Tumor suppressor protein Pdcd4 interacts with Daxx and modulates the stability of Daxx and the Hipk2-dependent phosphorylation of p53 at serine 46.

The tumor suppressor protein Pdcd4 is a nuclear/cytoplasmic shuttling protein that has been implicated in the development of several types of human cancer. In the nucleus, Pdcd4 affects the transcription of specific genes by modulating the activity of several transcription factors. We have identified the Daxx protein as a novel interaction partner of Pdcd4. Daxx is a scaffold protein with roles in diverse processes, including transcriptional regulation, DNA-damage signaling, apoptosis and chromatin remodeling. We show that the interaction of both proteins is mediated by the N-terminal domain of Pdcd4 and the central part of Daxx, and that binding to Pdcd4 stimulates the degradation of Daxx, presumably by disrupting the interaction of Daxx with the de-ubiquitinylating enzyme Hausp. Daxx has previously been shown to serve as a scaffold for protein kinase Hipk2 and tumor suppressor protein p53 and to stimulate the phosphorylation of p53 at serine 46 (Ser-46) in response to genotoxic stress. We show that Pdcd4 also disrupts the Daxx-Hipk2 interaction and inhibits the phosphorylation of p53. We also show that ultraviolet irradiation decreases the expression of Pdcd4. Taken together, our results support a model in which Pdcd4 serves to suppress the phosphorylation of p53 in the absence of DNA damage, while the suppressive effect of Pdcd4 is abrogated after DNA damage owing to the decrease of Pdcd4. Overall, our data demonstrate that Pdcd4 is a novel modulator of Daxx function and provide evidence for a role of Pdcd4 in restraining p53 activity in unstressed cells.

siRNA-mediated knockdown of Pdcd4 expression causes upregulation of p21(Waf1/Cip1) expression.

The transformation suppressor gene, programmed cell death gene 4 (Pdcd4), inhibits tumor-promoter-mediated transformation of mouse keratinocytes and has been implicated as a tumor suppressor gene in the development of human cancer. The Pdcd4 protein interacts with translation initiation factors eIF4A and eIF4G and binds to RNA, suggesting that it might be involved in regulating protein translation or other aspects of RNA metabolism. To study the function of Pdcd4 in more detail, we have downregulated Pdcd4 expression in HeLa cells by stable expression of shRNA. We have found that diminished Pdcd4 expression leads to increased expression of p21(Waf1/Cip1) and several other p53-regulated genes. Reporter gene studies demonstrate that Pdcd4 interferes with the activation of p53-responsive promoters genes by p53. Pdcd4 knockdown cells show decreased apoptosis and increased survival after UV irradiation. Taken together, our observations suggest a model in which low Pdcd4 expression after DNA damage favors the survival of cells, which would be eliminated by apoptosis under normal levels of Pdcd4 expression. Our results provide the first evidence that Pdcd4 is important role in the DNA-damage response and suggest that low levels of Pdcd4 expression observed in certain tumor cells contribute to tumorigenesis by affecting the fate of DNA-damaged cells.

Role of survivin splice variants in pediatric acute precursor B lymphoblastic leukemia.

BACKGROUND: Survivin, a member of the inhibitor of apoptosis protein (IAP) family is transiently expressed at low levels during normal hematopoesis but profoundly overexpressed in adult leukemia potentially contributing to leukemogenesis due to deregulated apoptosis and defective cell cycle control. Alternative splicing results in four different mRNA variants survivin, survivin2B, survivin-DeltaExon3 and survivin-3B, with distinct cellular localization patterns and anti-apoptotic potential. Due to co-localization of survivin and survivin-2B in the cytoplasm survivin-2B may permit interactive fine-tuning of survivin actions and moreover play an attenuating role in its anti-apoptotic function. Lack of survivin-2B is associated with disease progression of malignomas suggesting a differential role of these isoforms in tumorigenesis. PATIENTS AND METHODS: We therefore determined the expression of the functional survivin splice variants performing RT- and real-time PCR in a purely pediatric cohort of 20 patients suffering from precursor B-ALL (BCP-ALL). RESULTS: Here, we demonstrate for the first time in pediatric patients with precursor B-ALL an association between lower survivin-2B expression and affiliation to the high risk group. CONCLUSION: The idea that survivin-2B may act as natural antagonist of survivin could potentially be used in novel approaches of anti-cancer treatment by influencing the proportional expression of the different splice variants.

Disturbed balance of expression between XIAP and Smac/DIABLO during tumour progression in renal cell carcinomas.

Dysregulation of apoptosis plays an important role in tumour progression and resistance to chemotherapy. The X-linked inhibitor of apoptosis (XIAP) is considered to be the most potent caspase inhibitor of all known inhibitor of apoptosis-family members. Only recently, an antagonist of XIAP has been identified, termed Smac/DIABLO. To explore the relevance of antiapoptotic XIAP and proapoptotic Smac/DIABLO for tumour progression in renal cell carcinomas (RCCs), we analysed XIAP and Smac/DIABLO mRNA and protein expression in the primary tumour tissue from 66 RCCs of all major histological types by quantitative real-time PCR, Western blot and ELISA. X-linked inhibitor of apoptosis and Smac/DIABLO mRNA expression was found in all RCCs. Importantly, the relative XIAP mRNA expression levels significantly increased from early (pT1) to advanced (pT3) tumour stages (P=0.0002) and also with tumour dedifferentiation (P=0.04). Western blot analysis confirmed the tumour stage-dependent increase of XIAP expression on the protein level. In contrast, mRNA and protein expression levels of Smac/DIABLO did not significantly change between early and advanced tumour stages or between low and high tumour grades. Consequently, the mRNA expression ratio between antiapoptotic XIAP and proapoptotic Smac/DIABLO markedly increased during progression from early (pT1) to advanced (pT3) tumour stages. Moreover, RCCs confined within the organ capsule (pT1 and pT2) exhibited a significantly lower XIAP to Smac/DIABLO expression ratio when compared with RCCs infiltrating beyond the kidney (pT3; P=0.01). Thus, our investigation demonstrates that the delicate balance between XIAP and Smac/DIABLO expression is gradually disturbed during progression of RCCs, resulting in a relative increase of antiapoptotic XIAP over proapoptotic Smac/DIABLO, thereby probably contributing to the marked apoptosis resistance of RCC.

Transport of cytochrome c derivatives by the bacterial Tat protein translocation system.

An experimental system developed previously for the heterologous expression of c-type cytochromes in Escherichia coli Q1has been adapted to monitor protein transfer across the bacteria's cytoplasmic membrane. Apocytochrome, lacking the haem cofactor and probably in an unfolded state, was readily transferred across the cytoplasmic membrane when fused to a Sec-specific signal peptide. Furthermore, cytochrome fused to a signal peptide regarded as specific for the twin arginine transport (Tat) system was translocated in an unfolded state by the Sec apparatus. After maturation and folding in the cytoplasm, Tat-mediated transfer of holocytochrome to the periplasm occurred. We conclude that, in addition to the nature of the specific signal peptide, the folding state of a particular protein also governs its acceptance by a given transport system.