Adenovirus-p53 gene therapy in human nasopharyngeal carcinoma xenografts.

BACKGROUND: One major challenge to human cancer gene therapy, is efficient delivery of the gene-vector complex. METHODS AND RESULTS: Using two distinct human nasopharyngeal carcinoma (NPC) models, we demonstrate that intra-tumoural (IT) administration of adenoviral-mediated wild-type p53 gene therapy (Ad-p53) caused no greater inhibition of tumour growth as compared to ionizing radiation (XRT) alone. Detailed histologic examination of tumour sections demonstrated that <15% of tumour cells were transduced by IT adv-beta-gal. CONCLUSIONS: This report underscores the importance of developing gene transfer vectors, which can provide therapeutic levels of transgene expression efficiently in solid tumours.

The effects of combining ionizing radiation and adenoviral p53 therapy in nasopharyngeal carcinoma.

PURPOSE: Nasopharyngeal carcinoma (NPC) is a malignant disease of the head/neck region, with a 5-year survival level of approximately 65%. To explore gene therapy as a novel approach which might improve outcome, we have shown previously that introduction of human recombinant wild-type p53 mediated by the adenoviral vector (Ad5CMV-p53) was cytotoxic in two human nasopharyngeal carcinoma (NPC) cell lines (CNE-1 and CNE-2Z). The current work was designed to determine whether this strategy, combined with ionizing radiation (XRT), was more effective than either treatment alone. METHODS AND MATERIALS: CNE-1, CNE-2Z, and a normal human nasopharyngeal fibroblast strain, KS1, were infected with 2- and 6-plaque-forming units (pfu)/cell of Ad5CMV-p53, respectively. These doses were isoeffective for beta-galactosidase activity in the CNE-1 and CNE-2Z cells. XRT was administered 24 h post-infection, and Western blot analyses were conducted for p53, p21WAF1/CIP1, bax, and bcl-2 2 days after XRT. Cell survival was assessed using a clonogenic assay. Presence of DNA ladders reflecting apoptosis was detected using DNA agarose gel electrophoresis, and cell cycle was analyzed using flow cytometry. RESULTS: The combination of Ad5CMV-p53 plus XRT (2, 4, and 6 Gy) resulted in an approximately 1-log greater level of cytotoxicity compared to that observed with XRT alone for both NPC cell lines. The two modalities appear to be interacting in a synergistic manner in cancer cells, but not in KS1 fibroblasts. XRT alone stimulated minimal p53 expression in control cells; Ad5CMV-p53 alone induced significant recombinant p53 expression, which was not further enhanced by the addition of XRT. Similar observations were made for p21WAF1/CIP1 expression. No changes were observed for bax or bcl-2 expression with any of these treatments. Apoptosis was induced following 4 Gy of XRT alone, but was observed after only 2 Gy when combined with Ad5CMV-p53. Cell cycle analysis indicated that Ad5CMV-p53 infection did not perturb the cell cycle beyond that observed with XRT alone. CONCLUSION: p53 gene therapy and XRT appears to interact in a synergistic manner; underscoring the significant potential of this novel strategy in the treatment of NPC.

A second type-I PKS gene cluster isolated from Streptomyces hygroscopicus ATCC 29253, a rapamycin-producing strain.

Analysis of a 32.8-kb segment of DNA from the rapamycin (Rp) producer, Streptomyces hygroscopicus ATCC 29253, revealed a new type-I polyketide synthase (PKS) cluster consisting of four open reading frames (ORF 1-4), each encoding a single PKS module. The four ORFs are transcribed in the same direction and are flanked by several smaller ORFs (ORF 5-9), which may be related to the PKS cluster. The first PKS-containing ORF has a ligase domain at the N-terminus of the polypeptide. This domain has 55% aa identity to the CoA ligase domain of the Rp PKS (Schwecke et al., 1995. Proc. Natl. Acad. Sci. 92, 7839-7843) which is also encoded in this strain (Lowden et al., 1996. Angew. Chem. Int. Ed. Engl. 35, 2249-2251). ORF5 (340 aa) and ORF6 (924 aa) were found to be homologous to RapK (41% aa identity) and RapH (35% aa identity), which are hypothesized to be a pteridine-dependent dioxygenase and a regulatory protein, respectively (Molnar et al., 1996. Gene 169, 1-7). In addition, ORF7 (391 aa) was found to have up to 42% aa identity to a number of plant 3-deoxy-D-arabino-heptulosonate-7-phosphate synthases (DAHPS) and 47% aa identity to PhzF, a bacterial DAHPS involved in phenazine antibiotic synthesis. The proximity of the DAHPS-encoding gene to the PKS cluster containing a Rp-like ligase domain suggests that a derivative of shikimate may be used as the PKS starter. ORF8 (283 aa) was found to have homology (32% aa identity) to a Synechocystis sp. gene of unknown function. The N-terminal portion of ORF9 was found to be similar to a tetracycline 6-hydroxylase (34% aa identity) from Streptomyces aureofaciens.