Cyclooxygenase 2 and Prostaglandin E2 are not Involved in N-Nitrosodiethylamine-Initiated Early Rat Hepatocarcinogenesis

The present study was undertaken to investigate the effect of dietary supplementation with nimesulide or eugenol on N-nitrosodiethylamine (DEN)-initiated early hepatocarcinogenesis in F344 male rats. Both compounds did not alter the expression of cytochrome P450 (CYP) 2E1, the enzyme that plays a major role in the activation of DEN to genotoxic products; however, nimesulide induced the expression of CYP1A1. Western blot analysis revealed that COX-1 and COX-2 protein expressions were not modulated by DEN compared with normal controls. Furthermore, post-initiation feeding with nimesulide or eugenol did not modulate COX-2 protein expression in normal or DEN-treated rats, whereas eugenol significantly increased the liver prostaglandin E2 (PGE2) levels of DEN-injected animals compared with the DEN controls. Ultimately, nimesulide or eugenol did not modify DEN-induced hepatocarcinogenesis as evidenced by insignificant changes in the number and size of preneoplastic placental glutathione S-transferase (GST-P) positive liver foci compared with the DEN controls. These results suggest that COX-2, as well as prostaglandin E2, may play no role in the post-initiation development of DEN-induced rat hepatocarcinogenesis at an early stage

Telomerase immortalization of principal cells from mouse collecting duct

Recently, the use of overexpression of telomerase reverse transcriptase (TERT) has led to the generation of immortalized human cell lines. However, this cell immortalization approach has not been reported in well-differentiated mouse cells, such as renal epithelial cells. We sought to establish and then characterize a mouse collecting duct cell line, using ectopic expression of mTERT. Isolated primary cortical collecting duct (CCD) cell lines were transduced with mouse (m)TERT, using a lentiviral vector. mTERT-negative cells did not survive blasticidin selection, whereas mTERT-immortalized cells proliferated in selection media for over 40 subpassages. mTERT messenger RNA and telomerase activity was elevated in these cells, compared with an SV40-immortalized cell line. Flow cytometry with Dolichos biflorus agglutinin was used to select the CCD principal cells, and we designated this cell line mTERT-CCD. Cells were well differentiated and exhibited morphological characteristics typically found in renal epithelial cells, such as tight junction formation, microvilli, and primary cilia. Further characterization using standard immunofluorescence revealed abundant expression of aquaporin-2 and the vasopressin type 2 receptor. mTERT-CCD cells exhibited cAMP-stimulated/benzamil-inhibited whole cell currents. Whole cell patch-clamp currents were also enhanced after a 6-day treatment with aldosterone. In conclusion, we have successfully used mTERT to immortalize mouse collecting duct cells that retain the basic in vivo phenotypic characteristics of collecting duct cells. This technique should be valuable in generating cell lines from genetically engineered mouse models