蛋白激酶与细胞分化—两种分化诱导剂对酪氨酸蛋白激酶的抑制

在报道视黄酸(RA)是人肝癌细胞株SMMC-7721分化诱导剂的基础上,本文继续报道8-溴-环磷酸腺苷对该细胞也有分化诱导作用,两者都抑制细胞的增殖,降低γ-谷氨酰转肽酶(γ-GT)比活力和升高(ALP)碱性磷酸酶的比活力。在10μmol/LRA和0.5mmol/L-8-Br-cAMP处理细胞1、3、5天后,胞液和膜性组分中的酪氨酸蛋白激酶(TPK)的比活力均降低,其中RA对胞液TPK的作用在早期较明显,约降低30％,而对膜性TPK的影响则随培养天数而逐渐增加,至第5天下降达50％以上。8Br-cAMP则相反,对胞浆TPK的抑制主要发生在3天以后;约抑制43—53％,而对膜性组分则抑制率逐日降低,在第一天较为明显。因TPK是一个细胞增殖恶变的标志,故RA和8-溴-cAMP对TPK的抑制进一步证明这两种分化诱导剂对SMMC-7721细胞的逆转作用。     

诱发大鼠肝癌早期病变中蛋白激酶和磷脂的变化

<正> 近年来发现蛋白激酶不但参与细胞的代谢调节,还与细胞的增殖和分化密切相关。除酪氨酸蛋白激酶是某些癌基因的表达产物外,钙-磷脂依赖性蛋白激酶(又称蛋白激酶C,PK-C)是促癌剂TPA(12-O-tetradecanoyl-phorbol-13-aeetatc)的受体。作者在前文中曾报导:分化诱导剂视黄酸可使人肝癌细胞株SMMC-7721中蛋白激酶C的活力明显降低;而蛋白激酶A(cAMP依赖性蛋白激酶,PKA)则明显增加。本文研究化学诱发大鼠肝癌早期病变中这两种蛋白激酶的变化,观察有无和视黄酸处理时相反的改变。为了证实诱癌是否成     

佛波醇酯诱导HL-60细胞巨噬细胞样分化时蛋白激酶C活力及分布的改变

本文对佛波醇酶诱导人早幼粒白血病细胞系HL-60细胞分化为巨噬细胞样细胞对蛋白激酶c活力及其在亚细胞分布的变化进行了研究。蛋白激酶c活力在TPA处理1小时即明显降低,此低水平的酶活力持续整个实验时期。酶的亚细胞分布研究提示TPA处理细胞胞质组分酶活力剧烈降低,而颗粒组分存在一高盐浓度洗脱的酶活力峰。蛋白激酶c抑制剂三氟过(口了)嗪单独处理HL-60细胞导致胞质和颗粒组分酶活力升高,但并不诱导细胞分化;若与TPA合并处理细胞,酶活力又降低,此时细胞又分化为巨噬细胞样细胞。对上述结果的可能机理进行了讨论。     

视黄酸对人肝癌细胞的逆转作用——生物学研究

 SMMC-7721人肝癌细胞株在10μmol/L的视黄酸(又称维甲酸,Retinoic Acid,RA)处理后,增殖明显受到抑制。RA作用后的细胞~3H-TdR总参入量明显下降,而单个细胞的~3H-TdR参入率却变化不明显。流式细胞仪分析表明该细胞株G_0/G_1、S及G_2+M期的百分率分别为49.6％、16.7％、33.7％,而RA处理3天的细胞则相应地为59.1％、15.4％、25.5％。经RA处理3天后,细胞染色体的主流范围由对照细胞的48—56转变为44—50,其中二倍体细胞数由4％上升至15％。     

视黄酸对人肝癌细胞表面N糖链类型及天线数的影响

本文采用系列凝集素柱层析法,并配合外切糖苷酶处理研究了在视黄酸(RA)作用1—5天过程中人肝癌细胞株SMMC-7721细胞表面N糖链结构的变化。结果表明,RA促进3~H-甘露糖(Man)参入细胞表面N糖链,使高甘露糖型N糖链的百分比下降,复杂型百分比上升,并促进二天线N糖链的生物合成,使多天线特别是四天线和C_2,C_(21)b三天线N糖链的合成减少。结果提示,N糖链结构的这些变化可能是RA诱导SMMC-7721细胞向正常方向分化的结果。     

几种药物对HL-60细胞的诱导分化作用及其过程中蛋白激酶C活力分布的变化

本文就HHT、RA、WB_(852)对HL-60细胞的诱导分化作用及此过程中PKC活力在细胞胞浆部分及膜溶脱部分的变化进行研究。结果表明,在适当的用药浓度下,从细胞生长抑制情况、形态学观察及NBT还原能力测定判断,三种药物对HL-60细胞有明显的诱导分化作用。PKC活力分布变化的研究结果表明,用药组细胞胞浆部分酶活力有不同程度的下降,尤在用药早期(约6h以前)下降显著;而膜部分PKC活力则表现上升、或下降,或活力相差不大的结果。暗示在信息传递过程中起核心作用的PKC对不同的胞外刺激可能采取不同的应答方式。PKC的作用可能主要发生在信息传递的早期。     

几种药物对HL—60细胞的诱导分化作用及其过程中蛋白激酶C活力分…

本文就ＨＨＴ、ＲＡ、ＷＢ８５２对ＨＬ·６０细胞的诱导分化作用及此过程中ＰＫＣ活力在细胞胞浆部分及膜溶脱部分的变化进行研究。结果表明,在适当的用药浓度下,从细胞生长抑制情况,形态学观察及ＮＢＴ还原能力测定判断,三种药物对ＨＬ－６０细胞有明显的诱导分化作用。ＰＫＣ活力分布变化的研究结果表明,用药组细胞胞浆部分酶活力有不同程度的下降,尤在用药早期（约６ｈ以前）下降显著;而膜部分ＰＫＣ活力则表现上升,或下     

维甲酸对人肝癌细胞磷脂酰胆碱专一性磷脂酶D的作用

为了解细胞信号转导与细胞分化间的关系,研究了诱导分化剂全反式视黄酸(ATRA)和13顺视黄酸(13 cis-RA)对7721人肝癌细胞中磷脂酰胆碱专一性磷脂酶D(PC-PLD)的影响。发现ATRA和13 cis-RA除能抑制7721细胞生长,并在形态上向正常方向分化外,分别在第2或第4天使膜结合性PC-PLD的比活力升高,用每瓶细胞的总活力计算,ATRA的作用在第2天也高于13 cis-RA,但13 cis-RA在第4天的效应却高于ATRA,说明13 cis-RA较ATRA的效应滞后。每天更换培养液的条件较不更换时细胞生长速度较快,且更能引起PC-PLD比活力的上升,提示新鲜培养液中可能存在促进生长和增强维甲酸升高PC-PLD的化合物。ATRA在第2天对PC-PLD比活力的作用高于第4天,可能由于第4天细胞增殖加快,细胞中蛋白质含量上升,以至使以蛋白含量计算的酶比活力下降。进一步研究维甲酸使膜结合性PC-PLD升高与蛋白激酶的关系,发现ATRA不论在第2天和第4天均使膜结合性和胞液中蛋白激酶C(PKC)和酪氨酸蛋白激酶(TPK)的比活力下降,说明ATRA使膜结合性PC-PLD活力的升高不是通过PKC 或TPK对PC-PLD的激活作用来实现,其机理有待进一步研究。对维甲酸引起PC-PLD升高的生物学意义作了讨论。     

连香树精油对人肝癌SMMC-7721细胞抗肿瘤机制的研究

为探讨连香树精油的体外抗肿瘤活性。以人肝癌SMMC-7721细胞为受试细胞株,利用MTT法、流式细胞术、JC-1法和Western blot法评价连香树精油的体外抗肿瘤活性。结果表明:连香树精油处理24 h后可降低SMMC-7721细胞存活率;细胞中G0/G1期的比例下降,G2/M期的比例升高;细胞线粒体膜电位降低;自噬相关蛋白LC3-Ⅱ、Beclin-1表达上升,加入自噬抑制剂氯喹后LC3-Ⅱ蛋白与Bcl-2抗凋亡蛋白表达下降。综上说明连香树精油能抑制SMMC-7721细胞的增殖活性,通过阻滞G2/M期抑制细胞分裂,通过降低线粒体膜电位诱导SMMC-7721细胞凋亡,而自噬在SMMC-7721细胞凋亡过程中作为一种保护机制而存在。     

维甲酸与星状孢子素联合分化诱导HL-60细胞过程中酪氨酸蛋白质磷酸化系统的变化

以人早幼粒白血病细胞株（HL-60）为材料,对维甲酸与星状孢子素联合诱导HL-60细胞过程中胞浆和膜部分的蛋白质酪氨酸磷酸化水平变化进行了研究．结果表明,诱导后2～24h范围内,TPK活力出现波动,随时间延长（24～27h）,胞浆部分TPK活力下降,膜溶脱部分TPK活力上升;PTPP活力明显升高且上升幅度比TPK大得多．利用抗P-tyr-BSA抗体分析底物含量变化的结果与相应时相的TPK和PTPP活力变化趋势一致．     

Reduced Protein Kinase C Activity in Ischemic Spinal Cord

Protein phosphorylation was evaluated in a rabbit spinal cord ischemia model under conditions where cyclic AMP-dependent protein kinase (PK-A) and calcium/phospholipid-dependent protein kinase (PK-C) were activated. One hour of ischemia did not affect PK-A activity significantly; however, PK-C activity was reduced by more than 60%. In vitro phosphorylation of endogenous proteins by endogenous PK-C revealed that eight particulate and five cytosolic proteins showed stimulated phosphorylation by PK-C activators in control tissue, although this stimulation was virtually absent in ischemic samples. When control and ischemic particulate fractions were combined, the endogenous protein phosphorylation pattern under PK-C-activating conditions was similar to the ischemic sample, which suggests that inhibitory molecules may be present in the ischemic particulate fraction. In vitro phosphorylation of endogenous proteins under PK-A-activating conditions in ischemic tissue was similar to that in control tissue. The results suggest that the PK-C phosphorylation system is selectively impaired in ischemic spinal cord. In addition to reduced PK-C-dependent phosphorylation, an Mr 64,000 protein was phosphorylated in ischemic cytosolic samples, but not in control samples. The phosphorylation of the Mr 64,000 protein was neither PK-C-dependent nor PK-A-dependent. These altered phosphorylation reactions may play critical roles in neuronal death during the course of ischemia.     

Phosphorylation of Glial Fibrillary Acidic Protein and Vimentin by Cytoskeletal-Associated Intermediate Filament Protein Kinase Activity in Astrocytes

These studies describe a cytoskeletal-associated protein kinase activity in astrocytes that phosphorylated the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin and that appeared to be distinct from protein kinase C (PK-C) and the cyclic AMP-dependent protein kinase (PK-A). The cytoskeletal-associated kinase activity phosphorylated intermediate filament proteins in the presence of 10 mM MgCl2 and produced an even greater increase in 32P incorporation into these proteins in the presence of calcium/calmodulin. Tryptic peptide mapping of phosphorylated intermediate filament proteins showed that the intermediate filament protein kinase activity produced unique phosphopeptide maps, in both the presence and the absence of calcium/calmodulin, as compared to that of PK-C and PK-A, although there were some common sites of phosphorylation among the kinases. In addition, it was determined that the intermediate filament protein kinase activity phosphorylated both serine and threonine residues of the intermediate filament proteins, vimentin and GFAP. However, the relative proportion of serine and threonine residues phosphorylated varied depending on the presence or absence of calcium/calmodulin. The magnesium-dependent activity produced the highest proportion of threonine phosphorylation, suggesting that the calcium/calmodulin-dependent kinase activity acts mainly at serine residues. PK-A and PK-C phosphorylated mainly serine residues. Also, the intermediate filament protein kinase activity phosphorylated both the N-and the C-terminal domains of vimentin and the N-terminal domain of GFAP. In contrast, both PK-C and PK-A are known to phosphorylate the N-terminal domains of both proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phorbol Myristate Acetate and 8-Bromo-Cyclic AMP-Induced Phosphorylation of Glial Fibrillary Acidic Protein and Vimentin in Astrocytes: Comparison of Phosphorylation Sites

Both the protein kinase C (PK-C) activator, phorbol 12-myristate 13-acetate (PMA), and the cyclic AMP-dependent protein kinase (PK-A) activator, 8-bromo-cyclic AMP (8-BR), have been shown to increase 32P incorporation into glial fibrillary acidic protein (GFAP) and vimentin in cultured astrocytes. Also, treatment of astrocytes with PMA or 8-BR results in the morphological transformation of flat, polygonal-shaped cells into stellate, process-bearing cells, suggesting the possibility that signals mediated by these two kinase systems converge at the level of protein phosphorylation to elicit similar changes in cell morphology. Therefore, studies were conducted to determine whether treatment with PMA and 8-BR results in the phosphorylation of the same tryptic peptide fragments on GFAP and vimentin in astrocytes. Treatment with PMA increased 32P incorporation into all the peptide fragments that were phosphorylated by 8-BR on both vimentin and GFAP; however, PMA also stimulated phosphorylation of additional fragments of both proteins. The phosphorylation of vimentin and GFAP resulting from PMA or 8-BR treatment was restricted to serine residues in the N-terminal domain of these proteins. Studies were also conducted to compare the two-dimensional tryptic phosphopeptide maps of GFAP and vimentin from intact cells treated with PMA and 8-BR with those produced when the proteins were phosphorylated with purified PK-C or PK-A. PK-C phosphorylated the same fragments of GFAP and vimentin that were phosphorylated by PMA treatment. Additionally, PK-C phosphorylated some tryptic peptide fragments of these proteins that were not observed with PMA treatment in intact cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein kinase C and casein kinase II activities in two human colon carcinoma cell lines,HT-29 and CaCo-2: Possible correlation with differentiation

Protein kinase C (PK-C) and casein kinase II (CK-II) activities were studied in two human colon carcinoma cell lines (HT-29 and CaCO-2) undergoing differentiationin vitro resulting, in small-intestine-like cells. CaCo-2 cells, when grown under standard conditions, appear to undergo spontaneous differentiation. In these cells PK-C and CK-II activities were determined on day 5, 10 and 15. No significant differences in activities were seen either in PK-C or CK-II activity. HT-29 cells, when grown in glucose-free medium can be stimulated to undergo differentiation which is completed within 20 days. PK-C and CK-II activities were determined after 5, 10, 15, 20 and 25 days, respectively. PK-C activity rose from 7.9±3.5 pmole³²P/mg protein/min at day 5 to 37.5±14.8 pmole³²P/mg protein/min at day 20. After 25 days the activity was reduced to 20.0±7.8 pmole³²P/mg protein/min. CK-II activity did not change significantly during day 5 to 20, but on day 25 there was a significant decrease in CK-II activity from 94.9±6.4 pmole³²P/mg protein/min (day 20) to 62.6±3.9 pmole³²P/mg protein/min (day 25) p=0.003. The results in this study indicate a role for PK-C and CK-II in cell growth and differentiation.     

Activation by phorbol esters of protein kinase C in MCF-7 human breast cancer cells

Exposure of MCF-7 human breast cancer cells to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) leads to the inhibition of cell proliferation. We investigate here the short-term effects of TPA on subcellular distribution of protein kinase C, and on protein phosphorylation in cultured MCF-7 cells. We report a rapid and dramatic decrease in cytosolic protein kinase C activity after TPA treatment. Only 30% of the enzymatic activity lost in the cytosol was recovered in the particulate fraction. These data suggest that subcellular translocation of protein kinase C is accompanied by a rapid down-regulation of the enzyme (70%). Furthermore, TPA and other protein kinase C activators rapidly induce the phosphorylation of a 28 kDa protein in intact MCF-7 cells. Phorbol esters devoid of tumor-promoting activity are ineffective both for inducing these early biochemical events and for inhibiting cell proliferation.     

Effects of growth and differentiation inducing factors on protein kinase-C of cultured intestinal crypt cells

To assess the role of protein kinase-C (PK-C) in the growth and differentiation of small intestinal enterocytes, IEC-6 cells (a cell line derived from the crypts of rat small intestine) were incubated with factors known to induce growth (insulin, epidermal growth factor, gastrin, somatostatin and transferrin) or differentiation (transforming growth factor-beta, retinoic acid and phorbol 12-myristate 13-acetate (PMA)). Cell proliferation (3H-thymidine incorporation) and PK-C activity (Ca++/phospholipid dependent) were measured. Among growth promoting factors only epidermal growth factor, insulin and transferrin were associated with increased 3H-thymidine incorporation, and none of these agents induced PK-C activation as measured by its translocation from cytosol to membrane fraction. Of the differentiation inducing factors, only PMA translocated PK-C from cytosol to membrane. PMA also inhibited 3H-thymidine incorporation in a dose dependent manner. These results suggest that growth and proliferation of enterocytes occur independent of PK-C signal transduction.     

Effects of protein kinase inhibitors on pig oocyte maturation in vitro.

Normal oocyte maturation depends on signal transmission between granulosa cells and the oocyte. We have analysed the effects of inhibiting (I) cyclic AMP-dependent protein kinase (protein kinase A, PK-A), (II) Ca2+/phospholipid-dependent protein kinase (protein kinase C, PK-C) and (III) calmodulin (CaM) on pig oocyte maturation in vitro, protein synthesis and phosphorylation. The inhibition of PK-A using a specific inhibitor H8, decreased the maturation rate (rate of germinal vesicle breakdown, GVBD) of cumulus-enclosed pig oocytes in a dose-dependent manner by approximately 12%, reaching a plateau at 100 microM. The inhibition of PK-C with H7, an inhibitor with some side-effects on PK-A, decreased the maturation rate of cumulus-enclosed oocytes in a dose-dependent manner to a maximum of 20% at a concentration of 100 microM. The calmodulin antagonist W7 up to a concentration of 200 microM had no effects on maturation of cumulus-enclosed pig oocytes. None of the inhibitors (H7, H8 and W7) altered the patterns of protein synthesis of either pig oocytes and cumulus cells after maturation in vitro. Oocyte phosphoprotein patterns were, however, clearly changed by W7. Cumulus cell protein phosphorylation patterns were changed by all 3 agents. Since inhibition of cyclic AMP and Ca2+ phospholipid pathways by PK-A and PK-C blocking chemicals affected only a limited proportion of oocytes (12 and 20%, respectively) and inhibition of Ca2+ binding to CaM was without effect on oocyte maturation, we conclude that these pathways modulate rather than regulate oocyte maturation in the pig.     

Constitutively active PKB/Akt inhibited apoptosis and down-regulated beta1,4-galactosyltransferase 1 in hepatocarcinoma cells

Beta1,4-galactosyltransferase1 (beta1,4GT1) is localized both in the Golgi complex and on the cell surface. In our previous study, we first reported that beta1,4GT1 was associated with cycloheximide-induced apoptosis in human hepatocarcinoma cells. In this study, we transfected constitutively active protein kinase B (Gag-PKB), a central mediator of anti-apoptotic signals transduced by the PI3-kinase, into SMMC-7721 human hepatocarcinoma cells, and examined its effect on apoptosis and beta1,4GT1 activity. Flow cytometry analysis showed that apoptosis was inhibited in Gag-PKB transfected SMMC-7721 cells. At the same time, beta1,4GT1 mRNA level and enzyme activities were downregulated in these cells, consistent with which, the content of beta1,4 Gal branch in the glycoconjugates was decreased in stably transfected cells. Cotransfection of beta1,4GT1 promoter/luciferase reporter and Gag-PKB decreased the luciferase reporter activity in a dose-dependent manner, indicating that the differences in mRNA levels might be regulated through promoter function. All these findings suggested that changes of beta1,4GT1 activity might be involved in apoptotic pathway in hepatocarcinoma cells.     

HMBA诱导人肝癌SMMC-7721细胞分化的观察

本文研究HMBA对人肝癌SMMC-7721细胞的诱导分化作用.细胞生长曲线测定和细胞分裂指数观察显示HMBA可明显抑制细胞增殖,细胞生长抑制率达64.14%,分裂指数抑制率为53.88%.光镜和透射电镜观察可见HMBA能诱导人肝癌SMMG-7721细胞形态和超微结构发生恢复性改变.生化检测或免疫细胞化学方法观察显示,HM-BA处理后细胞γ.谷氨酸转肽酶(γ-GT)活性和甲胎蛋白(AFP)、增殖细胞核抗原(PCNA)表达均降低,而酪氨酸.酮戊二酸转氨酶(TAT)活性增强.流式细胞仪分析表明HMBA引起细胞发生G0/G1期阻滞.以上结果表明HMBA能有效抑制人肝癌细胞恶性增殖活性,逆转肝癌细胞恶性形态与超微结构特征,改变肝癌细胞相关酶活性和抗原表达,引发G0/G1期阻滞,从而对肝癌细胞具有明显的诱导分化作用.