Podcasts about g1 s

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.27.545208v1?rss=1 Authors: Oikawa, T., Hasegawa, J., Ohnishi, N., Onodera, Y., Hashimoto, A., Sasaki, J., Sasaki, T., Ueda, K., Sabe, H. Abstract: H3.1 is predominantly synthesized and enters the nucleus during the G1/S phase of the cell cycle, although the underlying mechanism remains unknown. Here we show that p53 is involved in this process. CTDNEP1 converts phosphatidic acid (PA) into diacylglycerol, and EZH2 generates H3K27me3. p53 increased CTDNEP1 and decreased EZH2 in the nuclear H3.1 interactome of the G1/S phase. Moreover, H3.1 bound robustly to PA but not to diacylglycerol. p53 deficiency caused perinuclear accumulation of EZH2-modified H3K27me3 of non-nucleosomal histones during the G1/S phase. p53 induced the expression of TMEM255A, which reduced nuclear PA levels by increasing CTDNEP1 levels. Therefore, H3.1 entering the nucleus in the absence of p53 may be trapped near the nuclear envelope (NE) and epigenetically marked as repressive without forming nucleosomes. Our study identified the NE as a novel target of p53, in which p53 downregulates nuclear PA levels to normalize H3.1 nuclear entry and epigenetic modification. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.08.535951v1?rss=1 Authors: Ling, Y. H., Chen, Y. Y., Leung, K. N., Chan, K. M., Liu, W. K. Abstract: CCHCR1 (coiled-coil alpha-helical rod protein 1) is a candidate gene of psoriasis and was recently identified as a novel component in P-bodies, the site for regulating mRNA turnover, and a protein in centrosomes. Little is known about its transcriptional regulation or its functions in cell cycle progression. In this study, CCHCR1 was found in a close (287-bp) head-to-head orientation with its neighboring gene, TCF19. The transcription of the CCHCR1/TCF19 gene pair was controlled by a shared bidirectional promoter and was induced at the G1/S transition of the cell cycle. The 287-bp intergenic sequence was sufficient for the G1/S expression of both genes, but the expression of CCHCR1 was further promoted by the presence of exon 1 of TCF19 and CCHCR1. The expression of the CCHCR1/TCF19 gene pair was dependent on the E2F1 transcription factor. E2F1 binding sites were predicted in the CCHCR1/TCF19 bidirectional promoter by in silico analysis. Overexpression of E2F1 induced the expression of CCHCR1 and TCF19. In addition, E2F1 knockdown inhibited both CCHCR1 and TCF19 expression. Knockdown of CCHCR1 or TCF19 reduced cell count but only depletion of CCHCR1 significantly induced p21 expression, implying that CCHCR1 and TCF19 may both regulate cell growth but in divergent pathways. Taken together, we revealed a bidirectional regulation of the CCHCR1/TCF19 gene pair in the G1/S transition and provide a new perspective to understand the role of CCHCR1 as a candidate gene of psoriasis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.17.533218v1?rss=1 Authors: Armstrong, C., Passanisi, V. J., Ashraf, H. M., Spencer, S. L. Abstract: Faithful DNA replication requires that cells fine-tune their histone pool in coordination with cell-cycle progression. Replication-dependent histone biosynthesis is initiated at a low level upon cell-cycle commitment, followed by a burst at the G1/S transition, but it remains unclear how exactly the cell regulates this change in histone biosynthesis as DNA replication begins. Here, we use single-cell timelapse imaging to elucidate the mechanisms by which cells modulate histone production during different phases of the cell cycle. We find that CDK2-mediated phosphorylation of NPAT at the Restriction Point triggers histone transcription, which results in a burst of histone mRNA precisely at the G1/S phase boundary. Excess soluble histone protein further modulates histone abundance by promoting the degradation of histone mRNA for the duration of S phase. Thus, cells regulate their histone production in strict coordination with cell-cycle progression by two distinct mechanisms acting in concert. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.12.12.520126v1?rss=1 Authors: Ader, N. R., Chen, L., Surovtsev, I. V., Chadwick, W. L., King, M. C., Lusk, C. P. Abstract: The reformation of the nuclear envelope (NE) at the end of metazoan mitosis requires the sealing of numerous fenestrations that recruit the endosomal sorting complexes required for transport (ESCRT) machinery. The molecular mechanisms by which ESCRT proteins drive NE sealing and their necessity to the reestablishment of the nuclear compartment have yet to be fully defined. Here, we leveraged the single NE hole generated by mitotic extrusion of the Schizosaccharomyces pombe spindle pole body to reveal two modes of ESCRT function carried out by unique complements of ESCRT-III proteins, both of which depend on the NE-specific CHMP7/Cmp7. The first is a grommet-like function, where we tie the presence of specific ESCRTs to a constriction of the NE hole from ~150 to ~50 nm in anaphase B. Consistent with a direct role for ESCRTs in restricting this NE hole diameter, the hole dilates a remarkable five-fold in cells lacking Cmp7. Second, in the subsequent G1/S-phases, a sealing module of ESCRT proteins replaces Cmp7 and is required to drive closure of the NE. Surprisingly, in the absence of Cmp7, nucleocytoplasmic compartmentalization remains intact despite discontinuities in the NE as large as 400 nm, suggesting a mechanism to establish a diffusion barrier(s) over persistent NE holes. Indeed, we demonstrate that the ortholog of the pericentriolar material protein, pericentrin (Pcp1), establishes such a barrier, likely through its ability to form a biomolecular condensate. NE remodeling mechanisms therefore cooperate with proteinaceous diffusion barriers beyond nuclear pore complexes to protect the nuclear compartment. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

本編 中央今年最初のG1フェブラリーS！ どの馬にもチャンス十分！穴を狙いましょう！ ブライトがnoteでサークルはじめました！一緒に競馬を楽しみましょう！(^^)→Mの法則研究同好会 ※フリーメールアドレスをお持ちの方...

In this week's cover paper of Oncotarget (Volume 12, Issue 12), entitled, "Frame-shift mediated reduction of gain-of-function p53 R273H and deletion of the R273H C-terminus in breast cancer cells result in replication-stress sensitivity," researchers used the CRISPR-Cas9 tool to analyze a p53 mutation in triple-negative breast cancer. The researchers from the City University of New York, Columbia University, and Weill Cornell Medical College wrote that both the C-terminal domain (CTD) and oligomerization domain (OD) of mtp53 R273H proteins are intact, and it is not clear if these regions are responsible for chromatin-based DNA replication activities. To examine the ability of mtp53 R273H to influence cell proliferation, DNA replication, and cell cycle progression of breast cancer cells, the researchers used the CRISPR-Cas9 tool to edit the C-terminal regions of the mtp53 gene. “CRISPR-Cas9 sgRNA editing of the C-terminal regions of the endogenous mtp53 gene were carried out so as to delete gene sequences that correspond to the OD and CTD regions.” The team generated breast cancer cells and edited CTD and OD regions of mtp53 R273H using the CRISPR-Cas9 tool. They then treated the cell populations with thymidine—to block cells at G1/S phase in the cell cycle. The researchers then compared the status and proliferation of the variants with the original cell line and observed changes in total cell lysates by western blot analysis. “We examined how changes in the level of mtp53 R273H level and/or deletion of the CTD, or OD plus CTD, region influenced cell proliferation, cell cycle progression, and chromatin association of mtp53, RPA, PCNA and MCM2.” Sign up for free Altmetric alerts about this article - https://oncotarget.altmetric.com/details/email_updates?id=10.18632%2Foncotarget.27975 DOI - https://doi.org/10.18632/oncotarget.27975 Full text - https://www.oncotarget.com/article/27975/text/ Correspondence to - Jill Bargonetti - bargonetti@genectr.hunter.cuny.edu Keywords - mutant p53, gain-of-function, oligomerization, DNA replication, frame-shift, breast cancer About Oncotarget Oncotarget is a bi-weekly, peer-reviewed, open access biomedical journal covering research on all aspects of oncology. To learn more about Oncotarget, please visit https://www.oncotarget.com or connect with: SoundCloud - https://soundcloud.com/oncotarget Facebook - https://www.facebook.com/Oncotarget/ Twitter - https://twitter.com/oncotarget YouTube - https://www.youtube.com/c/OncotargetYouTube/ LinkedIn - https://www.linkedin.com/company/oncotarget Pinterest - https://www.pinterest.com/oncotarget/ Reddit - https://www.reddit.com/user/Oncotarget/ Oncotarget is published by Impact Journals, LLC please visit https://www.ImpactJournals.com or connect with @ImpactJrnls Media Contact MEDIA@IMPACTJOURNALS.COM 18009220957

My AP Biology Thoughts  Unit 4 Cell Communication and Cell CycleWelcome to My AP Biology Thoughts podcast, my name is Nidhi and I am your host for episode #95 called Unit 4 Cell Communication and Cell Cycle: CDK and Cyclins. Today we will be discussing what cyclins and CDK are and why they're important.  Segment 1: Introduction to CDK and CyclinsCyclins are a group of related proteins, and there are four basic types found in humans and most other eukaryotes. These include G1cyclins, G1/S cyclins, S cyclins, and M cyclins. Each cyclin is associated with a particular phase, transition, or set of phases in the cell cycle and helps drive the events of that phase or period. For instance, M cyclin promotes the events of the Mitosis phase, such as nuclear envelope breakdown and chromosome condensation. Cyclin-dependent kinases, or CDKs, are enzymes that catalyze the phosphorylation of target proteins in the cell cycle. The attached phosphate makes the target protein more or less active. The CDKs are activated when attached to cyclin because the cyclin changes the shape of the enzyme. When a cyclin attaches to a Cdk, it has two important effects: it activates the Cdk as a kinase, but it also directs the Cdk to a specific set of target proteins ensuring that those are proteins appropriate to the cell cycle period controlled by the cyclin. After the phosphorylation of proteins is complete, cyclin breaks down and CDK is inactive. CDK-Cyclins also act as a control or regulator for the cell cycle. Cdk activity and target proteins change as levels of the various cyclins rise and fall. In addition to needing a cyclin, Cdks must also be phosphorylated on a particular site in order to be active. Segment 2: More About CDK and Cyclins An example of how cyclins and cdks work is the mitosis-promoting factor. A MPF molecule is a CDK bound to an M cyclin. As the cell approaches the G2/ Mitosis transition phase in the cycle, the levels of the M cyclin increase. It then binds to CDKs present in the cell and together they cause the Mitosis phase to begin. The MDF adds phosphate to protein in the nuclear envelope, causing it to break down, and activates chromosome condensation promoting targets. In addition to driving the events of M phase, MPF also triggers its own destruction by activating the anaphase-promoting complex/cyclosome or APC/C, a protein complex that causes M cyclins to be destroyed starting in anaphase. The destruction of M cyclins pushes the cell out of mitosis, allowing the new daughter cells to enter G1.  CDKs and cyclins often respond to cues from the cell to regulate. Positive cues, like growth factors, increase activity of Cdks and cyclins, while negative ones, like DNA damage, usually decrease activity. When DNA damage occurs, a protein called p53 triggers the production of CDK inhibitor proteins. These proteins bind to Cdk-cyclin complexes and block their activity, allowing time for DNA repair to occur. By ensuring that cells don't divide when their DNA is damaged, mutations are not being passed onto daughter cells. When p53 is defective or missing, mutations can accumulate, potentially leading to cancer  Segment 3: Connection to the Course Cylins and CDK can connect to the principles of evolution. Cyclins and Cdks are found in many different types of species, from yeast to frogs to humans. They vary slightly in each organism. For example, yeast has just one Cdk, while humans and other mammals have multiple Cdks that are used at different stages of the cell cycle. These common enzymes and proteins can provide evidence of a common ancestor between these species.  Thank you for listening to this episode of My AP Biology Thoughts. For more student-ran podcasts and digital content, make sure that you visit http://www.hvspn.com (www.hvspn.com)!  Music Credits: "Ice Flow" Kevin MacLeod (incompetech.com) Licensed under Creative Commons: By Attribution 4.0 License...

The cover for issue 31 of Oncotarget features Figure 4, "Concentration dose-response curves of sirolimus effect [55 nM–1 nM] on the number of cells per surviving colony in U2OS cell line after 2 weeks exposure," by Vasuri, et al. which reported that the authors evaluated the long-term effects of sirolimus on three different cell in vitro models, cultured in physiological conditions mimicking sirolimus-eluted stent, in order to clarify the effectiveness of sirolimus in blocking cell proliferation and survival. Three cell lines were selected and growth in 10 ml of Minimum Essential Medium for 5 weeks with serial dilutions of sirolimus. The number of colonies and the number of cells per colony were counted. As a result, the number of WPMY-1 surviving colonies increased in a dose-dependent manner when treated with sirolimus, while the number of U2OS colonies progressively decreased. In conclusion sirolimus showed the well-known cytostatic effect, but with an effect on clonogenic potential different among the different cell types. Dr. Gianandrea Pasquinelli from The Bologna University said, "Rapamycin (sirolimus) is a widely used cytostatic drug blocking the cell cycle in the phase G1/S through the inhibition of the mammalian target of Rapamycin (mTOR) pathway, that has found several clinical applications, from immunosuppression in diabetes and organ transplantation to cancer therapy and drug-eluting stents (DES)" Beside to its cytostatic activity, sirolimus was also discovered to protect normal human oral keratinocytes from apoptosis by activating autophagy, and to act as a basal stem cell keratinocyte-protecting drug in irradiated mice. The effect of sirolimus on mesenchymal cells is unknown, but it is an important issue, since mesenchymal cells such as myofibroblasts and cells promoting vascular calcification play an important role in atherogenesis and vascular restenosis. Sirolimus seems to block the proliferation and the migration of vascular smooth muscle cells, but we lack information concerning the effects on other cells composing atherosclerotic plaques. The aim of the present paper is to evaluate the long-term effects of sirolimus, rather than short-term cell survival, on three different cell in vitro models, cultured in Minimum Essential Medium, which simulates physiological conditions (w/o CO2 and glucose, in order to clarify the effectiveness of sirolimus in blocking cell proliferation and survival. The Pasquinelli Research Team concluded in their Oncotarget Research Paper that the plaque typology and the different cell composition of the plaque, e. g., the presence of inflammatory cells, angiogenesis, prevalence of fibrosis, presence of osteogenic progenitors, may influence the response to sirolimus. Moreover, it is known that the clonal capacity varies between cells and we should consider this matter when evaluating the effectiveness of eluted stent. Finally, additional mechanisms can have a role, such as amitotic cell division. These mechanisms were also observed in human atherogenesis and could be fundamental to evaluate the in vivo effect of sirolimus too. Full text - https://www.oncotarget.com/article/27554/text/ Correspondence to - Gianandrea Pasquinelli - gianandr.pasquinelli@unibo.it Keywords - atherosclerosis, cell proliferation, sirolimus, stents About Oncotarget Oncotarget is a weekly, peer-reviewed, open access biomedical journal covering research on all aspects of oncology. To learn more about Oncotarget, please visit https://www.oncotarget.com or connect with: SoundCloud - https://soundcloud.com/oncotarget Facebook - https://www.facebook.com/Oncotarget/ Twitter - https://twitter.com/oncotarget LinkedIn - https://www.linkedin.com/company/oncotarget Pinterest - https://www.pinterest.com/oncotarget/ Reddit - https://www.reddit.com/user/Oncotarget/ Oncotarget is published by Impact Journals, LLC please visit http://www.ImpactJournals.com or connect with @ImpactJrnls

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.05.08.084129v1?rss=1 Authors: Veerasammy, S., Van Steenwinckel, J., Le-Charpentier, T., Seo, J. H., Fleiss, B., Gressens, P., Levison, S. W. Abstract: Meta-analyses have revealed associations between the incidence of maternal infections during pregnancy, premature birth, smaller brain volumes, and subsequent cognitive, motor and behavioral deficits as these children mature. Inflammation during pregnancy in rodents produces cognitive and behavioral deficits in the offspring that are similar to those reported in human studies. These deficits are accompanied by decreased neurogenesis and proliferation in the subgranular zone (SGZ) of the dentate gyrus (DG) of the hippocampus. As systemically administering interleukin-1{beta} (IL-1{beta}) to neonatal mice recapitulates many of the brain abnormalities seen in premature babies including developmental delays, the goal of this study was to determine whether IL-1-mediated neuroinflammation would affect hippocampal growth during development to produce cognitive and behavioral abnormalities. For these studies, 10 ng/g IL-1{beta} was administered twice daily to Swiss Webster mice during the first 5 days of life, which increased hippocampal levels of IL-1 and acutely reduced the proliferation of Tbr2+ neural progenitors in the DG. In vitro , both IL-1 and IL-1{beta} produced G1/S cell cycle arrest that resulted in reduced progenitor cell proliferation within the transit amplifying progenitor cell cohort. By contrast, IL-1{beta} treatment increased neural stem cell frequency. Upon terminating IL-1{beta} treatment, the progenitor cell pool regained its proliferative capacity. An earlier study that used this in vivo model of perinatal inflammation showed that mice that received IL-1{beta} as neonates displayed memory deficits which suggested abnormal hippocampal function. To evaluate whether other cognitive and behavioral traits associated with hippocampal function would also be altered, mice were tested in tasks designed to assess exploratory and anxiety behavior as well as working and spatial memory. Interestingly, mice that received IL-1{beta} as neonates showed signs of anxiety in several behavioral assays during adolescence that were also evident in adulthood. Additionally, these mice did not display working memory deficits in adulthood, but they did display deficits in long-term spatial memory. Altogether, these data support the view that perinatal inflammation negatively affects the developing hippocampus producing behavioral deficits that persist into adulthood. These data provide a new perspective into the origin of the cognitive and behavioral impairments observed in prematurely-born sick infants. Copy rights belong to original authors. Visit the link for more info

In westlichen Industrieländern stellt Schlaganfall eine der häufigsten Todesursachen dar und ist hauptursächlich für körperliche Behinderung. In einem hohen Maße kann Schlaganfall auf genetische Faktoren zurückgeführt werden, weshalb kürzlich eine genomweite Assoziationsstudie (GWAS) in der METASTROKE-Kohorte für ischämischen Schlaganfall durchgeführt wurde. Hierbei konnte die Chromosomenregion 7p21.1 als bisher stärkster Risikolokus für atherosklerotischen Schlaganfall identifiziert werden. Diese Region umfasst das Ende des HDAC9-Gens und den benachbarten intergenischen Bereich stromaufwärts der Gene TWIST1 und FERD3L. Der Haupt-SNP rs2107595 kolokalisiert mit einem DNase I-hypersensitiven Bereich sowie Histonmodifikations-Hotspots und könnte somit genregulatorische Konsequenzen besitzen. Im Rahmen der vorliegenden Arbeit durchgeführte Genexpressionsstudien in humanen Blutzellen ergaben eine Dosis-abhängige Korrelation der HDAC9 mRNA-Spiegel mit dem rs2107595-Risikoallel. HDAC9 gehört zur Familie der Histondeacetylasen, die v.a. bei der Transkription eine wichtige Rolle spielen. Das rs2107595-Risikoallel verändert ein bioinformatisch vorhergesagtes Bindemotiv für E2F-Transkriptionsfaktoren, das beim häufigen Allel intakt ist. Untersuchungen der transkriptionellen Kapazität dieser Bindungsstelle zeigten eine erhöhte Aktivität des häufigen Allels im Vergleich zum Risikoallel. Zur Identifizierung dafür verantwortlicher DNA-interagierender Proteine wurde in Kollaboration mit dem Max-Planck-Institut für Biochemie, Martinsried eine proteomweite Analyse von SNPs (PWAS) durchgeführt. Hierbei wurden die Proteine E2F3, E2F4, TFDP1 und Rb1 mit präferentieller Bindung an das häufige rs2107595-Allel identifiziert. E2F/TFDP/Rb-Komplexe sind ein zentraler Bestandteil des G1/S-Übergangs im Zellzyklus und regulieren somit die Zellproliferation. In gain- und loss-of-function-Experimenten von E2F3, E2F4 und der Rb-Proteinfamilie in humanen Zelllinien mit verschiedenen Genotypen für rs2107595 konnte eine unterschiedliche Regulation der HDAC9-Expression beobachtet werden. Nach Zellzyklus-Synchronisation konnten erhöhte HDAC9-Spiegel im Zeitraum der aktiven Phase von E2F3 gezeigt werden, was den Befund einer Regulation durch den E2F3/TFDP1/Rb1-Komplex stützt. Ein erhöhtes Risiko für Schlaganfall könnte also durch eine Risikoallel-vermittelte Störung dieses genregulatorischen Mechanismus entstehen.

Das Mantelzelllyphom wird analog zu den indolenten NHL als nicht kurabel eingestuft, weist jedoch ein signifikant verkürztes medianes Gesamtüberleben auf und stellt somit für die Klinik eine Kombination aus den negativen Eigenschaften von indolenten und aggressiven Lymphomen dar. Das MCL stellt trotz intensiver Therapie aufgrund seiner frühzeitigen Rezidive eine große Herausforderung an die Medizin. Eine große Anzahl neuer Substanzen werden z.Z. auf ihre Wirksamkeit beim MCL geprüft. Zwei dieser Stoffe sind Flavopiridol, ein molekularer Serin/Threonin-Kinase-Inhibitor, und Bendamustin, ein bereits in der Klinik etabliertes Zytostatikum. Gegenstand dieser Arbeit ist zum einen die bessere Charakterisierung der Wirkung beider Substanzen auf MCL-Zelllinien in vitro, sowohl als Monotherapie als auch in Kombination mit anderen Medikamenten. Hauptziel ist es, einen potentiellen Synergismus der untersuchten Medikamente aufzudecken und den genaueren Effekt auf die Tumorzellen zu charakterisieren. Die in vitro Untersuchungen der vorliegenden Arbeit weisen eine dosis- und zeitabhängige zytotoxische Aktivität von Bendamustin nach, und spiegeln somit die hohe klinische Effektivität bei der Behandlung des MCL wieder. Ebenso konnte anhand der hier gezeigten Versuchsreihen demonstriert werden,dass der Cyclin-abhängige-Kinasen-Inhibitor Flavopiridol als Monosubstanz hochwirksam gegen MCL-Zelllinien in vitro ist. Nach Behandlung mit Flavopiridol konnte in allen untersuchten Zelllinien in klinisch realisierbaren Dosierungen Apoptose induziert werden, Verringerungen der CDK-Expression nachgewiesen, und darüber hinaus eine potente Inhibition des Zellzyklus im Sinne eines G1/S und G2/M Arrest demonstriert werden. Die selektive Hemmung der CDKs stellt somit einen attraktiven, zielgerichteten Ansatz in der Tumortherapie dar, denn diese Enzyme sind in den meisten malignen Zellen zur Aufrechthaltung einer unbegrenzten Proliferation notwendig. Für die Kombination von Flavopiridol mit Enzastaurin und Rad001 konnte, ins besonders in resistenten Zellreihen, ein mehr als additiver Effekt gezeigt werden; diese Erkenntnis spricht für eine Komplementarität in der antineoplastischen Wirkung dieser Substanzen bei zeitgleicher Inhibition der jeweiligen zellulären Zielstrukturen. Andererseits konnte für Kombinationen von Flavopiridol mit antimetabolischwirkenden Chemotherapeutika und Bendamustin, bei gleichzeitiger Anwendung, nur antagonistische Effekte beobachtet werden. Hier scheint der durch Flavopiridol verursachte potente Zyklusarrest am G1/SÜbergang der Grund für die verminderte Wirksamkeit der verwendeten phasenspezifischen Medikamente zu sein. Diese Resultate untersteichen die enorme Bedeutung der Sequenz bei der Verabreichung von zytostatisch wirkenden Stoffen in der Therapie von Malignomen. Die Erkenntnisse über edikamentenwirkungen aus in vitro Experimenten lassen sich allerdings nicht ohne weiteres auf komplexe Systeme in vivo übertragen und müssen deshalb berücksichtigt werden. Folgende Gründe sind hierfür ursächlich: Zum einen sind viele wichtige antineoplastische Mechanismen in der Zellkultur nicht messbar bzw. quantifizierbar, etwa eine Hemmung der Angioneogenese, Veränderungen des Mikromilieus der Tumorzellen oder zelluläre Immunantworten. Zum anderen sind die Biodistribution, die Pharmakodynamik und die Pharmakokinetik in vivo entscheidend, ob überhaupt eine Wirkung des Medikaments an der Tumorzelle entfaltet werden kann. Die Auswirkungen dieser Faktoren in komplexen biologische Systemen sind jedoch in vitro nicht einwandfrei zu beurteilen und können, am Beispiel von Flavopiridol, zu falschen Rückschlüssen bei der klinischen Anwendung eines Medikaments führen.

T cells directed against brain antigens are generally held to play a crucial role in the initiation of multiple sclerosis (MS). This was deduced from experimental autoimmune encephalomyelitis (EAE). In this model for MS, T cells reactive for myelin antigens induced a severe paralytic disease upon transfer to healthy syngeneic recipients. Intriguingly, the disease does not start immediately upon transfer of the pathogenic effector T cells. Instead, as earlier studies have shown, the effector T cells attack their target organ only after having migrated in the periphery through secondary lymphoid organs. The aim of the project was to characterize the functional properties of these migrating encephalitogenic T cells during the course of EAE and to identify biological pathways which determine their migratory behaviour and pathogenic potential. To this end, average linkage hierarchical clustering, pathway and gene ontology (GO) analyses of transcriptomes from cultured and ex vivo-isolated myelin basic protein-reactive T cells (TMBP cells) were performed. At the time of transfer, encephalitogenic T cells in vitro are maximally activated, i.e. they exhibit a prominent upregulation of cell cycle genes such as cyclin A2 (CCNA2) and cyclin B2 (CCNB2) among others. In contrast, T cells isolated from spleen 3 days post transfer, downregulated activation markers such as interleukin 2 receptor (IL2R) and interferon γ (IFNγ), and at the same time upregulated migration specific genes such as CC-chemokine receptor 1 (CCR1), CC-chemokine receptor 2 (CCR2) and CC-chemokine receptor 5 (CCR5). Hierarchical cluster analysis revealed that several transcription regulators known for inhibiting cell cycle progression such as krüppel-like factor 4 (KLF4), B-cell translocation gene 2 (BTG2) and transducer of ERBB2, 1 (TOB1) were clustered together with cell cycle and migration genes. Overexpression of KLF4 in T cells not only inhibited G1/S phase progression of the cell cycle but additionally induced upregulation of CCR2 and CCR5. A novel tetraspan membrane protein called epithelial membrane protein (EMP1), was found to be up regulated in ex vivo-isolated effector T cells. Overexpression of EMP1 in encephalitogenic T cells influenced the migratory behaviour of effector T cells both in vitro and in vivo. EMP1 enhanced T cell motility within the extracellular matrix milieu in vitro and promoted T cell migration from the connective tissue to lymph nodes in vivo resulting in an accelerated onset of EAE. In conclusion, gene expression profiling of encephalitogenic T cells revealed interesting genome wide transcriptomic changes and established a correlation between cell cycle progression and cell migration. As a result, in silico analysis put forth several interesting candidate genes that hold promise as potential targets for therapeutic intervention.

Pituitary adenomas are benign neoplasms accounting for 15% of all intracranial tumors. They are associated with significant clinical syndromes due to the hormonal excess they produce or to visual/cranial disturbances because of their considerable intracranial mass. Surgery is the primary means for the management of pituitary tumor mass, but it comes with considerable side effects to the patients and their quality of life. Tumor shrinkage by pharmacological agents currently used in neuroenocrinology, such as, somatostatin analogs (SSA) is not observed in a large fraction of pituitary adenomas. Therefore, efforts are taken to investigate how to overcome the resistance to existing treatments and to identify new cytostatic therapeutic agents. The PI3K/Akt/mTOR signaling pathway is frequently overactivated in a variety of tumors rendering them resistant to chemo- and radiotherapy. The present study shows that Akt overactivation confers resistance to the antiproliferative action of the SSA octreotide in pituitary tumor cells. Blocking the Akt pathway downstream with rapamycin rendered those cells sensitive to octeotide’s antiproliferative action. However, the efficacy of the combined treatment was not due to the antiproliferative action of rapamycin, since most of the tumors did not respond to this pharmacological agent. Rapamycin and its analogs (rapalogs) have a cytostatic effect in various tumors. However, resistance to rapalog treatment is reported with increasing frequency due to the elimination of the negative feedback loop exerted by the mTOR substrate p70/S6K on IRS-1. Rapamycin, by inhibiting mTOR and p70/S6K, decreases the inhibitory IRS-1 serine phosphorylation, activates IRS-1 and increases Akt-Ser473 phosphorylation. The present study shows for the first time that activating the G protein-coupled receptor Sstr2 with octreotide blocks the rapamycin-induced IRS-1 activation by increasing its inhibitory serine and decreasing its stimulatory tyrosine phosphorylation. This leads to decreased Akt-Ser473 phosphorylation in a mechanism involving the phosphotyrosine phosphatase SHP-1. Both octreotide and rapamycin are cytostatic agents blocking the G1/S cell cycle transition and herein it is seen that their potent antiproliferative action depends on the more potent upregulation of the Cdk2 inhibitor p27/Kip1. A novel drug able to co-target the PI3K pathway up- and downstream is the dual class PI3K/mTOR inhibitor, NVP-BEZ235, which has shown high antiproliferative efficacy in tumors with the overactivated PI3K pathway. In the present study NVP-BEZ235 treatment dramatically decreased cell viability by suppressing the cell cycle activators cyclin E and Cdk2 and upregulating the cell cycle progression inhibitor p27/Kip1. The remarkable sensitivity of pituitary adenomas to NVP-BEZ235 highlights the importance of the Akt dysregulation in their tumor maintenance. Altogether, these data provide new therapeutic cytostatic schemes that could prove beneficial for the management of pituitary macroadenomas. In addition they provide the biochemical basis for combating resistance to rapalog treatment also in other tumor types by concomitant administration of biologicals able to inhibit the PI3K pathway upstream.

The hallmark of embryonic stem (ES) cells is their ability for self-renewal (capability of unlimited cell division without the loss of pluripotency) as well as for differentiation into all cell types of the adult organism. One factor supposed to be involved in self-renewal is the rapid proliferation rate of ES cells, which is coupled to an unusual cell cycle distribution with the majority of cells in S-phase and a very short G1-phase. This is linked to the lack of a functional G1/S-phase checkpoint, which allows the cells to enter the S-phase almost directly after mitosis. Generally, cells have to closely coordinate growth and cell cycle progression during proliferation to prevent premature division. One important factor for cell growth is ribosome biogenesis. In mature cells, disruptions in ribosome biogenesis are directly linked to the cell cycle machinery by a p53-dependent activation of the G1/S-phase checkpoint, leading to an arrest of cells in G1-phase. During this work, the function of the proteins Pes1, Bop1 and WDR12, which were shown previously to be involved in ribosome biogenesis of mature cell lines, was investigated in mouse ES cells. Moreover, a putative crosstalk between ribosome biogenesis and proliferation of ES cells was assessed. A high expression of Pes1, Bop1 and WDR12 was observed in ES cells, which strongly decreased during in vitro differentiation. Localization of the proteins was predominantly nucleolar and the formation of a stable complex (PeBoW-complex), including all three proteins, was experimentally validated in mature mouse cells as well as in mouse ES cells. The function and stability of the proteins seems to be dependent on incorporation into the PeBOW-complex, as protein levels were interdependent on each other and no free, non-incorporated proteins were observed, except for WDR12. According to their nucleolar localization, depletion of Pes1 and Bop1 were shown to inhibit maturation of the 28S rRNA and thereby the large 60S ribosomal subunit. Further, impaired proliferation of ES cells was observed. Thus, the PeBoW-complex seems to be an essential factor for the rapid proliferation of ES cells and might therefore also be involved in self-renewal. However, first results suggest that the complex is not directly involved in the maintenance of pluripotency. No changes in the expression levels of pluripotency-genes like Nanog, KLF4 and Sox2 were observed. Moreover, alkaline phosphatase activity was equally detectable after depletion of Pes1 or Bop1 and no morphological changes within the ES cell colonies were observed. Impaired ribosome biogenesis is known to activate a p53-dependent checkpoint in mature cell lines, which leads to an arrest of cells in G1-phase. Treatment of mouse NIH3T3 cells with 5FU, a potent inhibitor of rRNA maturation, confirmed an activation of this checkpoint, leading to weak induction of the tumor suppressor p53, induction of the Cdk-inhibitor p21, an increase in active, hypo-phosphorylated Rb, and to accumulation of cells in the G1- and S-phase with an increase of cells in G1-phase. In contrast, ES cells showed strong induction of p53, but no induction of its target gene p21. The overall levels of Rb were strongly induced, but the ratio between inactive, hyper-phosphorylated Rb and active, hypo-phosphorylated Rb was not changed towards the active form. These results were observed upon 5FU treatment and upon depletion of Pes1 or Bop1. Hence, ribosomal stress does not lead to checkpoint activation via the p53-p21-Rb pathway in ES cells. Moreover, no robust accumulation of cells in G1-phase was observed. 5FU treated ES cells showed an accumulation of cells in S-phase instead. Whether this effect is regulated by the induced p53 needs further investigation. Overall, the results suggest that ES cells use different mechanisms as mature cells to coordinate their proliferation rate with ribosome biogenesis.

Human cells have evolved protective mechanisms such as DNA repair and cell cycle checkpoints in order to promote stability of the genome. Studies on hereditary instability syndromes associated with a higher incidence of malignancies like Xeroderma pigmentosum or Nijmegen breakage syndrome demonstrated that genetic defects and subsequent dysfunction of a specific DNA repair mechanism trigger the development of cancer. Within the last years, the investigation of cell cycle checkpoints gained increasing importance in cancer research. Checkpoints are signaling cascades that halt the cell cycle in response to DNA damage, thereby providing time for repair and preventing accumulation of DNA alterations. While the p53-dependent G1-S checkpoint has been extensively investigated, little is known about other checkpoints in humans such as the G2-M or the S-phase progression checkpoint. Studies on the human cancer syndrome ataxia telangiectasia (AT) showed that AT cells fail to induce several checkpoints in response to ionizing radiation (IR), indicating that a checkpoint gene defect is responsible for the AT-associated cancers. The responsible gene (ATM) has significant sequence homology to the checkpoint kinase gene sprad3 in the fission yeast Schizosaccharomyces pombe (S. pombe). In S. pombe, spRad3 regulates G2-M checkpoint activation in response to DNA damage. Defects in the sprad3 gene, like defects in ATM, sensitize the organisms to radiation and radiomimetic drugs, suggesting conservation of checkpoint pathways from yeast to humans as well as a potential role of the G2-M checkpoint in carcinogenesis. The discovery of G2-M checkpoint-deficient yeast mutants led to the cloning of additional checkpoint genes in yeast and their human homologs. This group of novel human genes includes homologs of sprad9 (hRAD9), sphus1 (hHUS1), and sprad1 (hRAD1). In S. pombe, these genes are required for activation of spRad3, and defects in one or more of these genes render the yeast more sensitive to genotoxic agents. Mutations within the human rad genes may bring about an increased rate of mutations and genomic instability as shown for p53 or AT and may be responsible for inherited predisposition to cancer. In view of this potential importance of human rad genes in the process of carcinogenesis, we have undertaken a cellular and molecular analysis of the novel human checkpoint proteins hRad9, hHus1, and hRad1 in the leukemia cell line K562 and in human keratinocytes. Using specific antibodies to the hRad9, hHus1, and hRad1 proteins we demonstrated with co-immunoprecipitation and Western-blot experiments that the human checkpoint proteins hRad9, hHus1, and hRad1 associate in a biochemical complex similar to the spRad9-spHus1-spRad1 complex reported in fission yeast. To generate a model system of checkpoint protein function amenable to biochemical analysis, we prepared epitope-tagged expression vectors for hRad9, hHus1, and hRad1, which were transfected into K562 cells by electroporation, resulting in transient expression of epitope-tagged protein. By simultaneous expression of hRad9, hHus1, and hRad1 we showed that transiently expressed epitope-tagged checkpoint proteins hRad9, hHus1, and hRad1 recapitulate complex formation of endogenous proteins. Immunoprecipitation studies with lysates of hRad9-overexpressing cells revealed that hRad9 undergoes complex post-translational modifications. Co-expression of hRad9 with hHus1, and hRad1 resulted in a large increase of the amount of a highly modified form of hRad9, suggesting that hRad1 and hHus1 either promote formation of, or stabilize the modified form of hRad9. Previously, a direct correlation between checkpoint protein phosphorylation and activation of DNA damage checkpoints in yeast was proposed. In this study, we show that hRad9 is phosphorylated in response to DNA damage, and that phosphorylated hRad9 interacts with hHus1 and hRad1 as well. The present results suggest that the hRad9-hHus1-hRad1 complex actively participates in an evolutionarily conserved DNA damage-induced signaling cascade. hRad1 seems to possess exonuclease activity. The presence of a putative DNA-metabolizing protein in the multimolecular checkpoint complex, coupled with genetic data that place spRad9, spHus1, and spRad1 early in the response pathway of checkpoint activation suggests that the complex may function as a sensor that scans the genome for damaged DNA. Once damaged DNA is detected, this complex may initiate endonucleolytic processing of the lesions and trigger interactions with downstream signaling elements, or may link unknown damage recognition components to downstream signal-transducing pathways that include the ATM kinase, which is implicated in actively enforcing cell cycle arrest after DNA damage. Potential goals of checkpoint research include the implementation of screening tests to identify familial cancer predisposition and treatment of checkpoint gene defects by gene transfer. Another aim of checkpoint research is the development of checkpoint-based cancer therapy. More than 50% of all human malignant tumors contain mutated p53, and p53-deficient tumor cells lack induction of the G1-S checkpoint in response to DNA damage. One emerging hypothesis is that selective inhibitors of the compensating G2-M checkpoint would preferentially radiosensitize p53-deficient tumor cells. Thus, the investigation of checkpoint function in humans provides further targets for chemotherapeutic agents and will help to design future strategies in cancer therapy.

Cardiomyocytes cease to divide shortly after birth and an irreversible cell cycle arrest is evident accompanied by the downregulation of cyclin-dependent kinase activities. To get a better understanding of the cardiac cell cycle and its regulation, the effect of functional recovery of the mitosis-promoting factor (MPF) consisting of cyclin B1 and the cyclin-dependent kinase Cdc2 was assessed in primary cultures of postmitotic ventricular adult rat cardiomyocytes ( ARC). Gene transfer into ARC was achieved using the adenovirus-enhanced transferrinfection system that was characterized by the absence of cytotoxic events. Simultaneous ectopic expression of wild-type versions of cyclin B1 and Cdc2 was sufficient to induce MPF activity. Reestablished MPF resulted in a mitotic phenotype, marked by an abnormal condensation of the nuclei, histone H3 phosphorylation and variable degree of decay of the contractile apparatus. Although a complete cell division was not observed, the results provided conclusive evidence that cell cycle-related events in postmitotic cardiomyocytes could be triggered by genetic intervention downstream of the G1/S checkpoint. This will be of importance to design novel strategies to overcome the proliferation arrest in adult cardiomyocytes.

The timing of commitment during stenotele differentiation in Hydra was determined. Regeneration of isolated distal regions of the body column induces stenotele differentiation. The kinetics of appearance of committed stenotele precursors was determined in such regenerating pieces. Using [3H]thymidine labeling and hydroxyurea sensitivity, the G1/S and the S/G2 boundaries of the precursor population was also determined. Comparison of these results indicates that stenotele commitment is localized near the S/G2 boundary in the terminal cell cycle of nests of precursor cells.