Up-regulation of the redox mediators thioredoxin and apurinic/apyrimidinic excision (APE)/ref-1 in hypoxic microregions of invasive cervical carcinomas, mapped using multispectral, wide-field fluorescence image analysis

Thioredoxin and apurinic/apyrimidinic excision (APE)/ref-1 are important redox mediators in biochemical pathways that promote cell survival under adverse conditions including hypoxia and oxidative stress. For example, elevated levels occur surrounding vascular infarcts and protect from reperfusion injury. Because elevated thioredoxin or APE/ref-1 is also associated with resistance to certain forms of cancer treatment, we examined their tissue distribution in a series of 110 cervical carcinoma biopsies. Analysis was done using a quadruple fluorescence imaging technique, incorporating carbonic anhydrase IX (CAIX) immunofluorescence to outline hypoxic microregions and 4',6-diamidino-2-phenylindole to localize nuclear staining of thioredoxin and APE/ref-1. A scanning autostage was used to image the entire tissue section. Thioredoxin and APE/ref-1 levels were expressed as the average pixel brightness in tumor tissue, subdivided based on CAIX and 4',6-diamidino-2-phenytindole staining. Results showed that the nuclear and cytoplasmic levels of thioredoxin were similar, whereas APE/ref-1 expression was greater in nuclei. Neither of these markers was predictive of outcome in this series of patients treated with radical radiotherapy. Both proteins showed highly significant elevations in CAIX-positive regions compared to CAIX-negative regions, and there was a nonsignificant trend for this effect to be greater in adenocarcinomas compared to squamous cell carcinomas. Levels of APE/ref- 1 decreased with increasing tumor grade, but the expression was similar in CAIX-positive regions of poorly differentiated tumors compared to moderately or well-differentiated tumors. Elevated expression of thioredoxin and APE/ref-1 might promote cancer cell survival in hypoxic micro-environments of cervical carcinomas.