expression of insulin-like growth factor binding proteins 2 (IGFBP2) and 5 (IGFBP5) appears to play a critical role in regulating apoptosis

4054 Background: Apoptosis is an essential biological process that has been shown to be deregulated in many human cancers. Several signaling pathways regulating apoptosis, and thus potential therapeutic targets, have been identified. Of these the IGF pathway, and IGFBPs are particularly interesting. Overexpression of IGFBP2 has been associated with tumor progression, while expression of IGFBP5, may have a proapoptotic effect. Another gene, clusterin has also been identified which may promote cell survival. The objective of this study is to better understand gene expression during apoptosis. Methods: Apoptosis was induced in a pheochromocytoma (PC12) cell line by treatment with 5uM of the topoisomerase inhibitor teniposide (VM26). Cell viability was determined using trypan blue positive staining. Pulse field gel electropheresis (PFGE) showing DNA fragmentation, and immunofluorescence microscopy characterizing morphological changes of apoptosis were used. expression of IGFBP2, IGFBP5, Clusterin, Cathepsin B, and RSG3 (Embigin) during the time course of apoptosis was determined using RTPCR. Hypoxanthine phosphoribosyl transferase (HPRT) a housekeeping gene was the internal control. To determine if IGF-1 had a protective effect on PC12 cells undergoing apoptosis, cell cultures were pretreated with IGF-1, and cell viability was assessed. Results: Following exposure to VM26, PC12 cells underwent apoptosis as assessed by trypan blue staining, pulse field gel electrophoresis and microscopy. Four hours after VM26 treatment 6.4% of cell had undergone cell death, by 8h 30% had died and 24h 42% of cells had died, in contrast to control. Using PFGE, the characteristic DNA laddering pattern was evident by 18h. Microscopy showed nuclear changes by 4h, including chromatin condensation and by 24h apoptotic bodies were evident.Gene expression studies showed IGFBP2 levels dramatically upregulated at 8h, then downregulated by 24 h. Levels of IGFBP5, decreased by 8h and remained low after 24h. The clusterin gene expression pattern paralleled that of IGFBP2, showing a peak level at 8h. Cathepsin B and RSG3 did not show appreciable changes. Finally pretreatment with increasing concentrations of IGF-1 lead to increased cell viability from 35.8% to 44.8%, 49.1% and 52.1% with 50, 100 and 200 ng/mL IGF-1 respectively. IGF-1 did not however exert proliferative effects on the PC12 cells. Conclusions: The IGF pathway and IGFPBP play a critical role in apoptosis. This study suggests that IGF-1 protects cells from apoptosis. Increased IGFBP2 and decreased IGFBP5 expression may be important survival signals for cells undergoing apoptosis in this model system. Taken together these results suggest that IGF and IGFBP may be important therapeutic anticancer targets.