A431 cells escape EGF-induced apoptosis by forming cell aggregates. We show that these clusters migrate and merge with neighboring ones, resulting in larger structures composed of a multilayer central (3D) population surrounded by a cell monolayer (2D). We found that after 48 hr of 10 nM EGF treatment, 3D structure formation correlates with ␣2␤1 integrin upregulation. Blockade of ␣2 integrin impairs 3D structure formation. We studied the involvement of reactive oxygen species (ROS) in this process. We show that A431 cells express the NADPH oxidase catalytic subunits Nox1. EGF-induced dose-dependant ROS production was inhibited by the NADPH oxidase inhibitor, diphenylene iodonium (DPI), in these cells while rotenone was ineffective. Inhibition of ROS level in A431 cells with DPI or ebselen (glutathione peroxydase mimic) as well as P38 MAP kinase inhibition by SB203580 decreases ␣2 integrin subunit expression and induces a shift to 3D versus 2D populations. Cell cycle analysis of 2D cells shows that DPI, ebselen and SB203580 decrease the number of cells in S/G2 phase without affecting the cell number in mitosis phase. On the contrary, for 3D cells, these treatments increased the proportion of cells in mitosis without modification of the cell number in S/G2 phase. For both populations, apoptosis was increased by DPI and ebselen. Resistance of cell aggregates by paclitaxel to cell death is usually described. We show that DPI abolishes paclitaxel resistance of 3D cell aggregates. We observed a greater than additive effect between paclitaxel and DPI resulting in an increased proportion of cells in S/G2 phase for 3D populations. These results suggested that the ROS-P38 MAP kinase-␣2␤1 integrin pathway was implicated in the A431 survival process by modulating the balance between 2D/3D cells. © 2004 Wiley-Liss, Inc. Key words: reactive oxygen species; ␣2␤1 integrin; P38 MAP kinase; EGF receptor; adenocarcinoma A431 cell; apoptosis resistance; paclitaxel Epidermal growth factor (EGF) stimulates growth of a great variety of normal and tumoral cells. 1 Nevertheless, a number of tumor cells that overexpress EGF receptors (EGFR) are growthinhibited upon EGF treatment. 2,3 This paradoxical phenomenon has been best characterized in human squamous carcinoma A431 cells, which express a high number of EGFR. 4 These cells have thus been shown to exhibit a biphasic response to EGF in that they are weakly stimulated by picomolar concentrations of EGF but exhibit marked inhibition of proliferation in the presence of nanomolar EGF. 3,5 The growth-inhibitory action of EGF can lead to cell cycle block due to elevation of cyclin-dependent kinase inhibitor p21/CIP1 protein level correlated to G1/S arrest 6 -8 and/or to apoptosis. 9 -11 In A431 cells and in MDA-MB-468, another human cancer cell line overexpressing EGFR, the p21/CIP1-dependent STAT signaling pathway was shown to be required for apoptosis. 12,13 Nevertheless, a proportion of A431 cells escape the EGF-induced apoptosis process and these survival cells were characteriz...