p38 MAP kinase enhances EGF-induced apoptosis in A431 carcinoma cells by promoting tyrosine phosphorylation of STAT1.

While epidermal growth factor (EGF) is a well known mitogen, high doses of EGF result in a paradoxical apoptotic response in the cells that overexpress EGF receptor such as A431 epidermoid carcinoma cells. EGF-induced apoptosis in A431 cells is dependent upon activation of transcription factor STAT1. In this study, we demonstrate that p38 MAP kinase is another important mediator of EGF-dependent pro-apoptotic response in A431 cells. By utilizing p38 MAP kinase inhibitors, SB203580 and BIRB0796, we significantly reduced the integral growth-inhibiting as well as pro-apoptotic effects of EGF. Moreover, we observed that inhibition of p38 MAP kinase markedly decreased phosphorylation of tyrosine 701 in STAT1, while neither EGF-induced accumulation nor serine phosphorylation of STAT1 was decreased. We propose that p38 MAP kinase mediates STAT1 tyrosine phosphorylation, thereby enforcing EGF-induced apoptosis.

EGF-induced apoptosis in A431 cells is dependent on STAT1, but not on STAT3.

EGF in high concentrations has a growth-inhibitory effect on human epidermoid carcinoma cells A431. The transcription factor STAT1 is the most probable candidate for mediating this effect. In the present study, we demonstrated a strong reduction of the expression level of STAT1 in EGF-resistant sub-clones of A431 cells. EGF resistance was reversed by introducing wild-type STAT1, but not its Y701F mutant. Moreover, blocking the activity of Src family kinases reduced tyrosine phosphorylation of STAT1 and STAT3 and protected A431 cells from the EGF-induced growth inhibition. To further elucidate roles of STATs in A431 cell growth and survival, clones of A431 cells expressing short hairpin RNA (shRNA) against STAT1 or STAT3 were generated. Neither STAT1 nor STAT3 knockdown exerted any effect on growth rate or apoptotic death of A431 cells in the absence of EGF. However, upon EGF treatment A431 cells with knocked down STAT1 continued to grow and demonstrated a significantly lower level of apoptosis as compared to A431 cells. The knockdown of STAT3 did not alter cell growth or apoptosis. Taken together, our experiments prove the essential role of tyrosine phosphorylated STAT1, but not of STAT3, in EGF-induced apoptosis in A431 cells.

Downregulation of peroxiredoxin V stimulates formation of etoposide-induced double-strand DNA breaks.

Antioxidant protein Peroxiredoxin V (PrxV) is located in mitochondria and peroxisomes but is also present in the nucleus. Here, we show that nuclear PrxV associates with coilin-containing bodies suggesting possible interaction of this protein with transcription complexes. We also studied etoposide-induced phosphorylation of histone H2AX (gamma-H2AX) in human cells in which PrxV activity was downregulated (knockdown, KD-clones) or compromised by overexpression of redox-negative (RD) protein. In KD clones, but not in RD-clones, formation of etoposide-induced gamma-H2AX was increased, indicating that PrxV inhibits conversion of topoisomerase II cleavage complexes into double-strand DNA breaks but this inhibition is not caused by its antioxidant activity.