Browsing by Subject "cell cycle"

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  • Filippou, Artemis; Pehkonen, Henna; Karhemo, Piia-Riitta; Väänänen, Juho; Nieminen, Anni I.; Klefström, Juha; Grenman, Reidar; Mäkitie, Antti; Joensuu, Heikki; Monni, Outi (2021)
    Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous group of tumors that derive from the mucosal epithelium of the upper aerodigestive tract and present high mortality rate. Lack of efficient targeted-therapies and biomarkers towards patients' stratification are caveats in the disease treatment. Anoctamin 1 (ANO1) gene is amplified in 30% of HNSCC cases. Evidence suggests involvement of ANO1 in proliferation, migration, and evasion of apoptosis; however, the exact mechanisms remain elusive. Aim of this study was to unravel the ANO1-dependent transcriptional programs and expand the existing knowledge of ANO1 contribution to oncogenesis and drug response in HNSCC. We cultured two HNSCC cell lines established from primary tumors harboring amplification and high expression of ANO1 in three-dimensional collagen. Differential expression analysis of ANO1-depleted HNSCC cells demonstrated downregulation of MCL1 and simultaneous upregulation of p27Kip1 expression. Suppressing ANO1 expression led to redistribution of p27Kip1 from the cytoplasm to the nucleus and associated with a cell cycle arrested phenotype. ANO1 silencing or pharmacological inhibition resulted in reduction of cell viability and ANO1 protein levels, as well as suppression of pro-survival BCL2 family proteins. Collectively, these data provide insights of ANO1 involvement in HNSCC carcinogenesis and support the rationale that ANO1 is an actionable drug target.
  • Gruzdaitis, Päivi (Helsingfors universitet, 2011)
    Complications of atherosclerosis such as myocardial infarction and stroke are the primary cause of death in Western societies. The development of atherosclerotic lesions is a complex process, including endothelial cell dysfunction, inflammation, extracellular matrix alteration and vascular smooth muscle cell (VSMC) proliferation and migration. Various cell cycle regulatory proteins control VSMC proliferation. Protein kinases called cyclin dependent kinases (CDKs) play a major role in regulation of cell cycle progression. At specific phases of the cell cycle, CDKs pair with cyclins to become catalytically active and phosphorylate numerous substrates contributing to cell cycle progression. CDKs are also regulated by cyclin dependent kinase inhibitors, activating and inhibitory phosphorylation, proteolysis and transcription factors. This tight regulation of cell cycle is essential; thus its deregulation is connected to the development of cancer and other proliferative disorders such as atherosclerosis and restenosis as well as neurodegenerative diseases. Proteins of the cell cycle provide potential and attractive targets for drug development. Consequently, various low molecular weight CDK inhibitors have been identified and are in clinical development. Tylophorine is a phenanthroindolizidine alkaloid, which has been shown to inhibit the growth of several human cancer cell lines. It was used in Ayurvedic medicine to treat inflammatory disorders. The aim of this study was to investigate the effect of tylophorine on human umbilical vein smooth muscle cell (HUVSMC) proliferation, cell cycle progression and the expression of various cell cycle regulatory proteins in order to confirm the findings made with tylophorine in rat cells. We used several methods to determine our hypothesis, including cell proliferation assay, western blot and flow cytometric cell cycle distribution analysis. We demonstrated by cell proliferation assay that tylophorine inhibits HUVSMC proliferation dose-dependently with an IC50 value of 164 nM ± 50. Western blot analysis was used to determine the effect of tylophorine on expression of cell cycle regulatory proteins. Tylophorine downregulates cyclin D1 and p21 expression levels. The results of tylophorine's effect on phosphorylation sites of p53 were not consistent. More sensitive methods are required in order to completely determine this effect. We used flow cytometric cell cycle analysis to investigate whether tylophorine interferes with cell cycle progression and arrests cells in a specific cell cycle phase. Tylophorine was shown to induce the accumulation of asynchronized HUVSMCs in S phase. Tylophorine has a significant effect on cell cycle, but its role as cell cycle regulator in treatment of vascular proliferative diseases and cancer requires more experiments in vitro and in vivo.
  • Mogollon, Isabel; Ahtiainen, Laura (2020)
    Embryonic development of ectodermal organs involves a very dynamic range of cellular events and, therefore, requires advanced techniques to visualize them. Ectodermal organogenesis proceeds in well-defined sequential stages mediated by tissue interactions. Different ectodermal organs feature shared morphological characteristics, which are regulated by conserved and reiterative signaling pathways. A wealth of genetic information on the expression patterns and interactions of specific signaling pathways has accumulated over the years. However, the conventional developmental biology methods have mainly relied on two-dimensional tissue histological analyses at fixed time points limiting the possibilities to follow the processes in real time on a single cell resolution. This has complicated the interpretation of cause and effect relationships and mechanisms of the successive events. Whole-mount tissue live imaging approaches are now revealing how reshaping of the epithelial sheet for the initial placodal thickening, budding morphogenesis and beyond, involve coordinated four dimensional changes in cell shapes, well-orchestrated cell movements and specific cell proliferation and apoptosis patterns. It is becoming evident that the interpretation of the reiterative morphogenic signals takes place dynamically at the cellular level. Depending on the context, location, and timing they drive different cell fate choices and cellular interactions regulating a pattern of behaviors that ultimately defines organ shapes and sizes. Here we review how new tissue models, advances in 3D and live tissue imaging techniques have brought new understanding on the cell level behaviors that contribute to the highly dynamic stages of morphogenesis in teeth, hair and related ectodermal organs during development, and in dysplasia contexts.
  • Liu, Wei; Lu, Xiangyi; He, Guangyang; Gao, Xiang; Xu, Maonian; Zhang, Jingkai; Li, Meiling; Wang, Lifeng; Li, Zhenjing; Wang, Likui; Luo, Cheng (2013)
  • Korsisaari, Nina Kristiina (Helsingfors universitet, 1998)
    Cells in tissues have only three serious options in life; they can grow and divide, remain static, or die by apoptosis. Upon growth factor stimulation a cell enters the so called cell cycle which will eventually lead to the division of the cell. Cell cycle can be divided into four phases; G1, S, G2 and M. The current model of the cell cycle control holds that the transitions between different cell cycle states are regulated by cyclin dependent kinases (CDK) with their activator subunits, the cyclins. CDK regulation can be separated into four distinct mechanisms, one of which being phosphorylation on the so called T-loop leading to complete activation. This phosphorylating activity is mediated by apparently a single enzymatic activity termed the CDK activating kinase, CAK. CAK activity was originally isolated as a biochemical purification extract and the enzyme was surprisingly noticed to be structurally related to CDKs. Since a novel cyclin was identified to be associated to it, the enzyme exercising CAK activity was named CDK7 and the cyclin was designated cyclin H. An entirely new perspective on CDK7 function was opened when CDK7 was identified as a subunit of transcription factor IIH (TFIIH) and shown to phosphorylate the carboxy-terminal domain (CTD) of RNA polymerase II (RNAPII). CDK7 has also been suggested to be involved in irradiation sensitivity pathways and nucleotide excision repair functions. To elucidate the intriguing in vivo role of CDK7, proteins interacting with CDK7 were screened for using the yeast two-hybrid method as part of previous studies of the laboratory. The results showed that 15 out of 144 (10,4%) positive clones were identified to encode a peptide sequence of a protein previously known as the inhibitor/interactor of protein kinase C (PKCI). These yeast colonies had an unexpected phenotype; contradictory to a dark blue color of the colonies, indicating strong interaction, the size of the PKCI colonies was small compared to others, indicating a possible growth inhibition effect. Several DNA open reading frames (ORF) coding for proteins related to human PKCI have been identified in a broad range of species representing mammalian, plant, fungal and bacterial kingdoms, all these forming a HIT (conserved triad of histidines) protein family. Another human member, part of this now super family, named FHIT (fragile triad of histidines) was identified with a dinucleoside 5’,5’’’-P1,P3-triphosphate hydrolase activity. These molecules; substrates of FHIT and related enzymes have been proposed to have various intracellular functions, including signalling stress responses. The aim of this study was to extend the investigation of the interaction between CDK7 and PKCI observed in yeast two-hybrid by means of several genetic and biochemical approaches to determine if this observed interaction and growth phenotype has any physiological significance. Investigations included performing yeast two-hybrid screening for PKCI, developing yeast three-hybrid system and carrying out growth rate assays for yeast liquid cultures. These studies also included performing biochemical purifications of over-expressed proteins, immunoprecipitations, western blot analysis and kinase activity assays. Protein extracts originated from transformed yeast cells, transfected mammalian cells or from in vitro transcription and translation reactions. On basis of growth rate assays it can be concluded that PKCI has an inhibitory growth effect in yeast. The preliminary finding of a specific PKCI-CDK7 interaction in yeast two-hybrid, however could not be conclusively verified by the other methods that were used in this study. Studies of PKCI characterisation also included examination of the subcellular localisation of PKCI in mammalian cells by immunofluorescence labelling of HA-PKCI. Results showed PKCI to localize both in the nucleus and in the cytoplasm. Also, studies to elucidate the function of PKCI were performed; whether it possesses enzymatic activity related to that of FHIT. By NMR spectroscopy using bacterially produced GST-PKCI, hydrolase activity towards ADP was indeed observed. Future studies will include elucidation of possible links between growth inhibition and hydrolase activity, in the form of stress signalling functions. The main focus of our future studies will be the generation of mice with targeted PKCI alleles offering powerful means to reveal the function of PKCI through observing phenotypes and through detailed analysis of these mice harbouring wild type, hypomorphic or null alleles.
  • Virkunen, Ekaterina (Helsingin yliopisto, 2018)
    The majority of cancers, such as breast cancer, originate from epithelial structures. Highly organized epithelial tissues are comprised of cells which manifest apico-basal polarization. Factors regulating apico-basal polarity and epithelial integrity are often observed deregulated in cancer cells and loss of polarity is often observed in tumors. However, the importance and the specific role of epithelial integrity regulators in tumorigenesis are still not fully defined. This study shows that the key regulators of epithelial cell polarity Par6B and Par6G proteins have a role in the restriction of cell proliferation in mammary epithelial cell lines. Using the shRNA silencing approach, downregulation of PARD6B and PARD6G in the cells lead to the impediment of the cell-cycle exit, verified in proliferation suppressive conditions in which cells normally would enter quiescence. Par6 proteins were shown to regulate cell proliferation via the canonical PI3K/Akt pathway, which is one of the most commonly deregulated cell proliferation promoting pathways in cancer cells. The results demonstrate the unknown function of Par6B and Par6G proteins as cell proliferation regulators and a previously unrecognized relation between Par6 proteins and PI3K/Akt pathway. However, the detailed interaction between Par6 proteins and the PI3K/Akt pathway ought to be investigated further. In addition, the results revealed that Par6 proteins have different effects on cell proliferation suggesting biological differences between Par6 proteins and that certainly bears further investigation. In conclusion, the study presents a previously unknown connection between epithelial integrity regulators and cancer-relevant cell proliferation promoting pathways, which may provide new targets for therapeutic intervention in the future.
  • Fagerholm, Rainer; Schmidt, Marjanka K.; Khan, Sofia; Rafiq, Sajjad; Tapper, William; Aittomaki, Kristiina; Greco, Dario; Heikkinen, Tuomas; Muranen, Taru A.; Fasching, Peter A.; Janni, Wolfgang; Weinshilboum, Richard; Loehberg, Christian R.; Hopper, John L.; Southey, Melissa C.; Keeman, Renske; Lindblom, Annika; Margolin, Sara; Mannermaa, Arto; Kataja, Vesa; Chenevix-Trench, Georgia; Lambrechts, Diether; Wildiers, Hans; Chang-Claude, Jenny; Seibold, Petra; Couch, Fergus J.; Olson, Janet E.; Andrulis, Irene L.; Knight, Julia A.; Garcia-Closas, Montserrat; Figueroa, Jonine; Hooning, Maartje J.; Jager, Agnes; Shah, Mitul; Perkins, Barbara J.; Luben, Robert; Hamann, Ute; Kabisch, Maria; Czene, Kamila; Hall, Per; Easton, Douglas F.; Pharoah, Paul D. P.; Liu, Jianjun; Eccles, Diana; Blomqvist, Carl; Nevanlinna, Heli; kConFab Investigators (2015)
    We have utilized a two-stage study design to search for SNPs associated with the survival of breast cancer patients treated with adjuvant chemotherapy. Our initial GWS data set consisted of 805 Finnish breast cancer cases (360 treated with adjuvant chemotherapy). The top 39 SNPs from this stage were analyzed in three independent data sets: iCOGS (n=6720 chemotherapy-treated cases), SUCCESS-A (n=3596), and POSH (n=518). Two SNPs were successfully validated: rs6500843 (any chemotherapy; per-allele HR 1.16, 95% C.I. 1.08-1.26, p=0.0001, p((adjusted))=0.0091), and rs11155012 (anthracycline therapy; per-allele HR 1.21, 95% C.I. 1.08-1.35, p=0.0010, p((adjusted))=0.0270). The SNP rs6500843 was found to specifically interact with adjuvant chemotherapy, independently of standard prognostic markers (p((interaction))=0.0009), with the rs6500843-GG genotype corresponding to the highest hazard among chemotherapy-treated cases (HR 1.47, 95% C.I. 1.20-1.80). Upon trans-eQTL analysis of public microarray data, the rs6500843 locus was found to associate with the expression of a group of genes involved in cell cycle control, notably AURKA, the expression of which also exhibited differential prognostic value between chemotherapy-treated and untreated cases in our analysis of microarray data. Based on previously published information, we propose that the eQTL genes may be connected to the rs6500843 locus via a RBFOX1-FOXM1 -mediated regulatory pathway.