Integration Sites of Adenovirus Type 12 DNA in Transformed Hamster Cells and Hamster Tumor Cells

The patterns and sites of integration of adenovirus type 12 (Ad12) DNA were determined in three lines of Ad12-transformed hamster cells and in two lines of Ad12-induced hamster tumor cells. The results of a detailed analysis can be summarized as follows. (i) All cell lines investigated contained multiple copies (3 to 22 genome equivalents per cell in different lines) of the entire Ad12 genome. In addition, fragments of Ad12 DNA also persisted separately in non-stoichiometric amounts. (ii) All Ad12 DNA copies were integrated into cellular DNA. Free viral DNA molecules did not occur. The terminal regions of Ad12 DNA were linked to cellular DNA. The internal parts of the integrated viral genomes, and perhaps the entire viral genome, remained colinear with virion DNA. (iii) Except for line HA12/7, there were fewer sites of integration than Ad12 DNA molecules persisting. This finding suggested either that viral DNA was integrated at identical sites in repetitive DNA or, more likely, that one or a few viral DNA molecules were amplified upon integration together with the adjacent cellular DNA sequences, leading to a serial arrangement of viral DNA molecules separated by cellular DNA sequences. Likewise, in the Ad12-induced hamster tumor lines (CLAC1 and CLAC3), viral DNA was linked to repetitive cellular sequences. Serial arrangement of Ad12 DNA molecules in these lines was not likely. (iv) In general, true tandem integration with integrated viral DNA molecules directly abutting each other was not found. Instead, the data suggested that the integrated viral DNA molecules were separated by cellular or rearranged viral DNA sequences. (v) The results of hybridization experiments, in which a highly specific probe (143-base pair DNA fragment) derived from the termini of Ad12 DNA was used, were not consistent with models of integration involving true tandem integration of Ad12 DNA or covalent circularization of Ad12 DNA before insertion into the cellular genome. (vi) Evidence was presented that a small segment at the termini of the integrated Ad12 DNA in cell lines HA12/7, T637, and A2497-3 was repeated several times. The exact structures of these repeat units remained to be determined. The occurrence of these units might reflect the mechanism of amplification of viral and cellular sequences in transformed cell lines.     

Characterization of the Tumorlike (T) Antigen Induced by Type 12 Adenovirus : I. Purification of the Antigen from Infected KB Cells and a Hamster Tumor Cell Line

The T antigen induced by type 12 adenovirus was purified from KB cells infected in the presence of 10(-6)m 5-fluoro-2-deoxyuridine to inhibit synthesis of viral capsid antigens. The antigen was purified approximately 200-fold, and the purified product contained only negligible amounts of host-cell contaminants, as judged by the residual radioactivity from (14)C-labeled uninfected cells which had been added to infected cells at the initiation of the purification. Immunoelectrophoresis indicated that the purified T-antigen preparation contained a single antigenic species. The T antigen from a hamster cell line (HT-1) derived from a type 12 adenovirus-induced tumor was purified by the same procedure. The T antigens from the two different sources were shown to be immunologically similar by use of a rabbit antiserum prepared against the purified T antigen from infected KB cells and sera from hamsters bearing tumors induced by type 12 adenovirus.     

Isolation of Immunogenic and Suppressogenic Glycoproteins from Adenovirus Type 12 Hamster Tumor Cells

Two types of glycoproteins were isolated from the membrane fraction of adenovirus type 12 (Ad12) hamster tumor cells by recovering detergent-solubilized glycoproteins using concanavalin A-affinity chromatography and gel filtration. One of the glycoproteins consisted of a polypeptide of 130,000 daltons (130K) with a pI value of 4.7–5.1, and the other consisted of a polypeptide of 18,500 daltons (18.5K) with a pi value of 6.3–6.6. The glycoproteins were immunologically different. The 18.5K glycoprotein induced in vivo resistance to tumor growth and anti-tumor cytotoxic T cells, while the 130K glycoprotein induced in vivo suppressor T cells which inhibited the activity of anti-tumor cytotoxic T cells.     

Nature of Hamster Complement-fixing Antibody to Adenovirus 12 Tumor Antigen

The presence of complement-fixing (CF) antibody reactive with T antigen and with viral C antigen in hamsters bearing adenovirus 12-induced tumors has been confirmed. Antibody activity in serum obtained at a time when the host was bearing large tumors was found to be associated exclusively with 7S immunoglobulins. Two populations of 7S immunoglobulins showing CF reactivity were distinguished by electrical charge, as determined by diethylaminoethyl cellulose chromatography and immunoelectrophoresis. No antibodies of the 19S IgM type were detected in the serum of hamsters bearing large tumors.     

腺病毒携带IL-12增强抗原致敏树突细胞在肝癌基因治疗中的研究

目的:近年来通过应用白介素-12治疗肿瘤取得良好效果,因此对国内外应用腺病毒携带IL-12增强抗原致敏树突细胞在肝癌基因治疗中的研究进展进行总结,以探索更为可行治疗方法。方法:运用Pubmed、Elsevier Sciencedirect、CNKI及万方全文数据库检索系统,以腺病毒,IL-12,肝癌,树突细胞为关键词,检索2008-01至2012-11月发表的文献。纳入标准:1)IL-12的生物学特性,在抗肿瘤过程中的免疫作用,2)应用腺病毒携带IL-12对抗肝癌治疗研究,3)肿瘤抗原致敏树突细胞对肿瘤的影响。根据纳入标准分析文献26篇。结果:通过腺病毒携带IL-12可以增强肿瘤抗原致敏树突细胞的免疫应答。并通过诱导肿瘤细胞的凋亡,减少新生血管的生成而对肿瘤产生直接抑制,有效抑制肝癌的生长和转移。结论:本文通过对IL-12的生物学特征、抗肿瘤通路、作用机制及在腺病毒介导下肿瘤抗原致敏树突细胞研究进展的概述,为腺病毒携带IL-12作为肝癌的基因治疗进一步提供理论依据和探索,期待在将来应用IL-12为基础的基因治疗一定会为包括肝癌在内的肿瘤治疗提供新的途径。     

Close Association of Virus-Specific Cell Surface (S) and H-2 Antigens in Ad12-Infected and -Transformed Mouse Cells

A virus-specific cell surface (S) antigen in adenovirus type 12 (Ad12)-transformed mouse cells has been assumed to be a direct target for cytotoxic thymus-derived lymphocytes (CTL). In this study, the spatial proximity between the S and H-2 antigens was determined by three different methods, the proximity and co-capping tests, and the test for blocking of CTL-mediated lysis by anti-H-2 serum. In the proximity test with Ad12-infected thymic and splenic lymphocytes, and an Ad12-transformed line of C3H/He (H-2^k) mouse cells, anti-H-2^k and anti-S sera reciprocally inhibited fluorescent-antibody staining of the opposite antigens. By contrast, anti-Thy-1, 2 serum as well as anti-Ia and anti-Ig sera failed to show any appreciable effect in this test, when paired with anti-S serum. In addition, the S and H-2 antigens co-capped in the infected thymic lymphocytes, and CTL-mediated lysis of the transformed cells was abrogated equally by treatment of cells with anti-S and anti-H-2 sera. These results clearly demonstrate that there is a close proximity between the S and H-2 antigens on the surface of Ad12-infected and -transformed mouse cells.     

Revertants of Adenovirus Type 12-Transformed Hamster Cell Line T637 as Tools in the Analysis of Integration Patterns

Spontaneously arising morphological revertants of the adenovirus type 12 (Ad12)-transformed hamster cell line T637 had been previously isolated, and it had been demonstrated that in these revertants varying amounts of the integrated Ad12 genome were eliminated from the host genome. In this report, the patterns of persistence of the viral genome in the revertants were analyzed in detail. In some of the revertant cell lines, F10, TR3, and TR7, all copies of Ad12 DNA integrated in line T637 were lost. In lines TR1, -2, -4 to -6, -8 to -10, and -13 to -16, only the right-hand portion of one Ad12 genome was preserved; it consisted of the intact right segment of Ad12 DNA and was integrated at the same site as in line T637. In revertant lines G12, TR11, and TR12, one Ad12 DNA and varying parts of a second viral DNA molecule persisted in the host genome. These patterns of persistence of Ad12 DNA molecules in different revertants supported a model for a mode of integration of Ad12 DNA in T637 hamster cells in which multiple (20 to 22) copies of the entire Ad12 DNA were serially arranged, separated from each other by stretches of cellular DNA. The occurrence of such revertants demonstrated that foreign DNA sequences could not only be acquired but could also be lost from eucaryotic genomes. There was very little, if any, expression of Ad12-specific DNA sequences in the revertant lines TR7 and TR12. Moreover, Ad12 DNA sequences which were found to be undermethylated in line T637 were completely methylated in the revertant cell lines G12, TR11, TR12, and TR2. These findings were consistent with the absence of T antigen from the revertant lines reported earlier. Hence it was conceivable that the expression of integrated viral DNA sequences was somehow dependent on their positions in the cellular genome. In cell line TR637, the early segments of Ad12 DNA were expressed and undermethylated; conversely, in the revertant lines G12, TR11, TR12, and TR2, the same segments appeared to be expressed to a limited extent and were strongly methylated.     

Characterization of the Tumorlike (T) Antigen Induced by Type 12 Adenovirus : II. Physical and Chemical Properties

T antigen, purified from KB cells infected with type 12 adenovirus, was characterized by chemical and physical methods. The immunologically active molecule had an ultraviolet-absorption spectrum characteristic of a protein, and was partially inactivated (i.e., approximately 85%) by trypsin (final concentration of 150 mug/ml). Centrifugation analyses indicated that the purified antigen was a single molecular species with an average sedimentation coefficient of 2.40S (measured by zonal centrifugation in sucrose gradients) and an S(20,w) of 2.58 determined by analytical centrifugation. The antigen was inactivated at temperatures above 40 C, it was stable between pH 5.7 and 8.8, and it had an isoelectric point of approximately 5.0, as determined by electrophoresis on cellulose polyacetate membranes.     

Chromosomal Aberrations and Cloning Efficiency in Adenovirus Type 12-infected Hamster Cells

The fate of hamster cells, abortively infected with adenovirus type 12, has been studied by correlation of chromosomal aberrations with induction of T antigens and cloning efficiency. The incidence of chromosomal changes paralleled to some extent the T antigen formation, but was inversely related to the cloning efficiency of the cells. At an input multiplicity of 100, within 24 hr after infection, nearly all of the cells or metaphases revealed the presence of T antigens and chromosomal lesions, respectively, but no clones of cells were obtained. Inhibition of cellular deoxyribonucleic acid synthesis was not noted during this period. Increasing doses of ultraviolet irradiation reduced, successively, the capacity of the virus to induce chromosomal aberrations and correspondingly improved cloning efficiency of the exposed cells. It is concluded that most, if not all, cells revealing chromosomal lesions 24 hr after infection fail to enter further mitoses.     

Characterization of a Tumorlike Antigen in Type 12 and Type 18 Adenovirus-Infected Cells.

Gilead, Zvee (University of Pennsylvania, Philadelphia), and Harold S. Ginsberg. Characterization of a tumor-like antigen in type 12 and type 18 adenovirus-infected cells. J. Bacteriol. 90:120-125. 1965.-An antigen that reacts with antibody from type 12 adenovirus tumor-bearing hamsters was identified in extracts of KB cells infected with type 12 or 18 adenovirus. In contrast, viral structural proteins separated by chromatography on diethylaminoethyl-cellulose did not react with the sera from tumorous hamsters. The tumorlike (T) antigen in infected cells was found to be smaller than the viral structural antigens and, therefore, could be separated from them by centrifugation in a linear sucrose gradient. Investigation of the production of the T antigen in virus-infected cells further distinguished it from viral structural proteins by the following properties: (i) the T antigen was first detected 3 to 4 hr after infection, whereas viral antigens were synthesized 17 to 20 hr after infection; and (ii) the T antigen was produced when deoxyribonucleic acid (DNA) biosynthesis was inhibited by 5-fluorodeoxyuridine (10(-6)m), but viral proteins were not synthesized in the absence of viral DNA replication.     

Persistence of the Viral Genome in Adenovirus Type 12-infected Hamster Cells

Induction of T antigen by adenovirus type 12 was studied in growing and growth-inhibited cultures of the Nil-2 line of Syrian hamster cells. At a viral input multiplicity of 10, neoantigen was present in 100% of the cells by 24 hr. T antigen gradually disappeared in descendants of these cells so that 2 weeks after infection only 1% gave specific immunofluorescence. When cellular replication was prevented by addition of fluorodeoxyuridine, T antigen persisted in all cells for the 2-week period. Upon infection of growing cultures with purified (3)H-labeled adenovirus type 12 and autoradiographic analysis of the cells at various times thereafter, a gradual reduction in labeled nuclear loci was noted which paralleled the decrease in T antigen-containing cells. In nongrowing cultures, no change in labeled loci was noted. Correlation of T antigen and labeled loci revealed that fluorescent cells contained, on the average, about 10 times more silver grains than nonfluorescent cells. All of 92 preselected fluorescent cells showed labeled loci, whereas, of 100 nonfluorescent cells, 18 were free of silver grains. The implications of these findings are discussed.     

Electron Microscopy of Adenovirus 12 Replication 1. Fine Structural Changes in the Nucleus of Infected KB Cells

The ultrastructure of KB cells infected with oncogenic adenovirus 12 was studied at various intervals from 4 to 72 hr after viral inoculation. At 12 hr after infection, the nucleus and the nucleolus became hypertrophic. At 16 hr, bundles of fibers digestable by proteolytic enzymes were seen in the nucleus; they are considered as the early viral antigens identified immunologically by others. Between 24 and 26 hr, four types of nuclear inclusions appeared. Their sequence of appearance and fine structure are described. On the basis of their sensitivity to proteolytic digestion in thin sections, and the results of immunoferritin studies made by others, some of these inclusions are believed to represent viral structural antigens. Throughout the cycle of viral replication, the nucleolus displayed prominent and constant changes in the form of focal condensations and loosening of the nucleolonema, followed by atrophy and fragmentation. It is suggested that the early nucleolar changes reflect an active participation of the nucleolus in the synthesis of adenovirus 12. A hitherto unknown striated structure with definite periodicity, which is easily digested by proteolytic enzymes, was found in the nuclei during the late stages of adenovirus 12 replication.     

Induction of Specific Chromosomal Aberrations by Adenovirus Type 12 in Human Embryonic Kidney Cells

Nonrandom chromosomal breaks in chromosomes 1 and 17 were provoked in human embryonic kidney cells 24 hr after infection with adenovirus type 12. These chromosomal changes disappeared in persistently infected cultures. Neutralization of the virus with type-specific antiviral serum prior to infection prevented the occurrence of chromosomal aberrations. No viral deoxyribonucleic acid (DNA) synthesis, as determined by autoradiography, was seen in metaphases containing adenovirus type 12-induced chromosomal aberrations. Ultraviolet irradiation of the virus reduced chromosomal aberrations linearly. This reduction in aberrations was fourfold slower than the inactivation of viral infectivity. At 24 hr after infection of cells with purified (3)H-labeled adenovirus type 12, the isotope was found to be associated with the nuclei. The uptake of isotope was reduced ninefold when the labeled virus was neutralized with type-specific antiviral serum. This difference is considered to account for neutralization of labeled virions. In metaphases infected with labeled viruses, most of the clustered grains were seen only on one arm of the chromatid, even after 72 hr. Isochromatid labeling was found, however, in a small percentage of chromosomes, and increased with time after infection. This increase was threefold between 24 and 72 hr after infection, whereas the mean grain counts decreased twofold during the same period. This has been tentatively interpreted to mean that most of the viral DNA molecules or parts thereof are merely attached to cellular chromatin, but a small fraction of them becomes gradually integrated as time proceeds. Certain chromosomal sites appeared to be preferentially labeled when chromosome 2 was used as a model for evaluation.     

Changes in Expression of Fibroblast Surface Antigen (SFA) during Cytodifferentiation and Heterokaryon Formation

Fibroblast surface antigen (SF antigen, SFA) is a major glycoprotein antigen detected in connective tissue cells (primitive mesenchymal cells, fibroblasts, and astroglial cells). In this study the expression of SFA was followed during differentiation of the mesenchymal cells of the mouse metanephros and during heterokaryon formation produced by Sendai-virus induced fusion of human fibroblasts and chick red blood cells. It was demonstrated by immunofluorescence that SFA was lost from the kidney mesenchymal cells when they differentiate into epithelial cells of the secretory tubuli. During this process SFA became detectable in the basement membrane formed around the tubuli. In cell fusion experiments human SFA which was present as fibrillar network on the surface of cultured fibroblasts, was gradually lost from the heterokaryons when the incorporated chick nuclei became activated. These two sets of experiments indicate that SFA can be used as a phenotypic marker of Cytodifferentiation.     

Human Adenovirus Type 2 but Not Adenovirus Type 12 Is Mutagenic at the Hypoxanthine Phosphoribosyltransferase Locus of Cloned Rat Liver Epithelial Cells

Using resistance to the base analog 8-azaguanine as a genetic marker, we showed that adenovirus type 2, but not adenovirus type 12, is mutagenic at the hypoxanthine phosphoribosyltransferase locus of cloned diploid rat liver epithelial cells. Adenovirus type 2 increased the frequency of 8-azaguanine-resistant colonies by up to ninefold over the spontaneous frequency, depending on expression time and virus dose.     

Adenovirus as a System for Delivering and Expressing Tumor Suppressor Genes in Tumor Cells

The strategy for tumor suppressor gene therapy for cancer is to suppress the malignant phenotype of tumor cells by replacing the inactivated gene with a normal (wild-type) one to restore control of cell growth and differentiation. To effectively carry out this strategy, the therapeutic genes must be delivered efficiently and expressed at an adequate level in the tumor. Adenoviral vectors have rapidly developed into one of the major systems now in use to effect this delivery and expression, primarily because of their advantages over other viral vectors, such as their ease of manipulation, their wide host cell range with high infectivity, their relative stability with high obtainable titers (10¹⁰–10¹²plaque-forming units/ml), and their episomal expression with low genotoxicity. Adenoviral vectors are a good technical approach to delivering tumor suppressor genes for cancer therapy; they have demonstrated effectiveness in preclinical animal models. This chapter organizes and describes a series of methods for developing a preclinical model for adenovirus-mediated tumor suppressor gene therapy of cancer. The disadvantages of adenoviral vectors and the possibilities for improving this vector system to enhance tumor suppression efficacy are also discussed.     

癌胚抗原(CEA)启动子在肿瘤细胞中的特异表达及介异bak基因诱发凋亡

构建由癌胚抗原 (CEA)启动子控制报道基因增强型绿色荧光蛋白 (EGFP)表达的重组表达质粒pCEA EGFP .用转染细胞后检测荧光的方法对CEA阳性细胞进行简便、直观的检测 ,并结合流式细胞计数对CEA启动子在人结直肠腺癌细胞LS 1 74T、结肠癌细胞SW 4 80、肺腺癌细胞A5 49、人宫颈癌细胞HeLa和人喉癌细胞HEp 2中的活性进行了分析 ,发现其在SW4 80、LS 1 74T、A5 49中活性较强 ,而在HeLa和HEp 2中无活性 .构建由CEA启动子控制凋亡基因bak表达的重组表达质粒pCEA bak ,转染HeLa及SW 4 80细胞 ,用Hoechst332 5 8染色及PI染色 流式细胞计数分析的方法证明 ,pCEA bak转染能够特异性引起SW 4 80细胞的凋亡 .结果表明 ,CEA启动子具有很好的特异性 ,CEA介导bak基因的方法可望用于CEA阳性癌细胞的靶向性基因治疗 .     

Immunodetection of a Tumor Associated Antigen (TAG-12): Comparison of Microwave Accelerated and Conventional Method

The microwave stimulated immunodetection of a tumor associated antigen (TAG-12) by monoclonal antibody 7A9 and an avidin-biotinylated alkaline phosphatase kit was compared with the conventional staining method. No difference in the staining pattern of antibody 7A9 was noticed in serial paraffin sections of 50 specimens including normal, benign and malignant breast tissues after microwave irradiated and conventional immunostaining. The results demonstrate that microwave stimulated immunostaining gives reliable results and can remarkably reduce the time of the staining procedure.     

Combined Therapy with Cytokine-Induced Killer Cells and Oncolytic Adenovirus Expressing IL-12 Induce Enhanced Antitumor Activity in Liver Tumor Model

Both adoptive immunotherapy and gene therapy hold a great promise for treatment of malignancies. However, these strategies exhibit limited anti-tumor activity, when they are used alone. In this study, we explore whether combination of cytokine-induced killer (CIK) adoptive immunotherapy with oncolytic adenovirus-mediated transfer of human interleukin-12 (hIL-12) gene induce the enhanced antitumor potency. Our results showed that oncolytic adenovirus carrying hIL-12 (AdCN205-IL12) could produce high levels of hIL-12 in liver cancer cells, as compared with replication-defective adenovirus expressing hIL-12 (Ad-IL12). AdCN205-IL12 could specifically induce cytotoxocity to liver cancer cells. Combination of CIK cells with AdCN205-IL12 could induce higher antitumor activity to liver cancer cells in vitro than that induced by either CIK or AdCN205-IL12 alone, or combination of CIK and control vector AdCN205-GFP. Furthermore, treatment of the established liver tumors with the combined therapy of CIK cells and AdCN205-IL12 resulted in tumor regression and long-term survival. High level expression of hIL-12 in tumor tissues could increase traffic of CIK cells to tumor tissues and enhance their antitumor activities. Our study provides a novel strategy for the therapy of cancer by the combination of CIK adoptive immunotherapy with oncolytic adenovirus-mediated transfer of immune stimulatory molecule hIL-12.