Figure 1.

Polyamine catabolic enzyme paox regulates DNp73 stability. (a) Schematic showing the polyamines (putrescine, spermidine and spermine) biosynthesis pathway and the involvement of anabolic (ODC, SAMDC, SPDS and SPMS) and catabolic enzymes (PAOX and SSAT; upper panel) and the effects of polyamines on Az1 frameshifting process (lower panel). N-acetylspermine (NASp) is the intermediate in the catabolic conversion from spermine to spermidine. (b) Semi-quantitative RT-PCR analysis of the expression of enzymes in the polyamine biosynthesis pathway in p53 null H1299 cells exposed to doxorubicin (5μM) for the indicated times. (c) Immunoblot analysis of steady-state DNp73β levels in H1299 cells transfected with or without Flag-tagged PAOX for 24 h followed by exposure to doxorubicin for 8 h. Expression of exogenous PAOX and DNp73 was detected by anti-Flag and anti-p73 (GC15) antibodies, respectively. (d) Immunoblot and semi-quantitative RT-PCR (top and middle panels), and quantitative real-time PCR (lower bar charts) analysis of overexpressed DNp73β or endogenous cyclin D1 in H1299 cells was analyzed 48 h after siRNA-mediated silencing of paox. All values are means±S.D. of duplicate experiments, reflected by error bars. (e) H1299 cells inducibly expressing DNp73β were induced by doxycycline for 24 h before treatment with the PAOX inhibitor, MDL72527, at the indicated doses for 16 h and harvested for immunodetection. All experiments were repeated at least thrice and representative data are shown throughout. (f and g) Intracellular levels of spermine and putrescine were measured in H1299 cells (left bars) or HCT116 cells (right bars) without (Untted) or with cisplatin (25 μM CDDP) or doxorubicin (5 μM Doxo) treatment for 6 h by HPLC and the ratio (see Supplementary Figure 1D for raw data) is presented (f). The levels of the intermediate product in the conversion of spermine back to spermidine, NASp was also measured and depicted (g). All values are means±S.D. of triplicate experiments for g