Gene Therapy For Pleomorphic Adenoma Of Salivary Gland In Vitro

Objectives:Salivary pleomorphic adenoma (SPA) is one of the most common tumors of salivary gland, accounting for 50% of the total epithelial tumors. This tumor usually involves the oral maxillofacial regions and affects the facial features. SPA occurred in parotid could destroy the facial nerve and result in facial paralysis. Although benign and slow-growing painless masses, some tumors have no an entire or even no capsule. So, the tumor is easy to relapse and implant after surgery, furthermore it tends to malignant transformation. Up to now, the surgery is not reasonable for the recurrence. A new approach for the treatment of SPA has to be developed, especial for the recurrence. With the progress of research for oncogene, it has been realized that tumourigenesis is a complicated procedure involved multiple factor and polygene mutations. Current studies showed that some oncogenes such as H-ras, wt-p53, bcl-2 was nearly associated with development of SPA and mutation of anti-oncogene wt-p53 was most common occurred. The p53 protein was investigated by using polymerase chain reaction (PCR), immunohistochemical staining and single strand conformational polymorphism (SSCP) analysis. The mutation rate of wt-p53 in SPA was 83% immunohistochemically and 41% by SSCP. The sequencing analysis revealed that the mutation of wt-p53 in SPA showed poly-codon mutation and poly-pattern mutations, chiefly showed point mutations in exon 5⁸. Gene therapy mainly introduced exogenous gene into tumors by using gene transfer technique to compensate deficiency of endogenous gene. The tumor suppressor activity of wt-p53 is mainly mediated through its ability to cause cell growth arrest or apoptosis, whereas a lack of functional wt-p53 usually leads to increased genomic instability, deregulated cell proliferation, accelerated tumor progression. Several reports have suggested that the p53 status in tumor cells is responsible for the biological behavior. The tumors with deficiency of functional wt-p53 transduced by adenovirus mediated human wt-p53 gene has been shown a significant suppressing effect in the pro-clinical studies and in clinical trials. Herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) method is one of the most frequently utilized forms of gene therapy. GCV, a kind of antiviral drug, can be phosphorylated by HSV-tk protein, and finally converted into triphosphates, which are potent inhibitors of DNA polymerase, leading to the disruption of cellar DNA synthesis and ultimately cell death. Experiments showed that HSV-tk gene causes efficiently anti-tumor effect in transduced tumor cells. However, suicide gene therapy alone cannot completely eliminate tumor cells, so it is necessary to combine suicide gene therapy with other therapy to improve performance. In order to explore the feasibility of combing HSV-tk and wt-p53 gene in SPA therapy, we used adenoviral vector containing HSV-tk and wt-p53 gene to transfer SPA cells and to evaluate the possibility of such a strategy as combing gene therapy in vitro. Methods 1. Culture of SPA cells and identification:Specimens of 4 SPA were obtained from surgical resection, tumor tissues were placed in RPMI1640 supplemented with 15% fetal calf serum (FCS), streptomycin (500ug/ml) and penicillin (1000U/ml), for 30 minutes. They were rid of necrotic tissue, plasma clot and mucosa, then minced into fine pieces and washed with PBS. Fragmental tissues were planted in culture flasks and incubated in 5%CO2 and 37℃. The medium was added or changed according to the variation of pH. When the cells reached 80% confluency, they were harvested by trypsinization which was adopted to get rid of fibroblasts through 2³ times. The pure tumor cells were obtained and evaluated by Giemsa stain. Immunohistochemistry and transmission electronic micoscope (TEM) were used to identify the histological features of SPA cells. 2. Exogenous HSV-tk and wt-p53 expression analysis:SPA cells weretransduced with 102 MOI Ad-wt-p53 and HSV-tk gene at 37℃and 5%CO₂ incubation. After 24h incubation, cells were harvested and washed with PBS. Total RNA was extracted from 5×10⁶ using SK1351 (Gibco). Then cDNA was generated by using the QuantiTectTM SYBR Green RT-PCR (Qiagen, germany), PCR was performed by an initial heating step at 50°C for 30 min, followed by 40 cycles at 94°C for 15 second, at 55°C for 30 second and at 72°C for 30 second. The PCR products were separated on 1.5% agarose gels and visualized by ethidium bromide staining and UV light. 3. Endogenous p53 gene status of SPA after wt-p53 infection:Cells were infected with Ad-wt-p53 at an MOI of 200 for 24h. After 24h incubation period, the cells were harvested and washed with cold PBS, then centrifuged for 10min at 8000r/min, DNA was extracted from 5×10⁶ cultured cells. Simultaneously, the corresponding tumor tissue DNA was extracted, the oligonucleotide primers of wt-p53 were designed. The condition for amplification was 94℃45 seconds,56℃45 seconds and 72℃45 seconds for 35 cycles. The PCR products were loaded onto 10% nondenaturing polyacrylamidedel gels and run at room temperature for 10¹2h. Staining of the single-and-double-stranded DNA was performed according to a silver staining protocol. Then DNA sequencing analysis was used to determined. 4. The Effects of HSV-tk suicide gene and wt-p53 gene on SPA:SPA cells were seeded in 96 well plates at a density of 4×10³cells per well, transfection were performed after the cells adhered, the cells were divided into 4 groups treated by wt-p53, HSV-tk /GCV, wt-p53+HSV-tk /GCV respectively. After 5 days, the plates were taken out and cells were added with 20μl of 5g/L MTT to each well, culturing was continued for 4h and the supernatant was removed. Add 150μl DMSO to react for 15 minutes. Absorbance was measured at 570nm by using a spectrophotometer. The growth inhibition ratio was calculated. Bystand effect was determined by mixing no infected cells and infected cells at varying rations. Thepercentage of infected cells was 0%, 25%, 50%, 75% and 100%. Cell mixtures were plated at 4×10³ cells per well. The survival ratios were measured. Results 1. The growth status and morphologic feature of SPA :Tumor tissue had the histologic features of SPA, they were characterized by a lobular architecture consisting of loose chondromyxoid stroma. The cellular component was made up of small rounded epithelial cells and slightly spindled myoepithelial cells. Both these cell types could either lie loose in the stroma or form cysts and ducts with a lining of epithelial cells surrounded by a more solid mass of myoepithelial cells. Under inverted microscope, tumor cells were large spindle-shaped or polygonal, nuclear was round or oval. In primary culture, cells overlapped growth and formed "cell clusters". Growth curve of the cells was gentle and doubling time was longer than that of malignancy tumors. On the 5th, 6th days, the curve reached a peak. Histologic observation: Cells were stained by Giemsa stain, adenoepithelial cells were round or polygonal, the cells were arranged cluster, and formed gland lumen, there were much mucus in cytoplasm of the cells. Myoepithelial cells were spindle, arranged in mass with some mucoid substance around them. Immunohistochemical stain showed that cytokeratin was labelled in columnar-shaped cells, and spindle -shaped cells were positive for the smooth muscle actin. It suggested adenoepithelial and myoepithelial natures. 2. Detection of HSV-tk and gene expression:Amplification fragment 1134 bp (wt-p53) and 1150 bp (TK) could be seen in the experimental group, while none was found in the control group. The results suggested that the exogenous HSV-tk and wt-p53 gene had been transferred and expressed by SPA cells. 3. Endogenous wt-p53 gene status of SPA after wt-p53 infection:PCR-SSCP analysis showed that 2 out of 4 SPA with abnormal exon 8. Comparison of SSCP with DNA sequencing analysis, the results showed that all samples...