Abstract
The p73 gene, a member of the p53 family, encodes several variants through differential splicing and use of alternative promoters. At the NH2 terminus, two different promoters generate the full-length and the DeltaN isoforms, with or without the transactivating domain. At the COOH terminus, seven isoforms generated through alternative splicing have been cloned. Previous studies have demonstrated that DeltaNp73 isoforms exert a dominant-negative effect on p73 by blocking their transactivation activity and hence the ability to induce apoptosis. Considerable efforts are made to identify the functional diversity of the COOH-terminal p73 variants. In this study, we found that p73alpha inhibited drug-induced apoptosis in small cell lung carcinoma cells, whereas p73beta promoted it. p73alpha prevented Bax activation, mitochondrial dysfunction, and caspase activation. In addition, p73alpha was also able to reduce apoptosis induced by the BH3-only protein PUMA (p53 up-regulated modulator of apoptosis). Furthermore, we discovered that p73alpha is able to inhibit the pro-apoptotic effect of p73beta, demonstrating the existence of equilibrium between these two p73 isoforms. In conclusion, the reported overexpression of p73alpha in certain tumor types, and our findings that p73alpha exerts anti-apoptotic functions, indicate a potential oncogenic activity for p73.
Highlights
GADD4, IGF-BP3, and 14-3-3 [7], and induces apoptosis irrespective of p53 status [1, 6]
Alternative splicing generates at least seven transcripts with different carboxyl termini (␣, , ␥, ␦, ⑀, , and ). p73␣ is the longest form of the p73 proteins, and contains in its carboxyl-terminal extension a sterile ␣ motif (SAM) domain. p73 is a smaller polypeptide, missing the extreme carboxylterminal region and most of the SAM domain present in p73␣ [6, 10]
Their impact on cell proliferation, differentiation, and cell death might depend on the balance between “pro” p73 isoforms and the “anti” ⌬Np73 isoforms. p73␣, which is most abundantly expressed in many tissues and cells among the alternatively spliced forms of p73, has an additional long carboxyl-terminal tail that might explain the distinct function of p53 and p73␣ or other p73 splicing variants
Summary
Reagents—Etoposide (VP16) and cisplatin were both from BristolMyers. Staurosporine (STS) and MitoTracker Red CM-H2XRos were purchased from Sigma and Molecular Probes, respectively. Transfections were performed in 24-well plates with Lipofectamine PLUS for Saos-2 and H1299 cells or with Lipofectamine 2000 for H82 and U1285 cells, according to the manufacturer’s protocol. Assessment of Mitochondrial Depolarization—After treatment at the indicated time points, the mitochondrial transmembrane potential was determined using MitoTracker Red CM-H2XRos (Molecular Probes), a cationic, lipophilic fluorochrome dye that is retained in the negatively charged mitochondrial matrix but is lost if the mitochondrial inner membrane potential is lost. Following TMRE exposure (added 30 min before harvesting to a final concentration of 25 nM), cells were centrifuged and resuspended in a TMRE-containing buffer (10 mM HEPES-NaOH, pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, and 25 nM TMRE). After incubation with Alexa Fluor 488-conjugated antimouse antibody for 30 min, cells (10,000 per sample) were analyzed on a FACS-Calibur flow cytometer, using Cell Quest software. Values shown are average of triplicates, with error bars representing S.D
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