Receptor for activated C-kinase (RACK1) plays a central role in Ras-mediated signal transduction

Presented at the 16th International Charles Heidelberger Symposium on Cancer Research, Coimbra, Portugal. 26–28 September 2010Peer ReviewedpublishedVersio

SIX3 and SIX6 interact with GEMININ via C-terminal regions

The histoarchitecture and function of eye and forebrain depend on a well-controlled balance between cell proliferation and differentiation. For example, the binding of the cell cycle regulator GEMININ to CDT1, which is a part of the pre-replication complex, promotes cell differentiation. Homeodomain transcription factors SIX3 and SIX6 also interact with GEMININ of which SIX3-GEMININ interaction promotes cell proliferation, whereas the nature of SIX6-GEMININ interaction has not been studied to date. We investigated SIX3/SIX6 and GEMININ interactions using bimolecular fluorescence complementation, surface plasmon resonance and isothermal titration calorimetry. Interactions between SIX3/SIX6 and GEMININ were detected in mammalian cells in culture. The presence of the C-terminal regions of SIX3 and SIX6 proteins, but not their SIX domains or homeodomains as previously thought, were required for interaction with GEMININ. Interestingly, the disordered C- and N- terminal regions of GEMININ were involved in binding to SIX3/SIX6. The coiled-coil region of GEMININ, which is the known protein-binding domain and also interacts with CDT1, was not involved in GEMININ-SIX3/SIX6 interaction. Using SPR and ITC, SIX3 bound GEMININ with a micromolar affinity and the binding stoichiometry was 1:2 (SIX3 - GEMININ). The present study gives new insights into the binding properties of SIX proteins, especially the role of their variable and disordered C-terminal regions.publishedVersio

Two tales of Annexin A2 knock-down: One of compensatory effects by antisense RNA and another of a highly active hairpin ribozyme

Besides altering its own expression during cell transformation, Annexin A2 is upregulated during the progression of many cancer types and also plays key roles during viral infection and multiplication. Consequently, there has been great interest in Annexin A2 as a potential drug target. The successful design of efficient in vivo delivery systems constitutes an obstacle in full exploitation of antisense and RNA-cleaving technologies for the knock-down of specific targets. Efficiency is dependent on the method of delivery and accessibility of the target. Here, hairpin ribozymes and an antisense RNA against rat annexin A2 mRNA were tested for their efficiencies in a T7-driven coupled transcription/translation system. The most efficient ribozyme and antisense RNA were subsequently inserted into a retroviral vector under the control of a tRNA promoter, in a cassette inserted between retroviral Long Terminal Repeats for stable insertion into host DNA. The Phoenix package system based on defective retroviruses was used for virus-mediated gene transfer into PC12 cells. Cells infected with the ribozyme-containing particles died shortly after infection. However, the same ribozyme showed a very high catalytic effect in vitro in cell lysates, explained by its loose hinge helix 2 region. This principle can be transferred to other ribozymes, such as those designed to cleave the guide RNA in the CRISPR/Cas9 technology, as well as to target specific viral RNAs. Interestingly, efficient down-regulation of the expression of Annexin A2 by the antisense RNA resulted in up-regulation of Annexin A7 as a compensatory effect after several cell passages. Indeed, compensatory effects have previously been observed during gene knock-out, but not during knock-down of protein expression. This highlights the problems in interpreting the phenotypic effects of knocking down the expression of a protein. In addition, these data are highly relevant when considering the effects of the CRISPR/Cas9 approach.publishedVersio

Expression of biomarkers (p53, transforming growth factor alpha, epidermal growth factor receptor, c-erbB-2/neu and the proliferative cell nuclear antigen) in oropharyngeal squamous cell carcinomas

Using immunohistochemistry, expression of p53, transforming growth factor-alpha (TGF-α), epidermal growth factor receptor (EGFR), c-erbB-2/neu and proliferating cell nuclear antigen (PCNA) was examined in 26 fresh frozen tissue specimens of oropharyngeal squamous cell carcinomas (SCCs). p53 gene mutations were examined by polymerase chain reaction (PCR)/DNA sequencing methods in 22 carcinomas. The findings were examined for correlations with patients’ clinicopathological parameters. Expressions of p53 and PCNA were also examined in 21 formalin-fixed corresponding tissues. Of the fresh frozen tissue specimens, 77% (20/26) showed expression and 68% (15/22) showed mutations (substitutions) of the p53, with significant clustering of the mutations in exons 5 (8/22; 36%), 7 (4/22; 18%) and 8 (5/22; 23%). No mutations were found in exon 6. There was a discordance between expression of p53 protein and mutations of the gene. Parallel to expression and mutations of the p53 found in most of the specimens, expression of TGF-α, EGFR, c-erbB-2/neu and PCNA was found in 88% (22/25), 92% (23/25), 58% (14/24) and 91% (21/23) of the specimens, respectively. For the formalin-fixed tissue specimens, 62% (13/21) and 90% (19/21) expressed p53 and PCNA, respectively. Examining for correlations with patients’ clinicopathological parameters, expression of p53, TGF-α, EGFR and c-erbB-2/neu seemed to negatively correlate with the increase of the tumour grade. The present work suggests that: (1) lack of negative growth regulation due to inactivation of the p53 gene together with activation of other proto-oncogenes are necessary genetic events in the carcinogenesis of oropharyngeal SCCs; (2) in oropharyngeal SCCs, p53 gene mutations were clustered in exons 5 (codons 130–186), 7 (codons 230–248) and 8 (codons 271–282) which perhaps suggests that tobacco carcinogens probably affect the mutational hot spots of the p53 gene at codons 157, 175, 186, 248, 273 and 282; and (3) fresh frozen and formalin-fixed tissue specimens give similar results when an immunohistochemical method is applied. The importance of p53, TGF-α, EGFR, c-erbB-2/neu and PCNA as biomarkers in oropharyngeal SCCs deserves particular attention because it might offer further understanding of the development of these carcinomas

Inverse correlation between PDGFC expression and lymphocyte infiltration in human papillary thyroid carcinoma

Background: Members of the PDGF family have been suggested as potential biomarkers for papillary thyroid carcinomas (PTC). However, it is known that both expression and stimulatory effect of PDGF ligands can be affected by inflammatory cytokines. We have performed a microarray study in a collection of PTCs, of which about half the biopsies contained tumour-infiltrating lymphocytes or thyroiditis. To investigate the expression level of PDGF ligands and receptors in PTC we measured the relative mRNA expression of all members of the PDGF family by qRT-PCR in 10 classical PTC, eight clinically aggressive PTC, and five non-neoplastic thyroid specimens, and integrated qRT-PCR data with microarray data to enable us to link PDGF-associated gene expression profiles into networks based on recognized interactions. Finally, we investigated potential influence on PDGF mRNA levels by the presence of tumour-infiltrating lymphocytes. Methods: qRT-PCR was performed on PDGFA, PDGFB, PDGFC, PDGFD, PDGFRA PDGFRB and a selection of lymphocyte specific mRNA transcripts. Semiquantitative assessment of tumourinfiltrating lymphocytes was performed on the adjacent part of the biopsy used for RNA extraction for all biopsies, while direct quantitation by qRT-PCR of lymphocyte-specific mRNA transcripts were performed on RNA also subjected to expression analysis. Relative expression values of PDGF family members were combined with a cDNA microarray dataset and analyzed based on clinical findings and PDGF expression patterns. Ingenuity Pathway Analysis (IPA) was used to elucidate potential molecular interactions and networks. Results: PDGF family members were differentially regulated at the mRNA level in PTC as compared to normal thyroid specimens. Expression of PDGFA (p = 0.003), PDGFB (p = 0.01) and PDGFC (p = 0.006) were significantly up-regulated in PTCs compared to non-neoplastic thyroid tissue. In addition, expression of PDGFC was significantly up-regulated in classical PTCs as compared to clinically aggressive PTCs (p = 0.006), and PDGFRB were significantly up-regulated in clinically aggressive PTCs (p = 0.01) as compared to non-neoplastic tissue. Semiquantitative assessment of lymphocytes correlated well with quantitation of lymphocyte-specific gene expression. Further more, by combining TaqMan and microarray data we found a strong inverse correlation between PDGFC expression and the expression of lymphocyte specific mRNAs. Conclusion: At the mRNA level, several members of the PDGF family are differentially expressed in PTCs as compared to normal thyroid tissue. Of these, only the PDGFC mRNA expression level initially seemed to distinguish classical PTCs from the more aggressive PTCs. However, further investigation showed that PDGFC expression level correlated inversely to the expression of several lymphocyte specific genes, and to the presence of lymphocytes in the biopsies. Thus, we find that PDGFC mRNA expression were down-regulated in biopsies containing infiltrated lymphocytes or thyroiditis. No other PDGF family member could be linked to lymphocyte specific gene expression in our collection of PTCs biopsies

Interaction between HIF-1α (ODD) and hARD1 does not induce acetylation and destabilization of HIF-1α

AbstractHypoxia inducible factor-1α (HIF-1α) is a central component of the cellular responses to hypoxia. Hypoxic conditions result in stabilization of HIF-1α and formation of the transcriptionally active HIF-1 complex. It was suggested that mammalian ARD1 acetylates HIF-1α and thereby enhances HIF-1α ubiquitination and degradation. Furthermore, ARD1 was proposed to be downregulated in hypoxia thus facilitating the stabilization of HIF-1α. Here we demonstrate that the level of human ARD1 (hARD1) protein is not decreased in hypoxia. Moreover, hARD1 does not acetylate and destabilize HIF-1α. However, we find that hARD1 specifically binds HIF-1α, suggesting a putative, still unclear, connection between these proteins

A novel human NatA Nα-terminal acetyltransferase complex: hNaa16p-hNaa10p (hNat2-hArd1)

<p>Abstract</p> <p>Background</p> <p>Protein acetylation is among the most common protein modifications. The two major types are post-translational N^ε-lysine acetylation catalyzed by KATs (Lysine acetyltransferases, previously named HATs (histone acetyltransferases) and co-translational N^α-terminal acetylation catalyzed by NATs (N-terminal acetyltransferases). The major NAT complex in yeast, NatA, is composed of the catalytic subunit Naa10p (<b>N a</b>lpha <b>a</b>cetyltransferase <b>10 p</b>rotein) (Ard1p) and the auxiliary subunit Naa15p (Nat1p). The NatA complex potentially acetylates Ser-, Ala-, Thr-, Gly-, Val- and Cys- N-termini after Met-cleavage. In humans, the homologues hNaa15p (hNat1) and hNaa10p (hArd1) were demonstrated to form a stable ribosome associated NAT complex acetylating NatA type N-termini <it>in vitro </it>and <it>in vivo</it>.</p> <p>Results</p> <p>We here describe a novel human protein, hNaa16p (hNat2), with 70% sequence identity to hNaa15p (hNat1). The gene encoding hNaa16p originates from an early vertebrate duplication event from the common ancestor of h<it>NAA15 </it>and h<it>NAA16</it>. Immunoprecipitation coupled to mass spectrometry identified both endogenous hNaa15p and hNaa16p as distinct interaction partners of hNaa10p in HEK293 cells, thus demonstrating the presence of both hNaa15p-hNaa10p and hNaa16p-hNaa10p complexes. The hNaa16p-hNaa10p complex acetylates NatA type N-termini <it>in vitro</it>. hNaa16p is ribosome associated, supporting its potential role in cotranslational N^α-terminal acetylation. h<it>NAA16 </it>is expressed in a variety of human cell lines, but is generally less abundant as compared to h<it>NAA15</it>. Specific knockdown of h<it>NAA16 </it>induces cell death, suggesting an essential role for hNaa16p in human cells.</p> <p>Conclusion</p> <p>At least two distinct NatA protein N^α-terminal acetyltransferases coexist in human cells potentially creating a more complex and flexible system for N^α-terminal acetylation as compared to lower eukaryotes.</p

Characterization of hARD2, a processed hARD1 gene duplicate, encoding a human protein N-α-acetyltransferase

BACKGROUND: Protein acetylation is increasingly recognized as an important mechanism regulating a variety of cellular functions. Several human protein acetyltransferases have been characterized, most of them catalyzing ε-acetylation of histones and transcription factors. We recently described the human protein acetyltransferase hARD1 (human Arrest Defective 1). hARD1 interacts with NATH (N-Acetyl Transferase Human) forming a complex expressing protein N-terminal α-acetylation activity. RESULTS: We here describe a human protein, hARD2, with 81 % sequence identity to hARD1. The gene encoding hARD2 most likely originates from a eutherian mammal specific retrotransposition event. hARD2 mRNA and protein are expressed in several human cell lines. Immunoprecipitation experiments show that hARD2 protein potentially interacts with NATH, suggesting that hARD2-NATH complexes may be responsible for protein N-α-acetylation in human cells. In NB4 cells undergoing retinoic acid mediated differentiation, the level of endogenous hARD1 and NATH protein decreases while the level of hARD2 protein is stable. CONCLUSION: A human protein N-α-acetyltransferase is herein described. ARD2 potentially complements the functions of ARD1, adding more flexibility and complexity to protein N-α-acetylation in human cells as compared to lower organisms which only have one ARD

LEDGF/p75 has increased expression in blasts from chemotherapy-resistant human acute myelogenic leukemia patients and protects leukemia cells from apoptosis in vitro

<p>Abstract</p> <p>Background</p> <p>Relapse due to chemoresistant residual disease is a major cause of death in acute myelogenous leukemia (AML). The present study was undertaken to elucidate the molecular mechanisms of chemoresistance by comparing differential gene expression in blasts from patients with resistant relapsing AML and chemosensitive AML.</p> <p>Results</p> <p>About 20 genes were identified as preferentially expressed in blasts pooled from patients with resistant disease, as compared to chemosensitive AML blasts, based on differential gene expression screening. Half of these genes encoded proteins related to protein translation, of these a novel protein related to the ribosomal stalk protein P0. Other upregulated mRNAs coded for cytochrome C oxidase III, the transcription factors ERF-2/TIS11d, and the p75 and p52 splice variants of Lens Epithelial Derived Growth Factor (LEDGF). Analysis of blasts from single patients disclosed that LEDGF/p75 was the most consistently upregulated mRNA in resistant AML. Transfection experiments demonstrated that LEDGF/p75 and p52b antagonized daunorubicin-induced and cAMP-induced apoptosis in an AML cell line. Also HEK-293 cells were protected against daunorubicin by LEDGF/p75 and p52b, whereas LEDGF/p52 splice variants lacking exon 6 had proapoptotic effects. Interestingly, full length LEDGF/p75 protected against truncated pro-apoptotic LEDGF/p75.</p> <p>Conclusion</p> <p>Our results provide evidence for an association between the overexpression of genes encoding survival proteins like LEDGF/p75 and chemo-resistance in acute myelogenous leukemia. LEDGF/p75 has previously not been shown to protect against chemotherapy, and is a potential drug target in AML.</p