The application of adenoviral molecular chemotherapy for systemic malignant disease using herpes simplex virus thymidine kinase has been limited by ectopic transgene expression in the liver due to the vector hepatotropism. The aim of this study was to mitigate this hepatotoxicity using the promoter of cyclooxygenase-2, inactive in liver but active in many gastrointestinal cancers. To analyze the specificity of transgene expression driven by cyclooxygenase-2 (cox-2) promoters, promoters of two different lengths were incorporated into adenoviral vectors to drive luciferase expression. The specific cytocidal effect and in vivo toxicity were analyzed with thymidine kinase expression vectors. The specificity of the cox-2 promoter was well preserved in the adenoviral vector. In vivo, the cox-2 promoter (-1432/+59) showed very little activity in the liver but attained high activity, comparable to that of the cytomegalovirus promoter, in cyclooxygenase-2-positive subcutaneous tumors. The cox-2 promoter-driven thymidine kinase-expressing vectors showed a cytocidal effect specifically in cyclooxygenase-2-positive cells. When mice were treated with the thymidine kinase-expressing vector and ganciclovir, the cox-2 promoter successfully mitigated the fatal hepatotoxicity, which was observed with the cytomegalovirus promoter-driven vector. The cox-2 promoter successfully mitigated the adverse effects of adenoviral suicide gene therapy by minimizing transgene expression in the liver.
Malignant mesothelioma (MM), an incurable tumor, is reportedly an interleukin‐6 (IL‐6) secreting tumor. The pathological significance of IL‐6 overexpression in this tumor, however, has remained unclear. We investigated the biological functions of IL‐6 in mesotheliomas. Five mesothelioma cell lines were analyzed for IL‐6 production and IL‐6 receptor (IL‐6R) expression. Of them, 2 produced high levels of IL‐6, 2 produced intermediate levels and 1 cell line showed no secretion. All mesothelioma cell lines used in this study expressed very small amounts of IL‐6R mRNA. We compensated for this low level of IL‐6R expression in mesotheliomas by adding recombinant soluble IL‐6R (sIL‐6R) to mediate the IL‐6 signal. IL‐6 together with sIL‐6R was found to promote cell growth of H2052 and H226 MMs classified as high‐level IL‐6 producers in a dose‐dependent manner. Moreover, a humanized anti‐IL‐6R antibody (MRA) capable of blocking IL‐6 signaling suppressed the cell growth of mesotheliomas induced by IL‐6/sIL‐6R. These findings demonstrate that IL‐6 serves as an autocrine growth factor in the development of mesothelioma. In addition, IL‐6/sIL‐6R stimulation increased the expression of vascular endothelial growth factor (VEGF) in 4 out of 5 cell lines, and this induction was inhibited by MRA treatment. The involvement of the signal transducer and activator of transcription 3 (STAT3) pathway in both cell growth and VEGF induction by IL‐6/sIL‐6R was verified by dominant negative STAT3 transduction combined with adenovirus gene‐delivery methods. Although IL‐6 induces VEGF through the JAK2/STAT3 pathway, anti‐VEGF antibody could not inhibit the IL‐6‐induced cell growth observed in H2052 and H226. We concluded that IL‐6‐dependent growth does not occur via VEGF induction. These results suggest that treatment with anti‐IL‐6R antibody may constitute a potential molecular targeting therapy for MMs. © 2006 Wiley‐Liss, Inc.
Previously, we showed that nasal administration of a naked cDNA plasmid expressing Flt3 ligand (FL) cDNA (pFL) enhanced CD4(+) Th2-type, cytokine-mediated mucosal immunity and increased lymphoid-type dendritic cell (DC) numbers. In this study, we investigated whether targeting nasopharyngeal-associated lymphoreticular tissue (NALT) DCs by a different delivery mode of FL, i.e., an adenovirus (Ad) serotype 5 vector expressing FL (Ad-FL), would provide Ag-specific humoral and cell-mediated mucosal immunity. Nasal immunization of mice with OVA plus Ad-FL as mucosal adjuvant elicited high levels of OVA-specific Ab responses in external secretions and plasma as well as significant levels of OVA-specific CD4(+) T cell proliferative responses and OVA-induced IFN-gamma and IL-4 production in NALT, cervical lymph nodes, and spleen. We also observed higher levels of OVA-specific CTL responses in the spleen and cervical lymph nodes of mice given nasal OVA plus Ad-FL than in mice receiving OVA plus control Ad. Notably, the number of CD11b(+)CD11c(+) DCs expressing high levels of costimulatory molecules was preferentially increased. These DCs migrated from the NALT to mucosal effector lymphoid tissues. Taken together, these results suggest that the use of Ad-FL as a nasal adjuvant preferentially induces mature-type NALT CD11b(+)CD11c(+) DCs that migrate to effector sites for subsequent CD4(+) Th1- and Th2-type cytokine-mediated, Ag-specific Ab and CTL responses.
Midkine ( MK ), a heparin binding growth factor, and cyclooxygenase -2 ( COX -2 ), a key enzyme in the conversion of arachidonic acid to prostaglandin, are both up -regulated at the mRNA or protein level in many human malignant tumors. Here, we investigated the tumor specificity of both MK and COX -2 promoters in human pancreatic cancer, with the aim to improve the selectivity of therapeutic gene expression. We constructed recombinant adenoviral ( Ad ) vectors containing either the luciferase ( Luc ) reporter gene under the control of the COX -2 or MK promoter or the herpes simplex virus thymidine kinase ( HSV Tk ) gene under the control of the COX -2 promoter and compared the expression with the cytomegalovirus ( CMV ) promoter. AdMKLuc achieved moderate to relatively high activity upon infection to both primary and established pancreatic carcinoma cells. Of the two COX -2 promoter regions ( COX -2M and COX -2L ), both revealed a high activity in primary pancreatic carcinoma cells, whereas in the established pancreatic carcinoma cell lines, COX -2L has an approximately equal high activity compared to CMV. In addition, both AdCOX -2M Tk and AdCOX -2L Tk induced marked cell death in response to ganciclovir ( GCV ) in three of four established pancreatic carcinoma cell lines. From these results, and because it has been reported that AdMKTk and AdCOX -2L Tk in combination with GCV did not reveal significant liver toxicity, we conclude that the MK as well as the COX -2 promoters are promising tumor -specific promoters for Ad vector -based gene therapy of pancreatic cancer. Cancer Gene Therapy ( 2001 ) 8, 990 -996
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