The mRNA expression of inducible nitric oxide synthase in DMBA-induced hamster buccal-pouch carcinomas: an in situ RT-PCR study

Abstract

Three isoforms of nitric oxide synthase (NOS) have been identified: endothelial NOS, neuronal NOS, and inducible NOS (iNOS). The enhanced expression of iNOS at the protein level using immunohistochemical technique has been reported previously in chemically induced oral carcinomas in hamster buccal-pouch mucosa. However, the corresponding expression of iNOS at the mRNA level has not yet been demonstrated using in situ reverse transcription-polymerase chain reaction (IS RT-PCR). The purpose of the present study is to assess the iNOS mRNA expression level in 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal-pouch carcinomas using IS RT-PCR. Thirty outbred, young (6-weeks old), male, Syrian golden hamsters (Mesocricatus auratus) were randomly divided into one experimental group (10 animals), and two control groups (10 animals each). The pouches of a group of 10 animals of the experimental group were painted bilaterally with a 0.5% DMBA solution three times a week for 15 weeks. Each animal of one of the control groups was similarly treated with only mineral oil. Another control group of 10 animals remained untreated throughout the experiment. Invasive squamous-cell carcinomas with a 100% tumour incidence developed in all of the DMBA-treated buccal pouches. The mineral oil-treated and untreated pouches revealed no obvious changes. Inducible NOS mRNA was demonstrated amongst all the 15-week DMBA-treated hamster buccal-pouch mucosa animals, but not in the untreated animals and not in the animals for which the buccal-pouch was treated with mineral oil. Further study is necessary to evaluate the mechanism(s) which contribute to the increased iNOS mRNA expression for experimentally induced oral carcinogenesis.

Figure 1

Inducible NOS mRNA as shown by the purple coloration was identified in the invasive tumour islands of a representative sample of hamster buccal-pouch tissue specimen treated with DMBA for 15 weeks. Note that iNOS mRNA staining was also present in cells of the tumour stroma, presumed to be macrophages (×100).

Figure 2

Inducible NOS mRNA activity could not be found in a representative sample of non-treated pouch tissue specimen (×100).

Figure 3

Inducible NOS mRNA activity could not be detected in a representative sample of mineral oil-treated pouch tissue specimen (×100).

Figure 4

Omission of the primers in control sections showed negative finding for iNOS mRNA activity (×100).

Figure 5

A band corresponding to 499 bp was observed for all the selected samples of hamster buccal-pouch tissue specimens treated with DMBA for 15 weeks (lanes 1–10). Lane M is the DNA molecular-weight marker.