PTEN is a tumor suppressor gene encoding a phosphatase that negatively regulates cell survival mediated by the PI3-kinase-Akt pathway. The gene for transcription factor EGR-1 is an early response gene essential for cellular growth, proliferation, and differentiation. Protein phosphatase inhibitors including calyculin A and okadaic acid are potent inducers of apoptosis in several cell lines; however, the molecular mechanisms underlying their action are unknown. The purpose of this study was to examine the expression of PTEN and EGR-1 and the phosphorylation status of EGR-1 and Akt in calyculin A-treated human squamous carcinoma cells (SCCTF). Phosphorylation of EGR-1 and upregulation of PTEN expression were observed to occur in SCCTF cells treated with calyculin A in time- and dose-dependent fashions. The level of phosphorylated Akt decreased as the expression of PTEN protein increased in the calyculin A-treated SCCTF cells. Calyculin A-stimulated expression of FGR-1 and PTEN might be p53 independent, because the expression of them was also detected in p53-null Saos-2 cells. RNA interference using double-stranded RNA specific for the EGR-1 gene inhibited not only EGR-1 expression but also PTEN expression in SCCTF cells treated or not with calyculin A. Calyculin A induced nuclear fragmentation and chromatin condensation in SCCTF cells. The present results suggest that the level of PTEN expression and the phosphorylation status of Akt were associated with apoptosis induced by calyculin A. These observations also support the view that EGR-1 regulates PTEN expression in the initial steps of the apoptotic pathway. (C) 2004 Wiley-Liss, Inc.