The DNA damaging agent VP16 induces the expression of a subset of ligands from the EGF system in bladder cancer cells, whereas none of the four EGF receptors are induced.

Increased activity of the EGF system exerts a cell survival function in the presence of cytotoxic agents. The aim of our investigation was to identify the ligands and receptors from the EGF system, that are induced by the chemotherapeutic DNA damaging agent VP16 in bladder cancer cell lines. By use of real-time RT-PCR assays for all four receptors and six ligands from the EGF system we demonstrate that in HCV29 bladder cancer cells, amphiregulin, HB-EGF, and epiregulin mRNA levels are elevated (more than 100, 5, and 4 fold, respectively) by VP16. The remaining ligands (EGF, TGFalpha and betacellulin) are uninduced. The same was found for T24A bladder cancer cells, except that TGFalpha also was induced. The four receptors were reduced by VP16 in both cell lines. This demonstrates that the induction of the EGF system is mediated by an increased expression of a subset of the ligands, whereas the four receptors are reduced. For amphiregulin and HER1 we investigated with ELISA assays if the effects of VP16 also were observed at the protein level. We found that VP16 increase the amount of amphiregulin peptide both in the cell membrane and the culture medium. Similarly, the reduced EGF receptor mRNA expression correlated with reduced HER1 protein. Several investigations have shown that labile protein factors can be involved in the regulation of stress inducible growth factors and cytokines. We investigated if a labile protein regulates the expression of the subset of ligands that were induced with VP16. Blocking of protein neosynthesis with cycloheximide resulted in induced mRNA expression of exactly the same subset of ligands as observed with VP16 treatment of both HCV29 and T24A cells. This suggests that a labile protein factor regulates either the transcription or degradation of these mRNA's, and it can be speculated that VP16 also operate by inhibiting the activity of this factor. This is further stressed by the observation that combined treatment with cycloheximide and VP16 show no additive effect. In conclusion, we show that a subset of ligands from the EGF system is upregulated by VP16, whereas none of the four receptors are induced. This might represent a physiological response aimed at rescuing the cells.