db-cAMP对转化细胞钙调素基因表达与细胞骨架的影响

转化的C_3H_(10)T_(1/2)细胞表现增殖速度加快、表面微绒毛增加,细胞变圆,叠层生长,ConA受体呈帽状分布,微管、微丝、纤粘蛋白分布明显减少。与增殖有关的癌基因c-fos表达增强,同时发现与细胞增殖、转化和细胞骨架调节有关的钙调素(CaM)基因表达加强。用1mmo/Ldb-cAMP处理转化细胞,观察到CaM基因和原癌基因c-fos的表达分别在处理后1小时和2小时急剧下降。处理后4—5天,转化细胞表型趋正常化,大部分细胞恢复单层生长。细胞表面微绒毛和泡状物减少,ConA受体帽状分布消失,恢复分散分布在细胞膜上的特点。细胞生长明显被抑制,用优先在G_1期表达的4F_1 cDNA为探针进行分子杂交,证实了经db-cAMP处理后的细胞被阻抑在G_1期。经db-cAMP处理6天的转化细胞中微管、微丝、纤粘蛋白基本恢复正常分布。实验表明CaM的表达增强与转化细胞表型变化和细胞骨架组装减弱密切相关,db-cAMP作用后CaM表达下降是抑制转化细胞增殖并使细胞表型和细胞骨架分布趋于正常的关键事件之一。     

Nuclear control of neurite induction in neuroblastoma cells

Induction of neurite formation in neuroblastoma cells by dibutyryl cyclic 3':5'-AMP (db-cAMP) or prostaglandin EI (PGE1) was enhanced after enucleation. Cells selected for resistance to db-cAMP were induced to form neurites by db-cAMP or PGE1 only after, but not before enucleation. Inhibition of protein synthesis inhibited neurite induction in nucleated, but not in enucleated cells, and enucleated cells were less sensitive to inhibition of neurite formation by concanavalin A (ConA). Colchicine, vinblastine and cytochalasin B (CB), compounds that interfere with the assembly of microtubules and microfilaments, inhibited induction in both types of cells. It is suggested that enucleation removes a nuclear inhibitor of neurite induction by db-cAMP and PGE1, and that neurite induction in nucleated cells requires that cAMP activates the assembly of microtubules and microfilaments and inactivates the nuclear inhibitor.     

Up-regulation of c-fos expression is a component of the mIg signal transduction mechanism but is not indicative of competence for proliferation

Control of entry into and progression through the early phases of cell cycle in B lymphocytes is poorly understood at the molecular level. Products of the c-fos proto-oncogene have been implicated in regulation of G0 to G1 cell cycle phase transition and cell proliferation in other systems. In view of these observations, the relationship between signals generated through receptor Ig which alter the B cells position in cell cycle and relative level of c-fos expression was investigated. Not unexpectantly, anti-Ig under conditions which promote G0-G1 and G1-S phase transition was observed to selectively up-regulate expression of c-fos. More interestingly, however, anti-Ig-induced cross-linking of surface Ig on the WEHI-231 B lymphoma also caused rapid and transient up-regulation of c-fos mRNA levels although it was associated with inhibition of proliferation of these cells. These results are important because they show that 1) c-fos expression is inducible in both normal and transformed B lymphocytes as a consequence of signals generated through receptor Ig, and 2) up-regulation of c-fos expression is not positively linked to B cell proliferation but rather appears to be a component of the surface Ig signal transduction mechanism. Finally, studies utilizing phorbol diesters suggest that pathways leading through protein kinase C are involved in both the growth inhibition and c-fos expression WEHI-231 following membrane-associated Ig cross-linking.     

Induction of reverse transformation and normal cell cycle regulation by dibutyryl cAMP in a chemically transformed cell line

The objective of this study was to determine whether N6, O2-dibutyryl 3',5'-adenosine monophosphate (db-cAMP)-induced reverse transformation in a chemically transformed mouse cell line, AKR-MCA, would restore normal cell cycle regulation, particularly with regard to their growth arrest in the early G1 period. The AKR-MCA cells were grown to confluency in the presence or absence of db-cAMP (0.5 mM) plus theophylline (1 mM). The confluent cultures were trypsinized and a portion of the cells were fused with mitotic HeLa cells to induce premature chromosome condensation, while the remaining cells were used to study the kinetics of initiation of DNA synthesis. The prematurely condensed chromosomes (PCC) of the control and the treated cultures were classified into G1, S, or G2 types on the basis of their morphology. The G1 PCC were further subclassified into six groups (+1 - +6); +1 being the most condensed and +6 the most decondensed. The cyclic AMP (cAMP)-treated cells exhibited better attachment to the culture dish, were blocked in early G1 period at confluency, and entered S phase about 4 h later than the control following subculturing. In contrast, a majority of cells in the control cultures were arrested in S phase at confluency. These data indicate that the db-cAMP-induced reverse transformation in AKR-MCA cells at least partially restores normal cell cycle regulation in these chemically transformed cells.     

Cell cycle dependent growth factor regulation of gene expression

The expression of the proto-oncogenes c-fos and c-myc is a rapid response of G0-arrested fibroblasts to serum and peptide growth factors; however, the role of the c-fos and c-myc gene products in subsequent cell cycle transit is not understood. We examined the expression of c-fos and c-myc mRNA in Balb/c 3T3 murine fibroblasts in response to platelet-derived growth factor (PDGF) and platelet-poor plasma, using arrest points associated with density dependent growth inhibition or metabolic inhibition to synchronize cells in S phase of the cell cycle. The expression of c-fos and c-myc mRNA in Balb/c 3T3 cells was differentially regulated with respect to growth factor dependence and cell cycle dependence. c-fos expression was induced in the presence of PDGF and was unaffected by plasma. The induction of c-fos expression in response to PDGF was cell cycle independent, occurring in cells transiting S phase and G2 as well as in G0 arrest. In contrast, c-myc expression was both growth factor and cell cycle dependent. In G0 arrested cells, c-myc expression was PDGF-dependent and plasma-independent, and PDGF was required for maintenance of elevated c-myc levels during G1 transit. In cells transiting S phase, c-myc mRNA was induced in response to PDGF, but was also plasma-dependent in S phase cells that had been "primed" by exposure to PDGF during S phase.     

Expression of c-fos oncogene during hepatocarcinogenesis, liver regeneration and in synchronized HTC cells

We have studied the expression of c-fos gene in rat hepatoma induced by DENA. An increase of c-fos mRNA concentration was observed after 8 days, but the maximal 5- to 6-fold increase was observed after 70 weeks. This increase was found in perinodular hepatocytes as well as in cancer nodules. c-fos expression was also enhanced during liver regeneration at a period corresponding to cell proliferation. In HTC cells the arrest of the cell cycle at early G1 phase by addition of sodium butyrate was accompanied by a strong increase of c-fos gene expression. However the c-fos mRNA rapidly decreased after removal of sodium butyrate during the progression of the cells in the cell cycle and increased transiently when the cells entered again in G1 phase.     

Modulation of serum-stimulated DNA synthesis in cultured human fibroblasts by cAMP

Density-dependent inhibition of growth of cultured human fibroblasts was associated with a 3- to 4-fold rise in the intracellular concentration of cyclic AMP (cAMP). Serum lowered cAMP levels in 2–5 min, with the low levels persisting for several hours. When quiescent fibroblast cultures were treated with 10% serum, the incorporation of [³H]TdR into DNA increased after a 10–16 h lag, reaching a peak by 20–24 h. Dibutyryl cyclic AMP (db-cAMP), when present throughout serum treatment, produced a dose-dependent inhibition of [³H]TdR incorporation. Half-maximal inhibition was seen with 0.1 mM db-cAMP. When db-cAMP or another cyclic nucleotide phosphodiesterase inhibitor, l-methyl-3-isobutylxanthine (SC-2964), was added together with serum to maintain elevated cAMP levels and after 4 h was replaced with fresh serum-containing medium, the wave of DNA synthesis induced by serum was not delayed. This implied that stimulation by serum could occur without an initial decrease in cAMP concentration. In contrast, db-cAMP added 8 h later than serum and not removed, inhibited [³H]TdR incorporation at the peak to the same extent as db-cAMP added together with serum. The inhibition decreased progressively when db-cAMP was added more than 8 h after serum. These results suggested that a cAMP-sensitive step occurred approx. 8 h after the addition of serum in mid-G1 of the cell cycle. Results obtained using fibroblasts synchronized at the G1/S boundary with hydroxyurea or exposed to db-cAMP for 24 h suggested that db-cAMP also inhibited TdR incorporation at the G1/S interphase or during S phase. Thus, whereas reduced cAMP concentrations did not appear to serve as an initial trigger for serum-stimulated DNA synthesis in human fibroblasts, db-cAMP and SC-2964, presumably by elevating cAMP levels, appeared to act in mid-G1 and possibly at the G1/S boundary or within S phase to inhibit thymidine incorporation.     

cAMP对转化细胞中几种基因表达及CREB DNA结合活性的影响

 从癌基因、抑癌基因及转录因子 CREB(c AMP反应序列结合蛋白 )对 CRE DNA序列结合活性的相关性 ,对 db- c AMP处理的小鼠 C3H10 T1 /2转化细胞增殖抑制作用进行了研究 .实验结果表明 ,转化细胞中 PKA(蛋白激酶 A)活性显著低于正常细胞 ,而 PKC(蛋白激酶 C)活性则显著高于正常细胞 .斑点印迹和 Northern印迹分析显示转化细胞中 c- myc和 Ca M(钙调素 )基因表达明显高于正常细胞 ,而 p53基因和 Rb基因表达则明显低于正常细胞 ,这些差别与 C3H10 T1/ 2 转化细胞增殖失控有关 .转化细胞经 db- c AMP(1 mmol/L)处理后 ,细胞增殖受到明显抑制 ,db- c AMP处理0 .5h后 ,转化细胞中 PKA活性便明显增强 ,PKC活性则被显著抑制 ,处理 2 h后 ,c- myc和 Ca M基因表达下降 ,而 p53和 Rb基因表达则增强 ,这些变化与 c AMP抑制 C3H10 T1/ 2 转化细胞增殖有密切联系 .凝胶阻滞电泳分析显示 db- c AMP(1 mmol/L )处理短时间内 ,CREB对 CRE DNA序列无结合活性 ,1 2 h后开始出现较弱的结合活性 ,2 4 h后才明显加强 ,表明在 db- c AMP处理的早期 ,调控区中含有 CRE序列的基因不参与 db- c AMP对细胞增殖抑制的调节 ,即与 CREB磷酸化及其相应的 DNA结合活性无相关性 .     

细胞松驰素B促微丝解聚对DNA合成的作用

利用微丝（ｍｉｃｒｏｆｉｌａｍｅｎｔ,ＭＦ）解聚药物细胞松驰素Ｂ（ｃｙｔｏｃｈａｌａｓｉｎＢ,ＣＢ）处理Ｇ_０期小鼠Ｃ_３Ｈ_（１０）Ｔ_（１／２）成纤维细胞,对Ｇ_０至Ｓ期ＤＮＡ合成,胸腺嘧啶核苷激酶（ｔｈｙｍｉｄｉｎｅｋｉｎａｓｅ,ＴＫ）活性、ＴＫ基因表达、钙调素（ｃａｌｍｏｄｕｌｉｎ,ＣａＭ）水平和一些细胞周期早期基因的表达进行了观察,Ｇ_０期细胞经３ｍｇ／ＬＣＢ处理２ｈ,促ＭＦ解聚增强了血清对Ｓ期细胞ＴＫ活性、ＴＫ基因表达和ＤＮＡ合成的刺激作用,并促进细胞提前进入Ｓ期．血清刺激Ｇ_０期细胞进入晚Ｇ_１期和Ｓ期时,ＣａＭ水平明显升高,而ＣＢ预处理则使ＣａＭ含量进一步增加,特别是ＣＢ处理促使Ｓ期ＣａＭ增加向核内转移．ＣＢ处理明显增强血清对ｃ－ｊｕｎ、ｃ－ｆｏｓ和ｃ－ｍｙｃ基因表达的刺激作用,而ＰＫＣ抑制剂Ｈ_７则抑制ＣＢ处理对这些基因转录的刺激作用,说明ＣＢ使Ｇ_０期细胞ＭＦ解聚刺激ｃ－ｊｕｎ、ｃ－ｆｏｓ和ｃ－ｍｙｃ的转录活性与ＰＫＣ的作用有关．结果表明Ｇ_０至Ｓ期早期ＭＦ的重组可促进细胞进入Ｓ期,增强ＤＮＡ合成．     

丁酸对体外培养的人鼻咽癌上皮细胞的影响

无论是自发的、病毒引起的或致癌物诱发的恶性转化的哺乳类细胞的体外培养,其形态多发生改变,总是变得近似圆形,边缘突起短而少,细胞致密和折光性强,同时失去生长接触抑制,降低细胞与细胞之间和细胞与生长底物之间的粘着性等特性。近年报道了关于短链脂肪酸如丁酸(或丁酸钠)对细胞能产生明显的影响,能抑制培养细胞的分裂,可诱发一些上皮性细胞产生形态的改变,可使转化的细胞