Control of growth and differentiation of normal human epithelial cells through the manipulation of reactive nitrogen species

Abstract

In this work, we addressed the issue of whether exogenous NO and ONOO- (peroxynitrite) are able to alter growth, viability and/or differentiation of normal epithelial cells using cultured normal human keratinocytes as a model. 3-Morpholino-sydnonimine (SIN-1), a donor of both NO and O2(-)., leading to the production of ONOO-, dose-dependently inhibited growth of human keratinocytes without loss of viability. This inhibitory effect was lowered when SIN-1 was transformed into a pure NO donor by scavenging O2(-). with superoxide dismutase/catalase. Finally, scavenging NO release from SIN-1 with carboxy-1H-imidazol-1-yloxy,2-(4-carboxyp henyl)-4,5-dihydro-4,4,5,5 -tetramethyl-3-oxide (PTIO) resulted in a loss of the inhibitory effect of SIN-1. Together these findings suggest that both ONOO- and NO exert a growth inhibitory effect on human keratinocytes without cytotoxicity. Further support for this conclusion came from the treatment of human keratinocytes with the NO. donor propanamine 3-(2-hydroxy-2-nitroso-1-propyl hydrazino) or with authentic peroxynitrite. Moreover, only SIN-1 or peroxynitrite, and not NO, was able to trigger the expression of markers of terminal differentiation in human keratinocytes. From a physiological perspective, the ability of peroxynitrite, a known genotoxic and potentially carcinogenic agent, to direct proliferating keratinocytes towards terminal differentiation may be crucial to protect the genomic stability of this barrier epithelium.

Evidence That Peroxynitrite Affects Human Osteoblast Proliferation and Differentiation

“…This strategy has been used already in a previous study showing that incubation of normal human keratinocytes in the presence of 1 mM of SIN-1 inhibited their growth, an effect that was reversed by a combined treatment with SIN-1 ϩ SOD ϩ CAT. (25) It has been shown that adding only SOD to SIN-1 does not inhibit PN generation, as could be expected. In addition to the unfavored kinetics of O 2 Ϫ scavenging by SOD as compared with the faster NO ⅐ and O 2 Ϫ reaction, the presence of SOD progres-…”

Keratinocyte serum-free medium maintains long-term liver gene expression and function in cultured rat hepatocytes by preventing the loss of liver-enriched transcription factors

“…We therefore sought to develop a protocol for long-term maintenance of hepatic functions in vitro by the use of a simple medium. Several groups previously used KSFM for culture of oesophageal keratinocytes, corneal epithelial cells and foreskin keratinocytes (Andl et al, 2003; Chen, Chang, Lee, Javier, & Azar, 2002; Vallette et al, 1998). Moreover, Katsura et al (2002) previously used KSFM medium along with serum to culture adult human hepatocytes (but in the absence of the EGF and PGE supplements).…”

“…Studies of liver function are therefore generally confined to the first few days of culture and this precludes longer-term studies. Conventional approaches to maintaining the differentiated properties of isolated hepatocytes in culture include supplementation of the medium with hormones such as dexamethasone (Dex) (Agius, Chowdhury, & Alberti, 1986; Enat et al, 1984), co-factors such as nicotinamide, pyruvate, DMSO and phenobarbital (LeCluyse, Bullock, Parkinson, & Hochman, 1996; Waxman, Morrissey, Naik, & Jauregui, 1990); the application of extracellular matrix components (Bissell, Arenson, Maher, & Roll, 1987; Gomez-Lechon et al, 1998) and co-culture with non-parenchymal epithelial cell-types (Rogiers & Vercruysse, 1993; Vallette et al, 1998). …”

The in vitro manipulation of carbohydrate metabolism: a new strategy for deciphering the cellular defence mechanisms against nitric oxide attack

“…The overwhelming of the cellular defence mechanisms by overproduction of ROS or RNOS is thought to play a pivotal role in the breakdown of the epithelial barrier during an inflammatory reaction. Surprisingly, primary cultures of epithelial cells or epithelial cell lines are highly resistant to a variety of oxidant challenges [3][4][5][6][7]. Nutritional conditions met by cells maintained in standard media, containing high concentrations of glucose (up to 25 mM in Dulbecco's modified Eagle's medium, DMEM), could strengthen their defence against an oxidative attack.…”