Differential mRNA expression of the human DNA methyltransferases (DNMTs) 1, 3a and 3b during the G(0)/G(1) to S phase transition in normal and tumor cells

DNA methylation is essential for mammalian development, X-chromosome inactivation, and imprinting yet aberrant methylation patterns are one of the most common features of transformed cells. One of the proposed causes for these defects in the methylation machinery is overexpression of one or more of the three known catalytically active DNA methyltransferases (DNMTs) 1, 3a and 3b, yet there are clearly examples in which overexpression is minimal or non-existent but global methylation anomalies persist. An alternative mechanism which could give rise to global methylation errors is the improper expression of one or more of the DNMTs during the cell cycle. To begin to study the latter possibility we examined the expression of the mRNAs for DNMT1, 3a and 3b during the cell cycle of normal and transformed cells. We found that DNMT1 and 3b levels were significantly downregulated in G₀/G₁ while DNMT3a mRNA levels were less sensitive to cell cycle alterations and were maintained at a slightly higher level in tumor lines compared to normal cell strains. Enzymatic activity assays revealed a similar decrease in the overall methylation capacity of the cells during G₀/G₁ arrest and again revealed that a tumor cell line maintained a higher methylation capacity during arrest than a normal cell strain. These results reveal a new level of control exerted over the cellular DNA methylation machinery, the loss of which provides an alternative mechanism for the genesis of the aberrant methylation patterns observed in tumor cells.     

Human Tendon Stem Cells Better Maintain Their Stemness in Hypoxic Culture Conditions

Tissues and organs in vivo are under a hypoxic condition; that is, the oxygen tension is typically much lower than in ambient air. However, the effects of such a hypoxic condition on tendon stem cells, a recently identified tendon cell, remain incompletely defined. In cell culture experiments, we subjected human tendon stem cells (hTSCs) to a hypoxic condition with 5% O₂, while subjecting control cells to a normaxic condition with 20% O₂. We found that hTSCs at 5% O₂ had significantly greater cell proliferation than those at 20% O₂. Moreover, the expression of two stem cell marker genes, Nanog and Oct-4, was upregulated in the cells cultured in 5% O₂. Finally, in cultures under 5% O₂, more hTSCs expressed the stem cell markers nucleostemin, Oct-4, Nanog and SSEA-4. In an in vivo experiment, we found that when both cell groups were implanted with tendon-derived matrix, more tendon-like structures formed in the 5% O₂ treated hTSCs than in 20% O₂ treated hTSCs. Additionally, when both cell groups were implanted with Matrigel, the 5% O₂ treated hTSCs showed more extensive formation of fatty, cartilage-like and bone-like tissues than the 20% O₂ treated cells. Together, the findings of this study show that oxygen tension is a niche factor that regulates the stemness of hTSCs, and that less oxygen is better for maintaining hTSCs in culture and expanding them for cell therapy of tendon injuries.     

A simulation study of cellular hypertrophy and connexin lateralization in cardiac tissue

Many cardiac diseases coincide with changes in cell size and shape. One example of such a disease is cardiac hypertrophy. It is established that cardiac impulse propagation depends on the cell size, as well as other factors, but interrelations between conduction velocity (CV), cell size, and gap junction (GJ) conductance (g_GJ) are complex. Furthermore, cardiac diseases are often accompanied by connexin (Cx) lateralization. To analyze the effects of cell size and Cx lateralization in cardiac disease, a two-dimensional computer simulation of ventricular myocytes based on the Luo-Rudy model was used. Control cells (80 μm/20 μm (length/diameter)), long cells (160 μm/20 μm), and wide cells (80 μm/40 μm) were simulated as was a redistribution of lateral GJs (constant lateral g_GJ and increased lateral g_GJ). CV in long cells showed high stability, i.e., it declined very slowly when g_GJ was gradually reduced. Wide cells, however, were more affected by reduced g_GJ, resulting in early transition to discontinuous propagation and low CV. Conduction block occurred earlier in enlarged cells than in control cells due to increased cell capacitance. Increased lateral g_GJ stabilized longitudinal CV, which was a result of two-dimensional effects during planar wave propagation. Therefore, Cx lateralization may compensate for cardiac inhomogeneities. High lateral g_GJ and enhanced cell diameter increased the susceptibility to conduction block at tissue expansion, providing a substrate for arrhythmia.     

Low C18 to C20 fatty acid elongase activity and limited conversion of stearidonic acid, 18:4(n–3), to eicosapentaenoic acid, 20:5(n–3), in a cell line from the turbot, Scophthalmus maximus

The TF cell line, derived from a top predatory, carnivorous marine teleost, the turbot (Scophthalmus maximus), is known to have a limited conversion of C₁₈ to C₂₀ polyunsaturated fatty acids (PUFA). To illuminate the underlying processes, we studied the conversions of stearidonic acid, 18:4(n–3), and its elongation product, 20:4(n–3), in TF cells and also in a cell line, AS, derived from Atlantic salmon (Salmo salar), by adding unlabelled (25 μM), U-¹⁴C (1 μM) or deuterated (d5; 25 μM) fatty acids. Stearidonic acid, 18:4(n–3), was metabolised to 20:5(n–3) in both cells lines, but more so in AS than in TF cells. Δ5 desaturation was more active in TF cells than in AS cells, whereas C₁₈ to C₂₀ elongation was much reduced in TF as compared to AS cells. Only small amounts of docosahexaenoic acid (22:6(n–3)) were produced by both cell lines, although there was significant production of 22:5(n–3) in both cultures, especially when 20:4(n–3) was supplemented. We conclude that limited elongation of C₁₈ to C₂₀ fatty acids rather than limited fatty acyl Δ5 desaturation accounts for the limited rate of conversion of 18:3(n–3) to 20:5(n–3) in the turbot cell line, as compared to the Atlantic salmon cell line. The results can account for the known differences in conversions of C₁₈ to C₂₀ PUFA by the turbot and the Atlantic salmon in vivo.     

Regulation and Functional Contribution of Thymidine Kinase 1 in Repair of DNA Damage

Cellular supply of dNTPs is essential in the DNA replication and repair processes. Here we investigated the regulation of thymidine kinase 1 (TK1) in response to DNA damage and found that genotoxic insults in tumor cells cause up-regulation and nuclear localization of TK1. During recovery from DNA damage, TK1 accumulates in p53-null cells due to a lack of mitotic proteolysis as these cells are arrested in the G₂ phase by checkpoint activation. We show that in p53-proficient cells, p21 expression in response to DNA damage prohibits G₁/S progression, resulting in a smaller G₂ fraction and less TK1 accumulation. Thus, the p53 status of tumor cells affects the level of TK1 after DNA damage through differential cell cycle control. Furthermore, it was shown that in HCT-116 p53^−/− cells, TK1 is dispensable for cell proliferation but crucial for dTTP supply during recovery from DNA damage, leading to better survival. Depletion of TK1 decreases the efficiency of DNA repair during recovery from DNA damage and generates more cell death. Altogether, our data suggest that more dTTP synthesis via TK1 take place after genotoxic insults in tumor cells, improving DNA repair during G₂ arrest.     

On the genetic control of cell cycles during morphogenesis in Ulva mutabilis

Development of the wild type and two temperature-sensitive mutants of the multicellular green alga Ulva mutabilis is compared. The mutants develop normal phenotypes at 22°C and abnormal phenotypes at 15°C. Normal development starts by formation of a filament consisting of a row of cells. The growth rate, the generation times, and the cell length at division change in a coordinated manner according to the positions of the cells within the filament. In the mutant cs₂ transfer to 15°C inhibits all cytoplasmic divisions during early development. In the mutant cs₆ the first three divisions proceed normally. Then cytoplasmic division is blocked in the most distal cells, while the proximal cells continue to divide according to a branched pattern. In the cs₂ mutant cell determination seems to occur at 15°C, while the differentiation of the determined cells can only occur at 22°C. In the mutant cs₆ the cells are not determined at 15°C. The cs₆⁺ gene, as well as the previously described Slender-like genes, presumably has a short period of activity and is concerned with more fundamental epigenetic processes than the cs₂⁺-gene and the previously described precocious-like genes, which seem to have more prolonged periods of activity.     

Changes of Saccharomyces cerevisiae cell membrane components and promotion to ethanol tolerance during the bioethanol fermentation

During bioethanol fermentation process, Saccharomyces cerevisiae cell membrane might provide main protection to tolerate accumulated ethanol, and S. cerevisiae cells might also remodel their membrane compositions or structure to try to adapt to or tolerate the ethanol stress. However, the exact changes and roles of S. cerevisiae cell membrane components during bioethanol fermentation still remains poorly understood. This study was performed to clarify changes and roles of S. cerevisiae cell membrane components during bioethanol fermentation. Both cell diameter and membrane integrity decreased as fermentation time lasting. Moreover, compared with cells at lag phase, cells at exponential and stationary phases had higher contents of ergosterol and oleic acid (C_18:1) but lower levels of hexadecanoic (C_16:0) and palmitelaidic (C_16:1) acids. Contents of most detected phospholipids presented an increase tendency during fermentation process. Increased contents of oleic acid and phospholipids containing unsaturated fatty acids might indicate enhanced cell membrane fluidity. Compared with cells at lag phase, cells at exponential and stationary phases had higher expressions of ACC1 and HFA1. However, OLE1 expression underwent an evident increase at exponential phase but a decrease at following stationary phase. These results indicated that during bioethanol fermentation process, yeast cells remodeled membrane and more changeable cell membrane contributed to acquiring higher ethanol tolerance of S. cerevisiae cells. These results highlighted our knowledge about relationship between the variation of cell membrane structure and compositions and ethanol tolerance, and would contribute to a better understanding of bioethanol fermentation process and construction of industrial ethanologenic strains with higher ethanol tolerance.     

Lysophosphatidic acid receptor-5 negatively regulates cellular responses in mouse fibroblast 3T3 cells

Lysophosphatidic acid (LPA) signaling via G protein-coupled LPA receptors (LPA₁–LPA₆) mediates a variety of biological functions, including cell migration. Recently, we have reported that LPA₁ inhibited the cell motile activities of mouse fibroblast 3T3 cells. In the present study, to evaluate a role of LPA₅ in cellular responses, Lpar5 knockdown (3T3-L5) cells were generated from 3T3 cells. In cell proliferation assays, LPA markedly stimulated the cell proliferation activities of 3T3-L5 cells, compared with control cells. In cell motility assays with Cell Culture Inserts, the cell motile activities of 3T3-L5 cells were significantly higher than those of control cells. The activity levels of matrix metalloproteinases (MMPs) were measured by gelatin zymography. 3T3-L5 cells stimulated the activation of Mmp-2, correlating with the expression levels of Mmp-2 gene. Moreover, to assess the co-effects of LPA₁ and LPA₅ on cell motile activities, Lpar5 knockdown (3T3a1-L5) cells were also established from Lpar1 over-expressing (3T3a1) cells. 3T3a1-L5 cells increased the cell motile activities of 3T3a1 cells, while the cell motile activities of 3T3a1 cells were significantly lower than those of control cells. These results suggest that LPA₅ may act as a negative regulator of cellular responses in mouse fibroblast 3T3 cells, similar to the case for LPA₁.     

Alphavbeta3 integrin blocking inhibits apoptosis and induces autophagy in murine breast tumor cells

Integrins are cell receptors that mediate adhesion to the extracellular matrix (ECM) and regulate cell migration, a crucial process in tumor invasion. The α_vβ₃ integrin recognizes the arginine-glycine-aspartic acid (RGD) motif in ECM proteins and it can be antagonized by RGD-peptides, resulting in decreased cell migration and invasion. RGD-based drugs have shown disappointing results in clinical trials; however, the reasons for their lack of activity are still obscure. Aiming to contribute to a better understanding of the molecular consequences of integrin inhibition, we tested a recombinant RGD-disintegrin (DisBa-01) in two types of murine cell lines, breast tumor 4T1BM2 cells and L929 fibroblasts. Only tumor cells showed decreased motility and adhesion, as well as morphologic alterations upon DisBa-01 treatment (100 and 1000 nM). This result was attributed to the higher levels of α_vβ₃ integrin in 4T1BM2 cells compared to L929 fibroblasts making the former more sensitive to DisBa-01 blocking. DisBa-01 induced cell cycle arrest at the S phase in 4T1BM2 cells, but it did not induce apoptosis, which was consistent with the decrease in caspase-3, 8 and 9 expression at mRNA and protein levels. DisBa-01 increases PI3K, Beclin-1 and LC3B expression in tumor cells, indicators of autophagic induction. In conclusion, α_vβ₃ integrin blocking by DisBa-01 results in inhibition of adhesion and migration and in the activation of an autophagy program, allowing prolonged survival and avoiding immediate apoptotic death. These observations suggest new insights into the effects of RGD-based inhibitors considering their importance in drug development for human health.     

Regional difference in dynamical property of sinoatrial node pacemaking: role of na+ channel current

To elucidate the regional differences in sinoatrial node pacemaking mechanisms, we investigated 1), bifurcation structures during current blocks or hyperpolarization of the central and peripheral cells, 2), ionic bases of regional differences in bifurcation structures, and 3), the role of Na⁺ channel current (I_Na) in peripheral cell pacemaking. Bifurcation analyses were performed for mathematical models of the rabbit sinoatrial node central and peripheral cells; equilibrium points, periodic orbits, and their stability were determined as functions of parameters. Structural stability against applications of acetylcholine or electrotonic modulations of the atrium was also evaluated. Blocking L-type Ca²⁺ channel current (I_Ca,L) stabilized equilibrium points and abolished pacemaking in both the center and periphery. Critical acetylcholine concentration and gap junction conductance for pacemaker cessation were higher in the periphery than in the center, being dramatically reduced by blocking I_Na. Under hyperpolarized conditions, blocking I_Na, but not eliminating I_Ca,L, abolished peripheral cell pacemaking. These results suggest that 1), I_Ca,L is responsible for basal pacemaking in both the central and peripheral cells, 2), the peripheral cell is more robust in withstanding hyperpolarizing loads than the central cell, 3), I_Na improves the structural stability to hyperpolarizing loads, and 4), I_Na-dependent pacemaking is possible in hyperpolarized peripheral cells.     

Ultrastructure of secretory cells in the gut of the cattle-tick Boophilus microplus

The ultrastructural study of the secretory cells type 1 and 2 confirmed the separate identities of two secretory cell types in the gut of female B. microplus. Secretory cell type 1 (s₁) synthesized and secreted large, spherical, uniformly electrondense granules. Secretory cell type 2 (s₂) synthesized smaller, irregularly shaped and more complex granules. Another cell type, the basophilic cell, was shown to be the reorganized basal remnant of secretory cell s₂. A few of the basophilic cells retained remnant s₂ granules within their cytoplasm. In these cells the reorganized cisternae of rough endoplasmic reticulum were arranged in whorls and parallel arrays. The cells synthesized granules with a different ultrastructure and position in the cell from the earlier granules. The new secretory material may be egg proteins which are released into the haemolymph, and transported to the ovary. Another secretory cell type with smaller spherical granules was seen in the gut caeca of only two female ticks and more evidence is needed to prove its separate identity.     

Growth Response of Clostridium perfringens to Oxidative Stress

The sensitivity of Clostridium perfringens strain 13 to oxygen and its toxic derivatives was investigated in a new, defined medium (MMP). Exponentially growing cells in MMP medium were very sensitive to exposure to air by vigorous shaking. When exposed to air, the cells survived only 1hour and then rapidly died. Addition of cysteine, ascorbic acid, or yeast extract to the medium significantly increased vegetative cell survival without inducing sporulation. The level of toxicity of peroxyl and hydroperoxyl radicals, generated by H₂O₂, t-butyl hydroperoxide or ethanol, was very similar with and without air exposure. By contrast, plumbagin or menadione, which generate superoxide radicals in the presence of oxygen, caused high levels of cell death only in aerobiosic culture. Growth-arrested cells were more resistant to H₂O₂and to redox-cycling agents than were exponentially growing cells, but the resistance required de novo synthesis of proteins. An adaptive response to oxidative stress was also suggested by the higher level of cell resistance to H₂O₂and to ethanol when cells were pretreated with sublethal doses of these oxidants.     

Is Cell Rheology Governed by Nonequilibrium-to-Equilibrium Transition of Noncovalent Bonds?

A living cell deforms or flows in response to mechanical stresses. A recent report shows that dynamic mechanics of living cells depends on the timescale of mechanical loading, in contrast to the prevailing view of some authors that cell rheology is timescale-free. Yet the molecular basis that governs this timescale-dependent behavior is elusive. Using molecular dynamics simulations of protein-protein noncovalent interactions, we show that multipower laws originate from a nonequilibrium-to-equilibrium transition: when the loading rate is faster than the transition rate, the power-law exponent α₁ is weak; when the loading rate is slower than the transition rate, the exponent α₂ is strong. The model predictions are confirmed in both embryonic stem cells and differentiated cells. Embryonic stem cells are less stiff, more fluidlike, and exhibit greater α₁ than their differentiated counterparts. By introducing a near-equilibrium frequency f_eq, we show that all data collapse into two power laws separated by f/f_eq, which is unity. These findings suggest that the timescale-dependent rheology in living cells originates from the nonequilibrium-to-equilibrium transition of the dynamic response of distinct, force-driven molecular processes.     

Effects of lysophosphatidic acid (LPA) receptor-2 (LPA2) and LPA3 on the regulation of chemoresistance to anticancer drug in lung cancer cells

Lysophosphatidic acid (LPA) mediates a variety of biological functions via the binding of G protein-coupled LPA receptors (LPA receptor-1 (LPA₁) to LPA₆). This study aimed to investigate the roles of LPA₂ and LPA₃ in the modulation of chemoresistance to anticancer drug in lung cancer A549 cells. In cell survival assay, cells were treated with cisplatin (CDDP) every 24 h for 2 days. The cell survival rate to CDDP of A549 cells was significantly elevated by an LPA₂ agonist, GRI-977143. To evaluate the roles of LPA₂-mediated signaling in cell survival during tumor progression, highly migratory (A549-R10) cells were generated from A549 cells. In the presence of GRI-977143, the cell survival rate to CDDP of A549-R10 cells were markedly higher than that of A549 cells, correlating with LPAR2 expression level. Moreover, to assess the effects of long-term anticancer drug treatment on cell survival, the long-term CDDP treated (A549-CDDP) cells were established from A549 cells. The cell survival rate to CDDP of A549-CDDP cells was elevated by GRI-977143. Since LPAR3 expression level was significantly higher in A549-CDDP cells than in A549 cells, we investigated the roles of LPA₃ in the cell survival to CDDP of A549 cells, using an LPA₃ agonist, 1-oleoyl-2-methyl-sn-glycero-3-phosphothionate ((2S)-OMPT). The cell survival rate to CDDP of A549 cells was significantly reduced by (2S)-OMPT treatment. In the presence of (2S)-OMPT, the cell survival rate to CDDP of A549 cells was elevated by LPA₃ knockdown. These results suggest that LPA signaling via LPA₂ and LPA₃ is involved in the regulation of chemoresistance in A549 cells treated with CDDP.     

Hydrogen Sulfide Represses Androgen Receptor Transactivation by Targeting at the Second Zinc Finger Module

Androgen receptor (AR) signaling is indispensable for the development of prostate cancer from the initial androgen-dependent state to a later aggressive androgen-resistant state. This study examined the role of hydrogen sulfide (H₂S), a novel gasotransmitter, in the regulation of AR signaling as well as its mediation in androgen-independent cell growth in prostate cancer cells. Here we found that H₂S inhibits cell proliferation of both androgen-dependent (LNCaP) and antiandrogen-resistant prostate cancer cells (LNCaP-B), with more significance on the latter, which was established by long term treatment of parental LNCaP cells with bicalutamide. The expression of cystathionine γ-lyase (CSE), a major H₂S-producing enzyme in prostate tissue, was reduced in both human prostate cancer tissues and LNCaP-B cells. LNCaP-B cells were resistant to bicalutamide-induced cell growth inhibition, and CSE overexpression could rebuild the sensitivity of LNCaP-B cells to bicalutamide. H₂S significantly repressed the expression of prostate-specific antigen (PSA) and TMPRSS2, two AR-targeted genes. In addition, H₂S inhibited AR binding with PSA promoter and androgen-responsive element (ARE) luciferase activity. We further found that AR is post-translationally modified by H₂S through S-sulfhydration. Mutation of cysteine 611 and cysteine 614 in the second zinc finger module of AR-DNA binding domain diminished the effects of H₂S on AR S-sulfhydration and AR dimerization. These data suggest that reduced CSE/H₂S signaling contributes to antiandrogen-resistant status, and sufficient level of H₂S is able to inhibit AR transactivation and treat castration-resistant prostate cancer.     

Gender disparities in torsade de pointes ventricular tachycardia

Background Gender disparities in the incidence of torsade de pointes (TdP) ventricular tachycardia exist, but the mechanisms in humans are unresolved. We addressed this issue using a mathematical model of a human ventricular cell. MethodsWe implemented gender differences in the Priebe-Beuckelmann model cell by modifying the amplitudes of the L-type Ca²⁺ current (I_Ca,L), transient outward K₊ current (I_to), and rapid component of the delayed rectifier K₊current (I_Kr), according to experimental data from animal male and female hearts. Gender disparities in electrical heterogeneity between transmural layers (subepicardium, midmyocardium, subendocardium) were implemented by modifying various ion currents according to experimental data. ResultsAction potentials in female cells have longer durations and steeper duration versus frequency relationships than male cells. In the female cells, electrical heterogeneity between transmural layers is larger and the susceptibility to early afterdepolarisations is higher than in male cells. ConclusionGender-related differences in I_Ca,L, I_to, and I_Kr may explain the gender disparities in human cardiac electrophysiology. Female cells have an increased susceptibility to early afterdepolarisations following mild reductions in net repolarising forces. Combined with their greater electrical heterogeneity, this renders them more vulnerable to TdP. (Neth Heart J 2007;15:405-11.)     

The Role of Calcium in the Regulation of the Steady-State Levels of Sodium and Potassium in the HeLa Cell

Studies on HeLa cells in spinner culture at pH 7.0 and 37° have shown that [Na]_i decreased and [K]_i increased with increasing [Ca]_o. In Na-free (choline) medium [K]_i remained high whether or not Ca was present in the medium. [Na]_i and [K]_i approached a new steady state within 1 min after transfer to Ca-free medium and returned to the initial values within 15 min upon readdition of Ca. 40% of the cell Ca exchanged within 1 min followed by a slow exchange of the remaining Ca over several hours. [Ca]_i increased with decreasing [Na]_o but was independent of [K]_o. Equimolar Mg did not substitute for Ca in maintaining low [Na]_i and high [K]_i. Under steady-state conditions about 50% of the cell Na exchanged in accordance with a single rate constant. The initial Na influx was 270, 100, and 2.5 µM/liter of cell water/sec for 0, 0.10, and 1.0 mM [Ca]_o, respectively. When Na transport was inhibited with strophanthidin and [Na]_i and [K]_i allowed to reach a steady state, Na influx was more rapid for cells incubated in Ca-free medium than for cells incubated in medium containing 1.0 mM Ca. These results suggest that Ca competes with Na at the cell membrane and thus controls the passive diffusion of Na into the cell.     

The human LT system. V. A comparison of the relative lytic effectiveness of various MW human LT classes on 51Cr-labeled allogeneic target cells in vitro: enhanced lysis by LT complexes associated with Ig-like receptor(s).

The present studies examine the in vitro cell-lytic capacity of various molecular weight (MW) human lymphotoxin (LT) classes obtained from lectin-activated normal or immune lymphocytes on allogeneic target cells. The findings reveal that the high-MW complex class of LT is up to 100 times more effective than the smaller MW LT forms (α, β, and γ) in causing lysis of various allogeneic cell types including lymphoid cells in vitro. Moreover, the data suggest that lectin-stimulated alloimmune cells (MLC sensitized) release complex LT forms in association with a specific antigen-binding receptor(s), and that these complexes are from 3 to 10 times more effective on the sensitizing target cell than complexes obtained from lectin-stimulated nonimmune cells. Positive evidence that complex-induced lysis involved LT was indicated by the finding that lysis was completely neutralized by incubation with heterologous antisera directed against a refined human α₂-LT subclass (anti-α₂) and partially neutralized with anti-human Fab₂′ serum. These findings support the concept that LT molecules may represent a system of related cell-lytic molecules. While the smaller MW forms are only weakly lytic by themselves, they can be assembled into highly lytic complexes which may be focused or directed by an antigen-binding receptor(s).