Figure 5.

Alternatively spliced forms of MDM2 and MDM4 are induced by genotoxic stress in RMS cell lines. SMS-CTR and Rh30 RMS cell lines were treated with increasing amounts of UV light (0, 10, 30, and 50 J/m²) or increasing concentrations of cisplatin (0, 25, and 50 µM) as represented by the corresponding increase in height of the triangle from left to right. Total RNA was harvested at 24 hours and subjected to nested RT-PCR for MDM2 and MDM4. In response to increasing doses of UV and cisplatin, the SMS-CTR and Rh30 cell lines show increased induction of (A) MDM2-ALT1 and (B) MDM4-ALT2. (C) For the ribosome profile analysis, RH3 cells were exposed to 0 or 50 J/m² UV and harvested 12 hours after treatment. Cytoplasmic extracts were separated on 15% to 50% linear sucrose density gradients. RNA from each fraction was taken and subjected to nested RT-PCR for MDM2 and MDM4. Damage promoted enhanced association of the MDM2-ALT1 and MDM4-ALT2 transcripts with polysomes in RH3 cells. Absorbance (A254) of RNA from each fraction and the corresponding ribosome profile in normal (-UV) and treated (+UV) cells are represented in the graph.