Prostaglandin F2alpha-F-prostanoid receptor signaling promotes neutrophil chemotaxis via chemokine (C-X-C motif) ligand 1 in endometrial adenocarcinoma.

The prostaglandin F(2alpha) (PGF(2alpha)) receptor (FP) is elevated in endometrial adenocarcinoma. This study found that PGF(2alpha) signaling via FP regulates expression of chemokine (C-X-C motif) ligand 1 (CXCL1) in endometrial adenocarcinoma cells. Expression of CXCL1 and its receptor, CXCR2, are elevated in cancer tissue compared with normal endometrium and localized to glandular epithelium, endothelium, and stroma. Treatment of Ishikawa cells stably transfected with the FP receptor (FPS cells) with 100 nmol/L PGF(2alpha) increased CXCL1 promoter activity, mRNA, and protein expression, and these effects were abolished by cotreatment of cells with FP antagonist or chemical inhibitors of Gq, epidermal growth factor receptor, and extracellular signal-regulated kinase. Similarly, CXCL1 was elevated in response to 100 nmol/L PGF(2alpha) in endometrial adenocarcinoma explant tissue. CXCL1 is a potent neutrophil chemoattractant. The expression of CXCR2 colocalized to neutrophils in endometrial adenocarcinoma and increased neutrophils were present in endometrial adenocarcinoma compared with normal endometrium. Conditioned media from PGF(2alpha)-treated FPS cells stimulated neutrophil chemotaxis, which could be abolished by CXCL1 protein immunoneutralization of the conditioned media or antagonism of CXCR2. Finally, xenograft tumors in nude mice arising from inoculation with FPS cells showed increased neutrophil infiltration compared with tumors arising from wild-type cells or following treatment of mice bearing FPS tumors with CXCL1-neutralizing antibody. In conclusion, our results show a novel PGF(2alpha)-FP pathway that may regulate the inflammatory microenvironment in endometrial adenocarcinoma via neutrophil chemotaxis.

Generation and use of a tailored gene array to investigate vascular biology.

Vasculogenesis, angiogenesis and vascular remodelling are complex processes where the fate of several cell types is determined by different signalling networks. Many of these networks ultimately function by changing the abundance of RNA transcripts within the cells which constitute blood vessel walls. Researchers can now map these transcript abundance changes using gene array technology. In this review, we describe the design, production and use of a gene array specifically tailored to investigate vascular biology. We describe the advantages of tailored gene arrays, and give detailed protocols based on our experience to allow the reader to use such gene arrays to generate meaningful data. We list the issues to consider when choosing and verifying the genes and splice variants included in an array, and describe our use of Arabidopsis sp. RNA spikes for quality control. We present data that illustrates the absolute necessity for both technical and biological replicates to be incorporated in the design of gene array experiments using primary cells such as HUVECS. Finally, we describe methods for the normalisation and interpretation of the data that gene arrays produce. The approach to gene array technology described here is easily within reach of the budget and expertise of most academic research groups.