The effect of various aldehydes on the respiration of rat liver and hepatoma AH-130 cells

Some aldehydes, produced during lipid peroxidation of liver lipids, are able to inhibit the respiration of mitochondria and of intact cells both in normal hepatocytes and in Yoshida hepatoma. In mitochondria, the respiratory stimulation produced by addition of ADP and dinitrophenol is decreased more in hepatoma than in normal liver. Two- to four-fold higher concentrations of aldehydes are needed to obtain the same degree of inhibition in normal liver mitochondria as in tumorous organs. The effect of aldehydes on intact cell respiration is absent or very low in hepatocytes, but it is consistently observed in hepatoma cells.     

Amiloride and glucose effects on the intracellular pH of Yoshida rat ascites hepatoma AH-130 cells grown in vivo

The equilibrium distribution of 5,5-dimethyloxazolidine-2,4-dione (DMO) between intra- and extracellular volume was used to estimate the intracellular pH in Yoshida rat ascites hepatoma AH-130 cells under different growth conditions (log, midlog and stationary). The cells were suspended in a Krebs-Ringer 25 mM phosphate buffer and the effects of variation of external pH, of glucose and amiloride addition on intracellular pH were measured. Proliferating cells had higher intracellular pH than stationary phase cells and this difference was inhibited by amiloride. On addition of glucose the fall in external pH was similar in all conditions and corresponded to lactate production. However, the intracellular pH decreased only in proliferating cells. Stationary phase cells showed an amiloride-sensitive cytoplasmic alkalinization with glucose. Glucose addition also caused prompt recovery to a normal polysomal pattern in these cells that might suggest increased efficiency of the initiation step of protein synthesis under these conditions. The data thus suggest that the increased intracellular pH of proliferating and of glucose-treated stationary phase cells is linked to the rate of protein synthesis and is mediated by the amiloride-sensitive Na+/H+ exchange system. This could lead to increased intracellular Na+ concentration under these conditions and to initiation of growth.     

Cell cycle-related transformation of the E2F4-p130 repressor complex

During G0 phase the p130, member of the pRb tumor suppressor protein family, forms a repressor complex with E2F4 which is inactivated in G1/S by hyperphosphorylation of the p130. The role of p130 after G1/S remains poorly investigated. We found that in nuclear extracts of T98G cells, the p130-E2F4-DNA (pp-E2F4) complex does not dissociate at G1/S transition, but instead reverts to the p130-E2F4-cyclin E/A-cdk2 (cyc/cdk-pp-E2F4) complex, which is detected in S and G2/M phases of the cell cycle. Hyperphosphorylation of the p130 at G1/S transition is associated with a decrease of its total amount; however, this protein is still detected during the rest of the cell cycle, and it is increasingly hyperphosphorylated in the cytosol, but continuously dephosphorylated in the nucleus. Both nuclear and cytosol cell fractions in T98G cells contain a hyperphosphorylated form of p130 in complex with E2F4 at S and G2/M cell cycle phases. In contrast to T98G cells, transformation of the p130 containing cyc/cdk-pp-E2F4 complex into the p130-pp-E2F4 repressor does not occur in HeLa cells under growth restriction conditions.     

Synthesis of native 60S and 40S ribosomal subunits in Yoshida rat ascites hepatoma AH-130 cells: correlation with the rate of cell growth

The 60Sn and 40Sn subunit ribosome synthesis declined significantly in Yoshida rat ascites hepatoma AH-130 cells from the log phase to the plateau phase of the in vivo growth. Two main classes of 40Sn particles with protein/RNA ratios of 1.82 (p2) and 1.20 (p3) and a minor "heavy" one with protein/RNA ratio of 0.96 (p1) could be distinguished reproducibly by their ultraviolet absorption after sucrose zone sedimentation. The p2 particles appeared the dominating class in log phase cells. In plateau phase cells a decrease of p2 and an increase of p3 particles was observed. Under these conditions the p1 particles and the peaks corresponding to 60Sn subunits and to 80S ribosomes were also increased. Newly synthetized 40Sn particles banded in the p3 region of the gradient and p2 particles originated from them. These particles entered into the ribosomal cycle and contained poly(A) RNA. Formation of radioactive 80S couples by subunits entering into the ribosomal cycle was markedly stimulated in log phase cells and almost completely blocked in cells at the plateau phase of growth.     

Effect of essential fatty acid deficiency on the lipid composition of the Yoshida ascites hepatoma (AH 130) and of the liver and blood plasma from host and normal rats.

In order to study the response of a poorly differentiated tumor to nutritional manipulation, the Yoshida ascites hepatoma (AH 130) was grown in rats fed an essential fatty acid (EFA)-deficient diet and in rats fed a control diet. Hepatomas, livers, and blood plasma from host rats and normal rats were studied as to the effects of EFA deficiency on the lipid composition. Normal rats fed an EFA-deficient diet showed an increased concentration of triglycerides and cholesteryl esters in the liver and a reduced level of total phospholipids in plasma. Host rats fed the EFA-deficient diet showed a lower concentration of triglycerides in the liver when compared with the host rats fed a control diet. In addition, EFA-deficient host rats had reduced levels of plasma free fatty acids and triglycerides. These latter were markedly high in host rats under normal dietetic conditions. As compared to the livers of either host rats or normal rats fed the control diet, the Yoshida hepatoma cells had a lower content of total phospholipids and free fatty acids as well as a higher level of free cholesterol; they also showed a typical fatty acid pattern in their phospholipids. The main characteristics of this pattern were a high content of oleic and palmitoleic acids and a low level of C20 and C22 polyunsaturated fatty acids. Exposure of Yoshida hepatoma cells to an EFA-deficient environment resulted in a decrease in the concentration of total phospholipids and free fatty acids and in changes in the fatty acid composition similar to those observed in the livers of normal and host rats. These changes suggest that, under the experimental conditions used, the Yoshida hepatoma cells are responsive to EFA deficiency.     

CKI p15INK4B高表达对人黑色素瘤细胞cyclinDl和c-myc表达的影响及生长特性的改变

通过 DNA体外重组和转染技术 ,将已构建好的含有 p1 5基因的质粒 p XJ- 41 - p1 5转染 p1 5缺失的人黑色素瘤细胞 A 375,经 G 41 8筛选出阳性单克隆 ,并经 PCR、Western等检测 ,证明建立了 p1 5稳定高表达的细胞模型 .生长曲线和 FCM实验表明 ,与对照组细胞相比 ,实验组细胞增殖能力减弱 ,G1期细胞增加 ,S期细胞减少 .同时 ,细胞血清依赖性有所恢复 ,在软琼脂的集落形成能力下降 ,显示出细胞部分恶性表型逆转 .进一步免疫印迹实验表明 ,癌基因 c- myc的蛋白表达水平明显下降 .Cyclin D1的表达受到抑制 ,而 CDK4的表达则基本不变 .以上结果显示出 ,p1 5基因高表达能够抑制人黑色素瘤细胞的增殖及部分恶性表型 ,负调细胞周期进程 ,而 c- Myc蛋白和 G1期引擎分子 Cyclin D1表达的被抑制可能是 p1 5负调细胞增殖的分子机理之一     

CKIp15~(INK4B)高表达对人黑色素瘤细胞cyclinD1和c-myc表达的影响及生长特性的改变

通过 DNA体外重组和转染技术 ,将已构建好的含有 p1 5基因的质粒 p XJ- 41 - p1 5转染 p1 5缺失的人黑色素瘤细胞 A 375,经 G 41 8筛选出阳性单克隆 ,并经 PCR、Western等检测 ,证明建立了 p1 5稳定高表达的细胞模型 .生长曲线和 FCM实验表明 ,与对照组细胞相比 ,实验组细胞增殖能力减弱 ,G1期细胞增加 ,S期细胞减少 .同时 ,细胞血清依赖性有所恢复 ,在软琼脂的集落形成能力下降 ,显示出细胞部分恶性表型逆转 .进一步免疫印迹实验表明 ,癌基因 c- myc的蛋白表达水平明显下降 .Cyclin D1的表达受到抑制 ,而 CDK4的表达则基本不变 .以上结果显示出 ,p1 5基因高表达能够抑制人黑色素瘤细胞的增殖及部分恶性表型 ,负调细胞周期进程 ,而 c- Myc蛋白和 G1期引擎分子 Cyclin D1表达的被抑制可能是 p1 5负调细胞增殖的分子机理之一     

Effects of growth phase,diel cycle and macronutrient stress on the quantification of Heterosigma akashiwo using qPCR and SHA

The development of molecular probe technologies over the last several decades has enabled more rapid and specific identification and enumeration of phytoplankton species compared to traditional technologies, such as light microscopy. Direct comparisons of these methods with respect to physiological status, however, are sparse. Here we directly compare quantitative real-time PCR (qPCR) and sandwich hybridization assay (SHA) for enumerating the raphidophyte Heterosigma akashiwo at several points during its growth phase, over a diel cycle and with macronutrient stress in laboratory cultures. To ensure consistency between comparisons, a single cellular homogenate was generated from each culture and split for analysis by qPCR and SHA. Since the homogenate was generated from the same number of cells during each experiment, results reflect changes in nucleic acid content (rRNA and DNA) at each time point or in response to environmental conditions relative to a reference sample. Results show a greater level of precision in SHA results which contributed to significant (2–3 fold) differences in rRNA content per cell in several of these analyses. There was significantly greater rRNA content during lag and exponential phases compared to stationary phase cultures, and a significant decrease in rRNA content during the light cycle compared to cells harvested in the dark. In contrast, there were no significant differences in DNA content per cell as determined by qPCR over a diel cycle or during different growth phases. There was also no decrease in either rRNA or DNA content for cultures under low P conditions compared to nutrient replete conditions. However, both rRNA and DNA content were significantly lower under N stress when compared to nutrient replete conditions. Results of this study suggest that growth stage, nutrient stress and cell cycle may impact molecular analyses, and that physiological status should be taken into account when using these methods for HAB monitoring.     

Effect of weak combined static and extremely low‐frequency alternating magnetic fields on tumor growth in mice inoculated with the Ehrlich ascites carcinoma

It has been shown that the ultralow‐frequency extremely weak alternating component of combined magnetic fields (MFs) exhibits a marked antitumor activity. The parameters of this component have been found (frequency 1, 4.4, 16.5 Hz or the sum of these frequencies; intensity 300, 100, 150–300 nT, respectively) at which this MF in combination with a collinear static MF of 42 µT inhibits or suppresses the growth of Ehrlich ascites carcinoma (EAC) in mice. It was shown that the exposure of mice with EAC to combined MFs causes structural changes in some organs (liver, adrenal glands), which are probably due to the total degradation of the tumor tissue. In mice with transplanted EAC, the tumor tissue after exposure to weak MFs was practically absent, as distinct from control animals in which the invasion of the tumor into the adipose tissue surrounding the kidneys, mesenteric lymph nodes, and spermatic appendages was observed. In animals without tumors, no pathological deviations from the norm in the structure of organs and tissues occurred after exposure to weak MF, indicating that this factor per se is not toxic to the organism. Bioelectromagnetics 30:343–351, 2009. © 2009 Wiley‐Liss, Inc.     

In vitro differential effects of sodium selenite on the growth of human hepatoma cells and human embryonic liver cells

The effects of various concentrations of Na₂SeO₃ on human hepatoma cells and human embryonic liver cells was investigated in vitro. For human hepatoma cells, mitotic index and cell count decreased with increasing selenium concentrations. At 1 μg/mL Na₂SeO₃, mitotic activity of human hepatoma cells were partially arrested. In human embryonic liver cells continuously treated with Na₂SeO₃, (1 μg/mL) cell count of the treated group decreased only by d 7; mitotic index, labeled index, and mean silver grain number per 50 labeled nuclei were the same as in the control group on exposure to 1, 3, and 5 μg/mL for up to 72 h. In mixed cultures of human hepatoma and embryonic liver cells treated with 3 and 5 μg/mL of Na₂SeO₃ for 24 h, hepatoma cells showed vacuolated cytoplasms, distorted nuclei, condensed chromatin, and even pyknosis, whereas the embryonic liver cells retained a normal morphology under the same treatment.     

PAK4 is required for regulation of the cell-cycle regulatory protein p21, and for control of cell-cycle progression

The serine/threonine kinase PAK4 regulates cytoskeletal architecture, and controls cell proliferation and survival. In most adult tissues PAK4 is expressed at low levels, but overexpression of PAK4 is associated with uncontrolled proliferation, inappropriate cell survival, and oncogenic transformation. Here we have studied for the first time, the role for PAK4 in the cell cycle. We found that PAK4 levels peak dramatically but transiently in the early part of G1 phase. Deletion of Pak4 was also associated with an increase in p21 levels, and PAK4 was required for normal p21 degradation. In serum-starved cells, the absence of PAK4 led to a reduction in the amount of cells in G1, and an increase in the amount of cells in G2/M phase. We propose that the transient increase in PAK4 levels at early G1 reduces p21 levels, thereby abrogating the activity of CDK4/CDK6 kinases, and allowing cells to proceed with the cell cycle in a precisely coordinated way.     

CDK4基因沉默对非小细胞肺癌A549 增殖和代谢的影响

目的:通过特异性小干扰RNA(small interfering RNA,si RNA),使CDK4基因沉默,探讨该基因沉默对肺癌A549细胞增殖和代谢的影响及其可能的作用机制。方法:将靶向CDK4小干扰RNA(si RNA-CDK4)和阴性对照干扰片段(si RNA-control)成功转染A549细胞后,利用实时荧光定量PCR和蛋白质免疫印迹法分别检测CDK4在m RNA和蛋白水平的变化;细胞计数法、CCK-8法和软琼脂糖克隆形成实验检测A549增殖的变化和克隆形成能力;FCM法检测A549细胞的细胞周期;18F-FDG摄取实验、乳酸检测试剂盒及海马技术检测A549细胞中葡萄糖、乳酸的量及氧耗的变化;利用RT-PCR检测CDK4基因沉默后A549细胞中糖代谢相关酶m RNA水平的变化。结果:将靶向CDK4小干扰RNA(si RNA-CDK4)转染A549细胞后,可明显抑制CDK4的m RNA和蛋白表达(P0.001,P0.01)。CDK4蛋白抑制后,细胞增殖在48、72和96 h均明显降低(P值均0.05),G1期细胞比例明显增多,S期细胞比例明显减少(P值均0.05);18F-FDG摄取量下降(42.21±1.90)%(P0.05),乳酸的生成量减少(29.39±5.35)%(P0.05),而细胞的基础耗氧量增加(67.17±3.58)%(P0.01);糖酵解相关酶PFKFB3、PKM2、LDHA在m RNA水平均明显减低(P0.001,P0.01,P0.001)。结论:抑制CDK4表达可明显降低糖酵解水平,并增加耗氧量;同时可引起细胞周期阻滞,抑制肿瘤细胞增殖。其机制可能与CDK4直接或间接调节糖酵解相关酶的表达有关。     

转录因子Foxo3a高表达对小鼠T淋巴瘤EL-4细胞周期和凋亡的影响

为了研究转录因子Foxo3a高表达对小鼠T淋巴瘤EL-4细胞周期和凋亡的影响,采用电穿孔法将真核表达载体pEGFP-N1/Foxo3a转染小鼠T淋巴瘤细胞系EL-4细胞,并通过聚合酶链式反应和免疫印迹法分别检测Foxo3a mRNA及蛋白表达。转录因子Foxo3a高表达后,采用细胞计数法绘制其细胞生长曲线;采用荧光显微镜法及流式细胞仪定性和定量观察典型EL-4细胞凋亡形态特征、细胞凋亡百分率及细胞周期变化情况。结果表明,转录因子Foxo3a真核表达质粒pEGFP-N1/Foxo3a经酶切鉴定及测序检测序列正确。转染pEGFP-N1/Foxo3a的小鼠EL-4细胞表达Foxo3a mRNA和蛋白水平显著升高。Foxo3a高表达明显抑制EL-4细胞的增殖能力,并使EL-4细胞发生明显G2期阻滞(P<0.001)。Foxo3a基因高表达后,荧光显微镜可以观察到典型凋亡的细胞形态。同时,EL-4细胞凋亡百分率显著升高(P<0.01)。结果提示,Foxo3a高表达可以有效抑制小鼠T淋巴瘤细胞体外细胞增殖,使细胞周期G2时相阻滞,并具有诱导细胞凋亡的作用。     

Identification and characterization of P15RS, a novel P15(INK4b) related gene on G1/S progression

To screen genes involved in P15(INK4b) regulation during cell cycle, differential display method was applied to compare mRNAs from G(1) synchronized cells of MLIK6, which overexpressed P15(INK4b) gene, and its control MLC2. By using this approach, 15 cDNA fragments that were preferentially expressed in MLIK6 cells, but not in MLC2 cells, were screened out. A novel gene named P15RS was identified with further analysis. Combining the sequence from DD-PCR, homology analysis against EST database and RACE, a 4,404 bp complete cDNA sequence of P15RS was generated. Sequence analysis revealed that P15RS cDNA encoded a 312-amino-acid peptide containing a RAR domain that is involved in regulation of nuclear pre-mRNA, which suggests that P15RS may be a nuclear regulation protein. Genomic sequence analysis demonstrated that human P15RS gene was localized on chromosome 18q12 with seven exons and six introns. Expressing antisense P15RS in MLIK6 cells can up-regulate the expression of cyclinD1 and cyclinE. These data indicate that P15RS may act as a negative regulator in G(1) phase.     

Studies on the Metabolism and Toxicity of l-Methyl-4-Phenyl-l,2,3,6-Tetrahydropyridine in Cultures of Embryonic Rat Mesencephalon

The metabolism of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was studied in dissociated cell cultures prepared from embryonic rat mesencephalon. MPTP was added to the feeding medium and at the end of the incubation period MPTP was separated from the water-soluble metabolite by ether extraction. Our results demonstrate that, in cultures, MPTP is oxidized predominantly by monoamine oxidase B, since deprenyl but not clorgyline had an inhibitory effect on its metabolism. The metabolite of MPTP diffuses freely in the feeding medium (99% of the total) and its concentration increases with time. The concentration of the metabolite can be increased by increasing the number of cells plated into the tissue culture dish and the toxicity to dopamine neurons depends on the amount of metabolite accumulated in the feeding medium. The metabolism of MPTP is reduced by decreasing the number of glial cells present in the cultures.     

Effect of calcium,its inhibitors,and heavy metals on the growth cycle of peanut cell aggregates

During the normal growth cycle of peanut (Arachis hypogaea L.) cells, cultured in suspension medium, cell aggregates of <0.5 mm were formed during the log phase and grew to aggregates of >0.5 mm during late growth phase. Calmodulin rose to its original level during <0.5 mm aggregate formation following an initial 50% drop. Observations by UV microscopy showed that calmodulin. Ca²⁺ was centered in intense fluorescent sites. Calmodulin antagonists and a calcium chelator inhibited <0.5 mm aggregate formation as well as protein accumulation. The chelator suppressed cationic peroxidase isozyme release, while the antagonists had some partial effect on the anionic isozyme. Some heavy metals such as cadmium, mercury, lead and cobalt at low concentrations would allow continued growth of >0.5 but not of the <0.5 populations. At high (1 mM) concentrations these ions caused arrested growth. At low (10 M) levels and in the presence of 3 mM calcium they had a synergistic effect.Abbreviations AOA amino oxy acetic acid - BCA big cell aggregates - CPZ chlorpromazine - EGTA ethylene glycol bis (-aminoethyl ether) - N, N, N, N tetra acetic acid - SCA small cell aggregates - TFP trifluoperazine