Posttranscriptional stabilization underlies p53-independent induction of p21WAF1/CIP1/SDI1 in differentiating human leukemic cells.

p21WAF/CIP1/SDI1 is a recently identified gene expressed in cells harboring wild-type but not mutant p53 gene. It encodes a nuclear protein of 21 kD which inhibits cyclin-dependent kinase activity. Constitutive p21WAF1/CIP1/SDI1 mRNA expression was detected in neoplastic cells from patients with various hematological malignancies as well as in normal bone marrow mononuclear cells and in myeloid and lymphoid cell lines independent of their p53 status. Induced differentiation of the p53-deficient promyelocytic HL-60 cells along the monocytic lineage by phorbol ester or 1a,25 dihydroxyvitamin D3 resulted in a marked increase of both p21WAF1/CIP1/SDI1 mRNA and protein expression due to enhanced mRNA stability. Differentiation towards the granulocytic lineage by all-trans retinoic acid or dimethylsulfoxide failed to produce this effect. p21WAF1/CIP1/SDI1 is an immediate early gene since its upregulation occurred independently of de novo protein synthesis. The induction of p21WAF1/CIP1/SDI1 expression and its regulation in p53-deficient differentiating leukemic cells support the idea of an additional, p53-independent role of p21WAF1/CIP1/SDI1 in human hematopoiesis.     

Expression of p21(WAF1/CIP1/SDI1) and p53 in apoptotic cells in the adrenal cortex and induction by ischemia/reperfusion injury.

p21(WAF1/CIP1/SDI1), an inhibitor of cyclin-dependent kinases, is expressed at varying levels in human adrenal glands removed during surgery or organ recovery. In glands with p21 mRNA, nuclear p21 immunoreactivity, which was occasionally extensive, colocalized with p53 immunoreactivity and DNA damage, as evidenced by in situ end-labeling. Many cells showed morphological features of apoptosis when observed by fluorescent DNA dye staining and electron microscopy. This pattern was also associated with high levels of cytoplasmic heat shock protein 70. To address the question of the origin of p21 expression in some human adrenal glands, rat adrenal glands were subjected to 30 min of ischemia followed by 8 h of reperfusion. Cells with nuclear p21 and p53 appeared in the adrenal cortex together with DNA damage detected by in situ end-labeling. Nuclear p21 immunoreactivity was also produced in adrenal tissue fragments incubated at 37 degrees C in vitro. However, in this case, p21 expression was confined to the cut edge of the tissue. In contrast, p21 in human adrenal glands, as in ischemic rat glands, was within the inner regions of the cortex, supporting an origin of the protein in vivo rather than postmortem. The p53/p21 pathway of reaction to cellular injury, potentially leading to apoptosis, may play a role in tissue damage such as that resulting from ischemia/reperfusion. In the human adrenal cortex this process may be a precursor of adrenal failure.     

p53 suppression of arsenite-induced mitotic catastrophe is mediated by p21CIP1/WAF1

Arsenic trioxide, an acute promyelocytic leukemia chemotherapeutic, may be an efficacious treatment for other cancers. Understanding the mechanism as well as genetic and molecular characteristics associated with sensitivity to arsenite-induced cell death is key to providing effective chemotherapeutic usage of arsenite. Arsenite sensitivity correlates with deficient p53 pathways in multiple cell lines. The role of p53 in preventing arsenite-induced mitotic arrest-associated apoptosis (MAAA), a form of mitotic catastrophe, was examined in TR9-7 cells, a model cell line with p53 exogenously regulated in a tetracycline-off expression system. Arsenite activated G1 and G2 cell cycle checkpoints independently of p53, but mitotic catastrophe occurred preferentially in p53- cells. Cyclin B/CDC2(CDK1) stabilization and caspase-3 activation persisted in arsenite-treated p53- cells consistent with MAAA/mitotic catastrophe. N-Benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, a pan-caspase inhibitor, completely abolished arsenite-induced MAAA/mitotic catastrophe and greatly increased the mitotic index. WEE1 and p21CIP1/WAF1 inhibit cyclin B/CDC2 by CDC2 tyrosine-15 phosphorylation and direct binding, respectively. CDC2-Y15-P was transiently elevated in arsenite-treated p53+ cells but persisted in p53- cells. Arsenite induced p53-S15-P and p21CIP1/WAF1 only in p53+ cells. P21CIP1/WAF1-siRNA-treated p53+ cells were similar to p53- cells in mitotic index and cell cycle protein levels. p53-inducible proteins GADD45alpha and 14-3-3sigma are capable of inhibiting cyclin B/CDC2 but did not play a p53-dependent role in mitotic escape in TR9-7 cells. The data indicate that p53 mediates cyclin B/CDC2 inactivation and mitotic release directly via p21CIP1/WAF1 induction.     

AML1-ETO对p21 WAF1/CIP1启动子转录活性作用的研究

目的观察AML1-ETO融合基因对p21WAF1／CIP1基因启动子转录活性的影响，探讨AML1-ETO促进白血病发生的机制。方法构建p21WAF1／CIP1基因启动子的报告质粒，与AML1-ETO、AML1b和AML1a的表达质粒共转染非洲绿猴肾细胞系CV—1细胞，测定荧光素酶的活性，分析AML1-ETO、AML1b和AML1a对p21WAF1／CIP1基因启动子转录活性的影响。结果在CV—1细胞中，AML1-ETO对p21WAF1／CIP1基因启动子的转录具有明显的抑制作用，在pCMV5-AML1-ETO的剂量为1000ng时，p21WAF1／CIP1启动子的转录活性下降为对照组的（19±4）％，这种作用具有序列特异性和剂量依赖性；AML1b和AML1a对p21WAF1／CIP1基因启动子转录活性的抑制作用不明显，在剂量为1000ng时，p21WAF1／CIP1启动子的转录活性分别下降为对照组的（61±16）％和（594-16）％。结论AML1在与ETO形成融合基因后，其产物由于ETO蛋白能够更有效地募集转录共抑制复合物，其转录抑制活性要比AML1a和AML1b更强；外源的AML1-ETO对p21WAF1／CIP1的作用可能也与细胞系有关。     

Suppression of p53 activity and p21WAF1/CIP1 expression by vascular cell integrin alphaVbeta3 during angiogenesis.

Induction of p53 activity in cells undergoing DNA synthesis represents a molecular conflict that can lead to apoptosis. During angiogenesis, proliferative endothelial cells become apoptotic in response to antagonists of integrin alphavbeta3 and this leads to the regression of angiogenic blood vessels, thereby blocking the growth of various human tumors. Evidence is presented that administration of alphavbeta3 antagonists during angiogenesis in vivo selectively caused activation of endothelial cell p53 and increased expression of the p53-inducible cell cycle inhibitor p21WAF1/CIP1. In vitro studies revealed that the ligation state of human endothelial cell alphavbeta3 directly influenced p53 activity and the bax cell death pathway. Specifically, agonists of endothelial cell alphavbeta3, but not other integrins, suppressed p53 activity, blocked p21WAF1/CIP1 expression, and increased the bcl-2/bax ratio, thereby promoting cell survival. Thus, ligation of vascular cell integrin alphavbeta3 promotes a critical and specific adhesion-dependent cell survival signal during angiogenesis leading to inhibition of p53 activity, decreased expression of p21WAF1/CIP1, and suppression of the bax cell death pathway.     

Sensitizing HER2-overexpressing cancer cells to luteolin-induced apoptosis through suppressing p21(WAF1/CIP1) expression with rapamycin

HER2 overexpression, which confers resistance to various therapeutic regimens, correlates with a poor clinical prognosis. In this study, we showed that luteolin, a naturally occurring flavonoid, is a potent stimulator of HER2 degradation. Luteolin effectively inhibited cell proliferation and induced apoptosis in HER2-overexpressing cancer cells. Furthermore, we found that low doses of luteolin up-regulated p21 expression and high doses of luteolin down-regulated its expression. Examination of the Akt/mammalian target of rapamycin (mTOR) signaling revealed that this signaling was only transiently inhibited by low doses of luteolin, which suggested that the inability to cause sustained Akt/mTOR inhibition may contribute to p21 induction and provide a survival advantage to HER2-overexpressing cancer cells. To test this hypothesis, we showed that the combined use of luteolin and mTOR inhibitor rapamycin prevented low doses of luteolin from inducing p21 expression, and HER2-overexpressing cancer cells would be sensitized toward luteolin-induced apoptosis. In addition, p21 small interfering RNA also increased the luteolin-induced cell death. In nude mice with xenografted SKOV3.ip1-induced tumors, luteolin significantly inhibited HER2 expression and tumor growth in a dose-dependent manner, and rapamycin further enhanced the effect of luteolin with a concomitant p21 inhibition. These results reveal an intriguing finding that suppressing p21 expression might have therapeutic implications and further suggest that combination of mTOR inhibitors may be a promising strategy to help increase the efficacy of preventive or therapeutic compounds against HER2-overexpressing tumors.     

苯丁酸钠通过上调p21 WAF1/CIP1基因抑制白血病细胞系的细胞周期

目的研究组蛋白脱乙酰化酶(HDAC)抑制剂苯丁酸钠(PB)对白血病细胞系细胞周期的影响,探讨其分子机制.方法PB处理白血病细胞系Kasumi-1、U937和NB4细胞,分别于处理后24,48和72 h收集细胞.碘化丙锭DNA染色,流式细胞术分析细胞周期的变化.半定量逆转录-聚合酶链反应(RT-PCR)分析细胞周期相关基因p21WAF1/CIP1表达的变化.在人肾上皮细胞系293T细胞用荧光素酶报告基因分析PB对p21WAF1/CIP1基因启动子活性的影响.结果PB可以抑制Kasumi-1、U937和NB4细胞的细胞周期,作用呈时间和剂量依赖关系.3 mmol/L PB作用72 h,分别使Kasumi-1、U937和NB4细胞的G0/G1期细胞比例增加42.03%、44.36%和26.82%,S期细胞比例减少31.86%、38.91%和26.77%.PB使Kasumi-1、U937和NB4细胞p21WAF1/CIP1表达增高.PB处理后,p21WAF1/CIP1的表达水平较处理前增高(2.06±0.27),(2.78±0.40)和(1.78±0.20)倍.PB可以上调p21WAF1/CIP1启动子的转录活性,且呈剂量依赖关系.3 mmol/L PB处理48 h使转录活性增高(5.74±0.93)倍.PB上调p21WAF1/CIP1启动子转录活性主要是依赖于转录起始位点上游101 bp的序列.结论PB可以抑制白血病细胞系的细胞周期,这种作用可能是通过上调细胞周期相关基因p21WAF1/CIP1的表达实现的.     

The cyclin-dependent kinase inhibitor flavopiridol disrupts sodium butyrate-induced p21WAF1/CIP1 expression and maturation while reciprocally potentiating apoptosis in human leukemia cells

Interactions between the cyclin-dependent kinase inhibitor flavopiridol (FP) and the histone deacetylase inhibitor sodium butyrate (SB) have been examined in human leukemia cells (U937) in relation to differentiation and apoptosis. Whereas 1 mM of SB or 100 nM of FP minimally induced apoptosis (4% and 10%, respectively) at 24 h, simultaneous exposure of U937 cells to these agents dramatically increased cell death (e.g., approximately 60%), reflected by both morphological and Annexin/propidium iodide-staining features, procaspase 3 activation, and poly(ADP-ribose) polymerase cleavage. Similar interactions were observed in human promyelocytic (HL-60), B-lymphoblastic (Raji), and T-lymphoblastic (Jurkat) leukemia cells. Coadministration of FP opposed SB-mediated accumulation of cells in G0G1 and differentiation, reflected by reduced CD11b expression, but instead dramatically increased procaspase-3, procaspase-8, Bid, and poly(ADP-ribose) polymerase cleavage, as well as mitochondrial damage (e.g., loss of mitochondrial membrane potential and cytochrome c release). FP also blocked SB-related p21WAF1-CIP1 induction through a caspase-independent mechanism and triggered the caspase-mediated cleavage of p27KIP1 and retinoblastoma protein. The latter event was accompanied by a marked reduction in retinoblastoma protein/E2F1 complex formation. However, FP did not modify the extent of SB-associated acetylation of histones H3 and H4. Treatment of cells with FP/SB also resulted in the caspase-mediated cleavage of Bcl-2 and caspase-independent down-regulation of Mcl-1. Levels of cyclins A, D1, and E, and X-linked inhibitor of apoptosis also declined in SB/FP-treated cells. Finally, FP/SB coexposure potently induced apoptosis in two primary acute myelogenous leukemia samples. Together, these findings demonstrate that FP, when combined with SB, induces multiple perturbations in cell cycle and apoptosis regulatory proteins, which oppose leukemic cell differentiation but instead promote mitochondrial damage and apoptosis.     

Roles of the Tumor Suppressor p53 and the Cyclin-dependent Kinase Inhibitor p21WAF1/CIP1 in Receptor-mediated Apoptosis of WEHI 231 B Lymphoma Cells

Treatment of WEHI 231 immature B lymphoma cells with an antibody against their surface immunoglobulin M (anti-IgM) induces apoptosis and has been studied extensively as a model of self-induced B cell tolerance. Since the tumor suppressor protein p53 has been implicated in apoptosis in a large number of cell types and has been found to be mutated in a variety of B cell tumors, here we sought to determine whether p53 and the p53 target gene cyclin-dependent kinase inhibitor p21^WAF1/CIP1 were involved in anti-IgM–induced cell death. Anti-IgM treatment of WEHI 231 cells increased expression of p53 and p21 protein levels. Ectopic expression of wild-type p53 in WEHI 231 cells induced both p21 expression and apoptosis. Ectopic expression of p21 similarly induced apoptosis. Rescue of WEHI 231 cells from apoptosis by costimulation with CD40 ligand ablated the increase in p21 expression. Lastly, a significant decrease in anti-IgM–mediated apoptosis was seen upon downregulation of endogenous p53 activity by expression of a dominant-negative p53 protein or upon microinjection of an antisense p21 expression vector or antibody. Taken together, the above data demonstrate important roles for p53 and p21 proteins in receptor-mediated apoptosis of WEHI 231 B cells.     

P21WAF1/CIP1和细胞周期蛋白E在脂溢性角化病中的表达及意义

目的研究脂溢性角化病皮损组织中P21WAF1/CIP1和细胞周期蛋白E(Cyclin E)表达和意义.方法采用免疫组织化学SP法对45例脂溢性角化病皮损组织及20例正常皮肤组织中P21WAF1/CIP1和Cyclin E表达进行检测.结果脂溢性角化病皮损组织P21WAF1/CIP1和Cyclin E 阳性表达率及表达强度均明显高于正常皮肤组织(P<0.05);P21WAF1/CIP1和Cyclin E 表达与患者年龄、性别、肿瘤组织学类型均不相关(P>0.05).结论P21WAF1/CIP1和Cyclin E过度表达与脂溢性角化病的发生发展密切相关.     

Effect of the ANF analog A68828 in cisplatin-induced acute renal failure.

Experiments were conducted to determine the effects of the reduced-size atrial natriuretic factor (ANF) analog, A68828, on renal function in rats with cisplatin (CP)-induced acute renal failure. CP was given as a single intraperitoneal injection (7.5 mg/kg) 3 days before experiments. In separate groups of rats, the renal response to intravenous infusion of A68828 at 3, 10 or 30 micrograms/kg/min or ANF[1-28] at 0.03, 0.1 or 0.3 micrograms/kg/min for 2 hr was evaluated. Another group of CP-treated rats were infused with the vehicle (0.1% bovine serum albumin in 0.9% NaCl). CP treatment resulted in a marked decline in glomerular filtration rate (GFR), arterial pressure, heart rate and reabsorption of water and electrolytes compared to untreated control animals. Infusion of A68828 produced a dose-dependent improvement in the glomerular filtration rate. The highest dose of A68828 produced a nearly 3-fold increase in the glomerular filtration rate, whereas arterial pressure was decreased; heart rate was unchanged. Despite producing a significant diuresis and natriuresis, net tubular reabsorption of water and sodium was also increased. Similar dose-dependent effects were observed with the native peptide, ANF[1-28]. These data indicate that infusion of the reduced-sized analog of ANF, A68828, can significantly improve glomerular and tubular function in rats with acute renal failure induced by CP.     

Coexpression of p21(WAF1/CIP1) in adenovirus vector transfected human primary hepatocytes prevents apoptosis resulting in improved transgene expression

Replication-deficient adenovirus (Ad vector) is one of the most effective gene transfer systems. However, its employment in human gene therapy trials is hampered by Ad vector associated cytotoxicity and induction of apoptosis of the infected cells. Here, we identify one underlying mechanism as uncoupling of S phase and mitosis of the cell cycle leading to apoptosis and decline of transgene expression. Moreover, we demonstrate a strategy to avoid Ad vector associated cytotoxicity and induction of apoptosis in human primary hepatocytes by coinfection of Ad vector carrying the cDNA of choice and the cell cycle regulator p21(WAF1/CIP1) (p21). In addition, animal experiments were performed using Ad vector directed coexpression of p21 and human alpha 1-antitrypsin. As serum analysis of alpha 1-antitrypsin after Ad vector mediated gene transfer to the liver of mice revealed, this strategy resulted also in the improvement of transgene expression by two orders of magnitude. These data suggest that coexpression of p21 and Ad vector carrying a therapeutic gene may be a promising strategy to avoid cytotoxicity and induction of apoptosis leading to improved safety in human gene therapy.     

人脑胶质瘤组织中P21WAF/CIP1的异常表达及其病理学意义

目的探讨人脑胶质瘤组织P21WAF/CIP1的表达水平与胶质瘤恶性度的关系。方法免疫组化方法检测人脑胶质瘤标本41例,并对其表达水平进行评价。结果P21WAF/CIP1表达水平随胶质瘤恶性程度的升高呈下降趋势,但与组织学分级无相关性,全部受检标本P21WAF/CIP1染色强度无差异。结论胶质瘤组织P21WAF/CIP1表达呈异质性,与胶质瘤分级无相关性,但可参与胶质瘤的发生、发展。     

p21对缺血-再灌注损伤后肾小管上皮细胞演变的影响

目的 探讨p21对缺血-再灌注损伤（IRI）后肾小管上皮细胞演变的影响。方法 选择低龄（2个月龄）和高龄（12个月龄）p21（＋／＋）和p21（-／-）鼠，建立左肾IRI模型。于IRI后0、1、3、7d及1、3、6个月光镜下观察肾小管组织学变化，采用免疫组化法检测肾小管上皮细胞增殖细胞核抗原（PCNA）表达，组织化学染色观察肾小管上皮细胞衰老相关β-半乳糖苷酶（SA-β-gal）活力，末端脱氧核糖转移酶介导的生物素化脱氧尿嘧啶缺刻标记技术（TUNEL）检测肾小管上皮细胞凋亡。结果 IRI后0d，肾小管以坏死为主，高龄鼠比低龄鼠严重、p21（-／-）鼠比p21（＋／＋）鼠严重（P均〈0．05）。肾小管上皮细胞凋亡在IRI 1d后出现，7d达高峰，且高龄鼠比低龄鼠明显、p21（-／-）鼠比p21（＋／＋）鼠明显（P均d0．05）。低龄鼠IRI后1个月出现SA—β-gal染色阳性的肾小管上皮细胞，而对侧肾此时未见衰老细胞，3和6个月时衰老的肾小管上皮细胞显著增多，且p21（＋／＋）鼠比p21（-／-）鼠明显（P〈0．05）；p21（＋／＋）高龄鼠IRI后0d双肾即可见大量的SA-β-gal染色阳性肾小管上皮细胞，且较p21（-／-）鼠显著增多（P〈O．05），但1d后，p21（＋／＋）和p21（-／-）鼠IRI肾衰老细胞均明显减少（P均〈0．05），1个月后又呈进行性增加，且p21（＋／＋）鼠始终比p21（-／-）鼠严重。高龄和低龄p21（＋／＋）鼠PCNA阳性染色细胞出现的几率差异无显著性（P〉0．05），但低龄鼠细胞增殖能力要强于高龄鼠；而p21（-／-）鼠的细胞增殖能力明显强于p21（＋／＋）鼠，低龄鼠更为显著（P均〈0．05）。对高龄鼠IRI后1d细胞衰老和凋亡进行相关分析显示，二者呈显著负相关Cp21（＋／＋）鼠：r=-0．82，P〈0．001,p21（-／-）鼠：r=-0．76，P〈0．0013。结论 ①IRI可促进正常肾小管上皮细胞衰老的进程；②已经进入衰老状态的肾小管上皮细胞在遭受IRI刺激后，更易走向死亡[坏死和（或）凋亡]；③p21在IRI所致肾小管上皮细胞演变过程中发挥重要的调控作用。     

Tautomycetin inhibits growth of colorectal cancer cells through p21cip/WAF1 induction via the extracellular signal-regulated kinase pathway

Tautomycetin is an antifungal antibiotic retaining potent immunosuppressive function. We have identified the roles of tautomycetin on cellular proliferation and transformation of colorectal cancer cells. The proliferation and anchorage-independent growth of HCT-15, HT-29, and DLD-1 colorectal cancer cells were efficiently inhibited without induction of apoptosis by 150 nmol tautomycetin. These growth inhibitory effects were dependent on p21Cip/WAF induction via the extracellular signal-regulated kinase pathway, and the tautomycetin effects were abolished in HCT-116 colon cells and eight other types of cells that did not induce p21Cip/WAF by 150 nmol tautomycetin. The crucial role of p21Cip/WAF1 in the extracellular signal-regulated kinase pathway-dependent antiproliferative responses by tautomycetin was confirmed by using p21Cip/WAF1 gene-deleted HCT-116 cells. The growth inhibitory effect of tautomycetin was acquired by regulation of Raf-1 activity through inhibition of protein phosphatase type 1 and protein phosphatase type 2A with high preference toward protein phosphatase type 1. Tautomycetin could be a potential drug for colorectal cancer.     

骨髓间充质干细胞移植对顺铂诱导的急性肾损伤的保护作用

目的探讨外源性骨髓间充质干细胞（MSCs）移植对顺铂诱导的急性肾损伤的保护作用。方法取C57BL/6小鼠骨髓,梯度离心法分离MSCs,采用PKH-26标记。24只雄性小鼠建立顺铂诱导的肾损伤模型后随机分为正常组,对照组和移植组,移植组于顺铂注射后24 h经尾静脉注入MSC,对照组注入等量生理盐水。于术后3 d处死小鼠,留取血标本,观察血清肌酐（Cr）和尿素氮（BUN）水平。留取肾组织,荧光显微镜及HE染色方法观察移植MSC在肾组织中的分布及肾组织结构。结果术后第3天,移植组小鼠血清BUN,血清Cr水平明显低于生理盐水对照组;HE染色肾小管坏死和管型在移植组小鼠也明显低于生理盐水对照组。结论 MSC移植可促进急性肾损伤的修复。     

骨髓间充质干细胞移植对顺铂诱导的急性肾损伤的保护作用

目的探讨外源性骨髓间充质干细胞(MSCs)移植对顺铂诱导的急性肾损伤的保护作用。方法取C57BL/6小鼠骨髓,梯度离心法分离MSCs,采用PKH-26标记。24只雄性小鼠建立顺铂诱导的肾损伤模型后随机分为正常组,对照组和移植组,移植组于顺铂注射后24 h经尾静脉注入MSC,对照组注入等量生理盐水。于术后3 d处死小鼠,留取血标本,观察血清肌酐(Cr)和尿素氮(BUN)水平。留取肾组织,荧光显微镜及HE染色方法观察移植MSC在肾组织中的分布及肾组织结构。结果术后第3天,移植组小鼠血清BUN,血清Cr水平明显低于生理盐水对照组;HE染色肾小管坏死和管型在移植组小鼠也明显低于生理盐水对照组。结论 MSC移植可促进急性肾损伤的修复。