| Bladder cancer is one of the conditions most frequently treated by urologists. Most bladder tumors are superficial transitional cell carcinomas , which can be treated with transurethral resection of the bladder tumor (TUR- BT) and with intravesical instillation of Bacilli Calmette -Guerin (BCG).Unfortunately , tumors recur in the majority of patients .Although a number of new treatment strategies have been tested recently , the combination of the herpes simplex virus thymidine kinase (HSV-tk) and ganciclovir (GCV) is most studied because HSV-tk, as suicide genes, can induce cell death only in dividing cells such as those found in tumors .HSV-tk converts the protoxic nucleoside analog GCV into a highly toxic phosphorylated GCV that acts as a chain terminator of DNA synthesis and an inhibitor of DNA polymerase , selectively killing dividing cells . Many studies have examined gene therapy for cancer using the HSV-tk/GCV system in vitro and in vivo.The bladder would seem to be an especially appropriate organ for this technique because it is easily approached from outside the body . Accordingly, the transurethral intravesical instillation is less invasive than either direct puncture of the tumor or organ or systemic administration .Most animal studies are currently performed by using transplanted tumors produced by implantation of cell lines . Subcutaneous heterotopic graft models are often used because they are relatively easy to generate. Unfortunately , this approach completely ignores the histologic and anatomic specificity of the organs under investigation .In contrast , N-methyl -N-nitrosourea (MNU )-induced tumors are very similar to human bladder transitional cell carcinomas and are considered to be excellent models of clinical bladder cancer.In this study , we describe our use of the MNU-induced rat bladder tumor to evaluate in vivo retrovirus -mediated and Liposome-mediated gene transfer .An retrovirus vector containing HSV-tk gene or Liposome/DNA complexes was transurethrally instilled into the bladder , after which the distribution of the gene was examined by reverse transcriptase -poly-merase chain reaction ( RT-PCR).The methods and results are listed as follows:1. The orthotopic rat bladder cancer were induced in femal Wistar rats by intravascular administration of the carcinogen ,MNU. The administration way of a total dose of 10 mg on 4 fraction of 2.5 mg at 2 -weekly intervals produced a 100% incidence of bladder tumors after 8 weeks . Histopathological characteristics of bladder tumor induced by MNU were basically similar to those of human bladder tumor .2. The orthotopic rat bladder tumor models were instillated retrovirus vector containing HSV-tk gene or Liposome/DNA complexes or naked DNA plasmid , saline. GCV was given the next day after instillation for 6 days. 3. We harvested RNA from kidney tissue and bladder tumor after 48 hours since the rats' instillation and subjected the RNA to RT-PCR ,using HSV-tk -specific primers . A 681 -base pair HSV-tk -specific band was obtained exclusively, which conformed that the distribution of gene after instillation into the bladder was transferred into the tumor cells by retrovirus vector or liposome and expressed successfully.4. The tumor volume were examinated by ultrasound before and after HSV-tk/GCV system administration. Tumor incidence and tumor weight , defined as the weight of the tumored bladder , were determined on day 6 and 10 after GCV intraperitoneal injection for 6 days . Significant difference was observed between retrovirus vector or liposome -treated rats and control rats .5. DNA extraction, electrophoresis and immunohistochemistry technique were used to observe apoptosis of the bladder tumor cells treated by HSV-tk /GCV system . Inducing apoptosis of tumor cells might be the important mechanism of HSV-tk/ GCV system.Our study show that retrovirus and liposome could transfer the HSV-tk gene effectively in the orthotopic rat bladder tumor models and HSV-tk/GCV system can acts as a candidate tr... |
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