a virus validation study of antibody solutions.

We aimed to investigate the effect of virus-spiking conditions on the filter performance (flux, flux decay, and parvovirus reduction) of the small virus filter Planova™ 20N. We used three kinds of porcine parvovirus (PPV) stocks: serum, serum-free, and purified. The flux profile with PPV spiking was similar to that without spiking for normal load filtration of about 250-300 L/m(2) . High volume (3 vol %) of serum-free PPV and 1 vol % serum PPV reduced the flux to some extent for high-load filtration (over 10 h, ca., 500 L/m(2) , 5 mg/mL IgG solution). Log reduction value (LRV) of PPV was maintained at a high level (>5) over the filtration volume. Flux for Planova™ 20N was only minimally affected by the use of different virus stocks for spiking. Transmission electron microphotography showed that the distribution of PPV particles captured inside the membrane wall was reached until the -60% thickness of the membrane, showing that the membrane of Planova™ 20N has a thick effective layer for virus removal. These results provided evidence for the robustness of the filter performance of Planova™ 20N, showing that it was not easily affected by virus spiking conditions and that it has a large capacity for high-load conditions.

Inconsistency between hepatic expression and serum concentration of transthyretin in mice humanized at the transthyretin locus.

Human transthyretin (TTR) has about 110 variants, more than 90 of which are associated with human amyloidosis. Several groups have generated transgenic mice that carry various mutant TTR genes. However, formation of mouse/human TTR heterotetramers has been shown to be inhibitory to dissociation and subsequent amyloid formation. To avoid the effect of mouse Ttr and produce humanized mice carrying different TTR variants at high efficiency, we first produced a null allele in the mouse transthyretin locus using targeting vector that contained a neomycin resistance gene flanked by lox71 and loxP. Then, through Cre-mediated recombination, we created a replacement allele that carried either a human normal (Val30) or mutant (Met30) TTR cDNA. This replacement resulted in a humanized TTR mouse with similar tissue-specific profile of human TTR as that of the endogenous mouse Ttr gene. The expression levels of human TTR mRNA and protein in the liver of homozygous human TTR (Val30/Val30) mice were about twice those of heterozygous mouse/human TTR (+/Val30) mice. However, the serum human TTR levels in the Val30/Val30 mice were much less than those in the +/Val30 mice. This contradictory expression was due to unstable Val30 tetramers caused by low binding affinity to mouse retinol binding protein.

CD44-chondroitin sulfate interactions mediate leukocyte rolling under physiological flow conditions.

CD44 on leukocytes binds to its glycosaminoglycan (GAG) ligand, hyaluronic acid, and mediates the rolling of leukocytes on vascular endothelial cells. We previously reported that the recombinant CD44 protein binds to other GAGs, including chondroitin sulfates (CS), although the physiological significance of this interaction has remained unclear. Here we report that the CD44 expressed on mouse lymphoma BW5147 cells supports cell binding to immobilized CS under static conditions and mediates cell rolling in CS-coated glass capillary tubes under shear stresses ranging from 0.5 to 1.5 dyn/cm(2), which is within the physiological range of forces in venules. Both interactions were completely inhibited by pretreating the cells with an anti-CD44 antibody or by pretreating the CS with chondroitinase ABC, but not hyaluronidase. To address the role of the CD44-CS interaction in vivo, we examined the tissue localization of the CS that interacts with CD44. Interestingly, a recombinant CD44 fusion protein bound to hepatic sinuosoidal endothelial cells where CS was also expressed, as assessed by immunohistochemistry. These findings support the involvement of the CD44-CS interaction in the primary adhesion of lymphocytes to endothelial cells and raise the possibility that this interaction plays a role in the capture of CD44-positive cells, such as activated T cells and certain tumor cells, by the hepatic sinusoidal vasculature.

Phosphoinositide 3-kinase accelerates autophagic cell death during glucose deprivation in the rat cardiomyocyte-derived cell line H9c2.

We investigated cell death during glucose deprivation in rat cardiomyocyte-derived H9c2 cells. Electron microscopic analysis revealed accumulation of autophagic vacuoles during glucose deprivation. The addition of 3-methyladenine or LY294002, which are known to inhibit autophagosome formation, reduced cell death while Z-VAD-FMK, a caspase inhibitor, slightly affected cell death. Thus, cell death during glucose deprivation is not type I programmed cell death (apoptotic cell death) but type II programmed cell death (autophagic cell death). Moreover, we found that both insulin-like growth factor-I and the adenovirus-mediated overexpression of wild-type class I PI 3-kinase accelerated cell death as well as accumulation of autophagic vacuoles during glucose deprivation while dominant-negative PI 3-kinase reduced these phenomena. The results indicate that IGF-I/PI 3-kinase accelerates the accumulation of autophagic vacuoles and subsequent autophagic cell death during glucose deprivation, revealing the opposing role of IGF-I/PI 3-kinase in two distinct types of programmed cell death (apoptotic and autophagic cell death).

Cutting edge: the B cell chemokine CXC chemokine ligand 13/B lymphocyte chemoattractant is expressed in the high endothelial venules of lymph nodes and Peyer's patches and affects B cell trafficking across high endothelial venules.

While CCR7 ligands direct T cell trafficking into lymph nodes (LNs) and Peyer's patches (PPs), chemokines that regulate B cell trafficking across high endothelial venules (HEVs) remain to be fully elucidated. Here we report that CXC chemokine ligand (CXCL)13 (B lymphocyte chemoattractant) is detected immunohistologically in the majority of HEVs in LNs and PPs of nonimmunized mice. Systemically administered anti-CXCL13 Ab bound to the surface of approximately 50% of HEVs in LNs and PPs, but not to other types of blood vessels, indicating that CXCL13 is expressed in the HEV lumen. In CXCL13-null mice, B cells rarely adhered to PP HEVs, whereas T cells did efficiently. Superfusion of CXCL13-null PPs with CXCL13 restored the luminal presentation of CXCL13 and also B cell arrest in PP HEVs at least partially. Collectively, these results indicate that CXCL13 expressed in the HEV lumen plays a crucial role in B cell trafficking into secondary lymphoid tissues such as PPs.

Protein kinase C-epsilon protects PC12 cells against methamphetamine-induced death: possible involvement of suppression of glutamate receptor.

The involvement of PKC isoform in the methamphetamine (MA)-induced death of neuron-like PC12 cell was studied. The death and the enhanced terminal dUTP nick end labeling (TUNEL) staining were inhibited by a caspase inhibitor, z-Val-Ala-Asp- (OMe)-CH(2)F (z-VAD-fmk). However, the cell death shows neither morphological nor biochemical features of apoptosis or necrosis. The cell death was suppressed by a protein kinase C (PKC) activator, 12,13-phorbol myristate acetate, but was enhanced by PKC specific inhibitor calphostin C or bisindolylmaleimide, not by PKC inhibitor relatively specific for PKC-alpha (safingol) or PKC-delta (rottlerin). Western blotting demonstrated the expression of PKC-alpha, gamma, delta, epsilon and zeta, of which PKC-epsilon translocated from the soluble to the particulate fraction after MA-treatment. Antisense to PKC-epsilon enhanced MA-induced death. A glutamate receptor antagonist MK801 abrogated the cell death, which is reversed by PKC inhibition. These data suggest that PKC-epsilon promotes PC12 cell survival through glutamate receptor suppression.

Effects of moderate hypothermia on proinflammatory cytokine production in a rat model of caerulein-induced pancreatitis.

INTRODUCTION: Proinflammatory cytokines act as mediators of the local and systemic manifestations of acute pancreatitis (AP). AIMS: To investigate whether moderate hypothermia (MH) (32 degrees C) can reduce the severity of AP by inhibiting cytokine production in a rat model of caerulein-induced pancreatitis. METHODOLOGY: Rats were divided into three groups: control rats (Group I), AP rats treated with normothermia (38 degrees C) (Group II), and AP rats treated with MH (Group III). AP was induced by intramuscular injection of caerulein and intraperitoneal infusion of lipopolysaccharide. MH was induced 4 hours after the first caerulein injection. Serum interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, IL-6, amylase, and lipase levels were determined 8 hours after the first injection. The pancreas and lung were examined histologically. RESULTS: MH in comparison with normothermia significantly reduced serum levels of IL-1beta, TNF-alpha, IL-6, amylase, and lipase. Histologically, the MH group showed less vacuolization of the acinar cells and cellular infiltration into the interacinar areas of the pancreas than were shown in the normothermia group, but these effects were not evident in the lung. CONCLUSION: Our results suggest that MH may be clinically applicable for reducing the severity of AP.

Characterization of mac25/angiomodulin expression by high endothelial venule cells in lymphoid tissues and its identification as an inducible marker for activated endothelial cells.

Previous results have indicated that mac25/angiomodulin (AGM) is expressed in lymph node (LN) high endothelial venules (HEV), the specialized venules that support efficient lymphocyte transendothelial migration. How mac25/AGM's endothelial expression pattern is regulated in situ remains unknown. Here, we demonstrate that in mouse LN blood vessels, including HEV, mac25/AGM is localized, unlike previous reports, not to the luminal or lateral regions bordering the endothelial cells (EC), but exclusively to the basal lamina that is in direct association with EC. In the spleen, mac25/AGM was expressed in the vascular basal lamina, in direct association with smooth muscle cells and pericytes, but not with EC. In addition, we report herein that mac25/AGM is an inducible marker for activated EC. In inflamed tissues, mac25/AGM expression was strongly induced in the abluminal region of blood vessels. In vitro, mac25/AGM was readily induced in EC upon activation with pro-inflammatory cytokines such as tumor necrosis factor-alpha, indicating that mac25/AGM is an activated EC marker. mac25/AGM binds vascular endothelial growth factor and, together with its strict abluminal localization, it is suggested that mac25/AGM has a specific function(s) in the subendothelium of activated blood vessels such as capturing humoral factors produced in the vicinity of HEV.