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Sp1 and p73 activate PUMA following serum starvation.

ABSTRACT: p53-upregulated modulator of apoptosis (PUMA) plays an essential role in p53-dependent apoptosis following DNA damage. PUMA also mediates apoptosis independent of p53. In this study, we investigated the role and mechanism of PUMA induction in response to serum starvation in p53-deficient cancer cells. Following serum starvation, the binding of Sp1 to the PUMA promoter significantly increased, whereas inhibition of Sp1 completely abrogated PUMA induction. p73 was found to be upregulated by serum starvation and mediate PUMA induction through the p53-binding sites in the PUMA promoter. Sp1 and p73beta appeared to cooperatively activate PUMA transcription, which is inhibited by the phosphoinsitide 3-kinase (PI3K)-protein kinase B (AKT) pathway. Furthermore, knockdown of PUMA suppressed serum starvation-induced apoptosis in leukemia cells. Our results suggest that transcription factors Sp1 and p73 mediate p53-independent induction of PUMA following serum starvation to trigger apoptosis in human cancer cells.

SUBMITTER: Ming L

PROVIDER: S-EPMC2722853 | biostudies-literature | 2008 Oct

REPOSITORIES: biostudies-literature

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Sp1 and p73 activate PUMA following serum starvation.

Ming Lihua L Sakaida Tsukasa T Yue Wen W Jha Anupma A Zhang Lin L Yu Jian J

Carcinogenesis 20080625 10

p53-upregulated modulator of apoptosis (PUMA) plays an essential role in p53-dependent apoptosis following DNA damage. PUMA also mediates apoptosis independent of p53. In this study, we investigated the role and mechanism of PUMA induction in response to serum starvation in p53-deficient cancer cells. Following serum starvation, the binding of Sp1 to the PUMA promoter significantly increased, whereas inhibition of Sp1 completely abrogated PUMA induction. p73 was found to be upregulated by serum ...[more]

PMID: 18579560

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Project description:Thymosin beta-4 (T?4) is an ubiquitous multi-functional regenerative peptide, related to many critical biological processes, with a dynamic and flexible conformation which may influence its functions and its subcellular distribution. For these reasons, the intracellular localization and trafficking of T?4 is still not completely defined and is still under investigation in in vivo as well as in vitro studies. In the current study we used HepG2 cells, a human hepatoma cell line; cells growing in normal conditions with fetal bovine serum expressed high levels of T?4, restricted to the cytoplasm until 72 h. At 84 h, a diffuse T?4 cytoplasmic immunostaining shifted to a focal perinuclear and nuclear reactivity. In the absence of serum, nuclear reactivity was localized in small granules, evenly dispersed throughout the entire nuclear envelop, and was observed as earlier as at 48 h. Cytoplasmic immunostaining for T?4 in HepG2 cells under starvation appeared significantly lower at 48 h and decreased progressively at 72 and at 84 h. At these time points, the decrease in cytoplasmic staining was associated with a progressive increase in nuclear reactivity, suggesting a possible translocation of the peptide from the cytoplasm to the nuclear membrane. The normal immunocytochemical pattern was restored when culture cells submitted to starvation for 84 h received a new complete medium for 48 h. Mass spectrometry analysis, performed on the nuclear and cytosolic fractions of HepG2 growing with and without serum, showed that T?4 was detectable only in the cytosolic and not in the intranuclear fraction. These data suggest that T?4 is able to translocate from different cytoplasmic domains to the nuclear membrane and back, based on different stress conditions within the cell. The punctuate pattern of nuclear T?4 immunostaining associated with T?4 absence in the nucleoplasm suggest that this peptide might be localized in the nuclear pores, where it could regulate the pore permeability.

Project description:Elucidation of the molecular mechanisms by which cancer cells overcome hypoxia is potentially important for targeted therapy. Complexation of hypoxia-inducible factors (HIFs) with aryl hydrocarbon receptor nuclear translocators can enhance gene expression and initiate cellular responses to hypoxia. However, multiple molecular mechanisms may be required for cancer cells to adapt to diverse microenvironments. We previously demonstrated that a physical interaction between the ubiquitously expressed transcription factor Sp1 and HIF2 is a major cause of FVII gene activation in poor prognostic ovarian clear cell carcinoma (CCC) cells under hypoxia. Furthermore, it was found that FVII activation is synergistically enhanced when serum-starved cells are cultured under hypoxic conditions. In this study, we investigated whether HIFs and transcription factor Sp1 cooperate to activate multiple genes in CCC cells under conditions of serum starvation and hypoxia (SSH) and then contribute to malignant phenotypes.To identify genes activated under hypoxic conditions in an Sp1-dependent manner, we first performed cDNA microarray analyses. We further investigated the molecular mechanisms of synergistic gene activations including the associated serum factors by various experiments such as real-time RT-PCR, western blotting and chromatin immunoprecipitation. The study was further extended to animal experiments to investigate how it contributes to CCC progression in vivo.ICAM1 is one such gene dramatically induced by SSH and is highly induced by SSH and its synergistic activation involves both the mTOR and autonomously activated TNFα-NFκB axes. We identified long chain fatty acids (LCFA) as a major class of lipids that is associated with albumin, a serum factor responsible for synergistic gene activation under SSH. Furthermore, we found that ICAM1 can be induced in vivo to promote tumor growth.Sp1 and HIFs collaborate to activate genes required for the adaptation of CCC cells to severe microenvironments, such as LCFA starvation and hypoxia. This study highlights the importance of transcriptional regulation under lipid starvation and hypoxia in the promotion of CCC tumor growth.

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Sp1 and p73 activate PUMA following serum starvation.

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Sp1 and p73 activate PUMA following serum starvation.

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