Preclinical Studies On Gene Therapy For Laryngeal Carcinoma

Laryngeal carcinoma is a very common malignant tumor, which is seriously detrimental to human health. It has been estimated that the incidence of laryngocarcinoma is 2% of all human tumors and 11-12% of the head and neck tumors. Conventional therapeutic methods such as surgical treatment ,radiotherapy and chemotherapy are being used currently, but all have certain limitations. It therefore ,is needed to develop a new kind of therapy for effective treatment of laryngeal carcinoma.In Department of Experimental Hematology of Beijing Institute of Radiation Medicine, they constructed a recombinant adenovirus carrying human wild -type p53 gene and tumor immunity-related genes -B7-1 and GM-CSF, named.as BB-102, and observed that proliferation of the human laryngocarcinoma cells was inhibited markedly and apoptosis was induced after infection with BB-102.Besides,such infected cells could also induce in vitro autologous peripheral blood lymphocytes to transform into tumor-killing cytotoxic T lymphocyte (CTL).Hence, in the present study the effect on cotransduction of human wide-type p53,B7-l and GM-CSF genes mediated by recombinant adenovirus on nude mice model bearing laryngocarcinoma were studied to explore the biological value in the therapy of human laryngeal carcinoma. Along with the study, we also explore the inhibitory effect of p!4ARF on the cell growth of laryngeal carcinoma and the expression on endogenous p53 to intensify the autologous level of p53 , thus providing experimental evidence for new therapeutic measures of laryngeal carcinoma.Construction and production of recombinant adenovirusesRecombinant plasmids pBB-102 (containing human wild-type p53, GM-CSF and B7-1 genes) were constructed by molecular cloning. Recombinant adenoviruses BB-102 were constructed by homologous recombination in 293 cells. BB-102 and Ad-GFP (control virus) were amplified in 293 cells on a large scale and purified by ultra-centrifugation in CsCl step gradient solutions. The viral particles were determined by OD260nm and the purity was evaluated by the ratio of OD260 nm/OD280 nm. The viral liters were determined by plaque assays.Preclinical study of recombinant adenovirus in the treatment of human laryngeal squamous carcinomaNude mice model bearing laryngocarcinoma were established using human laryngeal squamous carcinoma cell line (Hep-2). Large amounts of recombinant adenovirus were injected into the tumor. Changes in carcinoma administrated with recombinant adenovirus were observed under light and electron microscopes. The difference between experimental and control groups was statistically significant. The necrosis of the tumor cells and inflammatory cellular infiltration were found under light and electron microscopes. The morphology of cells infected with BB-102 was analyzed for evidence of apoptosis by transmission electron microscopy. It has been shown that wild type P53 protein can inhibit cell growth and induce apoptosis, while B7-1 and GM-CSF have no such effects. So the high level expression of p53 gene mediated by BB-102 is reasonably the major cause of cell growth inhibition and apoptosis.Effect of p!4ARF gene on cell growth of human laryngeal tumor cells and expression of endogenous p53 proteinp!4ARFcDNA was transferred to the cell line Hep-2 of squamous cell carcinoma of the larynx by gene transfer to study the cell cycles and the expression of endogenous wild type p53 by using flow cytometry, RT-PCR and Western-blotting. Expression of p!4ARF significantly affected the Hep-2 cell growth. The clone-forming efficiency of the Hep-2 cells transferred p!4ARF was 57%,compared with empty vector pcDNA3. The numbers of cells transferred p!4ARF at the Go/G,,G2/M phase as twice as the control after 48 hours transferred with p!4ARF cDNA . The expression of endogenous wild type p53 significantly enhanced.As mentioned above, the results showed that BB-102 has significant efficacy on suppressing tumor cell growth and inducing their apoptosis, which suggested that BB-102 might be further devel...