一种新的重组腺病毒的构建及其对人肿瘤细胞的影响

腺病毒E1A基因诱导细胞凋亡．E1B19K基因及E1B55K基因抑制细胞凋亡,前者被克隆到腺病毒转移载体pCA13的HCMVIE启动子下游．构建成转移载体pCAE1A。采用lipofectin法将PCAE1A和含腺病毒基因组（E1、E3区缺失）的质粒pBHG11共转染293细胞,7～10d后得到重组病毒v5Ad4。用v5Ad4感染人肺腺癌细胞系A549,结果表明v5Ad4有明显杀伤和裂解肿瘤细胞功能。在人胚肺正常二倍体细胞中,v5Ad4没有表现出可见的细胞毒效应。     

Adenovirus type 5 E1A immortalizes primary rat cells expressing wild-type p53

Adenovirus (Ad) E1A induces apoptosis in cells expressing wild-type p53, and stable transformation by Ad E1A requires the co-introduction of an anti-apoptotic gene such as Ad E1B 19K. Thus, cells immortalized by Ad E1A alone might have lost functional p53. In order to analyze the p53 in rat cells expressing Ad E1A, we established rat cell lines by transfecting primary rat embryo fibroblast (REF) and baby rat kidney (BRK) cells with cloned Ad5 E1A. By using a yeast functional assay, we analyzed p53 in six primary REF and three BRK cell lines immortalized by Ad5 E1A as well as five spontaneously immortalized rat cell lines (REF52, NRK, WFB, Rat-1 and 3Y1). The yeast functional assay revealed that all of the spontaneously and Ad5 ElA-immortalized rat cell lines except for 3Y1 expressed wild-type p53. All of the Ad5 E1A-immortalized rat cell lines contained p53 detectable by immunoprecipitation. Recombinant adenovirus expressing rat p53 cloned from a REF cell line immortalized by Ad5 E1A, as well as that expressing murine wild-type p53, induced apoptosis in p53-null cells in collaboration with E1A. Thus, it is suggested that the mutation of p53 appears to be not frequent in the spontaneous immortalization of primary rat cells, and that the functional loss of wild-type p53 is not a prerequisite of E1A-mediated immortalization.     

Alterations in cellular phenotype induced by the type 5 adenovirus E1A and the beta 1 protein kinase C genes in cloned rat embryo fibroblast cells.

The E1A gene of adenovirus type 5 (Ad5) induces morphological transformation and anchorage-independent growth in cloned rat embryo fibroblast (CREF) cells. In contrast, CREF cells transfected with a beta 1 protein kinase C (PKC) gene and expressing low-levels of beta 1 PKC display a CREF-like morphology and do not form colonies when grown in agar. The combination of Ad5 E1A and low-level beta 1 PKC expression in the same CREF cell line results in an enhanced ability to grow when suspended in agar. In Ad5 E1A and Ad5 E1A + low-level beta 1 PKC expressing CREF clones, the tumor promoting agent 12-0-tetradecanoyl-phorbol-13-acetate (TPA) further enhances anchorage-independence. In contrast, TPA does not induce CREF cells or transfected CREF cells expressing low-levels of beta 1 PKC to grow in agar. Low-level beta 1 PKC expression in transfected CREF cells is associated with a modest 1.2 to 1.6-fold increase in binding of [3H]-phorbol-12,13-dibutyrate (PDBu) and only a 2.3-fold increase in PKC enzymatic activity. In contrast, specific beta 1 PKC-retroviral vector transformed CREF clones (CREF-RV-PKC) display higher levels of PKC mRNA, PDBu binding and PKC enzymatic activity. A majority of CREF-RV-PKC clones exhibit a transformed morphology and grow more rapidly in monolayer culture, form macroscopic colonies in agar in the absence of TPA and in many independent clones TPA further enhances anchorage-independent growth. This effect is not directly related to the level of enhanced [3H]-PDBu binding. The present study indicates that the effect of beta 1 PKC on cellular phenotype in immortal rat embryo cells is complex and is affected by its mode of insertion into CREF cells, i.e. transfection versus retroviral insertion. In addition, the combination of a transfected Ad5 E1A and a beta 1 PKC gene in the same CREF clone results in an enhanced expression of the transformed phenotype in both the absence and presence of TPA.     

Virology- and immunology-based gene therapy for cancer

Current strategies for cancer gene therapy consist mainly of direct inhibition of tumor cell growth and activation of systemic host defense mechanisms. Conventional chemotherapy and radiotherapy, even considered to be temporally suppressing tumor growth, suppress immune responses; therefore, we examined potential clinical feasibility of virus-mediated tumor destruction, which can rather enhance immunity. We showed that human tumors were more susceptible to adenoviruses (Ad) in which the E1A expression was controlled by a putative tumor promoter than normal cells, and that a replication of the Ad was greater in tumor cells than in normal cells. We also demonstrated that the intratumoral injection of the Ad bearing a tumor promoter inhibited the subsequent tumor growth in vivo. The E1A expression was detected in the tumors injected with the Ad but not in non-tumorous tissues of the same mice. The Ad modified to show the regulated E1A expression is thereby oncolytic in nature. Antitumor immune responses are initiated after the acquisition of putative tumor antigen(s) by dendritic cells (DCs); therefore, enhanced antigen presentation is a crucial step for the early phase of cell-mediated immunity. Destruction of tumors can release the tumor antigens and DCs come to recognize them thereafter. We found that the stimulation of Fas expressed on DCs with Fas ligand (FasL) did not induce apoptosis of DCs but rather enhanced the antigen presentation. Activation of DCs induced production of a number of cytokines, and we showed that the interleukin-12 family secreted from tumors could induce systemic antitumor immunity. We presume that the administration of oncolytic Ad, which can destroy local tumors and subsequently make the putative tumor antigen(s) released from the tumors, stimulation of DCs with the Fas/FasL signal pathway and secretion of DCs-derived cytokines coordinately produce synergistic antitumor effects and that a combinatory application of these procedures can be a possible therapeutic strategy for cancer treatment.This article is a symposium paper from the Annual Meeting of the “International Society for Cell and Gene Therapy of Cancer” held in Shenzhen, China, on 9–11 December 2005.     

A pair of adjacent genes, cry5Ad and orf2-5Ad, encode the typical N- and C-terminal regions of a Cry5Aδ-endotoxin as two separate proteins in Bacillus thuringiensis strain L366

A new DNA sequence cry5Ad/orf2-5Ad (GenBank accession number EF219060 ) was isolated from Bacillus thuringiensis strain L366. This DNA sequence contains two ORFs: cry5Ad (a previously unreported member of the cry5A gene family) and orf2-5Ad . cry5Ad is unique among cry5A genes in that it encodes only the N-terminal region of a typical Cry5Aδ-endotoxin. The cry5Ad sequence includes homology blocks 1–5, which are present in most B. thuringiensis δ-endotoxins. The usual C-terminal region of a Cry5Aδ-endotoxin (including homology blocks 6–8) is encoded by orf2-5Ad . Both proteins encoded by cry5Ad and orf2-5Ad were found in IPTG-induced Escherichia coli , after a copy of cry5Ad/orf2-5Ad was cloned into the pQE32 expression vector and transformed into pREP4 E. coli cells. Both proteins were also found in parasporal crystal inclusions of B. thuringiensis L366. Sequencing of cDNA derived from transformed E. coli cells showed that the two ORFs are transcribed as a single mRNA. Extracts prepared from the recombinant E. coli expressing Cry5Ad and Orf2-5Ad were not toxic to nematode larvae ( Haemonchus contortus ), indicating that these two proteins are most likely not responsible for the nematocidal activity seen previously in the B. thuringiensis strain L366.     

E1A 12S and 13S of the transformation-defective adenovirus type 12 strain CS-1 inactivate proteins of the RB family, permitting transactivation of the E2F-dependent promoter.

The transformation-defective Vero cell host range mutant CS-1 of the highly oncogenic adenovirus type 12 (Ad12) (Ad12-CS-1) has a 69-bp deletion in the early region 1A (E1A) gene that removes the carboxy-terminal half of conserved region 2 and the amino-terminal half of the Ad12-specific so-called spacer that seems to play a pivotal role in the oncogenicity of the virus. Despite its deficiency in immortalizing and transforming primary rodent cells, we found that the E1A 13S protein of Ad12-CS-1 retains the ability to bind p105-RB, p107, and p130 in nuclear extract binding assays with glutathione S-transferase-E1A fusion proteins and Western blot analysis. Like wild-type E1A, the mutant protein was able to dissociate E2F from retinoblastoma-related protein-containing complexes, as judged from gel shift experiments with purified 12S and 13S proteins from transfection experiments with an E1A expression vector or from infection with the respective virus. Moreover, in transient expression assays, the 12S and 13S products of wild-type Ad12 and Ad12-CS-1 were shown to transactivate the Ad12 E1A promoter containing E2F-1 and E2F-5-motifs, respectively, in a comparable manner. The same results were obtained from transfection assays with the E2F motif-dependent E2 promoter of adenovirus type 5 or the human dihydrofolate reductase promoter. These data suggest that efficient infection by Ad12 and the correlated virus-induced reprogramming of the infected cells, including the induction of cell cycle-relevant mechanisms (e.g. E2F activation), can be uncoupled from the transformation properties of the virus.     

Enhanced TRAIL sensitivity by E1A expression in human cancer and normal cell lines: inhibition by adenovirus E1B19K and E3 proteins

TNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily of cytokines that induces apoptosis in a variety of cancer cells, but not in normal cells. However, more and more tumor cells remain resistant to TRAIL, which limited its application for cancer therapy. Expression of the adenovirus serotype 5 (Ad5) E1A sensitizes tumor cells to apoptosis by TNF-alpha, Fas-ligand, and TRAIL. Here we asked whether E1A overcomes this resistance and enhances TRAIL-induced apoptosis in the tumor cells. Our results revealed that the tumor cell lines, HeLa and HepG2, with infection by Ad-E1A, were highly sensitive to TRAIL-induced apoptosis. Importantly, we found that in normal primary human lung fibroblast cells (HLF) TRAIL is capable of inducing apoptosis in combination with E1A as efficiently as in some tumor cell lines. The adenovirus type 5 encoding proteins, E1B19K and E3 gene products, have been shown to inhibit E1A and TRAIL-induced apoptosis of HLF cells by using the recombinant adenovirus AdDeltaE1B55K, with mutation of E1B55K, containing E1B19K and complete E3 region. Further results demonstrated that the expression of DR5 and TRAIL was down-regulated in the AdDeltaE1B55K co-infected HLF cells. These findings suggest that TRAIL may play an important role in limiting virus infections and the ability of adenovirus to inhibit killing may prolong acute and persistent infections. The results from this study have also suggested the possibility that the combination of E1A with TRAIL could be used in the treatment of human malignancy, or in the selection of the optimal adenovirus mutant as effective delivering vector for cancer therapy.     

How reliable is amino acid sequence homology in predicting similarity of structure and function of c-myc and Ad12 E1A oncogenic proteins?

Summary Adenovirus E1A and c-myc genes are known to be capable of transforming primary rat cells when they occur in combination with either polyoma middle-T or T24 Harvey-ras 1 genes. There was a low level of amino acid sequence homology between the nuclear adenovirus-12 (Ad12) E1A protein product (289 amino acids) and the c-myc protein based on optimal alignment and percentage identity. In contrast to others [Ralston R, Bishop JM (1983) Nature 306:803–806], we concluded that this low level of amino acid sequence homology was not significant, since rabies glycoprotein (RGP), which has no transforming function and localizes to the cell surface, had a similar low level of amino acid sequence homology to the c-myc protein. Furthermore, dot-matrix analysis, when used to test the overall level of amino acid sequence homology, showed no significant homology between c-myc and Ad12 E1A, E1B, or RGP. Thus, low levels of amino acid sequence homology between two proteins may not be sufficient to predict structural and functional similarities between them reliably, even if the two proteins appear to share a common function.     

E1A基因转导抑制小鼠血管内皮细胞的激活

延缓性排斥反应 (delayedxenograftrejection ,DXR)是进行异种器官移植亟待解决的问题之一 .在DXR过程中 ,核心事件之一是核转录因子NF κB的激活 .人腺病毒 5 (Ad5 )的早期转录产物E1A蛋白可抑制以NF κB为核心的信号转导系统 .利用转基因技术向小鼠的受精卵导入了E1A基因 ,PCR和Southern印迹检测了 4 4只仔鼠 ,其中有 8只整合了E1A基因 .RT PCR检测发现 ,3只小鼠F1代的心、肝、肾等脏器都有E1AmRNA的表达 ,小鼠表型正常 .通过尾静脉向转基因阳性小鼠体内注射人灭活血清 ,模拟DXR发生的生理过程并用免疫荧光检测脏器细胞表面炎症分子E 选择素的表达水平 .结果表明 ,E1A基因的转导显著抑制了小鼠脏器细胞表面E 选择素的表达 ,为解决DXR的发生提供了可行的途径     

腺病毒5型E1A基因对永生化人支气管上皮细胞生长的抑制作用及其机理

腺病毒 5型早期区 1 A( Ad5E1 A)基因是新近发现的一个肿瘤抑制基因 .其产物 E1 A蛋白是多功能转录因子 ,它能从正、负 2个途径调控多种细胞基因的转录 ,具有降低体内致瘤性及抗转移等活性 .为了探讨 E1 A基因对代表肺癌癌前病变的永生化人支气管上皮细胞的生长是否具有抑制作用 ,构建了在真核细胞高表达 E1 A基因的重组质粒 p CEP4- E1 A.通过脂质体介导将 E1 A基因转入永生化人支气管上皮细胞第 1 68代 ( MP1 68)中 ,经潮霉素筛选 ,获得稳定表达 E1 A的永生化人支气管上皮细胞 ( MP1 68- E1 A) .结果表明 :E1 A基因的稳定表达抑制了 HER- 2 / neu基因的表达 .转染细胞 ( MP1 68- E1 A)回复扁平形态、恢复细胞生长的接触性抑制 ,细胞群体生长缓慢 (倍增时间是 MP1 68- vect细胞的 1 .41倍 ) ,细胞周期 G1期阻滞并出现凋亡 ,软琼脂集落形成抑制率达73.86% .结果说明 E1 A基因的稳定表达明显抑制了永生化人支气管上皮细胞的生长 .该作用可能与 E1 A抑制 HER- 2 / neu基因的表达及诱导永生化人支气管上皮细胞凋亡有关 .     

Tumor promoters alter the temporal program of adenovirus replication in human cells.

In this study we evaluated the effect of phorbol ester tumor promoters on the kinetics of adenovirus type 5 (Ad5) replication in human cells. When added at the time of infection, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) accelerated the appearance of an early virus antigen (72,000-molecular-weight [72K] deoxyribonucleic acid-binding protein), the onset of viral deoxyribonucleic acid synthesis, and the production of infectious virus. The appearance of an Ad5-specific cytopathic effect (CPE) was also accelerated in infected cultures exposed to TPA, whereas phorbol, 4 alpha-phorbol-12,13-didecanoate and 4-OmeTPA, which are inactive as tumor promoters, were ineffective in inducing this morphological change. The acceleration of the CPE seen in TPA-treated Ad5-infected cells was not caused by TPA induction of the protease plasminogen activator, since the protease inhibitors leupeptin and antipain do not inhibit the earlier onset of this CPE and, in contrast, epidermal growth factor, which induces plasminogen activator in HeLa cells, does not induce an earlier CPE. Evidence for a direct effect of TPA on viral gene expression was obtained by analyzing viral messenger ribonucleic acid (mRNA) synthesis. TPA accelerated the appearance of mRNA from all major early regions of Ad5, transiently stimulated the accumulation of region III mRNA, and accelerated the appearance of late Ad5 mRNA. Thus, TPA altered the temporal program of Ad5 mRNA production and accelerated the appearance of at least some Ad5-specific polypeptides during lytic infection of human cells. These effects presumably explain the earlier onset of the Ad5-specific CPE in TPA-treated cells and may have relevance to the effects of TPA on viral gene expression in nonpermissive cells carrying integrated viral deoxyribonucleic acid sequences.