Adenovirus Mediated Suicide Gene Therapy With HTERT Promoter In Head & Neck Cancer And Breast Cancer

The incidence of head and neck cancer is about 14/10 million, accounting for 16%-40% of systemic cancer. The number of new cases is about 500,000 each year. Head and neck cancer has become the fifth cancer and the sixth fatal cancer in the world. The incidence rate of breast cancer in the female is higher compared with head and neck cancer. According to WHO statistics reported a few years ago: new cases is up to about 1.2 million, death cases is about 400,000 and the incidence of breast cancer increases 5%-20% in the world each year. Breast cancer has become the most common malignant tumor in the female.At present, the main means of therapy is surgery, radiotherapy and chemotherapy for head&neck cancer and breast cancer. However, all therapeutic methods are usually accompanied with many adverse reactions, it is very important to find a new treatment approach to lower or eliminate the side effects and enhance inhibition effects for these cancers. According to the position of head&neck cancer and recurrence and metastasis of breast cancer, gene therapy, especially suicide gene therapy may be the development trend for the cancers. Suicide gene therapy is the majority of gene therapy for these cancers. Until Mar. 2008, among the 896 cases of gene therapy entered clinical trials for cancers, suicide gene therapy cases occupy up to 108. Although there are many advantages of single suicide gene system, its shortcomings is not ignorable. Combination of two gene therapy is a main method to overcome the shortages for cancer therapy.The herpes simplex thymidine kinase/ganciclovir (HSV-TK/GCV) system is the most widely used suicide genes, it is also the first one to be applied in clinical trial. But gap junction-mediated bystander effect and activating against cycling cells of HSV-TK/GCV system restricted its inhibition effect for cancer cells. Coli.NTR/CB1954 system has strong bystander effect independent gap-junction, which can activate against non-cycling cells. However, little is known concerning the toxicity profile of CB1954, and CB1954 is expensive and soluble in 5% DMSO only, which restricted its wide application. We developed a therapeutic strategy for head&neck cancer and breast cancer by combining HSV-TK/GCV and Coli.NTR/CB1954. Our aims are to reduce the amount of pre-drugs and improve anti-tumor effect.Gene therapy of cancer relies on attractive molecular targets for specific trans-gene expression control. Tumor-specific promoters are well described for selective trans-gene expression in cancer cells. Numerous studies have demonstrated that telomerase is activated in >90% of malignant tumors, but it is stringently repressed in normal somatic cells, and a strong correlation was found between hTERT expression and telomerase activity in a variety of tumors. Targeted cancer gene therapy is done through concentrating the objected therapeutic gene expression into the specific target tissue. Then it can minimize secondary effects and maximize the therapeutic effects. Therefore, the feasibility of using the hTERT promoter to induce tumor-specific trans-gene expression in cancer gene therapy has been expected.Based on the above reasons, we use adenovirus to mediate HSV-TK and Coli. NTR gene therapy with hTERT promoter control in head&neck cancer and breast cancerWe detected the presence of the telomerase in normal lung fibroblast CCD-11Lu, head&neck cancer cell lines Fadu and KB, breast cancer cell lines ZR-75-30 and MCF7. Strong telomerase activity was shown in tumor cell lines, but it was not detected in normal cell line with the use of the TRAP assay and flow cytometry. We analyzed hTERT and hTR expression by RT-PCR, hTR mRNA was expressed in all cell lines, but hTERT mRNA was only detected in positive cancer cell lines. These results showed that telomerase activity and hTERT mRNA expression levels had a close relation in the experimental cell lines.Adenovirus is hard to handle for its long genome, so we detected transcriptional activity of hTERT promoter in experimental cell lines. A transient transfection of luciferase reporter plasmid pGl3 was performed with CMV promoter as negative control. The results showed that hTERT promoter has significant transcriptional activity in all tumor cell lines. In contrast, no significant transcriptional activity was detected in telomerase activity negative normal fibroblast cell line. These results showed that tumor cell lines had strong transcriptional activity of hTERT promoter.We constructed recombinant adenovirus vector to mediate HSV-TK with hTERT promoter, CMV promoter was set as negative control. Expression of HSV-TK was detected in experimental cell lines infected Ad-CMV-TK-enh or Ad-hTERT-TK-enh by Western blot. HSV-TK mRNA was detected in tissues of ZR-75-30 tumor and tissues of adjacent tumor injected intratumorally rAds by RT-PCR. To determine cancer specific cell death by Ad-hTERT-TK-enh, tumor and normal cell lines were infected with rAds and GCV in vitro and in vivo.The results of WB and RT-PCR showed that expression of HSV-TK was detected in tumor cell lines and tissues of tumor. In contrast, there was no expression of HSV-TK in normal fibroblast cell line and tissues of adjacent tumor. After cell lines were infected with Ad-hTERT-TK-enh, GCV was added, almost all tumor cells growth were inhibited, while normal cell line was not. However, Ad-hTERT-TK-enh/GCV has lower inhibition effect compared with Ad-CMV-TK-enh/GCV. These results gave us the hint that we can enhance tumoricidal activity of HSV-TK/GCV and not alter specific priming activity of hTERT promoter.To increase the anti-tumor activity of Ad-hTERT-TK-enh, we constructed Ad-hTERT-TK-IRES-NTR-enh which expressed TK and NTR in the same vector. The results of anti-tumorous test showed Ad-hTERT-TK-IRES -NTR-enh/ GCV+CB1954 system is superior to the Ad-hTERT-TK-enh/GCV and Ad-hTERT-NTR-enh/CB1954 systems. These additive effects supported the proposed difference in mode of action of the two activated prodrugs. Cells must be in S-phase for cytotoxic GCV-triphosphate incorporation into DNA, whereas activated CB1954, which acts as a crosslinking agent should not require cell division for cytotoxicity. Co-operation between the actions of CB1954 and GCV could also be exploited to achieve an improved bystander killing effect. Antitumorous effects of Ad-hTERT-TK-IRES-NTR-enh /GCV +CB1954 is lower compared with Ad-hTERT-TK-enh/GCV+ Ad-hTERT-NTR-enh/CB1954. These results may be due to activity of IRES is lower than hTERT promoter. But difference was not significant, it was maybe cause by strong bystander effect of NTR/CB1954. All in all, Ad-hTERT-TK-IRES-NTR-enh reduced amount of rAds and boost antitumoral activity.This paper showed the specific activity of hTERT promoter in head&neck cancer and breast cancer cell lines with positive telomerase activity. The additive effects of HSV-TK and Coli.NTR with prodrugs were detected in tumor cell lines in vitro and in vivo. Our Work offer prime foundation for use of adenovirus to mediate HSV-TK and Coli.NTR gene therapy with hTERT control in cancer gene therapy of head&neck cancer and breast cancer.