| Pituitary rumors are typically benign adenomas that may cause severe clinical symptoms. Although many pituitary tumors are treated successfully, there are several tumors that do not respond to currently available therapies. Gene therapy is always used in the treatment of cancer. However, as the technology improves, gene therapy applications will increase and gain clinical acceptance for the treatment of non-life-threatening diseases, such as pituitary adenoma. Gene therapy is a very attractive alternative to classic therapeutic modalities - chemotherapy, radiotherapy and surgery. Recently, gene therapy using adenoviral vectors to deliver the herpes simplex virus-thymidine kinase (HSV-TK) followed by ganciclovir (GCV) administration has been developed as a strategy for the treatment of pituitary adenomas by several research groups. However, one of the limitations of this type of strategy is that it may affect normal pituitary cells.Our group appraised the receptor-mediated GE7 gene delivery system, the cell -specific promoters-human growth hormone promoter (hGHp) and developed the strategy of gene therapy for pituitary adenoma using both of them at the same time. The main results of the in vitro and in vivo experimental studies were as follows: Expression of EGFR in cell linesThe GE7 system is a gene delivery system capable of delivering therapeutic gene to the target cells, organs or tissues that express epidermal growth hormone factor receptor (EGFR). So, we detected the expression of EGFR of the cultured GH3, U-OS and HO8910PM cell lines using immunohistochemical method initially. The result suggests that there is EGFR in the membrane of GH3 and HO8910PM cell line, and not in that of U-2OS cell line. So GH3 cell line can be act as the objective cells in the EGFR-mediated gene therapy, HO8910PM cell line as positive control cells, and U-2OS cell line as negative control cells.Generation of recombinant plasmids expressing transgenes under the control of the human growth hormone promoterThe growth hormone promoter (hGHp) fragment was amplified by PCR using the primer pair and the sperm DNA as a template. The transgene, HSV-TK, was excised from the plasmids pcDNA3.1/His A-TK and cloned into the plasmid pcDNA3.1/His A, whose CMV promoter was cut, generating pcDNA3.1/His A-hGHp-TK. The plasmid pGL3-Basic-hGHp was constructed using the hGHp promoter driving the expression of the marker gene luciferase. Cell type-specific expression of luciferase or HSV-TK in the pituitary tumor cell line, GH3When the GH3, U-2OS and HO8910PM cells were infected with GE7-deliveried pGL3-Basic-hGHp, only the somatotrophic GH3 cells expressed the luciferase enzyme. No expression of luciferase was observed in U-2OS or HO8910PM cells. These results show that the GE7 gene delivery system and hGHppromoter can restrict expression of the marker gene luciferase to established somatotrophic tumor cell lines in vitro.Simultaneous detection of HSV-TK protein within GH3 cells following GE7 infection with pcDNA3.1/His A-hGHp-TK was performed by Western blot techniques. Infection of these cells with pcDNA3.1/His A-hGHp-TK resulted in a 46KD staining line in the somatotrophic GH3 cells. In U-2OS and HO8910 cells, we observed no HSV-TK expression.To quantitate promoter strength of the hGHp promoter within pGL3-Basic-hGHp, luciferase enzyme activity was assessed in the somatotrophic GHs cells following GE7 infection. The levels of enzyme activity increased with increasing plasmid used, and peaked in 2.0ng/ml plasmid.Target cytotoxicity of HSV-TK driven by hGHp promoter in combination with GCV in vitroThe most widely used approach of gene therapy for tumor treatment is conditional cytotoxicity. Therapy with conditional cytotoxic genes, such as HSV-TK, has shown toxicity in the presence of GCV. Therefore, we assessed the effect of HSV-TK expression on GH3, U-2OS and HO8910PM cells after GE7 infection with pcDNA3.1/His A-hGHp-TK, in the absence or presence of 0-16ug/ml GCV. We detected cell exist rate using MTT methods. We...
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