缺氧/氧再灌注诱导心肌细胞衰老的机制及其干预研究
|
摘要
研究背景
衰老是细胞脱离细胞周期并不可逆地丧失增殖能力后进入一种相对稳定的状态。自Hayflick等首次从人类纤维原细胞发现衰老现象后，人们逐渐发现人类其它各类细胞和其它物种的纤维细胞也存在着衰老。细胞发生衰老以后主要表现在细胞周期的改变、β-半乳糖苷酶活性的变化、线立体的皱缩与衰老基因的表达增加等。细胞衰老可以促进生物衰老，不仅与肿瘤发生有关，而且与心力衰竭、动脉粥样硬化、心律失常等心血管疾病发生有着密切的关系。因此，深入研究衰老的机制有着重要意义。
缺氧／氧再灌注是细胞、组织与器官最常见的损伤因素，也是诱导细胞发生衰老的常见方法。最近国外有研究显示缺氧与氧再灌可以使骨髓造血细胞发生衰老。
他汀类药物通过抑制3-羟基-3-甲基戊二酰辅酶的活性而抑制胆固醇的合成，常用来治疗高胆固醇血症，他汀类药物还可降低冠心病患者的死亡率、提高心脏移植患者的生存率、改善心肌重构等作用。除了调节胆固醇代谢外，最近Assmus等报道他汀类药物可通过上调细胞周期蛋白，下调细胞周期抑制因子p27~(Kipl)，抑制内皮祖细胞的衰老。Pravastatin除了心血管保护作用给患者带来的益处外，是否同时具有抑制缺氧／氧再灌注诱导心肌细胞衰老，对心脏产生保护作用呢?
在本研究中，我们将了解缺氧／氧再灌注是否能诱导培养SD乳大鼠心肌细胞发生衰老；研究缺氧／氧再灌注诱导培养SD乳大鼠心肌细胞发生衰老的机制；并拟采用pravastatin对缺氧／氧再灌注诱导SD乳大鼠心肌细胞的衰老进行干预，观察细胞周期与β-半乳糖苷酶活性等经典的细胞衰老指标的变化，以探讨pravastatin是否对心肌细胞衰老有抑制作用及可能机制。
第一部分SD乳大鼠心肌细胞的培养、鉴定与缺氧／氧再灌注诱导建立SD乳大鼠心肌细胞衰老模型
目的
在成功分离、培养SD乳大鼠心肌细胞的基础上，探讨获得较纯心肌细胞的方法以及采用缺氧／氧再灌注诱导建立SD乳大鼠心肌细胞衰老模型的可行性。
方法
采用胰蛋白酶消化心肌组织分离单个心肌细胞，联合差速贴壁与培养基中加入BrdU杀死心肌成纤维细胞。心肌细胞在含1％O_2与99％N_2的孵育箱中缺氧培养6h，在21％O_2与5％CO_2孵育箱中进行氧再灌注培养24和48h，建立心肌细胞衰老模型。采用免疫组化检测培养细胞α-actin表达情况；透射电镜观察心肌细胞超微结构改变；BrdU掺入试验了解心肌细胞增殖情况；流式细胞仪检测细胞周期；β-半乳糖苷酶试剂盒检测β-半乳糖苷酶活性。
结果
经α-actin抗体鉴定显示分离培养的细胞中95％以上是心肌细胞。心肌细胞经缺氧／氧再灌注诱导后，细胞体积变大、线立体明显皱缩；细胞增殖结果显示缺氧／氧再灌注组心肌细胞BrdU阳性率较对照组有显著下降(P＜0.01)；细胞周期检测结果显示缺氧／氧再灌注组心肌细胞G0／G1期细胞较对照组有显著增多(P＜0.01)，S期细胞较对照组有显著减少(P＜0.01)；β-半乳糖苷酶活性检测结果显示氧再灌注组心肌细胞β-半乳糖苷酶活性较对照组与缺氧6 h有显著提高(P＜0.01)。
结论
1．采用胰蛋白酶消化心肌组织分离心肌细胞，联合差速贴壁与培养基中加入BrdU杀死心肌成纤维细胞，可以获得较纯的心肌细胞，经α-actin抗体鉴定显示分离培养的细胞中95％以上是心肌细胞。
2．我们首次采用缺氧／氧再灌注，成功诱导建立了SD乳大鼠心肌细胞衰老模型，为心肌细胞衰老机制的研究提供了稳定可靠的模型。
第二部分缺氧／氧再灌注诱导SD乳大鼠心肌细胞衰老的机制研究
目的
从细胞周期调控方面探讨缺氧／氧再灌注诱导SD乳大鼠心肌细胞衰老的分子机制
方法
采用real-time quantitative PCR与western blot方法检测培养SD乳大鼠心肌细胞在对照组、缺氧6h、氧再灌注24和48h，心肌细胞周期相关蛋白(D1)，细胞周期依赖激酶(CDK4)，细胞周期蛋白和细胞周期依赖激酶复合物抑制剂(p21WAF1，p16IN4Ka)，Rb蛋白，p53等基因的mRNA与蛋白水平的表达变化。
结果
(1) Cyclin D1与β-actin基因mRNA水平的比值在缺氧6h、氧再灌注24和48h分别为0.024，0.082，0.092，与对照组0.005相比有显著增高(P＜0.05或P＜0.01)。Cyclin D1与β-actin基因蛋白水平比值在缺氧6h、氧再灌注24和48h分别为0.735，0.993，1.181，与对照组0.437相比升高明显(P＜0.05或P＜0.01)。
(2) CDK4与β-actin基因mRNA水平比值在缺氧6h、氧再灌注24和48h，分别为0.008，0.005，0.005与对照组0.011相比有显著降低(P＜0.05或P＜0.01)。CDK4与β-actin基因蛋白水平比值在缺氧6h、氧再灌注24和48h，分别为0.854，0.432，0.171与对照组1.102相比有明显减少(P＜0.05或P＜0.01)。p21WAF1是通过降低CDK4的活性导致细胞分裂停止，p21WAF1与β-actin基因mRNA水平比值在缺氧6h、氧再灌注24和48h，分别为0.009，0.009，0.012与对照组0.006相比有显著增高(P＜0.05或P＜0.01)。p21WAF1与β-actin基因蛋白水平比值在缺氧6h、氧再灌注24和48h，分别为0.831，1.978，1.736与对照组0.456相比升高明显(P＜0.05或P＜0.01)。p53是通过其靶蛋白p21WAF1起作用与p21WAF1的变化相平行。p53与β-actin基因mRNA水平比值在缺氧6h、氧再灌注24和48h，分别为0.002，0.003，0.004与对照组0.001相比有显著增高(P＜0.05或P＜0.01)。p53与β-actin基因蛋白水平比值在缺氧6h、氧再灌注24和48h，分别为0.678，0.978，1.236与对照组0.213相比升高明显(P＜0.01)。
(3) Rb与β-actin基因mRNA水平比值在缺氧6h、氧再灌注24和48h，分别为0.003，0.004，0.003，与对照组0.002相比有显著增高(P＜0.05)。但pRb与β-actin基因蛋白水平比值在缺氧6h、氧再灌注24和48h，分别为0.926，1.231，0.351，与对照组2.502相比有显著降低(P＜0.01)，p16IN4Ka能阻止CDK4／6与cyclinD1的结合对Rb的磷酸化。p16IN4Ka与β-actin基因mRNA水平比值在缺氧6h、氧再灌注24和48h，分别为0.007，0.022，0.022与对照组0.001相比有显著增高(P＜0.05或P＜0.01)。p16IN4Ka与β-actin基因蛋白水平比值在缺氧6h、氧再灌注24和48h，分别为0.078，0.082，0.109与对照组0.001相比升高明显(P＜0.01)。
结论
缺氧／氧再灌注通过调节细胞周期相关蛋白(cyclin D1、CDK4、p16IN4Ka、p21WAF1、p53等)而抑制心肌细胞增殖，促进细胞衰老。
第三部分Pravastatin对缺氧／氧再灌注诱导SD乳大鼠心肌细胞衰老的干预研究
目的
了解pravastatin对缺氧／氧再灌注诱导SD乳大鼠心肌细胞衰老是否有抑制作用及其机制。
方法
Pravastatin干预浓度为10~(-7)-10~(-5)mol／l，方法参考第一部分方法。
结果
Pravastatin干预后对照组、缺氧6h、氧再灌注24和48h的G0／G1期细胞分别为71.98％，86.47％，84.98％，88.78％较实验组72.71％，87.45％，87.97％，89.78％没有明显减少(P＞0.05)，同样对照组、缺氧6h、氧再灌注24和48h的S期细胞分别为20.34％，9.02％，6.06％，5.79％较实验组20.03％，9.01％，5.06％，4.93％没有明显增多(P＞0.05)。Pravastatin干预后，对照组、缺氧6h、氧再灌注24和48h的半乳糖苷酶活性阳性细胞分别为9.13％，12.55％，42.15％，53.48％较实验组8.56％，12.37％，42.34％，58.28％没有明显减少(P＞0.05)，不同浓度pravastatin的干预，结果相似。
结论
Pravastatin不能抑制缺氧／氧再灌注诱导SD乳大鼠心肌细胞衰老。
BACKGROUND
Cellular senescence has been defined by Hayflick as the ultimate andirreversible loss of replicative capacity occurring in primary somatic cellculture. After Hayflick identifying senescence in human fibroblast firstly,cellular senescence was identified in diverse type cells in human orfibroblasts in other species. Different markers of the senescent phenotypeas, for instance, G0/G1 specificity of cell cycle arrest,senescence-associatedβ-galactosidase activity, mitochondrialdehydration, or senescence-associated gene expression. Cellularsenescence may not only result in the aging of organism, but induceoccurrence of tumors, heart failure, atherosclerosis or arrhythmia.Therefor, it is very important to study the mechanism of cellularsenescence.
Hypoxia/reoxygenation (H/R) was the most common injury factorfor cell, tissue or organism. It was also an effective way to induce cellularsenescence. Recetenly, a report has shown that premature senescencecould be induced by H/R in FA bone marrow hematopoietic cells.
HMG-CoA reductase inhibitors, i.e. statins, were reversibleinhibitors of the rate-limiting step in cholesterol biosynthesis and weregenerally used for the treatment of hypercholesterolemia. Statins also could reduce mortality for patients with coronary heart disease, increasesurvival for patients with heart transplantation, and improve ventricularremodeling. Besides regulating lipid metabolism, recently, Assmusreported that atorvastatin could reduce senescence and increaseproliferation of endothelial progenitor cells via regulation of modulationexpression of cell cycle genes including upregulation of cyclins anddownregulation of the cell cycle inhibitor p27Kipl. Apart from statinsplay very important role in protection for patients with cardiovasculardiseases, whether pravastatin could inhibit premature senescence ofneonatal SD rat cardiomyocytes induced by H/R or not?
In this study, we would investigate whether premature senescencecould be induced by H/R in neonatal SD rat cardiomyocytes. To explorecellular and molecular mechanisms of premature senescence in neonatalrat cardiomyocytes exposed to H/R. To investigate whether pravastatincould inhibit premature senescence of neonatal SD rat cardiomyocytesinduced by H/R and assosiated mechanisms.
PartⅠIsolation, culture, purification and identification ofneonatal cardiomyocytes from SD rat heart. Establishmentthe premature senescence model of cardiomyocytes inducedby H/R
AIM
On the base of succeeding in isolation, culture neonatalcardiomyocytes from SD rat heart, to explore how to get morecardiomyocytes. To investigate whether premature senescence could beinduced by H/R in neonatal SD rat cardiomyocytes.
METHODS
Trypsin was used to cut pieces of heart tissue into single cells.Combination diferent time for cells to stain wall and mediumsupplemented with BrdU to remove cardiac fibroblasts.
Cardiomyocytes were isolated from neonatal SD rat heart andidentified by a-actin immunohistochemistry. The control cultures wereincubated at 37℃in humidified atmosphere of 5％CO_2, 95％air. Thehypoxic cultures were (within a modular incubator chamber filled with 1％O2, 5％CO2, and balance N2) for 6 h. The reoxygenated cultures weresubjected to 1％O2, 5％CO2 for 6 h then 21％oxygen for 24 and 48 h,respectively. Cell proliferation was determined using BrdU labelling.Ultrastructure of cardiomyocytes was observed by using electronmicroscope. Flow cytometry were used to investigate alteration of cellcycle.β-galactosidase activity was determined by using Senescenceβ-galactosidase Staining Kit.
RESULTS
Most cells (＞95%) in the isolated cultures were positive forα-actin antibody. The percentage of BrdU positive cells reduced significantly inH/R treated group (P＜0.01). Under the condition of H/R, mitochondrialdehydration appeared. Most cardiomyocytes resided in G0 and G1 phase inthe group of hypoxia 6 h, reoxygenation 24 and 48 h compared withcontrol one (P＜0.01). Cardiomyocytes did not stain forβ-galactosidasein the group of control, hypoxia 6 h, but did intensely for it inreoxygenation group of 24,48 h, compared with control one (P＜0.01).
CONCLUSIONS
1. Combination diferent time for cells to stain wall and mediumssupplemented with BrdU to remove cardiac fibroblasts are a good wayto purify cardiomyocytes.
2. Premature senescence could be induced in neonatal SD ratcardiomyocytes exposed to H/R.
PartⅡA study on mechanism of premature senescence ofcardiomyocytes induced by H/R
AIM
To explore cellular and molecular mechanisms of prematuresenescence in neonatal rat cardiomyocytes exposed to H/R.
METHODS
Real-time quantitative PCR and western blot were used to analyzemRNA and protein level of cyclin D1, cyclin dependent kinases (CDK4),inhibitors of the cyclin-CDK complexes (p16IN4Ka, p21WAF1), pRb,p53 in cardiomyocytes of neonatal SD rat in the group of control, hypoxia6h, reoxygenation 24 and 48 h.
RESULTS
Cyclin D1 mRNA and protein levels significantly increased in H/Rtreated group compared with control one (P＜0.05 or P＜0.01,respectively).
CDK4 mRNA and protein levels significantly decreased in H/Rtreated group compared with control one (P＜0.05 or P＜0.01,respectively). p21WAF1 inhibited cell cycle through reducing expressionof CDK4. p21WAF1 mRNA and protein levels increased significantly inH/R treated group compared with control one (P＜0.05 or P＜0.01,respectively). Paralleled to the change of p21WAF1, the mRNA andprotein levels of p53 were rosen significantly in H/R treated groupcompared with control one (P＜0.05 or P＜0.01, respectively).
Rb mRNA level increased significantly in H/R treated groupcompared with control one (P＜0.05, respectively). But pRb leveldecreased significantly in H/R treated group compared with control one(P＜0.01, respectively), p16IN4Ka mRNA and protein levels, which prevented CDK4/6 to catalyze phosphorylation of Rb, were rosensignificantly in H/R treated group compared with control one (P＜0.01,respectively).
CONCLUSIOS
H/R inhibited cell proliferation and accelerated prematuresenescence through regulating protein related to cell cycle (cyclin D1,CDK4, p16IN4Ka, p21WAF1, p53).
PartⅢA study about the effect of pravastatin onpremature senescence of neonatal SD rat cardiomyocytesinduced by H/R
AIM
To investigate whether pravastatin could inhibit prematuresenescence of neonatal SD rat cardiomyocytes induced by H/R.
METHODS
Intervention concentration of pravastatin was 10~(-7)-10~(-5) Mol/L, othermethods were as same as partⅠ
RESULTS
Compared with cells resided in G0/G1 phase in experimental group (72.71％, 87.45％, 87.97％, 89.78％in control group, hypoxia 6 h,reoxygenation 24 h and 48 h, respectively), pravastatin could not reversecells resided in G0/G1 phase (71.98％, 86.47％, 84.98％, 88.78％incontrol group, hypoxia 6 h, reoxygenation 24 h and 48 h, respectively) (P＞0.05). Pravastatin also could not reverse cells disappeared in S phase (P＞0.05). Compared with the percentage ofβ-galactosidase positivestaining cells in experimental group (9.13％, 12.55％, 42.15％, 53.48％in control group, hypoxia 6h, reoxygenation 24h and 48h, respectively)pravastatin could not reduceβ-galactosidase activity, the percentage ofβ-galactosidase positive staining cells is 8.56％, 12.37％, 42.34％,58.28％, in control group, hypoxia 6 h, reoxygenation 24 h and 48 h,respectively (P＞0.05), different intervetion concertration of pravastatin,with similar outcome.
CONCLUSIONS
Pravastatin could not inhibit premature senescence of neonatal SDrat cardiomyocytes induced by H/R.
衰老是细胞脱离细胞周期并不可逆地丧失增殖能力后进入一种相对稳定的状态。自Hayflick等首次从人类纤维原细胞发现衰老现象后，人们逐渐发现人类其它各类细胞和其它物种的纤维细胞也存在着衰老。细胞发生衰老以后主要表现在细胞周期的改变、β-半乳糖苷酶活性的变化、线立体的皱缩与衰老基因的表达增加等。细胞衰老可以促进生物衰老，不仅与肿瘤发生有关，而且与心力衰竭、动脉粥样硬化、心律失常等心血管疾病发生有着密切的关系。因此，深入研究衰老的机制有着重要意义。
缺氧／氧再灌注是细胞、组织与器官最常见的损伤因素，也是诱导细胞发生衰老的常见方法。最近国外有研究显示缺氧与氧再灌可以使骨髓造血细胞发生衰老。
他汀类药物通过抑制3-羟基-3-甲基戊二酰辅酶的活性而抑制胆固醇的合成，常用来治疗高胆固醇血症，他汀类药物还可降低冠心病患者的死亡率、提高心脏移植患者的生存率、改善心肌重构等作用。除了调节胆固醇代谢外，最近Assmus等报道他汀类药物可通过上调细胞周期蛋白，下调细胞周期抑制因子p27~(Kipl)，抑制内皮祖细胞的衰老。Pravastatin除了心血管保护作用给患者带来的益处外，是否同时具有抑制缺氧／氧再灌注诱导心肌细胞衰老，对心脏产生保护作用呢?
在本研究中，我们将了解缺氧／氧再灌注是否能诱导培养SD乳大鼠心肌细胞发生衰老；研究缺氧／氧再灌注诱导培养SD乳大鼠心肌细胞发生衰老的机制；并拟采用pravastatin对缺氧／氧再灌注诱导SD乳大鼠心肌细胞的衰老进行干预，观察细胞周期与β-半乳糖苷酶活性等经典的细胞衰老指标的变化，以探讨pravastatin是否对心肌细胞衰老有抑制作用及可能机制。
第一部分SD乳大鼠心肌细胞的培养、鉴定与缺氧／氧再灌注诱导建立SD乳大鼠心肌细胞衰老模型
目的
在成功分离、培养SD乳大鼠心肌细胞的基础上，探讨获得较纯心肌细胞的方法以及采用缺氧／氧再灌注诱导建立SD乳大鼠心肌细胞衰老模型的可行性。
方法
采用胰蛋白酶消化心肌组织分离单个心肌细胞，联合差速贴壁与培养基中加入BrdU杀死心肌成纤维细胞。心肌细胞在含1％O_2与99％N_2的孵育箱中缺氧培养6h，在21％O_2与5％CO_2孵育箱中进行氧再灌注培养24和48h，建立心肌细胞衰老模型。采用免疫组化检测培养细胞α-actin表达情况；透射电镜观察心肌细胞超微结构改变；BrdU掺入试验了解心肌细胞增殖情况；流式细胞仪检测细胞周期；β-半乳糖苷酶试剂盒检测β-半乳糖苷酶活性。
结果
经α-actin抗体鉴定显示分离培养的细胞中95％以上是心肌细胞。心肌细胞经缺氧／氧再灌注诱导后，细胞体积变大、线立体明显皱缩；细胞增殖结果显示缺氧／氧再灌注组心肌细胞BrdU阳性率较对照组有显著下降(P＜0.01)；细胞周期检测结果显示缺氧／氧再灌注组心肌细胞G0／G1期细胞较对照组有显著增多(P＜0.01)，S期细胞较对照组有显著减少(P＜0.01)；β-半乳糖苷酶活性检测结果显示氧再灌注组心肌细胞β-半乳糖苷酶活性较对照组与缺氧6 h有显著提高(P＜0.01)。
结论
1．采用胰蛋白酶消化心肌组织分离心肌细胞，联合差速贴壁与培养基中加入BrdU杀死心肌成纤维细胞，可以获得较纯的心肌细胞，经α-actin抗体鉴定显示分离培养的细胞中95％以上是心肌细胞。
2．我们首次采用缺氧／氧再灌注，成功诱导建立了SD乳大鼠心肌细胞衰老模型，为心肌细胞衰老机制的研究提供了稳定可靠的模型。
第二部分缺氧／氧再灌注诱导SD乳大鼠心肌细胞衰老的机制研究
目的
从细胞周期调控方面探讨缺氧／氧再灌注诱导SD乳大鼠心肌细胞衰老的分子机制
方法
采用real-time quantitative PCR与western blot方法检测培养SD乳大鼠心肌细胞在对照组、缺氧6h、氧再灌注24和48h，心肌细胞周期相关蛋白(D1)，细胞周期依赖激酶(CDK4)，细胞周期蛋白和细胞周期依赖激酶复合物抑制剂(p21WAF1，p16IN4Ka)，Rb蛋白，p53等基因的mRNA与蛋白水平的表达变化。
结果
(1) Cyclin D1与β-actin基因mRNA水平的比值在缺氧6h、氧再灌注24和48h分别为0.024，0.082，0.092，与对照组0.005相比有显著增高(P＜0.05或P＜0.01)。Cyclin D1与β-actin基因蛋白水平比值在缺氧6h、氧再灌注24和48h分别为0.735，0.993，1.181，与对照组0.437相比升高明显(P＜0.05或P＜0.01)。
(2) CDK4与β-actin基因mRNA水平比值在缺氧6h、氧再灌注24和48h，分别为0.008，0.005，0.005与对照组0.011相比有显著降低(P＜0.05或P＜0.01)。CDK4与β-actin基因蛋白水平比值在缺氧6h、氧再灌注24和48h，分别为0.854，0.432，0.171与对照组1.102相比有明显减少(P＜0.05或P＜0.01)。p21WAF1是通过降低CDK4的活性导致细胞分裂停止，p21WAF1与β-actin基因mRNA水平比值在缺氧6h、氧再灌注24和48h，分别为0.009，0.009，0.012与对照组0.006相比有显著增高(P＜0.05或P＜0.01)。p21WAF1与β-actin基因蛋白水平比值在缺氧6h、氧再灌注24和48h，分别为0.831，1.978，1.736与对照组0.456相比升高明显(P＜0.05或P＜0.01)。p53是通过其靶蛋白p21WAF1起作用与p21WAF1的变化相平行。p53与β-actin基因mRNA水平比值在缺氧6h、氧再灌注24和48h，分别为0.002，0.003，0.004与对照组0.001相比有显著增高(P＜0.05或P＜0.01)。p53与β-actin基因蛋白水平比值在缺氧6h、氧再灌注24和48h，分别为0.678，0.978，1.236与对照组0.213相比升高明显(P＜0.01)。
(3) Rb与β-actin基因mRNA水平比值在缺氧6h、氧再灌注24和48h，分别为0.003，0.004，0.003，与对照组0.002相比有显著增高(P＜0.05)。但pRb与β-actin基因蛋白水平比值在缺氧6h、氧再灌注24和48h，分别为0.926，1.231，0.351，与对照组2.502相比有显著降低(P＜0.01)，p16IN4Ka能阻止CDK4／6与cyclinD1的结合对Rb的磷酸化。p16IN4Ka与β-actin基因mRNA水平比值在缺氧6h、氧再灌注24和48h，分别为0.007，0.022，0.022与对照组0.001相比有显著增高(P＜0.05或P＜0.01)。p16IN4Ka与β-actin基因蛋白水平比值在缺氧6h、氧再灌注24和48h，分别为0.078，0.082，0.109与对照组0.001相比升高明显(P＜0.01)。
结论
缺氧／氧再灌注通过调节细胞周期相关蛋白(cyclin D1、CDK4、p16IN4Ka、p21WAF1、p53等)而抑制心肌细胞增殖，促进细胞衰老。
第三部分Pravastatin对缺氧／氧再灌注诱导SD乳大鼠心肌细胞衰老的干预研究
目的
了解pravastatin对缺氧／氧再灌注诱导SD乳大鼠心肌细胞衰老是否有抑制作用及其机制。
方法
Pravastatin干预浓度为10~(-7)-10~(-5)mol／l，方法参考第一部分方法。
结果
Pravastatin干预后对照组、缺氧6h、氧再灌注24和48h的G0／G1期细胞分别为71.98％，86.47％，84.98％，88.78％较实验组72.71％，87.45％，87.97％，89.78％没有明显减少(P＞0.05)，同样对照组、缺氧6h、氧再灌注24和48h的S期细胞分别为20.34％，9.02％，6.06％，5.79％较实验组20.03％，9.01％，5.06％，4.93％没有明显增多(P＞0.05)。Pravastatin干预后，对照组、缺氧6h、氧再灌注24和48h的半乳糖苷酶活性阳性细胞分别为9.13％，12.55％，42.15％，53.48％较实验组8.56％，12.37％，42.34％，58.28％没有明显减少(P＞0.05)，不同浓度pravastatin的干预，结果相似。
结论
Pravastatin不能抑制缺氧／氧再灌注诱导SD乳大鼠心肌细胞衰老。
BACKGROUND
Cellular senescence has been defined by Hayflick as the ultimate andirreversible loss of replicative capacity occurring in primary somatic cellculture. After Hayflick identifying senescence in human fibroblast firstly,cellular senescence was identified in diverse type cells in human orfibroblasts in other species. Different markers of the senescent phenotypeas, for instance, G0/G1 specificity of cell cycle arrest,senescence-associatedβ-galactosidase activity, mitochondrialdehydration, or senescence-associated gene expression. Cellularsenescence may not only result in the aging of organism, but induceoccurrence of tumors, heart failure, atherosclerosis or arrhythmia.Therefor, it is very important to study the mechanism of cellularsenescence.
Hypoxia/reoxygenation (H/R) was the most common injury factorfor cell, tissue or organism. It was also an effective way to induce cellularsenescence. Recetenly, a report has shown that premature senescencecould be induced by H/R in FA bone marrow hematopoietic cells.
HMG-CoA reductase inhibitors, i.e. statins, were reversibleinhibitors of the rate-limiting step in cholesterol biosynthesis and weregenerally used for the treatment of hypercholesterolemia. Statins also could reduce mortality for patients with coronary heart disease, increasesurvival for patients with heart transplantation, and improve ventricularremodeling. Besides regulating lipid metabolism, recently, Assmusreported that atorvastatin could reduce senescence and increaseproliferation of endothelial progenitor cells via regulation of modulationexpression of cell cycle genes including upregulation of cyclins anddownregulation of the cell cycle inhibitor p27Kipl. Apart from statinsplay very important role in protection for patients with cardiovasculardiseases, whether pravastatin could inhibit premature senescence ofneonatal SD rat cardiomyocytes induced by H/R or not?
In this study, we would investigate whether premature senescencecould be induced by H/R in neonatal SD rat cardiomyocytes. To explorecellular and molecular mechanisms of premature senescence in neonatalrat cardiomyocytes exposed to H/R. To investigate whether pravastatincould inhibit premature senescence of neonatal SD rat cardiomyocytesinduced by H/R and assosiated mechanisms.
PartⅠIsolation, culture, purification and identification ofneonatal cardiomyocytes from SD rat heart. Establishmentthe premature senescence model of cardiomyocytes inducedby H/R
AIM
On the base of succeeding in isolation, culture neonatalcardiomyocytes from SD rat heart, to explore how to get morecardiomyocytes. To investigate whether premature senescence could beinduced by H/R in neonatal SD rat cardiomyocytes.
METHODS
Trypsin was used to cut pieces of heart tissue into single cells.Combination diferent time for cells to stain wall and mediumsupplemented with BrdU to remove cardiac fibroblasts.
Cardiomyocytes were isolated from neonatal SD rat heart andidentified by a-actin immunohistochemistry. The control cultures wereincubated at 37℃in humidified atmosphere of 5％CO_2, 95％air. Thehypoxic cultures were (within a modular incubator chamber filled with 1％O2, 5％CO2, and balance N2) for 6 h. The reoxygenated cultures weresubjected to 1％O2, 5％CO2 for 6 h then 21％oxygen for 24 and 48 h,respectively. Cell proliferation was determined using BrdU labelling.Ultrastructure of cardiomyocytes was observed by using electronmicroscope. Flow cytometry were used to investigate alteration of cellcycle.β-galactosidase activity was determined by using Senescenceβ-galactosidase Staining Kit.
RESULTS
Most cells (＞95%) in the isolated cultures were positive forα-actin antibody. The percentage of BrdU positive cells reduced significantly inH/R treated group (P＜0.01). Under the condition of H/R, mitochondrialdehydration appeared. Most cardiomyocytes resided in G0 and G1 phase inthe group of hypoxia 6 h, reoxygenation 24 and 48 h compared withcontrol one (P＜0.01). Cardiomyocytes did not stain forβ-galactosidasein the group of control, hypoxia 6 h, but did intensely for it inreoxygenation group of 24,48 h, compared with control one (P＜0.01).
CONCLUSIONS
1. Combination diferent time for cells to stain wall and mediumssupplemented with BrdU to remove cardiac fibroblasts are a good wayto purify cardiomyocytes.
2. Premature senescence could be induced in neonatal SD ratcardiomyocytes exposed to H/R.
PartⅡA study on mechanism of premature senescence ofcardiomyocytes induced by H/R
AIM
To explore cellular and molecular mechanisms of prematuresenescence in neonatal rat cardiomyocytes exposed to H/R.
METHODS
Real-time quantitative PCR and western blot were used to analyzemRNA and protein level of cyclin D1, cyclin dependent kinases (CDK4),inhibitors of the cyclin-CDK complexes (p16IN4Ka, p21WAF1), pRb,p53 in cardiomyocytes of neonatal SD rat in the group of control, hypoxia6h, reoxygenation 24 and 48 h.
RESULTS
Cyclin D1 mRNA and protein levels significantly increased in H/Rtreated group compared with control one (P＜0.05 or P＜0.01,respectively).
CDK4 mRNA and protein levels significantly decreased in H/Rtreated group compared with control one (P＜0.05 or P＜0.01,respectively). p21WAF1 inhibited cell cycle through reducing expressionof CDK4. p21WAF1 mRNA and protein levels increased significantly inH/R treated group compared with control one (P＜0.05 or P＜0.01,respectively). Paralleled to the change of p21WAF1, the mRNA andprotein levels of p53 were rosen significantly in H/R treated groupcompared with control one (P＜0.05 or P＜0.01, respectively).
Rb mRNA level increased significantly in H/R treated groupcompared with control one (P＜0.05, respectively). But pRb leveldecreased significantly in H/R treated group compared with control one(P＜0.01, respectively), p16IN4Ka mRNA and protein levels, which prevented CDK4/6 to catalyze phosphorylation of Rb, were rosensignificantly in H/R treated group compared with control one (P＜0.01,respectively).
CONCLUSIOS
H/R inhibited cell proliferation and accelerated prematuresenescence through regulating protein related to cell cycle (cyclin D1,CDK4, p16IN4Ka, p21WAF1, p53).
PartⅢA study about the effect of pravastatin onpremature senescence of neonatal SD rat cardiomyocytesinduced by H/R
AIM
To investigate whether pravastatin could inhibit prematuresenescence of neonatal SD rat cardiomyocytes induced by H/R.
METHODS
Intervention concentration of pravastatin was 10~(-7)-10~(-5) Mol/L, othermethods were as same as partⅠ
RESULTS
Compared with cells resided in G0/G1 phase in experimental group (72.71％, 87.45％, 87.97％, 89.78％in control group, hypoxia 6 h,reoxygenation 24 h and 48 h, respectively), pravastatin could not reversecells resided in G0/G1 phase (71.98％, 86.47％, 84.98％, 88.78％incontrol group, hypoxia 6 h, reoxygenation 24 h and 48 h, respectively) (P＞0.05). Pravastatin also could not reverse cells disappeared in S phase (P＞0.05). Compared with the percentage ofβ-galactosidase positivestaining cells in experimental group (9.13％, 12.55％, 42.15％, 53.48％in control group, hypoxia 6h, reoxygenation 24h and 48h, respectively)pravastatin could not reduceβ-galactosidase activity, the percentage ofβ-galactosidase positive staining cells is 8.56％, 12.37％, 42.34％,58.28％, in control group, hypoxia 6 h, reoxygenation 24 h and 48 h,respectively (P＞0.05), different intervetion concertration of pravastatin,with similar outcome.
CONCLUSIONS
Pravastatin could not inhibit premature senescence of neonatal SDrat cardiomyocytes induced by H/R.
引文
1. Harary Ⅰ, Farley B. In vitro studies on single beating rat heart cells. Ⅱ. Intercellular communication. Exp Cell Res, 1963; 29: 466-474.
2. Chlopcikova S, Psotova J, Miketova P. Neonatal rat cardiomyocytes--a model for the study of morphological, biochemical and electrophysiological characteristics of the heart. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub, 2001; 145: 49-55.
3. Taylor MR, Slavov D, Ku L, et al. Prevalence of desmin mutations in dilated cardiomyopathy. Circulation, 2007; 115: 1244-1251.
4. Cortassa S, Aon MA, O'Rourke B, et al. A computational model integrating electrophysiology, contraction, and mitochondrial bioenergetics in the ventricular myocyte. Biophys J, 2006; 91: 1564-1589.
5. Li Q, Ren J. Cardiac overexpression of metallothionein attenuates chronic alcohol intake-induced cardiomyocyte contractile dysfunction. Cardiovasc Toxicol, 2006; 6: 173-1782.
6. Burkard N, Rokita AG, Kaufmann SG, et al. Conditional neuronal nitric oxide synthase overexpression impairs myocardial contractility. Circ Res, 2007; 100:e32-44.
7. Demiralay R, Gursan N, Erdem H. The effects of erdosteine, N-acetylcysteine and vitamin E on nicotine-induced apoptosis of cardiac cells. J Appl Toxicol, 2007; 27: 247-254.
8. Sutherland FJ, Hearse DJ. The isolated blood and perfusion fluid perfused heart. Pharmacol Res, 2000; 41: 613-627.
9. Fu M, Wu M, Wang JF, et al. Disruption of the intracellular Ca2+ homeostasis in the cardiac excitation-contraction coupling is a crucial mechanism of arrhythmic toxicity in aconitine-induced cardiomyocytes. Biochem Biophys Res Commun, 2007; 354: 929-936.
10. Gu X, Cheng L, Chueng WL, et al. Neovascularization of ischemic myocardium by newly isolated tannins prevents cardiomyocyte apoptosis and improves cardiac function. Mol Med, 2006; 12: 275-283.
11.Blondel B, Roijem I, Cheneval JP. Heart cells in culture: a simple method for increasing the proportion of myoblasts. Experientia, 1971; 27: 356-358.
12. Hoshida S, Nishida M, Yamashita N, et al. Heme oxygenese-1 expressionand its relation to oxidative stress during primary culture of cardiomyocytes. J Mol Cell Cardiol, 1996; 28: 1845-1855.
13. Hayflick L, Moorhead PS, et al. The serial cultivation of human diploid cell strains. Exp Cell Res, 1961; 25: 585-621.
14. Schmaltz C, Hardenbergh PH, Wells A, et al. Regulation of proliferation-survival decisions during tumor cell hypoxia. Mol Cell Biol, 1998; 18: 2845-2854.
15. Iida T, Mine S, Fujimoto H, et al. Hypoxia-inducible factor-lalpha induces cell cycle arrest of endothelial cells. Genes ells, 2002; 7: 143-149.
16. Gardner LB, Li Q, Park MS, et al. Hypoxia inhibits G1/S transition through regulation of p27 expression. J Biol Chem, 2001; 276: 7919-7926.
17. Vonzglinicki T, Saretzki G, Docke W, et al. Mild hyperoxia shortens telomeres and inhibits proliferation of fibroblasts: a model for senescence? Exp Cell Res, 1995; 220: 186-193.
18. Galaris D, Hoijer B, Rydstrom J. Improved methods for automatic monitoring of contracting heart cells in culture. J Biochem Biophys Methods, 1980; 2: 213-225.
19. Rakhit RD, Mojet MH, Marber MS, et al. Mitochondria as Targets for Nitric Oxide-Induced Protection During Simulated Ischemia and Reoxygenation in Isolated Neonatal Cardiomyocytes. Circulation, 2001; 103:2617-2623.
20. Grynberg A, Athias P, Degois M. Effect of change in growth environment on cultured myocardial cells investigated in a standardized medium. In Vitro Cell Dev Biol, 1986; 22: 44-50.
21. Ichiba T, Matsuda N, Takemoto N, et al. Regulation of intracellular calcium concentrations by calcium and magnesium in cardioplegic solutions protects rat neonatal myocytes from simulated ischemia. J Mol Cell Cardiol, 1998; 30: 1105-1114.
22. Adderley SR, Fitzgerald DJ. Oxidative damage of cardiomyocytes is limited by extracellular regulated kinases 1/2-mediated induction of cyclooxygenase-2. J Biol Chem, 1999; 274: 5038-5046.
23. Simpson P, Savion S. Differentiation of rat myocytes in single cell cultures with and without proliferating nonmyocardial cells. Circ Res, 1982; 50: 101-116.
24. Hoshida S, Nishida M, Yamashita N, et al. Heme oxygenese-1 expression and its relation to oxidative stress during primary culture of cardiomyocytes. J Mol Cell Cardiol, 1996; 28: 1845-1855.
25. Zhang X, Li J, Sejas DP, et al. Hypoxia-reoxygenation induces premature senescence in FA bone marrow hematopoietic cells. Blood, 2005; 106: 75-85.
26. Dimri GP, Lee X, Basile G, et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci USA, 1995; 92: 9363-9367.
27. Hayashi S, Inoue A. Cardiomyocytes re-enter the cell cycle and contribute to heart development after differentiation from cardiac progenitors expressing Isll in chick embryo. Dev Growth Differ, 2007; 49: 229-239.
28. Camelliti P, McCulloch AD, Kohl P. Microstructured cocultures of cardiac myocytes and fibroblasts: a two-dimensional in vitro model of cardiac tissue. Microsc Microanal, 2005; 11: 249-259.
29. Adderley SR, Fitzgerald DJ. Oxidative damage of cardiomyocytes is limited by extracellular regulated kinases 1/2-mediated induction of cyclooxygenase-2. J Biol Chem, 1999; 274: 5038-5046.
30. Marengo FD, Wang S, Wang B, et al. Dependence of cardiac cell Ca2+ permeability on sialic acid-containing sarcolemmal gangliosides. J Mol Cell Cardiol, 1998; 30: 127-137.
31. Matos MJ, Post JA, Roelofsen B, et al. Composition and organization of sarcolemmal fatty acids in cultured neonatal rat cardiomyocytes. Cell Biol Int Rep, 1990; 14: 343-352.
32. Karliner JS, Honbo N, Epstein CJ, et al. Neonatal mouse cardiac myocytes exhibit cardioprotection induced by hypoxic and pharmacologic preconditioning and by transgenic overexpression of human Cu/Zn superoxide dismutase. J Mol Cell Cardiol, 2000; 32: 1779-1786.
33. Lepic E, Burger D, Lu X, et al. Lack of endothelial nitric oxide synthase decreases cardiomyocyte proliferation and delays cardiac maturation. Am J Physiol Cell Physiol, 2006; 291: C1240-1246.
34. Driesen RB, Verheyen FK, Dispersyn GD, et al. Structural adaptation in adult rabbit ventricular myocytes: influence of dynamic physical interaction with fibroblasts. Cell Biochem Biophys, 2006; 44: 119-128.
35. Clark WA, Decker ML, Janes DM, et al. Cell contact as an independent factor moduletating cardiac myocytes hytertrophy and survival in long-term primary culture. J Mol Cell C ardiol, 1998; 30: 139-155.
36. Simpson P, McGrath A, Savion S. Myocyte hypertrophy in neonatal rat heart cultures and its regulation by serum and by catecholamines. Circ Res, 1982; 51: 787-800.
37. Iwaki K, Chi SH, Dillmann WH, et al. Induction of HSP70 in cultured rat neonatal cardiomyocytes by hypoxia and metabolic stress. Circ Res, 1993; 87: 2023-2032.
38. Musters RJ, Post JA, Verkleij AJ. The isolated neonatal rat-cardiomyocyte used in an in vitro model for 'ischemia'. I. A morphological study. Biochim Biophys Acta, 1991; 1091: 270-277.
39. Ollinger K, Brunmark A. Effect of different oxygen pressures and N, N-diphenyl-p-phenylenediamine on adriamycin toxicity to cultured neonatal rat heart myocytes. Biochem Pharmacol, 1994; 48: 1707-1715.
40. Brooks G, Poolman RA, McGill CJ, et al. Expression and activities of cyclins and cyclin-dependent kinases in developing rat ventricular myocytes. J Mol Cell Cardiol, 1997; 29: 2261-2271.
41. Soonpaa MH, Kim KK, Pajak L, et al. Cardiomyocyte DNA synthesis and binucleation during murine development. American Journal of Physiology, 1996; 40: H2183-H2189.
42. Stolzing A, Coleman N, Scutt A. Glucose-induced replicative senescence in mesenchymal stem cells. Rejuvenation Res, 2006; 9: 31-35.
43. Lehmann BD, McCubrey JA, Jefferson HS, et al. A dominant role for p53-dependent cellular senescence in radiosensitization of human prostate cancer cells. Cell Cycle, 2007; 6: 595-605.
44. Hastings R, Qureshi M, Verma R, et al. Telomere attrition and accumulation of senescent cells in cultured human endothelial cells. Cell Prolif, 2004; 37: 317-324.
45. Diez C, Nestler M, Friedrich U, et al. Down-regulation of Akt/PKB in senescent cardiac fibroblasts impairs PDGF-induced cell proliferation. Cardiovasc Res, 2001; 49: 731-740.
46. Allen RG, Tresini M, Keogh BP, et al. Differences in electron transport potential, antioxidant defenses, and oxidant generation in young and senescent fetal lung fibroblasts (WI-38). J Cell Physiol, 1999; 180: 114-122.
47. Von Zglinicki T, Schewe C, et al. Mitochondrial water loss and aging of cells. Cell Biochem Funct, 1995; 13: 181-187.
48. Chen Q, Ames BN. Senescence-like growth arrest induced by hydrogen peroxide in human diploid fibroblast F65 cells. Proc Natl Acad Sci, 1994; 91: 4130-4134.
49. Saretzki G, Feng J, von Zglinicki T, et al. Similar gene expression pattern in senescent and hyperoxic-treated fibroblasts. J Gerontol A Biol Sci Med Sci, 1998; 53: B438-42.
50. Halterman MW, Federoff HJ. HIF-1 alpha and p53 promote hypoxia-induced delayed neuronal death in models of CNS ischemia. Exp Neurol, 1999; 159: 65-72.
51. Von Zglinicki T, Marzabadi MR, Roomans GM. Water and ion distributions in myocytes cultured under oxidative stress mimic changes found in the process of aging. Mech Ageing Dev, 1991; 58: 49-60.
52. Imanishi T, Hano T, Nishio I, et al. Angiotensin II accelerates endothelial progenitor cell senescence through induction of oxidative stress. J Hypertens, 2005; 23: 97-104.
53. Aliouat-Denis CM, Dendouga N, Van den Wyngaert I, et al. p53-Independent Regulation of p21WAFl/Cipl Expression and Senescence by Chk2. Mol Cancer Res, 2005; 3: 627-634.
54. Chai J, Charboneau AL, Betz BL, et al. Loss of the hSNF5 gene concomitantly inactivates p21CIP/WAFl and p16IN4Ka activity associated with replicative senescence in A204 rhabdoid tumor cells. Cancer Res, 2005; 65: 10192-10198.
55. Chen QM. Replicative senescence and oxidantinduced premature senescence: beyond the control of cell cycle checkpoints. Ann N Y AcadSci, 2000; 908:111-125.
56. Katsiki M, Trougakos IP, Chondrogianni N, et al. Alterations of senescence biomarkers in human cells by exposure to CrVI in vivo and in vitro. Exp Gerontol, 2004; 39: 1079-1087.
57. Serrano M, Blasco MA. Putting the stress on senescence. Curr Opin Cell Biol, 2001, 13:748-753.
58.Randle DH, Zindy F, Sherr CJ, et al. ifferential effects of p19(Arf) and p16IN4Ka(Ink4a) loss on senescence of murine bone marrow-derived preB cells and macrophages. Proc Natl Acad Sci, 2001; 98: 9654-9659.
1. Ferrer JL, Dupuy J, Borel F, et al. Structural basis for the modulation of CDK-dependent/independent activity of cyclin D1. Cell Cycle, 2006; 5: 2760-2768.
2. White J, Dalton S. Cell cycle control of embryonic stem cells. Stem Cell Rev, 2005; 1: 131-138.
3. Bockstaele L, Coulonval K, Kooken H, et al. Regulation of CDK4. Cell Div,2006; 1:25.
4. Okamoto K, Kato S, Arima N, et al. Cyclin-dependent kinase inhibitor, p21Wafl, regulates vascular smooth muscle cell hypertrophy. Hypertens Res, 2004; 27: 283-291.
5. Sherr CJ, Roberts JM. CDK inhibitors: positive and negative regulators ofG_1-phase progression. Genes Dev, 1999; 13: 1501-1512.
6. Chai J, Charboneau AL, Betz BL, et al. Loss of the hSNF5 gene concomitantly inactivates p21CIP/WAFl and p16IN4Ka activity associated with replicative senescence in A204 rhabdoid tumor cells. Cancer Res, 2005; 65: 10192-10198.
7. Kramer DL, Chang BD, Chen Y, et al. Polyamine depletion in human melanoma cells leads to G1 arrest associated with induction of p21WAFl/CIPl/SDIl, changes in the expression of p21WAF1-regulated genes, and a senescence-like phenotype. Cancer Res, 2001; 61: 7754-7762.
8. Palmero I, McConnell B, Parry D, et al. Accumulation of p16IN4Ka in mouse fibroblasts as a function of replicative senescence and not of retinoblastoma gene status. Oncogene, 1997; 15: 495-503.
9. Chen J, Huang X, Halicka D, et al. Contribution of p16IN4Kaand p21WAF1 pathways to induction of premature senescence of human endothelial cells: permissive role of p53. Am J Physiol Heart Circ Physiol, 2006; 290: H1575-1586.
10. Weebadda WK, Jackson TJ, Lin AW. Expression of p16IN4Ka variants in senescent human fibroblasts independent of protein phosphorylation. J Cell Biochem, 2005; 94: 1135-1147.
11.Alcorta DA, Xiong Y, Phelps D, et al. Involvement of the cyclin-dependent kinase inhibitor p16 (INK4a) in replicative senescence of normal human fibroblasts. Proc Natl Acad Sci, 1996; 93: 13742-13747.
12. Li S, Mawal-Dewan M, Cristofalo VJ, et al. Enhanced proliferation of human fibroblasts, in the presence of dexamethasone, is accompanied by changes in p21WAF1/Cip1/Sdil and the insulin-like growth factor type 1 receptor. J Cell Physiol, 1998; 177: 396-401.
13. Reinhardt HC, Aslanian AS, Lees JA, et al. p53-deficient cells rely on ATM- and ATR-mediated checkpoint signaling through the p38MAPK/MK2 pathway for survival after DNA damage. Cancer Cell, 2007; 11: 175-189.
14. Tang Y, Luo J, Zhang W, et al. Tip60-dependent acetylation of p53 modulates the decision between cell-cycle arrest and apoptosis. Mol Cell, 2006; 24: 827-839.
15. Davis T, Skinner JW, Faragher RG, et al. Replicative senescence in sheep fibroblasts is a p53 dependent process. Exp Gerontol, 2005; 40: 17-26.
16.Ye X, Zerlanko B, Zhang R, et al. Definition of pRB- and p53-Dependent and -Independent Steps in HIRA/ASF1a-Mediated Formation of Senescence-Associated Heterochromatin Foci. Mol Cell Biol, 2007; 27: 2452-2465.
17.Huang X, Di Liberto M, Cunningham AF, et al. Homeostatic cell-cycle control by BLyS: induction of cell-cycle entry but not G_1/S transition in opposition to p18~(INK4c) and p27~(Kip1). Proc Natl Acad Sci, 2004; 101: 17789-17794.
18.Dulic V., Lees E., Reed S. I. Association of human cyclin E with a periodic G_1-S phase protein kinase. Science, 1992; 257: 1958-1961.
19.Goodwish D, Wang NP, Qian YW, et al. The retinoblastoma gene product regulates progression through the Gl phase of the cell cycle. Cell, 1991; 67: 293-302.
20.Dowdy SF, Hinds PW, Louie K, AL. Physical interaction of the retinoblastoma protein with human D cyclins. Cell, 1993; 73: 499-511.
21.Ewen ME, Sluss HK, Whitehouse LL, ET AL. TGF-β inhibition of CDK4 synthesis is linked to cell cycle arrest. Cell, 1993; 74: 1009-1020.
22.Zhang X, Li J, Sejas DP, ET AL. Hypoxia-reoxygenation induces premature senescence in FA bone marrow hematopoietic cells. Blood, 2005; 106: 75-85.
23.Martinez-Romero R, Canuelo A, Martinez-Lara E, et al. Aging affects but does not eliminate the enzymatic antioxidative response to hypoxia/reoxygenation in cerebral cortex. Exp Gerontol, 2006; 41: 25-31.
24.Gasbarrini A, Simoncini M, Di Campli C, et al. Ageing affects anoxia/reoxygenation injury in rat hepatocytes. Scand J Gastroenterol, 1998; 33: 1107-1112.
25.Zhang FX, Chen ML, Shan QJ, et al. Hypoxia reoxygenation induces premature senescence in neonatal SD rat cardiomyocytes. Acta Pharmacol Sin, 2007; 28: 44-51.
26.Vonzglinicki T, Saretzki G, Docke W, et al. Mild hyperoxia shortens telomeres and inhibits proliferation of fibroblasts: a model for senescence? Exp Cell Res, 1995; 220: 186-193.
27.Chen Q, Ames BN. Senescence-like growth arrest induced by hydrogen peroxide in human diploid fibroblast F65 cells. Proc Natl Acad Sci, 1994; 91: 4130-4134.
28. Von Zglinicki T, Schewe C, et al. Mitochondrial water loss and aging of cells. Cell Biochem Funct, 1995; 13: 181-187.
29. Saretzki G, Feng J, von Zglinicki T, et al. Similar gene expression pattern in senescent and hyperoxic-treated fibroblasts. J Gerontol A Biol Sci Med Sci, 1998; 53: B438-442.
30.Dulic V, Drullinger LF, Lees E, et al. Altered regulation of G_1 cyclins in senescent human diploid fibroblasts: accumulation of inactive cyclin E/CDK2 and cyclin D1/CDK2 complexes. Proc Natl Acad Sci, 1993; 90: 11034-11038.
31.Lucibello FC, Sewing A, Brusselbach S, et al. Deregulation of cyclins D1 and E and suppression of CDK2 and CDK4 in senescent human fibroblasts. J Cell Sci, 1993; 105: 123-133.
32.Noda A, Ning Y, Venable SF, et al. Cloning of senescent cell-derived inhibitors of DNA synthesis using an expression screen. Exp Cell Res, 1994; 211: 90-98.
33.LaBaer J, Garrett MD, Stevenson LF, et al. New functional activities for the p21WAFl family of CDK inhibitors. Genes Dev, 1997; 11: 847-862.
34.Abdel-Wahab N, Weston BS, Roberts T, ET AL. Connective tissue growth factor and regulation of the mesangial cell cycle: role in cellular hypertrophy. J Am Soc Nephrol, 2002; 13: 2437-2445.
35.Gretchen HS, Linda FD, Alexandre Soulard, et al. Differential Roles for Cyclin-Dependent Kinase Inhibitors p21WAFl and p16IN4Ka in the mechanisms of senescence and differentiation in human fibroblasts. Mol CellBiol, 1999; 19:2109-2117.
36.Cheng M, Olivier P, Diehl JA, et al. The p21(Cipl) and p27(Kipl) CDK "inhibitors" are essential activators of cyclin D-dependent kinases in murine fibroblasts.EMBO J, 1999; 18: 1571-1583.
37.Serrano M, Henhon GJ, Beach D. A new regulatory-notfy in cell cycle control cousing specific inhibition of cyclin D/CDK4. Nature, 1993; 366: 704-707.
38.D'Amico M, Wu K, Fu M, ET AL.The inhibitor of cyclin-dependent kinase 4a/alternative reading frame (INK4a/ARF) locus encoded proteins p16INK4a and p19ARF repress cyclin Dl transcription through distinct cis elements. Cancer Res, 2004; 64: 4122-4130.
39.Quentin T, Henke C, Korabiowska M, et al. Altered mRNA expression of the Rb and pl6 tumor suppressor genes and of CDK4 in transitional cell carcinomas of the urinary bladder associated with tumor progression. Anticancer Res, 2004; 24: 1011-1023.
40.Ogino A, Yoshino A, Katayama Y, et al. The p15 (INK4b)/pl6(INK4a)/RB1 pathway is frequently deregulated in human pituitary adenomas. J Neuropathol Exp Neurol, 2005; 64: 398-403.
41.Reznitsky D, Reed SI. Different roles for cyclins Dl and E in regulation of the G_1-to-S phase transition. Mol Cell Biol, 1995; 15: 3463-3469.
42.Weinberg RA. The retinoblastoma protein and cell cycle control. Cell, 1995; 81: 323-330.
43.E1-Deiry WS, Harper JW, O'Connor PM, et al. WAF1/CIP1 is induced in p53-mediated G1 arrest and apoptosis. Cancer Res, 1994; 55: 1169-1174.
44.Dulic V, Kaufmann WK, Wilson SJ, et al. p53-dependent inhibition of cyclin-dependent kinase activities in human fibroblasts during radiation-induced G1 arrest. Cell, 1994; 76: 1013-1023.
45.Hara E, Smith R, Parry D, et al. Regulation of p16IN4Ka expression and its implications for cell immortalization and senescence. Mol Cell Biol, 1996; 16: 859-867.
46.Gatza C, Moore L, Dumble M, et al. Tumor suppressor dosage regulates stem cell dynamics during aging. Cell Cycle, 2007; 6: 52-55.
47.Horn HF, Vousden KH. Coping with stress: multiple ways to activate p53. Oncogene, 2007; 26: 1306-1316.
48.Liu G, Lozano G. p21 stability: linking chaperones to a cell cycle checkpoint. Cancer Cell, 2005; 7: 113-114.
1. Goldstein JL, Brown MS. Regulation of the mevalonate pathway. Nature, 1990; 343: 425-430.
2. Maron DJ, Fazio S, Linton ME Current perspectives on statins. Circulation, 2000; 101: 207-213.
3. Keech A, Colquhoun D, Best J, et al. LIPID Study Group.Secondary prevention of cardiovascular events with long-term pravastatin in patients with diabetes or impaired fasting glucose: results from the LIPID trial. Diabetes Care, 2003; 26: 2713-2721.
4. Kobashigawa JA, Moriguchi JD, Laks H, et al. Ten-year follow-up of a randomized trial of pravastatin in heart transplant patients. J Heart Lung Transplant, 2005; 24: 1736-1740.
5. Lee TM, Lin MS, Tsai CH, et al. Effects of pravastatin on ventricular remodeling by activation of myocardial K (ATP) channels in infarcted rats: role of 70-kDa S6 kinase. Basic Res Cardiol, 2007; 102: 171-182.
6. Assmus B, Urbich C, Aicher A, et al. HMG-CoA reductase inhibitors reduce senescence and increase proliferation of endothelial progenitor cells via regulation of cell cycle regulatory genes. Circ Res, 2003; 92: 1049-1055.
7. Gray-Bablin J, Rao S, Keyomarsi K. Lovastatin induction of cyclin-dependent kinase inhibitors in human breast cells occurs in a cell cycle-independent fashion. Cancer Res, 1997; 57: 604-609.
8. Lee SJ, Ha MJ, Lee J, et al. Inhibition of the 3-hydroxy-3-methylglutaryl-coenzyme A reductase pathway induces p53-independent transcriptional regulation of p21WAF1/CIP1 in human prostate carcinoma cells. J Biol Chem, 1998; 273: 10618-10623.
9. Rao S, Lowe M, Herliczek TW, et al. Lovastatin mediated G_1 arrest in normal and tumor breast cells is through inhibition of CDK2 activity and redistribution of p21WAFl and p27, independent of p53. Oncogene, 1998; 17: 2393-2402.
10. Hengst L, Reed SI. Translational control of p27Kip1 accumulation during the cell cycle. Science, 1996; 271: 1861-1864.
11.Terada Y, Inoshita S, Nakashima O, et al. Lovastatin inhibits mesangial cell proliferation via p27 Kip1. J Am Soc Nephrol, 1998; 9: 2235-2243.
12. Vogt A, Sun J, Qian Y, et al. The geranylgeranyltransferase-I inhibitor GGTI-298 arrests human tumor cells in G_0/G_1 and induces p21 WAF1/CIP1/SDI1 in a p53-independent manner. J Biol Chem, 1997; 272: 27224-27229.
13. Hayflick L, Moorhead PS. The serial cultivation of human diploid cell strains. Exp Cell Res, 1961; 25: 585-621.
14. Schmaltz C, Hardenbergh PH, Wells A, et al. Regulation of proliferation-survival decisions during tumor cell hypoxia. Mol Cell Biol, 1998; 18: 2845-2854.
15. Iida T, Mine S, Fujimoto H, et al. Hypoxia-inducible factor-lalpha induces cell cycle arrest of endothelial cells. Genes cells, 2002; 7: 143-149.
16. Gardner LB, Li Q, Park MS, et al. Hypoxia inhibits G1/S transition through regulation of p27 expression. J Biol Chem, 2001; 276: 7919-7926.
17. Vonzglinicki T, Saretzki G, Docke W, et al. Mild hyperoxia shortens telomeres and inhibits proliferation of fibroblasts: a model for senescence? Exp Cell Res, 1995; 220: 186-193.
18. Chen Q, Ames BN. Senescence-like growth arrest induced by hydrogen peroxide in human diploid fibroblast F65 cells. Proc Natl Acad Sci, 1994; 91: 4130-4134.
19. Von Zglinicki T, Schewe C, et al. Mitochondrial water loss and aging of cells. Cell Biochem Funct, 1995; 13: 181-187.
20. Saretzki G, Feng J, von Zglinicki T, et al. Similar gene expression pattern in senescent and hyperoxic-treated fibroblasts. J Gerontol A Biol Sci Med Sci, 1998; 53: B438-442.
21. Dimri GP. What has senescence got to do with cancer? Cancer Cell, 2005; 7: 505-512.
22. Serrono M, HenhonQ, Reach D. A new regulmory-otfy in cell cycle control cousing specific inhibition of cycliO/CDK4.Nature, 1993; 366: 704-707.
23. Kmnb A, Gntis NA, Waever-Feldhaus J, et al. A cell regulator potentially involved genesis of many types. Science, 1994; 264: 436-440.
24. Ohtani N, Zebegee Z, Huot TJ, et al. Opposing effects of Ets and Id proteins on p16EST4KaINK4a expression during cellular senescence. Nature, 2001; 409: 1067-1070.
25. Herbig U, Sedivy JM. Regulation of rrowh arrest in senescence; Telomere damage is not the end of the dory. Mech Ageing Dev, 2006; 127: 16-24.
26. Iwasa H, Han J, Ishikawa F. Mitogen-activated protein kinase p3M defines the common senescence-signalling pathway. Genes Cells, 2003; 8: 131-144.
27. Wang W, Chen JX, Liao R, et al. Sequential activation of the MEK-extracellular signal-regulated kinase and MKK3/6-p38 mitogen-activated protein kinase pathways mediates oncogenic rasinduced premature senescence. Mol Cell Biol, 2002; 22: 3389-3403.
28. Passegue E, Wagner EF. Jun B suppresses cell proliferation by transcriptional activation of pl6IN4Ka (INK4a) expression. EMBO J, 2000; 19:2969-2979.
29. Itahana K, Zou Y, Itahana Y, et al. Control of the replicative life span of human fibroblasts by p16IN4Ka and the polycomb protein Bmi-1. Mol Cell Biol, 2003; 23: 389-401.
30. Gil J, Bernard D, Martinez D, et al. Polycomb CBX7 has a unifying role in cellular lifespan. Nat Cell Biol, 2004; 6: 67-72.
31.Dellambra E, Golisano O, Bondanza S, et al. Downregulation of 14-3-3 sigma prevents clonal evolution and leads to immortalization of primary human keratinocytes. J Cell Biol, 2000; 149: 1117-1130.
32. Friend S, Bernards R, Rogelj S, et al. A human DNA segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma. Nature, 1986; 323: 643-646.
33. Goodwish D, Wang NP, Qian YW, et al. The retinoblastoma gene product regulates progression through the Gl phase of the cell cycle. Cell, 1991; 67: 293-302.
34. Adams PD, KaelinY. Transcriptional control by E_2F. Semin Cancer Biol, 1995; 6: 99-108.
35. Kourarides T. Transcriptional control by the retinoblastome protein. Semin Cancer Biol, 1995; 6: 91-98.
36. Sage J, Miller A, Perez-mancera PA, et al. Acute mutationof retinoblastoma gene function is sufficient for cell cycle reentry. Nature, 2003; 424: 223-228.
37. Wei W, Herbig U, Wei S, et al. Loss of retinoblastoma but not p16IN4Ka function allows bypass of replicative senescence in human fibroblasts. EMBO Rep, 2003; 4: 1061-1065.
38. Alexander K, Yang HS, Hinds PW. pRb inactivation in senescent cells leads to an E2F-dependent apoptosis requiring p73. Mol Cancer Res, 2003; 1: 716-728.
39. Stein GH, Drullinger LF, Soulard A, et al. Differential roles for cyclin-dependent kinase inhibitors p21WAFl and p16IN4Ka in the mechanisms of senescence and differentiation in human fibroblasts. Mol Cell Biol, 1999,19: 2109-2117.
40. Wong DJ, Foster SA, Galloway DA, et al. Progressive region-specific de novo methylation of the p16IN4Ka CpG island in primary human mammary epithelial cell strains during escape from M(0) growth arrest. Mol Cell Biol, 1999; 19: 5642-5651
41.Beausejour CM, Katolica A, Galimi F, et al. Reversal of human cellular senescence; Roles of the p53 and p16IN4Ka pathways. EMBO J, 2003; 22: 4212-4222.
42. LaBaer J, Garrett M D, Stevenson L F, et al. New functional activities for the p21WAFl family of CDK inhibitors. Genes Dev, 1997; 11: 847-862.
43. Zhang H, Hannon G J, Beach D. p21WAF1-containing cyclin kinases exist in both active and inactive states. Genes Dev, 1994; 8: 1750-1758.
44. Sugrue MM, Shin DY, Lee SW, et al. Wild type p53 triggers a rapid senescence program in human tumors lacking functional p53. Proc Natl Acad Sci USA, 1997; 94: 9648 -9653.
45. Polyak K, Xia Y, Zweier JL, et al. Amodel for p53-induced apoptosis. Nature, 1997; 389: 300-305.
46. Sharpless NE. INK4a/ARF; A multifunctional tumor suppressorlocus. Mutat Res, 2005; 576: 22-38.
47. Pearson M, Carbone R, Sebastiani C, et al. PML regulates p53 acetylation and premature senescence induced by oncogenic Ras. Nature, 2000; 406: 207-210.
48. Lanqley E, Pearson M, Fareta M, et al. Human SIR2 deacetylates p53 and antagonizes PMUp53-induced cellular senescence. EMBO J, 2002; 21:2383-2396.
49. Bischof O, Nacerddine K, Dejean A. Human papillomavirus oncoprotein E7 targets the promyelocytic leukemia protein and circumvents cellular senescence via the Rb and p53 tumor suppressor pathways. Mol Cell Biol, 2005; 25: 1013-1024.
50. In HK, Bergmann S, Pandolfi PP. Cytoplasmic PML function in TGF-beta signalling. Nature, 2004; 431: 205-211.
51. Colombo E, Marine JC, Danovi D, et al. Nucleophosmin regulates the stability and transcriptional activity of p53. Nat Cell Biol, 2002; 4: 529-533.
52. Colombo E, Martinellip P, Zamponi R, et al. Delocalization and destabilization of the Arf tumor suppressor by the leukemia-associated NPM mutant. Cancer Res, 2006; 66: 3044-3050.
53. Goeman F, Thormeyer D, Abad M, et al. Growth inhibition by the tumor suppressor p331NG1 in immortalized and primary cells; Involvement of two silencing domains and effect of Ras. Mal Cell Biol, 2005; 25: 422-431.
54. Noda A, Ning Y, Venable SF, et al. Cloning of senescent cell-derived inhibitors of DNA synthesis using an expression screen. Exp. Cell Res., 211: 90-98, 1994.
55. Gretchen H. Stein, Linda F. Drullinger, Alexandre Soulard, et al. Differential Roles for Cyclin-Dependent Kinase Inhibitors p21WAFl and p16IN4Ka in the Mechanisms of Senescence and Differentiation in Human Fibroblasts. Mol Cell Biol, 1999; 19: 2109-2117.
56. Cheng M, Olivier P, Diehl JA, et al. The p21 WAF1 (Cip1) and p27 (Kip1) CDK "inhibitors" are essential activators of cyclin D-dependent kinases in murine fibroblasts. EMBO J, 1999; 18: 1571-1583.
57. Said TK, Medina D. Cell cyclins and cyclin-dependent kinase activities. Carcinogenesis, 1995; 16: 823-830.
58. Hartwel LH, Kastan MB.Cell cycle control and cancer. S cience, 1994; 266: 1821-1828.
59. Bartkova J, Lukas J, G oldberg P, et al .The pl6-cycbnD/CDK4-pRB pathway as a functional unit frequently altered in melanoma pathogenesis. Cancer Res, 1996; 56: 5475-5483.
60. Herbigb U, Wei W, Dutriaux A, et al. Real-time imaging of transcriptional activation in live cells reveals rapid up-regulation of the cyclin-dependent kinase inhibitor gene CDKNl A in replicative cellular senescence. Aging Cell, 2003; 2: 295-304.
61. Herbigb U, Jobling WA, Chen BP, et al. Telomere shortening triggers senescence of human cells through a pathway involving ATM, p53, and p21WAF1, but not pl6IN4Ka. Mol Cell, 2004; 14: 501-513.
2. Chlopcikova S, Psotova J, Miketova P. Neonatal rat cardiomyocytes--a model for the study of morphological, biochemical and electrophysiological characteristics of the heart. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub, 2001; 145: 49-55.
3. Taylor MR, Slavov D, Ku L, et al. Prevalence of desmin mutations in dilated cardiomyopathy. Circulation, 2007; 115: 1244-1251.
4. Cortassa S, Aon MA, O'Rourke B, et al. A computational model integrating electrophysiology, contraction, and mitochondrial bioenergetics in the ventricular myocyte. Biophys J, 2006; 91: 1564-1589.
5. Li Q, Ren J. Cardiac overexpression of metallothionein attenuates chronic alcohol intake-induced cardiomyocyte contractile dysfunction. Cardiovasc Toxicol, 2006; 6: 173-1782.
6. Burkard N, Rokita AG, Kaufmann SG, et al. Conditional neuronal nitric oxide synthase overexpression impairs myocardial contractility. Circ Res, 2007; 100:e32-44.
7. Demiralay R, Gursan N, Erdem H. The effects of erdosteine, N-acetylcysteine and vitamin E on nicotine-induced apoptosis of cardiac cells. J Appl Toxicol, 2007; 27: 247-254.
8. Sutherland FJ, Hearse DJ. The isolated blood and perfusion fluid perfused heart. Pharmacol Res, 2000; 41: 613-627.
9. Fu M, Wu M, Wang JF, et al. Disruption of the intracellular Ca2+ homeostasis in the cardiac excitation-contraction coupling is a crucial mechanism of arrhythmic toxicity in aconitine-induced cardiomyocytes. Biochem Biophys Res Commun, 2007; 354: 929-936.
10. Gu X, Cheng L, Chueng WL, et al. Neovascularization of ischemic myocardium by newly isolated tannins prevents cardiomyocyte apoptosis and improves cardiac function. Mol Med, 2006; 12: 275-283.
11.Blondel B, Roijem I, Cheneval JP. Heart cells in culture: a simple method for increasing the proportion of myoblasts. Experientia, 1971; 27: 356-358.
12. Hoshida S, Nishida M, Yamashita N, et al. Heme oxygenese-1 expressionand its relation to oxidative stress during primary culture of cardiomyocytes. J Mol Cell Cardiol, 1996; 28: 1845-1855.
13. Hayflick L, Moorhead PS, et al. The serial cultivation of human diploid cell strains. Exp Cell Res, 1961; 25: 585-621.
14. Schmaltz C, Hardenbergh PH, Wells A, et al. Regulation of proliferation-survival decisions during tumor cell hypoxia. Mol Cell Biol, 1998; 18: 2845-2854.
15. Iida T, Mine S, Fujimoto H, et al. Hypoxia-inducible factor-lalpha induces cell cycle arrest of endothelial cells. Genes ells, 2002; 7: 143-149.
16. Gardner LB, Li Q, Park MS, et al. Hypoxia inhibits G1/S transition through regulation of p27 expression. J Biol Chem, 2001; 276: 7919-7926.
17. Vonzglinicki T, Saretzki G, Docke W, et al. Mild hyperoxia shortens telomeres and inhibits proliferation of fibroblasts: a model for senescence? Exp Cell Res, 1995; 220: 186-193.
18. Galaris D, Hoijer B, Rydstrom J. Improved methods for automatic monitoring of contracting heart cells in culture. J Biochem Biophys Methods, 1980; 2: 213-225.
19. Rakhit RD, Mojet MH, Marber MS, et al. Mitochondria as Targets for Nitric Oxide-Induced Protection During Simulated Ischemia and Reoxygenation in Isolated Neonatal Cardiomyocytes. Circulation, 2001; 103:2617-2623.
20. Grynberg A, Athias P, Degois M. Effect of change in growth environment on cultured myocardial cells investigated in a standardized medium. In Vitro Cell Dev Biol, 1986; 22: 44-50.
21. Ichiba T, Matsuda N, Takemoto N, et al. Regulation of intracellular calcium concentrations by calcium and magnesium in cardioplegic solutions protects rat neonatal myocytes from simulated ischemia. J Mol Cell Cardiol, 1998; 30: 1105-1114.
22. Adderley SR, Fitzgerald DJ. Oxidative damage of cardiomyocytes is limited by extracellular regulated kinases 1/2-mediated induction of cyclooxygenase-2. J Biol Chem, 1999; 274: 5038-5046.
23. Simpson P, Savion S. Differentiation of rat myocytes in single cell cultures with and without proliferating nonmyocardial cells. Circ Res, 1982; 50: 101-116.
24. Hoshida S, Nishida M, Yamashita N, et al. Heme oxygenese-1 expression and its relation to oxidative stress during primary culture of cardiomyocytes. J Mol Cell Cardiol, 1996; 28: 1845-1855.
25. Zhang X, Li J, Sejas DP, et al. Hypoxia-reoxygenation induces premature senescence in FA bone marrow hematopoietic cells. Blood, 2005; 106: 75-85.
26. Dimri GP, Lee X, Basile G, et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci USA, 1995; 92: 9363-9367.
27. Hayashi S, Inoue A. Cardiomyocytes re-enter the cell cycle and contribute to heart development after differentiation from cardiac progenitors expressing Isll in chick embryo. Dev Growth Differ, 2007; 49: 229-239.
28. Camelliti P, McCulloch AD, Kohl P. Microstructured cocultures of cardiac myocytes and fibroblasts: a two-dimensional in vitro model of cardiac tissue. Microsc Microanal, 2005; 11: 249-259.
29. Adderley SR, Fitzgerald DJ. Oxidative damage of cardiomyocytes is limited by extracellular regulated kinases 1/2-mediated induction of cyclooxygenase-2. J Biol Chem, 1999; 274: 5038-5046.
30. Marengo FD, Wang S, Wang B, et al. Dependence of cardiac cell Ca2+ permeability on sialic acid-containing sarcolemmal gangliosides. J Mol Cell Cardiol, 1998; 30: 127-137.
31. Matos MJ, Post JA, Roelofsen B, et al. Composition and organization of sarcolemmal fatty acids in cultured neonatal rat cardiomyocytes. Cell Biol Int Rep, 1990; 14: 343-352.
32. Karliner JS, Honbo N, Epstein CJ, et al. Neonatal mouse cardiac myocytes exhibit cardioprotection induced by hypoxic and pharmacologic preconditioning and by transgenic overexpression of human Cu/Zn superoxide dismutase. J Mol Cell Cardiol, 2000; 32: 1779-1786.
33. Lepic E, Burger D, Lu X, et al. Lack of endothelial nitric oxide synthase decreases cardiomyocyte proliferation and delays cardiac maturation. Am J Physiol Cell Physiol, 2006; 291: C1240-1246.
34. Driesen RB, Verheyen FK, Dispersyn GD, et al. Structural adaptation in adult rabbit ventricular myocytes: influence of dynamic physical interaction with fibroblasts. Cell Biochem Biophys, 2006; 44: 119-128.
35. Clark WA, Decker ML, Janes DM, et al. Cell contact as an independent factor moduletating cardiac myocytes hytertrophy and survival in long-term primary culture. J Mol Cell C ardiol, 1998; 30: 139-155.
36. Simpson P, McGrath A, Savion S. Myocyte hypertrophy in neonatal rat heart cultures and its regulation by serum and by catecholamines. Circ Res, 1982; 51: 787-800.
37. Iwaki K, Chi SH, Dillmann WH, et al. Induction of HSP70 in cultured rat neonatal cardiomyocytes by hypoxia and metabolic stress. Circ Res, 1993; 87: 2023-2032.
38. Musters RJ, Post JA, Verkleij AJ. The isolated neonatal rat-cardiomyocyte used in an in vitro model for 'ischemia'. I. A morphological study. Biochim Biophys Acta, 1991; 1091: 270-277.
39. Ollinger K, Brunmark A. Effect of different oxygen pressures and N, N-diphenyl-p-phenylenediamine on adriamycin toxicity to cultured neonatal rat heart myocytes. Biochem Pharmacol, 1994; 48: 1707-1715.
40. Brooks G, Poolman RA, McGill CJ, et al. Expression and activities of cyclins and cyclin-dependent kinases in developing rat ventricular myocytes. J Mol Cell Cardiol, 1997; 29: 2261-2271.
41. Soonpaa MH, Kim KK, Pajak L, et al. Cardiomyocyte DNA synthesis and binucleation during murine development. American Journal of Physiology, 1996; 40: H2183-H2189.
42. Stolzing A, Coleman N, Scutt A. Glucose-induced replicative senescence in mesenchymal stem cells. Rejuvenation Res, 2006; 9: 31-35.
43. Lehmann BD, McCubrey JA, Jefferson HS, et al. A dominant role for p53-dependent cellular senescence in radiosensitization of human prostate cancer cells. Cell Cycle, 2007; 6: 595-605.
44. Hastings R, Qureshi M, Verma R, et al. Telomere attrition and accumulation of senescent cells in cultured human endothelial cells. Cell Prolif, 2004; 37: 317-324.
45. Diez C, Nestler M, Friedrich U, et al. Down-regulation of Akt/PKB in senescent cardiac fibroblasts impairs PDGF-induced cell proliferation. Cardiovasc Res, 2001; 49: 731-740.
46. Allen RG, Tresini M, Keogh BP, et al. Differences in electron transport potential, antioxidant defenses, and oxidant generation in young and senescent fetal lung fibroblasts (WI-38). J Cell Physiol, 1999; 180: 114-122.
47. Von Zglinicki T, Schewe C, et al. Mitochondrial water loss and aging of cells. Cell Biochem Funct, 1995; 13: 181-187.
48. Chen Q, Ames BN. Senescence-like growth arrest induced by hydrogen peroxide in human diploid fibroblast F65 cells. Proc Natl Acad Sci, 1994; 91: 4130-4134.
49. Saretzki G, Feng J, von Zglinicki T, et al. Similar gene expression pattern in senescent and hyperoxic-treated fibroblasts. J Gerontol A Biol Sci Med Sci, 1998; 53: B438-42.
50. Halterman MW, Federoff HJ. HIF-1 alpha and p53 promote hypoxia-induced delayed neuronal death in models of CNS ischemia. Exp Neurol, 1999; 159: 65-72.
51. Von Zglinicki T, Marzabadi MR, Roomans GM. Water and ion distributions in myocytes cultured under oxidative stress mimic changes found in the process of aging. Mech Ageing Dev, 1991; 58: 49-60.
52. Imanishi T, Hano T, Nishio I, et al. Angiotensin II accelerates endothelial progenitor cell senescence through induction of oxidative stress. J Hypertens, 2005; 23: 97-104.
53. Aliouat-Denis CM, Dendouga N, Van den Wyngaert I, et al. p53-Independent Regulation of p21WAFl/Cipl Expression and Senescence by Chk2. Mol Cancer Res, 2005; 3: 627-634.
54. Chai J, Charboneau AL, Betz BL, et al. Loss of the hSNF5 gene concomitantly inactivates p21CIP/WAFl and p16IN4Ka activity associated with replicative senescence in A204 rhabdoid tumor cells. Cancer Res, 2005; 65: 10192-10198.
55. Chen QM. Replicative senescence and oxidantinduced premature senescence: beyond the control of cell cycle checkpoints. Ann N Y AcadSci, 2000; 908:111-125.
56. Katsiki M, Trougakos IP, Chondrogianni N, et al. Alterations of senescence biomarkers in human cells by exposure to CrVI in vivo and in vitro. Exp Gerontol, 2004; 39: 1079-1087.
57. Serrano M, Blasco MA. Putting the stress on senescence. Curr Opin Cell Biol, 2001, 13:748-753.
58.Randle DH, Zindy F, Sherr CJ, et al. ifferential effects of p19(Arf) and p16IN4Ka(Ink4a) loss on senescence of murine bone marrow-derived preB cells and macrophages. Proc Natl Acad Sci, 2001; 98: 9654-9659.
1. Ferrer JL, Dupuy J, Borel F, et al. Structural basis for the modulation of CDK-dependent/independent activity of cyclin D1. Cell Cycle, 2006; 5: 2760-2768.
2. White J, Dalton S. Cell cycle control of embryonic stem cells. Stem Cell Rev, 2005; 1: 131-138.
3. Bockstaele L, Coulonval K, Kooken H, et al. Regulation of CDK4. Cell Div,2006; 1:25.
4. Okamoto K, Kato S, Arima N, et al. Cyclin-dependent kinase inhibitor, p21Wafl, regulates vascular smooth muscle cell hypertrophy. Hypertens Res, 2004; 27: 283-291.
5. Sherr CJ, Roberts JM. CDK inhibitors: positive and negative regulators ofG_1-phase progression. Genes Dev, 1999; 13: 1501-1512.
6. Chai J, Charboneau AL, Betz BL, et al. Loss of the hSNF5 gene concomitantly inactivates p21CIP/WAFl and p16IN4Ka activity associated with replicative senescence in A204 rhabdoid tumor cells. Cancer Res, 2005; 65: 10192-10198.
7. Kramer DL, Chang BD, Chen Y, et al. Polyamine depletion in human melanoma cells leads to G1 arrest associated with induction of p21WAFl/CIPl/SDIl, changes in the expression of p21WAF1-regulated genes, and a senescence-like phenotype. Cancer Res, 2001; 61: 7754-7762.
8. Palmero I, McConnell B, Parry D, et al. Accumulation of p16IN4Ka in mouse fibroblasts as a function of replicative senescence and not of retinoblastoma gene status. Oncogene, 1997; 15: 495-503.
9. Chen J, Huang X, Halicka D, et al. Contribution of p16IN4Kaand p21WAF1 pathways to induction of premature senescence of human endothelial cells: permissive role of p53. Am J Physiol Heart Circ Physiol, 2006; 290: H1575-1586.
10. Weebadda WK, Jackson TJ, Lin AW. Expression of p16IN4Ka variants in senescent human fibroblasts independent of protein phosphorylation. J Cell Biochem, 2005; 94: 1135-1147.
11.Alcorta DA, Xiong Y, Phelps D, et al. Involvement of the cyclin-dependent kinase inhibitor p16 (INK4a) in replicative senescence of normal human fibroblasts. Proc Natl Acad Sci, 1996; 93: 13742-13747.
12. Li S, Mawal-Dewan M, Cristofalo VJ, et al. Enhanced proliferation of human fibroblasts, in the presence of dexamethasone, is accompanied by changes in p21WAF1/Cip1/Sdil and the insulin-like growth factor type 1 receptor. J Cell Physiol, 1998; 177: 396-401.
13. Reinhardt HC, Aslanian AS, Lees JA, et al. p53-deficient cells rely on ATM- and ATR-mediated checkpoint signaling through the p38MAPK/MK2 pathway for survival after DNA damage. Cancer Cell, 2007; 11: 175-189.
14. Tang Y, Luo J, Zhang W, et al. Tip60-dependent acetylation of p53 modulates the decision between cell-cycle arrest and apoptosis. Mol Cell, 2006; 24: 827-839.
15. Davis T, Skinner JW, Faragher RG, et al. Replicative senescence in sheep fibroblasts is a p53 dependent process. Exp Gerontol, 2005; 40: 17-26.
16.Ye X, Zerlanko B, Zhang R, et al. Definition of pRB- and p53-Dependent and -Independent Steps in HIRA/ASF1a-Mediated Formation of Senescence-Associated Heterochromatin Foci. Mol Cell Biol, 2007; 27: 2452-2465.
17.Huang X, Di Liberto M, Cunningham AF, et al. Homeostatic cell-cycle control by BLyS: induction of cell-cycle entry but not G_1/S transition in opposition to p18~(INK4c) and p27~(Kip1). Proc Natl Acad Sci, 2004; 101: 17789-17794.
18.Dulic V., Lees E., Reed S. I. Association of human cyclin E with a periodic G_1-S phase protein kinase. Science, 1992; 257: 1958-1961.
19.Goodwish D, Wang NP, Qian YW, et al. The retinoblastoma gene product regulates progression through the Gl phase of the cell cycle. Cell, 1991; 67: 293-302.
20.Dowdy SF, Hinds PW, Louie K, AL. Physical interaction of the retinoblastoma protein with human D cyclins. Cell, 1993; 73: 499-511.
21.Ewen ME, Sluss HK, Whitehouse LL, ET AL. TGF-β inhibition of CDK4 synthesis is linked to cell cycle arrest. Cell, 1993; 74: 1009-1020.
22.Zhang X, Li J, Sejas DP, ET AL. Hypoxia-reoxygenation induces premature senescence in FA bone marrow hematopoietic cells. Blood, 2005; 106: 75-85.
23.Martinez-Romero R, Canuelo A, Martinez-Lara E, et al. Aging affects but does not eliminate the enzymatic antioxidative response to hypoxia/reoxygenation in cerebral cortex. Exp Gerontol, 2006; 41: 25-31.
24.Gasbarrini A, Simoncini M, Di Campli C, et al. Ageing affects anoxia/reoxygenation injury in rat hepatocytes. Scand J Gastroenterol, 1998; 33: 1107-1112.
25.Zhang FX, Chen ML, Shan QJ, et al. Hypoxia reoxygenation induces premature senescence in neonatal SD rat cardiomyocytes. Acta Pharmacol Sin, 2007; 28: 44-51.
26.Vonzglinicki T, Saretzki G, Docke W, et al. Mild hyperoxia shortens telomeres and inhibits proliferation of fibroblasts: a model for senescence? Exp Cell Res, 1995; 220: 186-193.
27.Chen Q, Ames BN. Senescence-like growth arrest induced by hydrogen peroxide in human diploid fibroblast F65 cells. Proc Natl Acad Sci, 1994; 91: 4130-4134.
28. Von Zglinicki T, Schewe C, et al. Mitochondrial water loss and aging of cells. Cell Biochem Funct, 1995; 13: 181-187.
29. Saretzki G, Feng J, von Zglinicki T, et al. Similar gene expression pattern in senescent and hyperoxic-treated fibroblasts. J Gerontol A Biol Sci Med Sci, 1998; 53: B438-442.
30.Dulic V, Drullinger LF, Lees E, et al. Altered regulation of G_1 cyclins in senescent human diploid fibroblasts: accumulation of inactive cyclin E/CDK2 and cyclin D1/CDK2 complexes. Proc Natl Acad Sci, 1993; 90: 11034-11038.
31.Lucibello FC, Sewing A, Brusselbach S, et al. Deregulation of cyclins D1 and E and suppression of CDK2 and CDK4 in senescent human fibroblasts. J Cell Sci, 1993; 105: 123-133.
32.Noda A, Ning Y, Venable SF, et al. Cloning of senescent cell-derived inhibitors of DNA synthesis using an expression screen. Exp Cell Res, 1994; 211: 90-98.
33.LaBaer J, Garrett MD, Stevenson LF, et al. New functional activities for the p21WAFl family of CDK inhibitors. Genes Dev, 1997; 11: 847-862.
34.Abdel-Wahab N, Weston BS, Roberts T, ET AL. Connective tissue growth factor and regulation of the mesangial cell cycle: role in cellular hypertrophy. J Am Soc Nephrol, 2002; 13: 2437-2445.
35.Gretchen HS, Linda FD, Alexandre Soulard, et al. Differential Roles for Cyclin-Dependent Kinase Inhibitors p21WAFl and p16IN4Ka in the mechanisms of senescence and differentiation in human fibroblasts. Mol CellBiol, 1999; 19:2109-2117.
36.Cheng M, Olivier P, Diehl JA, et al. The p21(Cipl) and p27(Kipl) CDK "inhibitors" are essential activators of cyclin D-dependent kinases in murine fibroblasts.EMBO J, 1999; 18: 1571-1583.
37.Serrano M, Henhon GJ, Beach D. A new regulatory-notfy in cell cycle control cousing specific inhibition of cyclin D/CDK4. Nature, 1993; 366: 704-707.
38.D'Amico M, Wu K, Fu M, ET AL.The inhibitor of cyclin-dependent kinase 4a/alternative reading frame (INK4a/ARF) locus encoded proteins p16INK4a and p19ARF repress cyclin Dl transcription through distinct cis elements. Cancer Res, 2004; 64: 4122-4130.
39.Quentin T, Henke C, Korabiowska M, et al. Altered mRNA expression of the Rb and pl6 tumor suppressor genes and of CDK4 in transitional cell carcinomas of the urinary bladder associated with tumor progression. Anticancer Res, 2004; 24: 1011-1023.
40.Ogino A, Yoshino A, Katayama Y, et al. The p15 (INK4b)/pl6(INK4a)/RB1 pathway is frequently deregulated in human pituitary adenomas. J Neuropathol Exp Neurol, 2005; 64: 398-403.
41.Reznitsky D, Reed SI. Different roles for cyclins Dl and E in regulation of the G_1-to-S phase transition. Mol Cell Biol, 1995; 15: 3463-3469.
42.Weinberg RA. The retinoblastoma protein and cell cycle control. Cell, 1995; 81: 323-330.
43.E1-Deiry WS, Harper JW, O'Connor PM, et al. WAF1/CIP1 is induced in p53-mediated G1 arrest and apoptosis. Cancer Res, 1994; 55: 1169-1174.
44.Dulic V, Kaufmann WK, Wilson SJ, et al. p53-dependent inhibition of cyclin-dependent kinase activities in human fibroblasts during radiation-induced G1 arrest. Cell, 1994; 76: 1013-1023.
45.Hara E, Smith R, Parry D, et al. Regulation of p16IN4Ka expression and its implications for cell immortalization and senescence. Mol Cell Biol, 1996; 16: 859-867.
46.Gatza C, Moore L, Dumble M, et al. Tumor suppressor dosage regulates stem cell dynamics during aging. Cell Cycle, 2007; 6: 52-55.
47.Horn HF, Vousden KH. Coping with stress: multiple ways to activate p53. Oncogene, 2007; 26: 1306-1316.
48.Liu G, Lozano G. p21 stability: linking chaperones to a cell cycle checkpoint. Cancer Cell, 2005; 7: 113-114.
1. Goldstein JL, Brown MS. Regulation of the mevalonate pathway. Nature, 1990; 343: 425-430.
2. Maron DJ, Fazio S, Linton ME Current perspectives on statins. Circulation, 2000; 101: 207-213.
3. Keech A, Colquhoun D, Best J, et al. LIPID Study Group.Secondary prevention of cardiovascular events with long-term pravastatin in patients with diabetes or impaired fasting glucose: results from the LIPID trial. Diabetes Care, 2003; 26: 2713-2721.
4. Kobashigawa JA, Moriguchi JD, Laks H, et al. Ten-year follow-up of a randomized trial of pravastatin in heart transplant patients. J Heart Lung Transplant, 2005; 24: 1736-1740.
5. Lee TM, Lin MS, Tsai CH, et al. Effects of pravastatin on ventricular remodeling by activation of myocardial K (ATP) channels in infarcted rats: role of 70-kDa S6 kinase. Basic Res Cardiol, 2007; 102: 171-182.
6. Assmus B, Urbich C, Aicher A, et al. HMG-CoA reductase inhibitors reduce senescence and increase proliferation of endothelial progenitor cells via regulation of cell cycle regulatory genes. Circ Res, 2003; 92: 1049-1055.
7. Gray-Bablin J, Rao S, Keyomarsi K. Lovastatin induction of cyclin-dependent kinase inhibitors in human breast cells occurs in a cell cycle-independent fashion. Cancer Res, 1997; 57: 604-609.
8. Lee SJ, Ha MJ, Lee J, et al. Inhibition of the 3-hydroxy-3-methylglutaryl-coenzyme A reductase pathway induces p53-independent transcriptional regulation of p21WAF1/CIP1 in human prostate carcinoma cells. J Biol Chem, 1998; 273: 10618-10623.
9. Rao S, Lowe M, Herliczek TW, et al. Lovastatin mediated G_1 arrest in normal and tumor breast cells is through inhibition of CDK2 activity and redistribution of p21WAFl and p27, independent of p53. Oncogene, 1998; 17: 2393-2402.
10. Hengst L, Reed SI. Translational control of p27Kip1 accumulation during the cell cycle. Science, 1996; 271: 1861-1864.
11.Terada Y, Inoshita S, Nakashima O, et al. Lovastatin inhibits mesangial cell proliferation via p27 Kip1. J Am Soc Nephrol, 1998; 9: 2235-2243.
12. Vogt A, Sun J, Qian Y, et al. The geranylgeranyltransferase-I inhibitor GGTI-298 arrests human tumor cells in G_0/G_1 and induces p21 WAF1/CIP1/SDI1 in a p53-independent manner. J Biol Chem, 1997; 272: 27224-27229.
13. Hayflick L, Moorhead PS. The serial cultivation of human diploid cell strains. Exp Cell Res, 1961; 25: 585-621.
14. Schmaltz C, Hardenbergh PH, Wells A, et al. Regulation of proliferation-survival decisions during tumor cell hypoxia. Mol Cell Biol, 1998; 18: 2845-2854.
15. Iida T, Mine S, Fujimoto H, et al. Hypoxia-inducible factor-lalpha induces cell cycle arrest of endothelial cells. Genes cells, 2002; 7: 143-149.
16. Gardner LB, Li Q, Park MS, et al. Hypoxia inhibits G1/S transition through regulation of p27 expression. J Biol Chem, 2001; 276: 7919-7926.
17. Vonzglinicki T, Saretzki G, Docke W, et al. Mild hyperoxia shortens telomeres and inhibits proliferation of fibroblasts: a model for senescence? Exp Cell Res, 1995; 220: 186-193.
18. Chen Q, Ames BN. Senescence-like growth arrest induced by hydrogen peroxide in human diploid fibroblast F65 cells. Proc Natl Acad Sci, 1994; 91: 4130-4134.
19. Von Zglinicki T, Schewe C, et al. Mitochondrial water loss and aging of cells. Cell Biochem Funct, 1995; 13: 181-187.
20. Saretzki G, Feng J, von Zglinicki T, et al. Similar gene expression pattern in senescent and hyperoxic-treated fibroblasts. J Gerontol A Biol Sci Med Sci, 1998; 53: B438-442.
21. Dimri GP. What has senescence got to do with cancer? Cancer Cell, 2005; 7: 505-512.
22. Serrono M, HenhonQ, Reach D. A new regulmory-otfy in cell cycle control cousing specific inhibition of cycliO/CDK4.Nature, 1993; 366: 704-707.
23. Kmnb A, Gntis NA, Waever-Feldhaus J, et al. A cell regulator potentially involved genesis of many types. Science, 1994; 264: 436-440.
24. Ohtani N, Zebegee Z, Huot TJ, et al. Opposing effects of Ets and Id proteins on p16EST4KaINK4a expression during cellular senescence. Nature, 2001; 409: 1067-1070.
25. Herbig U, Sedivy JM. Regulation of rrowh arrest in senescence; Telomere damage is not the end of the dory. Mech Ageing Dev, 2006; 127: 16-24.
26. Iwasa H, Han J, Ishikawa F. Mitogen-activated protein kinase p3M defines the common senescence-signalling pathway. Genes Cells, 2003; 8: 131-144.
27. Wang W, Chen JX, Liao R, et al. Sequential activation of the MEK-extracellular signal-regulated kinase and MKK3/6-p38 mitogen-activated protein kinase pathways mediates oncogenic rasinduced premature senescence. Mol Cell Biol, 2002; 22: 3389-3403.
28. Passegue E, Wagner EF. Jun B suppresses cell proliferation by transcriptional activation of pl6IN4Ka (INK4a) expression. EMBO J, 2000; 19:2969-2979.
29. Itahana K, Zou Y, Itahana Y, et al. Control of the replicative life span of human fibroblasts by p16IN4Ka and the polycomb protein Bmi-1. Mol Cell Biol, 2003; 23: 389-401.
30. Gil J, Bernard D, Martinez D, et al. Polycomb CBX7 has a unifying role in cellular lifespan. Nat Cell Biol, 2004; 6: 67-72.
31.Dellambra E, Golisano O, Bondanza S, et al. Downregulation of 14-3-3 sigma prevents clonal evolution and leads to immortalization of primary human keratinocytes. J Cell Biol, 2000; 149: 1117-1130.
32. Friend S, Bernards R, Rogelj S, et al. A human DNA segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma. Nature, 1986; 323: 643-646.
33. Goodwish D, Wang NP, Qian YW, et al. The retinoblastoma gene product regulates progression through the Gl phase of the cell cycle. Cell, 1991; 67: 293-302.
34. Adams PD, KaelinY. Transcriptional control by E_2F. Semin Cancer Biol, 1995; 6: 99-108.
35. Kourarides T. Transcriptional control by the retinoblastome protein. Semin Cancer Biol, 1995; 6: 91-98.
36. Sage J, Miller A, Perez-mancera PA, et al. Acute mutationof retinoblastoma gene function is sufficient for cell cycle reentry. Nature, 2003; 424: 223-228.
37. Wei W, Herbig U, Wei S, et al. Loss of retinoblastoma but not p16IN4Ka function allows bypass of replicative senescence in human fibroblasts. EMBO Rep, 2003; 4: 1061-1065.
38. Alexander K, Yang HS, Hinds PW. pRb inactivation in senescent cells leads to an E2F-dependent apoptosis requiring p73. Mol Cancer Res, 2003; 1: 716-728.
39. Stein GH, Drullinger LF, Soulard A, et al. Differential roles for cyclin-dependent kinase inhibitors p21WAFl and p16IN4Ka in the mechanisms of senescence and differentiation in human fibroblasts. Mol Cell Biol, 1999,19: 2109-2117.
40. Wong DJ, Foster SA, Galloway DA, et al. Progressive region-specific de novo methylation of the p16IN4Ka CpG island in primary human mammary epithelial cell strains during escape from M(0) growth arrest. Mol Cell Biol, 1999; 19: 5642-5651
41.Beausejour CM, Katolica A, Galimi F, et al. Reversal of human cellular senescence; Roles of the p53 and p16IN4Ka pathways. EMBO J, 2003; 22: 4212-4222.
42. LaBaer J, Garrett M D, Stevenson L F, et al. New functional activities for the p21WAFl family of CDK inhibitors. Genes Dev, 1997; 11: 847-862.
43. Zhang H, Hannon G J, Beach D. p21WAF1-containing cyclin kinases exist in both active and inactive states. Genes Dev, 1994; 8: 1750-1758.
44. Sugrue MM, Shin DY, Lee SW, et al. Wild type p53 triggers a rapid senescence program in human tumors lacking functional p53. Proc Natl Acad Sci USA, 1997; 94: 9648 -9653.
45. Polyak K, Xia Y, Zweier JL, et al. Amodel for p53-induced apoptosis. Nature, 1997; 389: 300-305.
46. Sharpless NE. INK4a/ARF; A multifunctional tumor suppressorlocus. Mutat Res, 2005; 576: 22-38.
47. Pearson M, Carbone R, Sebastiani C, et al. PML regulates p53 acetylation and premature senescence induced by oncogenic Ras. Nature, 2000; 406: 207-210.
48. Lanqley E, Pearson M, Fareta M, et al. Human SIR2 deacetylates p53 and antagonizes PMUp53-induced cellular senescence. EMBO J, 2002; 21:2383-2396.
49. Bischof O, Nacerddine K, Dejean A. Human papillomavirus oncoprotein E7 targets the promyelocytic leukemia protein and circumvents cellular senescence via the Rb and p53 tumor suppressor pathways. Mol Cell Biol, 2005; 25: 1013-1024.
50. In HK, Bergmann S, Pandolfi PP. Cytoplasmic PML function in TGF-beta signalling. Nature, 2004; 431: 205-211.
51. Colombo E, Marine JC, Danovi D, et al. Nucleophosmin regulates the stability and transcriptional activity of p53. Nat Cell Biol, 2002; 4: 529-533.
52. Colombo E, Martinellip P, Zamponi R, et al. Delocalization and destabilization of the Arf tumor suppressor by the leukemia-associated NPM mutant. Cancer Res, 2006; 66: 3044-3050.
53. Goeman F, Thormeyer D, Abad M, et al. Growth inhibition by the tumor suppressor p331NG1 in immortalized and primary cells; Involvement of two silencing domains and effect of Ras. Mal Cell Biol, 2005; 25: 422-431.
54. Noda A, Ning Y, Venable SF, et al. Cloning of senescent cell-derived inhibitors of DNA synthesis using an expression screen. Exp. Cell Res., 211: 90-98, 1994.
55. Gretchen H. Stein, Linda F. Drullinger, Alexandre Soulard, et al. Differential Roles for Cyclin-Dependent Kinase Inhibitors p21WAFl and p16IN4Ka in the Mechanisms of Senescence and Differentiation in Human Fibroblasts. Mol Cell Biol, 1999; 19: 2109-2117.
56. Cheng M, Olivier P, Diehl JA, et al. The p21 WAF1 (Cip1) and p27 (Kip1) CDK "inhibitors" are essential activators of cyclin D-dependent kinases in murine fibroblasts. EMBO J, 1999; 18: 1571-1583.
57. Said TK, Medina D. Cell cyclins and cyclin-dependent kinase activities. Carcinogenesis, 1995; 16: 823-830.
58. Hartwel LH, Kastan MB.Cell cycle control and cancer. S cience, 1994; 266: 1821-1828.
59. Bartkova J, Lukas J, G oldberg P, et al .The pl6-cycbnD/CDK4-pRB pathway as a functional unit frequently altered in melanoma pathogenesis. Cancer Res, 1996; 56: 5475-5483.
60. Herbigb U, Wei W, Dutriaux A, et al. Real-time imaging of transcriptional activation in live cells reveals rapid up-regulation of the cyclin-dependent kinase inhibitor gene CDKNl A in replicative cellular senescence. Aging Cell, 2003; 2: 295-304.
61. Herbigb U, Jobling WA, Chen BP, et al. Telomere shortening triggers senescence of human cells through a pathway involving ATM, p53, and p21WAF1, but not pl6IN4Ka. Mol Cell, 2004; 14: 501-513.