Greater Induction Of Apoptosis Research Articles

Markedly Enhanced Cytolysis byE1B-19kD-Deleted Oncolytic Adenovirus in Combination with Cisplatin

Oncolytic adenoviruses are currently being developed as novel antitumor therapeutics. To enhance their therapeutic potential, adenoviruses are being administered in combination with standard chemotherapy. Adenoviral vectors used in these clinical trials, however, can be destructive as they encode intact E1B 19-kDa protein, which can block the apoptotic pathway induced by a variety of chemotherapeutic agents. Previously, we have shown that oncolytic adenovirus Ad-DeltaE1B19/55, deleted for sequence encoding E1B 19-kDa and E1B 55-kDa proteins, exhibits marked enhancement in cytolytic and apoptotic activity [Kim, J., Cho, J.Y., Kim, J.H., Jung, K.C., and Yun, C.O. (2002). Cancer Gene Ther. 9, 725-736]. In the current study, we assess the therapeutic value of Ad- DeltaE1B55 and Ad-DeltaE1B19/55 in combination with cisplatin. A marked increase in cytotoxicity was observed for both Ad-DeltaE1B55 and Ad-DeltaE1B19/55 when combined with cisplatin. Relative to each other in all cell lines examined, the combination of the double-deleted adenovirus, Ad-DeltaE1B19/55, plus cisplatin exhibited a greater cell-killing effect than did the single-deleted adenovirus, Ad-DeltaE1B55, plus cisplatin. Propidium iodide staining and TUNEL analysis also revealed that the combination of cisplatin with Ad-DeltaE1B19/55 caused greater induction of apoptosis than that with Ad-DeltaE1B55. Similarly, in vivo, the combination of Ad-DeltaE1B55 or Ad-DeltaE1B19/55 with cisplatin also induced greater antitumor effect in a human cervical xenograft model. TUNEL staining showed that the apoptotic level was significantly higher in tumor tissue treated with Ad-DeltaE1B19/55 plus cisplatin than with any other treatment. In addition, viral presence was confirmed by immunohistological staining, with increased numbers of adenoviral particles detected in wider areas of tumors treated with Ad-DeltaE1B19/55 oncolytic adenovirus plus cisplatin. Taken together, these findings demonstrate that cisplatin in combination with E1B- 19kD-deleted oncolytic adenovirus may enhance therapeutic efficacy (via active induction of apoptosis), eliciting a greater efficacy profile than that with E1B-19kD-expressing oncolytic adenovirus.

Cytotoxic Effects of Low- and High-LET Radiation on Human Neuronal Progenitor Cells: Induction of Apoptosis andTP53Gene Expression

The induction of apoptosis, TP53 expression, caspase activation and cell toxicity were investigated after exposure of cells of the human neuronal progenitor cell line Ntera2 (NT2) to low-LET radiation (gamma and X rays). The data indicates that irradiation of NT2 cells quickly induced TP53 expression, which was followed in time by an increase in caspase activity, and ultimately resulted in the induction of apoptosis. Induction of apoptosis was dependent on dose, and the highest levels were measured 48 h after exposure. For comparison, the level of apoptosis induced by high-LET particle radiation (1 GeV/ nucleon iron ions) was also determined and was found to be dependent on dose. The relative biological effectiveness (RBE) was estimated from the slopes of the dose-response curves for the induction of apoptosis. The RBE(max) for apoptosis 48 h after exposure was at least 3.4. In short, exposure to high-LET radiation results in a more efficient and greater induction of apoptosis in human neuronal progenitor cells than low-LET radiation.

945. E1B-Mutant Replication Competent Adenovirus Combined with Chemotherapeutic Agents Shows Increased Anti-Tumor Effect

Top of pageAbstract Gene attenuated replication competent adenoviruses are being developed as novel anti-tumor therapeutics. Clinical trials with the ONYX-015 (E1B 55kDa-deleted) adenovirus in combination with chemotherapeutic agents, cisplatin or 5-FU, have demonstrated a synergistically lethal effect. All replicating adenoviruses used in combination therapy to date have their E1B 19kDa gene in tact. The E1B 19kDa functions as an anti-apoptotic agent. Due to cell death induced by the apoptotic pathway, the E1B 19kDa gene may interfere with the mode of action of chemotherapeutic agents, underestimating the value of these combination studies. For the aim of improving adenoviral vectors for cancer gene therapy, we generated adenoviral vectors with different combination of E1B 19kDa and E1B 55kDa genes and investigated the possibility of enhanced oncolytic and replication ability of these adenoviruses (Ad-ΔE1B55, Ad-ΔE1B19/55) combined with various chemotherapeutic agents. The in vitro efficacy of adenovirus and chemotherapeutic agents (taxol, cisplatin, 5-FU, adriamycin) has been evaluated by comparison of CPE assay and MTT assay. Our results show that the degree of cytopathic effect of Ad-ΔE1B19/55 was higher than that of Ad-ΔE1B55. Furthermore, compared with a virus-only treatment, combination therapy with Ad-ΔE1B19/55 and chemotherapeutic agents led to improved cytopathic effect compared to combination therapy with Ad-ΔE1B55 and chemotherapeutic agents. PI staining and TUNEL assay also revealed that combination therapy with Ad-ΔE1B19/55 and chemotherapeutic agents caused greater induction of apoptosis compared to that with Ad-ΔE1B55 and chemotherapeutic agents. Through limiting titration experiments, the total yield of adenovirus was shown to be higher when the adenovirus was treated in combination with paclitaxel compared to virus-only treatment. In contrast, the total yield of adenovirus was shown to be lower or equal when the adenovirus was treated in combination with cisplatin, 5-FU, or adriamycin compared to virus-only treatment. E1B-mutant replication competent adenovirus combined with paclitaxel exhibited superior anti-tumor effect in C33A cervical xenograft tumor in vivo compared to individual treatments of the adenovirus or paclitaxel.Taken together, the E1B 19kDa-deleted (Ad-ΔE1B19/55) replication competent adenovirus may serve as an enhanced vector for anticancer gene therapy, especially when applied in combination with apoptotic chemotherapeutic agents such as paclitaxel.

A complex adenovirus vector that delivers FASL-GFP with combined prostate-specific and tetracycline-regulated expression.

Cell-type-restricted transgene expression delivered by adenovirus vectors is highly desirable for gene therapy of cancer, as it can limit cytotoxic gene expression to tumor cells. However, many tumor- and tissue-specific promoters are weaker than the constitutively active promoters and are thus less effective. To combine cell-type specificity with high-level regulated transgene expression, we have developed a complex adenoviral vector. We have placed the tetracycline transactivator gene under the control of a prostate-specific ARR2PB promoter, and a mouse Tnfsf6 (encoding FASL)-GFP fusion gene under the control of the tetracycline responsive promoter. We have incorporated both expression cassettes into a single construct. We show that FASL-GFP expression from this vector is essentially restricted to prostate cancer cells, in which it can be regulated by doxycycline. Higher levels of prostate-specific FASL-GFP expression were generated by this approach than by driving the FASL-GFP expression directly with ARR2PB. More FASL-GFP expression correlated with greater induction of apoptosis in prostate cancer LNCaP cells. Mouse studies confirmed that systemic delivery of both the prostate-specific and the prostate-specific/tet-regulated vectors was well tolerated at doses that were lethal for FASL-GFP vector with CMV promoter. This strategy should be able to improve the safety and efficacy of cancer gene therapy using other cytotoxic genes as well.