Protection of rats against oxygen toxicity by tracheal administration of plasmid DNA: role of endogenous tumor necrosis factor.

Recombinant plasmid DNA alone or in conjunction with a gene delivery system has been used increasingly for in vivo gene transfer. However, little is known about the direct biological effects of plasmid DNA. In this study, we demonstrated that tracheal administration of a number of plasmid DNA protected rats against oxygen toxicity. This protection required the presence of intact plasmid DNA, was not due to endotoxin contamination, and was not shared by salmon testis DNA. The plasmid DNA-induced protection against oxygen toxicity was associated with the production of tumor necrosis factor (TNF) and the enhancement of pulmonary Mn-superoxide dismutase (MnSOD), CuZnSOD, and glutathione peroxidase activities. Coadministration of plasmid DNA and anti-TNF antibody (but not nonspecific IgG) partially abolished the protective effect and reduced the pulmonary MnSOD activity, suggesting that the plasmid DNA-induced oxygen tolerance was in part mediated by the endogenous TNF and MnSOD. In view of these observations and the known immunostimulatory effects of bacterial DNA, caution should be exercised in interpreting the results of in vivo gene transfer using recombinant plasmid DNA.

Decreased levels of CD44 protein and mRNA in prostate carcinoma. Correlation with tumor grade and ploidy.

BACKGROUND: CD44, a transmembrane protein, is associated with cell-cell and cell-matrix interaction and with tumor growth and metastasis. Expression of both standard form and variant isoforms of CD44 protein has been associated with aggressive behavior and metastasis in various tumors, but has not been characterized in prostate adenocarcinoma (PAC). METHODS: The expression of CD44 standard (CD44s) and splice variant v3, v4/5, v6, v7/8, and v10 proteins were studied in 109 PACs and correlated with tumor grade, DNA ploidy, and mRNA levels. Monoclonal antibodies against the various CD44 proteins were applied to microwave irradiated, formalin fixed, paraffin embedded sections. The DNA content of the tumors was evaluated by the Feulgen method with the CAS200 Image Analyzer. Total RNA exhibiting 18s and 28s bands was derived from two benign prostatic tissues and 5 PACs exhibiting decreased levels of CD44 protein by immunohistochemistry. The RNA was analyzed with reverse transcriptase polymerase chain reaction using CD44 specific primers. RESULTS: The basal cells of the benign prostatic acini revealed uniform membranous staining for CD44s, v3, and v6 in 95-97% of cases. Similar staining was observed for v4/5, v7/8, and v10 in 40%, 30%, and 2% of cases, respectively. Secretory epithelial cells of the benign prostatic acini showed predominant expression of CD44s (97% of cases). Staining for CD44 variant proteins (v3, v4/5, v6, v7/8, and v10) in this location ranged from 9-22% of cases. Approximately 70% of the PACs showed significant loss of CD44s expression, which correlated with high tumor grade (Gleason > or = 7) (P = 0.01) and aneuploid status (P = 0.002). In 93-98% of the PACS, there was a complete lack of membranous expression for all CD44 variant isoforms. The metastatic PACS did not show preferential expression of either the standard form or any variant isoform. The cDNA from the normal prostates yielded a prominent CD44 standard form polymerase chain reaction product at 482 base pair (bp) and variant isoforms at approximately 650 and 850 bp. No CD44 products could be amplified from the subset of five PAC cDNAs, even when present at four-fold excess. CONCLUSIONS: PACS exhibit down-regulation of CD44 protein expression, which correlates with high tumor grade and aneuploidy. v6 and v3 isoforms were preferentially expressed in the basal cells of benign prostatic acini. Based on a subset of cases, loss of CD44 protein expression is associated with decreased abundance of CD44 mRNA.