Differential regulation of p21waf-1/cip-1 and Mdm2 by etoposide: etoposide inhibits the p53-Mdm2 autoregulatory feedback loop

The Mdm2 protein is frequently overexpressed in human non-seminomatous germ cell tumours and transitional carcinoma of the bladder where it may contribute to tolerance of wtp53. Mdm2 forms an autoregulatory feedback loop with p53; the Mdm2 gene is responsive to transactivation by p53 and once synthesized the Mdm2 protein terminates the p53 response. We show here that the topoisomerase poison etoposide, like ultra violet irradiation, inhibits Mdm2 synthesis. Cytotoxic concentrations of etoposide (IC90 for >3 h) result in inhibition of Mdm2 induction at both the RNA and protein level. Rapid apoptosis ensues. Global transcription is not inhibited: p21waf-1/cip-1 and GADD45 expression increase in a dose dependent manner. Inhibition of Mdm2 synthesis depends on the continuous presence of etoposide, suggesting the DNA damage may prevent transcription. Downregulation of Mdm2 transcript occurs in cells expressing HPV16-E6 suggesting that inhibition of Mdm2 transcription is p53-independent. When cells are treated with a pulse (1 h) of etoposide and reincubated in drug free medium, Mdm2 synthesis commences immediately after damage is repaired (3 h) and the p53 response is attenuated. Induction of apoptosis and loss of clonogenicity are 3 – 5-fold lower under pulse treatment conditions. This is the first observation of inhibition of Mdm2 transcription following treatment with topoisomerase (topo II) poisons, a feature that may be useful in tumour types where p53 is tolerated by overexpression of Mdm2.