Cell cycle-dependent inhibition of DNA synthesis by prostaglandin I2 in cultured rabbit aortic smooth muscle cells

The role of prostaglandin I2 (PGI2) in the control of DNA synthesis during the cell cycle was investigated in cultured rabbit aortic smooth muscle cells (SMC). SMC at confluency in the G0 state reached the S phase about 16 h after stimulation with serum, as judged by measurement of [3H]thymidine incorporation into DNA (DNA synthesis). Cyclooxygenase inhibitors such as indomethacin and aspirin enhanced DNA synthesis, suggesting that endogenously synthesized prostaglandins inhibit DNA synthesis. Added PGE1 or PGE2 had little effect on DNA synthesis. PGI2 inhibited DNA synthesis only when added from 10 to 16 h after stimulation of SMC in the G0 state with serum. Addition of CS-570, a stable PGI2 analogue, inhibited DNA synthesis at any time after serum stimulation. The endogenous syntheses of PGI2 and DNA were negatively correlated. These results suggest that PGI2 inhibits DNA synthesis by acting on the progression stage of the G1 state.     

Induction of DNA synthesis in BALB/c 3T3 cells by serum components: Reevaluation of the commitment process

Serum contains a growth factor derived from platelets and also growth factors derived from platelet-poor plasma. Extracts of heated (100 degrees ) human platelets function synergistically with platelet-poor plasma to induce DNA synthesis in quiescent, density-inhibited BALB/c 3T3 cells. Platelet-poor plasma alone did not induce DNA synthesis. Cells exposed to platelet extracts became competent to enter the cell cycle, but the rate of entry into the S phase depended upon the concentration of platelet-poor plasma. The time required for the induction of this competent state was a function of the concentration of the platelet extract. A 2-hr exposure to 100 mug of the platelet extract at 37 degrees caused the entire cell population to become competent to enter the S phase. At 4 degrees or 25 degrees the cells did not become competent to synthesize DNA. The platelet extract-induced competent state was stable for at least 13 hr after removal of the platelet extract; however, in the absence of platelet-poor plasma, these competent cells did not progress through the cell cycle. The addition of an optimal concentration of platelet-poor plasma (5%) to these competent cells initiated cell cycle traverse with a rapid, first-order entry of cells into the S phase beginning 12 hr after addition of the plasma. The addition of a suboptimal concentration of the plasma (0.25%) did not increase the rate of cell entry into the S phase. Thus, the induction of DNA synthesis in quiescent BALB/c 3T3 cells can be resolved into at least two phases, controlled by different serum components: (i) competence, induced by the platelet-derived growth factor; and (ii) progression of competent cells into the cell cycle, mediated by factors in platelet-poor plasma.     

Heparin regulates smooth muscle S phase entry in the injured rat carotid artery

Smooth muscle cell (SMC) proliferation in injured arteries is inhibited by heparin, but the mechanism of inhibition is unknown. In particular, it is not clear whether heparin prevents exit of quiescent SMC from the resting state, inhibits progression through the prereplicative (G1) sequence, or acts during DNA synthesis itself. In this study, induction of ornithine decarboxylase (ODC) activity was used as a marker of SMC entry into the cell cycle in an attempt to localize the site of heparin action during the initial hours after rat carotid injury. Rapid and transient induction of ODC activity was observed that reached a maximum (twenty-three-fold) 6 hours after wounding. Heparin failed to prevent ODC induction but greatly reduced frequencies of [3H]thymidine-labelled SMC nuclei 33 hours after injury. Moreover, heparin infusion could be delayed for up to 18 hours after the injury event with no significant loss of antiproliferative effect. Further delays resulted in marked loss of growth inhibition. The results of these studies show that SMC rapidly and synchronously leave the resting state after injury and suggest that heparin acts late in the prereplicative (G1) sequence or early in S phase to inhibit SMC proliferation in damaged arteries.     

Initial amplification of duck hepatitis B virus covalently closed circular DNA after in vitro infection of embryonic duck hepatocytes is increased by cell cycle progression.

The relationship between the cell cycle and early amplification of duck hepatitis B virus covalently closed circular (CCC) DNA was studied after in vitro infection of fetal hepatocytes. We first showed that embryonic hepatocytes proliferated for at least 6 days after plating and that complete viral replication including CCC DNA amplification occurred in these proliferating cells. Addition of sodium butyrate or aphidicolin reversibly blocked cells in the G1 phase and diminished CCC DNA synthesis, which was restored after drug withdrawal, concomitantly with the entry of cells into S phase. Cell cycle progression of fetal hepatocytes can be triggered by stimulation with epidermal growth factor (EGF), hepatocyte growth factor (HGF), and tumor growth factor alpha (TGF-alpha). CCC DNA synthesis increased with progression to the S phase induced by EGF, HGF, and TGF-alpha alone or in combination. By contrast, tumor growth factor beta (TGF-beta) alone or in combination with EGF inhibited cell proliferation and viral DNA synthesis. By double labeling, viral nucleocapsids were found predominantly in bromodeoxyuridine-positive hepatocytes, indicating that high viral replication occurs preferentially in proliferating hepatocytes. CCC DNA was also detected mainly in cells in the S and G2/M phases separated from cells in the G1 phase by cell sorting. Taken together, these results show that hepatocyte proliferation may positively regulate the initial amplification of CCC DNA of avian hepadnaviruses, and may explain why mitosis is not necessarily associated with loss of CCC DNA.     

Regulation of proliferation by vasopressin in aortic smooth muscle cells: function of protein kinase C.

AIM: To investigate the effect of arginine vasopressin-stimulated prostaglandin synthesis and the activation of protein kinase C on DNA synthesis in rat aortic smooth muscle cells. METHODS: The effects of arginine vasopressin on the release of arachidonic acid and the synthesis of prostaglandin (PG) E2 and prostacyclin (PGI2) were determined. The effects of 12-o-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C-activating phorbol ester, and of 1-oleoyl-2-acetylglycerol, a specific activator of protein kinase C, were evaluated in cultured rat aortic smooth muscle cells. The effects of arginine vasopressin and prostaglandins on the progression from the late G1 to the S phase of the cell cycle were evaluated by measuring the DNA synthesis, and the effects of TPA on them were evaluated. RESULTS: Arginine vasopressin dose-dependently stimulated arachidonic acid release. TPA and 1-oleoyl-2-acetylglycerol dose-dependently increased the vasopressin-induced arachidonic acid release. Vasopressin stimulated the synthesis of both PGE2 and PGI2. TPA increased the vasopressin-stimulated prostaglandin synthesis as well as the arachidonic acid release. Vasopressin, added at the G0/G1 phase of the cell cycle, stimulated DNA synthesis of aortic smooth muscle cells. Exogenous PGE2 and PGI2 inhibited the DNA synthesis and showed maximum inhibition when added at the late G1 phase. TPA alone, added at the late G1 phase, reduced the DNA synthesis stimulated by vasopressin at the G0/G1 phase to about 45%, but vasopressin alone, added at the late G1 phase, had little effect. However, with TPA pretreatment, vasopressin significantly suppressed the DNA synthesis by about 70%. Staurosporine, a protein kinase C inhibitor, reduced the suppression by TPA alone or by vasopressin with TPA pretreatment almost to the control level. Indomethacin, a cyclo-oxygenase inhibitor, reduced the suppression by vasopressin with TPA pretreatment almost to the level of TPA alone. CONCLUSIONS: These results suggest that arginine vasopressin has a suppressive effect on DNA synthesis in rat aortic smooth muscle cells by inhibiting progression from the late G1 into the S phase of the cell cycle through the synthesis of PGE2 and PGI2, and that protein kinase C acts as an amplifier of this mechanism.     

反义ras寡聚脱氧核苷酸对血管平滑肌细胞生物学行为的影响

为了研究调控小分子Ｇ蛋白（ｒａｓ蛋白）以抑制血管平滑肌细胞对生长因子的反应机制，用合成的反义ｒａｓ寡聚脱氧核苷酸作用碱性纤维母细胞生长因子以刺激培养的大鼠血管平滑肌细胞，通过氚标胸腺嘧啶脱氧核苷酸掺入法及免疫组织化学方法，来观察反义ｒａｓ寡聚脱氧核苷酸对在碱性纤维母细胞生长因子刺激下的大鼠血管平滑肌细胞ＤＮＡ合成和增殖细胞核抗原的影响。结果表明：反义ｒａｓ寡聚脱氧核苷酸可明显抑制血管平滑肌细胞ＤＮ     

Overexpression of p21Waf1 induces apoptosis in immortalized human vascular smooth muscle cells

To understand the role of the cell cycle regulatory protein in the control of smooth muscle cell (SMC) proliferation, we tested the overexpression of p21Waf1, a cyclin-dependent kinase inhibitor, in human normal (MS9) and immortalized SMCs (ISS10) transfected with ori-minus simian virus 40 DNA, using an adenovirus-mediated system. In MS9, overexpression of p21Waf1 resulted in the inhibition of cell cycle progression at the G1/S boundary without apoptosis. On the other hand, in ISS10, overexpression of p21Waf1 induced marked apoptosis. In these cells, immunohistochemistry revealed that overexpressed p21Waf1 was localized in the nucleus. No differential expression pattern of either p53 or SV40T was observed in p21Waf1- and control gene (beta-galactosidase)-infected cells. Old-passaged ISS10 cells eventually showed growth arrest and a senescent-like phenotype. Immunohistochemistry revealed that p21Waf1 was localized in the cytoplasm of the early-passaged cells, but was found in the nucleus of the old-passaged cells. Our data suggested that nuclear accumulation of p21Waf1 plays a role in the cell death of immortalized SMC, which carries dysfunction of the cell cycle regulatory proteins such as p53. This culture model may be useful for studying the process of SMC proliferation, cell death, senescence, and cell cycle regulation.     

反义ras寡聚脱氧核苷酸对血管平滑肌细胞生物学行为的影响

为了研究调控小分子G蛋白（ras蛋白）以抑制血管平滑肌细胞对生长因子的反应机制，用合成的反斗ras寡聚脱氧核苷酸作用于碱性纤维母细胞生长因子以刺激培养的大鼠血管平滑肌细胞，通过鼠标胸腺嘧啶脱氧核苷酸掺入法及免疫细胞化学方法，来观察反斗ras聚脱氧核苷酸对在碱性纤维母细胞生长因子刺激下的大鼠血管平滑肌细胞DNA合成和增殖细胞核抗原的影响。结果发现，反义ras寡策脱氧核苷酸可明显抑制血管平滑肌细胞DNA的合成，作用12h和24h的抑制率分别为92％和90％；而相同浓度的正义链组仅呈现较弱的抑制作用（P＜0．01）．反义ras寡聚脱氧核苷酸亦可显著减少血管平滑肌细胞增殖细胞核抗原蛋白的表达．提示该策略的应用有可能解决以生长因子为始因引起的细胞增殖性疾病、经文腔内冠状动脉成形术后再狭窄、动脉粥样硬化及高血压等问题的临床防治．     

Antiproliferative action of cyclic GMP-elevating vasodilators in cultured rabbit aortic smooth muscle cells

In cultured rabbit aortic smooth muscle cells (SMCs), sodium nitroprusside (SNP) (10(-7) to 10(-4) M), atrial natriuretic peptide (ANP) (10(-9) to 10(-6) M) and 8-bromo-cyclic GMP (10(-6) to 10(-3) M) inhibited the whole blood serum (WBS)-induced DNA synthesis by about 30%. The doses of SNP and ANP necessary for the inhibition of the WBS-induced DNA synthesis were similar to those necessary for the formation of cellular cyclic GMP (cGMP). These agents were effective even when added 6 h after stimulation of the cells with WBS. These results suggest that cGMP inhibits the proliferation of rabbit aortic SMCs by inhibiting the progression from the G1 into S phase of the cell cycle and raise the possibility that cGMP-elevating vasodilators may suppress the atherogenic process by inhibiting vascular SMC proliferation.     

肺表面活性物质对哮喘T细胞亚群细胞周期及其调节蛋白的影响

目的 观察肺表面活性物质(PS)对发作期哮喘患者T细胞亚群细胞周期及其调节蛋白的影响,揭示PS对CD4+、CD8+T细胞增殖周期的具体作用机制及其在哮喘治疗中的意义.方法 利用细胞培养技术,通过体外PS干预,流式细胞仪测定经PS干预和未经PS干预的CD4+、CD8+T细胞的细胞周期分布和CD4+、CD8+T细胞内细胞周期调节蛋白p27kipl、cyclinE、cyclinA、cyclinB的表达水平.结果 哮喘患者CD4+、CD8+T细胞分别与PS体外共同培养后,加PS组的S期、G2/M期的CD4+T比例显著低于对照组(P＜0.01);G0/G1期的CD4+T细胞比例显著高于对照组(P＜0.01).加PS组的S期CD8+T比例显著低于对照组(P＜0.01);G2期的CD8+T细胞比例略低于对照组,但差异无统计学意义(P＞0.05);G0/G1期的CD8+T细胞比例高于对照组(P＜0.05).CD4+T细胞内p27kipl的表达水平高于对照组(P＜0.05);CD4+T细胞内cyclinE表达水平显著低于对照组(P＜0.01);CD4+T细胞内cyclinA、cyclinB的表达水平低于对照组(P＜0.05).CD8+T细胞内p27kipl的表达水平高于对照组(P＜0.05);CD8+T细胞内cyclinE、cyclinA表达水平低于对照组(P＜0.01);CD8T细胞内cyclinB的表达水平与对照组差异无统计学意义(P＞0.05).结论 PS通过抑制CD4+T细胞的正性细胞周期调节蛋白cyclinE、cyclinA、cyclinB的表达和上调其负性细胞周期调节蛋白p27kipl的表达,抑制CD4+T细胞的DNA合成和细胞分裂,进而抑制CD4+T细胞增殖;通过下调CD8+T细胞内cyclinE、cyclinA的表达,上调p27kippl的表达,抑制CD8+T细胞DNA合成.

Abstract：

Objective The aim of this study was to observe the effect of pulmonary surfactant (PS)on the cell cycle distribution and expression of cell cycle regulatory proteins in T lymphocyte subsets in patients with asthma attack and evaluate the molecular regulatory mechanism of PS on T lymphocyte subsets cell proliferation cycle during asthma attack and its therapy significance. Methods The CD4+、CD8+ T lymphocytes obtained from peripheral blood of 30 patients with asthma attack were incubated with PS,the cell cycle and the expression of p27kipl , CyclinE, CyclinA, CyclinB in CD4+ , CD8+ T lymphocytes with PS and without PS were anasyzed by flow cytometry. Results CD4 + T lymphocytes with PS progressed into S phase and G2/M phase were significantly lower than those without PS( P ＜0.01). But remained in G0/G1 phase were significantly higher than those without PS (P ＜ 0.01). CD8+ T lymphocytes with PS progressed into S phase were significantly lower than those without PS( P ＜0.01),G2/M phase were little lower than those without PS( P ＞ 0.05), Go/G1 phase were higher than those without PS( P ＜0.05). The expression of p27kipl in CD4+T lymphocytes with PS was higher than that without PS( P ＜0.05). The expression of cyclinE in CD4+ T lymphocytes with PS was significantly lower than that without PS( P ＜0.01 ). The expression of cyclinA,cyclinB in CD4+ T lymphocytes with PS were lower than those without PS( P ＜0.05). The expression of p27kipl in CD8+ T lymphocytes with PS was higher than that without PS( P ＜0.05). The expression of cyclinE and cyclinA in CD8+ T lymphocytes with PS were significantly lower than that without PS( P ＜0. 01). The expression of cyclinB in CD8+ T lymphocytes with PS was little lower than that without PS( P ＞0.05). Conclusions During asthma attack,PS can inhibit the DNA synthesis, cell division and proliferation of CD4+T lymphocytes by inhibiting positive cell cycle regulatory proteins cyclinE, cyclinA, cyclinB expression and increasing negative cell cycle regulatory protein p27kipl expression in CD4+ T lymphocytes. PS can inhibit the DNA synthesis of CD8+ T lymphocytes by inhibiting positive cell cycle regulatory proteins cyclinE, cyclinA,expression and increasing negative cell cycle regulatory protein p27kipl expression in CD8+ T lymphocytes.     

G1 cyclin-dependent activation of p34CDC28 (Cdc28p) in vitro.

In Saccharomyces cerevisiae, transient accumulation of G1 cyclin/p34CDC28 (Cdc28p) complexes induces cells to traverse the cell cycle Start checkpoint and commit to a round of cell division. To investigate posttranslational controls that modulate Cdc28p activity during the G1 phase, we have reconstituted cyclin-dependent activation of Cdc28p in a cyclin-depleted G1 extract. A glutathione S-transferase-G1 cyclin chimera (GST-Cln2p) efficiently binds to and activates Cdc28p as a histone H1 kinase. Activation of Cdc28p by GST-Cln2p requires ATP, crude yeast cytosol, and the conserved Thr-169 residue that serves in other organisms as a substrate for phosphorylation by cyclin-dependent protein kinase-activating kinase. This assay may be useful for distinguishing genes that promote directly the posttranslational assembly of active Cln2p/Cdc28p kinase complexes from those that stimulate the accumulation of active complexes via a positive-feedback loop that governs synthesis of G1 cyclins.     

野生型p^53基因导入对培养的兔血管平滑肌细胞生长的抑制作用

为探索腺病毒载体重组野生型ｐ５３基因转染平滑肌细胞的效率及其应用于动脉粥样硬化和动脉再狭窄的基因治疗的可能性，构建了野生型ｐ５３基因的生长缺陷型重组腺病毒载体。在体外培养条件下转染兔血管平滑肌细胞。应用生化染色法、免疫组化法及聚合酶链技术等检测了外源基因的转染效率及表达效果。应用细胞计数及同位素参入技术测定了ｐ５３基因对平滑肌细胞的抑制效果。流式细胞仪检测了平滑肌细胞。结果显示：腺病毒载体可快速有效地将外源基因转导进入平滑肌细胞，在转染细胞内可检测到外源性ｐ５３ｃＤＮＡ，并可高效表达ｐ５３蛋白。ｐ５３重组腺病毒载体转染细胞后，可显著抑制细胞生长，细胞计数减少，ＤＮＡ合成减少，并可导致细胞死亡。流式细胞仪检测有部分细胞体积缩小，ＤＮＡ减少。结果提示，野生型ｐ５３基因重组腺病毒载体转染平滑肌细胞可有效抑制细胞生长，有可能成为动脉粥样硬化和再狭窄的基因治疗手段。     

XMCM7, a novel member of the Xenopus MCM family, interacts with XMCM3 and colocalizes with it throughout replication.

A minichromosome maintenance (MCM) protein complex has been implicated in restricting DNA replication to once per cell cycle in Xenopus egg extracts, based on the behavior of a single protein, XMCM3. Using a two-hybrid screen with XMCM3, we have identified a novel member of the MCM family in Xenopus that is essential for DNA replication. The protein shows strong homology to Saccharomyces cerevisiae MCM7 (CDC47) and has thus been named XMCM7. XMCM7 is present in a multiprotein complex with other MCM proteins. It binds to chromatin and is displaced from chromatin by the act of replication. XMCM7 does not preferentially colocalize with sites of DNA replication but colocalizes with XMCM3 throughout replication. Immunodepletion of the MCM complex from Xenopus egg extract by anti-XMCM7 antibodies inhibits DNA replication of sperm and permeable HeLa G2 nuclei but not permeable HeLa G1 nuclei. Replication capacity of the Xenopus egg extract immunodepleted of the MCM complex by anti-XMCM7 antibody can be rescued by MCM proteins eluted from anti-XMCM3 antibody. We conclude that both proteins are present in the same complex in Xenopus egg extract throughout the cell cycle, that they remain together after binding to chromatin and during DNA replication, and that they perform similar functions.     

Cloned T-cell proliferation and synthesis of specific proteins are inhibited by quinine.

Recombinant human interleukin 2 (rIL-2) drives the proliferation of the cloned murine T-helper line L2. The initial G1 activation occurs during the first 20 hr after stimulation, with DNA synthesis (S phase) beginning approximately 20 hr after rIL-2 stimulation. Three patterns of protein synthesis were observed during G1 activation. Type I proteins (e.g., p72 and p66) were synthesized at near maximal rates as early as 4 hr after stimulation, with little change in rates of synthesis through the G1 to S phase transition. Type II proteins (e.g., p52 and p36) were detectable early after stimulation, but their rates of synthesis continued to increase throughout G1 activation, becoming maximal 24-28 hr after stimulation. Type III proteins (e.g., p93, p89, and p63) were synthesized maximally 4 or 8 hr after rIL-2 stimulation, then their rates of synthesis declined markedly to prestimulation levels. Type II proteins, p52 and p36, were shown to be correlated with cell proliferation, since their rates of synthesis were maximal while L2 cells were proliferating and declined as the cells returned to a quiescent state. The potassium channel blocker quinine inhibited cell growth and the synthesis of p52 and p36 when added 0 or 2 hr after rIL-2 stimulation but not when added 6 hr after rIL-2 stimulation. Thus, a quinine-sensitive event occurring in L2 cells between 2 and 6 hr after rIL-2 stimulation is necessary for synthesis of type II proteins, DNA synthesis, and cell proliferation.     

肺表面活性物质对哮喘T细胞亚群细胞周期及其调节蛋白的影响

目的观察肺表面活性物质（PS）对发作期哮喘患者T细胞亚群细胞周期及其调节蛋白的影响,揭示PS对CD4＋、CD8＋T细胞增殖周期的具体作用机制及其在哮喘治疗中的意义。方法利用细胞培养技术,通过体外PS干预,流式细胞仪测定经PS干预和未经PS干预的CD4＋、CD8＋T细胞的细胞周期分布和CD4＋、CD8＋T细胞内细胞周期调节蛋白p27kip1、cyclinE、cyclinA、cyclinB的表达水平。结果哮喘患者CD4＋、CD8＋T细胞分别与PS体外共同培养后,加PS组的S期、G2/M期的CD4＋T比例显著低于对照组（P〈0.01）;G0/G1期的CD4＋T细胞比例显著高于对照组（P〈0.01）。加PS组的S期CD8＋T比例显著低于对照组（P〈0.01）;G2期的CD8＋T细胞比例略低于对照组,但差异无统计学意义（P〉0.05）;G0/G1期的CD8＋T细胞比例高于对照组（P〈0.05）。CD4＋T细胞内p27kip1的表达水平高于对照组（P〈0.05）;CD4＋T细胞内cyclinE表达水平显著低于对照组（P〈0.01）;CD4＋T细胞内cyclinA、cyclinB的表达水平低于对照组（P〈0.05）。CD8＋T细胞内p27kip1的表达水平高于对照组（P〈0.05）;CD8＋T细胞内cyclinE、cyclinA表达水平低于对照组（P〈0.01）;CD8＋T细胞内cyclinB的表达水平与对照组差异无统计学意义（P〉0.05）。结论 PS通过抑制CD4＋T细胞的正性细胞周期调节蛋白cyclinE、cyclinA、cyclinB的表达和上调其负性细胞周期调节蛋白p27kip1的表达,抑制CD4＋T细胞的DNA合成和细胞分裂,进而抑制CD4＋T细胞增殖;通过下调CD8＋T细胞内cyclinE、cyclinA的表达,上调p27kip1的表达,抑制CD8＋T细胞DNA合成。