Nox1 Induces Differentiation Resistance in Immortalized Human Keratinocytes Generating Cells that Express Simple Epithelial Keratins

Abstract

We have shown that superoxide radical-generating NADPH oxidase 1 (Nox1) is increased in intermediate human transformed cells. It was unknown whether Nox1 overexpression could accelerate early transformation steps. We demonstrated that Nox1 rendered human immortalized (GM16) keratinocytes resistant against Ca(2+)/serum-induced differentiation. Nox1-transfected cells produced fast dividing resistant cells within 7-10 days after DMEM exposure. Progenitor lines (or Nox1 lines) were reproducibly generated from Nox1-transfected cells, while no lines were obtained from control transfections. From several attempts to generate control cells, one resistant population was obtained from untransfected GM16 cells after a 6-week DMEM exposure. Prolonged passaging of the control line could induce Nox1. Compared with the control line, Nox1 lines showed greater expression of Nox1, Rac1, p47phox, p67phox, NOXO1, and NOXA1 with concomitant increased superoxide generation. All five Nox1 lines contained varying amounts of E-cadherin, involucrin, vimentin, and K8/K18, while the control line did not. Since vimentin and K8/K18 are associated with malignant progression in different types of human epithelial tumors, our data demonstrate that Nox1 accelerated neoplastic-like progression by inducing generation of progenitor cells. Our data also emphasize the importance of Nox1 in inducing resistance against differentiation-induced cell death, suggesting a contribution of Nox1 and its oxidants during early stage of cell transformation.