In vivo expression and antitumor activity of p53 gene transfer with naked plasmid DNA in an ovarian cancer xenograft model in nude mice.

INTRODUCTION: Abnormalities in the p53 and p16 tumor suppressor genes are one of the most common occurrences associated with human neoplasia. Consequently, restoration of wild-type p53 or p16 functions is seen as a particularly promising approach for cancer gene therapy. In vitro and in vivo data have demonstrated that virus-mediated p53 gene transfer can induce active cell death and ovarian tumor regression. AIM: To evaluate the efficiency of intratumoral injection of naked DNA in tumor growth inhibition in an ovarian xenograft model. For that purpose, plasmid vectors encoding wild-type p53 (wt-p53) or p16 alone or in combination were used. METHODS: Nude mice were injected subcutaneously with the human ovarian adenocarcinoma cell line SKOV3. Three weeks after xenograft, tumor-bearing mice were injected twice a week with plasmid vectors carrying WT-p53 and/or WT-p16 cDNA. Empty plasmids and saline buffer were used as control. Tumor growth was monitored to evaluate the inhibition potential with p53 and/or p16 restoration. RESULTS: When compared to the control, intratumoral repeated injections of naked plasmid DNA encoding wt-p53 were inhibiting tumor growth. This inhibition was not observed with p16 and no synergy could be obtained between p53 and p16. p53 expression was restored in 84% of mice injected with plasmid encoding wt-p53. p16 expression was restored in 63% of mice injected with plasmid encoding p16. CONCLUSIONS: In this report we demonstrated that: (i) naked DNA represents an efficient gene transfer in the SKOV3 xenograft model; (ii) restoration of wt-p53 gene allows tumor growth inhibition; and (iii) this inhibition could be correlated with p53 expression as seen in 84% of treated mice after repeated naked DNA injections. These results allow us to envisage naked DNA as a therapeutic adjuvant in ovarian cancer treatment, concomitantly with tumor resection and chemotherapy.

Short-term culture of myeloid leukemic cells allows efficient transduction by adenoviral vectors.

BACKGROUND: Ex vivo gene therapy of acute myeloid leukemia (AML) requires efficient transduction of leukemic cells. Recombinant adenovirus has been reported to be a poorly efficient vector in leukemic cells. We investigated leukemic cell culture as a possible method of improving the efficacy of this vector. METHODS: Leukemic cell lines and primary cultured AML cells were incubated with adenoviral vectors carrying GFP, LacZ, or IL-12 cDNA. Transduction efficiency was evaluated by measuring adenoviral genome copy number and transgene expression in leukemic cells. The expression of the coxsackie/adenovirus receptor (CAR), CD29, CD49e, and CD51/61 was measured, as was the effect of blocking integrin on adenoviral transduction. RESULTS: Increasing the multiplicity of infection (MOI) to 300 plaque-forming units per cell enhanced transduction of leukemic cell lines and to a lesser degree of AML cells. Analysis of adenoviral genome copy per cell showed only a partial correlation between gene transfer efficiency and transgene expression. Culture of AML cells for 3 days prior to adenoviral transduction increased both adenoviral copy number per cell and the percentage of transgene-expressing cells. CD29, CD49e, and CD51/61 but not CAR expression increased in cultured AML cells between days 0 and 3 and integrin-blocking experiments showed inhibition of transduction in two of four AML samples tested. CONCLUSIONS: Efficient ex vivo gene transfer in primary cultured AML cells can be achieved by short-term culture of leukemic cells prior to gene transfer with adenoviral vectors at a high MOI. This effect appears to be at least partially mediated by enhanced integrin expression.

Efficient generation of antileukemic autologous T cells by short-term culture and gamma-irradiation of myeloid leukemic cells.

Blasts from patients with acute myeloid leukemia (AML) can be differentiated in dendritic cells (DCs) using appropriate combinations of cytokines. However, generation of autologous antileukemic cytotoxic T cells using leukemic DCs remains difficult. We have previously reported that expression of costimulatory molecules in cultured AML cells could be induced by gamma-irradiation. In the present study, blasts from 21 patients with AML were cultured in vitro for 2 days, then cells were gamma-irradiated and antigen-presenting cell (APC) characteristics were assessed. gamma-Irradiation induced expression of several characteristics of APCs in AML blasts, including expression of CD80, CD86, and BDCA-4, and were stimulators of allogeneic mixed lymphocyte reactions. Autologous antileukemic cytotoxicity was induced in seven out of ten cases. This study shows that cells with APC characteristics and able to induce ex vivo stimulation of autologous antileukemic T cells can be generated from AML cells using the simple and rapid method of gamma-irradiation of cultured leukemic cells.

Chemotherapy increases transgene expression in leukemic cells.

BACKGROUND: Patients with acute myeloid leukemia (AML) often obtain complete remission with chemotherapy but the majority of patients relapse. Combining chemotherapy and gene therapy appears to be a promising approach; however, the effects of chemotherapy on transgene expression in leukemic cells have not yet been investigated. METHODS: DA1-3b leukemic cells were transfected with pCDNA3 plasmids carrying GM-CSF or LacZ cDNA. The leukemic K562 cell line and primary cultured AML cells were transduced with an Ad5.CMV-LacZ adenoviral vector. Cells were then incubated with various concentrations of daunorubicin (DNR) and cytosine arabinoside (Ara-C), and expression of the transgene was measured. Murine DA1-3b-pCDNA3/LacZ leukemic cells were also injected into syngeneic C3H/Hej mice. RESULTS: In the cells carrying pCDNA3, DNR and Ara-C dramatically increased expression of the LacZ and GM-CSF transgenes. Over-expression depended on drug dose and was due to increased transcription. Enhancement was also observed in K562 cells and in some primary cultured AML samples transduced with the Ad5.CMV-LacZ adenovirus. Addition of N-acetyl-L-cysteine inhibited the over-expression, suggesting that reactive oxygen species were involved in activating the CMV promoter. In the A549 lung carcinoma cell line transduced with Ad5.CMV-LacZ, Ara-C had only a minor effect, and DNR had a detrimental effect, suggesting that expression depends on cell type. In vivo experiments in which mice received DA1-3b-pCDNA3/LacZ leukemic cells, and were then treated with Ara-C, also showed increased transgene expression in these leukemic cells. CONCLUSIONS: In leukemic cells, chemotherapeutic agents can induce over-expression of transgenes. This suggests a promising combined strategy for the treatment of acute leukemia.