Abstract
Abstract Shape changes in cancer cells profoundly alter the expression of genes that affect malignant properties. We postulated that shape change may increase expression of NANOG, an embryonic stem cell transcription factor expressed in human colorectal carcinoma (CRC) that regulates pluripotency and proliferation. Since shape change also increases intracellular nitric oxide (iNO) expression, we further postulated that iNO modulates NANOG expression. Clone A and CX-1 human CRC lines were cultured in serum-containing medium in monolayer cultures or in spheroids in serum free medium in low attachment plates. NANOG promoter activity was assessed in stable transfectants expressing promoter-driven GFP. iNO concentrations were determined by fluorescence cytometry with DAF-FM diacetate while Nanog gene and protein expression was analyzed by qRT-PCR and densitometry of western blots, respectively. Spherogenicity was measured by the percentage of spheroids formed from single cells in serum-free medium. Monolayer cultures with flat, spreading cells had low NANOG gene and protein expression, promoter activity and iNO whereas spheroids displayed high NANOG gene expression, promoter activity and iNO. 7 day monolayer cultures initiated with 104 cells/ml (Sparse) grew horizontally compared to cultures initiated with 106 cells/ml (Dense) that grew vertically and formed colonies. Sparse cultures had low NANOG gene expression, low NANOG promoter activity and low iNO. Dense cultures had high NANOG gene expression, NANOG promoter activity and iNO. Since NO stimulates cGMP production, the effects of the NO synthase inhibitor L-NMMA, NOC (a NO donor), 8-Br-cGMP (cGMP agonist) and ODQ (cGMP inhibitor) were tested on CRC monolayers. Physiologic concentrations of NOC increased NANOG and Oct4 gene and protein expression 3-fold in highly metastatic CX-1 cells but not in weakly metastatic Clone A cells. NOC also increased CD44 in CX-1 but not Clone A. cGMP may regulate NANOG expression since 8-Br-cGMP increased NANOG and Oct4 protein expression up to 3-fold in CX-1 but not in Clone A and without affecting CD44 expression. ODQ inhibited NANOG expression. L-NMMA stimulated both NANOG protein expression and the spherogenicity of CX-1 but not Clone A. NOC and 8-Br-cGMP increased inducible NO synthase (NOS2) but not NOS3 gene expression 3-fold in both spheroids and monolayers while ODQ decreased NOS2 levels. Assays are in progress to test the effects of NOC and the cGMP modulators on spherogenicity. Shape change, iNO and cGMP enhance NANOG, NOS2 and CD44 expression to alter CRC cancer stem cell function. cGMP may be an important regulator of stemness genes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4249.
Published Version
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