Identification of potential human oncogenes by mapping the common viral integration sites in avian nephroblastoma

Gene deregulation is a frequent cause of malignant transformation. Alteration of the gene structure and/or expression leading to cellular transformation and tumor growth can be experimentally achieved by insertion of the retroviral genome into the host DNA. Retrovirus-containing host loci found repeatedly in clonal tumors are called common viral integration sites (cVIS). cVIS are located in genes or chromosomal regions whose alterations participate in cellular transformation. Here, we present the chicken model for the identification of oncogenes and tumor suppressor genes in solid tumors by mapping the cVIS. Using the combination of inverse PCR and long terminal repeat-rapid amplification of cDNA ends technique, we have analyzed 93 myeloblastosis-associated virus type 2-induced clonal nephroblastoma tumors in detail, and mapped >500 independent retroviral integration sites. Eighteen genomic loci were hit repeatedly and thus classified as cVIS, five of these genomic loci have previously been shown to be involved in malignant transformation of different human cell types. The expression levels of selected genes and their human orthologues have been assayed in chicken and selected human renal tumor samples, and their possible correlation with tumor development, has been suggested. We have found that genes associated with cVIS are frequently, but not in all cases, deregulated at the mRNA level as a result of proviral integration. Furthermore, the deregulation of their human orthologues has been observed in the samples of human pediatric renal tumors. Thus, the avian nephroblastoma is a valid source of cancer-associated genes. Moreover, the results bring deeper insight into the molecular background of tumorigenesis in distant species.     

Rearrangements of the Pim-1, c-myc, and p53 genes in Friend helper virus-induced mouse erythroleukemias

The Friend helper leukemia virus (F-MuLV) induces in mice leukemias of the erythroid, lymphoid, and myeloblastic lineages. Erythroleukemic cell DNAs were examined for genetic alterations at loci described as common proviral integration regions in MuLV-induced myeloid or lymphoid leukemias or in Friend complex-induced erythroleukemias. No alteration of the Fim-1, Fim-2, Fim-3, pvt-1, and Spi-1 loci were detected in 17 erythroleukemias, p53 gene rearrangement was observed in 6 (30%) erythroleukemias and was always associated with a loss of the germ line allele. Interestingly, genetic alterations were also detected at two loci, c-myc and Pim-1, previously described as common provirus integration regions in T lymphoid leukemias. Rearrangements of these two genes were often associated with p53 gene alteration within the same tumor.     

In vivo immunologic selection of proviral gene deletion variants from a nonproducer clone

The mechanism by which tumors recur at sites of injection of retrovirus-infected fibrosarcoma cell lines was investigated. Previously, it was established that tumor recurrences reflect outgrowth of rare cells that lack viral antigens and are susceptible to superinfection with the homologous retrovirus. In the present study clones isolated from a retrovirus-infected cell line were evaluated as precursors for tumor recurrence. Under conditions of in vivo immunologic selection, a clone that contained a single abbreviated copy of the provirus formed variants that lacked the proviral gene. Tumor variants lacking the proviral gene grew progressively in both nonimmune and virus-immune male Sewall Wright strain 2 guinea pigs. Tumor recurrence could be prevented by superinfection of the virus-infected fibrosarcoma cell line or by superinfection of the precursor for tumor recurrence. Cell lines infected with retroviruses varied in frequency of tumor recurrence formation. This model may be useful in analyzing gene deletion as a mechanism of tumor escape from host immunologic attack.     

双自杀基因对Raji淋巴瘤细胞杀伤的增强作用

目的 :观察反转录病毒介导的双自杀基因对 Raji淋巴瘤细胞的杀伤增强作用 ,探讨淋巴瘤的基因治疗方法。方法 :通过脂质体将含有双自杀基因的反转录病毒载体 p WZL neo CDglytk导入病毒包装细胞 PA317,经 G4 18筛选后大量培养产病毒的阳性克隆 PA317/ CD+ tk细胞株 ,收集病毒上清 ,浓缩后转染 Raji淋巴瘤细胞 ,再次经 G4 18筛选 ,获得稳定表达双自杀基因的 Raji/ CD+ tk细胞株。用RT- PCR检测双自杀基因的表达。给予前体药物 5 -氟胞嘧啶 (5 - flourocytosine,5 - FC)和 /或无环鸟苷(Ganciciovir,GCV)后 ,MTT法测定细胞的存活率及 CDglytk双自杀基因对 Raji细胞的杀伤作用。结果 :双自杀基因在 Raji细胞中可稳定表达 ,联合使用 5 - FC和 GCV时 Raji细胞的存活率 (13.83% )明显低于单独使用 GCV (5 0 .6 5 % )或 5 - FC(5 7.6 8% )时 ,各组比差异具有显著性 (P<0 .0 1)。结论 :反转录病毒介导双自杀基因对 Raji淋巴瘤的杀伤作用明显增强     

重组人GM—CSF逆转录病毒基因转移系统的建立及其在人肿瘤细胞中的稳定…

建立逆转录病毒介导的ＧＭ－ＣＳＦ基因转移系统,为ＧＭ－ＣＳＦ基因悠我肿瘤细胞疫苗研究奠定实验基础。应用基因重组技术将ＧＭ－ＣＳＦｃＤＮＡ克隆于逆转录病毒载体ｐＤＯＲｎｅｏ,获得重组载体ｐＤＯＲＧＭ。经脂质体介针其转染于病毒包装细胞系ＰＡ３１７细胞,经ＮＩＨ３Ｔ３细胞检测病毒滴度,ＮＩＨ３Ｔ３放大实验检测辅助病毒,并用同熵度病毒感染人乳腺癌细胞系ＭＣＦ－７。