Gene expression and cellular sources of inducible nitric oxide synthase during tumor promotion

Abstract

The present studies examined the temporal sequence of inducible nitric oxide synthase (iNOS) gene expression and the cellular sources of iNOS protein and of 3-nitrotyrosine, as a marker of production of nitric oxide-derived reactive nitrogen intermediates during murine multi-stage carcinogenesis. Levels of iNOS mRNA in dorsal skin isolated from acetone-treated female Sencar mice were 2.5-fold higher than iNOS gene expression detected in cutaneous tissue isolated from Sencar mice at 1, 3, 6, 10, 16 and 22 weeks after exposure to a single topical application of 25 nmol 7,12-dimethylbenz[a]anthracene (DMBA) followed by repetitive applications of 2 microgram 17-O-tetradecanoylphorbol-13-acetate (TPA). Papillomas isolated at 16 and 22 weeks of a tumor promotion protocol also had low levels of iNOS mRNA. The diminished levels of iNOS mRNA inversely correlated with the extent of TPA-induced epidermal hyperplasia. In acetone-treated mouse skin, iNOS immunospecific antibody binding was localized to the stratum corneum and suprabasal keratinocytes. In contrast, iNOS protein was present in lower amounts and was localized to the upper-most suprabasal keratinocytes in cutaneous tissue isolated at 22 weeks following a single exposure to either 25 nmol DMBA or acetone and repetitive applications of 2 microgram TPA. At all time points examined over the 22 week time period of papilloma growth, infiltrating neutrophils within the dermis bound significant levels of anti-iNOS antibodies. The production of iNOS by neutrophils within the dermis correlated with the formation of protein nitrotyrosination within the dermal tissue, as detected by 3-nitrotyrosine-specific antibodies. The present studies indicate that NOS and reactive nitrogen intermediates, including peroxynitrite, are produced specifically by dermal neutrophils during the tumor promotion process at time points that correspond to simultaneous production of reactive oxygen intermediates. Conversely, iNOS is simultaneously down-regulated in hyperplastic epidermis and in papillomas.

Neutrophils, Nitric Oxide Synthase, and Mutations in the Mutatect Murine Tumor Model

“…Immunohistochemical analysis of MN-11 tumors demonstrated iNOS in infiltrating neutrophils and biochemical measurements of tissue extracts showed NOS activity. The presence of iNOS in mouse neutrophils 52,53 and the presence of high NOS activity in some human cancers has been reported. 54 Further evidence of the presence of RNS in MN-11 tumors was provided by immunohistochemical analysis for nitrotyrosine; immunoreactivity was observed throughout the tumor, both in the cytoplasm and the nucleus of tumor cells.…”

Clinically Relevant Immunosuppressants Influence UVB-Induced Tumor Size Through Effects on Inflammation and Angiogenesis

“…One immediate physiologic consequence of UVB exposure is skin inflammation (20)(21)(22)(23)(24), characterized by edema (25) and dermal neutrophil infiltration (26,27). Neutrophils contribute to skin inflammation by producing myeloperoxidase (MPO), reactive oxygen intermediates and pro-inflammatory cytokines (reviewed in (24,(28)(29)(30)(31), which can contribute to tumor growth by enhancing angiogenesis (32)(33)(34). Reducing inflammation in these early stages reduces both angiogenesis (reviewed in (35)) and tumor growth (27,36).…”

Acute Toxicity Assessment of Break-Free CLP: A Weapons Cleaning and Maintenance Compound

“…There was a direct correlation between increased skin NO concentrations and gross and histopathological evidence of dermal irritation in CD-I skin treated with DMBA. The observed increase in NO concentrations is most likely due to the induction of skin NOS-2 or iNOS activity; DMBA has previously shown to cause increases in iNOS expression in dermal cells (Robertson et al 1996). It is not clear if NO plays a pro-or anti-inflammatory role in tissue response to DMBA exposure.…”

“…Repeated or prolonged dermal contact with isoparaffmic hydrocarbons causes dermatitis (Ueno et al 2002, Mullin et al 1990) and may elicit contact sensitization in rare instances (Mullin et al 1990). Dermal application of croton oil and DMBA produces dermal irritation reactions (Moon et al 2001, Robertson et al 1996, Ruben 1982 and repeat application of DMBA produces a contact hypersensitivity response in the skin (Klemme et al 1987). …”

“…During an inflammation reaction, local NO concentrations increase over several hours and can remain elevated for hours, days, or longer (Coleman 2001). It has been shown that local iNOS expression in skin is increased following topical exposure to hydrocarbons including JP-8 (Kabbir et al 2001), m-xylene (Gunasekar et al 2003), and 7,12-dimethylbenz[a]anthracene (Robertson 1996), indicating that NO may have a mechanistic role in hydrocarbon-induced dermal irritation. It is possible that increased local NO concentrations can be used as a marker for acute dermal irritation for other types of compounds and NO concentration levels may have utility for characterizing or discerning between responses of different degrees of severity.…”