Juha-Pekka Pursiheimo

RNA-Binding Protein L1TD1 Interacts with LIN28 via RNA and is Required for Human Embryonic Stem Cell Self-Renewal and Cancer Cell Proliferation

Human embryonic stem cells (hESC) have a unique capacity to self‐renew and differentiate into all the cell types found in human body. Although the transcriptional regulators of pluripotency are well studied, the role of cytoplasmic regulators is still poorly characterized. Here, we report a new stem cell‐specific RNA‐binding protein L1TD1 (ECAT11, FLJ10884) required for hESC self‐renewal and cancer cell proliferation. Depletion of L1TD1 results in immediate downregulation of OCT4 and NANOG. Furthermore, we demonstrate that OCT4, SOX2, and NANOG all bind to the promoter of L1TD1. Moreover, L1TD1 is highly expressed in seminomas, and depletion of L1TD1 in these cancer cells influences self‐renewal and proliferation. We show that L1TD1 colocalizes and interacts with LIN28 via RNA and directly with RNA helicase A (RHA). LIN28 has been reported to regulate translation of OCT4 in complex with RHA. Thus, we hypothesize that L1TD1 is part of the L1TD1‐RHA‐LIN28 complex that could influence levels of OCT4. Our results strongly suggest that L1TD1 has an important role in the regulation of stemness. STEM CELLS 2012;30:452–460

p62 degradation by autophagy: another way for cancer cells to survive under hypoxia.

Hypoxia is a common feature of advanced solid tumors causing cancer progression and resistance to treatment. Hypoxia activates mitophagy as well as macroautophagy that regulate carcinoma cell survival. p62/SQSTM1, a multi-functional protein that targets proteins to degradation by proteasomes and autophagy, is itself downregulated by hypoxia-activated autophagy in carcinoma cells. The hypoxic degradation of p62 is seen across several carcinoma cell lines. In contrast to hypoxic activation of mitochondrial autophagy, the hypoxia-induced degradation of p62 occurs partially independently from the HIF pathway. The finding argues that in addition to transcriptional gene regulation through HIF, autophagy has a central role in the regulation of hypoxic cancer cell survival responses.

Protein kinase A balances the growth factor-induced Ras/ERK signaling

Protein kinase A (PKA) has been proposed to regulate the signal transduction through the Ras/extracellular‐regulated kinase (ERK) pathway. Here we demonstrate that when the PKA activity was inhibited prior to growth factor stimulus the signal flow through the Ras/ERK pathway was significantly increased. Furthermore, the data indicated that this PKA‐mediated regulation was simultaneously targeted to the upstream kinase Raf‐1 and to the ERK‐specific phosphatase mitogen‐activated protein kinase phosphatase‐1 (MKP‐1). Moreover, our data suggested that the level of PKA activity determined the transcription rate of mkp‐1 gene, whereas the Ras/ERK signal was required to protect the MKP‐1 protein against degradation. These results point to a tight regulatory relationship between PKA and the growth factor signaling, and further suggest an important role for basal PKA activity in such regulation. We propose that PKA adjusts the activity of the Ras/ERK pathway and maintains it within a physiologically appropriate level.

Continuous hypoxic culturing of human embryonic stem cells enhances SSEA-3 and MYC levels.

Low oxygen tension (hypoxia) contributes critically to pluripotency of human embryonic stem cells (hESCs) by preventing spontaneous differentiation and supporting self-renewal. However, it is not well understood how hESCs respond to reduced oxygen availability and what are the molecular mechanisms maintaining pluripotency in these conditions. In this study we characterized the transcriptional and molecular responses of three hESC lines (H9, HS401 and HS360) on short (2 hours), intermediate (24 hours) and prolonged (7 days) exposure to low oxygen conditions (4% O2). In response to prolonged hypoxia the expression of pluripotency surface marker SSEA-3 was increased. Furthermore, the genome wide gene-expression analysis revealed that a substantial proportion (12%) of all hypoxia-regulated genes in hESCs, were directly linked to the mechanisms controlling pluripotency or differentiation. Moreover, transcription of MYC oncogene was induced in response to continuous hypoxia. At the protein level MYC was stabilized through phosphorylation already in response to a short hypoxic exposure. Total MYC protein levels remained elevated throughout all the time points studied. Further, MYC protein expression in hypoxia was affected by silencing HIF2α, but not HIF1α. Since MYC has a crucial role in regulating pluripotency we propose that induction of sustained MYC expression in hypoxia contributes to activation of transcriptional programs critical for hESC self-renewal and maintenance of enhanced pluripotent state.

Promoter-specific alterations of APC are a rare cause for mutation-negative familial adenomatous polyposis.

In familial adenomatous polyposis (FAP), 20% of classical and 70% of attenuated/atypical (AFAP) cases remain mutation‐negative after routine testing; yet, allelic expression imbalance may suggest an APC alteration. Our aim was to determine the proportion of families attributable to genetic or epigenetic changes in the APC promoter region. We studied 51 unrelated families/cases (26 with classical FAP and 25 with AFAP) with no point mutations in the exons and exon/intron borders and no rearrangements by multiplex ligation‐dependent probe amplification (MLPA, P043‐B1). Promoter‐specific events of APC were addressed by targeted resequencing, MLPA (P043‐C1), methylation‐specific MLPA, and Sanger sequencing of promoter regions. A novel 132‐kb deletion encompassing the APC promoter 1B and upstream sequence occurred in a classical FAP family with allele‐specific APC expression. No promoter‐specific point mutations or hypermethylation were present in any family. In conclusion, promoter‐specific alterations are a rare cause for mutation‐negative FAP (1/51, 2%). The frequency and clinical correlations of promoter 1B deletions are poorly defined. This investigation provides frequencies of 1/26 (4%) for classical FAP, 0/25 (0%) for AFAP, and 1/7 (14%) for families with allele‐specific expression of APC. Clinically, promoter 1B deletions may associate with classical FAP without extracolonic manifestations.

Expression of small nucleolar RNAs in leukemic cells

PurposeSmall nucleolar RNAs (snoRNAs) direct sequence-specific modifications to ribosomal RNA. We hypothesized that the expression of snoRNAs may be altered in leukemic cells.MethodsThe expression of snoRNAs was analyzed in various leukemic cell lines by massive parallel sequencing (SOLiD). Quantitative real-time PCR (RT-qPCR) was used to validate the expression profiles.ResultsOur results show characteristic differences in the expression patterns of snoRNAs between cell lines representing the main subgroups of leukemia, AML, pre-B-ALL and T-ALL, respectively. In RT-qPCR analyses, several snoRNAs were found to be differentially expressed in T-ALL as compared to pre-B-ALL cell lines.ConclusionssnoRNAs are differentially expressed in various leukemic cell lines and could, therefore, be potentially useful in the classification of leukemia subgroups.

p62/SQSTM1 regulates cellular oxygen sensing by attenuating PHD3 activity through aggregate sequestration and enhanced degradation

Summary The hypoxia-inducible factor (HIF) prolyl hydroxylase PHD3 regulates cellular responses to hypoxia. In normoxia the expression of PHD3 is low and it occurs in cytosolic aggregates. SQSTM1/p62 (p62) recruits proteins into cytosolic aggregates, regulates metabolism and protein degradation and is downregulated by hypoxia. Here we show that p62 determines the localization, expression and activity of PHD3. In normoxia PHD3 interacted with p62 in cytosolic aggregates, and p62 was required for PHD3 aggregation that was lost upon transfer to hypoxia, allowing PHD3 to be expressed evenly throughout the cell. In line with this, p62 enhanced the normoxic degradation of PHD3. Depletion of p62 in normoxia led to elevated PHD3 levels, whereas forced p62 expression in hypoxia downregulated PHD3. The loss of p62 resulted in enhanced interaction of PHD3 with HIF-&agr; and reduced HIF-&agr; levels. The data demonstrate p62 is a critical regulator of the hypoxia response and PHD3 activity, by inducing PHD3 aggregation and degradation under normoxia.

Genome Sequence of Coxsackievirus A6, Isolated during a Hand-Foot-and-Mouth Disease Outbreak in Finland in 2008

ABSTRACT Reports of hand-foot-and-mouth disease (HFMD) outbreaks caused by coxsackievirus A6 have increased worldwide after the report of the first outbreak in Finland in 2008. The complete genome of the first outbreak strain from a vesicle fluid specimen was determined.

Next generation sequencing of all variable loops of synthetic single framework scFv-Application in anti-HDL antibody selections.

Next generation sequencing (NGS) can be applied to monitoring antibody phage display library selection processes to follow the enrichment of each individual antibody clone. Utilising the recent development of the Illumina sequencing platform enabling sequencing up to 2×300bp, we have developed a method to deep sequence all complementarity determining regions (CDRs) in the clones obtained from a synthetic single framework antibody library. This was complemented by an in-house bioinformatics pipeline for efficient analysis of the sequencing results. The method was utilised to study antibody selections against high density lipoprotein (HDL) particles. Sequencing of the output from each selection round enabled extraction of useful information on both the total copy numbers as well as the relative enrichment rates of the clones. Ten antibody clones showing different ranking in terms of frequency were reproduced from synthetic DNA constructs and their capacity to bind HDL was verified by an immunoassay. The method thus facilitates the isolation of clones of interest, and in particular can assist retrieval of less efficiently enriched, yet interesting clones, which are unlikely to be identified by conventional, random colony picking based, screening.