Post-translational regulation enables robust p53 regulation

The tumor suppressor protein p53 plays important roles in DNA damage repair, cell cycle arrest and apoptosis. Due to its critical functions, the level of p53 is tightly regulated by a negative feedback mechanism to increase its tolerance towards fluctuations and disturbances. Interestingly, the p53 level is controlled by post-translational regulation rather than transcriptional regulation in this feedback mechanism

Unlocking the potential of RNA interference as a therapeutic tool

The existence of an intrinsic biochemical pathway enabling specified regulation of gene expression was unheard of until the final years of the last decade. The identification of ribonucleic acid interference (RNAi) in mammalian cells has nowadays become of extreme importance in the field of functional genomics and translational medicine. The advent of RNAi technology has brought to the scientific research and pharmaceutical communities the ability to regulate expression of any desired gene in a reproducible manner. Consequently, such technology may be utilised in the design of novel therapeutics for clinical conditions having dys-regulated gene expression. Since most RNAi-based therapies in the drug development pipeline of pharmaceutical companies utilise short interfering RNA (siRNA), this review will focus on the role of siRNA in drug development.peer-reviewe

Signaling from mTOR to eIF2α mediates cell migration in response to the chemotherapeutic doxorubicin.

After exposure to cytotoxic chemotherapeutics, tumor cells alter their translatome to promote cell survival programs through the regulation of eukaryotic initiation factor 4F (eIF4F) and ternary complex. Compounds that block mTOR signaling and eIF4F complex formation, such as rapamycin and its analogs, have been used in combination therapies to enhance cell killing, although their success has been limited. This is likely because the cross-talk between signaling pathways that coordinate eIF4F regulation with ternary complex formation after treatment with genotoxic therapeutics has not been fully explored. Here, we described a regulatory pathway downstream of p53 in which inhibition of mTOR after DNA damage promoted cross-talk signaling and led to eIF2α phosphorylation. We showed that eIF2α phosphorylation did not inhibit protein synthesis but was instead required for cell migration and that pharmacologically blocking this pathway with either ISRIB or trazodone limited cell migration. These results support the notion that therapeutic targeting of eIF2α signaling could restrict tumor cell metastasis and invasion and could be beneficial to subsets of patients with cancer.R.F.H. was supported by MRC studentship and Wellcome Trust (grant number 110071/Z/15/Z). A.E.W., T.A.A.P., and M.S. were supported by MRC Programme funding (MC_UP_A600_1023

Phenotypic and functional characterization of corneal endothelial cells during in vitro expansion.

The advent of cell culture-based methods for the establishment and expansion of human corneal endothelial cells (CEnC) has provided a source of transplantable corneal endothelium, with a significant potential to challenge the one donor-one recipient paradigm. However, concerns over cell identity remain, and a comprehensive characterization of the cultured CEnC across serial passages has not been performed. To this end, we compared two established CEnC culture methods by assessing the transcriptomic changes that occur during in vitro expansion. In confluent monolayers, low mitogenic culture conditions preserved corneal endothelial cell state identity better than culture in high mitogenic conditions. Expansion by continuous passaging induced replicative cell senescence. Transcriptomic analysis of the senescent phenotype identified a cell senescence signature distinct for CEnC. We identified activation of both classic and new cell signaling pathways that may be targeted to prevent senescence, a significant barrier to realizing the potential clinical utility of in vitro expansion

Rational engineering of microRNA-regulated viruses for cancer gene therapy

MicroRNAs (miRNAs) are small noncoding RNA molecules that have important regulatory roles in a wide range of biological processes. miRNAs are often expressed in a tissue- and/or differentiation state-specific patterns, and it is estimated that miRNAs can regulate the expression of more than 50% of all human genes. We have exploited these tissue-specific miRNA expression patterns in the modification of viral replicative tropism. In order to engineer the replicative tropism of oncolytic adenoviruses, we developed a recombinant adenovirus that in the 3 UTR of the critical E1A gene contains sequences complementary to the liver-specific miRNA miR122. This allowed us to generate a novel recombinant adenovirus that was severely attenuated in human liver, but replicated to high titres in colorectal cancer. Systemic injection of miR122-targeted adenovirus into mice did not induce liver toxicity. In a human lung cancer xenograft mouse model this miR122-targeted adenovirus showed potent antitumour activity. We also studied the possibility to exploit neuron-specific miRNA expression patterns in the modification of tissue tropism of an alphavirus Semliki Forest virus (SFV). We engineered SFV genome to contain sequences complementary to the neuron-specific miRNA miR124. In vitro characterization of this novel virus showed that the modification of the SFV genome per se did not affect polyprotein processing or oncolytic potency. Intraperitoneally administered miR124-targeted SFV displayed an attenuated spread into the central nervous system (CNS) and increased survival of infected mice. Also, mice pre-infected with miR124-targeted SFV elicited strong protective immunity against otherwise lethal challenge with a highly virulent wild-type SFV strain. In conclusion, these results show that miRNA-targeting is a potent new strategy to engineer viral tropism in development of safer and more efficient reagents for virotherapy applications.MikroRNA:t (miRNA) ovat pieniä ei-koodaavia RNA molekyylejä joilla on tärkeä tehtävä useiden erilaisten biologisten prosessien säätelyssä. MiRNA:t ekpressoituvat usein kudos- ja/tai kehitysvaihespesifisesti sekä säätelevät jopa yli 50 prosenttia kaikista ihmisen geeneistä. Tässä väitöskirjatutkimuksessa pyrimme käyttämään hyväksi miRNA:iden kudosspesifistä ekpressiota virusten kudostropismin muokkaamisessa vähentääksemme virusvektoreiden haitallista kudostoksisuutta. Muokataksemme adenovirusvektoreiden kudostropismia, kehitimme uudentyyppisen adenoviruksen jonka E1A-geenin 3 ei-koodaavalle alueelle lisäsimme ihmisen maksaspesifisen miRNA miR122:n tunnistussekvenssejä. Tunnistussekvenssien lisäyksellä saimme aikaan adenoviruksen (miR122-targetoitu adenovirus) jonka replikaatiokyky oli huomattavasti heikentynyt ihmisen maksassa, mutta pystyi replikoitumaan voimakkaasti perä- ja paksusuolisyöpäkudoksessa. Hiireen systeemisesti injisoitu miR122-targetoitu adenovirus ei aiheuttanut maksatoksisuutta. Ihmisen keuhkosyöpähiirimallissa miR122-targetoitu virus tappoi tehokkaasti syöpäsoluja. Tässä väitöskirjatutkimuksessa tutkimme myös hermosoluspesifisen miRNA miR124:n hyväksikäyttöä Semliki Forest-viruksen (SFV) kudostropismin muokkauksessa. Kehitimme SFV:n jonka genomiin oli sisällytetty miR124:n tunnistussekvenssejä. In vitro-kokeilla osoitimme tämän miR124-targetoidun SFV:n proteiinien prosessoituvan normaalisti sekä onkolyyttisen tehon säilyneen villityypin viruksen kaltaisena. Vatsaonteloon injisoitu miR124-targetoitu SFV levisi hyvin heikosti keskushermostossa joka johti vähentyneeseen neurotoksisuuteen. Osoitimme myös miR124-targetoidun viruksen toimivan tehokkaana rokotteena erittäin patogeeniselle L10 SFV-kannalle. Tässä väitöskirjatutkimuksessa pystyimme osoittamaan miRNA-targetoinnin olevan tehokas uusi tapa muokata virusten kudostropismia ja parantaa virusvektoreiden turvallisuutta

ATM in focus:a damage sensor and cancer target

The ability of a cell to conserve and maintain its native DNA sequence is fundamental for the survival and normal functioning of the whole organism and protection from cancer development. Here we review recently obtained results and current topics concerning the role of the ataxia-telangiectasia mutated (ATM) protein kinase as a damage sensor and its potential as therapeutic target for treating cancer. This monograph discusses DNA repair mechanisms activated after DNA double-strand breaks (DSBs), i.e. non-homologous end joining, homologous recombination and single strand annealing and the role of ATM in the above types of repair. In addition to DNA repair, ATM participates in a diverse set of physiological processes involving metabolic regulation, oxidative stress, transcriptional modulation, protein degradation and cell proliferation. Full understanding of the complexity of ATM functions and the design of therapeutics that modulate its activity to combat diseases such as cancer necessitates parallel theoretical and experimental efforts. This could be best addressed by employing a systems biology approach, involving mathematical modelling of cell signalling pathways

Structural characterization of intrinsically disordered proteins by NMR spectroscopy.

Recent advances in NMR methodology and techniques allow the structural investigation of biomolecules of increasing size with atomic resolution. NMR spectroscopy is especially well-suited for the study of intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) which are in general highly flexible and do not have a well-defined secondary or tertiary structure under functional conditions. In the last decade, the important role of IDPs in many essential cellular processes has become more evident as the lack of a stable tertiary structure of many protagonists in signal transduction, transcription regulation and cell-cycle regulation has been discovered. The growing demand for structural data of IDPs required the development and adaption of methods such as 13C-direct detected experiments, paramagnetic relaxation enhancements (PREs) or residual dipolar couplings (RDCs) for the study of 'unstructured' molecules in vitro and in-cell. The information obtained by NMR can be processed with novel computational tools to generate conformational ensembles that visualize the conformations IDPs sample under functional conditions. Here, we address NMR experiments and strategies that enable the generation of detailed structural models of IDPs

TTC5 is required to prevent apoptosis of acute myeloid leukemia stem cells

Using a screening strategy, we identified the tetratricopeptide repeat (TPR) motif protein, Tetratricopeptide repeat domain 5 (TTC5, also known as stress responsive activator of p300 or Strap) as required for the survival of human acute myeloid leukemia (AML) cells. TTC5 is a stress-inducible transcription cofactor known to interact directly with the histone acetyltransferase EP300 to augment the TP53 response. Knockdown (KD) of TTC5 induced apoptosis of both murine and human AML cells, with concomitant loss of clonogenic and leukemia-initiating potential; KD of EP300 elicited a similar phenotype. Consistent with the physical interaction of TTC5 and EP300, the onset of apoptosis following KD of either gene was preceded by reduced expression of BCL2 and increased expression of pro-apoptotic genes. Forced expression of BCL2 blocked apoptosis and partially rescued the clonogenic potential of AML cells following TTC5 KD. KD of both genes also led to the accumulation of MYC, an acetylation target of EP300, and the form of MYC that accumulated exhibited relative hypoacetylation at K148 and K157, residues targeted by EP300. In view of the ability of excess cellular MYC to sensitize cells to apoptosis, our data suggest a model whereby TTC5 and EP300 cooperate to prevent excessive accumulation of MYC in AML cells and their sensitization to cell death. They further reveal a hitherto unappreciated role for TTC5 in leukemic hematopoiesis