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Diagnostic Technologies and Molecular and Cellular Profiling: Biomarkers in Early Detection and Diagnosis

The P2X₇ receptor: A novel biomarker of uterine epithelial cancers

CASE (Case Western Reserve) University, Cleveland, OH and Uniformed Services University, Bethesda, MD

Abstract

A21

The receptor P2X₇ is membrane-bound, ligand-operated channel for which ATP is the naturally occurring ligand. In human uterine epithelial cells activation of the receptor by extracellular ATP stimulates formation of pores in the plasma membrane that allow massive Ca²⁺ influx, and induces apoptosis by a mechanism that involves formation of mitochondrial permeability transition pores and activation of the caspase-9 / caspase-3 pathway (Wang et al, Endocrinology 145:5568, 2004). Earlier studies showed that baseline constitutive apoptosis, and ligand induced P2X₇-mediated apoptosis is greater in normal human ectocervical epithelial cells than in cancer human cervical cells (Wang et al, Am J Physiol 287:C1349, 2004). Steady-state levels of extracellular ATP in conditioned media of human normal and cancer epithelial cells were similar, and in the range sufficient to activate the P2X₇ receptor (ibid). This, and our finding that P2X₇ receptors are preferentially expressed in normal than in cancer cervical cells (Feng et al, J Biol Chem 281:17228, 2006) suggest that low expression of P2X₇ receptors may be casually related to cancer development and progression. To test this hypothesis, we determined expression of the P2X₇ receptor in human normal and cancer uterine tissues. Normal (n = 42) and cancer uterine tissues (n = 47) were obtained from a total of 72 women, ages 25-75. Endpoints for P2X₇ mRNA were qPCR and In-Situ-Hybridization, and for P2X₇ protein were Western-blots and immunostaining using anti-P2X₇ antibody. In normal uteri, P2X₇ mRNA and protein were expressed predominantly in the epithelial component of the endometrium, endocervix, and ectocervix, as confirmed histologically and by co-staining with the epithelial marker E-Cadherin. P2X₇ mRNA and protein were absent, or only minimally expressed in endometrial and endocervical adenocarcinoma tissues and in cervical squamous-cell carcinoma tissues. In cervical dysplasia, P2X₇ protein was absent in the dysplastic lesions, as confirmed histologically, but it was present in superficial layers of the epithelium representing receptor trapped in nucleated and un-nucleated envelops through the process of terminal differentiation of the epithelium. Semi-quantitative analysis using P2X₇ mRNA normalized to GAPDH, and P2X₇ protein levels normalized to tubulin, revealed separation of the P2X₇ mRNA and protein levels according to normal / cancer histology, whereby the ratios of both mRNA P2X₇ / GAPDH and protein P2X₇ / tubulin were on the average 10 fold higher in normal than in cancer tissues. qPCR (Real-Time PCR) and densitometry analyses revealed that P2X₇ mRNA and / or protein levels can distinguish uterine normal from cancer tissues at sensitivity of 92-100% and specificity of 100-90%. These data indicate that levels of the P2X₇ are lower in uterine epithelial cancer tissues than in the corresponding normal tissues. We suggest that tissue P2X₇ mRNA and protein levels could be used as a novel biomarker to differentiate normal and cancer uterine epithelial tissues.