Gene expression profiling of p53-sensitive and -resistant tumor cells using DNA microarray

Overexpression of wild-type p53 in ECV-304 tumor cells induced extensive apoptosis and the eventual death of nearly all of the cells. We generated ECV-304 cells resistant to p53-induced apoptosis as a strategy to identify novel genes that might be relevant to p53-mediated apoptosis. ECV-304 cells resistant to p53 were isolated by repeated infections with a recombinant p53 adenovirus and were designated as DECV. The expression of 5,730 genes in p53-resistant (DECV) and p53-sensitive ECV-304 cells were profiled by DNA microarray analysis. We report here the expression of 80 genes that differed by 2-fold or more between sensitive and resistant cells upregulated for p53. Many of these differentially expressed genes are regulated by p53 in ECV-304 and H1299 p53-null cells. Our analysis identifies many new potential targets for p53 that play roles in cell cycle regulation, DNA repair, redox control, cell adhesion, apoptosis, and differentiation.     

Construction of NF-κB-targeting RNAi adenovirus vector and the effect of NF-κB pathway on proliferation and apoptosis of vascular endothelial cells

To construct a recombinant adenovirus vector expressing a RNAi for the Nuclear Factor kappa B (NF-κB)/p65 gene and use it to explore the role of the NF-κB pathway on the regulation of proliferation and apoptosis of vascular endothelial cells. A recombinant adenovirus containing a RNAi cassette targeting the p65 gene was constructed, and its silencing effect on p65 was detected by Western blot analysis in ECV304 cells. Expression of the p65 protein in ECV304 cells was efficiently down-regulated by the RNAi adenovirus for more than 6 days. ECV304 cells proliferation and apoptosis were measured using the MTT assay and flow cytometry, respectively. Blocking the NF-κB pathway with the RNAi adenovirus substantially decreased the proliferation of ECV304 cells, but only slightly affected cell apoptosis. We used a NF-κB/p65-targeting RNAi adenovirus to demonstrate the role of the NF-κB pathway in the regulation of ECV304 cell proliferation. This adenovirus may serve as an important tool to study the NF-κB pathway.     

siRNA靶向沉默p22phox表达对内皮细胞衰老抑制作用的研究

设计特异性siRNA(Short interference RNA)诱导人脐静脉内皮细胞株ECV-304细胞NAD(P)H氧化酶活性亚单位p22phox基因沉默, 探讨p22phox基因沉默在血管紧张素Ⅱ(AngⅡ)诱导的ECV-304衰老中的作用及机理。应用体外转录合成3种siRNA转染体外培养的ECV-304, RT-PCR鉴定对p22phox基因沉默的效率和特异性, 确立适宜的转染浓度和基因沉默的持续时间; ECV-304分为空白对照组、AngⅡ组、siRNA转染组、AngⅡ+siRNA转染组, 观察细胞衰老改变及活性氧水平, 分析各组细胞p22phox的mRNA及蛋白表达。结果表明: 3种siRNA中, 一种对p22phox mRNA表达抑制率达到83％, 在一定转染浓度范围内, siRNA诱导的基因沉默效率呈剂量依赖性, 抑制效率高峰期在24~36 h; 给予AngⅡ后, b-gal染色阳性细胞数显著增加, 出现衰老的特征性改变, 衰老细胞p22phox的 mRNA及蛋白表达增加, 伴有一氧化氮(NO)生成减少, 活性氧生成增加, siRNA诱导p22phox基因沉默后降低了活性氧水平, 增加NO生成, 改善了AngⅡ诱导的ECV-304细胞的衰老改变。siRNA干扰技术可成功诱导NAD(P)H氧化酶p22phox基因沉默, 从而减缓AngⅡ诱导体外培养的ECV-304衰老进程, p22phox是防治衰老有希望的分子靶点。     

Transduction of Apaf-1 or caspase-9 induces apoptosis in A-172 cells that are resistant to p53-mediated apoptosis

p53 replacement gene therapy has been carried out clinically for cancers with p53 mutations; however, some cancers are resistant to p53 gene therapy. In this study, we transduced A-172 and U251 cells harboring p53 mutations with wild-type p53 using adenovirus vectors to induce wild-type p53 protein at similar expression levels. A-172 cells did not undergo apoptosis after p53 transduction, whereas U251 cells were markedly sensitive to p53-mediated apoptosis. A-172 cells showed higher endogenous expression of Bcl-X(L) than U251, and transduction of Bcl-X(L) repressed p53-mediated apoptosis in U251 cells, suggesting that high endogenous expression of Bcl-X(L) renders A-172 cells, at least in part, resistant to p53-mediated apoptosis. We transduced A-172 cells and U251 cells with the Apaf-1 or caspase-9 genes; both are downstream components of p53-mediated apoptosis. We found that A-172 cells were highly sensitive to Apaf-1- and caspase-9-mediated apoptosis. The results indicate that A-172 cells harboring mutant p53 were not susceptible to p53-mediated apoptosis, possibly due to high endogenous expression of Bcl-X(L). Transduction of Apaf-1 or caspase-9 would override the resistance mechanism of apoptosis in A-172 cells. These findings provide potentially a novel approach in killing cancers that are resistant to p53 replacement gene therapy.     

Apoptosis induced by adenovirus-mediated p14ARF expression in U2OS osteosarcoma cells is associated with increased Fas expression

The INK4A/ARF locus on chromosome 9 is a tumor suppressor gene frequently mutated in human cancers. In order to study the effects of p14ARF expression in tumor cells, we constructed a recombinant adenovirus containing p14ARF cDNA (Adp14ARF). Adp14ARF infection of U2OS osteosarcoma cells which has wild type p53 and mutant p14ARF revealed high levels of p14 (ARF) expression within 24h. In addition, Adp14ARF-mediated expressing of p14 (ARF) was associated with increased levels of p53, p21, and mdm2 protein. Growth inhibition assays following Adp14ARF infection demonstrated that the growth of U2OS cells was inhibited relative to infection with control virus. Furthermore, TUNEL analysis as well as PARP cleavage assays demonstrated that Adp14ARF infection was associated with increased apoptosis in U2OS cell line and that it was associated with Adp14ARF induced overexpression of Fas and Fas-L. Addition of Fas-L neutralizing antibody NOK-1 decreased Adp14-mediated cell death, indicating that p14 (ARF) induction of the Fas pathway is associated with increased apoptosis. The finding that Adp14ARF infection did not induce Fas expression in U2OS/E6 and MCF/E6 cells suggests that wild type p53 expression may be necessary for Adp14ARF-mediated induction of Fas. The observation that overexpression of p53 by Adp53 infection in MCF-7 does not induce increased Fas protein levels nor apoptotic cell death suggests that p53 overexpression is required but not sufficient enough for apoptosis. These studies suggest there are other mechanisms other than induction of p53 in ARF-mediated apoptosis and gene therapy using Adp14ARF may be a promising treatment option for human cancers containing wild type p53 and mutant or deleted p14 expression.     

Preparation of isotopically labelled recombinant β-defensin for NMR studies

Apoptosis is a major problem in animal cell cultures during production of biopharmaceuticals, such as recombinant proteins or viral vectors. A 293 cell line constitutively expressing vMIA (viral mitochondria-localized inhibitor of apoptosis) was constructed and examined on production of a model recombinant protein, green fluorescent protein (GFP) in the adenovirus-293 expression system, and on production of a model infectious adenoviral vector. vMIA-293 cells were more resistant than the parental 293 cells to apoptosis induced by either oxidative stress, or by adenovirus infection. The yield of GFP produced in vMIA-293 cell cultures was consistently higher (140%) compared to that in the parental cells. vMIA reduced production of adenovirus infectious particles, which was not due to a decline of adenovirus replication, since adenoviral DNA replication rate in vMIA-293 cells was higher than that in the parental cells.In conclusion, introduction of the vMIA gene into the 293 cell line is a promising strategy to improve recombinant protein production in the adenovirus-293 expression system.     

Phenotypic alterations of small cell lung carcinoma induced by different levels of wild-type p53 expression

p53 induces both growth arrest and apoptosis in cancer cells. To clarify whether the level of p53 expression determines the response of small cell lung carcinoma (SCLC) cells, we assessed the effect of various p53 levels on a p53-null SCLC cell line, N417, using a tetracycline (Tc)-regulated inducible p53 expression system. Apoptosis was induced in SCLC cells with high p53 expression. Although low levels of p53 induced G1 arrest accompanied by p21 expression, cells with G1 arrest seemed to undergo apoptosis after further cultivation. Expression of exogenous p21 induced G1 arrest but not apoptosis in SCLC cells, suggesting that p53-mediated G1 arrest was induced through p21 expression. Moreover, high level of p53 expression down-regulated Bcl-2 expression in SCLC cells, while Bax was consistently expressed irrespective to the level of p53 expression. These results suggest that p53-mediated apoptosis and G1 arrest depend on level of p53 expression in SCLC cells and that the relative dominancy of Bax to Bcl-2 is involved in the induction of apoptosis by high level of p53 expression.     

异构体ΔASPP2可拮抗ASPP2对p53（细胞）凋亡功能的增强作用

p53 凋亡刺激蛋白2(apoptosis stimulating protein 2 of p53, ASPP2)能够与p53 蛋白结合特异性地增强其促细胞凋亡功能,进而发挥肿瘤抑制作用．我们发现的1个比ASPP2少300多个N端氨基酸的异构体ΔASPP2.目前,ΔASPP2对p53起何种作用尚不清楚．在本研究中,我们构建了rAd-ASPP2、rAd-ΔASPP2腺病毒,利用rAd-p53、rAd-ASPP2、rAd-ΔASPP2 感染p53缺失的细胞系H1299,在MMS的作用下研究ASPP2 和 ΔASPP2 对p53介导的细胞凋亡的影响．结果发现,p53自身过表达能明显促进肿瘤细胞的凋亡;ASPP2可显著增强p53介导的MMS引起的H1299细胞凋亡的作用;然而,ΔASPP2对p53介导的细胞凋亡没有明显影响但却显著抑制rAd-ASPP2 增强的rAd-p53的促细胞凋亡作用．p53-ASPP2 复合体可能改变p53 蛋白的构象,促进p53 和增强子Bax的结合活性．p53 转录调控基因的表达研究显示,ΔASPP2的存在可显著抑制ASPP2增强p53 介导的bax基因转录活性, 提示ΔASPP2可能与ASPP2结合后来抑制p53的凋亡基因转录活性．     

Placental transforming growth factor-beta is a downstream mediator of the growth arrest and apoptotic response of tumor cells to DNA damage and p53 overexpression

The p53 tumor suppressor gene and members of the transforming growth factor-beta (TGF-beta) superfamily play central roles in signaling cell cycle arrest and apoptosis (programmed cell death) in normal development and differentiation, as well as in carcinogenesis. Here we describe a distantly related member of the TGF-beta superfamily, designated placental TGF-beta (PTGF-beta), that is up-regulated in response to both p53-dependent and -independent apoptotic signaling events arising from DNA damage in human breast cancer cells. PTGF-beta is normally expressed in placenta and at lower levels in kidney, lung, pancreas, and muscle but could not be detected in any tumor cell line studied. The PTGF-beta promoter is activated by p53 and contains two p53 binding site motifs. Functional studies demonstrated that one of these p53 binding sites is essential for p53-mediated PTGF-beta promoter induction and specifically binds recombinant p53 in gel mobility shift assays. PTGF-beta overexpression from a recombinant adenoviral vector (AdPTGF-beta) led to an 80% reduction in MDA-MB-468 breast cancer cell viability and a 50-60% reduction in other human breast cancer cell lines studied, including MCF-7 cells, which are resistant to growth inhibition by recombinant wild-type p53. Like p53, PTGF-beta overexpression was seen to induce both G(1) cell cycle arrest and apoptosis in breast tumor cells. These results provide the first evidence for a direct functional link between p53 and the TGF-beta superfamily and implicate PTGF-beta as an important intercellular mediator of p53 function and the cytostatic effects of radiation and chemotherapeutic cancer agents.     

Human lung carcinoma (A-549) continuing cell line and human endothelial (ECV-304) continuing cell line responses to the influenza virus at different multiplicities of infection

The course of infection upon virus entry into the cell depends not only on the biological characteristics of the cells and of the virus itself, but also on the intensity of the cell infection by the virus, i.e., on the multiplicity of infection. The purpose of our work was to perform a comparative study of the responses of two human cell lines, the lung carcinoma cell line A-549 and the endothelium cell line ECV-304, to the infection with the influenza virus A at different multiplicities of infection. At the first passage, both cell lines responded by enhancement of proliferation and apoptosis induction only to the low doses of influenza virus (ID 1–10). In A-49 cells, the stimulatory effect of the low virus doses was observed 1–2 days earlier than in ECV-304 cells. Enhanced proliferation was observed in both cell lines from the second to the fourth passages, when cells were infected with higher virus doses (ID 100 and 1000). In addition, the response of the A-549 cells to low doses of the H3N2 strain of the influenza virus A depended on the virus propagation conditions—namely, no enhancement of cell proliferation was observed in response to the infection with the virus propagating in chicken embryonated eggs, in contrast to infection with the virus that propagated in cell culture. Immunocytochemistry of A-549 cells has demonstrated that, on the third day after infection, there could be observed a change (in the dose-dependent manner) in the intracellular localization of p53 and cyclin A, proteins involved in the cell cycle progression. At the low virus dose, cyclin A was predominantly detected in the nuclei (63%), while at the high virus dose it was p53 (54%), which was predominantly detected in this cellular compartment, this observation confirming that stimulation of cell proliferation in the case of very low multiplicity of infection and cell division arrest takes place in the case of high multiplicity of influenza virus infection. The study of the influenza virus A reproduction in A-549 and ECV-304 cells using a whole number of virology techniques showed low sensitivity of these cells to the influenza virus, which manifested in the gradual decrease in the viral RNA expression and the impairment of mature viral particles assembly during several passages. Therefore, the decrease in the multiplicity of infection is associated in the A-549 and ECV-304 cells with impairment of production of mature virus particles or certain virus protein synthesis, which is accompanied by cell proliferation enhancement and apoptosis induction. As a result of the comparative study of the two cell lines (A-549 and ECV-304) upon infection with different doses of influenza virus A, we have revealed common principles and specific features indicating the effects of the biological properties of the viruses and cells, as well as of the multiplicity of infection on the course of virus infection.     

A human endothelial cell feeder system that efficiently supports the undifferentiated growth of mouse embryonic stem cells

Feeder cells are commonly used to culture embryonic stem cells to maintain their undifferentiated and pluripotent status. Conventionally, mouse embryonic fibroblasts (MEFs), supplemented with leukemia inhibitory factor (LIF), are used as feeder cells to support the growth of mouse embryonic stem cells (mESCs) in culture. To prepare for fresh MEF feeder or for MEF-conditioned medium, sacrifice of mouse fetuses repeatedly is unavoidable in these tedious culture systems. Here we report the discovery of a human endothelial cell line (ECV-304 cell line) that efficiently supports growth of mESCs LIF-free conditions. mESCs that were successfully cultured for eight to 20 passages on ECV-304 feeders showed morphological characteristics similar to cells cultured in traditional feeder cell systems. These cells expressed the stem cell markers Oct3/4, Nanog, Sox2, and SSEA-1. Furthermore, cells cultured on the ECV-304 cell line were able to differentiate into three germ layers and were able to generate chimeric mice. Compared with traditional culture systems, there is no requirement for mouse fetuses and exogenous LIF does not need to be added to the culture system. As a stable cell line, the ECV-304 cell line efficiently replaces MEFs as an effective feeder system and allows the efficient expansion of mESCs.     

Recombinant adenovirus-mediated p14(ARF) overexpression sensitizes human breast cancer cells to cisplatin

p14(ARF), the alternative product from the human INK4a/ARF locus, is one of the major targets for alterations in the development of human cancers. Overexpression of p14(ARF) results in cell cycle arrest and apoptosis. To examine the potential therapeutic role of re-expressing p14(ARF) gene product in human breast cancer, a recombinant adenovirus expressing the human p14(ARF) cDNA (Adp14(ARF)) was constructed and used to infect breast cancer cells. Five days after infection, Adp14(ARF) had considerable cytotoxicity on p53-wild-type MCF-7 cells. A time-course study showed that Adp14(ARF) infection of MCF-7 cells at 100pfu/cell increased the number of cells in G0/G1 phase and decreased that in S and G2/M phases. The presence of apoptotic cells was confirmed using the TUNEL assay. Adp14(ARF)-mediated expression of p14(ARF) also resulted in a considerable increase in the amounts of p53 and its target proteins, p21(WAF1) and MDM2. Furthermore, the combination treatment of MCF-7 cells with Adp14(ARF) and cisplatin resulted in a significantly greater cell death. Together, we conclude that p14(ARF) plays an important role in the induction of cell cycle arrest and apoptosis in breast cancer cells and recombinant adenovirus-mediated p14(ARF) expression greatly increases the sensitivity of these cells to cisplatin. These results demonstrate that the proper combination of Adp14(ARF) with conventional chemotherapeutic drug(s) could have potential benefits in treating breast cancer that carries wild-type p53 gene.     

Bcl-2 blocks p53-dependent apoptosis.

Adenovirus E1A expression recruits primary rodent cells into proliferation but fails to transform them because of the induction of programmed cell death (apoptosis). The adenovirus E1B 19,000-molecular-weight protein (19K protein), the E1B 55K protein, and the human Bcl-2 protein each cause high-frequency transformation when coexpressed with E1A by inhibiting apoptosis. Thus, transformation of primary rodent cells by E1A requires deregulation of cell growth to be coupled to suppression of apoptosis. The product of the p53 tumor suppressor gene induces apoptosis in transformed cells and is required for induction of apoptosis by E1A. The ability of Bcl-2 to suppress apoptosis induced by E1A suggested that Bcl-2 may function by inhibition of p53. Rodent cells transformed with E1A plus the p53(Val-135) temperature-sensitive mutant are transformed at the restrictive temperature and undergo rapid and complete apoptosis at the permissive temperature when p53 adopts the wild-type conformation. Human Bcl-2 expression completely prevented p53-mediated apoptosis at the permissive temperature and caused cells to remain in a predominantly growth-arrested state. Growth arrest was leaky, occurred at multiple points in the cell cycle, and was reversible. Bcl-2 did not affect the ability of p53 to localize to the nucleus, nor were the levels of the p53 protein altered. Thus, Bcl-2 diverts the activity of p53 from induction of apoptosis to induction of growth arrest, and it is thereby identified as a modifier of p53 function. The ability of Bcl-2 to bypass induction of apoptosis by p53 may contribute to its oncogenic and antiapoptotic activity.     

Adenovirus-mediated gene transfer of the tumor suppressor, p53, induces apoptosis in postmitotic neurons

Programmed cell death is an ongoing process in both the developing and the mature nervous system. The tumor suppressor gene, p53, can induce apoptosis in a number of different cell types. Recently, the enhanced expression of p53 has been observed during acute neurological disease. To determine whether p53 overexpression could influence neuronal survival, we used a recombinant adenovirus vector carrying wild type p53 to transduce postmitotic neurons. A control consisting of the same adenovirus vector background but carrying the lacZ reporter expression cassette was used to establish working parameters for the effective genetic manipulation of sympathetic neurons. We have found that recombinant adenovirus can be used at titers sufficiently high (10 to 50 multiplicity of infection) to transduce the majority of the neuronal population without perturbing survival, electrophysiological function, or cytoarchitecture. Moreover, we demonstrate that overexpression of wild type p53 is sufficient to induce programmed cell death in neurons. The observation that p53 is capable of inducing apoptosis in postmitotic neurons has major implications for the mechanisms of cell death in the traumatized mature nervous system.     

重组p16腺病毒的构建及其对人白血病细胞的抑制作用

为了探讨腺病毒载体用于基因治疗的可行性及野生型 p1 6基因的抗肿瘤特性 ,构建了复制缺陷型重组 p1 6腺病毒 .首先将 p1 6全长 c DNA插入穿梭质粒 p Ad CMV产生重组质粒 p Ad-CMV- p1 6,然后通过脂质体介导与 p JM1 7共转染 2 93细胞 ,经同源重组产生 E1区缺失的重组腺病毒空斑 .用纯化后的腺病毒感染人白血病细胞株 HL- 60后 ,PCR及 Western blot分析显示在感染细胞中有外源性 p1 6 c DNA存在和 p1 6蛋白表达 ;被感染的 HL- 60细胞的生长受到明显抑制 ,而未感染细胞及对照腺病毒感染的细胞没有受到抑制 .结果表明 ,腺病毒作为一种新型基因转移载体 ,可有效地介导肿瘤抑制基因 p1 6的表达 ,在肿瘤基因治疗方面具有很大的应用前景 .