Novel role of TRPV2 in promoting the cytotoxicity of H2O2-mediated oxidative stress in Human hepatoma cells

Oxidative stress is important for the initiation and progression of cancers, which confers the cells with a survival advantage by inducing oxidative adaption and drug resistance. Therefore, developing strategies to promote oxidative stress-induced cytotoxicity could be important for cancer therapy. Herein, we found that H₂O₂-mediated oxidative stress increases TRPV2 expression in human hepatoma (HepG2 and Huh-7) cells. This occurred at the mRNA and protein levels in a dose-dependent manner. The significance of TRPV2 in promoting H₂O₂-induced cell death was demonstrated in gain and loss of function studies with overexpression and knockdown of TRPV2, respectively. Mechanistically, H₂O₂-induced cell death involves inhibition of pro-survival signaling proteins (Akt, Nrf2) and activation of pro-death signaling proteins (p38, JNK1). Overexpression of TRPV2 in H₂O₂-treated hepatoma cells aggravates the inhibition of Akt and Nrf2, while it enhances the activation of p38 and JNK1 at the early stage of cell death. Interestingly, increased expression of TRPV2 in HepG2 cells improved the efficacy of stress-associated chemicals to induce cell death. Our findings suggest that TRPV2 acts as an important enhancer for H₂O₂-induced cytotoxicity. This process occurred by the inhibition of Akt and Nrf2 as well as the early activation of p38 and JNK1. These findings have important implications for inhibition of oxidative adaption and drug resistance.     

Chromosomal and cell size analysis of cold tolerant maize

Summary C-band number, guard cell length, and chloroplast number per guard cell were determined for eight maize populations. These populations consisted of maize selected for cold tolerance at the University of Nebraska as well as the original unselected populations. The genome size of these populations had previously been determined. C-band number fluctuated concertedly with the changes in genome size indicating that deletions and additions of constitutive heterochromatin occurred during selection, resulting in altered genome sizes. Guard cell size of all the cold tolerant populations was greater than the cell size of the respective nonselected populations. Chloroplast number per guard cell was also higher in all the cold tolerant populations than in their parental populations, but the increases were not statistically significant. The results indicate that changes in genome size that occurred during selection for cold tolerance are the result of changes in amounts of C-band heterochromatin and that the selection process results in an increase in cell size in the cold tolerant populations.     

Point Mutations in Dimerization Motifs of the Transmembrane Domain Stabilize Active or Inactive State of the EphA2 Receptor Tyrosine Kinase

The EphA2 receptor tyrosine kinase plays a central role in the regulation of cell adhesion and guidance in many human tissues. The activation of EphA2 occurs after proper dimerization/oligomerization in the plasma membrane, which occurs with the participation of extracellular and cytoplasmic domains. Our study revealed that the isolated transmembrane domain (TMD) of EphA2 embedded into the lipid bicelle dimerized via the heptad repeat motif L⁵³⁵X₃G⁵³⁹X₂A⁵⁴²X₃V⁵⁴⁶X₂L⁵⁴⁹ rather than through the alternative glycine zipper motif A⁵³⁶X₃G⁵⁴⁰X₃G⁵⁴⁴ (typical for TMD dimerization in many proteins). To evaluate the significance of TMD interactions for full-length EphA2, we substituted key residues in the heptad repeat motif (HR variant: G539I, A542I, G553I) or in the glycine zipper motif (GZ variant: G540I, G544I) and expressed YFP-tagged EphA2 (WT, HR, and GZ variants) in HEK293T cells. Confocal microscopy revealed a similar distribution of all EphA2-YFP variants in cells. The expression of EphA2-YFP variants and their kinase activity (phosphorylation of Tyr⁵⁸⁸ and/or Tyr⁵⁹⁴) and ephrin-A3 binding were analyzed with flow cytometry on a single cell basis. Activation of any EphA2 variant is found to occur even without ephrin stimulation when the EphA2 content in cells is sufficiently high. Ephrin-A3 binding is not affected in mutant variants. Mutations in the TMD have a significant effect on EphA2 activity. Both ligand-dependent and ligand-independent activities are enhanced for the HR variant and reduced for the GZ variant compared with the WT. These findings allow us to suggest TMD dimerization switching between the heptad repeat and glycine zipper motifs, corresponding to inactive and active receptor states, respectively, as a mechanism underlying EphA2 signal transduction.     

Human Pluripotent Stem Cells Have a Novel Mismatch Repair-dependent Damage Response

Human pluripotent stem cells (PSCs) are presumed to have robust DNA repair pathways to ensure genome stability. PSCs likely need to protect against mutations that would otherwise be propagated throughout all tissues of the developing embryo. How these cells respond to genotoxic stress has only recently begun to be investigated. Although PSCs appear to respond to certain forms of damage more efficiently than somatic cells, some DNA damage response pathways such as the replication stress response may be lacking. Not all DNA repair pathways, including the DNA mismatch repair (MMR) pathway, have been well characterized in PSCs to date. MMR maintains genomic stability by repairing DNA polymerase errors. MMR is also involved in the induction of cell cycle arrest and apoptosis in response to certain exogenous DNA-damaging agents. Here, we examined MMR function in PSCs. We have demonstrated that PSCs contain a robust MMR pathway and are highly sensitive to DNA alkylation damage in an MMR-dependent manner. Interestingly, the nature of this alkylation response differs from that previously reported in somatic cell types. In somatic cells, a permanent G₂/M cell cycle arrest is induced in the second cell cycle after DNA damage. The PSCs, however, directly undergo apoptosis in the first cell cycle. This response reveals that PSCs rely on apoptotic cell death as an important defense to avoid mutation accumulation. Our results also suggest an alternative molecular mechanism by which the MMR pathway can induce a response to DNA damage that may have implications for tumorigenesis.     

The Tumor Suppressor pVHL Down-regulates Never-in-Mitosis A-related Kinase 8 via Hypoxia-inducible Factors to Maintain Cilia in Human Renal Cancer Cells

NEK8 (never in mitosis gene A (NIMA)-related kinase 8) is involved in cytoskeleton, cilia, and DNA damage response/repair. Abnormal expression and/or dysfunction of NEK8 are related to cancer development and progression. However, the mechanisms that regulate NEK8 are not well declared. We demonstrated here that pVHL may be involved in regulating NEK8. We found that CAK-I cells with wild-type vhl expressed a lower level of NEK8 than the cells loss of vhl, such as 786-O, 769-P, and A-498 cells. Moreover, pVHL overexpression down-regulated the NEK8 protein in 786-O cells, whereas pVHL knockdown up-regulated NEK8 in CAK-I cells. In addition, we found that the positive hypoxia response elements (HREs) are located in the promoter of the nek8 sequence and hypoxia could induce nek8 expression in different cell types. Consistent with this, down-regulation of hypoxia-inducible factors α (HIF-1α or HIF-2α) by isoform-specific siRNA reduced the ability of hypoxia inducing nek8 expression. In vivo, NEK8 and HIF-1α expression were increased in kidneys of rats subjected to an experimental hypoxia model of ischemia and reperfusion. Furthermore, NEK8 siRNA transfection significantly blocked pVHL-knockdown-induced cilia disassembling, through impairing the pVHL-knockdown-up-regulated NEK8 expression. These results support that nek8 may be a novel hypoxia-inducible gene. In conclusion, our findings show that nek8 may be a new HIF target gene and pVHL can down-regulate NEK8 via HIFs to maintain the primary cilia structure in human renal cancer cells.     

Pay attention to membrane tension: Mechanobiology of the cell surface

The cell surface is a mechanobiological unit that encompasses the plasma membrane, its interacting proteins, and the complex underlying cytoskeleton. Recently, attention has been directed to the mechanics of the plasma membrane, and in particular membrane tension, which has been linked to diverse cellular processes such as cell migration and membrane trafficking. However, how tension across the plasma membrane is regulated and propagated is still not completely understood. Here, we review recent efforts to study the interplay between membrane tension and the cytoskeletal machinery and how they control cell form and function. We focus on factors that have been proposed to affect the propagation of membrane tension and as such could determine whether it can act as a global or local regulator of cell behavior. Finally, we discuss the limitations of the available tool kit as new approaches that reveal its dynamics in cells are needed to decipher how membrane tension regulates diverse cellular processes.     

Plant-derived smoke solutions stimulate the growth of Lycopersicon esculentum roots in vitro

Aqueous extracts of smoke, derived from Themeda triandra, a fire-climax grass, and Passerina vulgaris, a fynbos plant, stimulated the growth of primary root sections of tomato roots in suspension culture. The optimal dilution for both extracts was 1:2000. Several of the fractions obtained from TLC separation of the Themeda and the Passerina extracts significantly promoted primary root growth. The auxins naphthaleneacetic acid (NAA), indolebutyric acid (IBA) and indoleacetic acid (IAA) were found to stimulate the growth of the primary root axis, with IAA and NAA significantly promoting lateral root number. Similarly, the naturally occurring cytokinins, zeatin and its derivatives (zeatin-O-glucoside; dihydrozeatin and zeatin riboside) stimulated primary root length. Zeatin and dihydrozeatin promoted secondary root growth, but only at very low concentrations.     

Detection of cellular responses to toxicants by dielectrophoresis

The dielectrophoretic (DEP) crossover method has been applied to the detection of cell responses to toxicants. Time and dose responses of the human cultured leukemia (HL-60) line were measured for paraquat, styrene oxide (SO), N-nitroso-N-methylurea (NMU) and puromycin. These toxicants were chosen because of their different predominant mechanisms of action, namely membrane free radical attack, simultaneous membrane and nucleic acid attack, nucleic acid alkylation, and protein synthesis inhibition, respectively. For all treatments, the specific membrane capacitance (C_mem) of the cells decreased while the specific membrane conductance (G_mem) increased in dose- and time-dependent manners. The DEP responses correlated sensitively with alterations in cell surface morphology, especially folds, microvilli, and blebs, observed by scanning electron microscopy. The DEP method was more sensitive to agents that had a direct action on the membrane than to agents for which membrane alterations were secondary. The responses to paraquat and SO, which directly damaged the cell membrane, could be detected 15 min after exposure, while those for puromycin and NMU, which acted on intracellular targets, could be detected after 30 min. The detection times and dose sensitivity results showed that the DEP method is much faster and more sensitive than conventional cell and higher organism viability testing techniques. The feasibility of producing small instruments for toxicity detection and screening based on cellular dielectric responses is discussed.     

Dictyosome vesicle production and plasma membrane turnover in auxin-stimulated outer epidermal cells of coleoptile segments fromAvena sativa (L.)

Summary Dark grown coleoptile segments were floated on solutions of IAA alone and of IAA and the secretion inhibitors cytochalasin and monensin. The secretion inhibitors prevented normal elongation of the tissue segments, the monensin inhibition being virtually complete while cytochalasin gave a 40% reduction over the first six hours with little further further elongation in the following 18 hours. Vesicle production was assessed in outer epidermal cells after 6 hours of IAA-stimulated elongation using the vesicle accumulation method following a cytochalasin-block of vesicle transport. The results were compared with the area of plasma membrane required to enable cell elongation to proceed at the observed rate. The area of vesicle membrane delivered to the cell surface exceeded this requirement to such an extent that at least 65% of the delivered membrane must be recycled back into the cytoplasm. Expressed in terms of the whole cell, the plasma membrane turnover rate was found to be once every 200 minutes. It is concluded that limitation of elongation by secretion inhibitors is more likely to reflect a requirement for the vesicle contents than the vesicle membrane. These results are compared with those obtained from other secretory systems using a similar approach.Abbreviations IAA indole acetic acid - DMSO dimethyl sulphoxide - D dictyosome - ER endoplasmic reticulum - V vesicle     

The effects of X-rays on the proliferation dynamics of cells in the imaginal wing disc ofDrosophila melanogaster

Summary We report on the size distribution of clones marked by mitotic recombination induced by several different doses of X-rays applied to 72 h oldDrosophila larvae. The results indicate that the radiation significantly reduces the number of cells which undergo normal proliferation in the imaginal wing disc. We estimate that 1000 r reduces by 40–60% the number of cells capable of making a normal contribution to the development of the adult wing. Part of this reduction is due to severe curtailment in the proliferative ability of cells which nevertheless remain capable of adult differentiation; this effect is possibly due to radiation-induced aneuploidy. Cytological evidence suggests that immediate cell death also occurs as a result of radiation doses as low as 100 r. The surviving cells are stimulated to undergo additional proliferation in response to the X-ray damage so that the result is the differentiation of a normal wing.