Twenty four kDa endogenous caveolin 1 e pressed in the A431 cell line, and recognized by an anti caveolin 1 antibody, was used as the positive control in Western blotting. We ne t clarified whether the recombinant caveolin 1 SB1518 was localized in cells in the same manner as endogenous caveolin 1. The distribution of endogenous caveolin 1 in A431 cells was determined by immunofluorescent staining with an anti caveolin 1 pol yclonal antibody. E ogenous Myc tagged caveolin 1 dis tribution in A431 cells was detected by double immunofluorescent staining with anti caveolin 1 and anti Myc polyclonal antibodies. Both endogenous and e ogenous caveolin 1 had the same punctate distribution in the perinuclear region. We then transiently e pressed the Myc tagged caveolin 1 in GH3 cells to e amine the effect of caveolin 1 on GH3 cells.

By 48 hours after caveolin 1 e pression in GH3 cells, apopto sis like nuclear condensation was visible upon staining with Hoechst 33342. In the con trol e periment, transient e pression of enhanced green fluorescent protein did not alter nuclear morphol ogy. These data indicate that transient e pression of Caveolin 1 induces apoptosis in GH3 cells. To test this, the TUNEL assay, which discriminates apoptosis from necrosis and from primary DNA strand breaks, was used to measure DNA fragmentation 48 hours after transfec tion. Recombinant caveolin 1 e pressing cells appeared shrunken with positive TUNEL labeling. In the control e periments, all cells were positively 2% and 3% of EGFP e pressing cells and 1. 6 2% vehicle treated cells e hibited apoptosis 24 and 48 hours after transfection.

This result revealed that even tran sient caveolin 1 e pression increased GH3 cell apoptosis. Caveolin 1 induced apoptosis of GH3 cells involves caspase 8 The activation of caspases plays a pivotal role in the e e cution of apoptosis by various signaling pathways. Cilengitide To e amine the role of caspases in apoptotic GH3 cells after transient caveolin 1 e pression, we sep arately treated the ectopic caveolin 1 and DsRed N1 e pressing GH3 cells with a general caspase inhibitor, as well as specific caspase inhibitors, for cas pase 3, caspase 8 or caspase 9 before determining the number of apop totic cells by TUNEL assay. Over e pression of caveolin 1 resulted in 62% of the transiently transfected cells becom ing apoptotic. This effect was inhibited by treating GH3 cells with the general caspase inhibitor Z VAD fmk and with the cas pase 8 specific inhibitor, Z IETD fmk. Treatment with caspase 3 or caspase 9 specific inhibitors did not inhibit caveolin 1 induced apoptosis. In nega tive control e periments, cells e pressing the red fluores cent protein, DsRed N1 had no significant increase in apoptosis compared to untreated GH3 cells.