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首页> 外文期刊>Gene Therapy and Molecular Biology >Recombinant adenoviruses as expression vectorsand as probes for DNA repair in human cells
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Recombinant adenoviruses as expression vectorsand as probes for DNA repair in human cells

机译：重组腺病毒作为表达载体和人类细胞DNA修复探针

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There is widespread interest in the use of recombinant adenovirus (Ad) vectors for gene therapy of cancer and as tools in molecular biology research. There are also potential benefits to be gained by combining strategies for Ad- based gene therapy of cancer with radiotherapy and chemotherapy. However, there is limited information available on the effects of cytotoxic agents on transgene expression which would allow a rational approach to combining these modalities. We have used recombinant nonrepilcating Ad expressing the lacZ gene under the control of the cytomegalovirus (CMV) immediate early promoter to assess the effects of cytotoxic agents on the expression of a reporter gene in human cells. Using this approach we are able to examine both constitutive and inducible expression of the reporter gene. Pretreatment of normal human cells with low UV fluence results in an enhanced expression of the reporter gene. The enhanced expression occurs at lower doses of the DNA damaging agent in cells deficient in the transcription coupled repair (TCR) pathway of nucleotide excision repair (NER) suggesting that the enhancement in human cells is triggered by persistent damage in actively transcribed genes. The enhancement is also reduced or absent in SV4O-transformed cells and cells expressing the human papilloma virus ('WV) E7 gene, but not in Li-Fraumeni syndrome (LFS) cells or cells expressing the 'WV E6 gene. Since SV4O-transformation and HPV E7 expression both abrogate the retinoblastoma (pRb) family of proteins, whereas 'WV E6 abrogates p53 and LFS cells express mutant p53, these results indicate that the enhanced expression depends on one or more of the pRb family of proteins, but not on p53. We have also used recombinant Ad expressing a lacZ reporter gene as a probe for DNA repair in human cells. Using this approach we have examined constitutive as well as inducible DNA repair of a UV-damaged reporter gene in human cells. We detected enhanced host cell reactivation (HCR) of a UV- damaged reporter gene in pre-heat-shock treated or pre-UV treated TCR proficient but not in pretreated TCR deficient human fibroblasts or LFS cells. These results suggest the existence of an inducible repair response for UV- damaged DNA in human cells which is dependent on the TCR pathway of NER and the wild type p53 tumour suppressor. These results have important implications for the use of recombinant Ad-based expression vectors under the control of the CMV promoter in gene therapy for cancer when used in combination with DNA damaging agents.
机译：使用重组腺病毒（Ad）载体进行癌症的基因治疗并作为分子生物学研究的工具引起了广泛的兴趣。通过将基于Ad的癌症基因疗法与放射疗法和化学疗法相结合的策略，还可以获得潜在的好处。但是，关于细胞毒性剂对转基因表达的影响的信息有限，这将允许采用合理的方法来组合这些方式。我们已经使用在巨细胞病毒（CMV）立即早期启动子的控制下表达lacZ基因的重组非再生Ad来评估细胞毒剂对人细胞中报道基因表达的影响。使用这种方法，我们能够检查报告基因的组成型和诱导型表达。用低紫外线通量对正常人细胞进行预处理会导致报告基因表达增强。在核苷酸切除修复（NER）的转录偶联修复（TCR）途径不足的细胞中，增强的表达在较低剂量的DNA损伤剂中发生，表明人细胞中的增强是由主动转录的基因中的持续损伤触发的。在SV4O转化的细胞和表达人乳头瘤病毒（'WV）E7基因的细胞中，增强作用也减少或不存在，但在Li-Fraumeni综合征（LFS）细胞或表达'WV E6基因的细胞中则没有增强作用。由于SV4O转化和HPV E7表达均消除了视网膜母细胞瘤（pRb）蛋白家族，而'WV E6消除了p53和LFS细胞表达突变型p53，这些结果表明增强的表达取决于一个或多个pRb蛋白家族。 ，但不适用于p53。我们还使用了表达lacZ报告基因的重组Ad作为人类细胞DNA修复的探针。使用这种方法，我们检查了人类细胞中紫外线损伤的报告基因的组成型和诱导型DNA修复。我们在预热休克处理或预UV处理的TCR精通物中检测到了UV损伤的报告基因的增强的宿主细胞激活（HCR），而在未处理TCR的人成纤维细胞或LFS细胞中则未检测到。这些结果表明人细胞中存在紫外线损伤的DNA的诱导型修复反应，这取决于NER的TCR途径和野生型p53肿瘤抑制剂。这些结果对于与DNA破坏剂组合使用时在CMV启动子控制下基于重组Ad的表达载体在癌症基因治疗中的应用具有重要意义。

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