Functional characterization of Cytoplasmic Polyadenylation Element Binding proteins in the developing and diseased brain

Gliomas are the most common primary brain tumors with aggressive progression and devastating prognosis. Therefore identification of new therapeutic and diagnostic factors is necessary to improve the dramatic situation of glioma patients. Thus far, cancer-related genes were thoroughly analyzed by transcript-based microarrays (van den Boom et al., 2003; Phillips et al., 2006; Rickman et al., 2001). However, recent research shed light on the importance of post-transcriptional modifications of mRNAs that alter gene expression under physiological conditions, but also gives rise to the pathology of gliomas. The main goal of the present study was to investigate CPEB expression in human glioma specimens. CPEBs are auxiliary regulators associating with consensus sequences present in 3’UTRs of mRNAs, which activate or repress their translation. Via this mechanism CPEBs regulate essential cellular processes, such as development (Groisman et al., 2002; Novoa et al., 2010), memories formation (Theis et al., 2003) and progression of cancer (Ortiz-Zapater et al., 2012). In the current work aberrant CPEB expression was found to be a frequent phenomenon in both, low- and high-grade gliomas. Decreased CPEB1 expression was associated with the rising grade of tumor malignancy, suggesting it being a putative tumor suppressor. One of the mechanisms potentially underlying transcriptional silencing of cancer-related genes might be DNA methylation. However, despite hypermethylation of the CPEB1 gene, DNA methylation proved not to be directly responsible for its downregulation in gliomas. Thus, the underlying mechanism remains elusive. Abundant expression of CPEB 2-4 was detected in numerous human glioma specimens. CPEB2 expression in endothelial tumor cells suggested that CPEB2-mediated protein synthesis takes place in the close proximity of blood vessels within tumor tissue. On the other hand, CPEB4 expression appeared to support tumorigenic properties by its putative entanglement into local protein synthesis in transformed cells. Intriguingly, only CPEB3 expression was correlated positively with tumor progression. Phosphorylation of CPEB3 within the alternatively spliced region was negatively correlated with tumor malignancy. The loss of CPEB3 activity in high-grade gliomas is likely caused by the expression of alternatively spliced variants. This suggests that a substantial part of the CPEB3 in GBM specimens does not undergo phosphorylation and indicates phosphorylation to be a biologically important mechanism regulating CPEB3 activity. Furthermore, a significant correlation between CPEB3 activity and survival was observed in less aggressive low-grade astrocytomas considered to be sGBM precursors. Consequently, CPEB3 may be considered as an attractive therapeutic target in gliomas. To further investigate the relationship between CPEB activity, growth properties and cancer-relevant parameters an in vitro overexpression study was performed. This revealed a striking link between CPEB1, enhanced apoptotic activity and enhanced migration. One explanation could be that through rapid changes in the cytoskeleton of migrating cell the chance of mistakes rises, turning GBM cells towards an apoptotic pathway. On the other hand, CPEB2 overexpression decreased apoptotic activity, enhanced migration and by this additionally strengthened the cancerous properties of the cells. Importantly, CPEBs were also found to regulate various cancer pathways, including the TGF-β signaling cascade. However, further studies are needed to fully understand the interaction between CPEBs translational control and the pathophysiology of cultured GMB cells and human gliomas. Finally, the study revealed that CPEB2 is expressed in different cellular populations, brain regions, and stages of development, which indicates that this protein plays an important role in regulation of local protein synthesis, synaptic plasticity, and neuronal morphogenesis. The present study does not only increase our understanding of the function of CPEBs but also shows the importance of post-transcriptional modifications of mRNAs as a pathophysiological mechanism in gliomas and potentially other cancers. Therefore, these results may serve as a valuable basis for the identification of new therapeutic and diagnostic factors in cancer treatment

Effect of epigallocatechin gallate on DNA damage induced by etoposide in human leukemic K562 cells.

Public interest in green tea has grown recently due to the potential health benefits from its consumption. Epigallocatechin gallate (EGCG), a principal polyphenolic component of green tea, is considered key to these healthful qualities. In recent years, many studies have shown strong chemopreventive and possibly cancer chemotherapeutic effects of EGCG against cancers of the blood, skin, lung, breast, colon, liver, stomach and prostate. In vivo and in vitro experiments have shown that high concentrations of EGCG are cytotoxic and trigger genotoxic events in human cells. Although this catechin affects a number of cellular systems, the genotoxic effects are believed to be mediated by their actions on topoisomerase II.Of the many commercially available anticancer drug etoposide was selected for testing, which, although discovered more than forty years ago, is still one of the most common chemotherapeutic drugs for the treatment of a wide spectrum of cancers.The present investigation was carried out to examine the effects of EGCG on the cytogenetic change and DNA damage induced by etoposide in human K562 cells.Incubation of cells K562 with epigallocatechin gallate (concentration range of 0-100 µM) caused an increase in DNA damage depending on the dosage used. The EGCG, especially in concentration 50 M increased levels of DNA strand breaks. Percentage of cells with the single apoptosis was also dependent on the concentration and incubation time of cells K562 with etoposide (concentration range of 0–1,5µM) and/or EGCG. Exposure of K562 cells by 24 and 48 h of action of EGCG and then apply the 1h etoposide dose of 0,5 M increased the percentage of apoptotic cells observed immediately after removal of the compounds. Number of micronuclei formed, reflecting damage to genetic material of chromosomes was dependent on dose and duration of EGCG. The obtained results demonstrate that EGCG stimulates DNA cleavage and induction of apoptosis in K562 cells.Galusan epigallokatechiny (EGCG) jest głównym związkiem polifenolowym wchodzącym w skład zielonej herbaty. Obecność EGCG stanowi czynnik kluczowy dla zdrowotnych właściwości tej odmiany herbaty oraz determinuje jej zdolności chemoprewencyjne oraz chemoterapeutyczne wykorzystywane w leczeniu białaczek, nowotworów skóry, płuc, piersi, prostaty, okrężnicy i wątroby. W badaniach in virto oraz in vivo wykazano, że wysokie stężenia EGCG są cytotoksyczne oraz indukują wiele procesów genotoksycznych w ludzkich komórkach. Pomimo iż katechiny oddziałują na wiele struktur komórkowych, uważa się że genotoksyczny efekt jest związany z ich oddziaływaniem na topoizomerazę II. Z pośród wielu dostępnych leków przeciwnowotworowych do badań wybrano etopozyd, który mimo iż został odkryty ponad czterdzieści lat temu nadal jest jednym z najczęściej stosowanych leków w terapii szerokiego spektrum nowotworów.Celem niniejszej pracy było określenie, czy EGCG wywiera wpływ na zmiany cytogenetyczne oraz uszkodzenia DNA indukowane etopozydem w komórkach linii K562.Przeprowadzone badania wykazują, że inkubacja komórek K652 z galusanem epigalloktechiny w zakresie stężeń 1-100 µM powoduje wzrost uszkodzeń DNA w stopniu zależnym od zastosowanej dawki. Galusan epigallokatechiny, szczególnie w wysokim stężeniu 50 µM zwiększa poziom uszkodzeń nici kwasu deoksyrybonukleinowego. Odsetek komórek wchodzących na drogę apoptozy również zależy od zastosowanej dawki EGCG oraz czasu inkubacji komórek z EGCG i/lub etopozydem. Ekspozycja komórek K562 przez 24 oraz 48 godzin na działanie EGCG w stężeniach 1-100 µM i 1 godzinne działanie 0,5 µM etopozydu zwiększa odsetek obserwowanych komórek apoptotycznych. Liczba powstałych mikrojąder, odzwierciedlających uszkodzenia materiału genetycznego i chromosomów jest także zależna od zastosowanej dawki i czasu inkubacji komórek K562 z EGCG. Uzyskane wyniki potwierdzają, że EGCG wpływa na uszkodzenia materiału genetycznego i indukcję procesu apoptozy w komórkach linii K562

Can epigallocatechin gallate be an effective polyphenol in combination therapy with etoposide for the treatment of chronic myelogenous leukemia?

Publikacja recenzowana / Peer-reviewed publicationWprowadzenie: Składnik zielonej herbaty – galusan epigallokatechiny (EGCG) – znany jest ze swoich właściwości chemoprewencyjnych i chemoterapeutycznych. Wykazuje silne właściwości antyoksydacyjne i przeciwzapalne, a w stosunku do komórek nowotworowych – działanie antyproliferacyjne lub proapoptotyczne. Etopozyd jest jednym z najczęściej stosowanych leków przeciwnowotworowych, wywołującym jednak wiele skutków ubocznych. Materiały i metody: W eksperymentach in vitro badano potencjalną rolę EGCG w terapii skojarzonej z etopozydem w leczeniu przewlekłej białaczki szpikowej. Komórki ustalonej linii białaczkowej K562 poddano działaniu etopozydu i/lub EGCG w celu określenia wpływu EGCG na przeżywalność komórek, poziom uszkodzeń DNA oraz częstość procesu apoptozy. Poziom uszkodzeń DNA mierzono przy pomocy elektroforezy pojedynczych komórek w żelu agarozowym (test kometowy), natomiast apoptozę oceniano pod mikroskopem fluorescencyjnym z użyciem barwnika Hoechst 33342. Wyniki: Uzyskane wyniki badań wskazują, że EGCG w stężeniu 50 i 100 μM uwrażliwia komórki białaczkowe na cytotoksyczne działanie etopozydu, zwiększając poziom uszkodzeń DNA i częstość apoptozy. Wnioski: Dane wskazują, że galusan epigallokatechiny może się okazać skutecznym polifenolem w terapii skojarzonej z etopozydem w leczeniu przewlekłej białaczki szpikowej.Introduction: Green tea ingredient – epigallocatechin gallate (EGCG) – is known for its chemopreventive and chemotherapeutic properties. It has strong antioxidant and anti-inflammatory properties, and antiproliferative or proapoptotic activity against cancer cells. Etoposide is one of the most commonly used anti-cancer drugs causing many side effects. Materials and methods: This study investigated the role of EGCG in combination therapy with etoposide in the treatment of chronic myeloid leukemia. K562 cells were treated with EGCG and / or etoposide to determine the effect of this polyphenol on cell survival, DNA damage or apoptosis. DNA damages were measured with single cell gel electrophoresis (comet assay) and the apoptosis were analyzed in fluorescence microscope with Hoechst 33342 staining. Results: Preliminary results suggest that EGCG at 50 and 100 μM sensitizes leukemic cells to the cytotoxic effect of etoposide, increases DNA damage that promotes removal cell and directs them to apoptosis. Conclusions: The data show that epigallocatechin gallate may prove to be an eff ective polyphenol in combination therapy with etoposide in the treatment of chronic myeloid leukemia. However, further research is needed to explain the EGCG interaction with chemotherapeutics

New Phosphospecific Antibody Reveals Isoform-Specific Phosphorylation of CPEB3 Protein.

Cytoplasmic Polyadenylation Element Binding proteins (CPEBs) are a family of polyadenylation factors interacting with 3'UTRs of mRNA and thereby regulating gene expression. Various functions of CPEBs in development, synaptic plasticity, and cellular senescence have been reported. Four CPEB family members of partially overlapping functions have been described to date, each containing a distinct alternatively spliced region. This region is highly conserved between CPEBs-2-4 and contains a putative phosphorylation consensus, overlapping with the exon seven of CPEB3. We previously found CPEBs-2-4 splice isoforms containing exon seven to be predominantly present in neurons, and the isoform expression pattern to be cell type-specific. Here, focusing on the alternatively spliced region of CPEB3, we determined that putative neuronal isoforms of CPEB3 are phosphorylated. Using a new phosphospecific antibody directed to the phosphorylation consensus we found Protein Kinase A and Calcium/Calmodulin-dependent Protein Kinase II to robustly phosphorylate CPEB3 in vitro and in primary hippocampal neurons. Interestingly, status epilepticus induced by systemic kainate injection in mice led to specific upregulation of the CPEB3 isoforms containing exon seven. Extensive analysis of CPEB3 phosphorylation in vitro revealed two other phosphorylation sites. In addition, we found plethora of potential kinases that might be targeting the alternatively spliced kinase consensus site of CPEB3. As this site is highly conserved between the CPEB family members, we suggest the existence of a splicing-based regulatory mechanism of CPEB function, and describe a robust phosphospecific antibody to study it in future

Multiple sequence alignment of the regions flanking S419/S420 of CPEBs-2-4.

<p>Phosphorylation site was marked with a black asterisk. Residue conservation score was calculated with Jalview software according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150000#pone.0150000.ref061" target="_blank">61</a>]. Color intensity and the score (1–11) in the “conservation index” (below the alignment) reflects the conservation of physicochemical properties of amino acids in the particular column of the alignment. *: conserved column (score 11, highest); +: all the amino acid physical properties conserved (score 10).</p

CPEB3a phosphorylation in cultured hippocampal neurons.

<p>Normalized ratio of phosphorylated CPEB3a (phospho-CPEB3a) fluorescence over total CPEB3a (CPEB3a-EGFP) fluorescence (n = 25–35, 3 experiments). (<b>A</b>) Immunofluorescence of neurons transfected with CPEB3a-mGFP subjected to a 40 mM KCl stimulation for 90 s, which leads to a marked increase in phospho-CPEB3a signal. One hour pretreatment with 10 μM KN93 or 10μM H89 in the stimulation medium reverts this increase. Co-transfection of the natural CaMKII inhibitor (CaMKIIN-HA) prevents the increase in CPEB3a phosphorylation. (<b>B</b>) Quantification of the ratio of phospho-CPEB3a over CPEB3a-mGFP signals in A, which shows a significant increase of CPEB3a phosphorylation following KCl stimulation only, which is not seen in neurons treated with KN93, H89, or those co-transfected with CaMKIIN-HA; n = 10–35, at least 3 experiments per condition. (<b>C</b>) Immunofluorescence of neurons transfected with CPEB3a-mGFP and subjected to either 5 min 50 μM FS stimulation or 1 h 300 nm OA stimulation, which both cause a robust increase in phospho-CPEB3a signal. Pretreatment with 10 μM H89 or 100 μM RP-cAMPS for 1 h partially reverts the effect of FS. (<b>A</b>, <b>C</b>) The scale bar represents 10 μm and applies to all photomicrographs. (<b>D</b>) Quantification of the ratio of phospho-CPEB3a over CPEB3a-mGFP signals in C, which shows a significant increase of CPEB3a phosphorylation following FS, OA and combined FS + OA treatments. Increase in phosphorylation induced by FS is partially reversed by cAMPS-Rp treatment; n = 22–59, 2–5 experiments per condition. (<b>A</b>, <b>B</b>, <b>C</b>, <b>D</b>) *p<0.05, Kruskal-Wallis followed by Tukey’s least-significant difference test.</p

Schematic diagram of CPEB domain structure and sequence comparison of the variable middle region of mouse CPEB3.

<p>Two regions, termed B and C, are alternatively spliced, leading to expression of four isoforms: a (containing both regions), b (lacking the B-region), c (lacking the C-region), and d (lacking both regions) [Uniprot Q7TN99-1, -2, -3, -4 respectively]. Consensus phosphorylation sites for PKA and CaMKII was shown above the alignment. The putative phosphorylated residue was marked with an asterisk.</p

Non-radioactive phosphorylation assays.

<p>(<b>A</b>) Sequences of the synthetic peptides used for phosphorylation experiments in B, C, D, E. Alternatively spliced exon 7 (the B-region) was highlighted. Putative phosphorylation site is in <b>bold</b>. (<b>B</b>, <b>C</b>, <b>D</b>, <b>E</b>) <i>In vitro</i> phosphorylation of CPEB3 by PKA and CaMKII. (<b>B</b>) MALDI-TOF-MS analysis of CPEB3-derived peptides phosphorylated by PKA-C. A mass shift corresponding to incorporation of 1 or 2 phosphates is observed, as compared with no-kinase control. (<b>C</b>) MALDI-TOF-MS spectra of CPEB3-derived peptides phosphorylated by CaMKII. (<b>B, C</b>) orange trace–spectrum after phosphorylation; black trace–no-kinase control; m/z–mass to charge ratio; AU—arbitrary units. (<b>D</b>) Peptide phosphorylation by PKA-C (PKLight assay). The a/c-isoform peptide, S419A, and S420A mutant peptides are phosphorylated at a significantly higher rate compared to the double mutant, while the b/d-isoform phosphorylation was not significantly different. S420 is phosphorylated preferentially, as shown by decreased phosphorylation rate in case of S420A mutant peptide compared with S419A substitution (p<0.01). Values showing relative levels of unused ATP after phosphorylation reaction were normalized to no-substrate control levels (not shown). Error bars are SEM. *P<0.005, **P<0.001 (student’s t-test). (<b>E</b>) Peptide phosphorylation by CaMKII (ADP-Glo assay). (<b>E1</b>) The a/c-isoform peptide is phosphorylated at significantly higher rate compared with b/d-isoform and S419A/S420A double mutant. (<b>E2</b>) Serine 420 is phosphorylated preferentially, as shown by decreased phosphorylation rate in case of S420A mutant peptide. Values show relative ADP levels after phosphorylation reaction, normalized to no-substrate control levels (not shown). Error bars are SEM. *P<0.01, **P<0.001, ***P<0.05 (student’s t-test, n = 4).</p

Summary of mass spectrometric evidence for phosphorylation of CPEB3a by PKA, CaMKII and endogenous kinases present in HEK-293 cells.

<p>Summary of mass spectrometric evidence for phosphorylation of CPEB3a by PKA, CaMKII and endogenous kinases present in HEK-293 cells.</p

qRT-PCR analysis of CPEB3 isoforms of hippocampal mRNA after intraperitoneal kainate injection.

<p>The mRNA levels of isoforms with the B-region increased 30 min after kainate-induced <i>status epilepticus</i>, while the levels of the isoforms lacking the B-region remained unchanged. n = 4 animals = 8 hippocampi (per condition). *p<0.05, student’s t test.</p