Abstract
Among five members of the NADPH oxidase (Nox) family, Nox1 confers mitogenic properties and is implicated to participate in the process of cell transformation. We have established two phenotypes of carcinogenesis model by ethanol treatment of human gingival keratinocytes immortalized with E6/E7 oncogenes of human papillomavirus type16: immortalized (EPI) nontransformed cells with epithelium-like morphology and more advanced transformed (FIB) cells with spindle fibroblastic-shape morphology. FIB membranes possessed a 63-kDa Nox1 protein at higher levels and exhibited 2.8-fold higher capability for superoxide and hydroxyl radical generation, compared with EPI membranes. Both EPI and FIB cells expressed more abundant Nox1 protein at a proliferating stage than that at a quiescent confluent phase. Immunofluorescence staining with an anti-Nox1 antibody showed that immunoreactive materials were distributed in the whole interior of both types of cells, while they were preferentially localized in the nuclei of FIB cells. Nuclei isolated from EPI and FIB cells contained a 63 kDa-Nox1 protein. Compared with EPI cells, FIB cells expressed elevated levels of Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase proteins. Furthermore, JNK2 was constitutively phosphorylated in FIB cells. Together, our data strongly implicate Nox1 in redox-mediated signaling related to cellular activation of human keratinocytes at a more advanced stage of transformation.
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Abbreviations
- DEPMPO:
-
5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide
- DTPA:
-
dithylenetriamine-pentaacetic acid
- EPI:
-
nontransformed cells with epithelium-like morphology
- ERK:
-
extracellular regulated kinase
- FIB:
-
transformed cells with spindle fibroblastic-shape morphology
- FLOAT:
-
cells with nonadherent free-floating state from FIB cultures
- HPV:
-
human papillomavirus
- JNK:
-
c-Jun N-terminal kinase
- KGM:
-
keratinocyte growth medium
- MAP kinase:
-
mitogen-activated protein kinase
- phox :
-
phagocyte oxidase
- •OH:
-
hydroxyl radical
- •OOH:
-
superoxide radical
- SOD:
-
superoxide dismutase
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Acknowledgements
We are grateful to Dr D Noethe (University of Heidelberg) for the use of an EPR spectrometer. We thank Dr H Spring of DKFZ for advice on Confocal microscopy. W Chunglok was supported by Thailand Research Fund for graduate studentship. A part of this study was supported by Grants-in-Aid Scientific Research from Japan Society for the Promotion of Science (No. 14370184) and Priority Areas Research from Monbu Kagakusho (No. 14021077) (to K Rokutan). This study was in part supported by Dietmar-Hopp Foundation (to W Stremmel).
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Chamulitrat, W., Schmidt, R., Tomakidi, P. et al. Association of gp91phox homolog Nox1 with anchorage-independent growth and MAP kinase-activation of transformed human keratinocytes. Oncogene 22, 6045–6053 (2003). https://doi.org/10.1038/sj.onc.1206654
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DOI: https://doi.org/10.1038/sj.onc.1206654
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