氯化锂抑制A549细胞增殖及其对Polo-like激酶1转录活性的影响

利用糖原合成酶激酶3的抑制剂氯化锂作用于A549细胞,观察细胞形态与增殖的改变及其对Polo－like激酶1转录活性的影响.采用细胞计数检测细胞增殖,流式细胞术分析细胞周期变化;Western印迹检测磷酸化GSK3以及细胞周期相关蛋白p53、cyclin B1和Plk1的表达变化;RT-PCR检测Plk1 mRNA的表达;荧光素酶报告基因分析氯化锂对Plk1启动子活性的影响.结果显示,5 mmol/L氯化锂作用48 h后,A549细胞即发生明显的形态学改变,细胞增殖减慢并发生G2/M期阻滞;Plk1 mRNA和蛋白表达均升高,p53蛋白表达增强,而cyclin B1的蛋白表达无明显变化.氯化锂作用24 h后,可见pGL2-Plk1转染组中荧光素酶活性增高（与对照质粒相比,P<0.05）,48 h后更明显.以上结果表明, 氯化锂减慢A549细胞增殖,导致G2/M期阻滞,并能增强Plk1的启动子活性,促进Plk1的表达.     

Inhibition of glycogen-synthase kinase 3 stimulates glycogen synthase and glucose transport by distinct mechanisms in 3T3-L1 adipocytes

The role of glycogen-synthase kinase 3 (GSK3) in insulin-stimulated glucose transport and glycogen synthase activation was investigated in 3T3-L1 adipocytes. GSK3 protein was clearly present in adipocytes and was found to be more abundant than in muscle and liver cell lines. The selective GSK3 inhibitor, LiCl, stimulated glucose transport and glycogen synthase activity (20 and 65%, respectively, of the maximal (1 microm) insulin response) and potentiated the responses to a submaximal concentration (1 nm) of insulin. LiCl- and insulin-stimulated glucose transport were abolished by the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor, wortmannin; however, LiCl stimulation of glycogen synthase was not. In contrast to the rapid stimulation of glucose transport by insulin, transport stimulated by LiCl increased gradually over 3-5 h reaching 40% of the maximal insulin-stimulated level. Both LiCl- and insulin-stimulated glycogen synthase activity were maximal at 25 min. However, insulin-stimulated glycogen synthase activity returned to basal after 2 h, coincident with reactivation of GSK3. After a 2-h exposure to insulin, glycogen synthase was refractory to restimulation with insulin, indicating selective desensitization of this pathway. However, LiCl could partially stimulate glycogen synthase in desensitized cells. Furthermore, coincubation with LiCl during the 2 h exposure to insulin completely blocked desensitization of glycogen synthase activity. In summary, inhibition of GSK3 by LiCl: 1) stimulated glycogen synthase activity directly and independently of PI3-kinase, 2) stimulated glucose transport at a point upstream of PI3-kinase, 3) stimulated glycogen synthase activity in desensitized cells, and 4) prevented desensitization of glycogen synthase due to chronic insulin treatment. These data are consistent with GSK3 playing a central role in the regulation of glycogen synthase activity and a contributing factor in the regulation of glucose transport in 3T3-L1 adipocytes.     

Growth effects of lithium chloride in BALB/c 3T3 fibroblasts and madin-darby canine kidney epithelial cells

The ability of LiCl to initiate DNA synthesis was studied in Madin-Darby canine kidney (MDCK) cells, and mouse BALB/c 3T3 fibroblasts. In a defined culture medium lacking serum, LiCl increased DNA synthesis in BALB/c 3T3 cells 100–200% over control values. Maximum DNA synthesis was observed with concentrations of LiCl between 10 and 25 mM and increases from 40–50% over control were observed with concentrations as low as 1 mM. Exposure of BALB/c 3T3 cultures to LiCl resulted in an increase in the percentage of cells initiating DNA synthesis, total DNA content and cell number. Lithium chloride, in combination with insulin or epidermal growth factor (EGF), had either an additive or synergistic effect upon the growth of BALB/c 3T3 fibroblasts. MDCK cells proved refractory to the growth actions of LiCl, although they responded to EGF and insulin with increased DNA synthesis. Lithium chloride appears to have a direct effect on cell proliferation in some but not all cell types.     

锂对小鼠高增殖潜能集落形成细胞和粒巨噬系祖细胞体外增殖…

本文观察了锂对ＢＡＬＢ／Ｃ小鼠骨髓高增殖潜能集落形成细胞和粒巨噬系祖细胞ＣＦＵ－ＧＭ体外增殖的影响。ＨＰＰ－ＣＦＣ集落由ＩＬ－１,ＩＬ－６,ＷＥＨＩ３条件培养液及Ｌ９２９条件培养液所支持,而ＣＦＵ－ＧＭ由ＷＥＨＩ３－ＣＭ所支持。结果显示,ＬｉＣｌ浓度在０．４－２ｍｍｏｌ／Ｌ时呈现剂量依赖性抑制ＨＰＰ－ＣＦＣ增殖;而在０．４－１ｍｍｏｌ／Ｌ的浓度范围内,则对ＣＦＵ－ＧＭ的增殖起剂量依赖性促进作用。     

锂对小鼠高增殖潜能集落形成细胞和粒巨噬系祖细胞体外增殖的影响

本文观察了锂对ＢＡＬＢ／Ｃ小鼠骨髓高增殖潜能集落形成细胞（ＨＰＰ－ＣＦＣ）和粒巨噬系祖细胞ＣＦＵ－ＧＭ体外增殖的影响。ＨＰＰ－ＣＦＣ集落由ＩＬ－１、ＩＬ－６、ＷＥＨＩ３条件培养液（ＷＥＨＩ３－ＣＭ,含有ＩＬ－３）及Ｌ９２９条件培养液（Ｌ９２９－ＣＭ,含有Ｍ－ＣＳＦ）所支持,而ＣＦＵ－ＧＭ由ＷＥＨＩ３－ＣＭ所支持。结果显示,ＬｉＣｌ浓度在０．４－２ｍｍｏｌ／Ｌ时呈现剂量依赖性抑制ＨＰＰ－ＣＦＣ增殖;而在０．４－１ｍｍｏｌ／Ｌ的浓度范围内,则对ＣＦＵ－ＧＭ的增殖起剂量依赖性促进作用。这些结果提示ＬｉＣｌ对ＨＰＰ－ＣＦＣ和ＣＦＵ－ＧＭ的作用不同,可能锂有诱导ＨＰＰ－ＣＦＣ向成熟细胞分化的作用     

Lithium enhances TRAIL-induced apoptosis in human lung carcinoma A549 cells

Non-small cell lung cancer (NSCLC) A549 cells are resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Therefore, combination therapy using sensitizing agents to overcome TRAIL resistance may provide new strategies for treatment of NSCLC. Here, we investigated whether lithium chloride (LiCl), a drug for mental illness, could sensitize A549 cells to TRAIL-induced apoptosis. We observed that LiCl significantly enhanced A549 cells apoptosis through up-regulation of death receptors DR4 and DR5 and activation of caspase cascades. In addition, G2/M arrest induced by LiCl also contributed to TRAIL-induced apoptosis. Concomitantly, LiCl strongly inhibited the activity of c-Jun N-terminal kinases (JNKs), and the inhibition of JNKs by SP600125 also induced G2/M arrest and augmented cell death caused by TRAIL or TRAIL plus LiCl. However, glycogen synthase kinase-3β (GSK3β) inhibition was not involved in TRAIL sensitization induced by LiCl. Collectively, these findings indicated that LiCl sensitized A549 cells to TRAIL-induced apoptosis through caspases-dependent apoptotic pathway via death receptors signaling and G2/M arrest induced by inhibition of JNK activation, but independent of GSK3β.     

Lithium inhibits hepatic gluconeogenesis and phosphoenolpyruvate carboxykinase gene expression.

Incubation of isolated hepatocytes from fasted rats with 20 mM LiCl for 1 h decreased glucose production from lactate, pyruvate, and alanine. In addition, phosphoenolpyruvate carboxykinase (PEPCK) gene expression in FTO-2B rat hepatoma cells was inhibited by treatment with LiCl. Lithium was also able to counteract the increased PEPCK mRNA levels caused by both Bt2cAMP and dexamethasone, in a concentration-dependent manner. A chimeric gene containing the PEPCK promoter (-550 to +73) linked to the amino-3-glycosyl phosphotransferase (neo) structural gene was transduced into FTO-2B cells using a Moloney murine leukemia virus-based retrovirus. In these infected cells, 20 mM LiCl decreased both the concentration of neo mRNA transcribed from the PEPCK-neo chimeric gene and mRNA from the endogenous PEPCK gene. Lithium also inhibited the stimulatory effect of Bt2cAMP and dexamethasone on both genes. The stability of neo mRNA was not altered by lithium, since in cells infected with retrovirus containing only the neo gene transcribed via the retroviral 5'-LTR and treated with 20 mM LiCl, no change in neo mRNA levels was observed. The intraperitoneal administration of LiCl to rats caused a decrease in hepatic PEPCK mRNA, indicating that lithium could also modify gene expression in vivo. The effects of lithium were not due to an increase in the concentration of insulin in the blood but were correlated with an increase in hepatic glycogen and fructose 2,6-bisphosphate levels. These results indicate that lithium ions, at concentrations normally used therapeutically for depression in humans, can inhibit glucose synthesis in the liver by a mechanism which can selectively modify the expression of hepatic phosphoenolpyruvate carboxykinase.     

Lithium chloride potentiates tumor necrosis factor-induced and interleukin 1-induced cytokine and cytokine receptor expression.

The antimalignant cell activity of tumor necrosis factor (TNF) in many cell types can be enhanced by lithium chloride (LiCl). This study shows the in vitro effect of LiCl on the TNF-induced or interleukin 1 (IL-1)-induced expression of IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-3, IL-2, and the IL-2 receptor-alpha (IL-2R alpha). The levels of IL-6 and GM-CSF in the medium of TNF-treated L929 fibrosarcoma cells were increased by cotreatment with LiCl. In contrast, enhancement of IL-6 production by dibutyryl cyclic AMP or cycloheximide was not affected by LiCl. The production of IL-6 and GM-CSF was not correlated with sensitivity to TNF-mediated cell killing. IL-1 by itself had no measurable effects on L929 cells. However, LiCl potentiated the IL-1-induced synthesis of IL-6, GM-CSF, IL-3, and IL-2 in PC60 murine T-cell hybridoma cells. TNF alone induced only GM-CSF production in these cells, but in the presence of LiCl, increased amounts of GM-CSF as well as small amounts of IL-2 and IL-6 could be detected. It is also shown that in these PC60 cells the expression of the IL-2R alpha was induced by TNF + LiCl treatment but not by TNF alone. IL-2R alpha expression was likewise considerably enhanced by IL-1 + LiCl treatment, as compared with treatment with IL-1 alone. The effects of LiCl on the TNF-induced and the IL-1-induced gene expression seem to be independent of the protein kinase A and C pathways. These results show that LiCl can modulate both TNF-mediated cytotoxicity and TNF-induced and IL-1-induced cytokine expression, suggesting that Li+ acts early in the TNF-signaling pathway, but at a step shared with the IL-1-signaling pathway.     

Differential sensitivity of fungi to lithium chloride in culture media

Forty species of fungi, representing a range of ecological and taxonomic groups, were tested for their ability to grow on agar media amended with lithium chloride (LiCl) at 1.5, 3 and 6 g l⁻¹. Species of Trichoderma varied considerably in their sensitivity to LiCl; at one week on 6 g l⁻¹ LiCl medium, the growth of seven species of Trichoderma was considerably inhibited; however, by three weeks at this level, four of the species tested were able to attain ≥30 % of control growth. Of the seven species tested, an isolate of T. viride was the most sensitive to LiCl in agar. Eleven other imperfect fungi also showed a range of ability to grow on agar amended with LiCl, from total inhibition to complete lack of inhibition. Six ascomycete fungi were greatly inhibited by LiCl at all levels; however, an isolate of Chaetomium globosum was highly tolerant of LiCl. Seven basidiomycete wood-decay fungi were quite sensitive to LiCl in agar, showing total to nearly total inhibition even at the lowest level; however, after three weeks, an isolate of Postia placenta was nearly uninhibited except at 6 g l⁻¹. Five ectomycorrhizal basidiomycete fungi were totally inhibited by all levels of LiCl; however, one ectomycorrhizal imperfect fungus (Cenococcum graniforme) was able to grow at 3 g l⁻¹ and was uninhibited at 1.5 g l⁻¹. Four zygomycete fungus isolates were nearly unaffected in their growth by all levels of LiCl.     

Lithium chloride inactivates the 20S proteasome from WEHI-3B D+ leukemia cells

LiCl interacts synergistically with all-trans-retinoic acid, promoting the terminal differentiation of WEHI-3B D(+) cells, a phenomenon partially due to the ability of the monovalent lithium cation to inhibit the proteasome-dependent degradation of retinoic acid receptor alpha protein. In this report, the 20S proteasome was purified from WEHI-3B D(+) cells and the effects of LiCl on chymotrypsin-like (Chtl) activity and peptidyl-glutamyl peptide hydrolyzing (PGPH) activity were determined. LiCl functions to inactivate both proteasomal activities in a time-dependent manner, without affecting non-proteasomal proteases. The half-lives for inactivation of Chtl and PGPH hydrolyzing activities were approximately 23 and 36min, respectively, at 10mM LiCl. Both SDS and peptide substrate increased the rate of inactivation. Partial enzymatic activity was recovered after dialysis in the absence of SDS, indicating that the off-rate for lithium was extremely slow. The findings suggest that the inactivation of Chtl and PGPH activities by LiCl occurs through a proteasomal conformational change.     

The involvement of glycogen synthase kinase-3 and protein phosphatase-2A in lactacystin-induced tau accumulation

Here, we demonstrated that lactacystin inhibited proteasome dose-dependently in HEK293 cells stably expressing tau. Simultaneously, it induces accumulation of both non-phosphorylated and hyperphosphorylated tau and decreases the binding of tau to the taxol-stabilized microtubules. Lactacystin activates glycogen synthase kinsase-3 (GSK-3) and decreases the phosphorylation of GSK-3 at serine-9. LiCl inhibits GSK-3 and thus reverses the lactacystin-induced accumulation of the phosphorylated tau. Lactacystin also inhibits protein phosphase-2A (PP-2A) and it significantly increases the level of inhibitor 1 of PP-2A. These results suggest that inhibition of proteasome by lactacystin induces tau accumulation and activation of GSK-3 and inhibition of PP-2A are involved.     

Studies of lipoprotein lipase during the adipose conversion of 3T3 cells.

Lipoprotein lipase activity is negligible in exponentially growing 3T3-L1 cells and 3T3-F442A cells, but develops in both lines when they reach a confluent state and undergo adipose conversion. 3T3-C2 cells, which undergo adipose conversion with extremely low frequency, do not develop the enzyme. The lipase activity of 3T3-L1 and 3T3-F442A is greatly enhanced by insulin and increases 80–180 fold during the adipose conversion. The lipase has the following characteristics in common with lipoprotein lipase from adipose and other tissues: it is dependent upon serum, is inhibited by 0.5–1.0 M sodium chloride, is recovered from acetone powders, has an alkaline pH optimum and is released from the cells by heparin. Like the lipoprotein lipase of tissue adipose cells, the enzyme of 3T3-L1 decays in the presence of cycloheximide with a half-time of about 25 min at 37°C.The ability of 3T3-F442A and 3T3-L1 to take up triglyceride from the medium depends almost completely upon lipoprotein lipase. They incorporate the fatty acids of a large fraction of a triglyceride emulsion added to the medium, and this utilization is stimulated by heparin. Very little of the glycerol portion of the triglyceride is incorporated. 3T3-C2, which lacks lipoprotein lipase, utilizes very little of either the fatty acid or the glycerol portion of triglyceride.The relevance of external lipid or lipoprotein to both the adipose conversion and the appearance of lipoprotein lipase was tested using confluent cultures in medium depleted of these components. In the presence of serum whose lipoproteins have been removed by flotation, lines 3T3-F442A and 3T3-L1 undergo adipose conversion as completely as in the presence of untreated serum, and lipoprotein lipase activity appears at essentially the same rate. In medium whose serum supplement has been extracted with acetone:ethanol, 3T3-F442A cells undergo adipose conversion to nearly the same extent as in untreated serum, and develop nearly the same increase in lipoprotein lipase activity.Unless even very low concentrations of lipids or lipoprotein are saturating it can be concluded that the adipose conversion does not depend upon external lipids or lipoproteins for its induction; rather the differentiation program is built into the cell type and comes into operation when growth is arrested even in their absence. The source of fatty acids utilized for triglyceride synthesis, however, may be affected by the amount of lipid provided to the cells.     

The dephosphorylation of inositol 1,4-bisphosphate to inositol in liver and brain involves two distinct Li+-sensitive enzymes and proceeds via inositol 4-phosphate.

1. Hydrolysis of both enantiomers of inositol 1-phosphate and both enantiomers of inositol 4-phosphate to inositol is inhibited by LiCl in liver and brain. 2. The phosphatase activity is predominantly soluble. 3. Inositol 1,4-bisphosphate is also hydrolysed by the soluble fraction of liver and brain. 4. Bisphosphatase activity is inhibited by LiCl, but is less sensitive than monophosphatase activity. 5. The product of bisphosphatase in liver and brain is inositol 4-phosphate.     

Lithium fluxes in Paramecium and their relationship to chemoresponse.

Paramecia respond to environmental stimuli by altering swimming behavior to disperse from or accumulate in the vicinity of the stimulus. We have found, using the T-maze assay, that treatment of paramecia with LiCl in a time- and concentration-dependent manner modifies the normal response to folate, acetate, and lactate from attraction to no response or even repulsion. Responses to NH4Cl were unaffected and to cAMP were variably affected by LiCl. Cells incubated in the presence of K+, or both Na+ and K+, but not Na+ alone reliably recovered attraction to folate. Treatment of cells with 4 mM LiCl for 1 h dramatically slowed swimming speed from about 1 mm/s in NaCl or KCl (control) to 0.18 mm/s in LiCl. Li-treated cells subsequently incubated in 4 mM NaCl, KCl or sequentially in KCl and NaCl for a total of 20 min increased their swimming speed to 0.35, 0.45 and 0.67 mm/s, respectively. Paramecia readily took up Li+ in Na(+)- and K(+)-free media reaching intracellular concentrations of 5-10 mM in 10 mM extracellular Li+. Efflux of intracellular Li+ was stimulated 35% by extracellular 10 mM NaCl and 185% by 10 mM KCl over 10 mM choline chloride. Incubation of cells in 10 mM LiCl for 1 h inhibited the rate of Ca2+ efflux by 44% compared to cells in 10 mM NaCl. This may relate to the mechanism by which Li+ perturbs chemoresponse. A mutant with defects in Ca homeostasis responds normally to NH4Cl, but not to any of the stimuli that are affected by LiCl.     

Regulation of ProMMP-1 and ProMMP-3 activation by tissue factor pathway inhibitor-2/matrix-associated serine protease inhibitor

Tissue factor pathway inhibitor-2 (TFPI-2)/matrix-associated serine protease inhibitor (MSPI), a 32- to 33-kDa Kunitz-type serine protease inhibitor, inhibits plasmin and trypsin. Because plasmin and trypsin are involved in the activation of promatrix metalloproteases proMMP-1 and proMMP-3, we investigated the role of TFPI-2/MSPI in the activation of these proenzymes. Both plasmin and trypsin activated proMMP-1 by converting the 53-kDa proenzyme to the partially active 43-kDa polypeptide; this activity was inhibited by TFPI-2/MSPI. Similarly, TFPI-2/MSPI inhibited the conversion of 66-kDa proMMP-3 to the activated 45- and 30-kDa polypeptides by plasmin and trypsin. Because plasmin is involved in the physiological activation of proMMP-3, we tested whether TFPI-2/MSPI inhibits the activation of proMMP-3 by HT-1080 fibrosarcoma cells and urokinase-charged HeLa cells. We found that the inhibitor inhibited proMMP-3 activation by HT-1080 cells and urokinase-charged HeLa cells. Collectively, our results suggest that TFPI-2/MSPI indirectly regulates MMP-1- and MMP-3-catalyzed matrix proteolysis by regulating the activation of proMMP-1 and proMMP-3.     

Chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells treated by GSK-3 inhibitors

A study of the cartilage differentiation of mesenchymal stem cells (MSCs) would be of particular interest since one strategy for cell-based treatment of cartilage defects emphasizes the use of cells that are in a differentiated state. The present study has attempted to evaluate the effects of two well-known glycogen synthase kinase-3 inhibitors, including lithium chloride (LiCl) and SB216763 on a human marrow-derived MSC (hMSC) chondrogenic culture. Passaged-3 MSCs were condensed into small pellets and cultivated in the following groups based on the supplementation of chondrogenic medium: transforming growth factor (TGF)-β1, TGF-β1 + LiCl, TGF-β1 + SB216763, TGF-β3, TGF-β3 + LiCl, and TGF-β3 + SB216763. The cultures were maintained for 21 days and then analyzed for expression of Sox9, aggrecan, collagen II, β-catenin, and axin genes. Deposition of glycosaminoglycan (GAG) in the cartilage matrix was also measured for certain cultures. The presence of both LiCl and SB216763 along with TGF-β in the MSC chondrogenic culture led to the up-regulation of cartilage-specific genes. TGF-β3 appeared much better than TGF-β1. Based on our findings, SB216763 was more effective in up-regulation of cartilage-specific genes. These chondrogenic effects appeared to be mediated through the Wnt signaling pathway since β-catenin and axin tended to be up-regulated and down-regulated, respectively. In the culture with SB216763 + TGF-β3, significantly more GAG was deposited (P < 0.05). In conclusion, addition of either SB216763 or LiCl to hMSC chondrogenic culture up-regulates cartilage-specific gene expression and enhances GAG deposition in the culture.