COX-2 expression is increased by Pontin52/TIP49a and LEF-1 over-expression; colon cancer tissue shows increased expression and co-localization of Pontin52/TIP49a and COX-2. (A) Pontin52/TIP49a overexpression increases COX-2 promoter activity. Increasing amounts of an expression construct for Pontin52/TIP49a were transiently transfected into LoVo cells along with a COX-1840 reporter construct. A dose-dependent increase in COX-2 promoter activity was observed in co-transfections with Pontin52/TIP49a. (B) The COX-2 promoter is activated in an additive manner by co-overexpression of LEF-1 and Pontin52/TIP49a. cDNAs for LEF-1 (0.5 μg) and Pontin52/TIP49a (1 μg) were transfected into LoVo cells alone or in combination. Data are the means of assays performed in triplicate in each of two independent experiments. RLU: relative light units. (C) LEF-1 activation of the COX-2 promoter is independent of the β-catenin binding region (Δ N67) or the DNA-binding HMG domain (HMG; amino acids 300–359) of LEF-1. Transient transfection of LoVo cells with the -1840nt COX-2 reporter construct (+) and constructs containing wild type LEF-1 (WT) or the indicated constructs were completed. Controls included a COX-2 promoterless reporter, pGL3-basic (c) and LEF-1 vector controls, FLAG or EV3S (v). Data are the means of assays performed in triplicate in each of 3 independent experiments. In the LEF-1 transfected cells the fold-increase ranged from 1.2 to 2.3 and the average is shown. This response was observed in multiple other experiments, using different amounts of transfected cDNA, and in different cells. (D) Reverse transcription PCR was performed with cDNAs from paired samples of normal (N) colonic tissue and colon cancer tissue (CA) from each of four patients. The top panel shows Pontin52/TIP49a expression; the bottom panel shows COX-2 expression (upper band) relative to a control template (β-actin, lower band). The data shown are representative of two experiments. (E) An example of in situ hybridization using anti-sense (experimental) and sense (control) probes for Pontin52/TIP49a and COX-2, showing co-expression and up-regulation of mRNAs for both proteins in a colon cancer sample, compared to normal mucosa from the same patient.